Suppressive Effect Of Treg Cells Research Articles

Sirtuin4 suppresses the anti‐neuroinflammatory activity of infiltrating regulatory T cells in the traumatically injured spinal cord

The neuroinflammation following traumatic spinal cord injury (SCI) is a critical process that impacts both the injury and the recovery of spinal cord parenchyma. Infiltrating regulatory T (Treg) cells are potent anti-inflammatory cells that restrain post-SCI neuroinflammation. To understand the molecular mechanisms underlying the activity of infiltrating Treg cells, we used a mouse spinal cord compression injury model to analyze the role of Sirtuins (SIRTs) in the modulation of infiltrating Treg cell functions. We found that the expressions of SIRT4 and SIRT6 were up-regulated in infiltrating Treg cells. Using lentivirus-mediated gene expression or RNA interference, we revealed that SIRT4 substantially inhibited the expression of Foxp3, interleukin-10, and transforming growth factor-β in Treg cells, whereas SIRT6 had little effect on Treg cells. Consistently, SIRT4 overexpression weakened the suppressive effect of Treg cells on lipopolysaccharide-stimulated spinal cord CD11b+ myeloid cells. Knock-down of SIRT4 enhanced the anti-inflammatory activity of infiltrating Treg cells in the parenchyma of injured spinal cords. Additionally, SIRT4 overexpression blocked in vitro Treg cell generation from conventional T cells. Furthermore, SIRT4 down-regulated 5' AMP-activated protein kinase (AMPK) signaling in Treg cells, whereas the AMPK agonist AICAR restored the expression of Foxp3 and interleukin-10 in SIRT4-overexpressing Treg cells. In conclusion, our research unveils a new mechanism by which the post-SCI neuroinflammation is regulated.

Turning the Tide Against Regulatory T Cells.

Regulatory T (Treg) cells play crucial roles in health and disease through their immunosuppressive properties against various immune cells. In this review we will focus on the inhibitory role of Treg cells in anti-tumor immunity. We outline how Treg cells restrict T cell function based on our understanding of T cell biology, and how we can shift the equilibrium against regulatory T cells. To date, numerous strategies have been proposed to limit the suppressive effects of Treg cells, including Treg cell neutralization, destabilizing Treg cells and rendering T cells resistant to Treg cells. Here, we focus on key mechanisms which render T cells resistant to the suppressive effects of Treg cells. Lastly, we also examine current limitations and caveats of overcoming the inhibitory activity of Treg cells, and briefly discuss the potential to target Treg cell resistance in the context of anti-tumor immunity.

Regulatory T cells regulate the distribution of natural killer T cells through CD39 signal transduction in asthma.

Natural killer T cells (NKT cells) and regulatory T cells (Treg cells) are two important immune regulatory cells which both play critical roles in asthma. Our previous experiments revealed that activation of Treg cells suppressed NKT cells in asthma. However, the possible regulatory effects and the mechanisms linking Treg cells and NKT cells remain poorly understood. The current study was designed to further investigate the regulatory effect and its possible mechanisms of Treg cells on NKT cells function, especially the distribution of NKT cells. Regulatory T cell (Treg), responder T cell (Teff) and Natural killer T cell (NKT) were isolated and purified. After Lentivirus carrying CD39 (Le-CD39) was transfected into Treg cells, the immune phenotype of Treg cells was changed and the suppressive effect of Treg cells on Teff cells with an activation of Treg cells was enhanced, marking with a high expression level of interleukin 10 (IL-10) and transforming growth factor β (TGF-β). Up-regulation of CD39 expression led to lower ATP level in cell culture supernatant. To further explore its function in asthma, we introduced an ovalbumin (OVA)-induced mice model of asthma. And the data showed that up-regulation of CD39 remarkably alleviated OVA-induced hallmarks of the asthma and increased NKT cells in the spleen and peripheral blood; however, decreased NKT cells in the lung. Furthermore, up-regulation of CD39 decreased the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the lung of OVA-treated mice. Our results strongly suggest that Treg cells could be activated by CD39 signal transduction and then affected the distribution of NKT cells in the OVA-induced mice model of asthma.

In vivo activation of transferred regulatory T cells specific for third‐party exogenous antigen controls GVH disease in mice

Treg cells hold enormous promise for therapeutic application in GVH disease, a lethal complication of allogeneic HSC transplantation. Mouse studies showed that donor‐derived recipient‐specific Treg (rsTreg) cells are far more efficient than polyclonal Treg cells in suppressing GVH disease. However, clinical grade preparations of rsTreg cells carries the risk of containing significant numbers of highly pathogenic recipient‐specific effector T cells. We hypothesized that an alternative approach using Treg cells specific for an exogenous (i.e. nondonor, nonrecipient) Ag (exoTreg cells) can overcome this risk by taking advantage of the bystander suppressive effect of Treg cells. For this, we used a murine model for aggressive GVH disease. We expanded ex vivo exoTreg cells that are primed against the HY Ag, which is only expressed in males. ExoTreg cells supressed GVH disease as efficiently as rsTreg cells in recipient male mice. We also applied this strategy in female mice that do not express this Ag. While exoTreg cells were not effective in female recipients when applied alone, providing the cognate HY Ag in vivo along side effectively activated exoTreg cells and completely abrogated GVH disease, establishing a targeted on/off system to provide a suppressive effect on alloreactive effector T cells.

The imbalance between regulatory and IL-17-secreting CD4+T cells in multiple-trauma rat

BackgroundIt has been well recognised that a deficit of numbers and function of CD4+CD25+Foxp3+cells (Treg) is attributed to the development of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue; however, there are controversial data regarding the suppressive effect of Treg cells on the T-cell response in auto-immune diseases. Additionally, interleukin-17 (IL-17)-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue, but their role in multiple trauma remains unclear. ObjectiveThis study aims to investigate whether an imbalance of Treg and Th17 effector cells is characteristic of rats suffering from multiple trauma. Methods and subjectiveSixty Sprague-Dawley (SD) rats were randomly divided into three groups. The control group (n=20, group I) no received procedures (normal). The sham group (n=20, group II) only received anaesthesia, cannulation and observation. The bilateral femoral shaft fractures with haemorrhagic shock groups (n=20, group III). Rats in groups II and III were killed at the end of 4h after models were established. Peripheral blood samples were collected for assessment of Treg cells, Th17 cells and cytokines (IL-17, IL-6, IL-2, transforming growth factor beta (TGF-β)) and intestine tissue was collected for intestine histological analysis. ResultsWe observed decreased Treg/Th17 ratios in CD4+T cells in rats with multiple trauma and a strong inverse correlation with disease activity (intestinal histological scores). ConclusionWe suggest a role for immune imbalance in the pathogenesis and development of multiple trauma. The alteration of the index of Treg/Th17 cells likely indicates the therapeutic response and progress in the clinic.

Abstract P4-04-01: Combination of intratumoral CpG with systemic anti-OX40 and anti-CTLA4 mAbs eradicates established triple negative breast tumors in mice

Abstract Background: Evidence is mounting across diverse datasets and scientific techniques implicating immune dysregulation in poor outcomes of high grade and/or triple negative breast cancers. A multimodal strategy to stimulate the immune system could be an effective therapeutic approach for breast cancer. CpG oligodeoxynucleotides are single-stranded DNA that stimulate dendritic cells and B cells by binding to Toll-like receptor 9 (TLR9). OX40 (CD134) is predominantly expressed on activated T cells and the binding of OX40 with an agonistic antibody (anti-OX40) leads to the proliferation and survival of T cells. Agonistic anti-OX40 antibodies can also reverse the suppressive effects of Treg cells. A third avenue is to target Cytotoxic T lymphocyte associated antigen (CTLA4), which plays a negative regulatory role in T cell activation and is also constitutively expressed on Treg cells. Blocking CTLA4 with an antagonistic antibody (anti-CTLA4) has been shown to enhance anti-tumor responses. In this study, we tested the combination of intratumoral (IT) CpG with systemic anti-OX40 plus anti-CTLA4 in a mouse model of triple negative breast cancer. Methods: 4T1, an aggressive, triple-negative mammary carcinoma, was implanted orthotopically into syngeneic BALB/C mice. Treatment was initiated when tumors were ∼0.5 cm in the largest dimension. Mice were divided into 8 groups. Various combinations of PBS control, CpG, taxol, and anti-OX40+anti-CTLA4 mAbs were tested (Tables 1 and 2). CpG, 100 µg, was injected IT for 5 consecutive days (days 1–5); Taxol, 16 mg/kg, was administered IP on day 1; anti-OX40 (400 µg) and anti-CTLA4 (100 µg) were given IP on days 1 and 5. Mice were sacrificed once tumors became ulcerated or reached a diameter >2 cm. Results: As shown in Table 1, CpG alone, taxol alone, or the anti-OX40 + anti-CTLA4 combination each delayed the growth of established 4T1 tumors when compared to the untreated control group. However, the majority of the mice (except one in CpG treated group) died from tumor progression. The combination of CpG + anti-OX40 + anti-CTLA4 had the best anti-tumor effects, with 4 out of 5 mice having demonstrated complete regression of their tumors (Table 2). The addition of taxol to the combination of CpG + anti-OX40 + anti-CTLA4 did not improve the anti-tumor effect, and while there was a substantial delay in growth, only one animal achieved a complete regression. Conclusion: Our study demonstrated that the combination of IT CpG with systemic anti-OX40 + anti-CTLA4 mAbs can cure mice with established triple negative breast tumors, supporting the possibilities of applying immune modulation approach in human triple negative tumors. Studies are on-going to further dissect the immunological mechanisms involved. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-04-01.

CD4+ CD25− FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

The distribution and dynamic changes of CD4(+) CD25(+) FoxP3(+) and CD4(+) CD25(-) FoxP3(+) regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4(+) CD25(-) Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4(+) CD25(-) Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = -1, P < 0.0001). Hence, higher frequencies of functional CD4(+) CD25(-) Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4(+) CD25(-) Treg cells over CD4(+) CD25(+) Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4(+) CD25(-) Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4(+) CD25(-) Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.

Notch activation on effector T cells increases their sensitivity to Treg cell-mediated suppression through upregulation of TGF-βRII expression

Notch proteins play an important role in embryonic development and cell-fate decisions. Notch influences also the activation and differentiation of peripheral T cells. Here, we investigated whether Notch signaling modulates the response of effector T cells to regulatory T (Treg) cells. Pre-exposure of CD4(+) CD25(-) effector T cells to the Notch ligands Delta-4 and Jagged-1, but not Delta-1, increases significantly effector T-cell sensitivity to Treg cell-mediated suppression through upregulation of TGF-βRII expression and increased levels of the phosphorylated form of the Smad 3 protein. This effect is relieved by anti-TGF-β Abs. We demonstrate that HES (hairy and enhancer of split), the main transcription factor downstream of Notch, induces strong transactivation of TGF-ßRII by binding the TGF-βRII promoter through its DNA-binding domain. Thus, the crosstalk between Notch and the TGF-β pathway leads to potentiation of the suppressive effect of Treg cells.

Inhibiting effect of Astragalus polysaccharides on the functions of CD4+CD25 highTreg cells in the tumor microenvironment of human hepatocellular carcinoma.

Astragalus polysaccharides (APS), the main active extract from Astragalus membranaceus (a traditional Chinese medicinal herb), is associated with a variety of immunomodulatory activities. The purpose of the present study was to examine the effect of APS on the function of Treg cells in the tumor microenvironment of human hepatocellular carcinoma (HCC) and to identify the pharmacologic mechanism of APS responsible for the anti-chemotactic activity in CD4+CD25highTreg cells in tumor site of HCC. The prevalence of Treg in fresh tissue samples from 31 patients with HCC after radicalhepatectomy was detected. CD4, CD25 and CD127 were selected as Treg cell makers to phenotype cell populations. The expression of FOXp3 mRNA was also analyzed. The migration and proliferation of Treg cells were observed. Interleukin (IL)-4, IL-10, IFN-γ and SDF-1 in cell supernatant were detected. For all tests, functions of Treg cells were evaluated after treatment with APS. APS can inhibit the growth and proliferation of CD4+CD25+Treg cells in vitro in a dose- and time-dependent manner. APS may inhibit CD4+CD25+Treg cells through restoring the cytokine imbalance and reducing the expression of FOXp3 in local HCC microenvironments. SDF-1 played an important role in there recruitment of Treg cells into the tumor microenvironment of HCC. APS might have inhibiting effects on Treg cell migration by blocking SDF-1 or its receptor through the CXCR4/CXCL12 pathway. The increase in numbers of tumor associated Treg cells might play a role in modulation of the immune response against HCC. APS can restore the cytokine balance in the tumor micro environment and suppress the expression of FOXp3 mRNA to inhibit the immune suppressive effects of Treg cells. The application of APS in the tumor microenvironment might act to enhance the anti-tumor effects of the immunotherapy-based methods, and consequently to increase the survival rate in HCC.

Microbial Infection-Induced Expansion of Effector T Cells Overcomes the Suppressive Effects of Regulatory T Cells via an IL-2 Deprivation Mechanism

Foxp3(+) regulatory T (Treg) cells are a critical cell population that suppresses T cell activation in response to microbial and viral pathogens. We identify a cell-intrinsic mechanism by which effector CD4(+) T cells overcome the suppressive effects of Treg cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the expansion of pathogen-specific CD4(+) T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens.

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25- effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells. The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in (3) H-thymidine proliferation assays. Cytokine levels were analyzed using Bio-Plex Pro assay. Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA-DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells. Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro.

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).

Novel role of regulatory T cells in limiting early neutrophil responses in skin

It is clear that CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells inhibit chronic inflammatory responses as well as adaptive immune responses. Among the CD4(+) T-cell population in the skin, at least one-fifth express Foxp3. As the skin is constantly exposed to antigenic challenge and is a common site of vaccination, understanding the role of these skin-resident Treg cells is important. Although the suppressive effect of Treg cells on T cells is well documented, less is known about the types of innate immune cells influenced by Treg cells and whether the Treg cells suppress acute innate immune responses in vivo. To address this we used a mouse melanoma cell line expressing Fas ligand (B16FasL), which induces an inflammatory response following subcutaneous injection of mice. We demonstrate that Treg cells limit this response by inhibiting neutrophil accumulation and survival within hours of tumour cell inoculation. This effect, which was associated with decreased expression of the neutrophil chemoattractants CXCL1 and CXCL2, promoted survival of the inoculated tumour cells. Overall, these data imply that Treg cells in the skin are rapidly mobilized and that this activity serves to limit the amplification of inflammatory responses at this site.

The imbalance between regulatory and IL-17-secreting CD4+ T cells in lupus patients

It has been well recognized that a deficit of numbers and function of CD4+CD25+Foxp3+ cells (Treg) is attributed to the development of some autoimmune diseases; however, there are controversial data regarding the suppressive effect of Treg cells on the T cell response in systemic lupus erythematosus (SLE). Additionally, IL-17-producing cells (Th17) have been recently emerged as a new pathogenic cell, but their role in lupus remains unclear. In this study, we studied the connection between Treg and Th17 cells in lupus patients. We observed that, while Treg or Th17 cells alone were not correlated to SLE development, the ratio of Treg to Th17 cells in active SLE patients is significantly lower than that in inactive SLE patients and healthy controls, and we also found corticosteroid treatment increased the ratio of Treg to Th17 cells in active SLE patients. Moreover, this ratio is inversely correlated with the severity of active SLE. The present study indicates that active SLE appears to exist as an imbalance between Treg and Th17 cells. Correction of this Treg/Th17 imbalance may have therapeutic impact for patients with SLE.

Plasmodium vivax: Induction of CD4+CD25+FoxP3+ Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4+CD25+FoxP3+ regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-β) as well as pro-inflammatory (IFN-γ, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4+CD25+FoxP3+GITR+ or CD4+CD25+FoxP3+CTLA-4+ and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-Scid, IL-2Rγnull Mice In Vivo.

Abstract 726There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities, and it is a current crucial problem in the field of human ADCC research. To overcome this, we have established “humanized mice,” in which human immune cells from healthy individuals function as ADCC effector cells against allogeneic tumor cell lines, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients. In this model, the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to highly enhance ADCC, showed potent antitumor activity by human ADCC against CCR4 expressing tumor cell lines. In addition, KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in the tumor bearing humanized mice. These observations indicate that KM2760 could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. Using this humanized mouse model, we now have the opportunity to perform more appropriate preclinical evaluation of many types of mAb based immunotherapy, although in the initial study, we could not completely exclude nonspecific allogeneic immune responses because target and effector cells were obtained from different individuals. In addition, susceptibility to immunotherapy is likely to be different in established cell lines and primary tumor cells isolated directly ex vivo from patients, with the latter certainly being more relevant for evaluation of immunotherapeutic agents. To overcome the subsequent problems, we have established a primary human tumor bearing NOG mouse model, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T-cell leukemia/lymphoma (ATLL) cells. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in mAb based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. Disclosures:No relevant conflicts of interest to declare.

CD44 expression positively correlates with Foxp3 expression and suppressive function of CD4+ Treg cells

BackgroundCD4+CD25+ regulatory T (Treg) cells develop in the thymus and can suppress T cell proliferation, modulated by Foxp3 and cytokines; however, the relevance of CD44 in Treg cell development is less clear. To address this issue, we analyzed Foxp3 expression in CD44+ Treg cells by using multiple parameters, measured the levels of the immunoregulatory cytokine interleukin (IL)-10 in various thymocyte subsets, and determined the suppressor activity in different splenic Treg cell populations.ResultsWithin mouse thymocytes, we detected Treg cells with two novel phenotypes, namely the CD4+CD8-CD25+CD44+ and CD4+CD8-CD25+CD44- staining features. Additional multi-parameter analyses at the single-cell and molecular levels suggested to us that CD44 expression positively correlated with Foxp3 expression in thymocytes, the production of IL-10, and Treg activity in splenic CD4+CD25+ T cells. This suppressive effect of Treg cells on T cell proliferation could be blocked by using anti-IL-10 neutralizing antibodies. In addition, CD4+CD25+CD44+ Treg cells expressed higher levels of IL-10 and were more potent in suppressing effector T cell proliferation than were CD4+CD25+CD44- cells.ConclusionThis study indicates the presence of two novel phenotypes of Treg cells in the thymus, the functional relevance of CD44 in defining Treg cell subsets, and the role of both IL-10 and Foxp3 in modulating the function of Treg cells.ReviewersThis article was reviewed by Dr. M. Lenardo, Dr. L. Klein & G. Wirnsberger (nominated by Dr. JC Zungia-Pfluker), and Dr. E.M. Shevach.

Critical role of IFN-γ in CFA-mediated protection of NOD mice from diabetes development

IFN-gamma signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN-gamma signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN-gamma receptor-deficient (IFN-gammaR(-/-)) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice. These data clearly show that the pro-inflammatory cytokine IFN-gamma is necessary for the CFA-mediated protection of NOD mice from diabetes. There is no difference in the T(h)1/T(h)17 balance between IFN-gammaR(-/-) NOD and wild-type NOD mice. There is also no difference in the total numbers and percentages of regulatory T (Treg) cells in the lymph node CD4(+) T-cell populations between IFN-gammaR(-/-) NOD and wild-type NOD mice. However, pathogenic T cells lacking IFN-gammaR are resistant to the suppressive effect of Treg cells, both in vivo and in vitro. Therefore, it is likely that CFA-mediated protection against diabetes development depends on a change in the balance between Treg cells and pathogenic T cells, and IFN-gamma signaling seems to control the susceptibility of pathogenic T cells to the inhibitory activity of Treg cells.

Regulatory T Cells Sequentially Migrate from Inflamed Tissues to Draining Lymph Nodes to Suppress the Alloimmune Response

To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function.

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rgamma(null) mouse model.

There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.