Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens.

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

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.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

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.

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.

Multiscale engineering of immune cells and lymphoid organs.

Immunoengineering applies quantitative and materials-based approaches for the investigation of the immune system and for the development of therapeutic solutions for various diseases, such as infection, cancer, inflammatory diseases and age-related malfunctions. The design of immunomodulatory and cell therapies requires the precise understanding of immune cell formation and activation in primary, secondary and ectopic tertiary immune organs. However, the study of the immune system has long been limited to in vivo approaches, which often do not allow multidimensional control of intracellular and extracellular processes, and to 2D in vitro models, which lack physiological relevance. 3D models built with synthetic and natural materials enable the structural and functional recreation of immune tissues. These models are being explored for the investigation of immune function and dysfunction at the cell, tissue and organ levels. In this Review, we discuss 2D and 3D approaches for the engineering of primary, secondary and tertiary immune structures at multiple scales. We highlight important insights gained using these models and examine multiscale engineering strategies for the design and development of immunotherapies. Finally, dynamic 4D materials are investigated for their potential to provide stimuli-dependent and context-dependent scaffolds for the generation of immune organ models.

How Biophysical Forces Regulate Human B Cell Lymphomas.

The role of microenvironment-mediated biophysical forces in human lymphomas remains elusive. Diffuse large B cell lymphomas (DLBCLs) are heterogeneous tumors, which originate from highly proliferative germinal center B cells. These tumors, their associated neo-vessels, and lymphatics presumably expose cells to particular fluid flow and survival signals. Here, we show that fluid flow enhances proliferation and modulates response of DLBCLs to specific therapeutic agents. Fluid flow upregulates surface expression of B cell receptors (BCRs) and integrin receptors in subsets of ABC-DLBCLs with either CD79A/B mutations or WT BCRs, similar to what is observed with xenografted human tumors in mice. Fluid flow differentially upregulates signaling targets, such as SYK and p70S6K, in ABC-DLBCLs. By selective knockdown of CD79B and inhibition of signaling targets, we provide mechanistic insights into how fluid flow mechanomodulates BCRs and integrins in ABC-DLBCLs. These findings redefine microenvironment factors that regulate lymphoma-drug interactions and will be critical for testing targeted therapies.

Creating artificial lymphoid tissues to study immunity and hematological malignancies.

PURPOSE OF REVIEW: The specialized microenvironments of lymphoid tissue affect immune cell function and progression of disease. However, current animal models are low throughput and a large number of human diseases are difficult to model in animals. Animal models are less amenable to manipulation of tissue niche components, signalling pathways, epigenetics, and genome editing than ex vivo models. On the other hand, conventional 2D cultures lack the physiological relevance to study precise microenvironmental interactions. Thus, artificial tissues are being developed to study these interactions in the context of immune development, function, and disease. RECENT FINDINGS: New bone marrow and lymph node models have been created to, respectively, study microenvironmental interactions in hematopoiesis and germinal center-like biology. These models have also been extended to understand the effect of these interactions on the progression and therapeutic response in leukemia, multiple myeloma, and lymphoma. SUMMARY: 3D in-vitro immune models have elucidated new cellular, biochemical, and biophysical interactions as potential regulatory mechanisms, therapeutic targets, or biomarkers that previously could not be studied in animal models and conventional 2D cultures. Incorporation of advanced biomaterials, microfluidics, genome editing, and single-cell analysis tools will enable further studies of function, driver mutations, and tumor heterogeneity. Continual refinement will help inform the development of antibody and cell-based immunotherapeutics and patient-specific treatment plans.