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Hnatova, Martina, Micheline Wésolowski-Louvel, Guenaëlle Dieppois, Julien Deffaud i Marc Lemaire. "Characterization of KlGRR1 and SMS1 Genes, Two New Elements of the Glucose Signaling Pathway of Kluyveromyces lactis". Eukaryotic Cell 7, nr 8 (13.06.2008): 1299–308. http://dx.doi.org/10.1128/ec.00454-07.
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ABSTRACT The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Δsms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Δsms1 mutation.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen i M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis". Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882-3889.1993.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen i M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882.
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The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
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Naik, Abani Kanta, Aaron T. Byrd, Aaron C. K. Lucander i Michael S. Krangel. "Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes". Journal of Experimental Medicine 216, nr 1 (13.12.2018): 231–43. http://dx.doi.org/10.1084/jem.20181402.
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Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.
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Fisher, Megan, i Craig Bassing. "Pre-B cells suppress RAG expression in response to DNA double-strand breaks (HEM1P.225)". Journal of Immunology 194, nr 1_Supplement (1.05.2015): 50.8. http://dx.doi.org/10.4049/jimmunol.194.supp.50.8.
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Abstract The ability of the Rag1/Rag2 (RAG) endonuclease to assemble antigen receptor (AgR) genes is essential for adaptive immunity. However, aberrant induction or repair of RAG-induced DNA double strand breaks (DSBs) can lead to oncogenic AgR translocations. We have previously shown that RAG-induced DSBs in pre-B cells activate the ATM kinase to prevent RAG cleavage of the homologous allele, and to inhibit expression of Rag1 and Rag2. These breaks also suppress expression of Gadd45α, which promotes Rag1 and Rag2 transcription. Since DSBs induced by ionizing radiation (IR) signal through ATM to increase Gadd45α expression in all other cell types including mature B cells, these results indicate that RAG DSBs and/or pre-B cells use unique mechanisms to regulate Gadd45α expression. We now demonstrate that IR-induced DSBs in pre-B cells signal through the ATM kinase to down-regulate Gadd45α expression. We also demonstrate that these IR DSBs signal ATM-dependent suppression of Rag1 and Rag2 transcription, without affecting stability of Rag1 or Rag2 mRNA. Because the presence of multiple DSBs in a cell greatly increases the risk of translocation, we hypothesize that pre-B cells have developed a unique developmental stage-specific DSB response to suppress the induction of RAG DSBs in the presence of other DSBs. Our ongoing studies will determine the role of this pre-B cell specific DSB response in suppressing AgR translocations and enforcing mono-allelic assembly of immunoglobulin genes.
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Klemm, Lars, Srividya Swaminathan, Elli Papaemmanuil, Anthony M. Ford, Mel Greaves, Rafael Casellas, David Schatz, Michael R. Lieber i Markus Muschen. "Exposure to Inflammatory Immune Responses As Driver of Clonal Evolution in Childhood Acute Lymphoblastic Leukemia". Blood 126, nr 23 (3.12.2015): 166. http://dx.doi.org/10.1182/blood.v126.23.166.166.
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Abstract Background: Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1 gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear. Rationale: Activation Induced Cytidine Deaminase (AID) and Recombination Activation Genes 1 and 2 (RAG1-RAG2) are genetic modifiers of the immunoglobulin (Ig) genes that are expressed during normal B cell development. Although AID and RAG1/RAG2 are thought to be segregated to early (RAG1/RAG2) and late (AID) stages of B cell development, respectively, we found that the two enzymes can be concurrently expressed during early B-lymphopoiesis in the context of repeated inflammatory stimuli. Results: Our experiments identified transitional pre-B cells as the subset that is particularly vulnerable to concomitant expression of AID and RAG1-RAG2 with earlier B cells being protected by IL7 signaling.Human B cells from children lacking a functional IL-7 receptor (IL-7R) displayed both increased expression and activity of AID concurrently with RAG1-RAG2. These results demonstrated that AID activation in both mouse and human early B cell compartments increases genetic instability. Although concurrent activation of AID and RAG1-RAG2 in patient samples implicated a correlation between the two enzymes in the pathogenesis of leukemia, this as such did not prove that the enzymes causally induce overt leukemogenesis. Therefore, we next evaluated the requirement of AID and RAG1-RAG2 in leukemogenic transformation, and identified a condition that leads to massive activation of these enzymes in a pre-leukemic B cell. Importantly, AID and RAG1-RAG2 expression increased dramatically during inflammatory immune responses (e.g. infection), where both these enzymes diversify the antibody repertoire and improve its affinity to antigens from infectious pathogens. We therefore tested whether the pre-B cell subset that concurrently expresses AID and RAG1-RAG2 can respond to an inflammatory stimulus, such as LPS. We observed that pre-B cells require protection from IL7, which prevents aberrant activation of AID. In the absence of protective IL-7, these pre-B cells acquired responsiveness to LPS and strongly activated AID concurrently with RAG1-RAG2 enzymes. We developed IL7-dependent pre-B cell cultures as a disease model for ETV6-RUNX1 pre-leukemia and tested the role of AID and RAG1 in the progression of pre-leukemic clones. To this end, we expanded ETV6-RUNX1 pre-B cells from wildtype (AID and RAG1 expressing) mice, or from mice lacking AID (Aid-/-Rag1+/+) or RAG1 (Aid+/+Rag1-/-). We then challenged pre-B cell cultures by withdrawal of IL7 (loss of protection) and inflammatory stimuli (LPS) and transplanted pre-B cells that had undergone five cycles of -IL7/LPS challenge. Upon transplanting -IL7/LPS-treated Aid+/+Rag1+/+ or Aid-/-Rag1+/+ or Aid+/+Rag1-/- pre-B cells containing ETV6-RUNX1 into NOD-SCID recipient mice, we observed that loss of either Aid or Rag1 dramatically prolonged the latency and reduced the penetrance of leukemia in transplant recipients. This proved that AID and RAG1-RAG2 causally accelerate clonal evolution of a pre-leukemic B cell towards leukemia. Our findings provide a mechanism by which pre-leukemic clones carrying a prenatal genetic lesion such as ETV6-RUNX1 can evolve through infectious and inflammatory stimuli ultimately leading to full blown leukemia. Conclusion: The impact of inflammatory stimuli on leukemogenesis has been previously implicated in multiple epidemiological studies. For instance, day-care attendance primed the immune system during early childhood and is thought to protect against exacerbation of B cell responses and to prevent collateral damage driving clonal evolution towards leukemia. Although inflammation (LPS stimulation) seems to play a role in accelerating pre-B leukemogenesis in our model, further experiments testing actual infectious pathogens are needed to corroborate this concept. Moreover, it is crucial to test whether leukemogenesis is accelerated in individuals infected with restricted classes of pathogens, not all of which may activate AID in pre-B cells. Disclosures No relevant conflicts of interest to declare.
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Hao, Bingtao, Abani Kanta Naik, Akiko Watanabe, Hirokazu Tanaka, Liang Chen, Hunter W. Richards, Motonari Kondo i in. "An anti-silencer– and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development". Journal of Experimental Medicine 212, nr 5 (6.04.2015): 809–24. http://dx.doi.org/10.1084/jem.20142207.
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Rag1 and Rag2 gene expression in CD4+CD8+ double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.
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Swaminathan, Srividya, Lars Klemm, Eugene Park, Anthony M. Ford, Soo-mi Kweon, Daniel Trageser, Brian Hasselfeld i in. "Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia". Blood 124, nr 21 (6.12.2014): 861. http://dx.doi.org/10.1182/blood.v124.21.861.861.
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Abstract Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia. Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion. Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions. Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. Disclosures No relevant conflicts of interest to declare.
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Lee, Baeck-seung, Joseph D. Dekker, Bum-kyu Lee, Vishwanath R. Iyer, Barry P. Sleckman, Arthur L. Shaffer, Gregory C. Ippolito i Philip W. Tucker. "The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination". Molecular and Cellular Biology 33, nr 9 (25.02.2013): 1768–81. http://dx.doi.org/10.1128/mcb.00987-12.
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Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a lox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
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Bories, JC, JM Cayuela, P. Loiseau i F. Sigaux. "Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression". Blood 78, nr 8 (15.10.1991): 2053–61. http://dx.doi.org/10.1182/blood.v78.8.2053.2053.
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Abstract Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V- (D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.
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Bories, JC, JM Cayuela, P. Loiseau i F. Sigaux. "Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression". Blood 78, nr 8 (15.10.1991): 2053–61. http://dx.doi.org/10.1182/blood.v78.8.2053.bloodjournal7882053.
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Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V- (D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.
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Malshetty, Vidyasagar, Jian Chen, Mary Hanna i Patricia Cortes. "Role of Pax5 and YY1 in regulation of V(D)J recombination (111.1)". Journal of Immunology 188, nr 1_Supplement (1.05.2012): 111.1. http://dx.doi.org/10.4049/jimmunol.188.supp.111.1.
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Abstract The infinite variety of antigen-binding receptors originates from the rearrangement of B and T-cell receptor loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. Deregulation of this process, leads to a spectrum of diseases including immunodeficiency and leukemia. These leukemias are believed to arise from RAG1/2 mediated oncogenic chromosomal translocations. The transcription factor Pax5 is critical for B cell development and is known that, it interacts with RAG1/2 and mediates V to DJ recombination. Pax5 is also been implicated in human B cell malignancies. Another multifunctional transcription factor, YY1, is known to play a role in V to DJ recombination similar to Pax5. We were able to see an interaction of YY1 with RAG1/2 and Pax5, when these proteins were over-expressed in 293T cells. When we performed a plasmid based recombination assay in presence of Pax5, YY1 or both a consistent decrease in recombination levels was observed. To elucidate the mechanism of inhibition, we analyzed the recombination intermediates and found that the initial cleavage step is inhibited by these two factors. In addition, purified Pax5 and YY1 inhibit the in vitro cleavage activity of RAG1/2 recombinase. The expression of a mutant Pax5 increased the RAG recombinase activity, suggesting the importance of these two factors in regulation of RAGs mediated recombination.
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Swaminathan, Srividya, Lars Klemm, Anthony M. Ford, Klaus Schwarz, Rafael Casellas, Lothar Hennighausen, Huimin Geng i in. "Cooperation Between Aid and the Rag1/Rag2 V(D)J Recombinase Drives Clonal Evolution of Childhood Acute Lymphoblastic Leukemia". Blood 120, nr 21 (16.11.2012): 519. http://dx.doi.org/10.1182/blood.v120.21.519.519.
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Abstract Abstract 519 Background and Rationale: In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2. To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients. Conclusion: While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia. Disclosures: No relevant conflicts of interest to declare.
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Schabla, N. Max, i Patrick C. Swanson. "The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line". PLOS ONE 16, nr 10 (14.10.2021): e0258683. http://dx.doi.org/10.1371/journal.pone.0258683.
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The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.
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Schabla, N. Max, i Patrick C. Swanson. "The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line". PLOS ONE 16, nr 10 (14.10.2021): e0258683. http://dx.doi.org/10.1371/journal.pone.0258683.
Pełny tekst źródłaStreszczenie:
The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.
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Lassoued, Kaiss, Vincent Fuentes, Hussein Gamlouch, Eliane Bissac, Jean-Pierre Marolleau, Jacques Rochette i Hicham Bouhlal. "Role of the MAPK and PI3-Kinase/Akt Pathways in the Pre-B Cell Receptor (pre-BCR)-Induced NF-κb Activation and Rag1 and Rag2 Down Regulation." Blood 114, nr 22 (20.11.2009): 2667. http://dx.doi.org/10.1182/blood.v114.22.2667.2667.
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Abstract Abstract 2667 Poster Board II-643 The pre-BCR acts as a critical checkpoint in pre-B cell development and might be also involved in leukemogenesis. Using the 697 and Nalm6 human pre-B cell lines, we have previously shown that pre-BCR stimulation resulted in cell cycle progression associated with activation of number of adaptors and signaling pathways including the PI3-Kinase/Akt, Ras/MAPK, AP1 and the canonical NFkB pathway. We have also demonstrated that Src kinases together with Syk played a crucial role in controlling the pre-BCR-associated functions, acting upstream the above-mentioned signaling pathways. Pre-BCR crosslinking also induced down regulation of Rag1 and Rag2 transcription. In this study we aimed to evaluate the role of MAPK and Akt in the pre-BCR-induced NF-kB activation and Rag1/2 down modulation. For this purpose the 697 pre-B cells and normal bone marrow primary pre-B cells were treated with the U0126 and LY294002, and with MEK1/2 and Akt inhibitors, respectively. A dominant negative form of Akt fused to the HIV1 Tat peptide was also used to inhibit the PI3-Kinase/Akt pathway. We bring evidence that LY294002 could alter the pre-BCR-induced NF-kB activation by inhibiting : i) p105 degradation, ii) p50 NF-kB1 nuclear translocation and, iii) the binding of p50 to an oligonucleotide containing a specific consensus sequence. On the contrary, U0126 significantly enhanced p105 degradation, indicating that MAPK and Akt exerted antagonistic effects on the pre-BCR-induced NF-kB activation. Strikingly the baseline levels of Rag1 and Rag2 transcripts were increased in the LY294002 but not the U0126-treated pre-B cells. Futhermore, both inhibitors were shown to induce a strong increase in the expression of Rag1 and Rag2 transcripts upon pre-BCR crosslinking, suggesting that this receptor exerts dual effects on Rag1/2 expression with a predominant negative regulatory component mediated by both PI3-K and MAPK. No changes in the levels of Pax5, E2A, EBF, IFR4, IRF8, FOXO1, FOXO3, Myb, MAZ, LEF1 and SP1 (transcription factors implied in the regulation of Rag1 and Rag2 transcription) were observed in the pre-BCR stimulated or unstimulated-697 cells, treated or not with the MAPK and Akt inhibitors. Our results suggest that the pre-BCR signaling is a complex and tightly self-controlled process, which deregulation might alter cell growth and survival pathways via NF-kB as well as genomic stability trough Rag1/2 expression. Disclosures: No relevant conflicts of interest to declare.
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Meru, Nadine, Andreas Jung, Irith Baumann i Gerald Niedobitek. "Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils". Blood 99, nr 2 (15.01.2002): 531–37. http://dx.doi.org/10.1182/blood.v99.2.531.
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Abstract V(D)J recombination in lymphocytes is mediated by 2 recombination-activating genes, RAG1 and RAG2,which are expressed during lymphocyte development in bone marrow and thymus. Prompted by studies reporting re-expression of the RAGs in germinal center B cells, the expression of RAGs and terminal deoxynucleotidyl transferase (TdT) in human lymphoid tissues was examined using in situ hybridization and immunohistochemistry, respectively. Here it is shown that RAGs and TdT are not reinduced in germinal center reactions. However, RAG+/TdT+ cells are frequently present in extrafollicular areas of tonsils mainly at the boundary between lymphoid tissue and fibrous scaffold. Phenotypic analyses suggest that these cells are B cells. Finally, it is shown that RAG+/TdT+ cells are found more frequently in tonsils than in other peripheral lymphoid tissues. This may reflect an increased influx of RAG+/TdT+ cells as a result of higher antigenic stimulation at this site. Alternatively, this observation may indicate that the tonsils are an additional site of lymphocyte ontogeny.
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Miot, Charline, Rahul Arya, Thomas Burn, Edward M. Behrens i Craig Bassing. "Elucidating roles of the Rag1 N-terminus and RAG DSBs in shaping the cellular response to TCRa recombination". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 80.8. http://dx.doi.org/10.4049/jimmunol.204.supp.80.8.
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Abstract The RAG1/RAG2 (RAG) endonuclease generates lymphocyte antigen receptor gene diversity via V(D)J recombination. The large numbers of V, D, and J segments and inherent imprecision in repair of RAG DNA double strand breaks (DSBs) together establish a vast diversity of antigen receptor specificities, including self-reactive receptors. Mechanisms have evolved to negatively select self-reactive cells and inhibit autoimmunity. In humans, deletion of the RAG1 N-terminus causes Omenn Syndrome, a fatal immunodeficiency with ab T cell-based autoimmunity. We discovered impaired negative selection in a spontaneous homozygous Rag1 mutant mouse with loss of the Rag1 N-terminus. This Rag1 region, which is not required for V(D)J recombination, has intrinsic ubiquitin ligase activity and interacts with another ubiquitin ligase and a kinase. We hypothesize the RAG1 N-terminus signals from RAG DSBs induced during TCRa recombination to shape the thymocyte proteome and enhance negative selection. To test our hypothesis, we have made mice whose thymocytes lack RAG DSBs or harbor RAG DSBs induced by wild-type Rag1 or mutant Rag1 lacking the N-terminus or intrinsic ubiquitin ligase activity. Our preliminary data suggest that RAG DSBs and distinct RAG1 N-terminus activities up-regulate expression of Zap70, an intracellular protein that transmits TCR signals, and CD80, a transmembrane protein that co-stimulates T cells engaging antigen. These data are consistent with a model wherein the RAG1 N-terminus facilitates negative selection through stimulating TCR signaling in thymocytes that bind self-antigens presented by thymocytes, dendritic cells, or thymic epithelial cells.
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Kuwata, Naomi, Hideya Igarashi, Takafumi Ohmura, Shinichi Aizawa i Nobuo Sakaguchi. "Cutting Edge: Absence of Expression of RAG1 in Peritoneal B-1 Cells Detected by Knocking into RAG1 Locus with Green Fluorescent Protein Gene". Journal of Immunology 163, nr 12 (15.12.1999): 6355–59. http://dx.doi.org/10.4049/jimmunol.163.12.6355.
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Abstract It has been proposed that Ig gene rearrangement in the peritoneal cavity (Pc) B-1 cells might be involved in autoantibody generation. To study possible secondary B cell maturation, we prepared mice carrying a target integration of gfp gene into a rag1 locus (rag1/gfp mice). The GFP+ cells express rag1 mRNA and are undergoing Ig gene rearrangement. RAG1 expression was studied in Pc B-1 cells to detect cells during the stage of Ig gene rearrangement. In contrast to previous reports, Pc B-1 cells did not show RAG1 expression in adolescent or elderly mice. RAG1 expression was not induced in Pc B-1 cells in vivo after stimulation by oral or i.p. administration of LPS. Our results suggest that RAG1 expression in Pc B-1 cells is inhibited for a long period under normal condition and that this suppression is an essential state which maintains allelic exclusion of Ig genes.
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Dolence, Joseph J., Kimberly Gwin i Kay L. Medina. "Haploinsufficiency of Flt3-ligand limits RAG1 locus activation (87.2)". Journal of Immunology 182, nr 1_Supplement (1.04.2009): 87.2. http://dx.doi.org/10.4049/jimmunol.182.supp.87.2.
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Abstract Evidence is accumulating that the molecular circuitry initiating lymphoid lineage specification correlates with expression of the receptor tyrosine kinase Flt3. Recombination activating gene-1 (RAG1) locus activation is a hallmark of lymphoid lineage specification. Previous studies established that expression of a RAG1-GFP/+ reporter begins in a subset of lineage negative (Lin-) cells that express high levels of c-kit and Sca-1 (LSK+). To investigate a regulatory connection between Flt3 signaling and RAG1 locus activation from this stage, RAG1-GFP/+ knockin mice expressing wildtype, heterozygote, and knockout Flt3-ligand (FL) alleles were generated. Mono-allelic expression of the FL gene reduced FL transcripts by half, providing an in vivo model to evaluate threshold requirements for FL in lymphocyte development. Percentages and numbers of LSK+ cells were similar in FL+/+ and FL+/- mice, but significantly decreased in FL-/- animals. Analysis of GFP expression within the LSK+ compartment revealed a FL dose dependent reduction in frequencies and numbers of GFP+ cells. The requirement for threshold levels of FL in regulation of GFP expression extended into the B220- c-kitlo IL-7R+ lymphoid progenitor and CD43+ B220+ Pro-B compartments, but not in later stage B cell precursors. These findings establish a critical requirement for FL in regulating one event in lymphoid specification, RAG1 locus activation. These studies were supported by startup funds to K.L.M. from Mayo Clinic.
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Shaw, Albert C., Wojciech Swat, Roger Ferrini, Laurie Davidson i Frederick W. Alt. "Activated Ras Signals Developmental Progression of Recombinase-activating Gene (RAG)-deficient Pro-B Lymphocytes". Journal of Experimental Medicine 189, nr 1 (4.01.1999): 123–29. http://dx.doi.org/10.1084/jem.189.1.123.
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To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.
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Gollwitzer, Peter, Nina Grützmacher, Sabine Wilhelm, Daniel Kümmel i Constantinos Demetriades. "A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids". Nature Cell Biology 24, nr 9 (wrzesień 2022): 1394–406. http://dx.doi.org/10.1038/s41556-022-00976-y.
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AbstractAmino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.
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Igarashi, Hideya, Naomi Kuwata, Kumiko Kiyota, Kiminobu Sumita, Toshio Suda, Shiro Ono, Steven R. Bauer i Nobuo Sakaguchi. "Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfpknockin mice". Blood 97, nr 9 (1.05.2001): 2680–87. http://dx.doi.org/10.1182/blood.v97.9.2680.
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Abstract Secondary rearrangements of immunoglobulin gene segments that generate a new antibody repertoire in peripheral B cells have been described as receptor revision and occur by as yet unknown mechanisms. To determine the importance of recombination activating gene (RAG) expression in receptor revision, heterozygous rag1/green fluorescent protein (gfp) knockin mice were used to examine the location of RAG1 expression in the germinal centers (GCs) of lymphoid follicles after immunization with a variety of T-cell–dependent antigens. Immunization of rag1/gfp heterozygous mice orrag1 homozygous knockout mice reconstituted withrag1/gfp heterozygous spleen cells caused the down-regulation of RAG1/GFP signal in GCs. Although some RAG1/GFP+ cells appeared in regions surrounding the peanut agglutinin (PNA)+GL-7+ GC area, RAG1/GFP+ cells did not accumulate in the central region. In addition, the stimulation of spleen B cells with anti-μ antibody plus interleukin-4 (IL-4) or with anti-CD40 monoclonal antibody plus IL-7 did not induce GFP signals at detectable levels in vitro. These results clearly demonstrate that RAG1 re-expression either does not occur or is at extremely low levels in antigen-driven B cells in GCs of secondary lymphoid follicles, suggesting that other mechanisms may mediate the gene rearrangements observed in receptor revision.
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Schabla, N. Max, Greg A. Perry, Victoria L. Palmer i Patrick C. Swanson. "VprBP (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation". Journal of Immunology 201, nr 3 (20.06.2018): 930–39. http://dx.doi.org/10.4049/jimmunol.1800054.
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Pike-Overzet, Karin, Christopher Baum, Robbert G. M. Bredius, Marina Cavazzana, Gert-Jan Driessen, Willem E. Fibbe, H. Bobby Gaspar i in. "Successful RAG1-SCID gene therapy depends on the level of RAG1 expression". Journal of Allergy and Clinical Immunology 134, nr 1 (lipiec 2014): 242–43. http://dx.doi.org/10.1016/j.jaci.2014.04.033.
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Carroll, Virginia A., Mark K. Lafferty, Luigi Marchionni, Joseph L. Bryant, Robert C. Gallo i Alfredo Garzino-Demo. "Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice". Proceedings of the National Academy of Sciences 113, nr 46 (31.10.2016): 13168–73. http://dx.doi.org/10.1073/pnas.1615258113.
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HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear, but it may involve chronic B-cell activation, inflammation, and/or impaired immunity, possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly, because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region, thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model, and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined, only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells, and were CD19+IgM−IgD−CD93+CD43+CD21−CD23−VpreB+CXCR4+. Consistent with the pro-B-cell stage of B-cell development, microarray analysis revealed enrichment of transcripts, including Rag1, Rag2, CD93, Vpreb1, Vpreb3, and Igll1. We confirmed RAG1 expression in Tg26 tumors, and hypothesized that HIV-1 matrix protein p17 may directly induce RAG1 in B cells. Stimulation of human activated B cells with p17 enhanced RAG1 expression in three of seven donors, suggesting that intracellular signaling by p17 may lead to genomic instability and transformation.
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Xu, Mei, Brenda Reid i Chaim M. Roifman. "Identification of a novel RAG1 hypomorphic mutation in a child presenting with disseminated vaccine-strain varicella". LymphoSign Journal 8, nr 1 (1.03.2021): 5–10. http://dx.doi.org/10.14785/lymphosign-2021-0014.
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Background: Recombination-activating gene 1 (RAG1) and recombination-activating gene 2 (RAG2) encode unique lymphocyte endonuclease proteins that are crucial in T and B cell development through V(D)J recombination. RAG1 gene defects lead to variable phenotypes, ranging from immunocompetent to severe combined immunodeficiency (SCID). Curative therapy for severe manifestations can be achieved through hematopoietic stem cell transplantation (HSCT). Advances in genomic sequencing have led to the discovery of new variants and it is recognized that the level of recombinase activity correlates with disease severity. Aim: To report the clinical presentation, immunological work-up, decision process to undergo HSCT, and confirmatory genetic diagnosis in a patient who was well until her initial presentation with disseminated vaccine-strain varicella. Methods: Clinical data was gathered through retrospective chart review. Immunological investigations, targeted gene sequencing, and thymic biopsy results were reviewed. Further genetic analysis, including whole exome and whole genome sequencing was performed. Results: Whole exome sequencing identified a single missense mutation in RAG1, R474C (c.1420C>T), which would not account for the clinical presentation. Healthy individuals with only 1 mutation have been reported. Subsequently, whole genome sequencing revealed a novel second heterozygous missense variant, H945D (c.2833G>T) in the RAG1 gene. Conclusion: Hypomorphic RAG1 mutations with residual activity have a diverse phenotypic expression. Identifying and understanding the implications of these mutations is crucial for disease prognostication and tailoring management. Statement of novelty: We present a novel RAG1 missense variant, with likely complete or partial loss of function, in a patient with significant impairment in cellular immunity.
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Muire, Preeti Judith, Larry Hanson, Jeffrey Yoder i Lora Petrie-Hanson. "Transcript analysis of natural killer (NK) cell specific genes in the liver, kidney and spleen tissues of rag1 −/− mutant zebrafish in response to in vivo administration of TLR ligands". Journal of Immunology 196, nr 1_Supplement (1.05.2016): 216.4. http://dx.doi.org/10.4049/jimmunol.196.supp.216.4.
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Abstract T and B cells mediate specific protective immunity. NK cells can also mediate a memory response to murine cytomegalovirus in T and B cell deficient rag2−/− mice. T and B cell deficient rag1−/− mutant zebrafish mediate protective immunity to intracellular bacteria. NK cells have not been characterized in zebrafish due to a lack of tools to target specific markers. The aim of this study was to evaluate NK cell tissue distribution and transcript response to TLR ligands in rag1−/− mutant zebrafish. We accomplished this by determining the level of expression of ifnγ, t-bet, nitr9, and NK lysin (nkl) a, b, c and d transcripts following exposure to β glucan, Poly I:C, R-848 and VTX-2337. Expression levels were normalized to the basal level of expression and the fold changes were compared to that of endotoxin free PBS (carrier control) injected group. R848 and VTX-2337 exposure changed the gene expression profile in a tissue specific manner. Nitr9 protein expression was analyzed by western blot and detectable levels were found in the liver. Nitr9 expression correlated with the expression of ifnγ in liver and kidney. In another experiment, fish were injected with TLR ligand when vaccinated, were only vaccinated, or not vaccinated. After bacteria challenge, fish that received β glucan or R848 when vaccinated had the highest survival. This suggests the TLR ligands have an adjuvant activity on NK cell protective responses. Our results of NK cell stimulation indirectly demonstrate the presence of NK cells in zebrafish and suggest a possible means of enhancing NK cell based immunity. These findings demonstrate the clinical significance of the rag1−/− mutant zebrafish model.
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Han, S., B. Zheng, D. G. Schatz, E. Spanopoulou i G. Kelsoe. "Neoteny in Lymphocytes: Rag1 and Rag2 Expression in Germinal Center B Cells". Science 274, nr 5295 (20.12.1996): 2094–97. http://dx.doi.org/10.1126/science.274.5295.2094.
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Galler, Gunther R., Cornelia Mundt, Mathew Parker, Roberta Pelanda, Inga-Lill Mårtensson i Thomas H. Winkler. "Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components". Journal of Experimental Medicine 199, nr 11 (1.06.2004): 1523–32. http://dx.doi.org/10.1084/jem.20031523.
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Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre–B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible μHC transgene in Rag2−/− pro–B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-α. Furthermore, in wild-type mice and in mice lacking either λ5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in μHC+LC− pre–B cells. Surprisingly, μHC without LC is expressed on the surface of pro–/pre–B cells from λ5−/−, VpreB1−/−VpreB2−/−, and SLC−/− mice. Thus, SLC or LC is not required for μHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.
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Hauer, Julia, Charles Mullighan, Estelle Morillon, Gary Wang, Julie Bruneau, Nicole Brousse, Marc Lelorc'h i in. "Loss of p19Arf in a Rag1−/− B-cell precursor population initiates acute B-lymphoblastic leukemia". Blood 118, nr 3 (21.07.2011): 544–53. http://dx.doi.org/10.1182/blood-2010-09-305383.
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Abstract In human B-acute lymphoblastic leukemia (B-ALL), RAG1-induced genomic alterations are important for disease progression. However, given that biallelic loss of the RAG1 locus is observed in a subset of cases, RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf−/−Rag1−/− mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model, we identified a new, Rag1-independent leukemia-initiating mechanism originating from a Sca1+CD19+ precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM, a similar CD34+CD19+ population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans.
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Vaitaitis, Gisela, i David Wagner. "CD40 induced TCR revision promotes tolerance to self-antigen in Type I Diabetes pathogenic CD4+CD40+ T cells. (176.11)". Journal of Immunology 188, nr 1_Supplement (1.05.2012): 176.11. http://dx.doi.org/10.4049/jimmunol.188.supp.176.11.
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Abstract Biomarkers for effector cells have been difficult to find, but we described a pathogenic CD4 T cell subset expressing CD40. We demonstrated that peripheral CD4+CD40+ T cells are necessary and sufficient to transfer autoimmune disease. Here we demonstrate that CD40 engagement of peripheral CD4+CD40+ T cells in-vitro renders those cells unable to transfer disease. We have shown that CD40 signals primary peripheral CD4+CD40+ T cells to access the recombination machinery that causes revision of TCR Vα. We demonstrate here that CD40 signals also causes revision of TCR Vβ. While this constitutes a mechanism that could result in the generation of autoaggressive T cells in the periphery, we demonstrate that this mechanism can be exploited for tolerizing autoaggressive T cells. We show that CD40 interacts directly with recombination machinery proteins RAG1, RAG2 and Ku-70 in CD4+CD40+ T cells and that CD40 becomes translocated to the nucleus upon CD40 stimulation. We also show that while CD40 can induce RAG1 and RAG2 expression, Fas signals act as a silencing mechanism. It will be important to further understand the involvement of CD40 in TCR revision in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.
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Sathe, Priyanka, David Vremec, Li Wu, Lynn Corcoran i Ken Shortman. "Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells". Blood 121, nr 1 (3.01.2013): 11–19. http://dx.doi.org/10.1182/blood-2012-02-413336.
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Abstract The developmental origin of IFN-producing plasmacytoid dendritic cells (pDCs) has been uncertain. In the present study, we tracked the development of pDCs in cultures of BM precursors stimulated with Flt3 ligand. Common myeloid precursors (CMPs) produced both conventional DCs (cDCs) and pDCs via the DC-restricted common DC precursor. Common lymphoid precursors (CLPs) produced only a few cDCs with variable efficiency, but produced pDCs via a transient intermediate precursor with B-cell potential. The pDCs of both origins produced IFN-α when stimulated with CpG oligonucleotides. The pDCs of CLP origin showed evidence of past RAG1 expression and had D-J rearrangements in IgH genes. Most pDCs and all cDCs of CMP origin lacked these signs of a lymphoid past. However, in these cultures, some pDCs of CMP origin showed evidence of past RAG1 expression and had D-J IgH gene rearrangements; most of these derived from a subset of CMPs already expressing RAG1.
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Anbazhagan, Kolandaswamy, Vincent Fuentes, Eliane Bissac, Remy Nyga, Naomi Taylor, Jacques Rochette i Kaiss Lassoued. "The Human Pre-B Cell Receptor Signaling Cascade Is Regulated Via PI-3Kinase and MAPK Pathway". Blood 118, nr 21 (18.11.2011): 1314. http://dx.doi.org/10.1182/blood.v118.21.1314.1314.
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Abstract Abstract 1314 Background: Pre-B cell receptor (pre-BCR) constitutes a major check point in the early steps of mouse and human B cell development. Several functions have been attributed to this receptor which include a delivery of proliferation and survival signals, increased sensitivity to interleukin-7 (IL-7) and down modulation of recombinase activating genes (RAG) and surrogate light chain (SLC) encoding genes. Pre-BCR is also involved in shaping the VH repertoire and preventing autoimmunity. Finally, there is increasing evidence that pre-BCR might be implicated in leukemogenesis. Most of the functions of pre-BCR have been predicted based on studies in knockout mice and leukemic cell lines. In a previous study we have shown that pre-BCR aggregation resulted in the activation of src and Syk kinases which in turn activated the PI-3K/Akt, Btk, PLCγ-2 and Ras/MAPK. In this study, we examined the pre-BCR signalling cascade using human normal primary pre-B cells with a particular focus on transcription factors activation and Rag modulation and their regulatory aspects. Methods: Pre-B cells were sorted from adult human bone marrow samples, treated or not with inhibitors of Syk (BAY61–3606), Akt (LY294002) and MEKK1 (UO126) prior to crosslink the pre-BCR by means of F(ab')2 anti-μHC. The effect of Pre-BCR signaling was examined by quantifying the transcript levels of Rag1, Rag2, E2A, EBF1, Pax5, FoxO1 and FoxO3, IRF4/8. Activation of transcription factors such as NF-κB p50, c-Fos, IRF4 and FoxO3A, was assessed by analyzing their nuclear translocation by immunofluorescence microscopy. Results: We show that NF-κB p50 is translocated into nucleus within 3h after pre-BCR stimulation. Crosslinking of pre-BCR also resulted in an enhancement of nuclear c-Fos translocation. BAY61-3606 (Syk inhibitor) treatment resulted in complete apoptosis (100 % cell death within 48h). Although treatment of normal pre-B cells with LY294002 or U0126 did not alter cell survival, nuclear translocation of pre-BCR-induced p50 NF-κB was prevented by former and enhanced by later. Conversely, c-Fos nuclear expression was inhibited by U0126 and slightly but consistently enhanced by LY294002 in association with a decrease in its cytoplasmic location. Pre-BCR stimulation also induced IRF4 translocation to the nucleus. Pre-BCR stimulation also resulted in the down regulation of Rag1 (− 48 %, P<0.01), Pax5 (− 40%, P<0.01) and E2A (− 35 %, P< 0.01) transcripts, whereas EBF1 and FoxO1 and 3 expression remained unchanged. In LY294002-treated cells, Rag1/Rag2 expression was up regulated (+130%, P< 0.01 and +251%, P< 0.01, respectively) following pre-BCR crosslinking, whereas in the presence of U0126 the pre-BCR induced Rag1/Rag2 down modulation remained unchanged. Conclusion: Our results indicate that the pre-BCR has the potential to promote pre-B cell proliferation, survival and differentiation by activating NF-kB, c-Fos and IRF4. It also has the ability to protect pre-B cells from genome instability by down-regulating Rag1/2, probably through down modulation of Pax5 and E2A. We bring evidence that PI-3 K/Akt pathway plays a crucial role in the regulation of the pre-BCR signaling cascade and that Akt-mediated NF-kB and c-Fos activation is antagonized by MAPK. Up-regulation of Rag transcripts upon Akt inhibition suggests either a feed-back negative loop or a dual effect of pre-BCR on Rag expression with an Akt-dependent Rag down regulation and an accessory pathway that enhances Rag expression. Disclosures: No relevant conflicts of interest to declare.
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Yoshikawa, Genki, Kazuko Miyazaki, Hiroyuki Ogata i Masaki Miyazaki. "The Evolution of Rag Gene Enhancers and Transcription Factor E and Id Proteins in the Adaptive Immune System". International Journal of Molecular Sciences 22, nr 11 (31.05.2021): 5888. http://dx.doi.org/10.3390/ijms22115888.
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Adaptive immunity relies on the V(D)J DNA recombination of immunoglobulin (Ig) and T cell receptor (TCR) genes, which enables the recognition of highly diverse antigens and the elicitation of antigen-specific immune responses. This process is mediated by recombination-activating gene (Rag) 1 and Rag2 (Rag1/2), whose expression is strictly controlled in a cell type-specific manner; the expression of Rag1/2 genes represents a hallmark of lymphoid lineage commitment. Although Rag genes are known to be evolutionally conserved among jawed vertebrates, how Rag genes are regulated by lineage-specific transcription factors (TFs) and how their regulatory system evolved among vertebrates have not been fully elucidated. Here, we reviewed the current body of knowledge concerning the cis-regulatory elements (CREs) of Rag genes and the evolution of the basic helix-loop-helix TF E protein regulating Rag gene CREs, as well as the evolution of the antagonist of this protein, the Id protein. This may help to understand how the adaptive immune system develops along with the evolution of responsible TFs and enhancers.
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Yokota, Takafumi, Kenji Oritani, Stefan Butz, Koichi Kokame, Paul W. Kincade, Toshiyuki Miyata, Dietmar Vestweber i Yuzuru Kanakura. "The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life". Blood 112, nr 11 (16.11.2008): 727. http://dx.doi.org/10.1182/blood.v112.11.727.727.
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Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.
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Willett, Catherine E., Jason J. Cherry i Lisa A. Steiner. "Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish". Immunogenetics 45, nr 6 (9.04.1997): 394–404. http://dx.doi.org/10.1007/s002510050221.
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Niebergall, Emily R., Emily A. Beck, Susan Bassham i William A. Cresko. "Advancing threespine stickleback as an outbred immunogenetics model by pinpointing the onset of adaptive immunity". Journal of Immunology 202, nr 1_Supplement (1.05.2019): 53.25. http://dx.doi.org/10.4049/jimmunol.202.supp.53.25.
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Abstract The development of an outbred immunogenetics model system is needed to understand how genetic variation impacts phenotypic variation of disease states in humans. Threespine stickleback fish (Gasterosteus aculeatus) provide just such a model. Stickleback are genetically tractable laboratory organisms with a well-annotated genome, but with lines drawn from populations inhabiting vastly different habitats. Individuals from different populations show high levels of genetic variation. The onset of the adaptive immune system is currently unknown in stickleback, a significant hindrance in studies of immunodeficiency diseases. To characterize the early development of adaptive immunity, we will analyze the expression of known early indicators of adaptive immunity maturation. These include recombination activating genes, rag1 and rag2, which are essential to the maturation of T and B lymphocytes, and T cell receptor genes, tcr-β and tcr-γ, which are expressed in mature T lymphocytes. To analyze the expression of rag1, rag2, tcr-β, and tcr-γ, we will perform whole mount in situ hybridization throughout a developmental time series to detect when and where the genes are first expressed, with special focus on the head kidney and the thymus, followed by qPCR to quantify the expression of the early adaptive immunity genes. Knowing when adaptive immunity onset occurs in threespine stickleback advances threespine stickleback as an outbred disease model in immunogenetics studies, allowing manipulative studies of immunological disease phenotypes in the context of genetic variation.
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Dangoudoubiyam, Sriveny, Ramesh Vemulapalli, Kathy Hancock i Kevin R. Kazacos. "Molecular Cloning of an Immunogenic Protein of Baylisascaris procyonis and Expression in Escherichia coli for Use in Developing Improved Serodiagnostic Assays". Clinical and Vaccine Immunology 17, nr 12 (6.10.2010): 1933–39. http://dx.doi.org/10.1128/cvi.00404-10.
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ABSTRACT Larva migrans caused by Baylisascaris procyonis is an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of a B. procyonis third-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-length rag1 cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. The rag1 genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascaris species-specific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascarids Toxocara canis and Ascaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection of B. procyonis larva migrans.
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Jain, Pooja, Rashida Ginwala, Paige Charlins, Ramesh Akkina, Ronak Loonawat, Breanna Caruso3, Steven Jacobson, Sreesha Sreedhar i Zafar Khan. "HTLV-1 infection and neuropathogenesis in the context of Rag1−/−γc−/− (RAG1) and BLT mice". Journal of Immunology 196, nr 1_Supplement (1.05.2016): 217.30. http://dx.doi.org/10.4049/jimmunol.196.supp.217.30.
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Abstract HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a disabling chronic inflammatory disease of the central nervous system (CNS) with similarities to multiple sclerosis (MS). To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1 in an in vivo system. Previous work from others have established that host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34+ hematopoietic stem cells and followed HTLV-1 infection. Flow cytometry and histological analyses revealed reconstitution of Rag1 and BLT mice with human immune cells, including macrophages, dendritic cells, T cells and B cells. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs of neonatal and adult Rag1 and BLT hu-mice by droplet digital PCR. Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi) in Rag-1 and BLT hu-mice. Successful infection was followed by immune activation and Tax expression within lymphoid organs. Moreover, lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord of the infected mice. These data represents the first attempt to establish HTLV-1 neuropathogenesis in the context of RAG1 and BLT mice suggesting possibility of developing a small animal model of HAM/TSP in humanized mice.
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Nganga, Vincent K., Victoria L. Palmer, Hina Naushad, Michele D. Kassmeier, Dirk K. Anderson, Greg A. Perry, Nathan M. Schabla i Patrick C. Swanson. "Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1". Blood 121, nr 19 (9.05.2013): 3855–66. http://dx.doi.org/10.1182/blood-2012-08-446732.
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Key Points Expressing dominant-negative RAG1 to inhibit BCR editing of autoreactivity in CLL-prone Eμ-TCL1 mice accelerates disease onset. Gene expression profiling studies provide evidence of distinct but convergent pathways for CLL development.
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Madagula, Kiran Kumar, Rashida Ginwala, Breanna Caruso, Zafar K. Khan, Glen M. Chew, Ajinkya Pattekar, Michael J. Corley i in. "HTLV-1 infection and neuropathogenesis in the context of Rag1−/−γc−/− (RAG1-hu) and BLT mice". Journal of Immunology 198, nr 1_Supplement (1.05.2017): 78.29. http://dx.doi.org/10.4049/jimmunol.198.supp.78.29.
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Abstract To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1, the causative agent of chronic disabling neuroinflammatory disease HAM/TSP. Host immune response that plays a critical role in the outcome of HTLV-1 infection could be better tested in the context of humanized (hu) mice. Thus, we infected neonatal and adult Balb/c-Rag1−/−γc−/− (Rag1) as well as Bone marrow-Liver-Thymic (BLT) mice with HTLV-1. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs by droplet digital PCR. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi). Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Both PVL and Tax expression was considerably higher in the adult Rag1 mice as compared to the neonates with the latter showing less than 20% PVL in the peripheral blood, brain, and liver. Moreover, signs of lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord and brain of infected mice. Thus far, several members of the CD28:B7 family of co-signaling molecules have been associated with T-cell dysfunctions in HTLV-infected patients. We found increased expression of PD-1, TIGIT and TIM-3 on CD8+ T cells in the CNS of infected hu-mice. This represents the first attempt to establish HTLV-1 neuropathogenesis in the context of Rag-1 and BLT hu-mice suggesting the possibility of developing a small animal model of HAM/TSP for testing groundbreaking therapies.
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Glynn, Rebecca, i Craig Bassing. "Elucidating the Role of NEMO and SpiC in DNA Double Strand Break Induced Inhibition of V(D)J Recombination". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 223.12. http://dx.doi.org/10.4049/jimmunol.204.supp.223.12.
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Abstract V(D)J recombination is regulated such that most lymphocytes assemble and express an antigen receptor from only one allele (allelic exclusion). In pre-B cells, RAG-induced DNA double strand breaks (DSBs) at Igκ loci signal via ATM to rapidly repress Rag1/2 transcription, inhibit accessibility of Igk loci, and limit V recombination. We hypothesize that RAG DSB-induced repression of Rag1/2 is critical to transiently limit V recombination, ensure allelic exclusion, and inhibit oncogenic Ig translocations. Fittingly, Atm−/− mice have higher frequencies of developing B cells with RAG DSBs at both Igh and Igk loci and of mature B cells with bi-allelic expression of these genes. Yet, given the multifunctional roles of ATM in the DSB response, Atm−/− phenotypes cannot be directly attributed to Rag1/2 repression. To test our hypothesis, we study mouse models lacking downstream effectors of ATM: i) SpiC, which suppresses Igk accessibility in response to RAG DSBs, and ii) NFκB essential modulator (NEMO), which represses Rag1/2 in response to genotoxic DSBs. I show here that the frequency of surface bi-allelic Igκ expression is normal on SpiC−/− B cells and increased on Nemo−/− B cells. I also show that Nemo−/− pre-B cells exhibit impaired Rag1/2 repression in response to RAG DSBs and concomitant increased RAG cleavage at Igk. Our data support that RAG DSB-induced repression of Rag1/2 transiently inhibits additional Igk rearrangements to enforce Igκ allelic exclusion independent of ATM-mediated DSB repair. I am using Nemo−/− mouse models to determine if DSB-induced Rag1/2 repression requires Nemo in pro-B cells, and if this mechanism is critical to suppress oncogenic Ig translocations independent of defects in DSB repair/checkpoint activation.
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Girschick, Hermann J., Amrie C. Grammer, Toshihiro Nanki, Marlyn Mayo i Peter E. Lipsky. "RAG1 and RAG2 Expression by B Cell Subsets from Human Tonsil and Peripheral Blood". Journal of Immunology 166, nr 1 (1.01.2001): 377–86. http://dx.doi.org/10.4049/jimmunol.166.1.377.
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Lynch, Sara, Dermot Kelleher, Ross McManus i Cliona O'Farrelly. "RAG1 and RAG2 expression in human intestinal epithelium: evidence of extrathymic T cell differentiation". European Journal of Immunology 25, nr 5 (maj 1995): 1143–47. http://dx.doi.org/10.1002/eji.1830250502.
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Kurtz, Courtney C., Ioannis Drygiannakis, Makoto Naganuma, Sanford Feldman, Vasileios Bekiaris, Joel Linden, Carl F. Ware i Peter B. Ernst. "Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells". American Journal of Physiology-Gastrointestinal and Liver Physiology 307, nr 3 (1.08.2014): G338—G346. http://dx.doi.org/10.1152/ajpgi.00404.2013.
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Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A2A adenosine receptor (A2AAR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A2AAR−/− mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A2AAR controls. Comparison of T cell subsets in wild-type and A2AAR−/− mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A2AAR on CD45RBHI and CD45RBLO Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RBHI with CD45RBLO from wild-type mice into RAG1−/−/A2AAR−/− mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1−/− mice does not induce colitis. This suggests that the presence of A2AAR on recipient cells is also important for controlling colitis. To investigate the role of A2AAR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1−/− or RAG1−/−/A2AAR−/− mice into irradiated RAG1−/− mice. After adoptive transfer, these recipients did not develop colitis, regardless of A2AAR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A2AAR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.
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Jarvelainen, Harri, Wei-Hsiang Lin, Moorim Kang, Xiaojie Zhou, Robert F. Place i Long-Cheng Li. "Preclinical development of RAG1-40-31L: A novel small activating RNA-lipid conjugate targeting tumor suppressor gene p21 for treatment of non-muscle invasive bladder cancer." Journal of Clinical Oncology 41, nr 16_suppl (1.06.2023): e16620-e16620. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.e16620.
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e16620 Background: Loss-of-function of tumor suppressor genes are the dominant driving force in tumorigenesis. Restoration in function and/or expression of these genes holds tremendous therapeutic potential in cancer treatment. Bladder cancer is the 9th leading cause of cancer-related deaths in the United States in which ~75% of all newly diagnosed cases are non-muscle invasive bladder cancer (NMIBC). Standard-of-care includes intravesical installation of Bacillus Calmette-Guerin following tumor resection with an expected failure rate of ~50% within 6 months for patients with high-grade tumors. RNA activation (RNAa) is a mechanism in which short RNA duplexes termed small activating RNA (saRNA) induce gene transcription by targeting non-coding regulatory sequence ( i.e., promoters) embedded in chromatin. Thus, saRNA-induced overexpression of p21WAF1/CIP1 (p21) – a tumor suppressor of several anti-growth pathways – may hold therapeutic potential in bladder cancer. Through conjugation of a novel lipid ( i.e., C5X5), we have created a p21-targeting saRNA drug candidate referred to as RAG01-40-31L for delivery to bladder cancer cells in vivo. The aim of this work was to characterize the intravesical administration of RAG01-40-31L on p21 induction and its anti-tumor potential in NMBIC models. Methods: In vitro expression analyses were performed to investigate p21 induction via saRNA in KU7 and T24 bladder cancer cell lines by RT-qPCR and immunoblot analysis. Cell cycle analysis and tumorigenicity assays examined impact of RAG1-40-31L on cancer cell growth. Orthotopic human bladder tumors were established in female BALB/c nude mice using the recombinant cell line KU7 engineered to overexpress luciferase reporter. Tumor growth was monitored in live animals via luciferase bioluminescence using an in vivo imaging system. Plasma and tissue were harvested for drug exposure and expression analytics. Results: In vitro analyses indicated RAG1-40-31L activated p21 expression by approximately 6-fold and inhibited bladder cancer cell growth ( i.e., cell cycle arrest, apoptosis, and senescence) in a dose-dependent manner with low nanomolar potency. Intravesical administration of RAG1-40-31L (3 times per week for 2 weeks) inhibited growth of orthotopic tumors by 65% in mouse bladder models. Single-dose pharmacokinetics demonstrated a half-life (t1/2) of ~114 h with minimal systemic exposure (~0.05% of that of bladder tissue). Conclusions: These data constitute a preclinical proof-of-concept for saRNA as a novel p21-targeting modality in the treatment of cancer. The C5X5 lipid conjugate appears to be a viable approach to enhance oligonucleotide delivery and activity in bladder cancer. Preclinical safety assessment studies to enable clinical trials of RAG01-40-31L are currently underway.
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Sacramento, Lais A., Camila Farias Amorim, Claudia Lombana i Phillip Scott. "CD8 +T cells require CCR5 expression to mediate immunopathology in cutaneous leishmaniasis". Journal of Immunology 210, nr 1_Supplement (1.05.2023): 81.15. http://dx.doi.org/10.4049/jimmunol.210.supp.81.15.
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Abstract Cytolytic CD8 +T cells mediate immunopathology in cutaneous leishmaniasis by a mechanism dependent on degranulation and lysis of Leishmania-infected cells, culminating in NLRP3 activation and IL-1b release. Here, we sought to identify chemokine receptors involved in CD8 +T cell migration to the lesion that could be employed as a treatment target to ameliorate disease severity. A transcriptional study identified the profile of chemokine receptors that predicts treatment failure in Leishmania braziliensispatients and identifies possible targets that may be involved in CD8 +T cell migration. Using murine models of cutaneous leishmaniasis, we found that around 20% of CD8 +T-cells express CCR5 in lesions at the peak of the disease. In previous studies, we found that CD8 +T cells transferred to Rag1 −/−mice mediated increased NLRP3 and IL-1b dependent disease. Therefore, to test if CCR5 expression on CD8 +T cells was involved in their migration to lesions, we transferred wild-type or CCR5 −/−CD8 +T cells to L. braziliensisinfected Rag1 −/−mice. While infected Rag1 −/−mice reconstituted with wild-type CD8 +T cells developed severe pathology, Rag1 −/−mice that received CCR5 −/−CD8 +T cells developed smaller lesions accompanied by a significant reduction in the number of CD8 +T cells in the lesions. To test whether CCR5 blockade would control disease severity, we used maraviroc (MVC), a selective inhibitor of CCR5 approved by the FDA. MVC treatment significantly reduced lesion development without affecting parasite number in our murine models. Collectively, these results demonstrate that cytolytic CD8 +T cells migrate to leishmania lesions in a CCR5-dependent manner, and MVC treatment efficiently prevents CD8 +T-cell mediated pathology. R01 AI106842 and U01 AI08865006
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Koulnis, Miro, Ying Liu i Merav Socolovsky. "Negative Autoregulation by Fas Stabilizes the Erythroid Progenitor Pool and Accelerates the Erythropoietic Stress Response". Blood 116, nr 21 (19.11.2010): 2045. http://dx.doi.org/10.1182/blood.v116.21.2045.2045.
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Abstract Abstract 2045 Signaling and transcriptional networks frequently contain negative autoregulatory feedback loops, where gene products negatively regulate their own induction or activation. These negative autoregulatory motifs are predicted to exert dual functions, accelerating gene induction, and providing stable gene expression levels in the face of the random perturbations inherent to biological systems. These predictions were confirmed experimentally in synthetic transcriptional circuits [1,2], but it is unknown whether they also hold in naturally occurring higher level biological networks. Here we studied the role of negative autoregulation by erythroid progenitors in the control of erythropoiesis. Erythropoietic rate, which may increase ten fold its basal rate during hypoxic stress, is dependent on the size of the erythroid progenitor pool, in turn regulated by the hormone erythropoietin (Epo). We recently found that, in addition, early erythroblasts negatively regulate their own numbers, through their co-expression of the death receptor Fas and its ligand FasL. Here we investigated the role of this negative autoregulation using Fas or FasL-deficient mice. We used the naturally-occuring mutant mouse strains, lpr and gld, deficient in Fas and FasL, respectively, back crossed onto the Rag1-/- mutant background, in order to avoid the autoimmune syndrome associated with Fas mutation. We proceeded to examine basal and stress erythropoiesis in the gld-Rag1-/- and lpr-Rag1-/- mice, and in matched Rag1-/- controls. We found that, in the basal steady state, the average size of the spleen early erythroid progenitor pool in gld-Rag1-/- and lpr-Rag1-/- mice increased 1.5 to 2 fold, consistent with loss of a negative regulator. Further, gld-Rag1-/- mice had a significantly elevated hematocrit in spite of normal Epo blood levels. The hematocrit was normal in the lpr-Rag1-/- mice, but Epo levels in this strain were significantly lower than normal. Taken together, these genetic mouse models show that Fas-mediated apoptosis of early erythroblasts in spleen negatively regulates erythropoietic rate in the basal state. We also found that the size of the progenitor pool was highly variable between individual Fas-deficient mice, suggesting reduced ability to maintain a stable steady-state erythorpoietic rate. In addition, gld-Rag1-/- and lpr-Rag1-/- mice had a significantly delayed erythropoietic stress response. Following an injection of a single dose of Epo (300 U/25 g), the early erythroblast population in spleen, ‘EryA’ (Ter119highCD71highFSChigh, [3]) expanded 30 to 60 fold its basal size. However, this expansion was significantly delayed in gld-Rag1-/- and lpr-Rag1-/- mice. Specifically, on day 2 of the stress response, control Rag1-/- mice had a 30% larger EryA progenitor pool compared with lpr-Rag1-/- mice, a difference equivalent to 10 fold the size of the basal EryA pool. Consequently, control mice achieved a higher hematocrit 24 hours earlier than mutant gld-Rag1-/- and lpr-Rag1-/- mice. We propose that the larger expansion of EryA cells during the stress response in control mice is due to the recruitment of a reserve population of Fas-positive EryA. This reserve population, absent in mice deficient in the Fas pathway, undergoes Fas-mediated apoptosis in the basal steady state. However, high Epo levels during the stress response suppress Fas expression [3], rescuing these cells from apoptosis and accelerating the stress response. These findings show, using genetic mouse models, that the stability of stead-state erythropoietic rate and its rapid stress response are key outcomes of negative autoregulation within the erythroid progenitor pool. Furthermore, they show experimentally that dynamic properties of negative autoregulatory loops in simple low-level networks are also exerted in the context of complex inter-cellular, tissue level networks such as those that regulate erythroipoietic rate. References: 1. Becskei A, Serrano L (2000) Engineering stability in gene networks by autoregulation. Nature 405: 590–593. 2. Rosenfeld N, Elowitz MB, Alon U (2002) Negative autoregulation speeds the response times of transcription networks. J Mol Biol 323: 785–793. 3. Liu Y, Pop R, Sadegh C, Brugnara C, Haase VH, et al. (2006) Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo. Blood 108: 123–133. Disclosures: No relevant conflicts of interest to declare.
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Auer, Franziska, Deborah Ingenhag, Isidro Sánchez-García, Arndt Borkhardt i Julia Hauer. "Activation Induced Cytidine Deaminase (Aid) Acts As a Gate Keeper in Pro-B Cells and Prevents PB-ALL". Blood 128, nr 22 (2.12.2016): 1538. http://dx.doi.org/10.1182/blood.v128.22.1538.1538.
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Abstract Introduction: Activation induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination in splenic germinal center B cells and is implicated in retaining central B cell tolerance in the bone marrow (BM) (Cantaert et al., Immunity, 2015). Moreover, there is recent in vitro evidence that AID is upregulated in precursor B cells after exposure to LPS, contributing to the clonal evolution of pB-ALL (Swaminathan et al., Nat Immunol, 2015) (Greaves M. and Müschen M., Cancer Discovery, 2015). These studies were carried out in pre-BII / early immature B cells, which are the first B cell compartments with detectable intrinsic AID expression. However a functional role of AID in pro-B cells is still controversially discussed and a functional role of AID in leukemogenesis remains speculative. We designed an in vivo model which allowed us the investigation of intrinsic Aid expression in tumor prone pro-B cells. Our data indicate that Aid is a gate keeper at the early stage of B cell development and its loss of function facilitates the development of pB-ALL. Methods: We crossed a Rag1 deficient tumor prone mouse model (p19Arf-/-/Rag1-/-) (Hauer et al., Blood, 2011) on an Aid deficient background to obtain Aid knockout (p19Arf-/-/Rag1-/-/Aid-/-) and heterozygous (p19Arf-/-/Rag1-/-/Aid+/-) mice. Healthy and diseased mice were characterized by immunohistochemistry, Flow cytometric analysis, genome and transcriptome profiling. Cell cycle analysis was performed with pro-B cells of healthy mice. Results: P19Arf-/-Rag1-/- mice display a B cell developmental arrest at the pro-B stage and develop pB-ALL at an incidence of 26 %. Surprisingly, an additional loss of Aid in these cells accelerated the pB-ALL incidence to 98 % (44/45, median onset 25 weeks). Moreover our model reproduces the dose dependent effect of AID on regulating B cell tolerance in humans, since Aid+/- mice on the same background displayed significant disease reduction (83 %, 15/18, median onset 33 weeks, Mantel-Cox Test p=0.0175). The leukemia displayed a pro-B cell phenotype (CD19+B220+ckit+IgM-) and manifested with splenomegaly, dissemination of blast cells to the BM, peripheral blood (PB) and spleen. Pro-B tumors from p19Arf-/-Rag1-/- mice expressed Aid on transcript (qRT-PCR) and protein (western blot) level, indicating that Aid expression is not restricted to CD19+ BM cells with co-expression of a functional IgM heavy chain product but rather occurs at earlier stages of B cell development. Again this effect was dose dependent, since in pB-ALLs of p19Arf-/-Rag1-/-Aid-/+ mice Aid expression was significantly reduced. To identify the second hit we performed whole exome sequencing of murine tumors, which revealed accumulation of recurrent somatic Jak3 (R653H, V670A) and Dnm2 (G397R) mutations. To extend these findings further, Sanger sequencing of these regions displayed a mutational pattern of somatic Jak3 mutations in 60 % of Aid+/- and 80 % of Aid-/-pB-ALLs, while Dnm2 was somatically mutated in 96 % of all pB-ALLs analyzed. The detected Jak3 variants are known to induce a constitutive active downstream signaling. Loss of function mutations in DNM2 can increase the IL-7R cell surface expression, which highlight the relevance of the IL7R signaling in the context of tumor progression. However we did not observe detectable Aid expression in healthy pro-B cells of p19Arf-/-Rag1-/- animals in line with findings from Cantaert et al. On the other hand loss of Aid expression accelerates the repopulation capacity starting at the pro-B cell compartment (Kuraoka et al., Proc Natl Acad Sci, 2011). In our model Aid loss produces a dose dependent increase in proliferation and BrdU assays of B220+ sorted pro-B cells of healthy mice from the different cohorts (30 % cells in S-Phase in p19Arf-/-Rag1-/-compared to 50 % S-Phase with additional Aid loss), although Aid expression is below the detection limit. Conclusion: We present in vivo evidence that Aid has a gate keeper function in pro-B cells, which allows aberrant IL-7 dependent pro-B cells without a functional receptor to be eliminated through Aid induction. This further extends the observation that Aid mediates the clearance of autoreactive early immature B-cell clones and is required to prevent pB-ALL. In this regard Aid overexpression but also loss of Aid expression can facilitate pB-ALL development. Disclosures No relevant conflicts of interest to declare.