Administration Of anti-CD25 mAb Research Articles

Effects of regulatory T cells on glyceraldehyde-3-phosphate dehydrogenase vaccine efficacy against Schistosoma japonicum

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a candidate subunit vaccine that induces protective immunity and elicits partial resistance to Schistosoma japonicum upon mouse and livestock vaccination. This study aimed to evaluate the effect of regulatory T cells (Tregs), which were defined as CD4+CD25+Foxp3+ cells, on the efficacy of a GAPDH vaccine against S. japonicum. BALB/c female mice were randomly divided into five groups as follows: normal, infected control, anti-CD25 monoclonal antibody (anti-CD25 mAb), GAPDH group, and co-treated with anti-CD25 mAb and GAPDH group. The worm reduction and liver egg reduction rates in the GAPDH group were 32.46% and 35.43%, respectively, which increased to 60.09% and 58.78%, respectively, after anti-CD25 mAb administration. Compared with those in the infected control group, the percentage of Tregs in the spleen decreased significantly when GAPDH and anti-CD25 mAb were used either alone or in combination. Furthermore, secretions associated with the Th1 response increased in splenocytes of the anti-CD25 mAb group, whereas the Th1 and Th2 responses increased in splenocytes of the GAPDH and co-treated groups. Compared to that in the infected control group, granuloma diameter in the GAPDH and co-treated groups increased slightly, but there were no significant differences among the groups. Our results indicate that the protective effect of the GAPDH vaccines can be improved by decreasing Tregs and enhancing the Th1- and Th2-type immune responses. Therefore, anti-CD25 mAb and GAPDH might exert synergistic effects to clear parasites by decreasing the frequency of Tregs and increasing the Th1- and Th2-type immune responses.

Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4+CD25+Foxp3+ regulatory T cells.

The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb. Compared with the control group, the percentages of splenic CD4+CD25+Foxp3+ regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4+CD25+Foxp3+ Tregs, while anti-CD25 mAb can partially block CD4+CD25+Foxp3+ Tregs and thus enhance the protective efficacy of the GST vaccine.

Vaccination with a co-expression DNA plasmid containing GAD65 fragment gene and IL-10 gene induces regulatory CD4(+) T cells that prevent experimental autoimmune diabetes.

The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315 /IL-10). Female NOD mice at the age of 3-4weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315 /IL-10 (n = 32). The incidence of diabetes was monitored up to 30weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined. Administration with SGAD65190-315 /IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315 /IL-10 on diabetes and insulitis. Moreover, depletion of CD4(+) CD25(+) T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315 /IL-10 vaccination on adoptive transfer of diabetes. Our data suggested that SGAD65190-315 /IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John Wiley & Sons, Ltd.

Galectin-3 suppresses mucosal inflammation and reduces disease severity in experimental colitis.

Galectin-3, a member of the β-galactoside-binding lectin family, expresses in many different immune cells and modulates broad biological functions including cell adhesion, cell activation, cell growth, apoptosis, and inflammation. However, the role of galectin-3 in mucosal immunity or inflammatory bowel diseases is still not clear. We demonstrate here that galectin-3 knockout mice have more severe disease activity in the dextran sulfate sodium (DSS)-induced colitis model, indicating that galectin-3 may protect from inflammation in DSS-induced colitis. Furthermore, treating with galectin-3 reduced body weight loss, shortened colonic length, and ameliorated mucosal inflammation in mice having DSS-induced colitis. However, the protective effects of galectin-3 were eliminated by the administration of anti-CD25 mAb. In addition, primary T cells treated with galectin-3 ex vivo induced the expression of FOXP3, ICOS, and PD-1 with a Treg cell phenotype having a suppression function. Moreover, adoptive transfer of galectin-3-treated T cells reduced bowel inflammation and colitis in the T cell transfer colitis model. In conclusion, our results indicate that galectin-3 inhibited colonic mucosa inflammation and reduced disease severity by inducing regulatory T cells, suggesting that it is a potential therapeutic approach in inflammatory bowel disease. Galectin-3 offers protection from inflammation in experimental colitis. Galectin-3 knockout mice have more severe disease activity in DSS-induced colitis. Adoptive transfer of galectin-3-treated T cells reduced bowel inflammation. Galectin-3 inhibited colonic mucosa inflammation by inducing regulatory T cells. Galectin-3 is a potential therapeutic approach in inflammatory bowel disease.

An immunosufficient murine model for the study of human islets.

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6μMT-/- mice were used. For some anti-CD45RB-treated B6μMT-/- mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated μMT-/- mice. These μMT-/- mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

Long‐term tolerance to factor VIII is achieved by administration of interleukin‐2/interleukin‐2 monoclonal antibody complexes and low dosages of factor VIII

Regulatory T cells (Tregs) play a pivotal role in regulating anti-factor VIII (FVIII) immune responses. Interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb; JES6-1) can induce the selective expansion of Tregs in vivo. In the prevention experiments, we treated mice with hemophilia A with IL-2/IL-2mAb complexes (three times per week) and concurrently with FVIII protein (80 U kg(-1) per week) for 4 weeks. Generation of anti-FVIII immune responses was examined afterward. Next, to induce long-term tolerance to FVIII, a series of treatment dosages and schedules for administering IL-2/IL-2mAb complexes and FVIII protein in mice with hemophilia A was evaluated. Compared to control mice that were treated with only FVIII, which produced high-titer anti-FVIII antibodies, mice treated with IL-2/IL-2mAb complexes plus FVIII produced no antibodies. A marked seven-fold increase in CD4(+) CD25(+) Foxp3(+) Helios(+) natural Tregs was maintained for 4 weeks in blood, spleen, and lymph nodes and then dropped to normal levels within the next 10 days. The suppressive functions of expanded Tregs were demonstrated with suppressive, proliferative, and cytokine assays. The administration of anti-CD25 mAb (PC-61) blocked this protective effect, confirming the involvement of Tregs in suppressing anti-FVIII immune responses. Importantly, administration of IL-2/IL-2mAb complexes (three times per week for 8 weeks) combined with contiguous weekly injections of low-dosage FVIII protein (20 U kg(-1) per week for 18 weeks) not only abrogated the formation of anti-FVIII antibodies but also induced long-term tolerance to FVIII. Treatment with IL-2/IL-2mAb complexes is highly promising for the induction and maintenance of FVIII-specific tolerance after FVIII protein replacement therapy.

Impact of anti-CD25 monoclonal antibody on dendritic cell-tumor fusion vaccine efficacy in a murine melanoma model

BackgroundA promising cancer vaccine involves the fusion of tumor cells with dendritic cells (DCs). As such, a broad spectrum of both known and unidentified tumor antigens is presented to the immune system in the context of the potent immunostimulatory capacity of DCs. Murine studies have demonstrated the efficacy of fusion immunotherapy. However the clinical impact of DC/tumor fusion vaccines has been limited, suggesting that the immunosuppresive milieu found in patients with malignancies may blunt the efficacy of cancer vaccination. Thus, novel strategies to enhance fusion vaccine efficacy are needed. Regulatory T cells (Tregs) are known to suppress anti-tumor immunity, and depletion or functional inactivation of these cells improves immunotherapy in both animal models and clinical trials. In this study, we sought to investigate whether functional inactivation of CD4+CD25+FoxP3+ Treg with anti-CD25 monoclonal antibody (mAb) PC61 prior to DC/tumor vaccination would significantly improve immunotherapy in the murine B16 melanoma model.MethodsTreg blockade was achieved with systemic PC61 administration. This blockage was done in conjunction with DC/tumor fusion vaccine administration to treat established melanoma pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon Rank Sum Test. IFN-gamma ELISPOT assay was performed on splenocytes from treated mice.ResultsWe demonstrate that treatment of mice with established disease using mAb PC61 and DC/tumor fusion significantly reduced counts of pulmonary metastases compared to treatment with PC61 alone (p=0.002) or treatment with control antibody plus fusion vaccine (p=0.0397). Furthermore, IFN-gamma ELISPOT analyses reveal that the increase in cancer immunity was mediated by anti-tumor specific CD4+ T-helper cells, without concomitant induction of CD8+ cytotoxic T cells. Lastly, our data provide proof of principle that combination treatment with mAb PC61 and systemic IL-12 can lower the dose of IL-12 necessary to obtain maximal therapeutic efficacy.ConclusionsTo our knowledge, this is the first report investigating the effects of anti-CD25 mAb administration on DC/tumor-fusion vaccine efficacy in a murine melanoma model, and our results may aide the design of future clinical trials with enhanced therapeutic impact.

Administration of anti-CD25 mAb leads to impaired α-galactosylceramide-mediated induction of IFN-γ production in a murine model

CD4+CD25+Foxp3+ T regulatory cells (Tregs) and CD1d-restricted invariant natural killer T (iNKT) cells are two cell types that are known to regulate immune reactions. Depletion or inactivation of Tregs using specific anti-CD25 antibodies in combination with immunostimulation is an attractive modality especially in anti-tumour immunotherapy. However, CD25 is not expressed exclusively on Tregs but also on subpopulations of activated lymphocytes. Therefore, the modulatory effects of the specific anti-CD25 antibodies can also be partially attributed to their interactions with the effector cells. Here, the effector functions of iNKT cells were analysed in combination with anti-CD25 mAb PC61. Upon PC61 administration, α-galactosylceramide (α-GalCer)-mediated activation of iNKT cells resulted in decreased IFN-γ but not IL-4 production. In order to determine whether mutual interactions between Tregs and iNKT cells take place, we compared IFNγ production after α-GalCer administration in anti-CD25-treated and “depletion of regulatory T cell” (DEREG) mice. Since no profound effects on IFNγ induction were observed in DEREG mice, deficient in FoxP3+ Tregs, our results indicate that the anti-CD25 antibody acts directly on CD25+ effector cells. In vivo experiments demonstrated that although both α-GalCer and PC61 administration inhibited TC-1 tumour growth in mice, no additive/synergic effects were observed when these substances were used in combination therapy.

Treg cells may regulate interlukin-17 production by modulating TH1 responses in 1,3-β-glucan-induced lung inflammation in mice

1,3-β-glucan is considered a fungal biomarker and exposure to this agent can induce lung inflammation. Complement activation plays an important role in early immune responses to β-glucan. Previous studies showed that T-regulatory cells (Tregs) regulated 1,3-β-glucan-induced lung inflammation by modulating the maintenance of immune homeostasis in the lung. Both interleukin (IL)-17 and TH17 cells play pivotal roles in inflammation associated with lung disease and share reciprocal developmental pathways with Tregs. However, the effect of Tregs on IL-17 and TH17 responses in 1,3-β-glucan-induced lung inflammation remains unclear. In this study, mice were exposed to 1,3-β-glucan by intratracheal instillation. To investigate the effects of Tregs on IL-17 and TH17 cells in the induced lung inflammation, a Treg-depleted mice model was generated by administration of anti-CD25 mAb. The results indicated that Treg-depleted mice showed more severe pathological inflammatory changes in lung tissues. Tregs depletion reduced IL-17 expression in these tissues, and increased those of TH1 cytokines. The expression of IL-17 increased at the early phase of the inflammation response. There were no significant effects of the Tregs on expression of RORγt and IL-6 or the amount of CD4+IL-17+ cells in the lungs. When taken together, the late phase of the 1,3-β-glucan-induced inflammatory response in the mice was primarily mediated by TH1 cytokines rather than IL-17. In contrast, the early phase of the inflammatory response might be mediated in part by IL-17 along with activated complement. Tregs might be required for IL-17 expression during the late phase inflammatory response in mice. The increased IL-17 mRNA observed during the 1,3-β-glucan induced inflammatory response were attributed to cells other than TH17 cells.

Abstract A13: A combinatory treatment targeting CD25+ regulatory T cells and CTL-associated antigen-4 blockage enhanced anti-tumor immunity in the HLA-DR transgenic mouse model of prostate cancer

Abstract It is clearly established that the successful cancer immunotherapy will critically depend on the overcoming tolerance to “self” and negative immunoregulatory mechanisms. We have recently described a novel model of prostate cancer in double transgenic mice expressing human leukocyte antigen (HLA)-DR (HLA-DRB1*1501(DR2b)) and human prostate specific antigen (PSA) as a “self” antigen. In DR2bxPSA F1 mice, immunological tolerance to the transgenic mouse adenocarcinoma of prostate (TRAMP) tumor cells expressing PSA was established in a context of a “permissive” HLA-DR2b allele, and could be reversed by depleting CD25+ regulatory T cells (Tregs) prior to tumor inoculation. Several studies suggested that simultaneous targeting of both Tregs and negative regulatory pathway mediated by the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) may potentiate anti-tumor immune responses. We studied the effect of the combinatory treatment with anti-CD25 mAb and anti-CTLA-4 mAb on the growth of TRAMP-PSA tumor cells in DR2bxPSA F1 male mice. Single administration of anti-CD25 mAb (clone PC61) three days prior to tumor inoculation significantly increased PSA-specific CD8 T cell responses to the TRAMP-PSA tumor cells and delayed tumor growth compared to mice treated with isotype control antibodies. Similarly, treatment with anti-CTLA-4 mAb (clone 9D9) administered on days 3, 6 and 9 after tumor inoculation also significantly increased the levels CD8 T cells producing IFN-γ in response to the immunodominant H-2Db-restricted epitope PSA65–73 and delayed tumor growth. A combinatory treatment with both anti-CD25 and anti-CTLA-4 mAbs further enhanced anti-tumor immunity and caused a more profound delay in the tumor growth compared to each treatment alone. There were no tumor-free survivors among mice treated with anti-CD25 mAb alone (0 out of 21 mice), while 2 out of 21 mice treated with anti-CTLA-4 mAb alone (9.5%) and 3 out of 10 mice treated with a combination of anti-CD25 and anti-CTLA-4 mAbs (30%) survived tumor-free. Mice that survived a primary tumor challenge for 20 weeks demonstrated strong polyfunctional CD8 T cell memory responses to the secondary challenge with TRAMP-PSA tumors that was characterized by the significantly increased levels of PSA-specific IFN-γ+TNF-α+ CD8 T cells. Our data suggest that a combinatory treatment targeting Tregs and CTLA-4-mediated inhibitory pathways may be a promising approach for the prostate cancer immunotherapy. Citation Format: Elena N. Klyushnenkova, Richard B. Alexander. A combinatory treatment targeting CD25+ regulatory T cells and CTL-associated antigen-4 blockage enhanced antitumor immunity in the HLA-DR transgenic mouse model of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A13.

Asthma Prevention byLactobacillus Rhamnosusin a Mouse Model is Associated With CD4+CD25+Foxp3+T Cells

PurposeProbiotic bacteria can induce immune regulation or immune tolerance in allergic diseases. The underlying mechanisms have been recently investigated, but are still unclear. The aim of this study was to evaluate the protective effects of the probiotic Lactobacillus rhamnosus (Lcr35) in a mouse model of asthma and to identify its mechanism of action.MethodsLcr35 was administered daily by the oral route at a dosage of 1×109 CFU/mouse in BALB/c mice for 7 days before the first sensitization. Clinical parameters and regulatory T (Treg) cells were examined. The role of CD4+CD25+Foxp3+ Treg cells was analyzed using a Treg cell-depleting anti-CD25 monoclonal antibody (mAb).ResultsAirway hyperresponsiveness, total IgE production, pulmonary eosinophilic inflammation, and splenic lymphocyte proliferation were suppressed after Lcr35 treatment. Th1 (IFN-γ) and Th2 (IL-4, IL-5, and IL-13) cytokines in the serum were suppressed, and the percentage of CD4+CD25+Foxp3+ Treg cells in the spleen was significantly increased in the Lcr35 treatment group. Anti-CD25 mAb administration abolished the protective effects of Lcr35, indicating that CD4+ CD25+Foxp3+ Treg cells are essential in mediating the activity of Lcr35.ConclusionsOral administration of Lcr35 attenuated the features of allergic asthma in a mouse model and induced immune regulation by a CD4+CD25+Foxp3+ Treg cell-mediated mechanism.

Depletion of CD4+CD25+Foxp3+ regulatory T cells with anti-CD25 antibody may exacerbate the 1,3-β-glucan-induced lung inflammatory response in mice

1,3-β-Glucan was a major cell wall component of fungus. The existing studies showed that 1,3-β-glucan exposure could induce lung inflammation that involved both Th1 and Th2 cytokines. Regulatory T cells (Treg cells) played a critical role in regulating immune homeostasis by adjusting the Th1/Th2 balance. The role of Treg cells and regulatory mechanism in 1,3-β-glucan-induced lung inflammation is still unclear. In our study, mice were exposed to 1,3-β-glucan by intratracheal instillation. To investigate the role of Treg cells in response to 1,3-β-glucan, we generated Treg-depleted mice by intraperitoneal administration of anti-CD25 mAb. The Treg-depleted mice showed more inflammatory cells and severer pathological inflammatory change in lung tissue. Depletion of Treg cells led to increased Th1 cytokines and decreased Th2 cytokines. Treg-depleted mice showed a decreased expression of anti-inflammation cytokine and lower-level expression of CTLA-4. In all, our study indicated that Treg cells participated in regulating the 1,3-β-glucan-induced lung inflammation. Depletion of Treg cells aggravated the 1,3-β-glucan-induced lung inflammation, regulated the Th1/Th2 balance by enhancing Th1 response. Treg cells exerted their modulation function depending on both direct and indirect mechanism during the 1,3-β-glucan-induced lung inflammation.

Effect of CD4+CD25+ regulatory T cells on the immune evasion of Schistosoma japonicum

It has been known that parasites developed sophisticated strategies to escape from the host immune assault. More recently, one strategy to induce immune evasion involved CD4(+)CD25(+) regulatory T cells (Tregs). Mice were infected with Schistosoma japonicum cercariae and then injected intraperitoneally with anti-CD25 monoclonal antibody (anti-CD25 mAb). The results showed that the percentages of CD4(+)CD25(+) Tregs in mice were expanded by S. japonicum infection, and it could be partially blocked by anti-CD25 mAb. Worm burden in anti-CD25 mAb group (23.17 ± 6.94) was significantly lower than that in infected group (30.17 ± 5.85). The level of interferon gamma was increased with anti-CD25 mAb administration; meanwhile, lower concentration of interleukin 10 was observed in the same group. These results suggest that CD4(+)CD25(+) Tregs contribute to the escape of S. japonicum from the host immune responses, while anti-CD25 mAb can partially block CD4(+)CD25(+) Tregs and enhance the protective immunity to the parasite by Th1-type immune response.

Transient depletion of CD4+ T cells augments IL‐21‐based immunotherapy of disseminated neuroblastoma in syngeneic mice

IL-21 is a member of the IL-2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL-21-transduced neuroblastoma cells (Neuro2a/IL-21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL-21 vaccine. The administration of anti-CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti-CD4 mAb had a more potent effect leading to 80% cure rate. Anti-CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti-CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti-CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD-SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL-21 vaccination showed increased expression of IFN-alpha2, -beta1 and -gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti-CD4 mAb showed increased IFN-gamma serum levels and IFN-gamma-producing CD8+ T cells were found in spleen cells. In conclusion, anti-CD4 mAb potentiated IL-21-based IT by removing Treg cells and/or their precursors and other potentially immune-suppressive CD4+ cell subsets, thus allowing the development of an IL-21-driven CD8+ T cell response, which mediates NB rejection.

γδT Cells Are Involved in Liver Transplant Tolerance

The role of nonconventional T cells in innate and adaptive immunity is just emerging; γδT cells play important roles in anti-tumor and anti-infectious diseases. The involvement of γδT cells in immunologic responses to hematopoietic cell transplantation remains controversial; divergent results have been reported depending on the murine strains and model systems. Whether γδT cells are involved in solid organ transplantation is understudied. We have characterized the γδT cells in mouse livers and spleens to evaluate their contributions to liver transplant tolerance posttransplantation using a murine allogeneic liver transplant model which induces spontaneous T regulatory cell (Treg)-dependent tolerance. Our studies revealed that γδT cells comprised about 20% of the population of liver nonparenchymal cells (NPCs). In naïve C3H mice they were CD4, CD8, and NK1.1 negative. The percentage of γδT cells decreased in spontaneously tolerated liver grafts posttransplantation from 20% in naïve C3H livers to <10% in allografts throughout the time course. In contrast, they increased in liver grafts with rejection induced by anti-CTLA4 plus anti-CD25 mAb administration. CD4 and CD8 expression on γδT cells dramatically increased in the tolerated but not rejected livers posttransplantation to >20% of CD4 + and 30% of CD8 +. Our results suggested that γδT cells are involved in allogeneic immune responses. Whether γδT cells function as the causal or the effector cells in allograft tolerance rejection warrants further investigation.

A Combination of Systemic and Intracranial Anti-CD25 Immunotherapy Elicits a Long-Time Survival in Murine Model of Glioma

Abrogating the suppression of glioma-infiltrating Tregs in the periphery and the central nervous system is essential to successful glioma rejection. We sought to improve the immune response in glioma-bearing mice, by investigating new strategies using the anti-CD25 immunotherapy. We found a complete long-term survival of glioma-bearing mice treated with a combination of systemic and intracranial anti-CD25 mAb immunotherapy as compared to systemic administration of anti-CD25 mAb. In addition, the group of mice that had been cured by the combined anti-CD25 mAb showed long-term survival without late tumor relapse when challenged with the GL261 glioma. The antitumor immune response was investigated by analysis of antitumor immune response (CTL). Results showed that the use of the combined injections of anti-CD25 mAb induced efficient targeting of Tregs expansion inside and outside of the brain and altered Tregs trafficking in the bone marrow and brain areas where antitumor immunity was primed.

Anti-CD25 mAb, Anti-IL2 mAb, and IL2 Block Tolerance Induction Through Anti-CD154 mAb and Rapamycin in Xenogeneic Islet Transplantation

Background We have used anti-CD154 monoclonal antibody (mAb; MR1) and rapamycin (rapa) to induce tolerance to islet xenografts. The aim of this study was to investigate whether classical anergy and/or regulation by interleukin (IL)2-dependent CD25 + T regulatory cells played roles in the induction and maintenance of tolerance in this model. Methods Streptozotocin-induced diabetic mice were transplanted with rat islets. We performed the following groups: control group, islet transplantation without therapy; rapamycin group, 0.2 mg/kg by oral gavage on days 0, 1, 2, and every other day to day 14; anti-CD154 mAb (MR1) group, 0.5 mg intraperitoneally on days 0, 2, and 4; combination therapy group with rapa and MR1. We then administered in addition to the combination therapy with early (from days 0 to 14 [for IL2] or to 28 [for anti-IL2 mAb and anti-CD25 mAb] post-transplantation) or late (from days 100 to 114 [for IL2] or to 128 [for anti-IL2 mAb and anti-CD25 mAb] posttransplantation) recombinant IL2 (2000 U, intraperitoneally twice a day), a neutralizing anti-IL2 mAb (S4B6-1, 0.3 mg intraperitoneally twice weekly), and a depleting anti-CD25 mAb (PC61, 0.3 mg intraperitoneally twice weekly), respectively. Histology was performed at time of rejection. Results Rapa and MR1 therapy alone significantly prolonged xenograft survival compared to the control group: median graft survival was 34 days versus 17 days ( P < .05) and 98 days versus 17 days ( P < .05), respectively, but rejection still occurred. Combination therapy with MR1 and rapa allowed indefinite graft survival (median graft survival [MGS] >200 days, P < .001). When exogenous IL2 was administered early with MR1 and rapa, rapid rejection developed in 18 of 18 mice (MGS 7 days), whereas when IL2 was given late, only 3 of 10 developed rejection. Early administration of anti-IL2 mAb led to rejection in 10 of 10 mice (MGS 42 days), whereas late administration led to rejection in only one of four mice. Early administration of anti-CD25 mAb led to rejection in eight of nine mice (MGS 49 days), whereas late administration led to rejection in only three of seven mice. Conclusions Rapa and MR1 allowed indefinite graft survival of islet xenografts. Classical anergy and regulation by IL2-dependent CD25 + T regulatory cells were critical in the induction of tolerance in the immediate posttransplantation period and less important for maintenance of tolerance.

Anti-CD25 mAb Administration Prevents Spontaneous Liver Transplant Tolerance

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4 +CD25 + T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2 b) to C3H (H2 k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 μg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4 +CD25 + Treg were increased in the liver allografts and host spleens compared to naïve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4 +CD25 + Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-γ; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4 +CD25 + Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.

Neutralization of interleukin‐2 retards the growth of mouse renal cancer

To examine the significance of the thymus and the neutralization of interleukin-2 (IL-2) in treating renal cancer, as the involvement of immunoregulatory cells in tumour development in vivo is well known, naturally occurring CD25+ CD4+ T cells possess potent immunoregulatory functions, and they are of thymic origin dependent on IL-2. We first tested activity against mouse renal cell carcinoma (RENCA) cells by adoptively transferring splenocytes of euthymic Balb/c mice depleted of CD25+ cells into athymic Balb/c nude mice bearing established macroscopic RENCA tumours. Second, we tested the anti-RENCA activity in euthymic mice bearing macroscopic RENCA tumours by neutralizing IL-2. The intravenous administration of CD25+ cell-depleted splenocytes of euthymic Balb/c mice initiated the retardation of macroscopic RENCA tumours subcutaneously established in athymic Balb/c mice. The tumour site showed massive lymphocyte infiltration of mainly CD4+ T cells. By eliminating either the CD4+ cells, CD8+ cells, or natural killer (NK) cells with antibodies after the adoptive transfer of CD25+ cell-depleted splenocytes of euthymic Balb/c mice, macroscopic RENCA tumour retardation was abrogated. The growth of macroscopic RENCA tumour established in euthymic Balb/c mice was also retarded with IL-2 neutralization alone by anti-IL-2 monoclonal antibody (mAb), as well as co-administration of anti-IL-2 mAb and anti-CD25 mAb compared with that of the controls given vehicle. After tumour inoculation, peri- and intratumoral infiltration of CD4+ and CD8+ T cells was very prominent in RENCA tumours in hosts given anti-IL-2 mAb, regardless of the administration of anti-CD25 mAb. Two x 10(5) units of recombinant human IL-2 reverted the retardation of RENCA tumour growth caused by the anti-IL-2 mAb. IL-2 neutralization alone in euthymic Balb/c mice with no tumour inoculation did not suppress splenic CD25+ CD4+ T cells. Both the intravenous administration of CD25+ cell-depleted splenocytes of euthymic Balb/c mice into athymic Balb/c nude mice and IL-2 blocking with anti-IL-2 mAb in euthymic Balb/c mice retarded the growth of macroscopic RENCA tumours in vivo.

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer.

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.