Academic literature on the topic 'Type 1 regulatory T (Tr1) cells, Cytotoxicity, Immune regulation, granzyme B'

Abstract Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.

Abstract It is widely believed that the main function of B cells is antibody secretion, but not cellular cytotoxicity. Recently we found that human B cells activated with interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN) develop a phenotype similar to that of cytotoxic T lymphocytes. B cells treated in such a way start to secrete large amounts of granzyme B (GrB) instead of antibodies and, as in the case of B-chronic lymphocytic leukemia (B-CLL), acquire the capability to induce apoptosis in bystander B-CLL cells in a GrB-dependent manner. Using FACS and ELISpot analyses we could now demonstrate that GrB is actively secreted by B cells in a time-dependent manner and that IL-21 is not the only cytokine that induces GrB in B cells. Also cytokine combinations such as IL-10 and IL-4 as well as IL-10 and IFN-alpha induce GrB in normal B cells and various B cell lines including MEC-1 (CLL), ARH-77 (plasma cell leukemia) and Namalwa (Burkitts lymphoma). We conclude that IL-21 and further cytokines can induce B cells to produce functional granzyme B. Further studies are required to elucidate the interactions with B lymphocytes of cells producing these cytokines such as CD4+ T cells, regulatory T cells, NKT cells and plasmacytoid dendritic cells. Our unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.

e21081 Background: Matrix metalloproteinase 2 (MMP-2) cleaves many components of the extracellular matrix and is over-expressed in several cancers including melanoma. High levels of MMP-2 expressed by tumors are associated with later tumor stages, increased dissemination and poorer survival/prognosis. We recently identified a pathway whereby MMP-2 functions as a human endogenous “conditioner” that skews CD4+ T cells towards a detrimental/inefficient TH2 phenotype through OX40L expression and inhibition of IL-12p70 production. We unraveled the underlying mechanism: MMP-2 degrades the type I IFN receptor (IFNAR1), thereby preventing STAT1 phosphorylation necessary for IL-12 production. Here, we described IFNAR1 as a novel MMP-2 substrate. This finding opens the way to characterize a possible new immunosuppressive effect of MMP-2 on other IFNAR1+ immune cells such as NK cells and CD8+ T cells. Methods: CD8+ T cells and NK cells were purified from PBMCs and incubated with MMP-2 overnight. IFNAR1 expression was assessed by FACS and Western Blot. Granzyme B, TNF alpha and IFN gamma expression was determined by intracellular staining of memory T cells. NK cells cytotoxicity was measured by Lamp-1 expression after 4h of coculture with K562 cells. Results: We first found that IFNAR1 is expressed by NK and CD8+ T cells and confirmed that MMP-2 degraded it. Memory CD8+ T cells significantly down-regulate granzyme B expression in the presence of MMP-2. Furthermore, memory CD8+ T cells pre-incubated with MMP-2 were significantly impaired with respect to IFN-gamma and TNF-alpha production. Finally, our data suggest that NK cells’ cytotoxicity might also be affected by MMP-2 through modulation of Granzyme B production. Conclusions: Our data strongly suggest that MMP-2 regulates memory CD8+ T cells not only by down regulating cytolytic activity through inhibition of granzyme B, but also by inhibiting cytokine production such as IFN-gamma and TNF-alpha. Elucidating the underlying mechanisms of MMP-2-dependent immune regulation should lead to the development of innovative immune therapies bypassing tumor escape to treat cancer patients.

Abstract T regulatory cells (Tregs) play a pivotal role in promoting and maintaining tolerance. Several subsets of Tregs have been identified but, to date, the best characterized are the CD4+FOXP3+ Tregs (FOXP3+Tregs), thymic-derived or induced in the periphery, and the CD4+ IL-10-producing T regulatory type 1 (Tr1) cells. In the past decade much effort has been dedicated to develop methods for the in vitro induction and expansion of FOXP3+Tregs and of Tr1 cells for Treg-based cell therapy to promote and restore tolerance in T-cell mediated diseases, and for expanding antigen (Ag)-specific Tregs in vivo. FOXP3+Tregs constitutively express high levels of CD25 and of the transcription factor FOXP3. FOXP3+Tregs are distinguished from activated CD4+ T cells by the low expression of CD127, and by the DNA demethylation of a specific region of the FOXP3 gene called Treg-specific demethylated region (TSDR). FOXP3+Tregs suppress effector T-cell responses through cell-to-cell contact-dependent mechanisms and suppression requires activation via TCR and is IL-2 dependent. In vitro protocols to expand FOXP3+Tregs for adoptive transfer in vivo have been established. We demonstrated that rapamycin permits the in vitro expansion of FOXP3+Tregs while impairing the proliferation of non-Tregs. Moreover, rapamycin-expanded FOXP3+Tregs maintain their regulatory phenotype in a proinflammatory environment and Th17 cells do not expand in the presence of rapamycin. Despite the progress in FOXP3+Tregs expansion protocols, adoptive transfer of FOXP3+Tregs in humans remains a difficult experimental procedure due to the ability to expand a sufficient number of Ag-specific FOXP3+Tregs in vitro. To propagate a homogenous population of FOXP3+Tregs we developed a lentiviral vector (LV)-based strategy to ectopically express FOXP3 in CD4+ T cells. This method results in the development of suppressive cells that are super-imposable to FOXP3+Tregs. Conversion of effector T cells into FOXP3+Tregs upon LV-mediated gene transfer of wild-type FOXP3 was also obtained in CD4+T cells from immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. We also developed a LV platform, which selectively targets expression of the transgene in hepatocytes, to induce tolerance to self or exogenous Ags. Using this approach we showed that systemic administration of LV encoding for the gene of interest leads to the induction of Ag-specific FOXP3+ Tregs, which mediate tolerance even in pre-immunized hosts. Tr1 cells are identified by their cytokine profile (IL-10+TGF-b+IL-4-IL-17-). Tr1 cells express transiently FOXP3 upon activation; but FOXP3 expression never reaches the high levels characteristic of FOXP3+Tregs. Tr1 cell differentiation and function is independent of FOXP3 since suppressive Tr1 cells can be isolated or generated from peripheral blood of IPEX patients. Tr1 cells were first discovered in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant (HSCT). Since their discovery, Tr1 cells have proven to be important in mediating tolerance in several immune-mediated diseases. The immuno-regulatory mechanisms of Tr1 cells have been studied over the years thanks to the possibility to generate these cells in vitro. Tr1 cells suppress T-cell responses via the secretion of IL-10 and TGF-β and by the specific killing of myeloid APC via Granzyme B and perforin. Tr1 cells can be induced in vitro in an Ag-specific manner in the presence of IL-10 or of DC-10. Proof-of-principle clinical trials in allogeneic HSCT demonstrated the safety of Treg-based cell therapy with these polarized Tr1 cells. We are currently planning a phase I/II trial using in vitro polarized Tr1 cells with DC-10 in patients after kidney transplantation. An alternative strategy for the induction of high numbers of human Tr1 cells is the LV-mediated gene transfer of human IL-10 into conventional CD4+ T cells. Stable ectopic expression of IL-10 leads to the differentiation of homogeneous populations of Tr1-like cells displaying potent suppressive functions both in vitro and in vivo. A major hurdle, which limited the studies and the clinical use of Tr1 cells, was the lack of specific biomarkers. By gene expression profiling of human Tr1 cell clones we identified two surface markers (CD49b and LAG-3), which are stably and selectively co-expressed on murine and human Tr1 cells induced in vitro or in vivo. The co-expression of CD49b and LAG-3 enables the isolation of highly suppressive Tr1 cells from in vitro IL-10-polarized Tr1 cells and allows tracking of Tr1 cells in peripheral blood of patients who developed tolerance after allogeneic HSCT. The identification of CD49b and LAG-3 as Tr1-specific biomarkers will facilitate the study of Tr1 cells in vivo in healthy and pathological conditions and the use of Tr1 cells for forthcoming therapeutic interventions. In conclusion, Tregs play a key role in maintaining immunological homeostasis in the periphery. Several open questions regarding FOXP3+ Tregs or Tr1 cell-based therapy in humans remain: how long do Tregs survive after transfer? Is their phenotype stable in pathological conditions and inflammatory environments? Is their mechanism of suppression in vivo Ag-specific? Carefully designed and standardized future clinical protocols reflecting a concerted action among different investigators will help to address these questions and to advance the field. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2569 Introduction In AML, mutations in the nucleophosmin (NPM1) gene are one of the most frequent molecular alterations and predominantly occur in AML with normal cytogenetics. Patients with NPM1 mutation without FLT3-ITD mutation show a favourable prognosis of their disease. The functional role of mutated NPM1 for the improved clinical outcome is under evaluation. Immune responses might be involved in the clinical outcome of the disease. In this work, we demonstrate both CD4+ and CD8+ T cell responses against the mutated region of NPM1. Methods The entire amino acid sequences of the NPM1 wild type protein as well as of the mutated cytoplasmic NPM1 types A, B, C and D were screened for HLA-A*0201 binding T cell epitopes using the algorithms of the SYFPEITHI, the Rankpep and the HLA-Bind software programs. Ten peptides with most favourable characteristics were subjected to ELISpot analysis for interferon-γ and granzyme B in 22 healthy volunteers and 27 AML patients to test specific T cell responses of CD8+ T cells. Tetramer assays against the two most interesting epitopes have been performed and chromium release assays have been used to show the cytotoxicity of peptide-specific T cells to lyse T2 cells and leukemic blasts. Moreover, HLA-DR binding epitopes were screened in algorithmic analysis and HLA-DR*0701 binding peptides were exploited to stimulate CD4+ T cells. In the presence of overlapping peptide stimulated CD4+ T cells, NPM1-A specific CD8+ T cells revealed augmented interferon-γ and granzyme B secretion and up-regulation of intracellular interferon-γ. CD4+, CD4-CD8+, CD4-CD8- cell fractions were separated from PBMCs of HLA-A2+DR*0701+ healthy volunteers using a combination of CD4 and CD8 MicroBeads. Results Two epitopes (P3 and P9) derived from the NPM1-mutated protein showed specific T cell responses in healthy volunteers and AML patients. In NPM1-mutated AML patients 33% showed immune responses of CD8+ T cells against peptide P3 and 42% against peptide P9. Specific lysis was detected in chromium release assays NPM1 peptide-primed effector T cells generated from NPM1-mutated AML patients. Tetramer assays showed peptide-specific T cells. To obtain a robust and effective immune response against tumor cells, the activation of CD4 + helper T cells is crucial. Thus NPM1-peptide-A overlapping MHC class II epitopes were searched by primary structure analysis program. Based on plenary search, eight favourable overlapping peptides OL 1–8 were synthesized and exploited for CD4+ T cell stimulation. In granzyme B ELISPOT assay, OL8 co-pulsed NPM1-A CD8+ T cells indicated notable S.I., in contrast other OL1-7 disabled to increase granzyme B secretion. To ensure that Th1 cytokine secretion, under the condition of CD8+ and CD4+ T cells mixed culture, was resulted from NPM1-A CD8+ T cells but not HLA-DR epitope stimulated CD4+ T cells activation, HLA-A2 blocking effect was confirmed in ELISPOT assay. NPM1-A CD8+ T cells co-pulsed with OL6, 7 and 8 showed lesser interferon-γ secretion after HLA-A2 blocking antibody exposure as 73, 35 and 57%. Of note, 83–94% of granzyme B secretion levels were reduced by HLA-A2 blockade administration, and by which NPM1-A CD8+ T cells seemed to be the most probable IFN-gamma and granzyme B producers and CD4+ T cells to interfere with CD8+ T cells. Conclusion Taken together, mutated NPM1 is a promising target structure for specific immunotherapies in AML patients. Disclosures: No relevant conflicts of interest to declare.

11577 Background: Characterization of expression and function of immune regulatory molecules in tumor microenvironment will provide the framework for developing novel therapeutic strategies. Methods: We evaluated the expression and functional impact of various immuno-regulatory molecules, PD-1, PDL-1, PDL-2, LAG3, TIM3, OX40 and GITR, on the CD138+ tumor cells, myeloid derived suppressor cells (MDSC), and T cell subsets from patients with MGUS, SMM and active MM (newly diagnosed, relapsed, relapsed/refractory), and the myeloma-specific cytotoxic T lymphocytes (CTL) induced with XBP1/CD138/CS1 peptides. Results: PDL-1/PDL-2 was more highly expressed on CD138+ myeloma cells in active MM than SMM or MGUS. G-type MDSC (CD11b+CD33+HLA-DRlowCD15+). Treg cells (CD3+CD4+/CD25+FOXP3+) numbers were increased and expressed higher levels of PD1/PD-L1 in active MM than in MGUS, SMM or healthy donors. Among the checkpoint molecules (PD-1, PDL-1, PDL-2, LAG3, OX40, GITR) evaluated, PD-1 showed the highest expression on CD3+CD4+ and CD3+CD8+T cells in BMMC and PBMC from patients with active MM. Functionally, T cells from MM patients showed increased proliferation upon treatment with an individual immune agonist ( > 150%) or checkpoint inhibitor ( > 100%). Interestingly, each individual anti-checkpoint molecule induced proliferation of T cells expressing other checkpoint molecules. In addition, the blockade of PD1, LAG3 or TIM3 enhanced MM antigen-specific cytotoxicity, assessed by parameters including CD107a, granzyme B and IFN-g production, which was most prominent within the memory CTL subset of MM antigen-specific T cells. Conclusions: These results demonstrate an increased frequency of immune regulatory cells, which highly express checkpoint inhibitors in active MM. Direct stimulation with an immune agonist or blockade of a checkpoint inhibitor increased MM patients’ T cell proliferation and myeloma-specific CTL function, supporting development of combination immune regulatory therapies to improve patient outcome in MM.

Background: The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Rituximab, a monoclonal chimeric anti-CD20 antibody, has been widely used as a chemoimmunotherapeutic regimen in the frontline therapy for patients with CD20+ BL and diffuse large B-cell lymphoma. The addition of rituximab to the CHOP backbone or to standard FAB/LMB therapy has greatly improved outcomes without significantly increasing toxicity in patients with B-NHL (Goldman/Cairo, Leukemia, 2013, Coiffier et al, NEJM, 2002). However, patients who relapse have a poor clinical response to rituximab retreatment. Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully expanded functional and active peripheral blood NK cells PBNKwith irradiated feeder cells to target B-NHL (Chu/Cairo, et al, Can Imm Res 2015). We previously demonstrated that obinutuzumab has significantly enhanced expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and expand natural killer (NK) cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, and enhances long-term immunological memory which may lead to sustained anti-tumor immune response. Objective: To investigate the effects of NKTR-255 on the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Dean Lee, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFNg, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, ASH, 2018). MEC-1 (CLL), PGA-1 (CLL), DOHH2 (FL) and Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Results: NKTR-255 significantly enhanced the in vitro cytotoxicity of expanded NK cells when combined with rituximab against MEC-1 (E:T=3:1, p<0.001), PGA-1 (E:T=3:1, p<0.001), and DOHH2 (E:T=3:1, p<0.001) as compared to the control groups (Fig.1A). NKTR-255 also significantly enhanced granzyme and perforin release from expanded NK cells when combined with rituximab against MEC-1 (granzyme: p<0.05; perforin: p<0.001), PGA-1(granzyme: p<0.05; perforin: p<0.05), DOHH2 (granzyme: p<0.05; perforin: p<0.001) as compared to controls. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab agains rituximab-resistant BL cells like Raji-2R (E:T=3:1, p <0.01), and Raji-4RH (E:T=3:1, p<0.01) as compared to the control groups (Fig.1B). NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.001 and perforin: p<0.001) and Raji-4RH (E:T=3:1, IFN-g: p<0.001, granzyme: p<0.01 and perforin: p<0.01) as compared to controls. Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells in vitro with enhanced IFN-g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and anti-CD20 type I and type II antibodies against CLL, FL and rituximab-resistant BL cells using humanized NSG models are under investigation. Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Madakamutil:Nektar Therapeutics: Current Employment. Marcondes:Nektar Therapeutics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Cairo:Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding.

Abstract The role of interferon regulatory factor (IRF) 3 in initiating the type I interferon response in innate immune cells has been well studied in the context of viral infection. However, it is less clear what impact IRF3 has in shaping and maintaining adaptive immune responses. Previous reports have demonstrated that when restimulated, both CD4 and CD8 T cells from IRF3 knockout (KO) mice have significantly reduced expression of Granzyme B 30 days after infection or following secondary influenza A virus (IAV) challenge. To determine whether deficiencies in Granzyme B correlate with CTL function, we are examining the cytotoxic potential and specific antiviral lytic activity after primary and secondary challenge with IAV in CD4 and CD8 cells. Additionally, as early signals derived from antigen presenting cells (APCs) influence effector and memory T cell development, the innate immune response following IAV infection was examined. Our results demonstrate the innate response is not compromised in IRF3 KO mice at day 1 or 3 post IAV infection in the draining lymph node. Interestingly, macrophages and dendritic cells had decreased expression of MHCII and CD86 at both time points in the lung. These results suggest that while initial priming is sufficient in the draining lymph nodes, secondary factors in the lung could provide T cells with the signals needed for effective memory development. Together, this work provides insight into the molecular regulation of T cell responses to viral infections and highlights the role IRFs play in the development of high quality effector and memory T cells.

Several studies from the 1990s found that adverse effects on the fetal environment, such as poor maternal nutrition, can lead to an increased risk of developing chronic diseases in adulthood. These findings led to the fetal origins of disease hypothesis, commonly known as the Barker hypothesis, which proposes that exposures to insults during critical or sensitive windows of development can permanently reprogram physiological responses, thereby giving rise to metabolic and hormonal diseases and disorders later in life.1,2 Pregnancy remains one of the most vulnerable periods in terms of morbidity and mortality, certainly for the fetus, but also for the mother. According to the INEGI, in 2020 there were 22,637 fetal deaths, which corresponds to a national rate of 6.7 per 10,000 women of childbearing age. 82.9% of fetal deaths occurred before delivery, 15.6% during delivery, and in 1.5% of cases was not specified.3 The main complications, which cause 75% of maternal deaths, are: severe bleeding (mostly after childbirth); infections (usually after childbirth); gestational hypertension (preeclampsia and eclampsia); childbirth complications; unsafe abortions (performed clandestinely, in unsuitable conditions and by untrained personnel).4 Interactions between the product of conception and the mother are bidirectional: the fetoplacental tissues need nutrition and a suitable environment under homeostatic conditions, while the mother, influenced by placental factors, adapts her metabolism and immune system to ensure tolerance, which include embryonic and fetal, placental and maternal elements.5 At the maternal-fetal interface, the placenta contains cells of the immune system and mediators, such as uterine NK cells (uNK, 70%), macrophages (20%), T cells (including CD4+, CD8+, γδ T cells, and regulatory T cells) (10 %), dendritic cells, and few B cells. The numbers of these cells and the roles they play vary at different stages of pregnancy. It has been suggested that there may be several local and systemic modifications related to the protection of the developing fetus against attack by the maternal immune system, mainly the expression of unique human leukocyte antigens (HLA) by trophoblasts, the influence of female sex hormones and the bias of citokines.6 As soon as the embryo makes contact with the maternal endometrium, trophoblast cells fuse with the attachment site, forming a syncytium called a syncytiotrophoblast.7 This tissue and the cytotrophoblast do not express complex major histocompatibility (MHC) class I or class II molecules. In contrast, the extravillous cytotrophoblast does express non-classical MHC molecules (HLA-G or HLA-E), which inhibit the activation of uNK cells, favoring immunological tolerance.8 During the first trimester, uNK cells represent up to 50–70% of decidua lymphocytes. Differently from peripheral-blood NK, these are poorly cytolytic, and they release cytokines/chemokines that induce trophoblast invasion, tissue remodeling, embryonic development, and placentation. NK cells can also shift to a cytotoxic identity and carry out immune defense if infected in utero by pathogens. At late gestation, premature activation of NK cells can lead to a breakdown of tolerance of the maternal–fetal interface and, subsequently, can result in preterm birth.9 HLA-G interacts with receptors ILT2 and KIR2DL4 on macrophages and NK cells to enhance the production of proangiogenic cytokines and and enhance trophoblast integrity, invasion of decidua, thereby promoting spiral artery remodeling. In addition, HLA-G binds to ILT2, ILT4, and KIR2DL4 on NK cells, T cells and macrophages, inhibits the cytotoxicity of NK cells and CD8+ T cells, and causes an increase in the percentage of Treg cells in the population, and thereby contributes to immune tolerance. The abnormal expression and polymorphisms of HLA-G are related to adverse pregnancy outcomes such as preeclampsia (PE) and recurrent spontaneous abortion (RSA).10 Cytokine bias is associated with the predominance of Th2-type immunity, while Th1-type responses are considered potentially dangerous for the continuation of the pregnancy.11 Sex hormones have profound effects on the immune system and play a critical role in shaping Th cell immunity throughout stages of pregnancy. Androgens are considered to promote anti-inflammatory responses whereas estrogens can exhibit both pro- and anti-inflammatory roles depending on the relative expression of estrogen receptor isoforms.12 However, significantly high doses of estrogens such as those observed in pregnancy typically suppress immune responses. Pregnancy levels of estardiol also influence CD4+ T cell polarization through enhanced expression of Th2 associated (GATA3, IL-4) and Treg associated genes (Foxp3, PD-1, IL-10, and TGF-β) while suppressing the expression of Th1 associated (T-bet, IL-2, TNF-α, IFN-γ) and Th17 associated genes (ROR-γt, IL-6, IL-17, IL-23).12,13 During pregnancy progesterone induces anti-inflammatory responses and promotes tolerance through the selectively inducing the differentiation of naive CB T cells into Tregs, while suppressing their differentiation into inflammatory Th17 cells, potentially through suppression of the IL-6 receptor expression, and a systemic decline in its concentration prior to the onset of labor in most animal models.14 In conclusion, both allogeneic and hormonal stimulation are responsible for a harmonious regulation of the immune system leading to a successful pregnancy.

Immune responses that control tolerance to self, tolerance to conceptus during pregnancy, and defense against cancer and pathogens are governed by dendritic cells (DC), that are key immune cell types that integrate environment signals and direct T and B cell immune responses. Different DC subtypes exist in mice that each trigger a specific type of immune response, such as cytotoxicity, antibody production, and regulatory processes. Interestingly in mice, it is possible to target specific DC subtypes with antigenised antibodies and obtain a desired type of immune response. However this conceptual breakthrough in vaccinology and immune regulation manipulation may only be valid for laboratory mice, as unfortunately often encountered in the process of bench to bed side translation. Furthermore, whether DC subsets knowledge and manipulation can be translated to human and to animal of socio-economical importance is still not known. We adressed this question in pigs and ruminants. The interest of these species over mice are that (1) they are the direct target species for vaccines, (2) they present genetic diversities and live in an open environment, (3) they present physiological similarities with human such as skin for pigs with skin being a main site for vaccination, (4) skin migrated DC and DC subsets can be collected from lymph after surgical catheterisation of lymph ducts in these species, and not in mice. We studied the molecular characterisation profiles of DC subsets from skin in ruminant and swine and evaluated how they compare to mouse and human DC subsets, based on comparative transcriptomic analyses. We assessed whether ruminant and swine DC subsets share functional similarities and differences with the corresponding murine subsets, and whether these properties translate into novel vaccine developements. Overall our work unravel conserved molecular and functional features that allow characterization of dendritic cell subtypes across mammals and possibly across vertebrates. In the past few decades, a tremendous amount of effort has been invested in developing gene and cell therapies for inherited genetic diseases such as Huntington's disease (HD). However, progress in their clinical application has been very limited. One of the major barriers is the lack of appropriate animal models that allow precise prediction patterns in human patients. Most of the animal models used for gene and cell therapy study are primarily focused on safety and toxicity evaluation, while therapeutic efficacy cannot be fully addressed because they do not carry the same human diseases. Although mouse models of human diseases are available and have been widely used for the development of new therapies, mice are not good predictors for humans because of the fundamental differences (genome composition, body size, life span and metabolic mechanism) between humans and rodents. Although monkeys are one of the best models for studying pharmacokinetics and overall impact of treatment, they are primarily used for safety and toxicity evaluation. Even HD monkey models, created by chemical induction or focal gene transfer in the brain, develop similar cellular pathology, therapeutic efficacy and systemic evaluation cannot be determined, which is one of the major barriers in drug and therapeutic development. The development of transgenic HD monkeys has opened the door for a new paradigm of animal modeling for the advancement of novel gene and cell therapy. HD monkeys not only carry the genetic defect that leads to human HD, they also develop clinical features comparable to humans that no other animal model does. While testing in HD monkeys has yet to be achieved until a cohort of well characterized HD monkeys was established, iPS cell lines derived from HD monkeys with a board spectrum of HD pathology and clinical features are a unique in vitro model for studying HD pathogenesis and the development of novel therapeutic approaches. New knowledge and treatments generated from iPS cells can next be translated and applied in HD monkeys from whom the stem cells were derived, thus the goal of personalized medicine can also be evaluated. This work was funded by a grant from NCRR/NIH (R24RR018827).

IL-10-producing CD4+ type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T cell responses mainly via cytokine dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)- dependent mechanism that requires HLA class I recognition, CD54/Lymphocyte Function-associated Antigen (LFA)-1 adhesion, and activation via CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1 cell activation, is necessary for defining Tr1 cell target specificity. We also showed that high frequency of GZB expressing CD4+ T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10- producing CD4+ T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions which lead to selective killing of target cells and possibly bystander suppression. Adaptive type 1 regulatory T (Tr1) cells are suppressor cells characterized by the production of IL-10 in the absence of IL-4 and by the ability to suppress immune responses mainly by the release of IL- 10 and TGF-β. Despite several efforts for the detection of molecular markers of Tr1 cells have been made, so far Tr1 cell identification relies on cytokine production profile. Moreover, to date no master regulator gene for Tr1 cells have been defined. To identify Tr1 cell specific surface biomarkers, master regulator genes, and molecules involved in their suppressive functions, we performed a gene expression profiling to compare the gene expression of ex vivo isolated Tr1 cell clones compare to Th0 cell clones, in resting state and upon TCR activation. Results demonstrated that Tr1 cells signature is of anti-inflammation, anti-proliferation, and immuno-modulation. In addition, we identified surface molecules that could be useful to identify Tr1 cell population. Interestingly, several transcription factors resulted differentially expressed in Tr1 cells compared to Th0 cells, which may represent master regulators of Tr1 cells.