Veit R. Buchholz

The immune system integrates signals to regulate immune responses, forming complex context-dependent networks where identical cues produce distinct outcomes. Signal transducer and activator of transcription (STAT) transcription factors convert cytokine receptor engagement into transcriptional responses, serving as central regulatory hubs. How concurrent STAT activation across different environments shapes immune cell function remains unclear. Here, we show that STAT3 plays a context-dependent role in antiviral natural killer (NK) cells, either promoting or impairing adaptive responses depending on the inflammatory milieu during mouse cytomegalovirus (MCMV) infection. STAT3 is recruited to distinct genomic loci depending on IL-15 or type I interferon availability, thus driving alternative transcriptional programs. Mechanistically, STAT3 differentially regulates downstream transcription factors MYB and B lymphocyte–induced maturation protein-1 (BLIMP-1) to shape NK cell differentiation during homeostasis and infection. STAT3 relocation is governed by epigenetic mechanisms and preferred relocation toward distinct STAT motifs. Thus, STAT3 functions as a cytokine-sensing transcriptional switch integrating IL-15 and type I interferon signals to control lymphocyte adaptation during viral infection.

Cancer-specific TCF1⁺ stem-like CD8⁺ T cells can drive protective anticancer immunity through expansion and effector cell differentiation^1–4; however, this response is dysfunctional in tumours. Current cancer immunotherapies^2,5–9 can promote anticancer responses through TCF1⁺ stem-like CD8⁺ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1⁺CD8⁺ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE₂) restricts the proliferative expansion and effector differentiation of TCF1⁺CD8⁺ T cells within tumours, which promotes cancer immune escape. PGE₂ does not affect the priming of TCF1⁺CD8⁺ T cells in draining lymph nodes. PGE₂ acts through EP₂ and EP₄ (EP₂/EP₄) receptor signalling in CD8⁺ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1⁺ tumour-infiltrating CD8⁺ T lymphocytes (TILs). Ablation of EP₂/EP₄ signalling in cancer-specific CD8⁺ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE₂-mediated inhibition of TCF1⁺ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1⁺ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE₂–EP₂/EP₄ axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen¹. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP4². However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Localization is a crucial prerequisite for immune cell function and solid tumors evade immune control by modulating immune cell infiltration into the tumor stroma. Immunosuppressive cells like regulatory T cells are attracted, while cytotoxic CD8⁺ T cells are excluded. Engineering CD8⁺ T cells with chemokine receptors is a potent strategy to turn this mechanism of directed immune cell recruitment against the tumor. Here, we utilized fluorescent tagging to track the migratory behavior of tumor-specific T cells engineered with a library of all murine chemokine receptors in vivo. We then asked whether chemokine receptor-mediated redirection of antigen-specific T cells into tumors or tumor-draining lymph nodes showed superior anti-tumoral activity. We found that both targeting approaches showed higher therapeutic efficacy than control T cells. However, multiple receptors conveying the same homing pattern did not augment infiltration. Instead, in the MC38 colon carcinoma model, anti-tumoral efficacy as well as lymph node vs. tumor-homing patterns were mostly driven by CCR4 and CCR6, respectively. Overall, our data, based on fluorescent receptor tagging, identify the tumor-draining lymph node and the tumor itself as viable targets for chemokine receptor-mediated enhancement of adoptive T cell therapy.

CD8 ⁺ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine ⁺ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8 ⁺ T cells; however, it remains unclear whether they can actively modulate CD8 ⁺ T cell responses. In this study, we have developed a method to analyze cell-bound PS ⁺ EVs and their target cells in vivo. We show that EV ⁺ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8 ⁺ T cells. Superresolution imaging revealed that PS ⁺ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8 ⁺ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS ⁺ EVs provide Ag-specific adjuvant effects to activated CD8 ⁺ T cells in vivo.

T cell ignorance is a specific form of immunological tolerance. It describes the maintenance of naivety in antigen‐specific T cells in vivo despite the presence of their target antigen. It is thought to mainly play a role during the steady state, when self‐antigens are presented in absence of costimulatory signals and at low density or to T cells of low affinity. In how far antigen‐specific T cells can also remain clonally ignorant to foreign antigens, presented in the inflammatory context of systemic infection, remains unclear. Using single‐cell in vivo fate mapping and high throughput flow cytometric enrichment, we find that high‐affinity antigen‐specific CD8⁺ T cells are efficiently recruited upon systemic infection. In contrast, most low‐affinity antigen‐specific T cells ignore the priming antigen and persist in the naïve state while remaining fully responsive to subsequent immunization with a high‐affinity ligand. These data establish the widespread clonal ignorance of low‐affinity T cells as a major factor shaping the composition of antigen‐specific CD8⁺ T cell responses to systemic infection.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen–specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma—combined with transgenic reexpression of identified TCRs by CRISPR-Cas9–mediated orthotopic TCR replacement—revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

CD8⁺ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality—which is referred to as T cell exhaustion^1,2—is maintained by precursors of exhausted T (T_PEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1⁻ exhausted effector T cells^3–6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L⁺ T_PEX cells. The transcription factor MYB is not only essential for the development of CD62L⁺ T_PEX cells and maintenance of the antiviral CD8⁺ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L⁺ T_PEX cells and depends on MYB. Our findings identify CD62L⁺ T_PEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.