High levels of ecto-5′-nucleotidase (CD73) have been implicated in immune suppression and tumor progression, and have also been observed in cancer patients who progress on anti–PD-1 immunotherapy. Although regulatory T cells can express CD73 and inhibit T cell responses via the production of adenosine, less is known about CD73 expression in other immune cell populations. We found that tumor-infiltrating NK cells upregulate CD73 expression and the frequency of these CD73-positive NK cells correlated with larger tumor size in breast cancer patients. In addition, the expression of multiple alternative immune checkpoint receptors including LAG-3, VISTA, PD-1, and PD-L1 was significantly higher in CD73-positive NK cells than in CD73-negative NK cells. Mechanistically, NK cells transport CD73 in intracellular vesicles to the cell surface and the extracellular space via actin polymerization–dependent exocytosis upon engagement of 4-1BBL on tumor cells. These CD73-positive NK cells undergo transcriptional reprogramming and upregulate IL-10 production via STAT3 transcriptional activity, suppressing CD4-positive T cell proliferation and IFN-γ production. Taken together, our results support the notion that tumors can hijack NK cells as a means to escape immunity and that CD73 expression defines an inducible population of NK cells with immunoregulatory properties within the tumor microenvironment.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy.

Current methods for epigenomic profiling are limited in their ability to obtain genome-wide information with spatial resolution. We introduce spatial ATAC, a method that integrates transposase-accessible chromatin profiling in tissue sections with barcoded solid-phase capture to perform spatially resolved epigenomics. We show that spatial ATAC enables the discovery of the regulatory programs underlying spatial gene expression during mouse organogenesis, lineage differentiation and in human pathology.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell–to–myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell–derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m -knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell–mediated mechanism that drives the development of MDSCs during tumor immune escape.

Background: Adenosine is a metabolite that suppresses antitumor immune response of T and NK cells via extracellular binding to the two subtypes of adenosine-2 receptors, A₂ARs. While blockade of the A_2AARs subtype effectively rescues lymphocyte activity, with four A_2AAR antagonists currently in anticancer clinical trials, less is known for the therapeutic potential of the other A_2BAR blockade within cancer immunotherapy. Recent studies suggest the formation of A_2AAR/A_2BAR dimers in tissues that coexpress the two receptor subtypes, where the A_2BAR plays a dominant role, suggesting it as a promising target for cancer immunotherapy. Methods: We report the synthesis and functional evaluation of five potent A_2BAR antagonists and a dual A_2AAR/A_2BAR antagonist. The compounds were designed using previous pharmacological data assisted by modeling studies. Synthesis was developed using multicomponent approaches. Flow cytometry was used to evaluate the phenotype of T and NK cells on A_2BAR antagonist treatment. Functional activity of T and NK cells was tested in patient-derived tumor spheroid models. Results: We provide data for six novel small molecules: five A_2BAR selective antagonists and a dual A_2AAR/A_2BAR antagonist. The growth of patient-derived breast cancer spheroids is prevented when treated with A_2BAR antagonists. To elucidate if this depends on increased lymphocyte activity, immune cells proliferation, and cytokine production, lymphocyte infiltration was evaluated and compared with the potent A_2AAR antagonist AZD-4635. We find that A_2BAR antagonists rescue T and NK cell proliferation, IFNγ and perforin production, and increase tumor infiltrating lymphocytes infiltration into tumor spheroids without altering the expression of adhesion molecules. Conclusions: Our results demonstrate that A_2BAR is a promising target in immunotherapy, identifying ISAM-R56A as the most potent candidate for A_2BAR blockade. Inhibition of A_2BAR signaling restores T cell function and proliferation. Furthermore, A_2BAR and dual A_2AAR/A_2BAR antagonists showed similar or better results than A_2AAR antagonist AZD-4635 reinforcing the idea of dominant role of the A_2BAR in the regulation of the immune system.

Spatial and genomic heterogeneity of tumors are crucial factors influencing cancer progression, treatment, and survival. However, a technology for direct mapping the clones in the tumor tissue based on somatic point mutations is lacking. Here, we propose Tumoroscope, the first probabilistic model that accurately infers cancer clones and their localization in close to single-cell resolution by integrating pathological images, whole exome sequencing, and spatial transcriptomics data. In contrast to previous methods, Tumoroscope explicitly addresses the problem of deconvoluting the proportions of clones in spatial transcriptomics spots. Applied to a reference prostate cancer dataset and a newly generated breast cancer dataset, Tumoroscope reveals spatial patterns of clone colocalization and mutual exclusion in sub-areas of the tumor tissue. We further infer clone-specific gene expression levels and the most highly expressed genes for each clone. In summary, Tumoroscope enables an integrated study of the spatial, genomic, and phenotypic organization of tumors.