Treg-mediated Suppression Research Articles

Differential gene expression in G-MDSC and neutrophils from renal cell carcinoma patients

Myeloid-derived suppressor cells (MDSC), including undifferentiated, monocytic, and granulocytic subsets, are known to play a role in tumor progression via promotion of tumor invasion and angiogenesis, and suppression of Type 1 T cell responses. Elevated peripheral blood neutrophil counts have long been associated with poor outcome in multiple tumor types, and recently tumor-associated neutrophils (TAN) were shown to have similar properties as granulocytic MDSC (G-MDSC). The relationship between G-MDSC and neutrophils is unclear; although recent published data show that G-MDSC from tumor-bearing mice and activated neutrophils from non-tumor-bearing mice show differential expression of numerous genes by cDNA analysis. Here we have tried to dissect this relationship more deeply, in clear cell renal cell carcinoma (ccRCC) patients, by using microarray cDNA data to compare gene expression analysis in cancer patient G-MDSC and patient neutrophils. G-MDSC and neutrophils in patients were discriminated based on their density, and the ability of G-MDSC to co-precipitate with peripheral blood mononuclear cells. We found that genes upregulated in G-MDSC were mostly involved in protein translation initiation (EEF1A1, RPL15, LOC440595, RPL22, RPS3, and EIF4A1), chromatin remodeling (HNRNPR), and cell cycle, G1-S growth factor regulation through AP-1 signaling (JUN and PRKCB1). Additional upregulated genes of interest included DNAJA2, co-chaperone of Hsp70, which was recently implicated in Treg-mediated immune suppression, as well as the anti-apoptotic protein TMBIM6. Genes that were relatively upregulated in patient neutrophils compared to G-MDSC included those related to cell adhesion (FLOT2, TUBA4A, LLPH, and PCDHB9) and cellular response to external signals. These included S100A11 which, upon ligation with RAGE, leads to NFkB and MAPK activation involved in oxidative stress; FOXO4, involved in wound healing, stress response, and elaboration of anti-microbial peptides; VPS41, involved in lysosomal trafficking; and, finally, CFLAR, which is anti-apoptotic. The findings support the notion that G-MDSC in ccRCC patients represent relatively less mature neutrophils and neutrophil precursors which are actively cycling and making proteins, some of which may be necessary for their functional activity. Meanwhile, patient neutrophils which co-precipitate with red blood cells, appear to function mostly in response to stimulatory and adverse external signals, some of which are likely to be involved in the G-MDSC-like functions previously described for TAN. G-MDSC are likely to differentiate into the activated neutrophils described in cancer patients. Further studies will need to focus on the differences between those neutrophils found in cancer patients and those found in apparently healthy individuals.

Beyond regulatory T cells: the potential role for IL-2 to deplete T-follicular helper cells and treat autoimmune diseases.

Low-dose IL-2 administration suppresses unwanted immune responses in mice and humans, thus evidencing the potential of IL-2 to treat autoimmune disorders. Increased Tregs activity is one of the potential mechanisms by which low-dose IL-2 immunotherapy induces immunosuppression. In addition, recent data indicate that IL-2 may contribute to prevent unwanted self-reactive responses by preventing the developing of T-follicular helper cells, a CD4(+) T-cell subset that expands in autoimmune disease patients and promotes long-term effector B-cell responses. Here we discuss the mechanisms underlying the clinical benefits of low-dose IL-2 administration, focusing on the role of this cytokine in promoting Treg-mediated suppression and preventing self-reactive T-follicular helper cell responses.

Antigen targeting to dendritic cells combined with transient regulatory T cell inhibition results in long-term tumor regression

Therapeutic vaccinations against cancer are still largely ineffective. Major caveats are inefficient delivery of tumor antigens to dendritic cells (DCs) and excessive immune suppression by Foxp3+ regulatory T cells (Tregs), resulting in defective T cell priming and failure to induce tumor regression. To circumvent these problems we evaluated a novel combinatorial therapeutic strategy. We show that tumor antigen targeting to DC-SIGN in humanized hSIGN mice via glycans or specific antibodies induces superior T cell priming. Next, this targeted therapy was combined with transient Foxp3+ Treg depletion employing hSIGNxDEREG mice. While Treg depletion alone slightly delayed B16-OVA melanoma growth, only the combination therapy instigated long-term tumor regression in a substantial fraction of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8+ T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity.

Lactose inhibits regulatory T-cell-mediated suppression of effector T-cell interferon-γ and IL-17 production.

Our interest in lactose as an immunomodulatory molecule results from studies showing that lactose binds to galectin-9, which has been shown to have various regulatory functions in the immune system including regulation of T-cell responses. Impaired regulation of T helper (Th)1 and Th17 type immune responses and dysfunction of regulatory T cells (Treg) have been implicated in many human immune-mediated diseases. In the present study, we investigated the effects of lactose on immune regulation using co-cultures of human peripheral blood mononuclear cell (PBMC)-derived Treg and effector T cells (Teff) obtained from twenty healthy adults. Treg, i.e. CD4+CD25+CD127-, were isolated from PBMC by immunomagnetic separation. The fraction of CD4+CD127- cells that was depleted of CD25+ cells was used as Teff. Treg and Teff at a ratio 1:5 were activated and the effects of lactose on the secretion of interferon-γ (IFN-γ) and IL-17 were analysed using ELISA for protein and quantitative RT-PCR for mRNA. Treg down-regulated the secretion of both IFN-γ (8.8-3.9 ng/ml, n 20, P= 0.003) and IL-17 (0.83-0.64 ng/ml, n 15, P= 0.04) in co-cultures, while in the presence of lactose the levels of secreted IFN-γ and IL-17 remained high and no down-regulation was observed (16.4 v. 3.99 ng/ml, n 20, P< 0.0001, and 0.74 v. 0.64 ng/ml, n 15, P= 0.005, respectively). We showed that lactose inhibits human Treg-mediated suppression of Th1 and Th17 immune responses in vitro.

CD4+Foxp3+ Tregs protect against innate immune cell-mediated fulminant hepatitis in mice

Foxp3+ Tregs play important roles in maintaining homeostasis by suppressing excessive immune responses that result in serious tissue damage; yet, it is largely unknown about the impact of Tregs on innate immune cells in hepatitis models in vivo. In this study, we examined the effect of hepatic Tregs on innate immune-mediated liver injury by using the murine model of polyI:C and d-galactosamine (d-GalN)-induced hepatitis. Administration of polyI:C/d-GalN increased the number of CD4+Foxp3+ Tregs in the liver. Depletion of Tregs leaded to higher levels of proinflammatory cytokine expression and severer liver injury, whereas adoptive transfer of Foxp3+ Tregs attenuated liver injury in polyI:C/d-GalN-treated mice. In addition, depletion of Tregs leaded to a reduction in TGF-β and IL-10 expression in polyI:C/d-GalN-treated mice. Both of these cytokines were important for suppression of polyI:C/d-GalN-induced liver injury. TGF-β was derived from Tregs. IL-10 was derived from active Kupffer cells, and coincubation of Kupffer cells with Tregs increased IL-10 secretion. Furthermore, TGF-β blockade abrogated Treg-mediated suppression of proinflammatory cytokine production by innate immune cell in vitro. ConclusionCD4+Foxp3+ Tregs modify innate immune responses in polyI:C/d-GalN-induced fulminant hepatitis via producing TGF-β and enhancing IL-10 secretion by Kupffer cells.

CD4+Foxp3+ regulatory T cell differentiation mediated by endometrial stromal cell-derived TECK promotes the growth and invasion of endometriotic lesions.

Endometriosis is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissue. The proportion of CD4+Foxp3+ regulatory T cells (Tregs) is significantly increased in the peritoneal fluid of women with endometriosis. The thymus-expressed chemokine TECK/CCL25 directly promotes the invasiveness of endometrial stromal cells (ESCs). The aim of this study was to investigate the effects of ESC-derived TECK on the crosstalk between Tregs and ESCs in the progress of endometriosis. We determined that the percentage of Tregs and the concentration of TECK increased in the peritoneal fluid with the progression of endometriosis. The supernatant from co-cultured human ESCs and macrophages not only induced Treg differentiation and increased Treg expression of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and CD73 by activating the AKT/STAT3 signaling pathway but also repressed Treg apoptosis by downregulating Fas and FasL expression and enhanced the Treg-mediated suppression of CD4+CD25− T cells. In addition, in vitro and in vivo trials confirmed that these effects could be inhibited by anti-TECK neutralizing Abs. The secretion of IL-10 and TGF-β by Tregs increased MMP2 expression and decreased TIMP1 expression and further stimulated the proliferation and invasion of ESCs and the growth of ectopic lesions. These results indicate that TECK derived from ESCs and macrophages upregulates the number and function of Tregs in the ectopic milieu, which contributes to endometriotic immunotolerance and high levels of ESC proliferation and invasion, thereby facilitating the progression of endometriosis.

Gr-1⁺CD11b⁺ immature myeloid cells (IMC) promote resistance of pro-inflammatory T cells to suppression by regulatory T cells in atherosclerotic Apo E- deficient mice.

Accumulating evidence indicates that both defects in Treg numbers and/or function as well as resistance of effector T cells to suppression may contribute to the development of human chronic inflammatory diseases. However, which mechanism involved in the progression of atherosclerosis remains unclear. In this study, we evaluated the production and function of CD4+ inflammatory and regulatory T cells in atherosclerosis-prone mice. We found that the hyperactivity and unresponsiveness to Treg-mediated suppression of inflammatory CD4+ T cells occurred in the progression of atherosclerosis, though Treg cells were present in very large numbers and fully functional. We further found that Gr-1+CD11b+ immature myeloid cells were significantly accumulated in atherosclerotic Apo E−/− mice, and they promoted resistance of inflammatory CD4+ T cells to Treg-mediated suppression in vitro and in vivo. we further confirmed that Gr-1+CD11b+ immature myeloid cells produced high level of interleukin 6 which was at least partially responsible for inducing unresponsiveness of inflammatory CD4+ T cells to suppression via activation of Jak/Stat signaling pathway. Taken together, these findings might provide new insights to explore potential targets for immune therapeutic intervention in atherosclerosis.

Abstract 5023: Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy

Abstract Combination immunotherapy regimens incorporating agents that target more than a single regulatory pathway or immune inhibitory checkpoint can confer marked enhancement of anti-tumor immune responses when combined with active immunotherapy. We have tested the antitumor effect of the IDO pathway inhibitor NLG-919 (currently in Phase I clinical trial) in combination with indoximod (a different IDO pathway inhibitor currently in Phase II clinical trials), chemotherapy, vaccination and/or PD-1/PD-L1/PD-L2 blockade. Here we show that the highly potent, orally-bioavailable IDO pathway inhibitor NLG-919 is synergistic with blockade of the PD-1/PD-L1/PD-L2 pathway in mouse models of large established tumors. The combination of NLG-919 plus PD-1/PD-L1/PD-L2 blockade showed significantly enhanced anti-tumor effect compared to either agent alone. This synergy was particularly evident in the setting of large established tumors treated with normally ineffective immunotherapy regimens (e.g., a single dose of chemotherapy plus a vaccine against a poorly immunogenic shared-self antigen). In vivo, administration of NLG-919 enhanced anti-tumor vaccine responses against B16F10 melanoma treated with hgp100 vaccine plus resting, non-activated pmel-1 T cells NLG-919 also reduced Treg-mediated suppression in tumor-bearing hosts, and enhanced dendritic cell activation in tumors and TDLNs. In combination with chemotherapy, treatment with NLG-919 allowed effector T cell responses against endogenous tumor antigens released by chemotherapy. By each of the preceding readouts, the in vivo biologic effect of NLG-919 was qualitatively identical to that of indoximod, but the same immunologic effects could be achieved at lower plasma concentrations in vivo. In multiple models, a combination of oral NLG-919 with oral indoximod produced synergistic anti-tumor effects, which was further enhanced by blockade of the PD 1/PD L1/PD-L2 pathway. In a more stringent B16F10 melanoma tumor model that did not involve adoptive transfer of pmel-1 cells, a combination of immune checkpoint inhibition involving NLG-919, indoximod and anti-PD1/PD-L1/PD-L2 antibodies with chemotherapy and hgp100 peptide vaccine was able to achieve a significant antitumor effect. The current preclinical studies suggest a mechanistic rationale for a combining NLG919 with indoximod and with agents targeting the PD 1/PD L1/PD-L2 pathway. Citation Format: Mario R. Mautino, Charles J. Link, Nicholas N. Vahanian, James T. Adams, Clarissa Van Allen, Madhav D. Sharma, Theodore S. Johnson, David Munn. Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2014-5023

Systemic inflammatory response elicited by superantigen destabilizes T regulatory cells, rendering them ineffective during toxic shock syndrome.

Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti-IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.

Regulatory T cells suppress CD4+ T cells through NFAT-dependent transcriptional mechanisms.

Regulatory T cells (Tregs) control autoreactive T cells by inhibiting activation-induced proliferation and cytokine expression. The molecular mechanisms responsible for the inactivation of effector T cells by Tregs remain yet to be fully characterized. We report that T-helper cells stimulated in the presence of Tregs quickly activate NFAT1 and have increased NFAT1-dependent expression of the transcription repressor Ikaros. NFAT1 deficiency or dominant-negative Ikaros compromises Treg-mediated inhibition of T-helper cells in vitro and in vivo. Thus, our results place NFAT-dependent mechanisms as general regulators of T-cell tolerance and show that Treg-mediated suppression of T-helper cells results from the activation of NFAT-regulated gene expression.

Clematichinenoside AR induces immunosuppression involving Treg cells in Peyer׳s patches of rats with adjuvant induced arthritis

Ethnopharmacological relevanceClematichinenoside AR (AR) has been defined as a major active ingredient of triterpenoid saponins extracted from Clematidis Radix et Rhizoma, which is a traditional Chinese herbal medicine that has long been used in the treatment of rheumatoid arthritis (RA). To further explore the mechanism of AR in the treatment of RA, we investigated whether its immunomodulatory effects are related to Treg-mediated suppression derived from Peyer׳s patches (PPs) in adjuvant induced arthritis (AIA) rat model. Materials and methodsAR (8, 16, 32mg/kg) was orally administered daily from Day 18 to Day 31 after immunization. The effect of AR on AIA rats was evaluated by hind paw swelling and histopathological examination. Percentages of CD4+CD25+Foxp3+ T regulatory cells were determined by flow cytometry. Levels of IL-10, TGF-β1, IL-17A and TNF-α were measured by ELISA. Expressions of Foxp3 and RORγ in synovium were detected using immunohistochemical analysis. ResultsAR treatment significantly reduced paw swelling of AIA rats, and histopathological analysis confirmed it could suppress severity of established arthritis. AR treatment upregulated the percentages of CD4+CD25+Foxp3+ Treg cells among CD4+ T cells in PPs lymphocytes, and increased the levels of IL-10 and TGF-β1 secreted from ConA-activated PPs lymphocytes, whereas decreased the levels of IL-17 A and TNF-α. Similar tendency of circulating CD4+CD25+Foxp3+ Treg cells percentages and serum cytokine levels were observed. Moreover, AR decreased the expression levels of Foxp3 and RORγ in joint synovial membrane. ConclusionsIn conclusion, these results suggested AR has a potent protective effect on the progression of AIA, probably by augmenting CD4+CD25+Foxp3+ Treg cells in PPs to induce immunosuppression, and modulating the balance between Treg cells and Th17 cells systemically. These findings may help to develop AR as a potent immunosuppressive agent for the treatment of RA.

Impaired function of regulatory T-cells in patients with chronic obstructive pulmonary disease (COPD)

Anti-inflammatory pathways affecting chronic obstructive pulmonary disease (COPD) are poorly understood. Regulatory T-cells (Tregs) are important negative regulators of T-cell activity and hence were investigated in COPD patients in this study. We hypothesised that functional defects in Tregs may promote increased inflammation contributing to the pathogenesis of COPD.Peripheral blood mononuclear cells (PBMC) were isolated from patients with stable COPD and age-matched non-smoking controls. Treg-mediated suppression of memory non-Treg (Foxp3−CD45RO+) CD4+ T-cell activation was analysed by comparing PBMC responses to staphylococcal enterotoxin-B (SEB) pre- and post-depletion of Tregs (CD25+CD127lowCD4+ T-cells) by fluorescence-activated cell sorting (FACS). Activation of T-cells was assessed by HLA-DR expression. Levels of secreted cytokines were measured by ELISA.Depletion of Tregs increased SEB-induced activation of Foxp3−CD45RO+ CD4+ T-cells in samples from 15/15 healthy controls (demonstrating Treg-mediated suppression) and 9/14 COPD patients (Fisher's test, p=0.017). A screen of clinical data associated a failure of Treg-mediated suppression in the remaining five COPD patients with a higher body mass index (BMI) (33–38kg/m2) compared to patients with unimpaired Treg function (20–32kg/m2).In conclusion, we demonstrate impaired Treg-mediated suppression of CD4+ T-cell activation in a subset of COPD patients, all of whom had high BMI. Obesity and/or perturbed homeostasis of Treg subsets may explain this defect and therefore contribute to increased inflammation observed in COPD.

T cell-intrinsic role of IL-6 signaling in primary and memory responses.

Innate immune recognition is critical for the induction of adaptive immune responses; however the underlying mechanisms remain incompletely understood. In this study, we demonstrate that T cell-specific deletion of the IL-6 receptor α chain (IL-6Rα) results in impaired Th1 and Th17 T cell responses in vivo, and a defect in Tfh function. Depletion of Tregs in these mice rescued the Th1 but not the Th17 response. Our data suggest that IL-6 signaling in effector T cells is required to overcome Treg-mediated suppression in vivo. We show that IL-6 cooperates with IL-1β to block the suppressive effect of Tregs on CD4(+) T cells, at least in part by controlling their responsiveness to IL-2. In addition, although IL-6Rα-deficient T cells mount normal primary Th1 responses in the absence of Tregs, they fail to mature into functional memory cells, demonstrating a key role for IL-6 in CD4(+) T cell memory formation.DOI: http://dx.doi.org/10.7554/eLife.01949.001.

Regulatory T cells use LAG-3 as the “brake” in autoimmune diabetes but the “accelerator” in tumor environment (IRC4P.487)

Abstract Lymphocyte Activation Gene-3 (LAG-3, CD223) is an inhibitory receptor that is expressed upon TCR activation, is highly up-regulated on exhausted conventional T cells, and regulates T cell expansion, homeostasis and function via both cell intrinsic and extrinsic mechanisms. LAG-3 is also required for maximal regulatory T cell (Treg) function. Our lab has previously shown that LAG-3 modulates tolerance mechanisms, but the specific function of LAG-3 expressed by Tregs remains unknown. To elucidate the role of LAG-3 in Treg-mediated immune suppression, we generated mice in which active, membrane-expressed LAG-3 is specifically deleted in Foxp3+ Tregs. We used both the non-obese diabetes (NOD) and B6 mouse backgrounds to study the role of LAG-3-deficient Tregs in autoimmune diabetes and B16 melanoma, respectively. Surprisingly, the deletion of LAG-3 in Tregs both protected NOD mice from autoimmune diabetes and resulted in decreased B16 melanoma growth. In an autoimmune environment, the number of Tregs infiltrating the islets was limited by LAG-3, which facilitated the breakdown of self-tolerance. However, in the tumor microenvironment, LAG-3 activity was required for Treg-mediated immune suppression. These data suggest opposing roles for LAG-3 on Tregs in an inflammatory versus an immunosuppressive microenvironment. Therefore, specifically targeting LAG-3 on Tregs may provide a strategy for limiting tumor growth without subsequent development of autoimmunity.

CTLA-4+ regulatory T cells are increased in cetuximab treated head and neck cancer patients, suppress NK cell cytotoxicity and correlate with poor prognosis (TUM2P.916)

Abstract The EGFR-targeted antibody, cetuximab, is clinically effective against head and neck cancer (HNC) but only in 15 - 20% of patients. We hypothesized that regulatory T cells may have functional and clinical impact antitumor immunity induced in cetuximab-treated patients. Indeed, Treg frequency and expression of CTLA-4, CD39, and surface TGF-β were significantly increased after cetuximab-based therapy. These Treg were inversely associated with functional activity of intratumoral NK cells and with clinical outcome in cetuximab-treated HNC patients. Importantly, NK cells selectively eliminated intratumoral Treg in the presence of anti-CTLA mAb (ipilimumab), which overcame the Treg suppressive activity on cetuximab-mediated ADCC. These results suggest that differences in Treg-mediated suppression may play a role in the clinical response to cetuximab therapy, and that efficacy of cetuximab therapy could be enhanced by adding ipilimumab to eliminate Treg, thus promoting anti-tumor immunity.

Effects of regulatory T cells on peripheral blood mononuclear cell responses to live Mycobacterium avium subspecies paratuberculosis in cows with Johne’s disease. (VET2P.1035)

Abstract Johne’s disease (paratuberculosis) is a chronic wasting disease of wild and domestic ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Previously, we proposed that the chronic nature of Johne’s disease may result in development of a regulatory T cell (Treg) population in the host. These cells function to shift the immune balance away from a pro-inflammatory Th1 response to an unproductive Th2 response. Due to the inherently low abundance of Tregs in the periphery and the relatively low proportion of Tregs reactive toward any one particular infectious agent, we have developed an in vitro method to stimulate expansion of MAP-reactive Treg populations from CD4+CD25+ peripheral blood mononuclear cells (PBMCs). Enriched Tregs may subsequently be used in Treg-mediated suppression assays. A simplified version of this procedure is also used to determine differences in naïve T cell responses to MAP antigen stimulation, including induction of FoxP3 expression. Following PBMC stimulation with live MAP bacteria and with MAP-reactive Tregs, real-time PCR is used to analyze expression of Th1, Th2, and Treg transcription factors as well as pro- and anti-inflammatory cytokines including. Results are compared between cattle that are test-negative and test-positive for Johne’s disease, with results from test-positive cattle further subdivided between animals exhibiting symptoms of clinical vs subclinical disease.

Granzyme B plays a differential role in CD4+ T cell subsets versus CD8+ T cells in mediating the graft-versus-tumor effect and graft-versus-host disease (TRAN3P.888)

Abstract After allogeneic hematopoietic cell transplantation, donor-derived T cells can attack host tumor cells, producing a favorable graft-vs.-tumor (GVT) effect. However, graft-vs.-host disease (GVHD) may develop when donor T cells attack normal host tissue. We previously reported that while granzyme B (GzmB) is required for CD8+ T cells to cause GVHD, GzmB-mediated damage of CD8+ T cells impairs the GVT effect. In this study, we examined the role of GzmB in GVT and GVHD mediated by CD4+ T cell subsets, using an established A20 tumor model and bioluminescence imaging to monitor tumor growth in BALB/c hosts. We found that CD4+CD25+ regulatory T cells (Tregs) derived from C57BL/6 donors significantly suppressed GVT activity mediated by both CD8+ T cells and CD4+CD25- effector T cells (Teffs). However, GzmB-/- Tregs were as equally suppressive as WT Tregs, consistent with our previous report that GzmB is not required for Treg-mediated suppression of GVHD. Nonetheless, GzmB-/- Teffs were less effective in controlling tumor growth than WT Teffs; but surprisingly, GzmB-/- Teffs caused more severe GVHD than WT Teffs. Our data reveal that GzmB is essential for CD4+CD25- Teff-mediated optimal GVT effect but diminishes the ability of Teffs to cause GVHD, which contradicts its role in CD8+ T cells where GzmB is essential to cause GVHD but impairs GVT effect. These differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to separate GVT from GVHD.

TNFR2 expression by CD4 effector T cells is required to induce full-fledged experimental colitis (CCR3P.204)

Abstract There is now compelling evidence that TNFR2 is constitutively expressed on Tregs and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of CD4+Foxp3+ regulatory T cells (Tregs). However, we also showed that the expression of TNFR2 could be up-regulated on CD4+Foxp3- effector T cells (Teffs) upon TCR stimulation, and TNFR2-expressing Teff cells were more activated and more resistant to Treg-mediated inhibition. In order to further define the role of TNFR2 in the proliferative expansion of pathogenic CD4 T cells in a setting of autoimmune disorder, we transferred naïve CD4 cells from WT mice and TNFR2-/- mice into Rag1-/- recipients. The results showed that TNFR2-deficient Teff cells failed to induce full-fledged colitis unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to produce proinflammatory Th1 cytokines on a per cell basis. In vitro, TNF blockade inhibited the up-regulation of TNFR2 expression, as well as proliferative responses and cytokine production, by TCR-stimulated naïve CD4 cells. This effect of TNF blockade can be replicated by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Therefore, TNFR2 is critical for the proliferative expansion of pathogenic CD4 T cells, and thus may be a therapeutic target in the treatment of CD4 T cell-mediated autoimmune inflammation, although at a cost of attenuation of Treg activity.

FOXP3 is a direct target of miR15a/16 in umbilical cord blood regulatory T cells.

Exact mechanism of action of umbilical cord blood (CB)-derived regulatory T cells (Tregs) in the prevention of GVHD remains unclear. On the basis of selective overexpression of peptidase inhibitor 16 in CB Tregs, we explored the related p53 pathway, which has been shown to negatively regulate miR15a/16 expression. Significantly lower levels of miR15a/16 were observed in CB Tregs when compared with conventional CB T cells (Tcons). In a xenogeneic GVHD mouse model, lower levels of miR15a/16 were also found in Treg recipients, which correlated with a better GVHD score. Forced overexpression of miR15a/16 in CB Tregs led to inhibition of FOXP3 and CTLA4 expression and partial reversal of Treg-mediated suppression in an allogeneic mixed lymphocyte reaction that correlated with the reversal of FOXP3 demethylation in CB Tregs. On the other hand, miR15a/16 knockdown in CB Tcons led to expression of FOXP3 and CTLA4 and suppression of allogeneic lymphocyte proliferation. Using a luciferase-based mutagenesis assay, FOXP3 was determined to be a direct target of miR15a and miR16. We propose that miR15a/16 has an important role in mediating the suppressive function of CB Tregs and these microRNAs may have a 'toggle-switch' function in Treg/Tcon plasticity.

Costimulatory Blockade-Induced Allograft Survival Requires Beclin1

Autophagy is required for T cell homeostasis and activation-induced T cell expansion. Whether autophagy participates in tolerance induction to foreign antigens, including allografts, is unknown. We tested the role of an essential autophagy protein, Beclin1, in heart transplant survival in mice. We observed that long-term allograft survival induced by donor-specific transfusion plus anti-CD154 mAb required homozygous lymphocyte expression of Beclin1. Following adoptive transfer into allogeneic recipients, autophagy-deficient, Beclin1 heterozygous effector T cells (Teffs) exhibited enhanced proliferation with diminished cell death and increased production of interferon gamma. Whereas the induction and function of regulatory T cells (Tregs) in Beclin1 heterozygous mice were normal, Teffs from these mice were resistant to Treg-mediated suppression. Our findings identify a requisite role for Beclin1 in facilitating Teff death during tolerance induction.