Suppressor Cell Assays Research Articles

Induced CD8+FoxP3+ Treg cells in rheumatoid arthritis are modulated by p38 phosphorylation and monocytes expressing membrane tumor necrosis factor α and CD86.

Limiting the severity of inflammation and promoting its eventual resolution are vital for protecting host tissues both in autoimmunity and chronic infection. The aim of this study was to determine the suitability of repurposing anti-CD3 monoclonal antibody (mAb) therapy for rheumatoid arthritis (RA) by analyzing its ability to induce CD8+FoxP3+ Treg cells from peripheral blood mononuclear cells (PBMCs). Anti-CD3 mAb was cultured with RA PBMCs to induce CD8+FoxP3+ Treg cells, which were analyzed by flow cytometry to determine their phenotype. Treg cell induction was investigated via neutralization or blocking antibodies, cellular depletion, or ImageStream technology. Blotting was used to determine the signaling pathways involved in CD8+FoxP3+ Treg cell induction. Suppression of CD4+ T cell effector responses was assessed by Treg cell suppression assays and Mosaic enzyme-linked immunosorbent assay. Potent CD8+FoxP3+ Treg cells were induced from RA PBMCs by anti-CD3 mAb. Unlike their CD4+ counterparts, CD8+FoxP3+ Treg cells inhibited Th17 responses in a contact-dependent manner, thereby functioning to limit a wider range of inflammatory pathways. CD8+FoxP3+ Treg cell induction was supported both by p38 phosphorylation intrinsic to naive CD8+ T cells and by monocytes via CD86 and membrane tumor necrosis factor α (TNFα). Artificially increasing monocyte membrane TNFα or inhibiting CD8+ T cell p38 phosphorylation drove FoxP3 expression in a subset of initially unresponsive CD8+ T cells. These data define an unknown mechanism of CD8+FoxP3+ Treg cell induction by anti-CD3 mAb, which could be combined with a p38 inhibitor to improve therapeutic efficacy in RA patients and resolve chronic inflammation via the restoration of tolerance.

O6.04 * GRANULOCYTIC MYELOID-DERIVED CELLS OBTAINED FROM BLOOD MAINLY SUPPRESS T-CELL PROLIFERATION IN PATIENTS WITH GLIOBLASTOMA

OBJECTIVE: To study the importance of myeloid-derived suppressor cells (MDSC) in primary glioblastomas, we investigated the frequency, phenotype, and immune-suppressive function of different myeloid-derived cells (MDC) in the blood and freshly resected tumors of 40 patients. RESULTS: Multicolour flow cytometric analysis showed that the frequency of CD14lowCD15+ granulocytic CD11bhighCD33b+ MDC was higher in peripheral blood and tumor samples of glioblastoma patients compared to healthy controls and positively correlated with the tumor volume before resection. Granulocytic MDC expressed CD16, CD66b, CD114, CD195 and could be further divided into CD14lowCD15high neutrophilic and CD14lowCD15int eosinophilic MDC. Moreover, we could detect an intratumoral accumulation of CD14highCD15+ monocytic CD11bhighCD33b+ MDC expressing CD115, CD124, CD184, CD206 and CX3CR1. All MDC subsets stained positively for reactive oxygen species, but Arginase 1 was specifically expressed in tumor-infiltrating MDC. T cell suppression assays with FACS-sorted MDC revealed that granulocytic and most prominently eosinophilic MDC obtained from the blood, but not the tumor could efficiently suppress non-specific T cell proliferative responses. Contrary to blood-derived MDC, HLA-DR expression was significantly increased on tumor-infiltrating MDC. Upon LPS or R848 stimulation, blood-derived MDC showed an increased production of IL-10, in contrast to tumor-infiltrating MDC that were resistant to Toll-like-receptor (TLR) stimulation. Although the frequency of granulocytic MDC inversely correlated with CD4+ and CD8+ T memory cells in the blood, no association with time to tumor progression in glioblastoma patients could be found. CONCLUSION: Our findings provide a detailed characterization of the nature and suppressive function of MDC in glioblastoma patients and indicate that granulocytic MDC obtained from the blood but not from the tumor mainly suppress T-cell proliferation.

AB0142 Immunosuppressive effects of glucocorticoids and regulatory t cells on cd28null t cells in vitro

BackgroundCD28null T cells are terminally differentiated T cells lacking CD28 co-receptor. These cells display properties of proinflammatory killer cells1, 2 and are suggested to be resistant to apoptosis in vivo3....

Abstract 461: Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction.

Abstract Cancer immunotherapy aims to harness the power of the immune system to fight advanced stages of cancer. A main hindrance to cancer immunotherapy is tumor-induced immune suppression. Within the tumor microenvironment are a plethora of immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in nearly all types of cancer blocking T cell activation and shifting immunity towards a type 2 tumor-promoting phenotype through cross-talk with macrophages that enhances the secretion of pro-tumor molecules such as IL-10, and simultaneously drops IL-12 production by macrophages. MDSC accumulation is induced by pro-inflammatory cytokines including IL-1β, IL-6 and VEGF as well as alarmins such as S100A8/9. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Inhibition of HMGB1 in vitro by chemical inhibitors Glycyrrhizin and Ethyl Pyruvate decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages. Ethyl Pyruvate also reduced MDSC suppressive capacity in transgenic T cell suppression assays, suggesting that HMGB1 regulates multiple aspects of MDSC function. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin and Ethyl Pyruvate reduced lung metastases and did not impact primary tumor progression. Treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. HMGB1 consists of two domains, the pro-inflammatory B box domain and an anti-inflammatory A box domain. Treatment of C57BL/6 mice with MC38 tumor with A box significantly reduced primary tumor growth and decreased MDSC levels in the blood. These findings indicate that full-length HMGB1 drives inflammation and tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC. These results suggest that HMGB1 drives MDSC accumulation and tumor progression and that A box can be used to target MDSC-mediated tumor-induced immune suppression. (Research supported by NIH RO1CA115880 and RO1CA84232) Citation Format: Katherine Parker, Suzanne Rosenberg, Pratima Sinha, Huan Yang, Kevin Tracey, Jianhua Li. Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 461. doi:10.1158/1538-7445.AM2013-461

Monocyte-Derived Dendritic Cells Induce Functionally Active Regulatory T Cells Upon Exposure to BCR-ABL Tyrosine Kinase Inhibitors.

AbstractAbstract 2156Multiple approaches for treatment of malignant disease presently aim to combine targeted therapy with tyrosine kinase inhibitors (TKI) with immunotherapy. Dendritic cells (DC) are frequently used in such strategies due to their unique ability to initiate potent T cell anti-tumor immunity. Unfortunately, DC may also activate suppressive CD25+FOXP3+ regulatory T cells (Treg), which depends on the stimuli that influence DC in immature state and/or during development from precursor cells. High frequencies of Treg have been described in several types of tumors within the tumor microenvironment, which is associated with poor prognosis and reduced survival. DC development and function are moreover governed by various tyrosine kinases of which some are also inhibited by clinically used TKI. TKI thus may cause immunoinhibitory side effects, and we previously demonstrated that exposure of monocyte-derived DC to the BCR-ABL inhibitor imatinib causes up-regulation of the immunosuppressive type I transmembrane glycoprotein osteoactivin (GPNMB, DC-HIL) and reduces expression of activating surface antigens as well as T cell-stimulatory capacity of DC in vitro (Schwarzbich et al., 2012). Other investigators reported that imatinib induces functionally Treg in CML patients, but the underlying mechanisms are so far unknown. (Bachy et al., 2011). On the other hand, TKI may inhibit proliferation and suppressive capacity of regulatory T cells in vitro (Chen et al., 2007). Here we tried to solve this apparent discrepancy by analyzing the influence of TKI on DC-Treg interaction. Monocyte-derived DC (moDC) were generated over 7 days by exposing blood monocytes to GM-CSF and IL-4. TNF was added on day 6 of culture in case of maturation, and imatinib or nilotinib (3μM each) were added to the culture medium every second day starting from the first day of culture. Induction and functionality of Treg was determined by FACS and so called effector T cell suppression assays upon culture of moDC with autologous PBMC. We found that exposure of moDC to imatinib or nilotinib only slightly increased the frequency of Treg as compared to controls. However, these Treg strongly inhibited autologous T cell proliferation as assessed by T cell suppression assays. This was mediated by direct cellular interaction, as culture supernatants of TKI-treated DC did not alter Treg function and also did not contain elevated levels of the immunosuppressive (and Treg inducing) cytokines TGF-β and IL-10. Thus, our data indicate that the seemingly contradictory results of the in vivo and in vitro studies described above may be explained by the effects caused by exposure of moDC to BCR-ABL TKI which results in the induction of functionally active Treg. These findings are of special importance for future combinatory approaches using TKI and DC-based immunotherapy. Disclosures:No relevant conflicts of interest to declare.

The B10 Idd9.3 Locus Mediates Accumulation of Functionally Superior CD137+ Regulatory T Cells in the Nonobese Diabetic Type 1 Diabetes Model

CD137 is a T cell costimulatory molecule encoded by the prime candidate gene (designated Tnfrsf9) in NOD.B10 Idd9.3 congenic mice protected from type 1 diabetes (T1D). NOD T cells show decreased CD137-mediated T cell signaling compared with NOD.B10 Idd9.3 T cells, but it has been unclear how this decreased CD137 T cell signaling could mediate susceptibility to T1D. We and others have shown that a subset of regulatory T cells (Tregs) constitutively expresses CD137 (whereas effector T cells do not, and only express CD137 briefly after activation). In this study, we show that the B10 Idd9.3 region intrinsically contributes to accumulation of CD137(+) Tregs with age. NOD.B10 Idd9.3 mice showed significantly increased percentages and numbers of CD137(+) peripheral Tregs compared with NOD mice. Moreover, Tregs expressing the B10 Idd9.3 region preferentially accumulated in mixed bone marrow chimeric mice reconstituted with allotypically marked NOD and NOD.B10 Idd9.3 bone marrow. We demonstrate a possible significance of increased numbers of CD137(+) Tregs by showing functional superiority of FACS-purified CD137(+) Tregs in vitro compared with CD137(-) Tregs in T cell-suppression assays. Increased functional suppression was also associated with increased production of the alternatively spliced CD137 isoform, soluble CD137, which has been shown to suppress T cell proliferation. We show for the first time, to our knowledge, that CD137(+) Tregs are the primary cellular source of soluble CD137. NOD.B10 Idd9.3 mice showed significantly increased serum soluble CD137 compared with NOD mice with age, consistent with their increased numbers of CD137(+) Tregs with age. These studies demonstrate the importance of CD137(+) Tregs in T1D and offer a new hypothesis for how the NOD Idd9.3 region could act to increase T1D susceptibility.

Inhibitory Potential of Subpopulations of CD8+T Cells in HIV-1-Infected Elite Suppressors

Elite controllers or suppressors (ES) are HIV-1-infected individuals who suppress viral replication to clinically undetectable levels without antiretroviral therapy. Understanding the mechanisms by which ES control viral replication may prove informative for the design of a therapeutic vaccine. Qualitative differences in the CD8(+) T cell response have been implicated in control. Therefore, we isolated CD8(+) T cells from ES and characterized the ability of sorted memory and activation subpopulations to control viral replication at various effector-to-target cell ratios using a novel modification of a CD8(+) T cell suppression assay. The effector memory and terminal effector subpopulations of memory CD8(+) T cells had the highest inhibitory potential over the course of a 3-day in vitro infection. Interestingly, after 5 days of infection, central memory CD8(+) T cells were also very effective at suppressing viral replication. No significant correlation between the suppression of viral replication and the number of HIV-1-specific CD8(+) T cells was observed. HLA-DR(-) CD38(+) CD8(+) T cells possessed the lowest inhibitory potential of the activation subpopulations. Taken together, our data suggest that there are key differences in the magnitude and kinetics of the suppression of HIV-1 replication by different CD8(+) T cell subsets. These data should guide the development of an effective, cellular therapeutic vaccine that has the potential to elicit similar CD8(+) T cell responses.

Th17 cells are restrained by Treg cells via the inhibition of interleukin-6 in patients with rheumatoid arthritis responding to anti-tumor necrosis factor antibody therapy.

The importance of interleukin-17 (IL-17) is underscored both by its resistance to control by Treg cells and the propensity of Treg cells to produce this highly inflammatory cytokine. This study sought to address whether Th17 cells are inhibited by Treg cells in rheumatoid arthritis (RA) patients responding to anti-tumor necrosis factor (anti-TNF) therapy, and if so defining the underlying mechanisms of suppression. Inhibition of Th17 cell responses was determined by Treg cell suppression assays. The Treg cell phenotype was analyzed using flow cytometry and enzyme-linked immunosorbent assay. Mechanisms of suppression were tested by cytokine addition or antibody blockade. Th17 responses were inhibited by Treg cells from RA patients responding to the anti-TNF antibody adalimumab (Treg(ada) ), but not by Treg cells from healthy individuals or patients with active RA. Furthermore, Treg(ada) cells secreted less IL-17, even when exposed to proinflammatory monocytes from patients with active RA. Treg(ada) cells suppressed Th17 cells through the inhibition of monocyte-derived IL-6, but this effect was independent of IL-10 and transforming growth factor β, which mediated the suppression of Th1 responses. Adalimumab therapy led to a reduction in retinoic acid receptor-related orphan nuclear receptor C-positive Th17 cells and an increase in FoxP3+ Treg cells lacking expression of the transcription factor Helios. However, this acquisition of IL-17-suppressor function was not observed in RA patients responding to treatment with etanercept, a modified TNF receptor-Fc fusion protein. Indeed, there was no alteration in Treg cell number, function, or phenotype in etanercept-treated patients, and Th17 responses remained unchecked. Th1 and Th17 responses are controlled through distinct mechanisms by Treg cells from patients responding to anti-TNF antibody therapy. Adalimumab therapy, but not etanercept therapy, induces a potent and stable Treg cell population with the potential to restrain the progression of IL-17-associated inflammation in RA via regulation of monocyte-derived IL-6.

O038 Role of human tolerogenic dendritic cell derived IL-35 in regulating T cell responses

Introduction IL-35 is a novel cytokine of the IL-12 family, existing as a heterodimer of IL-12p35 and EBV-induced gene 3 (Ebi3). IL-35 is produced by natural and inducible regulatory T cells in humans and mice and seems required for optimal suppression. Dexamethasone (Dex) and 1, 25-dihydroxyvitamin D3 (D3) have previously been shown to inhibit IL-12 production in activated dendritic cells (DC) and suppress allogenic T cell responses. Tolerogenic DCs are potentially useful to specifically alleviate unwanted immune responses and induce immune tolerance in transplantation and autoimmunity. In this study, we investigated the regulation of production of various IL-12 family members in human tolerogenic DC (tDC), with a main focus on IL-35. Methods Monocytes were isolated from human PBMC and cultured for 6 days in the presence of IL-4 and GM-CSF to obtain immature DC. Tolerogenic dendritic cells were generated using the same culture conditions but with the addition of Dexamethasone for DexDC, or Dexamethasone with 1, 25-dihydroxyvitamin D3 for D3Dex. RT-PCR quantified IL-12A, IL-12B, IL-27A, IL-23A, EBI3 and IL-10. ELISA was used to measure IL-12p40, IL-12p70, IL-10 and IFN- γ in cellular supernatants. Intracellular expression of IL-12p35, Ebi3 and IL-12p40 was measured by flow cytometry. Expression of IL-12p35 and Ebi3 was evaluated using confocal microscopy and western blot. For T cell suppression assays naive CD4 + T cells were isolated form cord blood or PBMCs and stimulated with anti-CD3/28 in the presence or absence of DC derived supernatants. Proliferation was measured by 3 H- thymidine incorporation. Results We demonstrate by Q-PCR that, although tDC completely lack the expression of IL-12p40, they maintain mRNA expression of Ebi3 and IL-12p35. In line with this, tDC do not produce bioactive IL-12p70 or the homodimer IL-12p40. Using intracellular FACS and western blot we show that tDC maintain the protein expression of both Ebi3 and IL-12-p35. Expression of these proteins can be further enhanced upon stimulation with pro-inflammatory cytokines, TLR agonists and CD40 ligation. Functional inhibition studies show that tDC derived supernatants have the ability to suppress T cell proliferation. At present we are performing blocking experiments in tDC supernatants to fully evaluate the extent at which IL-35 contributes to the tolerogenic characteristic these cells possess in regulating T cell responses. Conclusion Taken together, our results suggest that aside from increased expression of previously described tolerance inducing markers (B7-H1, ILT3, etc.) and increased IL-10 production, tDC produce IL-35, providing an additional novel mechanism by which these cells elicit their tolerogenic potential.

The Comparison of Interleukin 6–Associated Immunosuppressive Effects of Human ESCs, Fetal-Type MSCs, and Adult-Type MSCs

Although human embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) from various sources display immunomodulatory effects, direct comparisons among these classes of stem cells have not been reported. Peripheral blood mononuclear cell suppression assays and carboxyfluorescein diacetate succinimidyl ester assays were used to assess the immunosuppressive effects of stem cells. Gene expression was measured using DNA microarrays. Enzyme-linked immunosorbent assays were used to determine interleukin (IL)-6 levels. We found that fetal-type MSCs proliferated significantly faster than adult-type MSCs. Compared with ESCs grown on feeder cells, ESCs grown in feeder cell-free conditions exhibited decreased immunosuppressive effects. The suppressive effects of ESCs were significantly stronger than those of MSCs, and the suppressive effects of fetal-type MSCs were significantly stronger than those of adult-type MSCs at each tested dose level. Analysis of gene expression by microarray and MetaCore pathway mapping revealed changes in eight different immune response pathways; we observed that IL-6 gene expression was highly significantly involved in all eight pathways. Significantly higher IL-6 elevation ratios (IL-6after:IL-6before) were found in ESCs compared with fetal-type MSCs, and these were also found in fetal-type MSCs compared with adult-type MSCs. Furthermore, IL-6 levels were found to correlate with cell dosages of MSCs and the suppressive effects. The ease of obtaining fetal-type MSCs and their rapid proliferation make these cells ideal candidates for cell-based therapies, especially for diseases associated with immune responses, given the immunosuppressive effects of these cells. IL-6 might play an important role in the immunosuppressive effects of various stem cells.

A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy

AbstractRegulatory T cells (CD4+CD25hiCD127loFOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

Use of SNARF-1 to measure murine T cell proliferation in vitro and its application in a novel regulatory T cell suppression assay

The green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track the proliferation of T cells in vitro. Such assays often incorporate more than one population of cells, but the paucity of alternative, spectrally distinct dyes suitable for measuring proliferation has hampered the simultaneous tracking of multiple cell populations; furthermore, CFSE is not compatible with green fluorescent protein (GFP), used to identify T cells in various transgenic mice. We have therefore validated the use of the far red dye seminaphthorhodafluor-1 (SNARF)-1 – originally developed to measure intracellular pH – to track murine T cell proliferation in vitro, demonstrating its ability to distinguish multiple cycles of proliferation over three days in a similar fashion to CFSE. The small changes in fluorescence emission attributed to intracellular alkalinisation of proliferating T cells have minimal impact on the ability of SNARF-1 to track cell division and this dye induces minimal cell death at the concentration used in this application. On the basis of these results, we have developed a novel in vitro murine T cell suppression assay, in which the proliferation of both conventional T cells (Tcons) stained with SNARF-1 and regulatory T cells (Tregs) stained with CFSE can be measured simultaneously. We have also demonstrated that SNARF-1 may be used to stain Tcons in assays of suppression involving ‘designer’ Tregs, generated by the transduction of CD4+ T cells with constructs encoding the Foxp3gfp fusion protein.

Superantigen-Induced Proliferation of Human CD4+CD25− T Cells Is Followed by a Switch to a Functional Regulatory Phenotype

Bacterial superantigens are potent T cell activators. In humans they cause toxic shock and scarlet fever, and they are implicated in Kawasaki's disease, autoimmunity, atopy, and sepsis. Their function remains unknown, but it may be to impair host immune responses increasing bacterial carriage and transmission. Regulatory (CD25(+)FOXP3(+)) T cells (Tregs) play a role in controlling inflammatory responses to infection. Approximately 2% of circulating T cells are naturally occurring Tregs (nTregs). Conventional Ag stimulation of naive FOXP3(-) T cells induces Ag-specific Tregs. Polyclonal T cell activation has been shown to produce non-Ag-specific Tregs. Because superantigens are unique among microbial virulence factors in their ability to trigger polyclonal T cell activation, we wanted to determine whether superantigen stimulation of T cells could induce non-Ag-specific Tregs. We assessed the effect of superantigen stimulation of human T cells on activation, regulatory markers, and cytokine production by flow cytometry and T cell suppression assays. Stimulation of PBMCs with staphylococcal exotoxin A and streptococcal pyrogenic exotoxins A and K/L resulted in dose-dependent FOXP3 expression. Characterization of this response for streptococcal pyrogenic exotoxin K/L confirmed its Vβ specificity, that CD25(+)FOXP3(+) cells arose from CD25(-) T cells and required APCs. These cells had increased CTLA-4 and CD127 expression, typical of the recently described activated converted Treg-like cells, and exhibited functional suppressor activity comparable to nTregs. Superantigen-stimulated CD25(+)FOXP3(+) T cells expressed IL-10 at lower superantigen concentrations than was required to trigger IFN-γ production. This study provides a mechanism for bacterial evasion of the immune response through the superantigen induction of Tregs.

Involvement of T helper type 17 and regulatory T cell activity in tumour immunology of bladder carcinoma

T helper type 17 (Th17) and regulatory T cells (T(reg) ) play an important role in the pathogenesis of inflammation and autoimmune disorders. Recent studies have suggested that they also had an impact on tumour immunology. However, the relationship between Th17 and T(reg) cells in the pathogenesis of bladder carcinoma is still unclear. Flow cytometry was used to analyse the numbers, phenotype and cytokine production of Th17 cells in peripheral blood and tumour tissue from bladder carcinoma patients, in parallel with analysis of T(reg) cells. The suppressor capacity of T(reg) and the potential effects of interleukin (IL)-2 on the differentiation of Th17 and T(reg) cells in vitro were studied in a T cell stimulation and suppression assays. The results were as follows: Th17 cells were enriched in the tumours of patients with bladder carcinoma compared with the peripheral blood of patients and controls; patients with bladder carcinoma had a higher proportion of T(reg) cells in peripheral blood compared with healthy controls and nearly all patients examined showed a relative enrichment of tumour-infiltrating T(reg) with respect to peripheral blood; there appeared to be an inverse relationship between tumour-infiltrating Th17 and T(reg) cells; IL-2 could convert tumour-infiltrating T(reg) cells cultured in the presence of the autologous irradiated CD3(-) fraction into Th17 cells, down-regulate forkhead box P2 expression and suppressive capacity of T(reg) cells. This study is the first to define the frequency and characteristics of Th17 cells in bladder carcinoma. We suggest that the balance between Th17 and T(reg) cells may be involved in the development or progression of bladder carcinoma.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Abstract 2424Poster Board II-401We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. [Display omitted] Disclosures:Bozulic:Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

BackgroundRegulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc).Methods/Principal FindingsPatients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25highCD127- and CD4CD25lowCD127high and CD3+ cells. Suppressive function was correlated with CD69 surface expression and TGFβ secretion/expression. The frequency of CD4+CD25+ and CD25highFoxP3highCD127neg T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma.Conclusions/SignificanceThese results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFβ expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.

The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors.

FoxP3(+) regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes. NOD Tregs did show a defect in standard in vitro T cell suppression assays, particularly at low suppressor/effector ratios. Gene expression profiling revealed the vast majority of transcripts constituting the "Treg signature" to be normally distributed in NOD Tregs versus CD4(+) T conventional (Tconv) cells, although there were a few differences affecting one or the other population. According to results from criss-cross experiments, the functional inefficacy was not rooted in NOD Tregs, which suppressed as well as their C57BL/6 (B6) counterparts, but rather in NOD Tconv, which were less prone to suppression than were B6 Tconv cells. They also responded more effectively to anti-CD3/28 monoclonal antibody (mAb) stimulation in vitro or to a natural pancreatic antigen in vivo. This difference was independent of autoimmune inflammation, did not map to the idd3 region, and was not due to the overproduction of interleukin-21 in NOD mice. That the immune dysregulation in this T1D model is rooted in the ability of effector T cells to be regulated, rather than in Tregs themselves, has implications for proposed therapeutic interventions.

Human FOXP3+ T regulatory cells suppress mouse T cell activation by targeting mouse dendritic cells via a human LFA‐1/mouse ICAM‐1 mediated interaction

We have developed a novel in vitro T suppressor cell assay in which the responders are mouse CD4+CD25‐ T cells (mCD4+) stimulated by mouse T‐depleted splenocytes and soluble anti‐mouse CD3, and the T suppressors are human CD4+FOXP3+CD25hi cells (nTreg) activated with plate‐bound anti‐human CD3/CD28. Human nTreg were almost as efficient as mouse nTreg in suppressing the responses of mCD4+ cells. Suppression was not abrogated by neutralizing human IL‐10 or TGFbeta, and was not observed when responders and suppressors were separated in a transwell. Cell contact between the human nTreg and the mouse cells was required as blocking human CD11a or CD18 completely abrogated suppression. Mouse mCD4+ cells deficient in ICAM‐1 were suppressible, suggesting that the nTreg targeted the mouse dendritic cells through the binding of human LFA‐1 with mouse ICAM‐1. Mouse nTreg could not suppress human CD4+ T cells most likely due to the fact that mouse LFA‐1 could not bind to human ICAM. Under suboptimal stimulatory conditions, hIL‐2 was required for activation of nTreg suppressor function. Since the human IL‐2R does not bind mouse IL‐2, IL‐2 consumption is ruled out as a mechanism of suppression. Our results indicate that one of the mechanisms of nTreg‐mediated suppression functions across species and that this cross‐species assay should prove to be a valuable tool for studying the mechanisms of action of human nTreg.

Conserved Regions fromNeisseria gonorrhoeaePilin Are Immunosilent and Not Immunosuppressive

PilE is the primary subunit of type IV pili from Neisseria gonorrhoeae and contains a surface-exposed hypervariable region thought to be one feature of pili that has prevented development of a pilin-based vaccine. We have created a three-dimensional structure-based antigen by replacing the hypervariable region of PilE with an aspartate-glutamine linker chosen from the sequence of Pseudomonas aeruginosa PilA. We then characterized murine immune responses to this novel protein to determine if conserved PilE regions could serve as a vaccine candidate. The control PilE protein elicited strong T-cell-dependent B-cell responses that are specific to epitopes in both the hypervariable deletion and control proteins. In contrast, the hypervariable deletion protein was unable to elicit an immune response in mice, suggesting that in the absence of the hypervariable region, the conserved regions of PilE alone are not sufficient for antibody production. Further analysis of these PilE proteins with suppressor cell assays showed that neither suppresses T- or B-cell responses, and flow cytometry experiments suggested that they do not exert suppressor effects by activating T regulatory cells. Our results show that in the murine model, the hypervariable region of PilE is required to activate immune responses to pilin, whereas the conserved regions are unusually nonimmunogenic. In addition, we show that both hypervariable and conserved regions of pilin are not suppressive, suggesting that PilE does not cause the decrease in T-cell populations observed during gonococcal cervicitis.

An Optimized Method for the Functional Analysis of Human Regulatory T Cells

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen-responsive T cells in a cell contact-dependent manner. In order to investigate the impact of soluble mediators and receptor-ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen-presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti-CD25 monoclonal antibody (MoAb)-coated Dynabeads followed by a detachment step. The remaining CD4+ CD25- responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T-cell Activation/Expansion Beads from Miltenyi Biotec pre-coated with anti-CD3 plus anti-CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 microg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti-CD3/anti-CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg-mediated T-cell suppression by some cytokines (IL-2, IL-15), soluble IL-6 receptor/IL-6 fusion protein and recombinant GITR-ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25- responder T cells.