Presence Of TGF-b Research Articles

Non-clinical evaluation of NT-175, an autologous T cell product engineered to express an HLA-A*02:01-restricted TCR targeting TP53 R175H and resistant to TGF-b inhibition.

2560 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in tumor suppressor genes such as TP53. TP53 is the most commonly mutated gene across all cancer types, with the R175H mutation being the most prevalent across different indications. NT-175 is an autologous engineered T-cell product expressing an HLA-A*02:01-restricted TCR that specifically targets the TP53 R175H mutation. NT-175 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-175, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-A*02:01-restricted TP53 R175H neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-175 product functionality was evaluated in vivo models and in vitro. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. The benefit of TGFBR2 knock-out (KO) in the presence of TGF-b was evaluated by measuring phosphorylation of SMAD2/3 proteins, the impact on cell viability and serial killing of target cells. For determination of NT-175 product safety, cross-reactivity and HLA-specificity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-175. Results: High reactivity of the NT-175 TCR against TP53 R175H and HLA-A*02:01 expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against TP53 WT, minimal cross-reactivity against antigens with up to 4 mismatches to the minimal TP53 R175H encoding epitope recognized by the NT-175 TCR, and lack of T-cell activation in the absence of HLA-A*02:01. In the presence of TGF-b, TGFBR2 KO TCR-edited T-cells displayed inhibition of SMAD2/3 phosphorylation, increased cell viability and increased cytotoxicity and proliferation in serial stimulation assays. In vivo, NT-175 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-175 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-175 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant TP53-targeted cancer immunotherapy.

Abstract 1768: Multiplexed shRNA cassettes targeting orthogonal pathways (FAS/PTPN2/TGFBR) enhance the potency of integrated circuit T cells (ICTs) in multiple solid tumor models

Abstract T cell exhaustion resulting from chronic antigen stimulation and immunosuppression in the tumor microenvironment (TME) limits CAR T efficacy in the solid tumor setting. We have previously shown that engineering therapeutic T cell products using CRISPR-based gene insertion of a dual shRNA cassette targeting Fas and PTPN2 significantly increased antitumor efficacy of Integrated Circuit T cells (ICTs) in ovarian cancer models. We sought to build on this finding to induce additional gene perturbations that improve the efficacy of ICTs. Transforming growth factor (TGF)-b is an immunosuppressive cytokine that potently inhibits T cell responses and is present at high levels in numerous solid tumors, including renal cell carcinoma (RCC). In order to render ICT cells less susceptible to TGF-b-mediated suppression, we developed a quadruple shRNA cassette that simultaneously targets Fas, PTPN2, and TGFBR. Candidate TGFBR-targeting shRNAs were selected for their ability to reduce surface TGFBR receptor expression and impair proximal (pSMAD) or distal (CD103, PD-1) signaling through TGFBR. While single shRNAs against TGFBR did not rescue ICT cell activity in the presence of TGF-b, likely due to partial knockdown of TGFBR signaling, a cassette encoding two shRNAs against TGFBR restored ICT function to similar levels observed in the absence of TGF-b. The quadruple shRNA cassettes targeting Fas/PTPN2/TGFBR significantly enhanced antitumor activity of ICT cells in multiple xenograft tumor models relative to Fas/PTPN2 cassettes. These results demonstrate the utility of multiplexed shRNA strategies to render therapeutic T cells resistant to orthogonal suppressive pathways in solid tumors. Citation Format: Thomas J. Gardner, Adam Litterman, Brenal K. Singh, Luisa Silva, Mukund Hari, Stanley Zhou, Colin Tang, Sahil Joshi, John Gagnon, Oliver Takacsi-Nagy, Jason Hall, Hans Pope, James Zhang, Alma Gomez, Jeremy Chen, Suchismita Mohanty, Vince Thomas, Nicholas Quant, Beatriz Millare, Amy-Jo Casbon, Natalie Bezman, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Multiplexed shRNA cassettes targeting orthogonal pathways (FAS/PTPN2/TGFBR) enhance the potency of integrated circuit T cells (ICTs) in multiple solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1768.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Abstract 4618: TGF-B upregulates GSK3B and mediates NK dysfunction in cancer

Abstract Transforming growth factor-B (TGF-B) is an important regulator of tumorigenesis. Initially, it was discovered to have anti-cancer activities by promoting apoptosis and preventing hyperproliferation of cells. However, abundant presence of TGF-B in the tumor microenvironment is known to hinder cancer eradication by suppressing anti-tumor activities of immune cells. The molecular switches that regulate the role of TGF-B are not fully understood. One main target of TGF-B-mediated immune cell dysfunction are natural killer (NK) cells. NK cells are innate immune cells that lyse virally-infected and cancer cells. Recently, we have discovered that glycogen synthase kinase 3-beta (GSK3B) upregulation in NK cells leads to NK dysfunction. Another study has indicated that GSK3 phosphorylates SMAD3 to inhibit TGF-B-mediated growth dysfunction in AML12 cells. In addition, GSK3B inhibition has been linked to increased sensitivity of cancer cells to NK killing but the role of GSK3B in NK function is understudied. This study examines how the role that GSK3B has on TGF-B-mediated NK cell dysfunction. Methods: NK cells were isolated from the peripheral blood of healthy donors and expanded in vitro for 14 days. NK cells were either treated with or without 5ng/mL TGF-B for 72 hours, and GSK3B expression, and the expression of NK activating receptors was measured via flow cytometry. In addition, GSK3B expression is abrogated using a siRNA in NK cells. The NK cells were treated with or without TGF-B, and NK cell cytotoxicity is measured with a fluorometric cytotoxicity assay. Results: TGF-B pretreatment of NK cells led to 2-fold GSK3B expression in NK cells. TGF-B pretreatment led to decreased expression of NK activating receptors NKp46 and NKG2D. Also, TGF-B led to decreased expression of lymphocyte function-associated antigen-1 (LFA-1), an adhesion molecule necessary for NK cells to adhere to target cells prior to lysis of the target cells. TGF-B pretreatment of NK cells led to 33.3% reduced killing of both leukemic and solid tumor targets. Pharmacologic and genetic abrogation of GSK3 minimized TGF-B-mediated NK dysfunction as measured by calcein-AM based cytotoxicity assays. Importantly, inhibiting GSK3B activity with TGF-B treatment rescued NK cell cytotoxic function. Discussion: Our data suggests that TGF-B leads to upregulated GSK3B expression, and downregulation of NK activating receptors. In addition, TGF-B reduced NK killing of cancer cells. We believe that TGF-B-mediated NK dysfunction is facilitated by GSK3B upregulation. Further studies are underway to determine how TGF-B affects the expression of other NK receptors, and to elucidate the mechanism by which TGF-B directs GSK3B upregulation. Citation Format: Evelyn Ojo, Folashade Otegbeye, Stephen Moreton, David Wald. TGF-B upregulates GSK3B and mediates NK dysfunction in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2017-4618

Upregulation of aryl hydrocarbon receptor expression in CD4 T lymphocytes during an immune response in vivo. (INM1P.431)

Abstract Activation of the aryl hydrocarbon receptor (AHR) can impact T-cell differentiation and the maintenance of tissue-resident T cells. AHR expression, measured by RT-PCR or Western blot has been shown to be low in most CD4 T-cell subsets with highest expression found after stimulation of naïve CD4 T cells under Th17 conditions in vitro. The current study measures AHR expression within CD4 T-cell subsets of naïve or skin-grafted mice using flow cytometry. Cells were freshly isolated from the spleen, lymph nodes or skin of either untreated mice or mice that underwent allogeneic skin transplant. AHR expression in cells from untreated mice was highest in IL-17A+ CD4 T cells, followed by FoxP3+ and IFNγ+ cells. Naïve, CD62L+ CD4 T cells had the lowest expression. AHR expression in splenic or lymph node CD4 T cells from skin-grafted mice was similar to naïve mice with 20 - 30 % of FoxP3+ and <10% of FoxP3- being AHR+. However, CD4 T cells within the skin grafts showed elevated AHR expression compared to spleen or LN with 75 - 85 % of FoxP3+ and 40 - 70% of FoxP3- being AHR+. This is comparable to the >90% AHR+ CD4 T cells found when splenocytes were stimulated in vitro with anti-CD3 antibody in the presence of TGFb and IL-6. We conclude that the up-regulation of AHR expression in CD4 T cells in vitro also happens during immune responses in vivo and is not restricted to a single T cell subset, and may be important in T cell differentiation, trafficking, and cytokine production.

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Immune Privilege and the Philosophy of Immunology

Like other scientists, immunologists use two types of approaches to research: one reduces the problem to its parts; the other studies the emergent phenomenon produced by the parts. Scientists that reduce the problem to its parts are sometimes called reductionists. The conclusions of reductionist experiments are often applied to the greater whole, when in actuality they may only apply to that particular experimental set. We, reductionists, are the ones who think our immune behavior exists solely because of genes, the presence of TGFb, the presence of inflammatory cytokines, and appearance of a receptor. We tend to interpret the outcome in the colors of our interests (ergo “to a hammer, everything is a nail”). We measure parts and from the parts, we draw parallels to farremoved outcomes in terms of health and disease. More often than not, however, the results from the study of the parts do not predict the whole, and often the whole becomes greater than the sum of its parts. For example, a toll-like receptor does one thing when there is a bacterial invasion but the same toll-like receptor may lead to a different outcome when activated by danger signals during injury. The approach that is perhaps more applicable to biology, and more specifically, to immunology, is the chaos theory. The chaos theory deals with the multiple layers of conditions and unexpected turns and restarts that can effect the outcome. It is applicable to studies of dynamic systems like the weather, and in our case, dynamic biological/immunological systems. The chaos theory points out that small differences in initial conditions make it impossible to predict the outcome. Thus, the behavior of the parts does not make the outcome predictable. Our point? Immune privilege is broadly understood as the ability of the tissue to actively regulate and direct immune responses that take place in its “territory.” The articles in this Research Topic in Frontiers in Immunology, “Good news‐bad news”, support the idea that to understand how immune privilege works, one has to understand the dynamics of the different tissues in terms of initiation, expression, regulation, and behavior, before one can begin to predict an outcome. This Research Topic contains a number of papers that deal with immune privilege in the eye: a review that explores the relationship of immune privilege to ocular disease (1); a contribution on local regulation of immune CD8 C T cells in the eye (2); a review discussing the intriguing parallels between the mechanisms of ocular immune privilege and uveal melanoma (3); a thoughtful contribution on how CD8 C Treg cells might enable ocular tumor growth (4); a review proposing that (paradoxically) immune privilege emerges as an enabler of immune cells that heal, as well as of regulator immune cells that promote tissue damage (5). While the eye is considered the prototypic immune privileged tissue, it is not the only one. We therefore shift to reviews of immune privilege and its mechanisms in other tissues and organs, underscoring the universality of the phenomenon: the reproductive tract (6), the testis (7), tumor environment (8), and finally chronic inflammatory diseases (9). While the eye, testes, reproductive tract, tumors, and chronic immune diseases all seem to share some “immune privileged” mechanisms, each has developed unique features of its own. Recent reports have repeatedly shown that immune regulation is “tailored” to the individual tissue in

Rare simultaneous occurrence of two supposedly antagonistic diseases: Vogt-Koyanagi-Harada disease and allergic conjunctivitis

Rare simultaneous occurrence of two supposedly antagonistic diseases: Vogt-Koyanagi-Harada disease and allergic conjunctivitis

High Efficacy of Alloantigen-Specific Induced Regulatory T Cells in the Prevention of Acute Graft-Versus-Host Disease in Mice

Abstract 4112 Introduction:Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took an alternative approach to generate antigen-specific induced Tregs (iTregs), and tested their efficacy and selectivity in the prevention of GVHD in pre-clinical models of bone marrow transplantation (BMT). Methods:We selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, naïve CD4+CD25− cells were isolated from MHC II-restricted HY-specific transgenic mice, and were stimulated with HY peptide and APCs, in the presence of TGFb and retinoic acid. Using similar protocol, we also generated polyclonal iTregs from CD4+CD25− cells of normal C57BL/6 (B6) mice with allogeneic dendritic cells (DCs). iTregs were isolated by positively selecting CD4+CD25hi cells 6–7 days after generation. Frequency of T cells in recognizing alloantigens was measured using a limited dilution assay. Various MHC-mismatched or matched murine BMT models were used, where polyclonal T cells (Teffs hereafter) were transplanted with donor bone marrow to induce GVHD in myeloablative allogeneic recipients. Results:We first assessed the effect of HY-specific iTregs on GVHD using an MHC II-mismatched B6 ® (B6 × bm12)F1 BMT model, and found that HY-specific iTregs prevented GVHD mortality in male (HY+) but not female (HY−) recipients. On the per-cell basis, HY-specific iTregs were significantly more potent than polyclonal Tregs in the prevention of GVHD. By measuring iTregs and Teffs in spleen and liver of the recipients, we found that HY-specific iTregs expanded extensively and significantly suppressed expansion, activation and infiltration of Teffs in male but not female recipients. To exclude the possibility the observation was model specific, we evaluated the efficacy of HY-specific iTregs and found that those iTregs were highly effective in the prevention of GVHD in two additional BMT models, including one MHC-matched but miHAg-mismatched B6 ® BALB.b model and one haplo-mismatched B6 ® B6D2F1 model. To increase translational potential of our approach, we extended our studies to alloantigen-specific polyclonal iTregs. After 7-day culture in iTreg- generating condition with BALB/c DCs, the frequency of B6 iTregs in recognizing BALB/c alloantigens was increased for 16-fold than that of naïve CD4+CD25− T cells. Furthermore, these iTregs were 64–128 fold more suppressive than nTregs to conventional T-cell response against BALB/c alloantigens. Their highly suppressive activity was antigen-specific, because the same alloreactive iTregs had significant lower suppressive activity to T-cell response against the third-party alloantigens. In vivo using an MHC-mismatched B6 ® BALB/c BMT model, we found that these alloreactive iTregs could effectively prevent GVHD at 2:1 ratio of Teff:Treg, at which polyclonal nTregs had a minimal effect. Conclusion:Using monoclonal HY-specific or polyclonal alloantigen-specific iTregs, these studies demonstrate that antigen-specific iTregs were highly effective in controlling GVHD in an activation-dependent manner. Because iTregs specific for a given miHAg (e.g. HY) can control polyclonal Teffs in response to multiple alloantigens and prevent GVHD in allogeneic recipient, these data also indicate that Tregs may control GVHD through a linked-suppression on Teffs in vivo. In conclusion, the current study presents a promising strategy to generate alloantigen-specific iTregs for effective GVHD prevention in human allogeneic hematopoietic cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Prevention of Graft-Versus-Host Disease by HY-Specific Induced Tregs Through Activation-Dependent Manner,

Abstract 4023Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took the alternative approach to induce antigen-specific Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in a pre-clinical model of bone marrow transplantation (BMT). We selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, naïve CD4+CD25− cells were isolated from MHC II-restricted, HY-specific transgenic and Foxp3/GFP knock-in mice, and were stimulated with HY peptide and APC, in the presence of TGFb and retinoic acid. Marrow plus polyclonal CD4 T effector cells (Teffs) from B6 donor splenocytes induced GVHD when transplanted into lethally irradiated (B6 x bm12)F1 recipients, and adoptive transfer of HY-specific iTregs (CD4+CD25+GFP+) significantly decreased GVHD mortality in male (HY+) but not female (HY−) recipients. On a per cell basis, HY-specific iTregs were significantly more potent than polyclonal Tregs in the prevention of GVHD. These data indicate that HY-specific iTregs were effective in controlling GVHD in an activation-dependent manner. Because polyclonal Teffs cells recognize bm12 alloantigen whereas iTregs recognize HY miHAg, these data suggest that Tregs may control GVHD through a linked-suppression on Teffs in vivo. Mechanistically, by measuring iTregs and Teffs in spleen and liver of the recipients, we found that HY-specific iTregs expanded extensively and significantly suppressed expansion and infiltration of Teffs in male but not female recipients. We finally generated alloreactive iTregs from polyclonal CD4 precursors, and found that these iTregs highly suppressed the Teffs alloresponse in vitro and in vivo. These results show that Ag-specific iTregs are promising cell therapy for effective GVHD prevention in human allogeneic hematopoietic cell transplantation. Disclosures:No relevant conflicts of interest to declare.

Regulatory T Cells as Potential Targets for Immunotherapy in Inflammatory Bowel Disease

intestine are particularly efficient for the peripheral conversion of Tregs via TGF-band retinoic acid-dependent mechanisms. Tregs that do not express FoxP3 but are functionally suppressive both in vitro and in vivo include Tr1 cells, which are defined by the production of IL-10 and can be induced by oral administration of antigen, in vitro culture with IL-10 or exposure to immature DCs. Another subset of Tregs, Th3 cells, suppresses immune responses in a TGF-b-dependent fashion. Recent data have suggested that Th3 cells may function by inducing de novo FoxP3 Tregs via TGF-b [4]. Indeed, we have reported that inhibition of TGF-b1 interferes with the induction of mucosal Tregs [5]. Disturbances in Treg number and function are associated with immune-mediated disorders. The inf lammatory bowel diseases (IBDs) include ulcerative colitis and Crohn’s disease, and are chronic diseases characterized by infiltration of inflammatory cells into the lamina propria of the intestinal tract. Data from patients and animal models suggest that IBD results from an aberrant immune response to the intestinal microflora [6]. In support of this hypothesis, genetic susceptibility to IBD in humans is associated with variations in genes that encode proteins relevant to both innate and adaptive immunity, including NOD2, ATG16L1, IL-12B, STAT3 and the IL-23 receptor (IL-23R) [7]. In addition, data from animal models demonstrate that intact intestinal flora are necessary for the development of IBD, and antibiotics and probiotics have been demonstrated to be beneficial under certain circumstances in the treatment of patients with IBD [8]. As Tregs are thought to play a critical role in limiting inflammation in response to these nonpathogenic antigens, defects in this T-cell subset The gut mucosal immune system is the largest lymphoid organ in the body. This site undergoes continuous antigenic challenges from food antigens, antigens of the abundant normal bacterial flora and pathogens. Despite this constant antigenic stimulation, controlled inf lammatory responses and suppression of inflammation appear to be the rule. Apparently, the gut immune system effectively differentiates the potentially harmful antigenic signals from the high background noise of food and bacterial antigens. This tight regulation required to maintain homeostasis is achieved through multiple nonimmune and immune factors [1]. Regulatory pathways mediated by regulatory T cells (Tregs) are essential homeostatic mechanisms. Distinct Treg subsets coexist in the intestinal mucosa and mesenteric lymph nodes. These include the ‘natural’ and ‘adaptive’ CD4 forkhead box p3 (FoxP3) Tregs, as well as Tr1 and Th3 cells. Tregs that develop in the thymus are referred to as ‘natural’ Tregs (nTregs). In addition to their natural counterparts, Tregs can develop in response to stimuli in the periphery and these have been termed ‘adaptive’ Tregs. A subset of adaptive Tregs express FoxP3, making them difficult to distinguish from their natural counterparts. In murine CD4 T cells, FoxP3 can be induced in response to activation of the T-cell receptor in the presence of TGF-b and confers suppressor function. TGF-b is also necessary for the induction of Th17 effector cells, but this requires IL-6 [2]. Interestingly, inducible Tregs (iTregs), but not nTregs, are resistant to Th17 conversion by IL-6 [3]. The intestinal environment appears to be an important site for the generation of Tregs, as dendritic cells (DCs) from the mesenteric lymph nodes and small

HIGH DOSES OF TCR STIMULI PREVENT CONVERSION OF NAÏVE CD4+ T CELLS INTO iTREGS IN AN NF-kB-DEPENDENT MANNER (139.10)

Abstract Conversion of naïve CD4+ T cells into FoxP3-expressing regulatory T cells (iTregs) can be achieved in the presence of TGF-b and IL-2 upon TCR triggering by intermediate concentrations of antigen or anti-CD3 mAb, but is prevented by high concentration of antigen. The mechanism for this high dose inhibition of FoxP3 induction has not been elucidated. As TCR stimulation promotes NF-kB signaling, we hypothesized that TCR-induced NF-kB may be responsible for inhibition of FoxP3 expression at high TCR occupancy. Whereas increasing doses of anti-CD3 mAb or antigenic peptide resulted in inhibition of FoxP3 induction in wildtype T cells under iTreg conditions, CD4 transgenic cells expressing an NF-kB superrepressor retained the ability to express FoxP3. Conversely, overactivation of the NF-kB pathway via a constitutively active IKKb reduced Foxp3 expression in wildtype T cells at lower TCR occupancy. Furthermore, CARMA1, a key molecule for NF-kB signaling downstream of the TCR, was critical for Foxp3 inhibition at high TCR occupancy. This was not due to increased TGF-b- or IL-2-mediated signals, or diminished Akt activation in CARMA1-KO T cells, as phosphorylation of Smad2, STAT5 and the Akt target S6 was similar to that in wildtype cells. Rather, the accessibility of the Foxp3 locus was impaired in wildtype but not CARMA1-deficient T cells. Our results suggest that higher levels of NF-kB can inhibit FoxP3 chromatin remodeling, thereby preventing its induction.

Induced Organ-Specific T Regulatory (Treg) Cells Prevent Autoimmunity by Reducing the Ability of Dendritic Cells (DC) to Present Self-Antigen. (131.4)

Abstract Foxp3− T cells can be converted to Foxp3+ T reg cells by T cell stimulation in the presence of TGFb. TGFb-induced Tregs (iTregs) have been shown to prevent the induction of autoimmunity although little is known about their mechanism of action in vivo. Here we demonstrate that naïve CD4+Foxp3− T cells specific for the gastric parietal cell antigen, H+/K+ ATPase, can be converted to Foxp3+ T regulatory cells following TCR activation in the presence of TGFb. The iTregs were anergic to TCR stimulation, failed to produce effector cytokines, and were suppressive in vitro. When co-transferred with disease inducing CD4+ T cells, the iTreg were long lived in vivo and were able to prevent organ-specific autoimmunity. iTregs prevented the initial activation and expansion of autoreactive T cells in response to H+/K+ ATPase presented by DC in the gastric lymph node. Gastric lymph node DC from animals that were treated with iTreg had a markedly reduced ability to stimulate H+/K+ ATPase-specific T cells in vitro. When stimulated by peptide in vitro, iTregs mediated the downregulation of the costimulatory molecules CD80 and CD86, but not MHC class II or CD40, on the surface of DC. These data demonstrate that the autoantigen presenting DC is the major target for Treg mediated suppression in vivo, and raise the possibility of using TGFb to generate large numbers of organ-specific Tregs to prevent and possibly treat autoimmunity.

127 P - TGF-b reduces nitric oxide production BT tumour associated macrophages

Previous studies have shown that the polyamine producing enzyme arginase, is increased in the serum of breast cancer patients. Tumour derived TGF-b has been found to inhibit nitric oxide production from macrophages in a methylcholanthrene induced tumour model. We hypothesised that TGF-b regulated arginine metabolism in the hypoxic environment of breast tumours. To determine the pathway of arginine utllilisation within tumours we measured nitric oxide production from both tumour associated macrophages and monocyte derived macrophages (MDMs). cultured in hypoxic conditions with TGF-b. Results TGF-b levels were significantly increased in the serum of malignant compared to benign breast cancer patients (101.17±14.9 vs 52.07±15.6 ng/ml p=.018), Tumour associated macrophages (TAMs) produce lower amounts of nitric oxide than normal tissue macrophages (2±3 vs 6.4±3 pM/Well/ug protein). MDMs cultured under hypoxic conditions in the presence of TGF-b produce less nitric oxide than those cultured with hypoxia alone (10±2 vs 30±7 pM/well/ug protein). Conclusions Taken together with evidence of increased arginase in these patients, this data indicates that arginine within breast tumours is diverted from the nitric oxide pathway, possibly to the polyamine producing arginase pathway. In this way increased arginase activity may promote tumour growth by providing polyamine substrates.

Functional Dichotomy between Langerhans Cells that Present Antigen to Naive and to Memory/Effector T Lymphocytes

The general thrust of this volume is to review the roles of accessory cells in regulating T and B lymphocytes. To that end, we have summarized the evidence that indicates the crucial role that Langerhans cells play in the induction and expression of immunity to antigens that gain access to, or arise within, skin. Langerhans cells accomplish this important goal by their abilities to (a) activate naive T cells to antigens not previously encountered by the host, and (b) activate memory/effector T cells specific for previously encountered antigens. Arguments have been advanced to support the view that the functional properties of Langerhans cells used to present antigens to naive T cells differ substantially from the properties that equip Langerhans cells to activate effector T cells. The arguments are based in part on the fact that Langerhans cells carry out these functions in two very different environments: in the epidermis, and in the draining lymph node. The arguments are also based on results of in vitro experiments that reveal distinct differences in antigen processing and presenting properties of Langerhans cells freshly obtained from mouse and human skin as compared to Langerhans cells that have been cultured in vitro for 2-3 days. We propose that freshly explanted Langerhans cells faithfully reflect the functional program of intraepidermal Langerhans cells, and are able to present antigen to memory/effector T cells that enter the epidermal compartment. To accomplish this task, epidermal LC pick up environmental antigens, process them with great efficiency, and then present them in situ, without further upregulation of "accessory" signals (cell-adhesion molecules, secretion of additional cytokines). They can carry out this function, even in the presence of TGFB--a a cytokine which is constitutively made by keratinocytes, and which we have found to profoundly inhibit antigen presentation by most other types of "professional" antigen-presenting cells. Intraepidermal Langerhans cells are also capable of carrying cutaneous antigens through the dermal epidermal junction and migrating to the draining lymph node. We further propose that cultured Langerhans cells are fated to present antigens to unprimed/naive T cells, and thereby to initiate immune responses to new cutaneous antigens. Cultured LC process antigens less efficiently than fresh cells, but their unique capacity to present antigen effectively to unprimed T cells rests chiefly on the fact that they have significantly upregulated cell surface adhesion molecules, expression of MHC molecules, and secretion of activating cytokines--the "accessory" signals that are required for arousing naive T cells.(ABSTRACT TRUNCATED AT 400 WORDS)