The role of mast cells in CD8+ T cell-mediated immune responses

A precisely regulated crosstalk between innate and adaptive immunity is a prerequisite for an optimal immune response and successful survival strategy. Important players of innate immunity are the mast cells (MCs). These are long-living cells at sites of host-environment interface and important effector players during allergic responses. Recently, MCs have been described as central regulatory cells not only in innate but also in adaptive immune responses. MCs interact with cells of the adaptive immune system and recruit CD8+ T cells upon different stimuli. The purpose of this study was to investigate the interaction between MCs and CD8+ T cells, identify the factors that modulate this interaction and examine its downstream effects. By using murine bone marrow-derived MCs, this study demonstrated that MCs promote the survival of naïve, primary CD8+ T cells in an antigen-independent and cell-cell contact-dependent manner. The investigation of the antigen-dependent interaction between MCs and CD8+ T cells showed that MCs induce antigen-specific activation, proliferation and cytokine secretion by TCR-transgenic CD8+ T cells in vitro. Furthermore, the increased intracellular content of granzyme B and enhanced CD8+ T cell degranulation indicated an increase in the cytotoxic potential of CD8+ T cells. This antigen-driven communication between MCs and CD8+ T cells required both direct cell-cell contact and the release of soluble factors by MCs. TLR-mediated activation of MCs augmented their capacity to activate CD8+ T cells, partially due to enhanced surface expression of MHC class I molecules. Remarkably, the adoptive transfer of antigen-pulsed MCs induced proliferation of antigen-specific CD8+ T cells in vivo, in wild-type as well as in 2-microglobulin-deficient mice, which lack functional MHC class I expression. Thus, MCs promote CD8+ T cell responses, inducing effector CD8+ T cells in vitro and in vivo. Furthermore, CD8+ T cells enhanced the expression of several genes in MCs, in an antigen-dependent as well as antigen-independent manner, as demonstrated by differential gene expression analysis of MCs. Many of these genes are implicated in the signal transduction pathway of interferons, suggesting that the MC-CD8+ T cell interaction may contribute significantly to host defense mechanisms. Additionally, upregulation of major histocompatibility complex-related molecules and of the co-stimulatory molecule 4-1BB suggests that the contact with CD8+ T cells enhances the potential of MCs to modulate adaptive immune responses. In conclusion, this study adds new insights into the physiological role of MCs in the context of adaptive immune responses, such as a CD8+ T cell-driven antiviral immune response. This novel understanding of MC biology foresees new promising approaches for a therapeutic manipulation of antiviral immunity.Ein exakt regulierter Dialog zwischen angeborener und adaptiver Immunität ist eine wesentliche Voraussetzung für eine optimale Immunantwort und somit für eine erfolgreiche Überlebensstrategie. Zu den bedeutenden Zellen der angeborenen Immunität zählen unter anderen die Mastzellen (MZ). MZ sind langlebige Zellen, welche überwiegend an Umwelt-exponierten Körperflächen lokalisiert sind und wichtige Effektorzellen während einer allergischen Reaktion darstellen. Kürzlich wurden MZ als zentrale, regulatorische Zellen sowohl der angeborenen, als auch innerhalb der adaptiven Immunantwort beschrieben. MZ interagieren mit Zellen des angeborenen Immunsystems und vermögen nach unterschiedlicher Stimulation CD8+ T-Zellen zu rekrutieren. Absicht der vorliegenden Studie war es, die Interaktion zwischen MZ und CD8+ T-Zellen zu untersuchen, die diese Interaktion modulierenden Faktoren zu identifizieren und deren weiterführende Auswirkungen zu bestimmen. Unter Verwendung aus murinem Knochenmark generierter MZ zeigte diese Studie, dass MZ das Überleben naiver Primär-CD8+ T-Zellen Antigen-unabhängig und Zell-Zell-Kontakt-abhängig unterstützen. Untersuchungen der Antigen-abhängigen Interaktionen zwischen MZ und CD8+ T-Zellen zeigten, dass MZ eine Antigen-spezifische Aktivierung, Proliferation und Zytokinproduktion TCR-transgener CD8+ T-Zellen in vitro induzieren. Desweiteren deuten ein erhöhter intrazellulärer Gehalt an Granzym B und ein Anstieg der CD8+ T-Zell-Degranulation auf ein gesteigertes zytotoxisches Potential der CD8+ T-Zellen hin. Diese Antigen-gesteuerte Kommunikation zwischen MZ und CD8+ T-Zellen benötigte sowohl Zell-Zell-Kontakt als auch die Freisetzung löslicher Faktoren durch Mastzellen. Eine Aktivierung der MZ durch die Toll-like-Rezeptoren erhöhte deren Fähigkeit CD8+ T-Zellen zu aktivieren, teilweise vermittelt durch eine gesteigerte Zelloberflächenexpression der MHC Klasse I Molküle. Bemerkenswerter Weise induzierte der direkte Transfer Antigen-stimulierter MZ die Proliferation Antigen-spezifischer CD8+ T-Zellen in vivo, sowohl in wildtypischen als auch in 2-Mikroglobulin-defizienten Mäusen, welchen eine funktionale MHC Klasse I Expression fehlt. Somit wurde deutlich, dass MZ die CD8+ T-Zelle-Antwort fördern und dabei Effektor-CD8+ T-Zellen in vitro und in vivo induzieren. Zudem waren CD8+ T-Zellen in der Lage die Expression verschiedener Gene in MZ Antigen-abhängig als auch Antigen-unabhängig deutlich zu verstärken, wie mittels differentieller Genexpressionsanalyse gezeigt werden konnte. Viele dieser Gene haben eine wichtige Funktion innerhalb der Signaltransduktionswege von Interferonen, was zu der Annahme führte, dass die MZ-CD8+ T-Zell-Interaktion wesentlich zu Abwehrmechanismen beitragen könnte. Zusätzlich lässt die Hochregulation des Major Histocompatibility Complex-verwandten Moleküls 4—1BB vermuten, dass der Kontakt mit CD8+ T-Zellen das Potential der MZ, die adaptive Immunantwort zu modulieren, erhöhen könnte. Zusammenfassend ist zu sagen, dass diese Studie neue Einsichten in die physiologische Rolle der MZ im Kontext der adaptiven, wie einer CD8+ T-Zelle-gesteuerten anti-viralen Immunantwort gibt. Dieses neue Verständnis der MZ-Biologie birgt vielversprechende Ansätze für eine Manipulation der anti-viralen Immunantwort im therapeutischen Sinne

Trimellitic anhydride-conjugated serum albumin activates rat alveolar macrophages in vitro

BACKGROUND: Occupational exposure to airborne low molecular weight chemicals, like trimellitic anhydride (TMA), can result in occupational asthma. Alveolar macrophages (AMs) are among the first cells to encounter these inhaled compounds and were previously shown to influence TMA-induced asthma-like symptoms in the Brown Norway rat. TMA is a hapten that will bind to endogenous proteins upon entrance of the body. Therefore, in the present study we determined if TMA and TMA conjugated to serum albumin induced the production of the macrophage mediators nitric oxide (NO), tumour necrosis factor (TNF), and interleukin 6 (IL-6) in vitro using the rat AM cell line NR8383 and primary AMs derived from TMA-sensitized and naïve Brown Norway rats. METHODS: Cells were incubated with different concentrations of TMA, TMA conjugated to bovine serum albumin (BSA), and BSA as a control for 24 h and the culture supernatant was analyzed for mediator content. RESULTS: TMA alone was not able to induce the production of mediators by NR8383 cells and primary AMs from sensitized and sham-treated rats. TMA-BSA, on the contrary, dose-dependently stimulated the production of NO, TNF, and IL-6 by NR8383 cells and of NO and TNF, but not IL-6, by primary AMs independent of sensitization. CONCLUSION: Results suggest that although TMA is a highly reactive compound, conjugation to a suitable protein is necessary to induce mediator production by AMs. Furthermore, the observation that effects of TMA-BSA were independent of sensitization suggests involvement of an immunologically non-specific receptor. In the discussion it is argued that a macrophage scavenger receptor is a likely candidate

Long-Term Persistence of Exhausted CD8 T Cells in Chronic Infection Is Regulated by MicroRNA-155

Persistent viral infections and tumors drive development of exhausted T (TEX) cells. In these settings, TEX cells establish an important host-pathogen or host-tumor stalemate. However, TEX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained TEX cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection. Genetic deficiency of miR-155 ablated CD8 T cell responses during chronic infection. Conversely, enhanced miR-155 expression promoted expansion and long-term persistence of TEX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal TEX cell subset. Transcriptional profiling identified coordinated control of cell signaling and transcription factor pathways, including the key AP-1 family member Fosl2. Overexpression of Fosl2 reversed the miR-155 effects, identifying a link between miR-155 and the AP-1 transcriptional program in regulating TEX cells. Thus, we identify a mechanism of miR-155 regulation of TEX cells and a key role for Fosl2 in T cell exhaustion. During persistent viral infections, exhausted T cells (TEX) erode quantitatively and qualitatively and therefore fail to provide protection. Stelekati et al. identified microRNA (miR)-155 as a key molecule that can enhance and sustain TEX responses long-term during chronic viral infection

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

Mast cells in allergy: Innate instructors of adaptive responses

The function of mast cells as effector cells in allergy has been extensively studied. However, increasing insight into mast cell physiology has revealed new mast cell functions and has introduced mast cells as key players in the regulation of innate as well as adaptive immunity. For example, mast cells have recently been found to express Toll-like receptors (TLRs), which enable them to participate in the innate immune response against pathogens. Furthermore, mast cells have been reported to interact with B cells, dendritic cells and T cells and thereby modulate the direction of an adaptive immune response. Finally, recent documentation that mast cells express functional MHC class II and costimulatory molecules and release immunologically active exosomes, has raised the possibility that mast cells also engage in (as yet) poorly understood antigen presentation functions. In this review, we explore the hypothesis that mast cells serve as central mediators between innate and adaptive immunity, rather as pure effector cells, during allergic innate responses

Abstract 3807: MicroRNA-29a synergizes with PD-1 therapy to regulate anti-tumor immunity

Abstract CD8 T cells initiate potent anti-tumor immune responses. However, tumor cells employ PD-1 ligand, (PD-L1) to obstruct CD8 T cell responses and drive exhausted T cells (TEX) differentiation. TEX are characterized by sub-optimal functionality and inability to efficiently eliminate tumor cells. Checkpoint inhibitor therapy such as anti-PD-1 can partially restore the effector functions of TEX, but the stable epigenetic programs of TEX lead to the loss of reinvigoration and tumor relapse, providing a challenge for the efficacy of checkpoint blockade therapy. We recently demonstrated that miR-29a acts as a key regulator of TEX differentiation, promotes memory-like differentiation and attenuates exhaustion during chronic infection. Importantly, miR-29a favors the expansion of a progenitor TEX subset expressing the transcription factor TCF-1 that responds to immunotherapy. Therefore, we hypothesized that miR-29a can substantially alter TEX differentiation and synergize with checkpoint blockade to enhance anti-tumor immune responses. A well-established mouse model of chronic infection (LCMV clone-13) was used to induce TEX differentiation, with transcriptional and epigenetic profiles similar to TEX from human tumors. We found that combination of miR-29a overexpression and anti-PD-L1 treatment enhanced CD8 T cell expansion > 20 fold, whereas anti-PD-L1 alone marginally increased the number of CD8 T cells, suggesting that miR-29a synergizes with anti-PD-L1 to enhance CD8 T cell responses. Immune reinvigoration induced by anti-PD-L1 therapy is characterized by a partial reduction of inhibitory receptors and increase in effector responses. Instead, miR-29a overexpression combined with anti-PD-L1 promoted memory-like differentiation and long-term persistence of CD8 T cells, without compromising effector functions. Mechanistically, miR-29a synergized with anti-PD-L1 by inducing expression of a key memory-associated transcription factor, TCF-1, and antagonizing the master epigenetic regulator of exhaustion, TOX. To further understand whether miR-29a promotes only expansion of a progenitor TCF1+ subset or fundamentally alters TEX subset differentiation, equal numbers of miR-29a overexpressing and control TCF1+ CD8 T cells were isolated and adoptively transferred to secondary host mice followed by PD-L1 blockade. TCF1+ CD8 T cells with enforced miR-29a expression responded to PD-L1 blockade more efficiently than control TCF+ CD8 T cells. These results suggest that miR-29a fundamentally alters TEX differentiation by regulating key exhaustion-related transcriptional and epigenetic circuits. Therefore, we suggest that miR29a has the potential to be used as a novel therapeutic inducing robust and durable immune responses upon PD-1 therapy. Citation Format: Xuebing Leng, Svetlana Ristin, Christine Rafie, Lance Buchness, Alejandro Villarino, Erietta Stelekati. MicroRNA-29a synergizes with PD-1 therapy to regulate anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3807

Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy

Abstract CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (T EX). T EXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial T EXreinvigoration. Some T EXregain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of T EXreinvigoration are not well understood. Moreover, a key unknown question is why only some T EXgain reinvigoration. Here, we used proliferation as a surrogate for T EXreinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated T EXbefore first cell division and ask what molecular signals induce T EXreinvigoration. Thus, we found that T EXreinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated T EXupon anti-PD-L1 compared to untreated T EXat 48 hours post treatment. Therefore, we identified the molecular signatures of T EXthat have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy

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

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

TET2 Regulates CD8+ T Cell Responses to Acute and Chronic Viral Infection

Abstract DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+ T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+ T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+ T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+ T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+ T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity. Disclosures No relevant conflicts of interest to declare