The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity.

The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues.

Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells.

Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges 1 . Here, we developed a high-throughput approach for high-efficiency and massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale screens editing 102 genes with central functions in T cells and full-length tiling mutagenesis of selected genes, and read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. We discovered a broad landscape of gain- and loss-of-function mutations, including in PIK3CD and its regulatory subunit encoded by PIK3R1, LCK , AKT1, CTLA-4 and JAK1 . We identified variants that affected several (e.g., PIK3CD C416R) or only selected (e.g. LCK Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation. Using primary human T cells in which we engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, we demonstrate that base edits identified in our screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. In summary, we present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at high efficiency. Coupled with multi-modal phenotypic mapping, we accurately nominate variants that produce a desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies.

Cutting Edge: Promoting T Cell Factor 1+ T Cell Self-Renewal to Improve Programmed Cell Death Protein 1 Blockade.

Immune checkpoint blockade is limited by resistance to treatment, with many patients not achieving durable antitumor responses. Self-renewing (T cell factor 1+ [TCF1+]) CD8+ T cells have recently been implicated in efficacy of anti-programmed cell death protein 1 (anti-PD-1). Mice challenged with syngeneic tumors were treated with anti-PD-1 and/or a reversible inhibitor of PI3K δ, designed to promote T cell self-renewal. Growth of tumors in untreated mice was characterized by waning proportions of TCF1+ T cells, suggesting self-renewing T cells become limiting for successful immunotherapy. Higher proportions of TCF1+ T cells in tumor and blood correlated with better control of tumor growth. Combining anti-PD-1 and inhibitor of PI3K δ conferred superior protection compared with either monotherapy and was associated with higher frequency of TCF1+ T cells in tumor and blood compared with anti-PD-1 alone. These findings reveal predictive importance of self-renewing T cells in anti-tumor immunity and suggest that resistance-directed strategies to enhance T cell self-renewal could potentiate the efficacy of PD-1 blockade.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.