CD4+ICOS+Foxp3+: a sub-population of regulatory T cells contribute to malaria pathogenesis.

BACKGROUND: Regulatory T cells are known to play a key role to counter balance the protective immune response and immune mediated pathology. However, the role of naturally occurring regulatory cells CD4+CD25+Foxp3+ in malaria infection during the disease pathogenesis is controversial. Beside this, ICOS molecule has been shown to be involved in the development and function of regulatory T cell enhance IL-10 production. Therefore, possible involvement of the ICOS dependent regulatory CD4+ICOS+Foxp3+ T cells in resistance/susceptibility during malaria parasite is explored in this study. METHODS: 5 × 105 red blood cells infected with non-lethal and lethal parasites were inoculated in female Balb/c mice by intra-peritoneal injection. Infected or uninfected mice were sacrificed at early (3rd day post infection) and later stage (10th day post infection) of infection. Harvested cells were analysed by using flow cytometer and serum cytokine by Bioplex assay. RESULTS: Thin blood films show that percentages of parasitaemia increases with disease progression in infections with the lethal malaria parasite and mice eventually die by day 14th post-infection. Whereas in case of non-lethal malaria parasite, parasitaemia goes down by 7th day post infection and gets cleared within 13th day. The number of CD4+ ICOS+ T cells increases in lethal infection with disease progression. Surprisingly, in non-lethal parasite, ICOS expression decreases after day 7th post infection as parasitaemia goes down. The frequency of CD4+ICOS+FoxP3+ Tregs was significantly higher in lethal parasitic infection as compared to the non-lethal parasite. The level of IL-12 cytokine was remarkably higher in non-lethal infection compared to the lethal infection. In contrast, the level of IL-10 cytokines was higher in lethal parasite infection compared to the non-lethal parasite. CONCLUSION: Taken together, these data suggest that lethal parasite induce immunosuppressive environment, protecting from host immune responses and help the parasite to survive whereas non-lethal parasite leads to low frequencies of Treg cells seldom impede immune response that allow the parasite to get self-resolved.

Follicular Helper CD4+ T Cells, Follicular Regulatory CD4+ T Cells, and Inducible Costimulator and Their Roles in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

Follicular helper CD4+ T (TFH) cells are a specialized subset of effector T cells that play a central role in orchestrating adaptive immunity. TFH cells mainly promote germinal center (GC) formation, provide help to B cells for immunoglobulin affinity maturation and class-switch recombination of B cells, and facilitate production of long-lived plasma cells and memory B cells. TFH cells express the nuclear transcriptional repressor B cell lymphoma 6 (Bcl-6), the chemokine (C-X-C motif) receptor 5 (CXCR5), the CD28 family members programmed cell death protein-1 (PD-1) and inducible costimulator (ICOS) and are also responsible for the secretion of interleukin-21 (IL-21) and IL-4. Follicular regulatory CD4+ T (TFR) cells, as a regulatory counterpart of TFH cells, participate in the regulation of GC reactions. TFR cells not only express markers of TFH cells but also express markers of regulatory T (Treg) cells containing FOXP3, glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T lymphocyte antigen 4 (CTLA-4), and IL-10, hence owing to the dual characteristic of TFH cells and Treg cells. ICOS, expressed on activated CD4+ effector T cells, participates in T cell activation, differentiation, and effector process. The expression of ICOS is highest on TFH and TFR cells, indicating it as a key regulator of humoral immunity. Multiple sclerosis (MS) is a severe autoimmune disease that affects the central nervous system and results in disability, mediated by autoreactive T cells with evolving evidence of a remarkable contribution from humoral responses. This review summarizes recent advances regarding TFH cells, TFR cells, and ICOS, as well as their functional characteristics in relation to MS.

Role of Inducible Co-Stimulator (ICOS) in cancer immunotherapy.

Introduction: The promotion of antitumor response by targeting co-stimulatory B7 superfamily members has become evident to create a new wave of cancer immunotherapy. Inducible Co-Stimulator (ICOS), which is expressed on activated T cells, gained interest in the translational medicine community.Areas covered: We performed an extensive literature review using the keywords 'ICOS' and 'cancer', and the Clinicaltrials.gov database for early phase clinical trials targeting ICOS. In this review, we highlight the dual role of ICOS in oncogenesis in different malignancies. We summarize the current state of knowledge about ICOS/ICOSL pathway targeting by immunotherapies.Expert opinion: Due to its multifaceted link with anti-tumor immunity, both antagonist and agonist antibodies might be of interest to target the ICOS/ICOSL pathway for tumor treatment. Indeed, ICOS activation might potentiate the effect of an inhibitory checkpoint blockade, while its neutralization could decrease the function of immunosuppressive Tregs and inhibit lymphoid tumor cells expressing Tfh markers.

Tolerogenic Dendritic Cells Attenuate Experimental Autoimmune Antimyeloperoxidase Glomerulonephritis.

Background Because of their capacity to induce antigen-specific immunosuppression, tolerogenic dendritic cells are a promising tool for treatment of autoimmune conditions, such as GN caused by autoimmunity against myeloperoxidase (MPO). METHODS: We sought to generate tolerogenic dendritic cells to suppress anti-MPO GN by culturing bone marrow cells with an NFκB inhibitor (BAY 11-7082) and exposing them to a pulse of MPO. After administering these MPO/BAY dendritic cells or saline to mice with established anti-MPO or anti-methylated BSA (mBSA) immunity, we assessed immune responses and GN. We also examined mechanisms of action of MPO/BAY dendritic cells. RESULTS: MPO/BAY dendritic cells decreased anti-MPO immunity and GN without inhibiting immune responses against mBSA; they also induced IL-10-producing regulatory T cells in MPO-immunized mice without affecting IL-10+ CD4+Foxp3- type 1 regulatory T cells or regulatory B cells. MPO/BAY dendritic cells did not inhibit anti-MPO immunity when CD4+Foxp3+ cells were depleted in vivo, showing that regulatory T cells are required for their effects. Coculture experiments with dendritic cells and CD4+Foxp3- or CD4+Foxp3+ cells showed that MPO/BAY dendritic cells generate Foxp3+ regulatory T cells from CD4+Foxp3- cells through several pathways, and induce IL-10+ regulatory T cells via inducible costimulator (ICOS), which was confirmed in vivo. Transfer of MPO/BAY dendritic cell-induced regulatory T cells in vivo, with or without anti-IL-10 receptor antibody, demonstrated that they suppress anti-MPO immunity and GN via IL-10. CONCLUSIONS: MPO/BAY dendritic cells attenuate established anti-MPO autoimmunity and GN in an antigen-specific manner through ICOS-dependent induction of IL-10-expressing regulatory T cells. This suggests that autoantigen-loaded tolerogenic dendritic cells may represent a novel antigen-specific therapeutic option for anti-MPO GN.

ICOS signal facilitates Foxp3 transcription to favor suppressive function of regulatory T cells.

Inducible costimulator (ICOS) plays an important role in the suppressive immunity mediated by regulatory T cells (Tregs), but the molecular regulation mechanism is not well known. Here we performed a study to explore the possible mechanism by which ICOS regulates the suppressive functions and survival of Tregs. This study showed that both the ICOS and CD28 signal could promote the survival of Tregs. However, ICOS but not CD28 improved the suppressive function of Tregs. Mechanistic studies demonstrated that ICOS could induce the transcription activity of Foxp3, by facilitating the nuclear factor of activated T cells (NFAT): Foxp3 over NFAT: activator protein 1 (AP-1). The results of Q-PCR showed that AP1 downstream regulatory genes (IL-2 and IL-6) were down-regulated, and Foxp3 downstream regulatory genes (IL-4, IL-10 and TGF-ß) were up-regulated. Further, ICOS promoted anti-apoptosis may be by activating protein kinase B (Akt) signal. These findings demonstrated that ICOS signal could facilitate Foxp3 transcription in favor of survival and suppressive function of Tregs.

Inducible T-cell co-stimulators regulate the proliferation and invasion of human hepatocellular carcinoma HepG2 cells.

BACKGROUND: This study determined the regulatory effects of inducible T-cell co-stimulators (ICOS) in human hepatocellular carcinoma HepG2 cells using a RNA interference (RNAi) technique. METHODS: A RNAi technique was used to knockdown the expression of ICOS. ICOS expression after knockdown was detected as mRNA and protein levels by RT-PCR and Western blot, respectively. A MTT colorimetric assay was used to detect cell proliferation, and the Transwell assay was used to detect cell invasion. Western blot was carried out to detect the level of Bcl-2, AKT, and PI3K protein expression in different groups. RESULTS: The proliferation of HepG2 cells were significantly decreased after ICOS siRNA transfection (EG group). Similarly, the results of the Transwell experiment showed that invasion of HepG2 cells in the EG group was clearly reduced compared to the negative control (NC) and blank control groups (CON). Western blot analysis showed that knockdown of ICOS expression reduced the levels of Bcl-2 and AKT, and also significantly up-regulated the level of PI3K phosphorylation (P < 0.01). CONCLUSION: Down-regulating ICOS expression in HepG2 cells suppressed cell proliferation and invasion. The underlying mechanism may be related to the expression of the downstream factor, PI3K/AKT.

Inducible T-cell co-stimulators regulate the proliferation and invasion of human hepatocellular carcinoma HepG2 cells

Abstract Background This study determined the regulatory effects of inducible T-cell co-stimulators (ICOS) in human hepatocellular carcinoma HepG2 cells using a RNA interference (RNAi) technique. Methods A RNAi technique was used to knockdown the expression of ICOS. ICOS expression after knockdown was detected as mRNA and protein levels by RT-PCR and Western blot, respectively. A MTT colorimetric assay was used to detect cell proliferation, and the Transwell assay was used to detect cell invasion. Western blot was carried out to detect the level of Bcl-2, AKT, and PI3K protein expression in different groups. Results The proliferation of HepG2 cells were significantly decreased after ICOS siRNA transfection (EG group). Similarly, the results of the Transwell experiment showed that invasion of HepG2 cells in the EG group was clearly reduced compared to the negative control (NC) and blank control groups (CON). Western blot analysis showed that knockdown of ICOS expression reduced the levels of Bcl-2 and AKT, and also significantly up-regulated the level of PI3K phosphorylation (P < 0.01). Conclusion Down-regulating ICOS expression in HepG2 cells suppressed cell proliferation and invasion. The underlying mechanism may be related to the expression of the downstream factor, PI3K/AKT.

Exploiting IL-17-producing CD4+ and CD8+ T cells to improve cancer immunotherapy in the clinic.

Cancer immunotherapy is one the most effective approaches for treating patients with tumors, as it bolsters the generation and persistence of memory T cells. In preclinical work, it has been reported that adoptively transferred CD4+ and CD8+ lymphocytes that secrete IL-17A (i.e., Th17 and Tc17 cells) regress tumors to a greater extent than IFN-γ(+)Th1 or Tc1 cells in vivo. Herein, we review the mechanisms underlying how infused Th17 and Tc17 cells regress established malignancies in clinically relevant mouse models of cancer. We also discuss how unique signaling cues--such as co-stimulatory molecules (ICOS and 41BB), cytokines (IL-12 and IL-23) or pharmaceutical reagents (Akt inhibitors, etc.)--can be exploited to bolster the therapeutic potential of IL-17(+) lymphocytes with an emphasis on using this knowledge to improve next-generation clinical trials for patients with cancer.

Follicular Helper CD4+ T Cells in Human Neuroautoimmune Diseases and Their Animal Models.

Follicular helper CD4(+) T (TFH) cells play a fundamental role in humoral immunity deriving from their ability to provide help for germinal center (GC) formation, B cell differentiation into plasma cells and memory cells, and antibody production in secondary lymphoid tissues. TFH cells can be identified by a combination of markers, including the chemokine receptor CXCR5, costimulatory molecules ICOS and PD-1, transcription repressor Bcl-6, and cytokine IL-21. It is difficult and impossible to get access to secondary lymphoid tissues in humans, so studies are usually performed with human peripheral blood samples as circulating counterparts of tissue TFH cells. A balance of TFH cell generation and function is critical for protective antibody response, whereas overactivation of TFH cells or overexpression of TFH-associated molecules may result in autoimmune diseases. Emerging data have shown that TFH cells and TFH-associated molecules may be involved in the pathogenesis of neuroautoimmune diseases including multiple sclerosis (MS), neuromyelitis optica (NMO)/neuromyelitis optica spectrum disorders (NMOSD), and myasthenia gravis (MG). This review summarizes the features of TFH cells, including their development, function, and roles as well as TFH-associated molecules in neuroautoimmune diseases and their animal models.

ICOS promotes group 2 innate lymphoid cell activation in lungs.

Group 2 innate lymphoid cells (ILC2s) are newly identified, potent producers of type 2 cytokines, such as IL-5 and IL-13, and contribute to the development of allergic lung inflammation induced by cysteine proteases. Although it has been shown that inducible costimulator (ICOS), a costimulatory molecule, is expressed on ILC2s, the role of ICOS in ILC2 responses is largely unknown. In the present study, we investigated whether the interaction of ICOS with its ligand B7-related protein-1 (B7RP-1) can promote ILC2 activation. Cytokine production in ILC2s purified from mouse lungs was significantly increased by coculture with B7RP-1-transfected cells, and increased cytokine production was inhibited by monoclonal antibody-mediated blocking of the ICOS/B7RP-1 interaction. ILC2 expansion and eosinophil influx induced by papain, a cysteine protease antigen, in mouse lungs were significantly abrogated by blocking the ICOS/B7RP-1 interaction. Dendritic cells (DCs) in the lungs expressed B7RP-1 and the number of DCs markedly increased with papain administration. B7RP-1 expression on lung DCs was reduced after papain administration. This downregulation of B7RP-1 expression may be an indication of ICOS/B7RP-1 binding. These results indicate that ILC2s might interact with B7RP-1-expressing DCs in allergic inflammatory lung, and ICOS signaling can positively regulate the protease allergen-induced ILC2 activation followed by eosinophil infiltration into the lungs.

[Effect of ICOS signaling on CD154/CD40 expressions in mice infected with Schistosoma japonicum].

OBJECTIVE: To explore the effect of ICOS signaling on the CD154/CD40 expressions and immunopathology in mice infected with Schistosoma japonicum. METHODS: ICOS transgenic (ICOS-Tg) mice and wildtype FVB/NJ mice were used as experimental schistosomiasis models. The expressions of CD154 and CD40 on splenocytes and on inflammatory cells around granulomatous infiltration of the liver in the mice infected with S. japonicuin were detected by flow cytometry and im- munohistochemical staining. HE staining was applied to observe the changes on the granulomatous of the mice liver. RESULTS: Compared with the wildtype FVB/NJ mice, the expressions of CD154 on CD4 T splenocytes and of CD40 on CD19' B splenocytes in the ICOS-Tg mice significantly increased in 12 and 16 weeks post-infection (all P < 0.05). Moreover, the expressions of CD40 and CD154 on inflammatory cells around granulomatous infiltration in the liver of the ICOS-Tg mice were significantly higher than those of the wildtype FVB/NJ mice in 7, 12, 16 and 20 weeks post-infection (all P < 0.05). The volumes of liver egg granulomas of the ICOS-Tg mice were significantly bigger than those of the wildtype mice (P < 0.05 or P < 0.01). CONCLUSIONS: In ICOS-Tg mice infected with S. japonicum, the ICOS signaling has a regulatory effect on CD154/CD40 expressions, and may play an important role in the hepatic egg granuloma formation of schistosomiasis.

T-cell costimulatory blockade in organ transplantation.

Before it became possible to derive T-cell lines and clones, initial experimentation on the activation requirements of T lymphocytes was performed on transformed cell lines, such as Jurkat. These studies, although technically correct, proved misleading as most transformed T cells can be activated by stimulation of the clonotypic T-cell receptor (TCR) alone. In contrast, once it became possible to study nontransformed T cells, it quickly became clear that TCR stimulation by itself is insufficient for optimal activation of naïve T cells, but in fact, induces a state of anergy. It then became clear that functional activation of T cells requires not only recognition of major histocompatibility complex (MHC) and peptide by the TCR, but also requires ligation of costimulatory receptors expressed on the cell surface.

Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice.

We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.

Plasmacytoid dendritic cells promote immunosuppression in ovarian cancer via ICOS costimulation of Foxp3(+) T-regulatory cells.

Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer death among women. Despite its immunogenicity, effective antitumor responses are limited, due, in part, to the presence of forkhead box protein 3-positive (Foxp3(+)) T regulatory (Treg) cells in the tumor microenvironment. However, the mechanisms that regulate the accumulation and the suppressive function of these Foxp3(+) Treg cells are poorly understood. Here, we found that the majority of Foxp3(+) Treg cells accumulating in the tumor microenvironment of EOCs belong to the subset of Foxp3(+) Treg cells expressing inducible costimulator (ICOS). The expansion and the suppressive function of these cells were strictly dependent on ICOS-L costimulation provided by tumor plasmacytoid dendritic cells (pDC). Accordingly, ICOS(+) Foxp3(+) Treg cells were found to localize in close vicinity of tumor pDCs, and their number directly correlated with the numbers of pDCs in the tumors. Furthermore, pDCs and ICOS(+) Foxp3(+) Treg cells were found to be strong predictors for disease progression in patients with ovarian cancer, with ICOS(+) Treg cell subset being a stronger predictor than total Foxp3(+) Treg cells. These findings suggest an essential role for pDCs and ICOS-L in immunosuppression mediated by ICOS(+) Foxp3(+) Treg cells, leading to tumor progression in ovarian cancer.

Basic science for the clinician 56: inducible T-cell costimulator--the world of costimulation gets more complicated and interesting.

They say that nothing is assured in this world but change. And this applies in a cynical way to immunology: nothing is assured--we can never rest with an assumption that we know it all because there are always more apparent complications when analyzing immune mechanisms (someday, perhaps we will arrive on a "grand scheme" that elegantly explains it all, just as our brethren in particle physics seek the Higgs boson and the completion of a better model--I am not holding my breath awaiting completion of our task!). In a recent article in this series, we explored CTLA4 as a counterregulator of the CD28-CD80/86 costimulatory pathway. However, treatment with CTLA4 does not entirely shutdown the immune system; engineering animals so that the CD28-CD80/86 pathway no longer functions does not prevent functional protective immune responses. Thus, there must be yet another pathway. And there is--an "inducible T-cell costimulator" (ICOS) is found on T cells (activated, not naive), which has a single ligand on antigen-presenting cells (ICOS ligand, B7-RP-1). The rapid induction of ICOS speaks to its importance in T-cell function; however, ICOS is more relevant to the stimulation of effector and memory T cells than is CD28 signaling. There are similarities and differences, interactions, and overlaps between the 2 pathways, some of which are very useful in understanding the immunopathogenesis of immune diseases.

Comprehensive analysis of CD4+ T cells in the decision between tolerance and immunity in vivo reveals a pivotal role for ICOS.

We have established a comprehensive in vivo mouse model for the CD4(+) T cell response to an "innocuous" versus "dangerous" exogenous Ag and developed an in vivo test for tolerance. In this model, specific gene-expression signatures, distinctive upregulation of early T cell-communication molecules, and differential expansion of effector T cells (Teff) and regulatory T cells (Treg) were identified as central correlates of T cell tolerance and T cell immunity. Different from essentially all other T cell-activation molecules, ICOS was found to be induced in the immunity response and not by T cells activated under tolerogenic conditions. If expressed, ICOS did not act as a general T cell costimulator but selectively caused a massive expansion of effector CD4(+) T cells, leaving the regulatory CD4(+) T cell compartment largely undisturbed. Thus, ICOS strongly contributed to the dramatic change in the balance between Ag-specific Teff and Treg from ∼1:1 at steady state to 21:1 at the height of the immune response. This newly defined role for the balance of Teff to Treg, together with its known key function in T cell help for B cells, establishes ICOS as a central mediator of immunity. Given its exceptionally selective induction on CD4(+) T cells under inflammatory, but not tolerogenic, conditions, ICOS emerges as a pivotal effector molecule in the early decision between tolerance and immunity to exogenous Ag.

Heterozygosity for Roquinsan leads to angioimmunoblastic T-cell lymphoma-like tumors in mice.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (T(FH)) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the "san" allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of T(FH) cells preceded tumor development, and clonal rearrangements in the TCR-ß genes were present in most tumors. Furthermore, T(FH) cells exhibited increased clonality compared with non-T(FH) cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent T(FH) development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a T(FH)-derived origin. Roquin(san/+) mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated T(FH) cells or their consequences.

ICOS-LICOS interaction is critically involved in TGN1412-mediated T-cell activation.

TGN1412, a superagonistic CD28-specific antibody, was shown to require Fc-cross-linking or immobilization as a prerequisite to mediate T-cell proliferation and cytokine release in vitro. We used primary human umbilical vein endothelial cells (HUVECs) to study their ability to induce activation of TGN1412-treated T cells. We confirmed that peripheral primary human T cells do not show activation upon stimulation with soluble TGN1412 alone. Nevertheless, cocultivation of TGN1412-treated T cells with HUVECs induced T-cell activation that was further enhanced using cytokine prestimulated HUVECs. Unexpectedly, Fc-FcγR interaction was dispensable for endothelial cell-mediated proliferation of TGN1412-treated T cells. Transwell-culture assays showed that TGN1412-treated T cells need direct cell-to-cell contact to HUVECs to induce proliferation. We found that costimulatory ICOS-LICOS interaction between T cells and endothelial cells is critically involved in TGN1412-mediated effects. Blocking LICOS reduced TGN1412-mediated T-cell proliferation significantly, whereas recombinant LICOS fully conferred TGN1412-mediated T-cell proliferation. Of note, cytokine stimulation enhanced LICOS expression on HUVECs and ICOS-LICOS interaction up-regulated ICOS expression on TGN1412-treated T cells. Hence, we provide a model of positive feedback conferred by ICOS-LICOS interaction between TGN1412-treated T cells and endothelial cells.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice.

A progressive waning in Foxp3(+) regulatory T cell (Treg) functions is thought to provoke autoimmunity in the NOD model of type 1 diabetes (T1D). A deficiency in IL-2 is one of the main triggers for the defective function of Tregs in islets. Notably, abrogation of the ICOS pathway in NOD neonates or BDC2.5-NOD (BDC2.5) mice exacerbates T1D, suggesting an important role for this costimulatory pathway in tolerance to islet Ags. Thus, we hypothesize that ICOS selectively promotes Foxp3(+) Treg functions in BDC2.5 mice. We show that ICOS expression discriminates effector Foxp3(-) T cells from Foxp3(+) Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand, secrete IL-10, and mediate suppressive activity in vitro and in vivo. Consistently, Ab-mediated blockade or genetic deficiency of ICOS selectively abrogates Treg-mediated functions and T1D protection and exacerbates disease in BDC2.5 mice. Moreover, T1D progression in BDC2.5 mice is associated with a decline in ICOS expression in and expansion and suppression by intra-islet Foxp3(+) Tregs. We further show that the ICOS(+) Tregs, in contrast to their ICOS(-) counterparts, are more sensitive to IL-2, a critical signal for their survival and functional stability. Lastly, the temporal loss in ICOS(+) Tregs is readily corrected by IL-2 therapy or protective Il2 gene variation. Overall, ICOS is critical for the homeostasis and functional stability of Foxp3(+) Tregs in prediabetic islets and maintenance of T1D protection.