Designing Cancer Immunotherapies That Engage T Cells and NK Cells.

T cells and natural killer (NK) cells have complementary roles in tumor immunity, and dual T cell and NK cell attack thus offers opportunities to deepen the impact of immunotherapy. Recent work has also shown that NK cells play an important role in recruiting dendritic cells to tumors and thus enhance induction of CD8 T cell responses, while IL-2 secreted by T cells activates NK cells. Targeting of immune evasion mechanisms from the activating NKG2D receptor and its MICA and MICB ligands on tumor cells offers opportunities for therapeutic intervention. Interestingly, T cells and NK cells share several important inhibitory and activating receptors that can be targeted to enhance T cell- and NK cell-mediated immunity. These inhibitory receptor-ligand systems include CD161-CLEC2D, TIGIT-CD155, and NKG2A/CD94-HLA-E. We also discuss emerging therapeutic strategies based on inhibitory and activating cytokines that profoundly impact the function of both lymphocyte populations within tumors.

Targeting of the CD161 inhibitory receptor enhances T-cell-mediated immunity against hematological malignancies.

ABSTRACT: The CD161 inhibitory receptor is highly upregulated by tumor-infiltrating T cells in multiple human solid tumor types, and its ligand, CLEC2D, is expressed by both tumor cells and infiltrating myeloid cells. Here, we assessed the role of the CD161 receptor in hematological malignancies. Systematic analysis of CLEC2D expression using the Cancer Cell Line Encyclopedia revealed that CLEC2D messenger RNA was most abundant in hematological malignancies, including B-cell and T-cell lymphomas as well as lymphocytic and myelogenous leukemias. CLEC2D protein was detected by flow cytometry on a panel of cell lines representing a diverse set of hematological malignancies. We, therefore, used yeast display to generate a panel of high-affinity, fully human CD161 monoclonal antibodies (mAbs) that blocked CLEC2D binding. These mAbs were specific for CD161 and had a similar affinity for human and nonhuman primate CD161, a property relevant for clinical translation. A high-affinity CD161 mAb enhanced key aspects of T-cell function, including cytotoxicity, cytokine production, and proliferation, against B-cell lines originating from patients with acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and Burkitt lymphoma. In humanized mouse models, this CD161 mAb enhanced T-cell-mediated immunity, resulting in a significant survival benefit. Single cell RNA-seq data demonstrated that CD161 mAb treatment enhanced expression of cytotoxicity genes by CD4 T cells as well as a tissue-residency program by CD4 and CD8 T cells that is associated with favorable survival outcomes in multiple human cancer types. These fully human mAbs, thus, represent potential immunotherapy agents for hematological malignancies.

Ralstonia solanacearum Effectors Localize to Diverse Organelles in Solanum Hosts.

Phytopathogenic bacteria secrete type III effector (T3E) proteins directly into host plant cells. T3Es can interact with plant proteins and frequently manipulate plant host physiological or developmental processes. The proper subcellular localization of T3Es is critical for their ability to interact with plant targets, and knowledge of T3E localization can be informative for studies of effector function. Here we investigated the subcellular localization of 19 T3Es from the phytopathogenic bacteria Ralstonia pseudosolanacearum and Ralstonia solanacearum. Approximately 45% of effectors in our library localize to both the plant cell periphery and the nucleus, 15% exclusively to the cell periphery, 15% exclusively to the nucleus, and 25% to other organelles, including tonoplasts and peroxisomes. Using tomato hairy roots, we show that T3E localization is similar in both leaves and roots and is not impacted by Solanum species. We find that in silico prediction programs are frequently inaccurate, highlighting the value of in planta localization experiments. Our data suggest that Ralstonia targets a wide diversity of cellular organelles and provides a foundation for developing testable hypotheses about Ralstonia effector function.

Increasing transposase abundance with ocean depth correlates with a particle-associated lifestyle.

Transposases are mobile genetic elements that move within and between genomes, promoting genomic plasticity in microorganisms. In marine microbial communities, the abundance of transposases increases with depth, but the reasons behind this trend remain unclear. Our analysis of metagenomes from the Tara Oceans and Malaspina Expeditions suggests that a particle-associated lifestyle is the main covariate for the high occurrence of transposases in the deep ocean, and this trend holds true for individual genomes as well as in a community-wide sense. We observed a strong and depth-independent correlation between transposase abundance and the presence of biofilm-associated genes, as well as the prevalence of secretory enzymes. This suggests that mobile genetic elements readily propagate among microbial communities within crowded biofilms. Furthermore, we show that particle association positively correlates with larger genome size, which is in turn associated with higher transposase abundance. Cassette sequences associated with transposons are enriched with genes related to defense mechanisms, which are more highly expressed in the deep sea. Thus, while transposons spread at the expense of their microbial hosts, they also introduce novel genes and potentially benefit the hosts in helping to compete for limited resources. Overall, our results suggest a new understanding of deep ocean particles as highways for gene sharing among defensively oriented microbial genomes.IMPORTANCEGenes can move within and between microbial genomes via mobile genetic elements, which include transposases and transposons. In the oceans, there is a puzzling increase in transposase abundance in microbial genomes as depth increases. To gain insight into this trend, we conducted an extensive analysis of marine microbial metagenomes and metatranscriptomes. We found a significant correlation between transposase abundance and a particle-associated lifestyle among marine microbes at both the metagenome and genome-resolved levels. We also observed a link between transposase abundance and genes related to defense mechanisms. These results suggest that as microbes become densely packed into crowded particles, mobile genes are more likely to spread and carry genetic material that provides a competitive advantage in crowded habitats. This may enable deep sea microbes to effectively compete in such environments.

Addressing Tumor Heterogeneity by Sensitizing Resistant Cancer Cells to T cell-Secreted Cytokines.

Tumor heterogeneity is a major barrier to cancer therapy, including immunotherapy. Activated T cells can efficiently kill tumor cells following recognition of MHC class I (MHC-I)-bound peptides, but this selection pressure favors outgrowth of MHC-I-deficient tumor cells. We performed a genome-scale screen to discover alternative pathways for T cell-mediated killing of MHC-I-deficient tumor cells. Autophagy and TNF signaling emerged as top pathways, and inactivation of Rnf31 (TNF signaling) and Atg5 (autophagy) sensitized MHC-I-deficient tumor cells to apoptosis by T cell-derived cytokines. Mechanistic studies demonstrated that inhibition of autophagy amplified proapoptotic effects of cytokines in tumor cells. Antigens from apoptotic MHC-I-deficient tumor cells were efficiently cross-presented by dendritic cells, resulting in heightened tumor infiltration by IFNγ-and TNFα-producing T cells. Tumors with a substantial population of MHC-I-deficient cancer cells could be controlled by T cells when both pathways were targeted using genetic or pharmacologic approaches. SIGNIFICANCE: Tumor heterogeneity is a major barrier to immunotherapy. We show that MHC-I-deficient tumor cells are forced into apoptosis by T cell-derived cytokines when TNF signaling and autophagy pathways are targeted. This approach enables T cell-mediated elimination of tumors with a substantial population of resistant, MHC-I-deficient tumor cells. This article is highlighted in the In This Issue feature, p. 1027.

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.

Vaccine efficacy against primary and metastatic cancer with in vitro-generated CD103+ conventional dendritic cells.

BACKGROUND: Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+ cDC1s in mice, CD141+ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied. METHODS: We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s. RESULTS: In vitro-generated CD103+ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+ CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. CONCLUSIONS: Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.