Follicular helper T cell profiles predict response to costimulation blockade in type 1 diabetes.

Follicular helper T (TFH) cells are implicated in type 1 diabetes (T1D), and their development has been linked to CD28 costimulation. We tested whether TFH cells were decreased by costimulation blockade using the CTLA-4-immunoglobulin (Ig) fusion protein (abatacept) in a mouse model of diabetes and in individuals with new-onset T1D. Unbiased bioinformatics analysis identified that inducible costimulatory molecule (ICOS)+ TFH cells and other ICOS+ populations, including peripheral helper T cells, were highly sensitive to costimulation blockade. We used pretreatment TFH profiles to derive a model that could predict clinical response to abatacept in individuals with T1D. Using two independent approaches, we demonstrated that higher frequencies of ICOS+ TFH cells at baseline were associated with a poor clinical response following abatacept administration. Therefore, TFH analysis may represent a new stratification tool, permitting the identification of individuals most likely to benefit from costimulation blockade.

Novel TCR-based biologics: mobilising T cells to warm 'cold' tumours.

Immunotherapeutic strategies have revolutionised cancer therapy in recent years, bringing meaningful improvements in outcomes for patients with previously intractable conditions. These successes have, however, been largely limited to certain types of liquid tumours and a small subset of solid tumours that are known to be particularly immunogenic. Broadening these advances across the majority of tumour indications, which are characterised by an immune-excluded, immune-deserted or immune-suppressed ('cold') phenotype, will require alternative approaches that are able to specifically address this unique biological environment. Several newer therapeutic modalities, including adoptive cell therapy and T cell redirecting bispecific molecules, are considered to hold particular promise and are being investigated in early phase clinical trials across various solid tumour indications. ImmTAC molecules are a novel class of T cell redirecting bispecific biologics that exploit TCR-based targeting of tumour cells; providing potent and highly specific access to the vast landscape of intracellular targets. The first of these reagents to reach the clinic, tebentafusp (IMCgp100), has generated demonstrable clinical efficacy in an immunologically cold solid tumour with a high unmet need. Here, we highlight the key elements of the ImmTAC platform that make it ideally positioned to overcome the cold tumour microenvironment in an off-the-shelf format.

Helicobacter pylori-Mediated Protection from Allergy Is Associated with IL-10-Secreting Peripheral Blood Regulatory T Cells.

Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients' colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4(+)CD25(hi) Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA(+) strains or those expressing the more active i1 form of VacA. The systemic IL-10(+) Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.

T-Regulatory Cells and Inflammatory and Inhibitory Cytokines in Malawian Children Residing in an Area of High and an Area of Low Malaria Transmission During Acute Uncomplicated Malaria and in Convalescence.

BACKGROUND: Malaria still infects many Malawian children, and it is a cause of death in some of them. Regulatory T cells (Tregs) help in negating immune-related pathology, it but can also favor multiplication of malaria parasites. The question remains whether children recovering from uncomplicated malaria (UCM) have higher Tregs and interleukin (IL)-10 levels in convalescence. METHODS: We recruited children between the ages of 6 and 60 months presenting with acute UCM in Blantyre (low transmission area) and Chikwawa (high transmission area). We observed the children after 1 month and 3 months and analyzed their blood samples for parasitemia, lymphocyte subsets, and levels of the cytokines interferon (IFN)-γ, IL-10, and transforming growth factor (TGF)-ß. Blood samples from age-matched controls were also analyzed for the same parameters. RESULTS: Compared with controls, acute UCM was associated with mild lymphopenia, splenomegaly, and high levels of IFN-γ, tumor necrosis factor-α, and IL-10, which normalized in convalescence. In Chikwawa, Treg counts were significantly (P < .0001) higher in convalescence compared with acute disease, whereas in Blantyre, these were as low as in healthy controls both during acute disease and in convalescence. Blantyre had a higher percentage of parasiteamic children (15% versus 12%) in convalescence compared with Chikwawa, but none of these developed symptomatic malaria during the study duration. Concentrations of TGF-ß were higher at time points for the study participants and in controls from Blantyre compared with those recruited in Chikwawa. CONCLUSIONS: The high transmission area was associated with high Tregs counts and IL-10 concentrations in convalescence, which could have an effect on parasite clearance. We recommend that children recovering from UCM, especially those from high transmission area, should sleep under insecticide-treated nets, be screened for parasitemia, and a provision of antimalarial prophylaxis should be considered.

Follicular helper T cell signature in type 1 diabetes.

The strong genetic association between particular HLA alleles and type 1 diabetes (T1D) indicates a key role for CD4+ T cells in disease; however, the differentiation state of the responsible T cells is unclear. T cell differentiation originally was considered a dichotomy between Th1 and Th2 cells, with Th1 cells deemed culpable for autoimmune islet destruction. Now, multiple additional T cell differentiation fates are recognized with distinct roles. Here, we used a transgenic mouse model of diabetes to probe the gene expression profile of islet-specific T cells by microarray and identified a clear follicular helper T (Tfh) cell differentiation signature. Introduction of T cells with a Tfh cell phenotype from diabetic animals efficiently transferred diabetes to recipient animals. Furthermore, memory T cells from patients with T1D expressed elevated levels of Tfh cell markers, including CXCR5, ICOS, PDCD1, BCL6, and IL21. Defects in the IL-2 pathway are associated with T1D, and IL-2 inhibits Tfh cell differentiation in mice. Consistent with these previous observations, we found that IL-2 inhibited human Tfh cell differentiation and identified a relationship between IL-2 sensitivity in T cells from patients with T1D and acquisition of a Tfh cell phenotype. Together, these findings identify a Tfh cell signature in autoimmune diabetes and suggest that this population could be used as a biomarker and potentially targeted for T1D interventions.

CTLA-4 controls follicular helper T-cell differentiation by regulating the strength of CD28 engagement.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4-deficient mice show spontaneous T-follicular helper (T(FH)) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti-CTLA-4 antibody in wild-type mice is sufficient to elicit T(FH) generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for T(FH) differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered T(FH) generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of T(FH) differentiation by graded control of CD28 engagement.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

IL-21 promotes CD4 T cell responses by phosphatidylinositol 3-kinase-dependent upregulation of CD86 on B cells.

The cytokine IL-21 is a potent immune modulator with diverse mechanisms of action on multiple cell types. IL-21 is in clinical use to promote tumor rejection and is an emerging target for neutralization in the setting of autoimmunity. Despite its clinical potential, the biological actions of IL-21 are not yet fully understood and the full range of effects of this pleiotropic cytokine are still being uncovered. In this study, we identify a novel role for IL-21 as an inducer of the costimulatory ligand CD86 on B lymphocytes. CD86 provides critical signals through T cell-expressed CD28 that promote T cell activation in response to Ag engagement. Expression levels of CD86 are tightly regulated in vivo, being actively decreased by regulatory T cells and increased in response to pathogen-derived signals. In this study, we demonstrate that IL-21 can trigger potent and sustained CD86 upregulation through a STAT3 and PI3K-dependent mechanism. We show that elevated CD86 expression has functional consequences for the magnitude of CD4 T cell responses both in vitro and in vivo. These data pinpoint CD86 upregulation as an additional mechanism by which IL-21 can elicit immunomodulatory effects.

Cutting edge: cell-extrinsic immune regulation by CTLA-4 expressed on conventional T cells.

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. Although early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinsic, has been an ongoing paradox in the field. Although CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. In this study, we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4(+/+) T cells, from DO11/rag(-/-) mice that lack regulatory T cells, were able to regulate the response of CTLA-4(-/-) T cells in cotransfer experiments. This observation provides a potential resolution to the above paradox and suggests CTLA-4 function on both Tconv and regulatory T cells can be achieved through cell-extrinsic mechanisms.

IL-7 receptor blockade reverses autoimmune diabetes by promoting inhibition of effector/memory T cells.

To protect the organism against autoimmunity, self-reactive effector/memory T cells (T(E/M)) are controlled by cell-intrinsic and -extrinsic regulatory mechanisms. However, how some T(E/M) cells escape regulation and cause autoimmune disease is currently not understood. Here we show that blocking IL-7 receptor-α (IL-7Rα) with monoclonal antibodies in nonobese diabetic (NOD) mice prevented autoimmune diabetes and, importantly, reversed disease in new-onset diabetic mice. Surprisingly, IL-7-deprived diabetogenic T(E/M) cells remained present in the treated animals but showed increased expression of the inhibitory receptor Programmed Death 1 (PD-1) and reduced IFN-γ production. Conversely, IL-7 suppressed PD-1 expression on activated T cells in vitro. Adoptive transfer experiments revealed that T(E/M) cells from anti-IL-7Rα-treated mice had lost their pathogenic potential, indicating that absence of IL-7 signals induces cell-intrinsic tolerance. In addition to this mechanism, IL-7Rα blockade altered the balance of regulatory T cells and T(E/M) cells, hence promoting cell-extrinsic regulation and further increasing the threshold for diabetogenic T-cell activation. Our data demonstrate that IL-7 contributes to the pathogenesis of autoimmune diabetes by enabling T(E/M) cells to remain in a functionally competent state and suggest IL-7Rα blockade as a therapy for established T-cell-dependent autoimmune diseases.

IL-21 inhibits T cell IL-2 production and impairs Treg homeostasis.

Modulation of regulatory T cell (Treg) suppression has important implications for vaccine development, the effectiveness of tumor surveillance, and the emergence of autoimmunity. We have previously shown that the cytokine IL-21 can counteract Treg suppression. However, whether this reflects an effect of IL-21 on Treg, conventional T cells, or antigen-presenting cells is not known. Here we have used lymphocyte populations from IL-21R-deficient mice to pinpoint which cell type needs to be targeted by IL-21 for Treg suppression to be overcome. We show that IL-21 counteracts suppression by acting on conventional T cells and that this is associated with inhibition of IL-2 production. Despite the lack of IL-2, conventional T-cell responses proceed unimpaired because IL-21 can substitute for IL-2 as a T cell growth factor. However, IL-21 is unable to substitute for IL-2 in supporting the Treg compartment. Thus, IL-21 signaling in conventional T cells indirectly impacts Treg homeostasis by decreasing IL-2 availability. These data demonstrate that IL-21 and IL-2 can have overlapping roles in promoting conventional T-cell responses but play distinct roles in controlling Treg homeostasis and function. The data also suggest a new paradigm whereby cytokines can promote immunity by inhibiting IL-2.

Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation.

CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface transmembrane ligands in that CTLA-4 is rapidly internalized from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analyzed in detail the internalization, recycling, and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalized from the plasma membrane in a clathrin- and dynamin-dependent manner driven by the well characterized YVKM trafficking motif. Furthermore, we show that once internalized, CTLA-4 co-localizes with markers of recycling endosomes and is recycled to the plasma membrane. Although we observed limited co-localization of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression; however, this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalized in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane; however, CTLA-4 endocytosis continues and is not stabilized during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.

Immune regulation by CTLA-4--relevance to autoimmune diabetes in a transgenic mouse model.

BACKGROUND: The importance of cytotoxic T lymphocyte antigen-4 (CTLA-4) in immune regulation is unquestioned, yet a precise understanding of which cells express it, and how it mediates immune inhibitory function, is lacking. Regulatory T cells are known to constitutively express CTLA-4 intracellularly, whereas conventional T cells require activation to trigger CTLA-4 expression. However comparative analysis of CTLA-4 trafficking in regulatory and conventional subsets has not been performed. METHODS: Here we assess CTLA-4 expression in antigen-specific conventional and regulatory cells responding to immunizing antigen in vivo and analyse the membrane trafficking of CTLA-4 using an in vitro recycling assay. We assess the expression of CTLA-4 on Treg infiltrating the pancreas in the DO11×RIP-mOVA diabetes model and the role of CTLA-4 in Treg function. RESULTS: Regulatory T cells show an enhanced capacity to traffic CTLA-4 following stimulation compared with conventional T cells. Treg infiltrating the pancreas in DO11×RIP-mOVA mice show high expression of CTLA-4. Furthermore CTLA-4-deficient Treg fail to control diabetes in an adoptive transfer model of diabetes, even in situations where they outnumber the disease-inducing conventional T cells. CONCLUSIONS: These data show that not only do regulatory T cells express higher levels of intracellular CTLA-4 than conventional T cells, but they also show an increased capacity to traffic CTLA-4 to the cell surface following stimulation. CTLA-4 is strongly upregulated in regulatory T cells infiltrating the target tissue in a mouse model of type 1 diabetes and expression of this protein is critical for effective regulation.

B1 cells promote pancreas infiltration by autoreactive T cells.

The entry of autoreactive T cells into the pancreas is a critical checkpoint in the development of autoimmune diabetes. In this study, we identify a role for B1 cells in this process using the DO11 x RIP-mOVA mouse model. In transgenic mice with islet-specific T cells, but no B cells, T cells are primed in the pancreatic lymph node but fail to enter the pancreas. Reconstitution of the B1 cell population by adoptive transfer permits extensive T cell pancreas infiltration. Reconstituted B1 cells traffic to the pancreas and modify expression of adhesion molecules on pancreatic vasculature, notably VCAM-1. Despite substantial pancreas infiltration, islet destruction is minimal unless regulatory T cells are depleted. These data identify a role for B1 cells in permitting circulating islet-specific T cells to access their Ag-bearing tissue and emphasize the existence of multiple checkpoints to regulate autoimmune disease.

Multiple cytokines regulate the NK gene complex-encoded receptor repertoire of mature NK cells and T cells.

Mature NK cells comprise a highly diverse population of lymphocytes that express different permutations of receptors to facilitate recognition of diseased cells and perhaps pathogens themselves. Many of these receptors, such as those belonging to the NKRP1, NKG2, and Ly49 families are encoded in the NK gene complex (NKC). It is generally thought that these NKC-encoded receptors are acquired by a poorly understood stochastic mechanism, which operates exclusively during NK cell development, and that following maturation the repertoire is fixed. However, we report a series of observations that demonstrates that the mature NK cell repertoire in mice can in fact be radically remodeled by multiple cytokines. Thus, both IL-2 and IL-15 selectively induce the de novo expression of Ly49E on the majority of mature NK cells. By contrast, IL-4 not only blocks this IL-2-induced acquisition of Ly49E, but reduces the proportion of mature NK cells that expresses pre-existing Ly49 receptors and abrogates the expression of NKG2 receptors while leaving the expression of several NKRP1 receptors unaltered. IL-21 also abrogates NKG2 expression on mature NK cells and selectively down-regulates Ly49F. IL-4 and IL-21 additionally cause dramatic and selective alterations in the NKC-encoded receptor repertoire of IL-2-activated T cells but these are quite different to the changes induced on NK cells. Collectively these findings reveal an unexpected aspect of NKC receptor expression that has important implications for our understanding of the function of these receptors and of the genetic mechanisms that control their expression.