Erietta Stelekati

Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

The microRNA miR-155 controls CD8 + T cell responses by regulating interferon signaling

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8+ T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8+ T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8+ T cells, as miR-155-deficient CD8+ T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8+ T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8+ T cells had enhanced type I interferon signaling and were more susceptible to interferons antiproliferative effect. Inhibition of the type I interferon–associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8+ T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8+ T cell responses to pathogens in vivo.

Mast Cell-Mediated Antigen Presentation Regulates CD8+ T Cell Effector Functions

The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8(+) T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8(+) T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-gamma, and macrophage inflammatory protein-1alpha by CD8(+) T cells. MCs regulated antigen-specific CD8(+) T cell cytotoxicity by increasing granzyme B expression and by promoting CD8(+) T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8(+) T cell proliferation, and MCs regulated CD8(+) T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8(+) T cells.

Chronic bystander infections and immunity to unrelated antigens.

Chronic infections with persistent pathogens such as helminths, mycobacteria, Plasmodium, and hepatitis viruses affect more than a third of the human population and are associated with increased susceptibility to other pathogens as well as reduced vaccine efficacy. Although these observations suggest an impact of chronic infections in modulating immunity to unrelated antigens, little is known regarding the underlying mechanisms. Here, we summarize evidence of the most prevalent infections affecting immunity to unrelated pathogens and vaccines, and discuss potential mechanisms of how different bystander chronic infections might impact immune responses. We suggest that bystander chronic infections affect different stages of host responses and may impact transmission and recognition of other pathogens, innate immune responses, priming and differentiation of adaptive effector responses, as well as the development and maintenance of immunological memory. Further understanding of the immunological effects of coinfection should provide opportunities to enhance vaccine efficacy and control of infectious diseases.

Bystander Chronic Infection Negatively Impacts Development of CD8+ T Cell Memory

Epidemiological evidence suggests that chronic infections impair immune responses to unrelated pathogens and vaccines. The underlying mechanisms, however, are unclear and distinguishing effects on priming versus development of immunological memory has been challenging. We investigated whether bystander chronic infections impact differentiation of memory CD8(+) T cells, the hallmark of protective immunity against intracellular pathogens. Chronic bystander infections impaired development of memory CD8(+) T cells in several mouse models and humans. These effects were independent of initial priming and were associated with chronic inflammatory signatures. Chronic inflammation negatively impacted the number of bystander CD8(+) T cells and their memory development. Distinct underlying mechanisms of altered survival and differentiation were revealed with the latter regulated by the transcription factors T-bet and Blimp-1. Thus, exposure to prolonged bystander inflammation impairs the effector to memory transition. These data have relevance for immunity and vaccination during persisting infections and chronic inflammation.

Cutting Edge: IL-4, IL-21, and IFN-γ Interact To Govern T-bet and CD11c Expression in TLR-Activated B Cells

T-bet and CD11c expression in B cells is linked with IgG2c isotype switching, virus-specific immune responses, and humoral autoimmunity. However, the activation requisites and regulatory cues governing T-bet and CD11c expression in B cells remain poorly defined. In this article, we reveal a relationship among TLR engagement, IL-4, IL-21, and IFN-γ that regulates T-bet expression in B cells. We find that IL-21 or IFN-γ directly promote T-bet expression in the context of TLR engagement. Further, IL-4 antagonizes T-bet induction. Finally, IL-21, but not IFN-γ, promotes CD11c expression independent of T-bet. Using influenza virus and Heligmosomoides polygyrus infections, we show that these interactions function in vivo to determine whether T-bet+ and CD11c+ B cells are formed. These findings suggest that T-bet+ B cells seen in health and disease share the common initiating features of TLR-driven activation within this circumscribed cytokine milieu.

Abstract CT137: Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma

Purpose: Combining immunostimulatory monoclonal (mAb) and checkpoint inhibition may improve response rates and overall survival in patients with metastatic melanoma. CP-870,893 is an agonistic fully human IgG2 mAb targeting the immune stimulatory molecule CD40 (αCD40). Tremelimumab (treme) is a fully human IgG2 mAb targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a negative co-stimulatory molecule found on T cells. Methods: Patients with metastatic melanoma were enrolled on a single-arm open-label phase I study. αCD40 and treme were dosed i.v. every 3 weeks and 12 weeks, respectively. Dose escalation followed a 3 + 3 strategy with alternating increases of αCD40 and treme in each cohort. Outcomes were scored using the Common Terminology Criteria for Adverse Events v3.0 and RECIST 1.0. Flow cytometric analysis of baseline blood samples was compared to samples obtained at least 3 weeks after the last dose of αCD40 and treme. Results: Toxicity: Twenty-four patients were enrolled and treated at 4 different dose levels. Two patients were replaced per protocol due to rapid, symptomatic progression of disease; these patients were not evaluated for response but were included in toxicity analysis. Dose-limiting toxicities (DLT), considered to be likely due to treatment, were colitis (n = 1) and hypophysitis (n = 1) and uveitis (n = 1). Cytokine release syndrome (CRS), grade 1-2, occurring within 24 hours of CP administration, was the most common treatment-related toxicity occurring in 19 (79.2%) patients. CRS symptoms were controlled with standard supportive care. All episodes of CRS resolved within 24 hours of αCD40 administration. The estimated maximum tolerated dose was 0.2 mg/kg of αCD40 and 10 mg/kg of treme. Outcomes: Overall objective response rate (ORR) was 27.3% (best response): two patients (9.1%) had complete responses to treatment and four (18.2%) patients had partial responses. The median follow-up was 22 months with a median progression free survival of 2.5 months and a median overall survival (OS) of 26.1 months. Correlative studies: Flow cytometric analysis of peripheral blood lymphocytes revealed changes in CD8+ T cell phenotypes. Double positive granzyme B+ and Ki-67+ cells increased from 0.56 ± 0.17% (mean ± standard error) for all CD8+ T cells at baseline to 1.01 ± 0.23% post-treatment (p = 0.03). CD8+ T cells expressing both programmed cell death 1 and eomesodermin were also higher after treatment, increasing from 7.7 ± 1.1% to 10.4 ±1.8% (p = 0.04) of all CD8+ T cells. Conclusion: Combination therapy with αCD40 and treme was well-tolerated in patients with metastatic melanoma, with rates of CRS and other toxicity similar to previously reported rates for αCD40 or treme treatments alone. Overall ORR was 27.3%, with median OS of 26.1 months. Given the tolerability, antitumor activity, and biomarker evidence of immune activation, expanded study of this combination should be pursued. Citation Format: David L. Bajor, Rosemarie Mick, Matthew J. Riese, Lee P. Richman, Xiaowei Xu, Drew A. Torigian, Erietta Stelekati, Martha Sweeney, Brendan Sullivan, Lynn M. Schuchter, Ravi Amaravadi, E. John Wherry, Robert H. Vonderheide. Combination of agonistic CD40 monoclonal antibody CP-870,893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT137. doi:10.1158/1538-7445.AM2015-CT137

ST2 contributes to T-cell hyperactivation and fatal hemophagocytic lymphohistiocytosis in mice

Cytokine storm syndromes, such as familial hemophagocytic lymphohistiocytosis (FHL), are lethal disorders caused by uncontrolled, systemic immune activation. In the murine model of FHL, in which perforin-deficient (Prf1(-/-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), disease is driven by overabundant interferon (IFN)γ-producing LCMV-specific CD8(+) T cells thought to arise from excessive antigen stimulation through the T-cell receptor. However, this paradigm is insufficient to explain several fundamental aspects of FHL, namely, the inability of many pathogenic antigens to induce hyperinflammation, and the previously identified role of MyD88 in the disease. We now show a novel role for the MyD88-dependent interleukin-33 (IL-33) receptor, ST2, in FHL. Expression of IL-33 and ST2 is upregulated in LCMV-infected Prf1(-/-) mice. Blockade of ST2 markedly improves survival of LCMV-infected Prf1(-/-) mice and reduces the severity of multiple disease parameters, including serum levels of IFNγ. This decrease in IFNγ corresponds to a reduction in both the frequency of IFNγ(+) LCMV-specific CD8(+) and CD4(+) T cells and the magnitude of IFNγ expression in these cells. These findings demonstrate that disruption of ST2 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ and suggest a revised paradigm in which danger signals such as IL-33 are crucial amplifiers of immune dysregulation in FHL. Furthermore, this study provides evidence to support blockade of ST2 as a novel therapeutic strategy for FHL.

Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood

Significance Herein we used peptide–MHC tetramer staining and enrichment to identify antigen-specific Tregs directly ex vivo and found that most CD4+ T-cell specificities, self or foreign, contain 5–15% Tregs. A notable exception is that two dominant influenza specificities contain very small proportions of Tregs. We demonstrated using human cord blood cells and murine lymphocytic choriomeningitis virus infection that antigen exposure can alter the balance between Tregs and conventional T cells in an antigen-specific and context-dependent manner. The ratio between specific Tregs and cognate effectors correlates with protective immunity in mice and T-cell dominance hierarchy in humans. Our finding provides insight into Treg specificity and highlights the importance of Treg and effector balance in an antigen-specific context. The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide–MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity.

The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation

T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.