Bertram Bengsch

Regulated Expression of Nuclear Receptor RORγt Confers Distinct Functional Fates to NK Cell Receptor-Expressing RORγt+ Innate Lymphocytes

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.

Coexpression of PD-1, 2B4, CD160 and KLRG1 on Exhausted HCV-Specific CD8+ T Cells Is Linked to Antigen Recognition and T Cell Differentiation

Exhausted CD8+ T cell responses during chronic viral infections are defined by a complex expression pattern of inhibitory receptors. However, very little information is currently available about the coexpression patterns of these receptors on human virus-specific CD8+ T cells and their correlation with antiviral functions, T cell differentiation and antigen recognition. We addressed these important aspects in a cohort of 38 chronically HCV infected patients and found a coexpression of inhibitory receptors such as 2B4, CD160 and KLRG1 in association with PD-1 in about half of the HCV-specific CD8+ T cell responses. Importantly, this exhaustive phenotype was associated with low and intermediate levels of CD127 expression, an impaired proliferative capacity, an intermediate T cell differentiation stage and absence of sequence variations within the corresponding epitopes, indicating ongoing antigen triggering. In contrast, a low expression of inhibitory receptors by the remaining HCV-specific CD8+ T cells occurred in concert with a CD127hi phenotype, an early T cell differentiation stage and presence of viral sequence variations within the corresponding epitopes. In sum, these results suggest that T cell exhaustion contributes to the failure of about half of HCV-specific CD8+ T cell responses and that it is determined by a complex interplay of immunological (e.g. T cell differentiation) and virological (e.g. ongoing antigen triggering) factors.

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

CD8+ T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8+ T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8+CD161+ T cells in healthy donors and those with hepatitis C virus and defined a population of CD8+ T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor γ-t, P = 6 × 10−9; RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 × 10−7; IL-18 receptor, P = 4 × 10−6), and chemokine receptors (e.g., CCR6, P = 3 × 10−8; CXCR6, P = 3 × 10−7; CCR2, P = 4 × 10−7). CD161+CD8+ T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-γ and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8+ T cells in normal humans and a substantial fraction of tissue-infiltrating CD8+ T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.

Expression of the Interleukin-7 Receptor Alpha Chain (CD127) on Virus-Specific CD8+ T Cells Identifies Functionally and Phenotypically Defined Memory T Cells during Acute Resolving Hepatitis B Virus Infection

ABSTRACT Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.

T-cell invigoration to tumour burden ratio associated with anti-PD-1 response

Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.

Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 T cells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory T cells (TMEM) is unclear. We found that reinvigoration of TEX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX became reexhausted if antigen concentration remained high and failed to become TMEM upon antigen clearance. TEX acquired an epigenetic profile distinct from that of effector T cells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

Antiviral and Regulatory T Cell Immunity in a Patient with Stromal Interaction Molecule 1 Deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

Human Th17 Cells Express High Levels of Enzymatically Active Dipeptidylpeptidase IV (CD26)

Dipeptidylpeptidase IV (CD26) is a multifunctional ectoenzyme involved in T cell activation that has been implicated in autoimmune pathophysiology. Because IL-17–producing CD4+ T cells (Th17 cells) are important mediators of autoimmune disease, we analyzed the expression of CD26 and its enzymatic function on human Th17 cells. Analysis of CD26 expression on different CD4+ T helper subsets showed that CD26 expression is highest on CD4+ T cells producing type 17 cytokines (e.g., IL-22, IL-17, GM-CSF, or TNF) compared with Th1, Th2, and regulatory T cells. Phenotypic analysis revealed that CD26++CD4+ T cells express the type 17 differentiation molecules CD161, CCR6, lL-23R, and retinoic acid-related orphan receptor-γt. Furthermore, sorted CD26++CD4+ T cells contain >90–98% of Th17 cells, indicating that CD26++ T cells harbor the Th17 lineage. A comparison with CD161 and CCR6 indicated that analysis of CD26 coexpression may improve the phenotypic characterization of Th17 cells. Of note, CD26++ Th17 cells are enriched in the inflamed tissue of patients with hepatitis and inflammatory bowel disease. Functional analysis in migration assays revealed that CD26 expressed on Th17 cells is enzymatically active. Indeed, CD26 negatively regulates the chemotactic CD4+ T cell response to the inflammatory chemokines CXCL9–12 that can be restored by pharmacological blockade of the enzymatic center of CD26. In summary, these results strongly suggest that CD26 may contribute to the orchestration of the immune response by Th17 cells in human inflammatory diseases. They also suggest that the phenotypic analysis of Th17 cells may be facilitated by determination of CD26 expression.

Analysis of CD127 and KLRG1 Expression on Hepatitis C Virus-Specific CD8+ T Cells Reveals the Existence of Different Memory T-Cell Subsets in the Peripheral Blood and Liver

ABSTRACT The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1− and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.

Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation

BACKGROUND & AIMS The upregulation of several inhibitory signalling pathways by exhausted HBV-specific CD8+ T cells in chronic infection is thought to contribute to viral persistence. Blockade of inhibitory receptors to reinvigorate exhausted T cell function is a promising novel therapeutic approach. However, little information is available regarding the relative contribution of individual inhibitory pathways to HBV-specific CD8+ T cell failure and the impact of inhibitory receptor blockade on restoration of T cell function in chronic HBV. METHODS 98 HLA-A2+ chronically infected patients were analysed ex vivo for HBV-specific CD8+ T cell responses, the expression of multiple inhibitory receptors and T cell differentiation markers. The effects of inhibitory receptor blockade targeting PD-1, 2B4, Tim-3, CTLA-4, and BTLA were assessed in vitro. RESULTS In our cohort, ex vivo HBV-specific CD8+ T cell responses were identified preferentially in HBeAg patients with low ALT and low viral load (inactive carriers). We observed a clear hierarchy of inhibitory receptor expression dominated by PD-1. The response to inhibitory receptor blockade was heterogeneous. Compared to the blockade of other inhibitory receptors, blockade of the PD-1 pathway resulted in the strongest increase in function. Of note, a positive effect of PD-1 blockade was linked to intermediate T cell differentiation. CONCLUSIONS Despite the expression of multiple inhibitory receptors by HBV-specific CD8+ T cells, expression and response to blockade was dominated by PD-1. However, PD-1 expression did not predict response to blockade. Rather, response to blockade was associated with intermediate T cell differentiation. These findings have important implications for our understanding of inhibitory receptor blockade as a novel therapeutic strategy.