Littérature scientifique sur le sujet « BAFF-R »

Abstract BAFF receptor (BAFFR), a member of the TNFR superfamily, binds to the pro-survival cytokine BAFF to promote B cell development and survival, and deficiencies of either BAFFR or BAFF result in inhibition of B cell development. BAFF-R engagement recruits TRAFs 2, 3 and 6 and activates the non-canonical NF-κB2 pathway. Current models propose that TRAF3 mediates degradation of the NF-κB activating kinase NIK, concluding that BAFFR-mediated TRAF3 degradation is necessary and sufficient for NF-κB2 activation. However, our recent studies of a mutant BAFFR indicate that this model is inadequate to fully explain how BAFFR activates the NF-κB2 pathway. The A/WySnJ mouse harbors a spontaneous mutation of BAFFR in which 8 residues of the cytoplasmic tail are replaced by 22 amino acids from a viral genome. Engagement of this mutant BAFF-R fails to activate the NF-κB2 pathway, and A/WySnJ mice have a substantial B cell deficiency. However, the mutant BAFF-R shows no decrease in recruitment or receptor-mediated degradation of TRAF3, demonstrating that TRAF3 degradation is not sufficient to allow NF-κB2 activation. Our recent data instead suggest that recruitment of TRAFs 2 and 6 and the signaling protein Act1 may play important roles in BAFFR-mediated NF-κB2 activation.

Abstract The results of clinical trials evaluating CD19-targeting chimeric antigen receptor (CAR) T cells are impressive, with overall response rates of up to 90% in B cell acute lymphoblastic leukemia (ALL) and 50-80% in lymphoma. Despite initial responses, antigen-negative relapse is common following treatment with CD19-targeted therapies and is estimated to occur in up to 39% of patients. One approach of addressing this problem is to utilize dual-targeting CAR T cells, a strategy that has recently been applied to CD19/CD22 for ALL, CS1/BCMA, and BCMA/GPRC5D for multiple myeloma. Dual-targeting CARs can simultaneously target two tumor antigens and, therefore, potentially eradicate heterogeneous tumors. The initial response to dual-targeted CAR T cells is expected to provide greater tumor coverage compared to single-targeted therapy, and can potentially circumvent antigen escape. Moreover, if one tumor antigen becomes downregulated during treatment, the second targeting domain will continue to be reactive to tumors. Therefore, it is critical to identify novel targets that can be combined with CD19 in a dual-targeted immunotherapeutic platform. To expand the potential for dual targeting of ALL, we developed a CAR T cell therapy against a novel target, B-cell activating factor receptor (BAFF-R), based on the remarkable specificity of anti-BAFF-R antibodies that we previously generated. BAFF-R is expressed almost exclusively on B cells, including in patients with CD19-negative relapse, making it an ideal immunotherapeutic target. Studies demonstrate that the role of BAFF-R in B cell function and survival is conclusive, an important feature that may mitigate the tumor's ability to escape therapy through antigen loss, particularly if a non-redundant role for BAFF-R is confirmed. BAFF-R-CAR T cells demonstrate in vitro effector function and in vivo therapeutic efficacy in CD19-negative models, including patient-derived xenograft models, and are currently being evaluated clinically for the treatment of ALL (NCT04690595). We hypothesized that simultaneous targeting of CD19 and BAFF-R in a bispecific CAR platform could confer a therapeutic advantage and avoid the challenges of sequential administration of CD19 and BAFF-R monospecific CAR T cells. We leveraged our experience with CD19- and BAFF-R-CAR T cells to develop a dual-targeting, bispecific CAR with a 41BB costimulatory domain (CD19/BAFF-R dual CAR). Here, we identified the optimal orientation of the single-chain variable fragment (loop scFv) domains within the dual construct and tested the CD19/BAFF-R dual CAR T cells for their in vitro effector function and in vivo anti-leukemia activity. To evaluate the specific targeting to CD19 and BAFFR, we developed Nalm-6-BAFF-R-knockout (KO) and Nalm-6-CD19-KO cell lines. CD19/BAFF-R dual CAR T cells specifically released IFN-g following incubation with Nalm-6 CD19-/- or BAFF-R-/- cells (P<0.001) compared with un-transduced mock T cells. Both CD4+ and CD8+ CAR T cell populations exhibited effector function. To evaluate the antigen-dependent targeting of the CD19/BAFF-R dual CAR T cells in vivo, we utilized a mixed B-cell leukemia model that simulates clinical tumor heterogeneity. NOD-scid IL2Rgammanull (NSG) mice were inoculated with 2-3x10 5 of mixed Nalm-6 BAFF-R-/- and CD19-/- cells at a 1:1 ratio with a single injection. 1x10 6 CD19/BAFF-R dual CAR, CD19, or BAFF-R single CAR-T cells were administered intravenously 9-10 days later. Tumor growth was monitored by bioluminescent imaging weekly. We observed superior tumor eradication (P<0.01) and survival (P<0.01) (Figure 1) by CD19/BAFF-R dual CAR T cells compared to either single-targeting CAR and mock T cells. The adoptively transferred CD19/BAFF-R dual CAR T cells were able to persist in vivo. Our unique CD19/BAFF-R dual-targeting CAR T cells will be the first to target this combination of tumor-associated antigens. Our study demonstrated the reliability of bispecific CD19/BAFF-R dual CAR T cell therapy in inducing remission in ALL consisting of CD19-/- and BAFF-R-/- tumors. We hypothesize that simultaneous immunotherapy targeting of heterogeneous leukemic cell populations may diminish the likelihood of antigen escape and may have a significant impact on leukemia treatment by improving the therapeutic benefits of CAR T cell therapy. Figure 1 Figure 1. Disclosures Wang: Pepromene Bio, Inc.: Consultancy. Forman: Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Kwak: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company. Qin: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company.

Abstract Abstract 2642 BAFF is essential for B cell maturation, and a lack of either BAFF or its primary receptor, BAFF-R, results in a severe depletion of T2 marginal zone and follicular B cells. Elevated serum BAFF levels have been correlated with an increased risk of developing non-Hodgkin's lymphoma (NHL), along with a more aggressive phenotype. A growing body of genetic evidence points toward an association between the development of human disease and variation in genes encoding BAFF and its receptors. Recently, we characterized a novel lymphoma-associated mutation in TNFRSF13C, the gene encoding BAFF-R. This mutation (BAFF-R H159Y) encodes a His159Tyr substitution in the C-terminus of BAFF-R adjacent to the TRAF3 binding motif. Signaling through BAFF-R H159Y results in increased NF-κB activity, elevated immunoglobulin production and increased association with TRAF2, TRAF3 and TRAF6 compared to wild type (WT) BAFF-R. We have detected this mutation in 6% of total NHL cases (n=129), and in 10% of follicular lymphoma (FL) cases (n=41) evaluated thus far. We previously reported that BAFF-R H159Y expressing mouse B cells exhibited significantly more resistance to Fas ligand (FasL) induced apoptosis compared to their cells expressing BAFF-R WT, and we propose here that BAFF-R H159Y mediated increases in PI3K activity may explain such an enhanced anti-apoptotic response. In this study we now show that BAFF stimulated HEK 293 cells stably expressing BAFF-R H159Y not only display significantly increased Akt phosphorylation when compared to BAFF-R WT expressing cells, but also demonstrate robust Akt phosphorylation in the absence of BAFF. BAFF-R H159Y-dependent Akt activation also led to activation of the downstream Akt targets mTOR and GSK3β and their phosphorylation was inhibited following treatment with the PI3- kinase inhibitor wortmannin. We next examined the impact of the BAFF-R H159Y mutation on expression of BAFF-target genes. Quantitative PCR analyses revealed that BAFF-R H159Y cells exhibited a pattern of gene expression indicative of promoting cell survival, displaying significantly higher levels of BCL2, BCL2L1 and PIN1, while down-regulating expression of the pro-apoptotic gene BIM. We recently reported that TRAF6 associates with BAFF-R, and that such binding is more pronounced in cells expressing BAFF-R H159Y. In order to investigate the role TRAF6 plays in mediating BAFF-R-dependent PI3K activity, we silenced TRAF6 expression in HEK 293 and Karpas 422 lymphoma cells using TRAF6 shRNA. Reduced TRAF6 protein expression resulted in a parallel decrease in BAFF-R WT mediated phosphorylation of mTOR in Karpas 422 cells and phosphorylation of both Akt and GSK3β was markedly reduced in BAFF-R H159Y expressing HEK 293 cells. Interestingly, TRAF6 knock-down did not affect NF-kB2 activation in either Karpas 422 or HEK BAFF-R expressing cells suggesting that Akt does not play a role in BAFF-R mediated activation of non-canonical NF-kB. Finally, preliminary co-precipitation studies indicate that Akt can be recruited to BAFF-R itself, and our initial observations suggest that such an association is significantly reduced in cells expressing BAFF-R H159Y. Taken together, these studies suggest that the BAFF-R H159Y mutation confers enhanced BAFF-R-dependent PI3K signaling and pro-survival gene expression independent of BAFF. Moreover, such enhanced P13K activation is partly dependent upon TRAF6, and decreased recruitment of Akt to BAFF-R H159Y may function to increase the amount of this PI3K target for activation. Thus, BAFF-R H159Y likely contributes to BAFF signaling irregularities in NHL patients harboring this mutation, and may predispose individuals to developing lymphoma regardless of their serum BAFF concentration. Disclosures: No relevant conflicts of interest to declare.

The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.

Abstract Abstract 468 The requirement for BAFF and BAFF-R in normal human and murine B cells is well studied, but there is also significant evidence to suggest that BAFF plays an important role in malignant B cell proliferation and survival. Serum BAFF levels are elevated in patients with non-Hodgkin lymphoma (NHL) and high BAFF levels correlate with aggressive disease and a poor response to therapy. There is also increasing genetic evidence suggesting an association between the development of human disease and genetic variation in genes encoding BAFF and its receptors. Mutations in TNFRSF13B (TACI) were identified in patients with familial common variable immunodeficiency (CVID) and IgA deficiency and we have found that single nucleotide polymorphisms (SNP) in TNFSF13B (BAFF) are associated with elevated BAFF levels and risk for developing NHL. To build upon these findings we sequenced BAFF and its receptors; TNFSF13B, TNFRSF13B, TNFRSF17(BCMA), and TNFRSF13C (BAFF-R) in NHL patients to identify novel genetic variants that may be associated with NHL risk. Among 40 individual samples (20 controls and 20 follicular lymphoma (FL) cases) that were bi-directionally sequenced we identified a heterozygous cytosine to thymidine transition in 1 patient specimen at position 475 (C475T) of TNFRSF13C. The C475T transition encodes a missense substitution of tyrosine for histidine in codon 159 (H159Y) in the highly conserved cytoplasmic tail of BAFF-R, adjacent to the TRAF3 binding motif PVPAT. We next expanded our analysis of BAFF-R H159Y and analyzed NHL tumor biopsies for the presence of the mutation. 4/41 (10%) follicular lymphomas (FL), 2/42 (5%) diffuse large B cell lymphomas, 1/22 (5%) lymphoplasmacytic lymphomas (LPL), and 1/24 (4%) mucosal associate lymphoid tissue lymphomas carried the heterozygous mutation. The BAFF-R H159Y mutation was not detected in any of the normal control DNA from healthy donors (n=100). Given its close proximity to the TRAF3 binding site in the cytoplasmic domain of BAFF-R we first wanted to determine if the H159Y mutation altered BAFF induced signaling. We generated cell lines that express HA-tagged wildtype BAFF-R, BAFF-R with the H159Y mutation, or BAFF-R with an ablated TRAF3 binding site as a negative control. Analysis of cells expressing H159Y BAFF-R demonstrates that this mutation results in increased BAFF-R-mediated NFκB1 and NF-κB2 activation. The enhanced signal activated by BAFF-R H159Y is coupled with a several fold increase in TRAF3, TRAF2, and TRAF6 recruitment to BAFF-R and increased IgM production. We further demonstrate that recruitment of TRAF6 to BAFF-R is not unique to the mutant H159Y BAFF-R, but is also an important and necessary feature of BAFF-R signaling in normal B cells. Collectively, our data identify a novel lymphoma-associated mutation in BAFF-R and describe exciting new aspects of BAFF-R signaling that are important for understanding normal B cell homeostasis and function, as well as pathogenic BAFF-R contributions to human disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we demonstrate a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate a potential of BAFF-R as a novel treatment target for PCNSL.

Abstract The cytokines BAFF (B-cell activating factor of the TNF family, BLyS) and APRIL (a proliferation-inducing ligand) activate several major signaling cascades responsible for B-cell survival and homeostasis. Made primarily by myeloid cells, BAFF binds and activates three cell membrane receptors; BCMA (B-cell maturation antigen), TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF Receptor, BR3), while APRIL binds TACI and BCMA. Studies of genetically altered mice demonstrate that TACI and BCMA perform niche roles in B-cell Ig isotype switching and plasma cell maintenance. In contrast, BAFF-R deletion in mice results in greater than 90% loss of mature B-cells, revealing it as an essential mediator of B-cell maturation and survival beyond the immature transitional (T1) stage. TNF receptor family proteins utilize characteristic combinations of TNF receptor associated factors (TRAFs) to bridge receptor activation with a multitude of downstream signaling pathways. To date, studies of TRAF recruitment by TACI and BAFFR and their roles in signaling have been limited, and TRAF3 was the only TRAF thought to associate with these receptors. Here we present the novel finding that TRAF6 is directly recruited by TACI and BAFFR in B-cells, and that it forms an essential link to the initiation of the canonical NF-kB pathway and other pro survival signals.

Abstract The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R–dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Abstract B-lymphocyte stimulator (BLyS) is a relatively newly described tumor necrosis factor (TNF) superfamily cytokine involved in cell survival and proliferation in normal and neoplastic B cells, particularly in the aggressive B cell non-Hodgkin’s lymphomas (NHL-B). BLyS binds to three receptors: BAFF-R (or BR3), BCMA and TACI. However, recent studies have shown that BLyS regulates B cell survival and proliferation predominately through BAFF-R. Mice with mutant BAFF-R show significant decrease in the peripheral blood B-lymphocyte compartment, similar to the phenotype of BLyS knockout mice. BLyS/BAFF-R signaling functions through activation of the critical transcription factor NF-kB pathways, especially the NF-kB2 pathway, which mainly involves the p52/ rel-B complex. Our previous studies have shown that BLyS is constitutively expressed in aggressive NHL-B cells leading to increased survival and proliferation of the malignant B cells. In this study, we found by western blotting and confocal microscopy that BAFF-R, the major B cell associated cell membrane receptor for BLyS, was also present in the B cell nucleus, in addition to its location in plasma membrane and cytoplasm in both normal peripheral blood B lymphocytes and NHL-B cells. Nuclear presence can be increased by anti-IgM and soluble CD154 treatment in normal peripheral blood B lymphocytes, and is constitutively expressed in aggressive NHL-B. Inhibition of BLyS expression by specific BLyS siRNA decreases nuclear BAFF-R level and survival in LBCL cells. Immunostaining experiments show that in the cytoplasm of normal and neoplastic B cells, BAFF-R interacts with the improtin a and b which are members of the classic karypherin pathway. A candidate nuclear localization sequence (NLS) was also identified in the BAFF-R protein sequence, and mutation of this putative NLS can block BAFF-R entering nucleus, which is consistent with our hypothesis that BAFF-R undergoes nuclear translocation. To further investigate the functional role of BAFF-R in nucleus, we performed confocal immunostaining analysis and co-immunoprecipitation, that shows that BAFF-R co-localizes with some NF-kB family members such as c-rel in the B cell nucleus. We also found that nuclear BAFF-R/c-rel complex can bind to the NF-kB binding site on the promoters of NF-kB target genes such as BLyS, CD154, Bcl-xL and Bfl-1 /A1 by chromatin immnoprecipitation (ChIP) assay. Luciferase reporter assays show that BAFF-R has transactivation activity on these NF-kB target genes. Furthermore, NLS mutant BAFF-R decreases NF-kB target gene promoter activity, compared to wild type BAFF-R, and NHL-B cell proliferation. These findings indicates that in addition to activating NF-kB pathways at the plasma membrane, BAFF-R may also promote survival and proliferation of both normal and NHL-B cell in the nucleus by directly regulating transcriptional activity of key NF-kB target genes and may functions as a transcriptional co- factor with NF-kB.

The tumor necrosis factor family members BAFF and APRIL induce Ig isotype switching in human B cells. We analyzed the ability of BAFF and APRIL to induce isotype switching in murine B cells to IgG1, IgA, and IgE. APRIL and BAFF each engage two receptors, transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), on B cells. In addition, BAFF engages a third receptor on B cells, BAFF-R. To determine the role of these receptors in isotype switching, we examined B cells from mice deficient in TACI, BCMA, and BAFF-R. The results obtained indicate that both TACI and BAFF-R are able to transduce signals that result in isotype switching.

La leucemia linfoblastica acuta B (LLA-B) è la leucemia più comune nei bambini (80%), ma ha anche un picco di incidenza in età adulta. Recentemente, gli approcci di immunoterapia diretti contro la molecola CD19 hanno dimostrato un notevole successo nel trattamento della LLA-B recidiva e refrattaria, che rimane una delle principali necessità cliniche. Svantaggi importanti di tali strategie sono la comparsa di recidive CD19-negative e aplasia delle cellule B come risultato della persistenza delle cellule anti-CD19 CAR. In questo contesto, abbiamo ipotizzato che il recettore per il fattore di attivazione delle cellule B (BAFF-R), una proteina transmembrana fondamentale nella maturazione delle cellule B e nella loro sopravvivenza, potrebbe essere una molecola interessante per strategie di targeting, avendo come vantaggio il fatto che questo recettore non è rilevabile nelle cellule B precursori del midollo osseo. Nel nostro lavoro abbiamo dimostrato che BAFF-R è altamente espresso in tutti i campioni primari di LLA-B sia all'insorgenza che alla ricaduta. Al fine di sviluppare un approccio mediato da un recettore chimerico (CAR) specifico per l’antigene BAFF-R, sono state sviluppate sei tipologie di CAR anti-BAFFR che si differenziano per l'inversione della VH e VL e la lunghezza del dominio cerniera. Cellule killer indotte da citochine (CIK), ingegnerizzate utilizzando un sistema non virale che sfrutta trasposoni Sleeping Beauty (SB), esprimono stabilmente anti-BAFFR.CARs e mantengono il loro caratteristico fenotipo. Tra i CAR generati, il CAR più breve VHVL esercita la massima attività anti-leucemica verso le cellule bersaglio, come NALM-6, con un'attività citotossica in vitro pari al 60%. Abbiamo valutato anche le funzioni effettrici a lungo termine di rilascio di citochine mediante colorazione intracellulare (8,9 ± 2% di cellule producesti IFN-γ e 16,4 ± 5,5% di cellule producesti IL-2). Inoltre, abbiamo anche rilevato una specifica attività citotossica nei confronti di blasti primari di LLA-B (media 65,6 ± 4,5%, n = 9). Combinando le cellule trasdotte Invsh.CAR con le cellule trasdotte CD19.CAR abbiamo rilevato una attività antitumorale superiore nei confronti di tutti le linee target. Infine, utilizzando un campione raccolto da un paziente con recidiva di malattia negativa per l’antigene CD19, abbiamo dimostrato la capacità del INVsh.CAR di lisare blasti CD19-negativi. Concludendo, questi risultati dimostrano come questo recettore sia un bersaglio sicuro e attraente per un trattamento immunoterapeutico di seconda linea per la LLA-B in caso di recidiva dopo terapia con approcci di targeting dell’antigene CD19 o per un approccio di targeting doppio. Essendo BAFF-R ristretto a cellule B mature e assente in precursori e plasmablast, la nostra strategia potrebbe avere una tossicità inferiore, per quanto riguarda l'emergere di aplasia delle cellule B osservata nei pazienti trattati con le cellule T anti-CD19 CAR.
B-cell Acute Lymphoblastic Leukemia (B-ALL) is most common in children (80%), but it has also a peak of incidence in adult age. Recently, immunotherapeutic approaches targeting the CD19 molecule have demonstrated remarkable success in the treatment of relapsed and refractory B-ALL, which remains a major clinical need. Important downsides of these strategies are the emergence of CD19-negative relapses and B-cell aplasia as a result of anti-CD19 CAR T-cell persistence. In this context, we hypothesized that the receptor for B-cell activating factor (BAFF-R), a transmembrane protein fundamental in B-cell maturation and survival, could be an interesting molecule to be targeted, taking the advantage that this receptor is undetectable on bone marrow B-cell precursors. Here we showed that BAFF-R is highly expressed in B-ALL primary samples at the onset and relapse In order to develop a chimeric antigen receptor (CAR) approach targeting BAFF-R molecule, six anti-BAFFR CAR genes that differ for the inversion of the VH and VL and the length of the spacer domain have been generated. Cytokine-induced Killer (CIK) cells, engineered using an improved Sleeping Beauty (SB) transposon system, stably expressed anti-BAFFR.CARs, and maintained their characteristic phenotype. Among the newly constructed CARs, the shortest VHVL CAR exerted the highest anti-leukemic activity towards target cells, such as NALM-6, with an in vitro killing activity of 60%. We also evaluated later effector functions in terms of cytokine release by intracellular staining (8,9±2% of IFN-γ and 16,4±5,5% of IL-2 producing cells). Importantly, we also detected a specific cytotoxic activity towards primary B-ALL blasts (average 65,6±4,5%, n=9). Combining the Invsh.CAR with CD19.CAR we detected a superior antitumor activity towards ALL targets. Furthermore, by using a sample collected from a patient relapsed with CD19 negative disease, we demonstrated the ability of the INVsh.CAR to lysate CD19-negative blasts. Taken together, these findings make this receptor a safe and attractive target for a second line B-ALL immunotherapy in case of relapse after CD19-targeting therapies or for a double targeted approach. Being restricted to mature B cells, but absent in precursors and plasmablasts, our strategy could have an inferior toxicity concerning the emergence of B-cell aplasia observed in patients treated with anti-CD19 CAR-modified T cells.

Trabalho final de mestrado integrado em Medicina (Biologia Aplicada/Medicina Interna), apresentado á Faculdade de Medicina da Universidade de Coimbra
Introdução: Atualmente, verifica-se um aumento de incidência tanto de doenças oncológicas como de doenças auto-imunes na população em geral. Há diversos mecanismos celulares e vias de sinalização celular envolvidos na doença autoimune e oncológica, no entanto o NF-κB, os ligandos BAFF e APRIL e seus recetores, BAFF-R e TACI, e as células reguladoras T reguladoras são os mais preponderantes. Objetivos: Com este trabalho pretende-se identificar e quantificar o NF-kB, os ligandos BAFF e APRIL e seus recetores, BAFF-R e TACI, e as células T reguladoras, em doentes com doenças auto-imunes e/ou neoplásicas, de modo a contribuir para clarificar o seu papel nestas patologias. Métodos: Foram quantificadas as células T reguladoras (Treg) e avaliada a expressão do BAFF, BAFF-R, TACI e APRIL em doentes com doenças auto-imunes e/ou neoplásicas de modo a analisar o seu papel na fisiopatogenia destas doenças. O estudo foi efetuado em Sangue Periférico (SP) de 11 controlos saudáveis (CTL) e de 76 doentes, dos quais 21 com doenças auto-imunes (DAI), 47 com neoplasias (NEO) e 8 com ambas as patologias (DAI e NEO). Após a colheita do SP, procedeu-se à análise das diferentes populações linfocitárias, com identificação das células Treg, e avaliação da expressão do BAFF, BAFF-R, TACI e APRIL por citometria de fluxo, utilizando anticorpos monoclonais marcados com sondas fluorescentes. Os resultados obtidos foram estatisticamente analisados pelo teste Anova, T-student e análise multivariada (p<0.05). Resultados: Na análise dos resultados do linfograma, os linfócitos totais dos doentes DAI e dos doentes NEO encontravam-se diminuídos em comparação com os controlos. Observou-se diminuição significativa de linfócitos B nos doentes com DAI e doença auto-imune e neoplásica (DAI+NEO) e aumento significativa dos linfócitos T dos doentes com DAI comparativamente aos controlos e aos doentes com neoplasia. As células nTreg dos indivíduos saudáveis (controlos) representam um pequeno nicho de células, no entanto, os diferentes grupos de doentes apresentam aumento significativo desta percentagem de células. Os doentes com neoplasia apresentam aumento da percentagem de células a expressar BAFF comparativamente aos controlos, contrariamente, os doentes com DAI+NEO e os doentes com DAI apresentam menor percentagem destas células. O recetor de superfície BAFF-R revelou maior percentagem de expressão nas células de doentes com DAI+NEO do que nas células dos controlos. A análise do ligando TACI revelou que o grupo de doentes com neoplasia se destacava dos restantes grupos de doentes devido à elevada percentagem de expressão. Por fim, a análise do ligando APRIL revelou que os grupos de doentes com DAI+NEO e os NEO apresentam cerca de 3 vezes mais células com expressão desta molécula comparativamente aos controlos. Discussão: Nos doentes analisados verificou-se aumento significativo das células nTreg nos doentes com DAI, com doença neoplásica e principalmente nos doentes com DAI e neoplasia, possivelmente uma forma das patologias oncológicas gerarem um ambiente imunossupressor e uma tentativa do sistema imune dos doentes com DAI de suprimir a resposta inflamatória anómala. Considerando os ligandos BAFF, BAFF-R, TACI e APRIL, o grupo de doentes com neoplasia, revelou aumento substancial de expressão destes ligandos, revelando que podem ser mecanismos adquiridos de sobrevivência celular em doenças neoplásicas. Conclusão: Comprovou-se que há distúrbios relevantes a nível dos linfócitos B e T, com alteração das suas populações em ambas as patologias. Além disso, os nossos resultados sugerem que as células T reguladoras e os ligandos e recetores relacionados com o NF-kB, BAFF, APRIL e BAFF-R e TACI, respetivamente, podem participar na fisiopatologia das doenças auto-imunes e neoplásicas, podendo eventualmente constituir novos alvos terapêuticos para o tratamento destas patologias
Introduction: Now a day, it’s verified that there is an increase of incidence in both oncologic and autoimmune diseases on general population. There are several cellular mechanisms and cellular pathways involved on autoimmune and oncologic pathologies, however, the NF-κB, the ligands BAFF and APRIL and their receptors BAFF-R and TACI and the T regulatory cells are the most preponderant. Objectives: With this work we want to identify and quantify the NF-κB, the ligands BAFF and APRIL, their receptors BAFF-R and TACI, and the T regulatory cells on patients with autoimmune and/or oncologic diseases, so that we can contribute to clarify their role on these pathologies. Methods: The T regulatory cells (Treg) were quantified and it was evaluated the expression of BAFF, BAFF-R, TACI and APRIL in patients with autoimmune and/or oncologic diseases so that his role could be assessed. The study was made on Peripheral Blood (SP) of 11 healthy controls (CTL) and of 76 patients, 21 with autoimmune diseases (DAI), 47 with cancers (NEO) and 8 with both diseases (DAI + NEO). After the SP harvest we proceeded to the analysis of the different lymphocytic populations, with identification of the Treg cells and evaluation of the expression of BAFF, BAFF-R, TACI and APRIL by flow cytometry using fluorescent bind monoclonal antibodies. The results were statistically analyzed by Anova, T-student and multivariate analysis (p<0,05). Results: Upon the linfogram analysis, total lymphocytes from the DAI and NEO patients were decreased in comparison to the controls. It was observed a significant decline of the B lymphocytes on the DAI and DAI+NEO patients and an increase on T lymphocytes on DAI individuals when compared to healthy controls and NEO. The Treg cells of the controls represented only and small amount of cells, however the different groups of patients presented a significant increase of these cells percentage. Patients with cancer had an increase on BAFF expressing cells comparing to controls, on the other hand, DAI+NEO and DAI individuals showed diminished percentage of these cells. The surface receptor BAFF-R revealed biggest expression on DAI+NEO’s cells than in healthy controls’ cells. The TACI analysis showed that the group with oncologic disease had high expression that stood alone from the rest of the groups. Finally the APRIL ligand analysis revealed that DAI+NEO and NEO groups of patients had three times more expression of this molecule than the controls. Discussion: On the analyzed patients we verify an significant increase on Treg cells on DAI patients, individuals with oncologic disease and mainly on patients with both diseases, possibly a way of the oncologic pathologies create an immunossupressor environment and an attempt of the immune system of the DAI patients to suppress the abnormal inflammatory response. Considering the ligands BAFF, APRIL and the receptors BAFF-R and TACI, the group of oncologic patients revealed a substantial increase on their expression, disclosing that they can be acquired mechanisms of cellular survival in oncologic diseases. Conclusion: It was concluded that there are relevant disturbances on B and T lymphocytes, with change on their populations on both pathologies. Furthermore, our results suggest that the T regulatory cells and ligands and receptors related to NF-κB, BAFF, APRIL and BAFF-R and TACI, respectively, can participate on the fisiopathology of autoimmune and oncologic diseases, and eventually can compose new therapeutic targets to the treatment of these illnesses