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Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force. Editor's summary: Immune cells apply mechanical forces to their surrounding environment, but whether their biomechanical output is tuned to support distinct immunological functions remains unclear. Using super-resolution traction force microscopy, de Jesus et al. compared the patterns of interfacial forces exerted by cytotoxic T lymphocytes (CTLs) with other immune cells. Within the immune synapse, CTLs formed a complex compressive crater, a biomechanical signature that was distinct from those of macrophages initiating phagocytosis and T cells engaging in nonlytic interactions. These findings demonstrate that immune cells exert specialized patterns of force on their targets to carry out distinct immunological functions. —Claire Olingy [ABSTRACT FROM AUTHOR]

Tumors evade attacks from the immune system through various mechanisms. Here, we identify a component of tumor immune evasion mediated by YTH domain–containing family protein 2 (YTHDF2), a reader protein that usually destabilizes m6A-modified mRNA. Loss of tumoral YTHDF2 inhibits tumor growth and prolongs survival in immunocompetent tumor models. Mechanistically, tumoral YTHDF2 deficiency promotes the recruitment of macrophages via CX3CL1 and enhances mitochondrial respiration of CD8+ T cells by impairing tumor glycolysis metabolism. Tumoral YTHDF2 deficiency promotes inflammatory macrophage polarization and antigen presentation in the presence of IFN-γ. In addition, IFN-γ induces autophagic degradation of tumoral YTHDF2, thereby sensitizing tumor cells to CD8+ T cell–mediated cytotoxicity. Last, we identified a small molecule compound that preferentially induces YTHDF2 degradation, which shows a potent antitumor effect alone but a better effect when combined with anti–PD-L1 or anti–PD-1 antibodies. Collectively, YTHDF2 appears to be a tumor-intrinsic regulator that orchestrates immune evasion, representing a promising target for enhancing cancer immunotherapy. Editor's Summary: Immune evasion mechanisms facilitate tumor growth and limit immunotherapy efficacy. Xiao et al. identified a role for YTH domain–containing family protein 2 (YTHDF2), a N6-methyladenosine reader protein, in regulating immune evasion in solid tumors. YTHDF2 can target CX3CL1 mRNA to limit expression, which prevents inflammatory macrophage recruitment to tumors and cytotoxic T cell responses. CD8 T cell–derived IFN-γ can mediate autophagic degradation of YTHDF2, thus sensitizing tumor cells to T cell–mediated destruction. They also identified a small molecule compound that degrades YTHDF2 in tumor cells, which exhibits antitumor effects that are further enhanced with PD-1/PD-L1 blockade. —Christiana Fogg [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade is a promising approach to activate antitumor immunity and improve the survival of patients with cancer. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an immune checkpoint target; however, the downstream signaling mechanisms are elusive. Here, we identify leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) as a VISTA binding partner, which acts as an inhibitory receptor by engaging VISTA and suppressing T cell receptor signaling pathways. Mice with T cell–specific LRIG1 deletion developed superior antitumor responses because of expansion of tumor-specific cytotoxic T lymphocytes (CTLs) with increased effector function and survival. Sustained tumor control was associated with a reduction of quiescent CTLs (TCF1+ CD62Lhi PD-1low) and a reciprocal increase in progenitor and memory-like CTLs (TCF1+ PD-1+). In patients with melanoma, elevated LRIG1 expression on tumor-infiltrating CD8+ CTLs correlated with resistance to immunotherapies. These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy. Editor's summary: The negative checkpoint regulator VISTA has emerged as a promising target for cancer immunotherapy. VISTA plays an important role in maintaining T cell quiescence; however, the molecular mechanisms through which VISTA regulates antitumor immunity are not well defined. In this study, Ta et al. identified LRIG1 as a VISTA-binding partner and co-inhibitory receptor. VISTA engagement by LRIG1 impaired murine antitumor immunity by promoting quiescence in tumor-specific CD8 T cells and limited the abundance of progenitor and memory-like cells with the ability to differentiate into effector T cells. In patients with melanoma, expression of LRIG1 on tumor-infiltrating lymphocytes correlated with resistance to immunotherapy. Together, these findings suggest that selectively inhibiting VISTA/LRIG1 may be an effective cancer immunotherapy. —Hannah Isles [ABSTRACT FROM AUTHOR]

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases. Editor's Summary: Programmed cell death-1 (PD-1) is well known as an immune checkpoint receptor on T cells that can inhibit activation upon engagement with its ligands PD-L1 or PD-L2. PD-1 has largely been considered to function as a monomer, but Philips et al. now show that PD-1 can form dimers in cis via transmembrane domain interactions. They observed that limiting PD-1 dimerization weakens T cell inhibition upon ligand engagement, which enhances antitumor and cytotoxic T cell function and promotes autoimmunity. These findings may shape ongoing efforts to more effectively block PD-1 function or, conversely, to develop PD-1 agonists. —Christiana Fogg [ABSTRACT FROM AUTHOR]

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus–based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus–triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)—whose deficiency causes the FHL subtype FHL3—in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells. Editor's summary: Familial hemophagocytic lymphohistiocytosis (FHL) is a spectrum of inherited, potentially fatal inflammatory diseases caused by loss-of-function mutations in the NK cell and T cell cytotoxic machinery. Although FHL can be treated with hematopoietic stem cell transplantation (HSCT), mortality is still high and more effective therapeutic approaches are needed. Li et al. developed an adeno-associated virus (AAV)–based CRISPR-Cas9 system combined with nonhomologous end joining (NHEJ) inhibition to repair T cells from a perforin (Prf1)–deficient Epstein-Barr virus (EBV)–triggered mouse model of FHL (FHL2). Repaired T cells could prevent or cure FHL2-like disease caused by EBV-driven B cell hyperproliferation. Moreover, this CRISPR-Cas9 system could be used to mend T cells from pediatric patients with FHL2 (PRF1 deficiency) and FHL3 (Munc13-4/UNC13D deficiency). This work demonstrates that gene repair of autologous T cells is a potentially promising treatment for human FHL. —Seth Thomas Scanlon [ABSTRACT FROM AUTHOR]

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor–α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II–dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies. Editor's summary: CD4+ T cells have been primarily appreciated for their helper functions in antitumor immune responses, but the extent to which they directly contribute to tumor killing remains unclear. Using a mouse model of cutaneous melanoma, Bawden et al. characterized the effector functions of CD4+ T cells in generating protective antitumor immunity. Melanoma-specific CD4+ T cells infiltrated tumors, adopted diverse effector states, and could provide tumor protection independent of other lymphocytes. Whereas MHC II expression by melanoma cells was sufficient to activate protective CD4+ T cell–mediated immunity, host antigen-presenting cells could also drive protection. CD4+ T cells provided protection through multiple partially redundant cytotoxicity pathways, including TNF and Fas ligand, but also by activating IFN-γ–dependent nitric oxide production in myeloid cells. Together, these results demonstrate that tumor-infiltrating CD4+ T cells are equipped to contribute to tumor control through multiple helper and effector modes. —Claire Olingy [ABSTRACT FROM AUTHOR]

Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)–restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8+ T cells engineered with the same TCR. TCR-iNKT cells achieved robust cancer control by simultaneously modulating intratumoral suppressive myeloid populations and killing malignant cells. This dual antitumor function was further enhanced when the iNKT cell agonist α-galactosyl ceramide (α-GalCer) was administered as a therapeutic booster through a platform that ensured controlled delivery at the tumor site, named multistage vector (MSV). These preclinical results support the combination of tumor-redirected TCR-iNKT cells and local α-GalCer boosting as a potential therapy for patients with cancer. TCR-engineered iNKT cells: Adoptive cell therapy has had success in treating various blood cancers. However, in solid tumors, most of these therapies show little efficacy due to exhaustion and poor migration to tumors. Here, Delfanti et al. leveraged natural killer T cells as an adoptive cell therapy platform, specifically producing them to express a TCR specific for a tumor-associated antigen. In mouse models, they found that these TCR-engineered iNKT robustly delayed tumor growth, leading to rejection and prolonged antitumor effects in some mice. These cells migrated well into tumors, killed tumor cells directly, and helped to shift intratumorally myeloid cells to an antitumor phenotype. Boosting TCR-engineered iNKTs with α-GalCer improved antitumor immune responses. Thus, TCR-engineered iNKT cells might be a promising therapy for patients with cancer. [ABSTRACT FROM AUTHOR]

Sex bias exists in the development and progression of nonreproductive organ cancers, but the underlying mechanisms are enigmatic. Studies so far have focused largely on sexual dimorphisms in cancer biology and socioeconomic factors. Here, we establish a role for CD8+ T cell–dependent antitumor immunity in mediating sex differences in tumor aggressiveness, which is driven by the gonadal androgen but not sex chromosomes. A male bias exists in the frequency of intratumoral antigen-experienced Tcf7/TCF1+ progenitor exhausted CD8+ T cells that are devoid of effector activity as a consequence of intrinsic androgen receptor (AR) function. Mechanistically, we identify a novel sex-specific regulon in progenitor exhausted CD8+ T cells and a pertinent contribution from AR as a direct transcriptional transactivator of Tcf7/TCF1. The T cell–intrinsic function of AR in promoting CD8+ T cell exhaustion in vivo was established using multiple approaches including loss-of-function studies with CD8-specific Ar knockout mice. Moreover, ablation of the androgen-AR axis rewires the tumor microenvironment to favor effector T cell differentiation and potentiates the efficacy of anti–PD-1 immune checkpoint blockade. Collectively, our findings highlight androgen-mediated promotion of CD8+ T cell dysfunction in cancer and imply broader opportunities for therapeutic development from understanding sex disparities in health and disease. Antitumor CD8+ T cells curtailed by androgen: Cancer epidemiology statistics reveal a male sex bias toward increased incidence and worse clinical outcomes. Kwon et al. used mouse models of bladder cancer showing male sex bias to investigate whether androgen acting on CD8+ T cells is part of the biological explanation. Single-cell RNA sequencing analysis of intratumoral CD8+ T cells revealed male-biased expression of a progenitor exhausted (PE) signature and higher levels of effector genes in female tumors. The skew toward a PE state in male T cells was dependent on both androgen availability and androgen receptor expression by T cells. These advances in understanding the immunological basis of sex bias in cancer create opportunities to design precision medicine therapies that factor in functional differences between male and female cytotoxic T cells. [ABSTRACT FROM AUTHOR]

Gammaherpesviruses (γHVs) have coevolved with their host, leading to a remarkably high infection prevalence and establishment of latency. The lifelong persistence of γHVs in hosts appears to broadly shape host immunity, and we show here that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment of Ly6Chi monocytes (MOs) into the airway, thereby modulating the host immune response. The absence of Ly6Chi MOs is associated with severe virus-induced immunopathology and the systemic release of inflammatory mediators. Mechanistically, MuHV-4–imprinted MOs recruit CD4 T cells to the airways and trigger immunosuppressive signaling pathways through the PD-L1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in modulating CD4 T cell functions and reveal pathways that could be targeted therapeutically to reduce detrimental immunopathological responses associated with respiratory viral infections. Monocyte mediator: Gammaherpesviruses (γHVs) coevolved with their hosts in a manner that allows for the lifelong persistence of these viruses in immunocompetent individuals. The presence of latent γHVs in host organisms has been shown to shape adaptive immunity, and here, Maquet et al. observed that pulmonary infection of mice with a murine γHV (MuHV-4) leads to recruitment of Ly6Chi monocytes (MOs) to airways. These γHV-imprinted Ly6Chi MOs recruit CD4 T cells to airways followed by engagement of the immunosuppressive PD-1/PD-L1 signaling pathway, which limits pathological cytotoxic CD4 T cell responses. These findings highlight a role for γHV-imprinted Ly6Chi MOs in shaping CD4 T cell functions and suggest a potential pathway for the development of therapeutics that target respiratory viral infections. [ABSTRACT FROM AUTHOR]

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques. Both routes of vaccination induced a strong envelope-specific IgG in serum and rectal secretions directed against V1V2 scaffolds from a global panel of viruses with polyfunctional activities. Envelope-specific IgG showed lower fucosylation compared with total IgG at baseline, and most of the vaccine-induced proliferating blood CD4+ T cells did not express CCR5 and α4β7, markers associated with HIV target cells. After SHIV challenge, both routes of vaccination conferred significant and equivalent protection, with 40% of animals remaining uninfected at the end of six weekly repeated challenges with an estimated efficacy of 68% per exposure. Induction of envelope-specific IgG correlated positively with G1FB glycosylation, and G2S2F glycosylation correlated negatively with protection. Vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CD4+ T cells expressing low levels of CCR5 in the rectum at prechallenge were associated with decreased risk of SHIV acquisition. These results demonstrate that the clade C MVA/CycP-gp120 vaccine provides heterologous protection against a tier2 SHIV rectal challenge by inducing a polyfunctional antibody response with distinct Fc glycosylation profile, as well as cytotoxic CD8 T cell response and CCR5-negative T helper response in the rectum. A clade C HIV-1 vaccine: Clade C HIV-1 infections are responsible for a large proportion of global HIV-1 burden, and a vaccine is urgently required. Sahoo et al. created a clade C HIV-1 vaccination regimen that they tested in rhesus macaques, consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120). To assess delivery methods, vaccines were administered by parenteral injection or oral administration using a needle-free injector. Both routes of MVA/CycP-gp120 administration provided equivalent protection against mucosal SHIV infection. Reduced risk of infection correlated with distinct env-specific IgG Fc glycosylation profiles, as well as with vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CCR5lo CD4+ T cells. These findings demonstrate that the MVA/CycP-gp120 immunization regimen provides significant protection against heterologous SHIV infection. [ABSTRACT FROM AUTHOR]

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses. Spacing of immune cells matters: A better understanding of the tumor immune microenvironment can determine biomarkers of immunotherapy effectiveness. Moldoveanu et al. used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to characterize the immune microenvironment of melanoma patient samples. CyTOF IMC identified the spatial arrangement and proximity of immune cells to each other. The researchers observed that a closer proximity of antigen-experienced cytotoxic T cells to melanoma cells intratumorally correlated to a patient's responsiveness to immune checkpoint blockade. Thus, spatial characterization of the tumor immune microenvironment using CyTOF IMC allows for the identification of prognostic biomarkers for immunotherapy. [ABSTRACT FROM AUTHOR]

Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy. Encoding human dendritic cell fate: Recent advances in human dendritic cell (DC) biology have been accelerated by single-cell technologies, but DCs remain challenging to study because of their rarity and localization within tissues. Rosa et al. used single-cell transcriptomics to guide reprogramming of human fibroblasts into induced DCs and benchmarked their function against blood-derived DCs. Enforced expression of the transcription factors PU.1, IRF8, and BATF3 induced a small population resembling type 1 conventional DCs (cDC1s), which could be improved to more than 75% efficiency with a constitutive promoter and the inflammatory cytokines IFN-γ, IFN-β, and TNF-α. PU.1 chromatin binding early during reprogramming recruited IRF8 and BATF3 to facilitate cDC1-associated gene expression. Systems to generate functional patient-specific DCs represent a promising tool for studying human DCs and developing novel immunotherapies. [ABSTRACT FROM AUTHOR]

Interleukin-12 (IL-12) plays a critical role in modulating the tumor immune environment, including activating and expanding tumor-infiltrating lymphocytes (TILs). (C) Tumor growth curves of C57BL/6N mice (n = 8 per group) bearing MC38 tumors that were depleted of T cells and then treated with PBS or 3.33 pmol of pro-IL-12 by intraperitoneal injection on days 13, 16, and 19. (D) Tumor growth curves of MC38 tumor-bearing mice (n = 11 per group) given FTY720 and then treated with 3.33 pmol of pro-IL-12 by intraperitoneal injection on days 11, 14, and 17. A next-generation tumor-targeting IL-2 preferentially promotes tumor-infiltrating CD8+ T-cell response and effective tumor control. [Extracted from the article]

keyimage.jpg Baseline killers aid immune checkpoint blockade: CD8+ T cells are crucial for the efficacy of immune checkpoint blockades (ICB), but it is unclear how specific T cell subsets or clones affect efficacy. Here, Watson et al. used transcriptomic approaches to phenotype the subsets and TCR clones of peripheral CD8+ T cells in eight patients with metastatic melanoma before and during ICB. They found that many of the expanded T cell clones during ICB were from a pool of cytotoxic T cell clones present pretreatment. In a larger independent clinical dataset of patients with melanoma, the authors found that the clone size and cytotoxicity of CD8+ T cells before treatment positively correlated to ICB efficacy. Together, these data suggest that targeting preexisting cytotoxic CD8+ T cell clones might improve the antitumor responses of ICB. The antitumor action of immune checkpoint blockade (ICB) is primarily mediated by CD8+ T cells. How sensitivity to ICB varies across CD8+ T cell subsets and clonotypes and the relationship of these with clinical outcome is unclear. To explore this, we used single-cell V(D)J and RNA-sequencing to track gene expression changes elicited by ICB across individual peripheral CD8+ T cell clones, identify baseline markers of CD8+ T cell clonal sensitivity, and chart how CD8+ T cell transcriptional changes vary according to phenotypic subset and clonal size. We identified seven subsets of CD8+ T cells with divergent reactivity to ICB and found that the cytotoxic effector subset showed the greatest number of differentially expressed genes while remaining stable in clonal size after ICB. At the level of CD8+ T cell clonotypes, we found a relationship between transcriptional changes and clone size, with large clones showing a greater number of differentially regulated genes enriched for pathways including T cell receptor (TCR) signaling. Cytotoxic CD8+ effector clones were more likely to persist following ICB and were more likely to correspond with public tumor-infiltrating lymphocyte clonotypes. Last, we demonstrated that individuals whose CD8+ T cell pretreatment showed low cytotoxicity and had fewer expanded clones typically had worse outcomes after ICB treatment. This work further advances understanding of the molecular determinants of ICB response, assisting in the search for peripheral prognostic biomarkers and highlighting the importance of the baseline CD8+ immune landscape in determining ICB response in metastatic melanoma. [ABSTRACT FROM AUTHOR]