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Immune checkpoint therapy (ICT) has the power to eradicate cancer, but the mechanisms that determine effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling, we interrogate whether the landscape of T cell states in the peripheral blood predict responses to combinatorial targeting of the OX40 costimulatory and PD-1 inhibitory pathways. Single-cell RNA sequencing and mass cytometry expose systemic and dynamic activation states of therapy-responsive CD4 + and CD8 + T cells in tumor-bearing mice with expression of distinct natural killer (NK) cell receptors, granzymes, and chemokines/chemokine receptors. Moreover, similar NK cell receptor-expressing CD8 + T cells are also detected in the blood of immunotherapy-responsive cancer patients. Targeting the NK cell and chemokine receptors in tumor-bearing mice shows the functional importance of these receptors for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use and targeting of dynamic biomarkers on T cells to improve cancer immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Expression of killer cell lectin-like receptor B1 ( KLRB1 ), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown., Methods: We examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies., Results: Central and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/ KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)β1., Conclusion: High levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells., Competing Interests: Competing interests: GS reports personal fees from Pieris Pharmaceuticals GmbH outside the submitted work. FF has received honoraria for lecturing at the ESMO Virtual Advanced Course on Biomarkers for Precision Medicine 2021. PtD reports grants from Oncode Institute and Cancer Genomics Center Netherlands (CGC.NL). VMB reports funding from Centre National de la Recherche Scientifique, French Government (National Research Agency, ANR) through the 'Investments for the Future' programs LABEX SIGNALIFE ANR-11-LABX-0028 and IDEX UCAJedi ANR-15-IDEX-01, Cancéropole PACA and Fondation ARC pour la recherche sur le Cancer. SHvdB reports grants from IO Biotech and DC Prime for immunemonitoring of trials and personal consulting fees from ISA Pharmaceuticals, PCI Biotech, DC Prime, CHDR consultancy services and AGLAIA outside the submitted work. The other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Tissue-resident memory CD8 + T cells (T RM ) constitute a noncirculating memory T cell subset that provides early protection against reinfection. However, how T RM arise from antigen-triggered T cells has remained unclear. Exploiting the T RM -restricted expression of Hobit, we used T RM reporter/deleter mice to study T RM differentiation. We found that Hobit was up-regulated in a subset of LCMV-specific CD8 + T cells located within peripheral tissues during the effector phase of the immune response. These Hobit + effector T cells were identified as T RM precursors, given that their depletion substantially decreased T RM development but not the formation of circulating memory T cells. Adoptive transfer experiments of Hobit + effector T cells corroborated their biased contribution to the T RM lineage. Transcriptional profiling of Hobit + effector T cells underlined the early establishment of T RM properties including down-regulation of tissue exit receptors and up-regulation of T RM -associated molecules. We identified Eomes as a key factor instructing the early bifurcation of circulating and resident lineages. These findings establish that commitment of T RM occurs early in antigen-driven T cell differentiation and reveal the molecular mechanisms underlying this differentiation pathway., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tissue-resident memory CD8 + T cells (T RM ) localize to barrier tissues and mediate local protection against reinvading pathogens. Circulating central memory (T CM ) and effector memory CD8 + T cells (T EM ) also contribute to tissue recall responses, but their potential to form mucosal T RM remains unclear. Here, we employed adoptive transfer and lymphocytic choriomeningitis virus reinfection models to specifically assess secondary responses of T CM and T EM at mucosal sites. Donor T CM and T EM exhibited robust systemic recall responses, but only limited accumulation in the small intestine, consistent with reduced expression of tissue-homing and -retention molecules. Murine and human circulating memory T cells also exhibited limited CD103 upregulation following TGF-β stimulation. Upon pathogen clearance, T CM and T EM readily gave rise to secondary T EM . T CM also formed secondary central memory in lymphoid tissues and T RM in internal tissues, for example, the liver. Both T CM and T EM failed to substantially contribute to resident mucosal memory in the small intestine, while activated intestinal T RM , but not liver T RM , efficiently reformed CD103 + T RM . Our findings demonstrate that circulating T CM and T EM are limited in generating mucosal T RM upon reinfection. This may pose important implications on cell therapy and vaccination strategies employing memory CD8 + T cells for protection at mucosal sites., (© 2020 Wiley-VCH GmbH.)

Factors that govern the complex formation of memory T cells are not completely understood. A better understanding of the development of memory T cell heterogeneity is however required to enhance vaccination and immunotherapy approaches. Here we examined the impact of pathogen- and tissue-specific cues on memory CD8 + T cell heterogeneity using high-dimensional single-cell mass cytometry and a tailored bioinformatics pipeline. We identified distinct populations of pathogen-specific CD8 + T cells that uniquely connected to a specific pathogen or associated to multiple types of acute and persistent infections. In addition, the tissue environment shaped the memory CD8 + T cell heterogeneity, albeit to a lesser extent than infection. The programming of memory CD8 + T cell differentiation during acute infection is eventually superseded by persistent infection. Thus, the plethora of distinct memory CD8 + T cell subsets that arise upon infection is dominantly sculpted by the pathogen-specific cues and further shaped by the tissue environment., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue-resident memory CD8 + T cells (T RM cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall responses is unclear. We developed a unique lineage tracer mouse model exploiting the T RM -defining transcription factor homolog of Blimp-1 in T cells (Hobit) to fate map the T RM progeny in secondary responses. After reinfection, a sizeable fraction of secondary memory T cells in the circulation developed downstream of T RM cells. These tissue-experienced ex-T RM cells shared phenotypic properties with the effector memory T cell population but were transcriptionally and functionally distinct from other secondary effector memory T cell cells. Adoptive transfer experiments of T RM cells corroborated their potential to form circulating effector and memory cells during recall responses. Moreover, specific ablation of primary T RM cell populations substantially impaired the secondary T cell response, both locally and systemically. Thus, T RM cells retain developmental plasticity and shape both local and systemic T cell responses on reinfection.

Tissue-resident memory CD8 + T (T RM ) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8 + T RM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8 + T cells. In contrast to CD8 + T RM cells at these sites, CD8 + T RM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8 + T RM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8 + T RM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8 + T RM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8 + T RM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8 + T RM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8 + T RM cells and inhibited the differentiation of central memory CD8 + T (T CM ) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8 + T RM cells in the lungs, potentially through control of the lineage choice between T CM and T RM cells during the differentiation of influenza-specific CD8 + T cells.

Tissue-resident memory T cells (T RM ) are noncirculating immune cells that contribute to the first line of local defense against reinfections. Their location at hotspots of pathogen encounter frequently exposes T RM to tissue damage. This history of danger-signal exposure is an important aspect of T RM -mediated immunity that has been overlooked so far. RNA profiling revealed that T RM from liver and small intestine express P2RX7, a damage/danger-associated molecular pattern (DAMP) receptor that is triggered by extracellular nucleotides (ATP, NAD + ). We confirmed that P2RX7 protein was expressed in CD8 + T RM but not in circulating T cells (T CIRC ) across different infection models. Tissue damage induced during routine isolation of liver lymphocytes led to P2RX7 activation and resulted in selective cell death of T RM P2RX7 activation in vivo by exogenous NAD + led to a specific depletion of T RM while retaining T CIRC The effect was absent in P2RX7-deficient mice and after P2RX7 blockade. TCR triggering down-regulated P2RX7 expression and made T RM resistant to NAD-induced cell death. Physiological triggering of P2RX7 by sterile tissue damage during acetaminophen-induced liver injury led to a loss of previously acquired pathogen-specific local T RM in wild-type but not in P2RX7 KO T cells. Our results highlight P2RX7-mediated signaling as a critical pathway for the regulation of T RM maintenance. Extracellular nucleotides released during infection and tissue damage could deplete T RM locally and free niches for new and infection-relevant specificities. This suggests that the recognition of tissue damage promotes persistence of antigen-specific over bystander T RM in the tissue niche., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA + effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)