Targeting TFH cells in human diseases and vaccination: rationale and practice.

The identification of CD4+ T cells localizing to B cell follicles has revolutionized the knowledge of how humoral immunity is generated. Follicular helper T (TFH) cells support germinal center (GC) formation and regulate clonal selection and differentiation of memory and antibody-secreting B cells, thus controlling antibody affinity maturation and memory. TFH cells are essential in sustaining protective antibody responses necessary for pathogen clearance in infection and vaccine-mediated protection. Conversely, aberrant and excessive TFH cell responses mediate and sustain pathogenic antibodies to autoantigens, alloantigens, and allergens, facilitate lymphomagenesis, and even harbor viral reservoirs. TFH cell generation and function are determined by T cell antigen receptor (TCR), costimulation, and cytokine signals, together with specific metabolic and survival mechanisms. Such regulation is crucial to understanding disease pathogenesis and informing the development of emerging therapies for disease or novel approaches to boost vaccine efficacy.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

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.

Contemplating autoimmunity in the Aegean islands.

The Greek island of Crete became host to lively discussions on immunoregulation as experts from around the world gathered for the 7th Aegean Conference on Autoimmunity in September 2015.

Dimers Aren't Forever: CD80 Breaks up with PD-L1.

Targeting the CTLA-4 and PD-1 "checkpoints" is an effective treatment for a number of cancers. In this issue of Immunity, Hui et al. reveal that interaction between a CTLA-4 ligand, CD80, and its counterpart in the PD-1 pathway, PD-L1, affects both PD-1 and CTLA-4 function, raising new questions about the biological effects of using checkpoint inhibitors alone and in combination.

T-cell help in the germinal center: homing in on the role of IL-21.

Interleukin 21 (IL-21) is a pleiotropic cytokine that is overproduced in multiple autoimmune settings. Provision of IL-21 from follicular helper T cells is an important component of T-cell help within germinal centers (GC), and the last few years have seen a resurgence of interest in IL-21 biology in the context of the GC environment. While it has been more than a decade since T cell-derived IL-21 was found to upregulate B-cell expression of the GC master transcription factor B-cell lymphoma 6 (Bcl-6) and to promote GC expansion, several recent studies have collectively delivered significant new insights into how this cytokine shapes GC B-cell selection, proliferation, and fate choice. It is now clear that IL-21 plays an important role in GC zonal polarization by contributing to light zone GC B-cell positive selection for dark zone entry as well as by promoting cyclin D3-dependent dark zone inertial cycling. While it has been established that IL-21 can contribute to the modulation of GC output by aiding the generation of antibody-secreting cells (ASC), recent studies have now revealed how IL-21 signal strength shapes the fate choice between GC cycle re-entry and ASC differentiation in vivo. Both provision of IL-21 and sensitivity to this cytokine are finely tuned within the GC environment, and dysregulation of this pathway in autoimmune settings could alter the threshold for germinal center B-cell selection and differentiation, potentially promoting autoreactive B-cell responses.

Type 1 diabetes.

Type 1 diabetes is a chronic disease caused by autoimmune destruction of pancreatic ß cells. Individuals with type 1 diabetes are reliant on insulin for survival. Despite enhanced knowledge related to the pathophysiology of the disease, including interactions between genetic, immune, and environmental contributions, and major strides in treatment and management, disease burden remains high. Studies aimed at blocking the immune attack on ß cells in people at risk or individuals with very early onset type 1 diabetes show promise in preserving endogenous insulin production. This Seminar will review the field of type 1 diabetes, highlighting recent progress within the past 5 years, challenges to clinical care, and future directions in research, including strategies to prevent, manage, and cure the disease.

Maintaining a competitive edge: new rules for peripheral T cell homeostasis.

The contraction of T cell populations after immune responses is poorly understood. In this issue of Immunity, Singh et al. show that "deletor" T cells regulate the frequency of antigen-specific T cells by competing for shared subthreshold ligands.

Therapeutic Myeloperoxidase Inhibition Attenuates Neutrophil Activation, ANCA-Mediated Endothelial Damage, and Crescentic GN.

BACKGROUND: Myeloperoxidase released after neutrophil and monocyte activation can generate reactive oxygen species, leading to host tissue damage. Extracellular glomerular myeloperoxidase deposition, seen in ANCA-associated vasculitis, may enhance crescentic GN through antigen-specific T and B cell activation. Myeloperoxidase-deficient animals have attenuated GN early on, but augmented T cell responses. We investigated the effect of myeloperoxidase inhibition, using the myeloperoxidase inhibitor AZM198, to understand its potential role in treating crescentic GN. METHODS: We evaluated renal biopsy samples from patients with various forms of crescentic GN for myeloperoxidase and neutrophils, measured serum myeloperoxidase concentration in patients with ANCA-associated vasculitis and controls, and assessed neutrophil extracellular trap formation, reactive oxygen species production, and neutrophil degranulation in ANCA-stimulated neutrophils in the absence and presence of AZM198. We also tested the effect of AZM198 on ANCA-stimulated neutrophil-mediated endothelial cell damage in vitro, as well as on crescentic GN severity and antigen-specific T cell reactivity in the murine model of nephrotoxic nephritis. RESULTS: All biopsy specimens with crescentic GN had extracellular glomerular myeloperoxidase deposition that correlated significantly with eGFR and crescent formation. In vitro, AZM198 led to a significant reduction in neutrophil extracellular trap formation, reactive oxygen species production, and released human neutrophil peptide levels, and attenuated neutrophil-mediated endothelial cell damage. In vivo, delayed AZM198 treatment significantly reduced proteinuria, glomerular thrombosis, serum creatinine, and glomerular macrophage infiltration, without increasing adaptive T cell responses. CONCLUSIONS: Myeloperoxidase inhibition reduced neutrophil degranulation and neutrophil-mediated endothelial cell damage in patients with ANCA-associated vasculitis. In preclinical crescentic GN, delayed myeloperoxidase inhibition suppressed kidney damage without augmenting adaptive immune responses, suggesting it might offer a novel adjunctive therapeutic approach in crescentic GN.

Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations.

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches.

Characterization of CTLA4 Trafficking and Implications for Its Function.

CTLA4 is an essential negative regulator of T-cell immune responses and a key checkpoint regulating autoimmunity and antitumor responses. Genetic mutations resulting in quantitative defects in the CTLA4 pathway are also associated with the development of immune dysregulation syndromes in humans. It has been proposed that CTLA4 functions to remove its ligands CD80 and CD86 from opposing cells by a process known as transendocytosis. A quantitative characterization of CTLA4 synthesis, endocytosis, degradation, and recycling and how these affect its function is currently lacking. In a combined in vitro and in silico study, we developed a mathematical model and identified these trafficking parameters. Our model predicts optimal ligand removal in an intermediate affinity range. The intracellular CTLA4 pool as well as fast internalization, recovery of free CTLA4 from internalized complexes, and recycling is critical for sustained functionality. CD80-CTLA4 interactions are predicted to dominate over CD86-CTLA4. Implications of these findings in the context of control of antigen-presenting cells by regulatory T cells and of pathologic genetic deficiencies are discussed. The presented mathematical model can be reused in the community beyond these questions to better understand other trafficking receptors and study the impact of CTLA4 targeting drugs.

Study of an extended family with CTLA-4 deficiency suggests a CD28/CTLA-4 independent mechanism responsible for differences in disease manifestations and severity.

The CTLA-4 checkpoint regulates the activation of T cells. Individuals with heterozygous mutations in CTLA-4 have a complex phenotype typically characterized by antibody deficiency alongside variable autoimmunity. Despite severe disease in some individuals, others remain largely unaffected with reasons for this variation unknown. We studied a large family carrying a single point mutation in CTLA-4 leading to an amino acid change R75W and compared both unaffected with affected individuals. We measured a variety of features pertaining to T cell and CTLA-4 biology and observed that at the cellular level there was complete penetrance of CTLA-4 mutations. Accordingly, unaffected individuals were indistinguishable from those with disease in terms of level of CTLA-4 expression, percentage of Treg, upregulation of CTLA-4 upon stimulation and proliferation of CD4 T cells. We conclude that the wide variation in disease phenotype is influenced by immune variation outside of CTLA-4 biology.

CTLA-4 and Autoimmunity: New Twists in the Tale.

CTLA-4 has long been associated with control of autoimmunity. A recent study by Sharpe and colleagues explores this relationship in a model that enables conditional deletion of CTLA-4 in adult mice, with some surprising new conclusions.

Confusing signals: recent progress in CTLA-4 biology.

The mechanism of action of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) remains surprisingly unclear. Regulatory T (Treg) cells can use CTLA-4 to elicit suppression; however, CTLA-4 also operates in conventional T cells, reputedly by triggering inhibitory signals. Recently, interactions mediated via the CTLA-4 cytoplasmic domain have been shown to preferentially affect Treg cells, yet other evidence suggests that the extracellular domain of CTLA-4 is sufficient to elicit suppression. Here, we discuss these paradoxical findings in the context of CTLA-4-mediated ligand regulation. We propose that the function of CTLA-4 cytoplasmic domain is not to transmit inhibitory signals but to precisely control the turnover, cellular location, and membrane delivery of CTLA-4 to facilitate its central function: regulating the access of CD28 to their shared ligands.

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.

Homeostasis and function of regulatory T cells (Tregs) in vivo: lessons from TCR-transgenic Tregs.

The identification of CD25 and subsequently Forkhead box protein 3 (Foxp3) as markers for regulatory T cells (Tregs) has revolutionized our ability to explore this population experimentally. In a similar vein, our understanding of antigen-specific Treg responses in vivo owes much to the fortuitous generation of T-cell receptor (TCR)-transgenic Tregs. This has permitted tracking of Tregs with a defined specificity in vivo, facilitating analysis of how encounter with cognate antigen shapes Treg homeostasis and function. Here, we review the key lessons learned from a decade of analysis of TCR-transgenic Tregs and set this in the broader context of general progress in the field. Use of TCR-transgenic Tregs has led to an appreciation that Tregs are a highly dynamic proliferative population in vivo, rather than an anergic population as they were initially portrayed. It is now clear that Treg homeostasis is positively regulated by encounter with self-antigen expressed on peripheral tissues, which is likely to be relevant to the phenomenon of peripheral repertoire reshaping that has been described for Tregs and the observation that the Treg TCR specificities vary by anatomical location. Substantial evidence has also accumulated to support the role of CD28 costimulation and interleukin-2 in Treg homeostasis. The availability of TCR-transgenic Tregs has enabled analysis of Treg populations that are sufficient or deficient in particular genes, without the comparison being confounded by repertoire alterations. This approach has yielded insights into genes required for Treg function in vivo, with particular progress being made on the role of ctla-4 in this context. As the prospect of manipulating Treg populations in the clinic becomes reality, a full appreciation of the rules governing their homeostasis will prove increasingly important.

Germinal centre frequency is decreased in pancreatic lymph nodes from individuals with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: Pancreatic lymph nodes (PLNs) are critical sites for the initial interaction between islet autoantigens and autoreactive lymphocytes, but the histology of PLNs in tissue from individuals with type 1 diabetes has not been analysed in detail. The aim of this study was to examine PLN tissue sections from healthy donors compared with those at risk of, or with recent-onset and longer-duration type 1 diabetes. METHODS: Immunofluorescence staining was used to examine PLN sections from the following donor groups: non-diabetic (n=15), non-diabetic islet autoantibody-positive (n=5), recent-onset (≤1.5 years duration) type 1 diabetes (n=13), and longer-duration type 1 diabetes (n=15). Staining for CD3, CD20 and Ki67 was used to detect primary and secondary (germinal centre-containing) follicles and CD21 and CD35 to detect follicular dendritic cell networks. RESULTS: The frequency of secondary follicles was lower in the recent-onset type 1 diabetes group compared with the non-diabetic control group. The presence of insulitis (as evidence of ongoing beta cell destruction) and diagnosis of type 1 diabetes at a younger age, however, did not appear to be associated with a lower frequency of secondary follicles. A higher proportion of primary B cell follicles were observed to lack follicular dendritic cell networks in the recent-onset type 1 diabetes group. CONCLUSIONS/INTERPRETATION: Histological analysis of rare PLNs from individuals with type 1 diabetes suggests a previously unrecognised phenotype comprising decreased primary B cell follicle frequency and fewer follicular dendritic cell networks in recent-onset type 1 diabetes.

A transendocytosis model of CTLA-4 function predicts its suppressive behavior on regulatory T cells.

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study, we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking Abs had no impact on the response. In contrast, in settings where CTLA-4(+) cells were present as "regulators," inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of APC. At low numbers of APC or low levels of ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the APC. These data reveal clear rules for the inhibitory function of CTLA-4 on regulatory T cells, which are predicted by its ability to remove ligands from APC.

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.