An autoimmune stem-like CD8 T cell population drives type 1 diabetes.

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis.

CD8(+) T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Here we developed a tamoxifen-inducible liver cancer mouse model with a defined oncogenic driver antigen (SV40 large T-antigen) to follow the activation and differentiation of naive tumor-specific CD8(+) T (TST) cells after tumor initiation. Early during the pre-malignant phase of tumorigenesis, TST cells became dysfunctional, exhibiting phenotypic, functional, and transcriptional features similar to dysfunctional T cells isolated from late-stage human tumors. Thus, T cell dysfunction seen in advanced human cancers may already be established early during tumorigenesis. Although the TST cell dysfunctional state was initially therapeutically reversible, it ultimately evolved into a fixed state. Persistent antigen exposure rather than factors associated with the tumor microenvironment drove dysfunction. Moreover, the TST cell differentiation and dysfunction program exhibited features distinct from T cell exhaustion in chronic infections. Strategies to overcome this antigen-driven, cell-intrinsic dysfunction may be required to improve cancer immunotherapy.

TOX is a critical regulator of tumour-specific T cell differentiation.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Chromatin states define tumour-specific T cell dysfunction and reprogramming.

Tumour-specific CD8 T cells in solid tumours are dysfunctional, allowing tumours to progress. The epigenetic regulation of T cell dysfunction and therapeutic reprogrammability (for example, to immune checkpoint blockade) is not well understood. Here we show that T cells in mouse tumours differentiate through two discrete chromatin states: a plastic dysfunctional state from which T cells can be rescued, and a fixed dysfunctional state in which the cells are resistant to reprogramming. We identified surface markers associated with each chromatin state that distinguished reprogrammable from non-reprogrammable PD1hi dysfunctional T cells within heterogeneous T cell populations from tumours in mice; these surface markers were also expressed on human PD1hi tumour-infiltrating CD8 T cells. Our study has important implications for cancer immunotherapy as we define key transcription factors and epigenetic programs underlying T cell dysfunction and surface markers that predict therapeutic reprogrammability.

Beyond Genomics: Multidimensional Analysis of Cancer Therapy Resistance.

Cancer resistance to therapy occurs through a selection process generally thought to be driven by mutations. In a recent study, Hugo et al. use multidimensional analysis of the dynamic genetic, transcriptional, epigenetic, and immune landscape alterations in baseline and MAPK inhibitor-resistant melanoma tumors, demonstrating a role for 'non-genomic' drivers in cancer evolution.

Tolerance and exhaustion: defining mechanisms of T cell dysfunction.

CD8 T cell activation and differentiation are tightly controlled, and dependent on the context in which naïve T cells encounter antigen, can either result in functional memory or T cell dysfunction, including exhaustion, tolerance, anergy, or senescence. With the identification of phenotypic and functional traits shared in different settings of T cell dysfunction, distinctions between such dysfunctional states have become blurred. Here, we discuss distinct states of CD8 T cell dysfunction, with an emphasis on: (i) T cell tolerance to self-antigens (self-tolerance); (ii) T cell exhaustion during chronic infections; and (iii) tumor-induced T cell dysfunction. We highlight recent findings on cellular and molecular characteristics defining these states, cell-intrinsic regulatory mechanisms that induce and maintain them, and strategies that can lead to their reversal.

IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner.

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Cell-intrinsic abrogation of TGF-ß signaling delays but does not prevent dysfunction of self/tumor-specific CD8 T cells in a murine model of autochthonous prostate cancer.

Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of antitumor activity of transferred T cells remain major problems. TGF-ß is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell-mediated antitumor activity. In this study, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell-intrinsic abrogation of TGF-ß signaling in self/tumor-specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and antitumor activity of adoptively transferred effector T cells deficient in TGF-ß signaling were significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate-infiltrating T cells were no longer functional. These findings reveal that TGF-ß negatively regulates the accumulation and effector function of transferred self/tumor-specific CD8 T cells and highlight that, when targeting a tumor Ag that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.

Intratumoral immune triads are required for immunotherapy-mediated elimination of solid tumors.

Tumor-specific CD8+ T cells are frequently dysfunctional and unable to halt tumor growth. We investigated whether tumor-specific CD4+ T cells can be enlisted to overcome CD8+ T cell dysfunction within tumors. We find that the spatial positioning and interactions of CD8+ and CD4+ T cells, but not their numbers, dictate anti-tumor responses in the context of adoptive T cell therapy as well as immune checkpoint blockade (ICB): CD4+ T cells must engage with CD8+ T cells on the same dendritic cell during the effector phase, forming a three-cell-type cluster (triad) to license CD8+ T cell cytotoxicity and cancer cell elimination. When intratumoral triad formation is disrupted, tumors progress despite equal numbers of tumor-specific CD8+ and CD4+ T cells. In patients with pleural mesothelioma treated with ICB, triads are associated with clinical responses. Thus, CD4+ T cells and triads are required for CD8+ T cell cytotoxicity during the effector phase and tumor elimination.

Multiplexed spatial profiling of Hodgkin Reed-Sternberg cell neighborhoods in classic Hodgkin lymphoma.

PURPOSE: Classic Hodgkin lymphoma (cHL) is a B cell lymphoma that occurs primarily in young adults and, less frequently, in elderly individuals. A hallmark of cHL is the exceptional scarcity (1-5%) of the malignant Hodgkin Reed-Sternberg (HRS) cells within a network of non-malignant immune cells. Molecular determinants governing the relationship between HRS cells and their proximal microenvironment remain largely unknown. EXPERIMENTAL DESIGN: We performed spatially resolved multiplexed protein imaging and transcriptomic sequencing to characterize HRS cell states, cellular neighborhoods, and gene expression signatures of 23.6 million cells from 36 newly diagnosed Epstein-Barr virus (EBV) positive and EBV-negative cHL tumors. RESULTS: We show that MHC-I expression on HRS cells is associated with immune inflamed neighborhoods containing CD8+ T cells, MHC-II+ macrophages, and immune checkpoint expression (i.e., PD-1 and VISTA). We identified spatial clustering of HRS cells, consistent with the syncytial variant of cHL, and its association with T cell excluded neighborhoods in a subset of EBV-negative tumors. Finally, a subset of both EBV-positive and EBV-negative tumors contained regulatory T cells high neighborhoods harboring HRS cells with augmented proliferative capacity. CONCLUSIONS: Our study links HRS cell properties with distinct immunophenotypes and potential immune escape mechanisms in cHL.

Induced sensitization of tumor stroma leads to eradication of established cancer by T cells.

Targeting cancer cells, as well as the nonmalignant stromal cells cross-presenting the tumor antigen (Ag), can lead to the complete destruction of well-established solid tumors by adoptively transferred Ag-specific cytotoxic T lymphocytes (CTLs). If, however, cancer cells express only low levels of the Ag, then stromal cells are not destroyed, and the tumor escapes as Ag loss variants. We show that treating well-established tumors expressing low levels of Ag with local irradiation or a chemotherapeutic drug causes sufficient release of Ag to sensitize stromal cells for destruction by CTLs. This was shown directly using high affinity T cell receptor tetramers for visualizing the transient appearance of tumor-specific peptide-MHC complexes on stromal cells. Maximum loading of tumor stroma with cancer Ag occurred 2 d after treatment and coincided with the optimal time for T cell transfer. Under these conditions, tumor rejection was complete. These findings may set the stage for developing rational clinical protocols for combining irradiation or chemotherapy with CTL therapy.

Antibody recognition of a unique tumor-specific glycopeptide antigen.

Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the "Tn antigen." Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen.

Intratumoral immune triads are required for adoptive T cell therapy-mediated elimination of solid tumors.

Tumor-reactive CD8 T cells found in cancer patients are frequently dysfunctional, unable to halt tumor growth. Adoptive T cell transfer (ACT), the administration of large numbers of in vitro-generated cytolytic tumor-reactive CD8 T cells, is an important cancer immune therapy being pursued. However, a limitation of ACT is that transferred CD8 T cells often rapidly lose effector function, and despite exciting results in certain malignancies, few ACT clinical trials have shown responses in solid tumors. Here, we developed preclinical cancer mouse models to investigate if and how tumor-specific CD4 T cells can be enlisted to overcome CD8 T cell dysfunction in the setting of ACT. In situ confocal microscopy of color-coded cancer cells, tumor-specific CD8 and CD4 T cells, and antigen presenting cells (APC), combined with functional studies, revealed that the spatial positioning and interactions of CD8 and CD4 T cells, but not their numbers, dictates ACT efficacy and anti-tumor responses. We uncover a new role of antigen-specific CD4 T cells in addition to the known requirement for CD4 T cells during priming/activation of naïve CD8 T cells. CD4 T cells must co-engage with CD8 T cells and APC cross-presenting CD8- and CD4-tumor antigens during the effector phase, forming a three-cell-cluster (triad), to license CD8 T cell cytotoxicity and mediate cancer cell elimination. Triad formation transcriptionally and epigenetically reprogram CD8 T cells, prevent T cell dysfunction/exhaustion, and ultimately lead to the elimination of large established tumors and confer long-term protection from recurrence. When intratumoral triad formation was disrupted, adoptively transferred CD8 T cells could not be reprogrammed, and tumors progressed despite equal numbers of tumor-infiltrating CD8 and CD4 T cells. Strikingly, the formation of CD4 T cell::CD8 T cell::APC triads in tumors of patients with lung cancers treated with immune checkpoint blockade was associated with clinical responses, but not CD4::APC dyads or overall numbers of CD8 or CD4 T cells, demonstrating the importance of triads in non-ACT settings in humans. Our work uncovers intratumoral triads as a key requirement for anti-tumor immunity and a new role for CD4 T cells in CD8 T cell cytotoxicity and cancer cell eradication.

GABA Regulates Electrical Activity and Tumor Initiation in Melanoma.

Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell-cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. SIGNIFICANCE: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma. See related commentary by Ceol, p. 2128. This article is featured in Selected Articles from This Issue, p. 2109.

CD8+ T cell differentiation and dysfunction in cancer.

CD8+ T cells specific for cancer cells are detected within tumours. However, despite their presence, tumours progress. The clinical success of immune checkpoint blockade and adoptive T cell therapy demonstrates the potential of CD8+ T cells to mediate antitumour responses; however, most patients with cancer fail to achieve long-term responses to immunotherapy. Here we review CD8+ T cell differentiation to dysfunctional states during tumorigenesis. We highlight similarities and differences between T cell dysfunction and other hyporesponsive T cell states and discuss the spatio-temporal factors contributing to T cell state heterogeneity in tumours. An important challenge is predicting which patients will respond to immunotherapeutic interventions and understanding which T cell subsets mediate the clinical response. We explore our current understanding of what determines T cell responsiveness and resistance to immunotherapy and point out the outstanding research questions.

Noninvasive Imaging of CD4+ T Cells in Humanized Mice.

Antibody-based PET (immunoPET) with radiotracers that recognize specific cells of the immune system provides an opportunity to monitor immune cell trafficking at the organismal scale. We previously reported the visualization of human CD8+ T cells, including CD8+ tumor-infiltrating lymphocytes (TIL), in mice using a humanized CD8-targeted minibody. Given the important role of CD4+ T cells in adaptive immune responses of health and disease including infections, tumors, and autoimmunity, we explored immunoPET using an anti-human-CD4 minibody. We assessed the ability of [64Cu]Cu-NOTA-IAB41 to bind to various CD4+ T-cell subsets in vitro. We also determined the effect of the CD4-targeted minibody on CD4+ T-cell abundance, proliferation, and activation state in vitro. We subsequently evaluated the ability of the radiotracer to visualize CD4+ T cells in T-cell rich organs and orthotopic brain tumors in vivo. For the latter, we injected the [64Cu]Cu-NOTA-IAB41 radiotracer into humanized mice that harbored intracranial patient-derived glioblastoma (GBM) xenografts and performed in vivo PET, ex vivo autoradiography, and anti-CD4 IHC on serial brain sections. [64Cu]Cu-NOTA-IAB41 specifically detects human CD4+ T cells without impacting their abundance, proliferation, and activation. In humanized mice, [64Cu]Cu-NOTA-IAB41 can visualize various peripheral tissues in addition to orthotopically implanted GBM tumors. [64Cu]Cu-NOTA-IAB41 is able to visualize human CD4+ T cells in humanized mice and can provide noninvasive quantification of CD4+ T-cell distribution on the organismal scale.

Ketohexokinase-mediated fructose metabolism is lost in hepatocellular carcinoma and can be leveraged for metabolic imaging.

The ability to break down fructose is dependent on ketohexokinase (KHK) that phosphorylates fructose to fructose-1-phosphate (F1P). We show that KHK expression is tightly controlled and limited to a small number of organs and is down-regulated in liver and intestinal cancer cells. Loss of fructose metabolism is also apparent in hepatocellular adenoma and carcinoma (HCC) patient samples. KHK overexpression in liver cancer cells results in decreased fructose flux through glycolysis. We then developed a strategy to detect this metabolic switch in vivo using hyperpolarized magnetic resonance spectroscopy. Uniformly deuterating [2-13C]-fructose and dissolving in D2O increased its spin-lattice relaxation time (T1) fivefold, enabling detection of F1P and its loss in models of HCC. In summary, we posit that in the liver, fructolysis to F1P is lost in the development of cancer and can be used as a biomarker of tissue function in the clinic using metabolic imaging.