Suppressive Activity Of CD4 Research Articles

CD4+ and CD8+ regulatory T cell characterization in the rat using a unique transgenic Foxp3-EGFP model

BackgroundRegulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged.ResultsWe generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5–10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP− cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2.ConclusionsThis new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.

IL-2-Anti-IL-2 Monoclonal Antibody Immune Complexes Inhibit Collagen-Induced Arthritis by Augmenting Regulatory T Cell Functions.

IL-2 induces regulatory T cells (Tregs) and reduces disease severity, such as in graft-versus-host disease and systemic lupus erythematosus. To investigate the regulatory network of IL-2 in rheumatoid arthritis, we examined the effects of IL-2-anti-IL-2 mAb immune complexes (IL-2ICs) in a rheumatoid arthritis model of collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice by two immunizations with type II collagen at 3-wk intervals. IL-2ICs were prepared by mixing 5 μg of an anti-IL-2 mAb (clone JES6-1D) with 1 μg of mouse IL-2 and were injected i.p. every day for 3 d. Mouse paws were scored for arthritis using a macroscopic scoring system. Th1, Th2, Th17, and Tregs were analyzed by flow cytometry. Joint histopathology was examined by H&E and immunohistochemical staining. Treg functions were examined by studying in vitro suppression using flow cytometry. IL-2IC administration effectively elicited a 1.6-fold expansion of CD4+Foxp3+ Tregs in peripheral blood cells relative to that found in control mice. IL-2IC treatment significantly inhibited arthritis in CIA mice. Histopathological examination of joints revealed inhibited synovial cell proliferation and IL-17, IL-6, and TNF-α levels but increased Foxp3+ Tregs after IL-2IC treatment. Flow cytometric examination of spleen cells revealed reduced IFN-γ- and IL-17-producing cells and increased IL-10-producing Tregs after IL-2IC treatment. The suppressive activities of CD4+CD25+ Tregs induced by IL-2ICs were stronger than those in untreated mice. IL-2ICs inhibited arthritis by augmenting not only Treg numbers but also Treg functions, which play regulatory roles in autoimmune arthritis.

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma.

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8+ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8+ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8+ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8+ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8+ T cells was observed in female recipient mice. Male CD8+ T cells produced higher levels of IFN-γ than female CD8+ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4+ T cells. Interestingly, IFN-γ receptor expression on CD4+ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8+ T cells and the lower expression of IFN-γ receptor on CD4+ T cells in females compared with males.

Peripheral blood regulatory T cells in patients with diffuse systemic sclerosis (SSc) before and after autologous hematopoietic SCT: a pilot study

The present pilot study aims to evaluate the frequency and the function of regulatory T (Treg) cells in patients with diffuse cutaneous SSc (dcSSc) before and after autologous hematopoietic SCT (aHSCT). Peripheral blood lymphocytes from seven dcSSc patients were analyzed before and 24 months after aHSCT and were compared with those from seven healthy donors (controls). Immunophenotyping of CD4(+)CD25(high)FoxP3(+) natural Treg (nTreg), CD4(+)CD25(+)TGF-β(+) and CD4(+)CD25(+)IL-10(+) adaptive Treg (aTreg) cell subsets was performed using four-color flow cytometry. Treg-suppressive capability was measured after coculture with autologous T effector cells by evaluation of T-cell proliferation using (3)H-thymidine incorporation. Peripheral CD4(+)CD25(high)FoxP3(+) (2±0.5 vs 4.2±1.1, P<0.01), CD4(+)CD25(+)TGF-β(+) (6.9±1.8 vs 14.6±5.0, P<0.05) and CD4(+)CD25(+)IL-10(+) (10.7±0.5 vs 16.1±3.2, P<0.01) Tregs as well as CD4(+)CD25(high)CD127(low) Tregs suppressive capacity (P<0.05) were decreased in dcSSc patients vs controls. After aHSCT (n=7), the percentages of CD4(+)CD25(high)FoxP3(+) (4.1±1.8) and CD4(+)CD25(+)IL-10(+) (15.7±2.2) Treg cells and the suppressive activity of CD4(+)CD25(high)CD127(low) were restored to the levels in controls. The decreased frequency and the functional defect of peripheral Treg cells from patients with dcSSc are reversed following aHSCT to reach those observed in controls. This pilot study brings evidence of an effective restoration of nTreg and aTreg subsets, and recovery of nTreg suppressive function following aHSCT.

Expression of Ets-1 and FOXP3 mRNA in CD4+CD25+ T regulatory cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complex genetic predisposing factors involved. Ets-1 transcription factor plays an important role in the suppressive activity of CD4(+)CD25(+) Treg cells and stable expression of FOXP3. To find its potential role in the pathogenesis of SLE, we investigate the mRNA expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from patients with SLE. Real-time transcription-polymerase chain reaction analysis was used to determine the expression of Ets-1 and FOXP3 mRNA in CD4(+)CD25(+) Treg cells from 36 patients with SLE and 18 sex-and-age-matched healthy controls. The Ets-1 mRNA expression level was decreased in patients with SLE [0.225 (0.135, 0.337)] than healthy controls [0.528 (0.303, 0.681)] (P < 0.001). The expression levels of FOXP3 mRNA were lower in SLE patients [0.608 (0.272, 1.164)] than healthy controls [0.919 (0.690, 1.223)], but the difference was not significant (P = 0.106). Significant reduction in Ets-1 and FOXP3 expression was also found in new-onset SLE subgroup when compared with healthy controls (P < 0.001). The level of Ets-1 and FOXP3 mRNA was not significantly different in hyperactive and lower active SLE group when compared with inactive SLE group, respectively (P > 0.05). There were no significant differences between SLE with lupus nephritis (LN) and SLE without LN either (P > 0.05). Associations of Ets-1 and FOXP3 mRNA expression levels with major clinical and laboratory parameters of SLE patients were also analyzed. However, no significant association was found. Significant positive correlation was found between Ets-1 and FOXP3 mRNA expression in CD4(+)CD25(+) Treg cells from SLE patients (r = 0.698, P < 0.001). Our results found that the expression levels of Ets-1 mRNA were decreased in SLE patients and Ets-1 expression was positively correlated with the expression of FOXP3. It indicated that Ets-1 may play an important role in the stable expression of FOXP3 in CD4(+)CD25(+) Treg cells.

Host‐derived CD4+ T cells attenuate stem cell–mediated transfer of autoimmune arthritis in lethally irradiated C57BL/6.g7 mice

In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients. Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer. Titers of anti-GPI antibodies in reconstituted B6.g7 mice were ∼60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased. Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.

Blockade of Myeloid-Derived Suppressor Cells after Induction of Lymphopenia Improves Adoptive T Cell Therapy in a Murine Model of Melanoma

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor-bearing mice. Reconstitution rate and suppressive activity of CD4(+)CD25(+)Foxp3(+) Tregs and CD11b(+)Gr1(+) MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor-bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8(+) T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.

CD8+CD45RA+CCR7+FOXP3+ T Cells with Immunosuppressive Properties: A Novel Subset of Inducible Human Regulatory T Cells

CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8+CCR7+ regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8+CCR7+ Tregs is not mediated by IL-10, TGF-β, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8+CCR7+ Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8+CCR7+ Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8+CCR7+ Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.

Strong Impact of CD4+Foxp3+ Regulatory T Cells and Limited Effect of T Cell-Derived IL-10 on Pathogen Clearance during Plasmodium yoelii Infection

It is well established that CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a crucial role in the course of different infectious diseases. However, contradictory results have been published regarding to malaria infection. In this study, we report that specific ablation of Foxp3(+) Tregs in Plasmodium yoelii-infected DEREG-BALB/c mice leads to an increase in T cell activation accompanied by a significant decrease in parasitemia. To better understand how Foxp3(+) Tregs orchestrate this phenotype, we used microarrays to analyze CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells in the course of P. yoelii infection. Using this approach we identified genes specifically upregulated in CD4(+)CD25(+)Foxp3(+) Tregs in the course of infection, such as G-protein-coupled receptor 83 and Socs2. This analysis also revealed that both CD4(+)CD25(+)Foxp3(+) Tregs and CD4(+)CD25(-)Foxp3(-) T cells upregulate CTLA-4, granzyme B, and, more strikingly, IL-10 during acute blood infection. Therefore, we aimed to define the function of T cell-derived IL-10 in this context by Cre/loxP-mediated selective conditional inactivation of the IL-10 gene in T cells. Unexpectedly, IL-10 ablation in T cells exerts only a minor effect on parasite clearance, even though CD8(+) T cells are more strongly activated, the production of IFN-γ and TNF-α by CD4(+)CD25(-) T cells is increased, and the suppressive activity of CD4(+)CD25(+) Tregs is reduced upon infection. In summary, these results suggest that CD4(+)Foxp3(+) Tregs modulate the course of P. yoelii infection in BALB/c mice. Moreover, CD4(+) T cell-derived IL-10 affects T effector function and Treg activity, but has only a limited direct effect on parasite clearance in this model.

Influence of donor specific transfusion with impaired ICOS/B7h allorecognition on CD4 + CD25+regulatory T cells of cardiac allograft recipient

Objective To investigate the possibility of CD4+CD25+ regulatory T cells (Treg) enhancement, which induced by donor specific transfusion combined with blockade of ICOS/B7h costimulation following an allogenic murine heterotopic cardiac transplantation. Methods Recipient mice were assigned to 3 groups. In allogenic group, donor hearts were harvested from BALB/C mice, and allografts were heterotopically transplanted in the neck of C57BL/6 using Chen' s technique, without treatment. In isogeneic group, donor hearts were harvested from C57BL/6 mice, and isografts were heterotopically transplanted in the neck of C57BL/6, without treatment. In treatment group, a combination of 5 × 106 ICOS-Fcrecipient C57BL/6 mice following a heterotopic cardiac allograft transplantation. Graft function was assessed daily by palpation, with rejection defined as the absence of detectable beating, then survival of grafts was recorded. CD4 + CD25 + Treg subsets in the peripheral blood of allograft recipients were analyzed by flow cytometry. The expression of FOXP3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). To assess the suppressive activity of CD4 + CD25 + Treg from tolerant C57BL/6 mice on CD4 + CD25- Teff from naive BALB/C mice, suppression allogeneic MLR assays were performed in vitro.Results In comparison with allogenic group, the survival of cardiac grafts could be prolonged by the comICOS-Fc on the day 0-6 [(84.38 ± 29. 14) days vs (7.00 ± 0. 76) days, P ＜ 0. 01]. Among 3 groups, a considerable proportion of CD4 + CD25 + Treg could be observed in treatment group [(15.60 ± 5.69 )% ,P ＜0. 01], meanwhile, high expression of FOXP3 mRNA was also detected within allografts in treatment group.Furthermore, in contrast to naive C57BL/6 mice, CD4+ CD25 + Treg harvested from tolerant C57BL/6 mice showed more potent suppressive effect on the proliferation of CD4 + CD25 - Teff. Conclusion Allograft tolerance can be induced by donor specific transfusion with impaired ICOS/B7h allorecognition. CD4 + CD25 + Treg played a critical role in the establishment and the maintenance of this allograft tolerance. Key words: Regulatory T cells; Donor specific transfusion; Inducible costimulator; Cardiac allograft tolerance

Abstract 5325: Inhibition of CD4+CD25+ regulatory T cell suppression to anti-tumor responses by OK-432 (lyophilized Streptococcus pyogenes)

Abstract It is important to control the suppressive activity to anti-tumor immune responses mediated by CD4+CD25+ regulatory T cell (Treg) for effective immunological cancer treatment. Recently, a number of studies have shed new light on recognition of pathogen-associated molecular patterns through toll like receptors (TLRs) to break the suppressive environment present at the tumor local site. TLR signals are not only able to block the suppressive activity of CD4+CD25+ Tregs but can also break CD8+ tolerance even in the presence of CD4+CD25+ Tregs. Recently, we reported that while high-avidity NY-ESO-1-specific CD4+ T cell precursors are indeed present in naïve T cell populations, their activation following peptide vaccination is kept under the strict control of Tregs, resulting in a selective activation/expansion of low-avidity NY-ESO-1 peptide-specific CD4+ T cells. Additionally, following DNA vaccination, high-avidity NY-ESO-1-specific CD4+ T cells can only be observed transiently and become rapidly suppressed by Tregs. Here, we focused on OK-432 (lyophilized preparation of Streptococcus pyogenes) that can stimulate TLRs and explored 1) whether OK-432 administrated into tumor-associated exudate fluids (chest or abdominal cavity) inhibits local Treg function and accumulation, and 2) whether OK-432 used as an adjuvant of cancer vaccine can break Treg suppression and induce the activation of high-avidity tumor antigen-specific T cell precursors. Levels of Tregs in cells collected from pleural effusion or ascites of cancer patients treated with OK-432 were assessed by staining with antibodies for CD4, CD25 and Foxp3. The frequency of Foxp3+ Tregs significantly decreased after OK-432 administration. Furthermore, suppressive function of Tregs from patients also decreased after OK-432 treatment. PBMCs were collected from patients receiving vaccination with tumor antigens (HER2 and NY-ESO-1), formulated in choresteryl hydrophobized pullulan (CHP) together with OK-432 and the presence of high-avidity NY-ESO-1-specific CD4+ T cells in effector/memory (CD25-CD45RO+) T cell population was examined. NY-ESO-1-specific CD4+ T cells were detected within CD45RO+ memory T cell population after vaccination. In contast to peptide vaccination, CD4+ T cell clones induced from these NY-ESO-1-protein vaccinated patients in the presence of OK-432 had high-affinity T cell receptors and recognized naturally-processed NY-ESO-1 protein presented by dendritic cells. Taken together, OK-432 appears to inhibit Treg function and contribute to the durable differentiation of high-avidity tumor antigen-specific naive T cell precursors into effector/memory T cells with relative resistance to Tregs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5325.

Immune-modifying properties of topical vitamin D: Focus on dendritic cells and T cells

Topical creams containing the active form of vitamin D (1,25-dihydroxyvitamin D 3; 1,25(OH) 2D 3) or analogues of this compound are currently used with some success to treat skin conditions including psoriasis and vitiligo. As well as targeting inflammatory processes in the skin, topical application of 1,25(OH) 2D 3 also affects the function of immune cells in the skin and draining lymph nodes. Topically applied 1,25(OH) 2D 3 reduces the number of dendritic cells in the skin, resulting in suppressed immunity and in particular reduced contact hypersensitivity (CHS) responses. Topical 1,25(OH) 2D 3 may also promote the migration of dendritic cells from the skin to the draining lymph nodes. Skin application of 1,25(OH) 2D 3 prevented the inflammatory effects of UVB irradiation on lymph node hypertrophy, when cell numbers were examined 4 days after skin treatment. In contrast, when 1,25(OH) 2D 3 was applied to UVB irradiated skin, there was no reversal in the suppression of CHS responses caused by UVB irradiation. Instead, 1,25(OH) 2D 3 had an additive effect with UVB to suppress CHS responses to a greater degree than UVB alone. In these studies, 1,25(OH) 2D 3 was applied to the treated skin of BALB/c mice immediately following UVB irradiation. Finally, topical 1,25(OH) 2D 3 also enhanced the number and suppressive activity of CD4+CD25+ regulatory T cells in the lymphatic tissue draining skin.

Clinical rejection and persistent immune regulation in kidney transplant patients

We evaluated whether the regulatory function of CD4 +CD25 high+FoxP3 + T-cells from patients on tacrolimus and mycophenolate mofetil (MMF) is affected by preceding steroid and anti-CD25 mAb induction therapy and whether this function is associated with rejection after kidney transplantation. Kidney recipients ( N = 15) were randomized to receive either anti-CD25 mAb induction (i.e., daclizumab) or steroids for 4 months. We analyzed the presence and suppressive activity of CD4 +CD25 high+FoxP3 + peripheral T-cells in samples obtained at pre and 4–6 months after transplantation. Anti-CD25 mAb therapy and treatment with steroids did not significantly affect protein expression of FoxP3. However, at the functional level, significant differences were found in the regulatory activities of CD4 +CD25 high+ T-cells from the anti-CD25 group vs those from the steroid group. At 4–6 months after transplantation, the regulatory activities of CD4 +CD25 high+ T-cells were comparable to those before anti-CD25 mAb therapy; 49 ± 13% (mean ± SEM) vs 40 ± 14% at a 1:20 ratio (CD25 high+:CD25 −/dim), respectively. In contrast, the regulatory capacities of CD +D25 bright+ T-cells from the steroid patient group became significantly impaired. The percentage inhibition of the anti-donor response decreased from 57 ± 12% before transplantation to 12 ± 7% after transplantation ( p < 0.01). Five out of 15 patients experienced a rejection episode. At 4–6 months after transplantation, the CD25 high+ cells from these rejectors (who all received daclizumab induction therapy) had clear regulatory function, while suppression by CD25 high+ cells from non-rejectors ( N = 10) was significantly lower. The percentage inhibition of the anti-donor response was 48 ± 14% (mean ± SEM) vs 10 ± 7%, respectively, p = 0.02. Anti-CD25 mAb induction therapy does not negatively influence the regulatory function of CD4 +CD25 high+FoxP3 + T-cells from kidney transplant recipients on tacrolimus and MMF. The majority of these patients experienced an acute rejection episode, which suggests that immune activation is required for persistent immunoregulatory function.

Tolerization with Hsp65 induces protection against adjuvant‐induced arthritis by modulating the antigen‐directed interferon‐γ, interleukin‐17, and antibody responses

Pretreatment of Lewis rats with soluble mycobacterial Hsp65 affords protection against subsequent adjuvant-induced arthritis (AIA). This study was aimed at unraveling the mechanisms underlying mycobacterial Hsp65-induced protection against arthritis, using contemporary parameters of immunity. Lewis rats were given 3 intraperitoneal injections of mycobacterial Hsp65 in solution prior to the initiation of AIA with heat-killed Mycobacterium tuberculosis. Thereafter, mycobacterial Hsp65-specific T cell proliferative, cytokine, and antibody responses were tested in tolerized rats. The roles of anergy and the indoleamine 2,3 dioxygenase (IDO)-tryptophan pathway in tolerance induction were assessed, and the frequency and suppressive function of CD4+FoxP3+ Treg cells were monitored. Also tested was the effect of mycobacterial Hsp65 tolerization on T cell responses to AIA-related mycobacterial Hsp70, mycobacterial Hsp10, and rat Hsp65. The AIA-protective effect of mycobacterial Hsp65-induced tolerance was associated with a significantly reduced T cell proliferative response to mycobacterial Hsp65, which was reversed by interleukin-2 (IL-2), indicating anergy induction. The production of interferon-gamma (but not IL-4/IL-10) was increased, with concurrent down-regulation of IL-17 expression by mycobacterial Hsp65-primed T cells. Neither the frequency nor the suppressive activity of CD4+FoxP3+ T cells changed following tolerization, but the serum level of anti-mycobacterial Hsp65 antibodies was increased. However, no evidence was observed for a role of IDO or cross-tolerance to mycobacterial Hsp70, mycobacterial Hsp10, or rat Hsp65. Tolerization with soluble mycobacterial Hsp65 leads to suppression of IL-17, anergy induction, and enhanced production of anti-mycobacterial Hsp65 antibodies, which play a role in protection against AIA. These results are relevant to the development of effective immunotherapeutic approaches for autoimmune arthritis.

Generation and Regulation of CD8+ Regulatory T Cells

Research into the suppressive activity of CD4(+)FoxP3(+) T regulatory cells (Treg) has defined a sublineage of CD4(+) cells that contribute to self-tolerance and resistance to autoimmune disease. Much less attention has been given to the potential contribution of regulatory sublineages of CD8(+) cells. Analysis of a small fraction of CD8(+) cells that target autoreactive CD4(+) cells through recognition of the MHC class Ib molecule Qa-1 in mouse and HLA-E in human has revitalized interest in CD8(+) Treg. Here we summarize recent progress and future directions of research into the role of this CD8(+) sublineage in resistance to autoimmune disease.

Involvement of CD4+CD25+ regulatory T cells in the pathogenesis of polycythaemia vera

Regulatory T cells (T(reg)) have been shown to play an important role in the regulation of hematopoietic activity. However, there is no information about the effect of T(reg) cells in the pathogenesis of polycythaemia vera (PV). In this study, we investigated the percentage and function of T(reg) cells in the peripheral blood of 21 PV patients and 25 healthy donors. T(reg) cells were identified and characterized as CD4+CD25+ FOXP3+ by flow cytometry. The suppressive activity of CD4+CD25+ T(reg) cells was assessed by the proliferation and cytokine secretion of the co-cultured CD4+CD25- fractions. The results showed that the percentage of T(reg) cells in the peripheral blood of PV patients significantly increased compared to healthy controls ((10.93 +/- 4.02)% vs (5.86 +/- 1.99)%, P < 0.05). Moreover, the mRNA and protein expression of FOXP3 was higher in CD4+CD25+ T(reg) cells. Coordinately, when co-cultured with the activated CD4+CD25- cells, the CD4+CD25+ T(reg) cells showed enhanced suppressive function in PV. Yet, the underlying mechanism for the increased frequency and function of CD4+CD25+ T(reg) cells is still to be clarified. T(reg) cells expansion might account for the abnormal T cell immunity in PV patients and thus contribute to the pathogenesis of PV.

Influence of high mobility group box-1 protein on the correlation between regulatory T cells and CD4+ CD25- T cells of spleen in mice

To investigate the influence of high mobility group box-1 protein (HMGB1) on the immunosuppression function of splenic regulatory T cells (Tregs) and its potential regulatory mechanism underlying the effect on CD4+ CD25- T cells in mice. CD4+ CD25+ Tregs isolated from the spleens of male BALB/c mice by magnetic beads were seeded on 96-well (1 x 10(5) cells/well) cell culture plates coated with 1 microg/ml anti-CD3 and soluble CD28. After being stimulated with HMGB1 for different time and concentrations, the secretions of IL-2 and IL-10 were analyzed by ELISA. Tregs stimulated for 72 hours were cultured with CD4+ CD25- T cells together. The suppressive activity of CD4+ CD25+ Treg to CD4+ CD25- T cells was analyzed by MTT test. IL-2, IL-10, IL-4, and interferon (IFN)-gamma in the cell suspensions were determined by ELISA. After stimulation with HMGB1, the suppressive activity of splenic Tregs in mice were significantly down-regulated at 72 hours, when the proportion of Tregs to CD4+ CD25- T cells was 1 : 1. The secretion of IL-2 of Tregs stimulated by HMGB1 was not markedly changed (P > 0.05), while a dose-dependent decrease between IL-10 induction and HMGB1 concentration was obviously (P < 0.05). When CD4+ CD25- T cells were cultured with stimulated Tregs, comparing with unstimulated-Treg group, levels of IL-2 and IFN-gamma were elevated following the increased concentration of HMGB1 (P < 0.05 or P < 0.01). Meanwhile the secretion of IL-4 and IL-10 significantly decreased when cultured with stimulated Tregs (P < 0.05). These data suggested that HMGB1 stimulation can result in significant down-regulation of immunosuppression of splenic Tregs in mice. HMGB1 might be a potential immunoregulatory signal that influences the proliferation of effector T cells, secretion of IL-2 and cells-polarization by inhibiting CD4+ CD25+ Tregs activity.

Immunological and clinical effects of a multiple peptide vaccine and low-dose cyclophosphamide in prostate carcinoma patients

13502 Background: Despite effective therapeutic strategies for the treatment of prostate cancer (PC), patients still recur and progress in a significant percentage. Here we describe the preliminary clinical and immunological data of a peptide-based strategy of vaccination in biochemical relapsed PC patients. Methods: We planned to enroll 30 HLA-A*0201 patients with biochemical failure after surgery, radiation or androgen deprivation therapy (ADT), according to the ASTRO criteria. On entering the study, patients discontinue ADT. No selections were made based on PSA levels at diagnosis, Gleason pattern score or other pathological features. Vaccination includes three peptides derived from proteins highly expressed in PC, PSMA (PSMA-P14–12 and PSMA-P2711–719) and Survivin (SVV96- 104/97M). Peptides, emulsified in Montanide ISA 51, are administered subcutaneously (8 injections) at two-weeks intervals. Vaccine is preceded by systemic administration of low dose cyclophosphamide (300 mg/m2/four weeks), aimed at selectively down- modulating regulatory T cells (Treg). Immunological monitoring includes evaluation of peptide and tumor-specific CD8+T cells in peripheral blood (IFN?-Elispot and HLA/multimer staining) and analysis of the frequency and suppressive activity of CD4+CD25+Foxp3+Treg. Results: So far, ten patients have been enrolled. Treatment is well tolerated, with mild cutaneous toxicity due to Montanide injection. A significant increase in the frequency of antigen-specific CD8+ T cells was observed in 8/10 patients. Treg, only marginally expanded in these patients, do not seem to decrease by cyclophosphamide treatment, although studies on the potential modulation of their suppressive activity are in progress. After a follow-up of 6–12 months, PSA stabilization was observed in 7 patients, 2 of which entered the trial after maximal androgen blockade discontinuation. Interestingly, PSA stabilization appears to directly correlate with the induction of antigen-specific T cell responses. Conclusions: Our vaccination strategy proved to be safe, non toxic and well tolerated. Initial monitoring data show an expansion of antigen specific T cells, with an apparent correlation between immunological and biochemical responses. No significant financial relationships to disclose.

Effect of immunosuppressants on the expansion and function of naturally occurring regulatory T cells

The induction of immune tolerance is one of the final therapeutic goals in clinical transplantation. Regulatory T lymphocytes are important for the induction and maintenance of immune tolerance to grafts. If immunosuppressive drugs used clinically to prevent immune rejection also inhibit regulatory T lymphocytes, tolerance would not be achieved. We therefore tested the effect of several immunosuppressants with different mechanisms of action on the proliferation and suppressive activity of CD4 +CD25 + regulatory T cells. Highly purified CD4 +CD25h + T cells from C57BL/6 (H-2 b) mice were stimulated with allogeneic T-depleted splenocytes (BALB/c; H-2 d) in the presence of various immunosuppressants. After one week in culture, viable T cells were recovered, their regulatory capacity was assessed by their ability to inhibit responder T cell proliferation in MLR, and their cytokine production profile was measured by ELISA. The immunosuppressants rapamycin, cyclosporine A, and methylprednisolone significantly inhibited the expansion of regulatory T cells upon stimulation with alloantigen, whereas mycophenolic acid and the costimulatory blockers, anti-CD40L and CTLA4Ig, did not. None of these immunosuppressants, however, reduced the suppressive capacity of regulatory T cells. Pretreatment with immunosuppressants did not induce significant changes in the cytokine production profile of regulatory T cells. Our results suggest that costimulatory blockers and mycophenolate mofetil can be utilized therapeutically in the induction of immune tolerance. In contrast, the use of rapamycin, cyclosporine A, and methylprednisolone should be reconsidered, due to their deleterious effects on the expansion of naturally occurring regulatory T cells.

Schistosoma japonicum egg antigens stimulate CD4+ CD25+ T cells and modulate airway inflammation in a murine model of asthma

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4(+) CD25(+) T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4(+) CD25(+) T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25(+) depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25(+) T cells from these mice to suppress T-cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4(+) CD25(+) T cells of OVA-treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0.05). Depletion of CD25(+) cells accelerated OVA-induced airway inflammation and increased the expression of interleukin (IL)-5 and IL-4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4(+) CD25(+) T cells, which made IL-10, but little IL-4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen-induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4(+) CD25(+) T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.