To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.

Immunotherapy is among the most rapidly evolving treatment strategies in oncology. The therapeutic potential of immune-checkpoint inhibitors is exemplified by the recent hail of Food and Drug Administration (FDA) approvals for their use in various malignancies. Continued efforts to enhance outcomes with immunotherapy agents have led to the formulation of advanced treatment strategies. Recent evidence from pre-clinical studies evaluating immune-checkpoint inhibitors in various cancer cell-lines has suggested that combinatorial approaches may have superior survival outcomes compared to single-agent immunotherapy regimens. Preliminary trials assessing combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 immune-checkpoint inhibitors have documented considerable advantages in survival indices over single-agent immunotherapy. The therapeutic potential of combinatorial approaches is highlighted by the recent FDA approval of nivolumab plus ipilimumab for patients with advanced melanoma. Presently, dual-immune checkpoint inhibition with anti-programmed death receptor-1/programmed cell death receptor- ligand-1 (anti-PD-1/PD-L1) plus anti-cytotoxic T lymphocyte associated antigen-4 (anti-CTLA-4) monoclonal antibodies (MoAbs) is being evaluated for a wide range of tumor histologies. Furthermore, several ongoing clinical trials are investigating combination checkpoint inhibition in association with traditional treatment modalities such as chemotherapy, surgery, and radiation. In this review, we summarize the current landscape of combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 MoAbs for patients with melanoma and non-small cell lung cancer (NSCLC). We present a synopsis of the prospects for expanding the indications of dual immune-checkpoint inhibition therapy to a more diverse set of tumor histologies.

In recent years, reagents have been developed that specifically target signals critical for effective T cell activation and function. Manipulation of the CD28/CD80/86 and CD40/CD154 pathways has exhibited extraordinary efficacy, particularly when the pathways are blocked simultaneously. Despite the reported efficacy of anti-CD154 in rodents and higher models, its future clinical use is uncertain due to reported thromboembolic events in clinical trials. To circumvent this potential complication, we developed and evaluated a chimeric Ab targeting CD40 (Chi220, BMS-224819) as an alternative to CD154. Although Chi220 blocks CD154 binding, it also possesses partial agonist properties and weak stimulatory potential. The anti-CD40 was tested alone and in combination with a rationally designed, high affinity variant of CTLA4-Ig, LEA29Y (belatacept), in a nonhuman primate model of islet transplantation. Although either agent alone only modestly prolonged islet survival (Chi220 alone: 14, 16, and 84 days; LEA29Y alone: 58 and 60 days), their combination (LEA29Y and Chi220) dramatically facilitated long term survival (237, 237, 220, >185, and 172 days). We found that the effects of Chi220 treatment were not mediated solely through deletion of CD20-bearing cells and that the combined therapy did not significantly impair established antiviral immunity.

BACKGROUND

Severe sepsis is characterized by an initial hyper-inflammatory response that may progress to an immune-suppressed state associated with increased susceptibility to nosocomial infection. Analysis of samples obtained from patients who died of sepsis has identified expression of specific inhibitory receptors expressed on lymphocytes that are associated with cell exhaustion. The objective of this study was to prospectively determine the pattern of expression of these receptors and immune cell function in patients with acute sepsis.

METHODS

Twenty-four patients with severe sepsis were enrolled within 24 hours of the onset of sepsis, as were 12 age-matched healthy controls. Peripheral blood was obtained at enrollment and again seven days later. Immune cell subsets and receptor expression were extensively characterized by quantitative flow cytometry. Lymphocyte function was assayed by stimulated cytokine secretion and proliferation assays. Results were also correlated to clinical outcome.

RESULTS

At the onset of severe sepsis, patients had decreased circulating innate and adaptive immune cells and elevated lymphocyte expression of receptors associated with cell activation compared to controls. Samples analyzed seven days later demonstrated increased expression of the inhibitory receptors CTLA4, TIM-3 and LAG-3 on T lymphocytes accompanied by decreased expression of the IL-7 receptor. Functional assays revealed impaired secretion of interferon γ following stimulation in vitro, which was reversible by incubation overnight in fresh media. Impaired secretion of IFNγ correlated with death or development of secondary infection.

CONCLUSIONS

Lymphocytes from patients with acute sepsis upregulate expression of receptors associated with cell exhaustion, which may contribute to the immune suppressed state that occurs in protracted disease. Therapy that reverses T cell exhaustion may restore immune function in immunocompromised patients and improve survival in sepsis.

A significant fraction of patients with advanced prostate cancer treated with androgen deprivation therapy experience relapse with relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint blockade using antibodies against cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) or programmed cell death 1/programmed cell death 1 ligand 1 (PD1/PD-L1) generates durable therapeutic responses in a significant subset of patients across a variety of cancer types. However, mCRPC showed overwhelming de novo resistance to immune checkpoint blockade, motivating a search for targeted therapies that overcome this resistance. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumour immune evasion. The abundance of circulating MDSCs correlates with prostate-specific antigen levels and metastasis in patients with prostate cancer. Mouse models of prostate cancer show that MDSCs (CD11b+Gr1+) promote tumour initiation and progression. These observations prompted us to hypothesize that robust immunotherapy responses in mCRPC may be elicited by the combined actions of immune checkpoint blockade agents together with targeted agents that neutralize MDSCs yet preserve T-cell function. Here we develop a novel chimaeric mouse model of mCRPC to efficiently test combination therapies in an autochthonous setting. Combination of anti-CTLA4 and anti-PD1 engendered only modest efficacy. Targeted therapy against mCRPC-infiltrating MDSCs, using multikinase inhibitors such as cabozantinib and BEZ235, also showed minimal anti-tumour activities. Strikingly, primary and metastatic CRPC showed robust synergistic responses when immune checkpoint blockade was combined with MDSC-targeted therapy. Mechanistically, combination therapy efficacy stemmed from the upregulation of interleukin-1 receptor antagonist and suppression of MDSC-promoting cytokines secreted by prostate cancer cells. These observations illuminate a clinical path hypothesis for combining immune checkpoint blockade with MDSC-targeted therapies in the treatment of mCRPC.

Despite the success of anti-programmed cell death protein 1 (PD1), anti-PD1 ligand 1 (PDL1) and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) therapies in advanced cancer, a considerable proportion of patients remain unresponsive to these treatments (known as innate resistance). In addition, one-third of patients relapse after initial response (known as adaptive resistance), which suggests that multiple non-redundant immunosuppressive mechanisms coexist within the tumour microenvironment. A major immunosuppressive mechanism is the adenosinergic pathway, which now represents an attractive new therapeutic target for cancer therapy. Activation of this pathway occurs within hypoxic tumours, where extracellular adenosine exerts local suppression through tumour-intrinsic and host-mediated mechanisms. Preclinical studies in mice with adenosine receptor antagonists and antibodies have reported favourable antitumour immune responses with some definition of the mechanism of action. Currently, agents targeting the adenosinergic pathway are undergoing first-in-human clinical trials as single agents and in combination with anti-PD1 or anti-PDL1 therapies. In this Review, we describe the complex interplay of adenosine and adenosine receptors in the development of primary tumours and metastases and discuss the merits of targeting one or more components that compose the adenosinergic pathway. We also review the early clinical data relating to therapeutic agents inhibiting the adenosinergic pathway.

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.

Human breast tumors are infiltrated by memory CD4(+) T cells along with increased numbers of regulatory T cells (Treg) and plasmacytoid dendritic cells (pDC) that facilitate immune escape and correlate with poor prognosis. Here, we report that inducible costimulatory molecule (ICOS), a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, is expressed mostly by tumor-associated Treg in primary breast tumors. A large proportion of these ICOS(+) Treg were Ki67(+) and this evident proliferative expansion was found to rely on interactions with tumor-associated pDC. Indeed, tumor-associated Treg highly expanded in presence of pDC but failed to proliferate under CD3/CD28 signal. In vitro experiments revealed that the addition of a neutralizing anti-ICOS antibody blocked pDC-induced Treg expansion and interleukin-10 secretion by memory CD4(+) T cells, establishing a pivotal role for ICOS in this process. Supporting these findings, the presence of ICOS(+) cells in clinical specimens of breast cancer correlated with a poor prognosis. Together, our results highlight an important relationship between Treg and pDC in breast tumors, and show that ICOS/ICOS-L interaction is a central event in immunosuppression of tumor-associated memory CD4(+) T cells. These findings strongly rationalize antibody-mediated ICOS blockade as a powerful clinical strategy to correct immune escape and promote therapeutic responses in breast cancer.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

The rejection of the transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD28. CTLA4-Ig is a fusion protein that has previously been shown to block the CD28-mediated costimulatory signal and inhibit immune responses in vitro and in vivo. In this report we show that treatment of the C3H/He recipient of a BALB/c vascularized cardiac allograft with a 12-day course of CTLA4-Ig produced indefinite graft survival >> 100 days) in the majority of recipients. In addition, these recipients demonstrated donor-specific transplantation tolerance when tested with donor-specific (BALB/c) and third-party (C57BL/10) skin grafts. These results demonstrate that CTLA4-Ig can induce transplantation tolerance in the adult murine cardiac allograft model.

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2(d)) mouse bone marrow progenitors in IL-10 and TGF-beta, then stimulated the cells with LPS. These "alternatively activated" (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

T cell activation requires at least two distinct signals, including signaling via the Ag-specific TCR and a costimulatory pathway. The best characterized costimulatory pathway involves the CD28 molecule, which is expressed constitutively on T cells and binds the family of B7 counter-receptors on APCs. Inhibition of this costimulatory pathway prevents T cell activation and can lead to long-term T cell unresponsiveness or anergy. In contrast, CTLA4, which is homologous to CD28, has been shown to be a negative regulator of T cell activation. The CTLA4 molecule is not expressed on resting T cells, but is induced after the initial steps of T cell activation. To address the regulation of CTLA4 expression, we have analyzed CTLA4 at the level of cell surface expression, mRNA, rate of transcription, and rate of decay of message. Nuclear runoff results show an increase in the rate of transcription following T cell activation. Our analyses of non-T cells, including B cells, mastocytoma, and fibroblasts, by Northern blot analysis detect only T cell expression of CTLA4. Reporter gene analysis indicates that 335 bp of upstream CTLA4 sequence are sufficient to control inducibility. We have identified important regulatory regions that control inducible and cell-specific CTLA4 expression. These results also suggest that both positive and negative response elements modulate the transcriptional regulation of CTLA4 gene expression. Understanding the regulation of CTLA4 should provide insight into the regulation of T cell activation at the molecular level.

Activation of the T-cell co-receptor cytotoxic T-lymphocyte antigen 4 (CTLA4) has a pivotal role in adjusting the threshold for T-cell activation and in preventing autoimmunity and massive tissue infiltration by T cells. Although many mechanistic models have been postulated, no single model has yet accounted for its overall function. In this Opinion article, I outline the strengths and weaknesses of the current models, and present a new 'reverse stop-signal model' to account for CTLA4 function.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized. In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors. We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation. Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB. Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data. Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.

Hypothyroidism is the most common thyroid disorder, affecting about 5% of the general population. Here we present the current largest genome-wide association study of hypothyroidism, in 3,736 cases and 35,546 controls. Hypothyroidism was assessed via web-based questionnaires. We identify five genome-wide significant associations, three of which are well known to be involved in a large spectrum of autoimmune diseases: rs6679677 near PTPN22, rs3184504 in SH2B3, and rs2517532 in the HLA class I region (p-values 2.8·10(-13), 2.6·10(-12), and 1.3·10(-8), respectively). We also report associations with rs4915077 near VAV3 (p-value 7.5·10(-10)) and rs925489 near FOXE1 (p value 2.4·10(-19)). VAV3 is involved in immune function, and FOXE1 and PTPN22 have previously been associated with hypothyroidism. Although the HLA class I region and SH2B3 have previously been linked with a number of autoimmune diseases, this is the first report of their association with thyroid disease. The VAV3 association is also novel. We also show suggestive evidence of association for hypothyroidism with a SNP in the HLA class II region (independent of the other HLA association) as well as SNPs in CAPZB, PDE8B, and CTLA4. CAPZB and PDE8B have been linked to TSH levels and CTLA4 to a variety of autoimmune diseases. These results suggest heterogeneity in the genetic etiology of hypothyroidism, implicating genes involved in both autoimmune disorders and thyroid function. Using a genetic risk profile score based on the top association from each of the five genome-wide significant regions in our study, the relative risk between the highest and lowest deciles of genetic risk is 2.0.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.

BACKGROUND

Autoimmune mechanisms are thought to have a major role in the pathogenesis of multiple sclerosis. We aimed to identify coexisting autoimmune phenotypes in patients with multiple sclerosis from families with several members with the disease and in their first-degree relatives.

METHODS

A total of 176 families (386 individuals and 1107 first-degree relatives) were characterised for a history of other autoimmune disorders. Family-based or case-control analyses were done to assess the association of cytotoxic T-lymphocyte-antigen 4 (CTLA4) and protein tyrosine phosphatase (PTPN22) variants with susceptibility to multiple sclerosis.

RESULTS

46 (26%) index cases reported at least one coexisting autoimmune disorder. The most common were Hashimoto thyroiditis (10%), psoriasis (6%), inflammatory bowel disease (3%), and rheumatoid arthritis (2%). 112 (64%) families with a history of multiple sclerosis reported autoimmune disorders (excluding multiple sclerosis) in one or more first-degree relatives, whereas 64 (36%) families reported no history of autoimmunity. Similar to index cases, Hashimoto thyroiditis, psoriasis, and inflammatory bowel disease were also the most common disorders occurring in family members. A common variant within CTLA4 was strongly associated with multiple sclerosis in families who had other autoimmune diseases (p=0.009) but not in families without a history of other autoimmune disorders (p=0.90).

CONCLUSIONS

The presence of various immune disorders in families with several members with multiple sclerosis suggests that the disease might arise on a background of a generalised susceptibility to autoimmunity. This distinct multiple-sclerosis phenotype, defined by its association with other autoimmune diseases, segregates with specific genotypes that could underlie the common susceptibility.

Common variable immunodeficiency (CVID) is a primary antibody deficiency characterised by hypogammaglobulinaemia, impaired production of specific antibodies after immunisation and increased susceptibility to infections. CVID shows a considerable phenotypical and genetic heterogeneity. In contrast to many other primary immunodeficiencies, monogenic forms count for only 2-10% of patients with CVID. Genes that have been implicated in monogenic CVID include ICOS, TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFSF12 (TWEAK), CD19, CD81, CR2 (CD21), MS4A1 (CD20), TNFRSF7 (CD27), IL21, IL21R, LRBA, CTLA4, PRKCD, PLCG2, NFKB1, NFKB2, PIK3CD, PIK3R1, VAV1, RAC2, BLK, IKZF1 (IKAROS) and IRF2BP2 With the increasing number of disease genes identified in CVID, it has become clear that CVID is an umbrella diagnosis and that many of these genetic defects cause distinct disease entities. Moreover, there is accumulating evidence that at least a subgroup of patients with CVID has a complex rather than a monogenic inheritance. This review aims to discuss current knowledge regarding the molecular genetic basis of CVID with an emphasis on the relationship with the clinical and immunological phenotype.

Many advances in the treatment of cancer have been driven by the development of targeted therapies that inhibit oncogenic signaling pathways and tumor-associated angiogenesis, as well as by the recent development of therapies that activate a patient's immune system to unleash antitumor immunity. Some targeted therapies can have effects on host immune responses, in addition to their effects on tumor biology. These immune-modulating effects, such as increasing tumor antigenicity or promoting intratumoral T cell infiltration, provide a rationale for combining these targeted therapies with immunotherapies. Here, we discuss the immune-modulating effects of targeted therapies against the MAPK and VEGF signaling pathways, and how they may synergize with immunomodulatory antibodies that target PD1/PDL1 and CTLA4. We critically examine the rationale in support of these combinations in light of the current understanding of the underlying mechanisms of action of these therapies. We also discuss the available preclinical and clinical data for these combination approaches and their implications regarding mechanisms of action. Insights from these studies provide a framework for considering additional combinations of targeted therapies and immunotherapies for the treatment of cancer.

Naive T cells require interleukin 4 (IL-4) to develop into IL-4-producing T cells and IL-4 blocks development of such cells into interferon gamma (IFN-gamma) producers. Prior studies in accessory cell-independent priming systems using antireceptor antibodies as agonists have demonstrated that IL-2 is also necessary for the development of IL-4-producing cells under these culture conditions. Here we have examined the role of IL-2 and the CD28 costimulation pathway in priming for IL-4 and IFN-gamma production using a more physiologic model. This involved antigen presentation by accessory cells to naive CD4+ T cells from transgenic mice whose cells express a T cell receptor (TCR) specific for a cytochrome c peptide in association with I-Ek. With splenic antigen-presenting cells (APCs), inhibition of CD28 costimulation by the fusion protein CTLA4-immunoglobulin (Ig) blocked effective priming. Similarly, transfected fibroblasts expressing both MHC class II and the CD28 ligand B7 could prime for IL-4 production and such priming also was blocked by CTLA4-Ig. However, APCs deficient in CD28 ligands also could prime TCR transgenic T cells to become IL-4 producers if an exogenous source of IL-2, as well as IL-4, was provided, and the inhibition of priming seen with splenic or transfected fibroblast APCs in the presence of CTLA4-Ig could be reversed by addition of IL-2. Likewise, priming for IFN-gamma production could be blocked by CTLA4-Ig and reversed by IL-2. Thus, we conclude that IL-2 plays a critical role in priming naive CD4+ T cells to become IL-4 or IFN-gamma producers. Engagement of the CD28 pathway, although normally important in such priming, is unnecessary in the presence of exogenous IL-2.

In pregnancy, the decidua is infiltrated by leukocytes promoting fetal development without causing immunological rejection. Murine regulatory T (Treg) cells are known to be important immune regulators at this site. The aim of the study was to characterize the phenotype and origin of Treg cells and determine the quantitative relationship between Treg, T-helper type 1 (T(H)1), T(H)2, and T(H)17 cells in first-trimester human decidua. Blood and decidual CD4(+) T cells from 18 healthy first-trimester pregnant women were analyzed for expression of Treg-cell markers (CD25, FOXP3, CD127, CTLA4, and human leukocyte antigen-DR [HLA-DR]), chemokine receptors (CCR4, CCR6, and CXCR3), and the proliferation antigen MKI67 by six-color flow cytometry. Treg cells were significantly enriched in decidua and displayed a more homogenous suppressive phenotype with more frequent expression of FOXP3, HLA-DR, and CTLA4 than in blood. More decidual Treg cells expressed MKI67, possibly explaining their enrichment at the fetal-maternal interface. Using chemokine receptor expression profiles of CCR4, CCR6, and CXCR3 as markers for T(H)1, T(H)2, and T(H)17 cells, we showed that T(H)17 cells were nearly absent in decidua, whereas T(H)2-cell frequencies were similar in blood and decidua. CCR6(+) T(H)1 cells, reported to secrete high levels of interferon gamma (IFNG), were fewer, whereas the moderately IFNG-secreting CCR6(-) T(H)1 cells were more frequent in decidua compared with blood. Our results point toward local expansion of Treg cells and low occurrence of T(H)17 cells. Furthermore, local, moderate T(H)1 activity seems to be a part of normal early pregnancy, consistent with a mild inflammatory environment controlled by Treg cells.

There is an increasing amount of knowledge on the functional properties of regulatory T cells (Treg) in the adult immune system, but data on the generation and function of these cells during human embryonic development are scarce. In this study, we show that in the fetal thymus, double-positive cells initiate expression of CD25, GITR, CTLA4 and CD122 during their transition from the CD27- to the CD27+ stage. Moreover, CD4+CD25+ fetal thymocytes already have the potential to suppress proliferation of CD25- cells. After leaving the thymus, FoxP3+CD4+CD25+ Treg enter the fetal lymph nodes and spleen, where they acquire a primed/memory phenotype. A model is proposed for the development of human fetal Treg that encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Upon activation, FoxP3+CD4+CD25+ Treg suppress potentially deleterious responses by autoreactive lymphocytes and maintain homeostasis within the developing fetus.

CD4+ T regulatory cells (Treg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of Treg or conventional CD4+FoxP3- T cells (Tconv) from mouse lymphoid organs and human blood. Treg and Tconv pools showed areas of overlap, as resting 'furtive' Tregs with overall similarity to Tconvs or as a convergence of activated states. All Tregs expressed a small core of FoxP3-dependent transcripts, onto which additional programs were added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasiconstant, inhibitory cytokines being more sparsely distributed. TCR signal intensity did not affect resting/activated Treg proportions but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations of Treg heterogeneity.