Most clinically applied cancer immunotherapies rely on the ability of CD8⁺ cytolytic T cells to directly recognize and kill tumour cells^1–3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment^4–6. The ability of CD4⁺ effector cells to contribute to antitumour immunity independently of CD8⁺ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified^7–10. Here, we describe a mechanism whereby a small number of CD4⁺ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8⁺ T cell targeting. The CD4⁺ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II⁺CD11c⁺ antigen-presenting cells. We show that T helper type 1 cell-directed CD4⁺ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4⁺ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4⁺ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8⁺ T cells and natural killer cells and advance cancer immunotherapies.

Rejection remains the main cause of premature graft loss after kidney transplantation, despite the use of potent immunosuppression. This highlights the need to better understand the composition and the cell-to-cell interactions of the alloreactive inflammatory infiltrate. Here, we performed droplet-based single-cell RNA sequencing of 35,152 transcriptomes from 16 kidney transplant biopsies with varying phenotypes and severities of rejection and without rejection, and identified cell-type specific gene expression signatures for deconvolution of bulk tissue. A specific association was identified between recipient-derived FCGR3A+ monocytes, FCGR3A+ NK cells and the severity of intragraft inflammation. Activated FCGR3A+ monocytes overexpressed CD47 and LILR genes and increased paracrine signaling pathways promoting T cell infiltration. FCGR3A+ NK cells overexpressed FCRL3, suggesting that antibody-dependent cytotoxicity is a central mechanism of NK-cell mediated graft injury. Multiplexed immunofluorescence using 38 markers on 18 independent biopsy slides confirmed this role of FcγRIII+ NK and FcγRIII+ nonclassical monocytes in antibody-mediated rejection, with specificity to the glomerular area. These results highlight the central involvement of innate immune cells in the pathogenesis of allograft rejection and identify several potential therapeutic targets that might improve allograft longevity.

Clinically relevant immunological biomarkers that discriminate between diverse hypofunctional states of tumor-associated CD8 ⁺ T cells remain disputed. Using multiomics analysis of CD8 ⁺ T cell features across multiple patient cohorts and tumor types, we identified tumor niche–dependent exhausted and other types of hypofunctional CD8 ⁺ T cell states. CD8 ⁺ T cells in “supportive” niches, like melanoma or lung cancer, exhibited features of tumor reactivity–driven exhaustion (CD8 ⁺ T _EX ). These included a proficient effector memory phenotype, an expanded T cell receptor (TCR) repertoire linked to effector exhaustion signaling, and a cancer-relevant T cell–activating immunopeptidome composed of largely shared cancer antigens or neoantigens. In contrast, “nonsupportive” niches, like glioblastoma, were enriched for features of hypofunctionality distinct from canonical exhaustion. This included immature or insufficiently activated T cell states, high wound healing signatures, nonexpanded TCR repertoires linked to anti-inflammatory signaling, high T cell–recognizable self-epitopes, and an antiproliferative state linked to stress or prodeath responses. In situ spatial mapping of glioblastoma highlighted the prevalence of dysfunctional CD4 ⁺ :CD8 ⁺ T cell interactions, whereas ex vivo single-cell secretome mapping of glioblastoma CD8 ⁺ T cells confirmed negligible effector functionality and a promyeloid, wound healing–like chemokine profile. Within immuno-oncology clinical trials, anti–programmed cell death protein 1 (PD-1) immunotherapy facilitated glioblastoma’s tolerogenic disparities, whereas dendritic cell (DC) vaccines partly corrected them. Accordingly, recipients of a DC vaccine for glioblastoma had high effector memory CD8 ⁺ T cells and evidence of antigen-specific immunity. Collectively, we provide an atlas for assessing different CD8 ⁺ T cell hypofunctional states in immunogenic versus nonimmunogenic cancers.

While immune checkpoint–based immunotherapy (ICI) shows promising clinical results in patients with cancer, only a subset of patients responds favorably. Response to ICI is dictated by complex networks of cellular interactions between malignant and nonmalignant cells. Although insights into the mechanisms that modulate the pivotal antitumoral activity of cytotoxic T cells (Tcy) have recently been gained, much of what has been learned is based on single-cell analyses of dissociated tumor samples, resulting in a lack of critical information about the spatial distribution of relevant cell types. Here, we used multiplexed IHC to spatially characterize the immune landscape of metastatic melanoma from responders and nonresponders to ICI. Such high-dimensional pathology maps showed that Tcy gradually evolve toward an exhausted phenotype as they approach and infiltrate the tumor. Moreover, a key cellular interaction network functionally linked Tcy and PD-L1+ macrophages. Mapping the respective spatial distributions of these two cell populations predicted response to anti-PD-1 immunotherapy with high confidence. These results suggest that baseline measurements of the spatial context should be integrated in the design of predictive biomarkers to identify patients likely to benefit from ICI. Significance: This study shows that spatial characterization can address the challenge of finding efficient biomarkers, revealing that localization of macrophages and T cells in melanoma predicts patient response to ICI. See related commentary by Smalley and Smalley, p. 3198

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.