T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche.

Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.

Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

Therapy-Induced Evolution of Human Lung Cancer Revealed by Single-Cell RNA Sequencing.

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.

Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation.

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

Adaptive Immune Resistance Emerges from Tumor-Initiating Stem Cells.

Our bodies are equipped with powerful immune surveillance to clear cancerous cells as they emerge. How tumor-initiating stem cells (tSCs) that form and propagate cancers equip themselves to overcome this barrier remains poorly understood. To tackle this problem, we designed a skin cancer model for squamous cell carcinoma (SCC) that can be effectively challenged by adoptive cytotoxic T cell transfer (ACT)-based immunotherapy. Using single-cell RNA sequencing (RNA-seq) and lineage tracing, we found that transforming growth factor ß (TGF-ß)-responding tSCs are superior at resisting ACT and form the root of tumor relapse. Probing mechanism, we discovered that during malignancy, tSCs selectively acquire CD80, a surface ligand previously identified on immune cells. Moreover, upon engaging cytotoxic T lymphocyte antigen-4 (CTLA4), CD80-expressing tSCs directly dampen cytotoxic T cell activity. Conversely, upon CTLA4- or TGF-ß-blocking immunotherapies or Cd80 ablation, tSCs become vulnerable, diminishing tumor relapse after ACT treatment. Our findings place tSCs at the crux of how immune checkpoint pathways are activated.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Inactivation of CDK12 Delineates a Distinct Immunogenic Class of Advanced Prostate Cancer.

Using integrative genomic analysis of 360 metastatic castration-resistant prostate cancer (mCRPC) samples, we identified a novel subtype of prostate cancer typified by biallelic loss of CDK12 that is mutually exclusive with tumors driven by DNA repair deficiency, ETS fusions, and SPOP mutations. CDK12 loss is enriched in mCRPC relative to clinically localized disease and characterized by focal tandem duplications (FTDs) that lead to increased gene fusions and marked differential gene expression. FTDs associated with CDK12 loss result in highly recurrent gains at loci of genes involved in the cell cycle and DNA replication. CDK12 mutant cases are baseline diploid and do not exhibit DNA mutational signatures linked to defects in homologous recombination. CDK12 mutant cases are associated with elevated neoantigen burden ensuing from fusion-induced chimeric open reading frames and increased tumor T cell infiltration/clonal expansion. CDK12 inactivation thereby defines a distinct class of mCRPC that may benefit from immune checkpoint immunotherapy.

An Immune Atlas of Clear Cell Renal Cell Carcinoma.

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.

Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.

CD300ld on neutrophils is required for tumour-driven immune suppression.

The immune-suppressive tumour microenvironment represents a major obstacle to effective immunotherapy1,2. Pathologically activated neutrophils, also known as polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), are a critical component of the tumour microenvironment and have crucial roles in tumour progression and therapy resistance2-4. Identification of the key molecules on PMN-MDSCs is required to selectively target these cells for tumour treatment. Here, we performed an in vivo CRISPR-Cas9 screen in a tumour mouse model and identified CD300ld as a top candidate of tumour-favouring receptors. CD300ld is specifically expressed in normal neutrophils and is upregulated in PMN-MDSCs upon tumour-bearing. CD300ld knockout inhibits the development of multiple tumour types in a PMN-MDSC-dependent manner. CD300ld is required for the recruitment of PMN-MDSCs into tumours and their function to suppress T cell activation. CD300ld acts via the STAT3-S100A8/A9 axis, and knockout of Cd300ld reverses the tumour immune-suppressive microenvironment. CD300ld is upregulated in human cancers and shows an unfavourable correlation with patient survival. Blocking CD300ld activity inhibits tumour development and has synergistic effects with anti-PD1. Our study identifies CD300ld as a critical immune suppressor present on PMN-MDSCs, being required for tumour immune resistance and providing a potential target for cancer immunotherapy.

TET2 guards against unchecked BATF3-induced CAR T cell expansion.

Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.

Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy.

Extracellular deposition of amyloid-ß as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles are two of the characteristic hallmarks of Alzheimer's disease1,2. The regional progression of brain atrophy in Alzheimer's disease highly correlates with tau accumulation but not amyloid deposition3-5, and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. So far, little is known about the extent or role of the adaptive immune response and its interaction with the innate immune response in the presence of amyloid-ß or tau pathology6. Here we systematically compared the immunological milieux in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not those with amyloid deposition developed a unique innate and adaptive immune response and that depletion of microglia or T cells blocked tau-mediated neurodegeneration. Numbers of T cells, especially those of cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the Alzheimer's disease brain. T cell numbers correlated with the extent of neuronal loss, and the cells dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of interferon-γ and PDCD1 signalling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy- and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in Alzheimer's disease and primary tauopathies.

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours.

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

Regulation and impact of tumor-specific CD4+ T cells in cancer and immunotherapy.

CD4+ T cells are crucial in generating and sustaining immune responses. They orchestrate and fine-tune mammalian innate and adaptive immunity through cell-based interactions and the release of cytokines. The role of these cells in contributing to the efficacy of antitumor immunity and immunotherapy has just started to be uncovered. Yet, many aspects of the CD4+ T cell response are still unclear, including the differentiation pathways controlling such cells during cancer progression, the external signals that program them, and how the combination of these factors direct ensuing immune responses or immune-restorative therapies. In this review, we focus on recent advances in understanding CD4+ T cell regulation during cancer progression and the importance of CD4+ T cells in immunotherapies.

Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model.

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.