Immunotherapies have had unprecedented success in the treatment of multiple cancer types, albeit with variable response rates. Unraveling the complex network of immune cells within the tumor microenvironment (TME) may provide additional insights to enhance antitumor immunity and improve clinical response. Many studies have shown that NK cells or innate lymphoid cells (ILC) have regulatory capacity. Here, we identified CD103 as a marker that was found on CD56+ cells that were associated with a poor proliferative capacity of tumor-infiltrating lymphocytes in culture. We further demonstrated that CD103+CD56+ ILCs isolated directly from tumors represented a distinct ILC population that expressed unique surface markers (such as CD49a and CD101), transcription factor networks, and transcriptomic profiles compared with CD103-CD56+ NK cells. Using single-cell multiomic and spatial approaches, we found that these CD103+CD56+ ILCs were associated with CD8+ T cells with reduced expression of granzyme B. Thus, this study identifies a population of CD103+CD56+ ILCs with potentially inhibitory functions that are associated with a TME that includes CD8+ T cells with poor antitumor activity. Further studies focusing on these cells may provide additional insights into the biology of an inhibitory TME.

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a molecularly distinct tyrosine kinase-driven cancer that has a high relapse rate and poor response toward combinatorial chemotherapy. Tyrosine kinase inhibitors (TKI) in the clinic improve the survival of patients with Ph-like ALL. Engineered antibody and cell-based immunotherapies can advance treatment for this genetic subtype of ALL. Allogeneic memory-like natural killer (ML-NK) cells have been used to treat leukemia and have shown a low risk of graft-versus-host reaction, which may be combined with leukemia epitope-targeting antibodies. However, mutation or pathway-directed TKI of Ph-like ALL can interfere with memory-function and antibody-dependent NK cell-mediated cytotoxicity (ADCC). Here, we explored the potential of ML-NK cells and Fc-enhanced CD19-ADCC in combination with TKI directed against kinase-driven leukemia models, including patient-derived xenografted Ph-like ALL. We demonstrate that receptor crosslinking in coculture with K562 feeder cells generated a robust memory-like state of NK cells than coactivation with soluble interleukins (ILs) 12, 15, and 18, as determined by genomic and functional studies. After receptor crosslinking and subsequent ILs-preactivation, the optimized ML-NK cells showed enhanced antileukemic effector functions which could compensate for exhausted B cell precursor leukemia-infiltrating primary NK cells. TKI differentially affected multiple features of NK cell biology including viability, expansion, metabolism, receptor repertoire and cytotoxicity. ADCC was maintained upon exposure to specific ABL or JAK-inhibitors, in contrast to the multi-target TKI dasatinib impeding SYK-dependent ADCC. In conclusion, optimized ML-NK cell and CD19 antibody-based immunotherapy combined with carefully selected TKI demonstrates significant in vitro treatment efficacy in kinase-driven leukemia.

Biomarkers of responsiveness to immune checkpoint blockade (ICB) are heavily sought given the breadth and depth of the use of ICB in cancer. PD-L1 expression was among the first biomarkers identified, but multiple factors have precluded more widespread use. In this issue, Galsky and colleagues utilize two separate PD-L1 assays to study urothelial carcinoma specimens and observe that SP142 (relative to 22C3) preferentially stains dendritic cells. These observations may help reconcile the discordant performance of the two PD-L1 assays in ICB-treated urothelial carcinoma while underscoring the role of dendritic cells in orchestrating ICB response. See related article by Galsky et al., p. 476 .

Memory-phenotype (MP) CD4+ T lymphocytes spontaneously develop in steady state from peripheral naïve precursors in a manner dependent on self-antigen recognition. While MP cells possess innate type 1 and 3 effector functions that can contribute to host defense and autoimmunity, their immunological functions in tumor immunity and graft-versus-host disease (GVHD), which results from therapeutic bone marrow transplantation (BMT) against hematological malignancies, remain unclear. Here we show that in mixed lymphocyte reactions, MP lymphocytes can generate T helper 1 (Th1), T helper 17 (Th17), and regulatory T (Treg) cell subsets, whereas naïve cells dominantly differentiate to Th1. Consistent with this, naïve lymphocytes mainly induce Th1 responses in the mouse EL4 model of malignant lymphoma and the B16 model of malignant melanoma, whereas MP cells efficiently give rise to Th1, Th17, and Treg subsets to exert mild, IFN--dependent antitumor activities in vivo. Moreover, we demonstrate using a mouse model of BMT that MP cells more efficiently differentiate into Treg cells to partially suppress GVHD as compared to naïve T lymphocytes. Furthermore, our data suggest that when used as donor T lymphocytes in BMT in tumor-bearing mice, MP cells give rise to Th1, Th17, and Treg cells to generate antitumor responses without inducing GVHD. Together these results identify MP cells as a unique T-cell population that has potential to generate multiple T helper subsets including Th1 and Treg cells, thereby contributing to tumor immunity while inhibiting development of BMT-associated GVHD.

Local immunotherapy stimulates immune responses against tumors while avoiding adverse effects associated with systemic administration. However, current strategies for tumor-targeted in situ immunotherapy are still limited. mRNA-based gene therapy represents a promising strategy. Gasdermin E (GSDME)-mediated pyroptosis is reported to exert anti-tumor immunity. Here, we synthetized mRNA encoding GSDME encapsulated by lipid nanoparticles (LNP-Gsdme). In situ delivery of LNP-Gsdme through intratumoral injection suppressed tumor growth, boosted monocyte infiltration and activated CD8+T cells. LNP-Gsdme induced immunogenic cell death (ICD) in tumor cells, releasing creatine as a metabolic damage-associated molecular pattern. Creatine elicited cGAMP-STING-type I interferon signaling pathway in monocytes and reprogrammed intratumoral monocytes toward an immunostimulatory phenotype, consequently potentiating CD8+T cell-mediated anti-tumor immune responses. Furthermore, creatine supplementation enhanced the antitumor efficacy of LNP-Gsdme. Our study uncovers creatine as an important metabolic biomarker of pyroptosis-induced ICD in tumors, providing new insights and a promising therapeutic approach for in vivo mRNA-based immunotherapies for cancer.

The advent of syngeneic mouse tumor models provided the scientific foundation for cancer immunotherapies now in widespread use. However, in many respects, these models do not faithfully recapitulate the interactions between cancer cells and the immune systems of human patients who have solid tumors because they represent a very early stage in the immune response to the newly transplanted cancer cells compared with the relatively mature stage found in human patients at the time of treatment. The lack of translatability of syngeneic models is probably responsible for many failed clinical trials conducted at considerable expense, involving far too many patients with cancer who received no benefit. Better mouse models would substantially accelerate the pace of discovery of new immunotherapies. Until these models emerge, a better understanding of the differences between the existing syngeneic models and human cancers may provide a more efficient path for moving experimental drugs into clinical development. To accomplish this, we must consider mice transplanted with syngeneic tumor cells to be in vivo assays, potentially useful for understanding the mechanism of action of immunotherapies rather than disease models.

Cytotoxic chemotherapy that kills cancer cells can also elicit anti-tumor immune responses. Therefore, understanding the immunogenic context of cytotoxic chemotherapy can improve combination immunotherapies. In this study, we sought to improve our understanding about dendritic cell (DC) dynamics in cytotoxic chemotherapy-treated tumor tissues by developing 3D microfluidic devices that enable high-resolution visualization of cellular dynamics. Specifically, microfluidic chips mimicking 3D tumor tissues were fabricated and used. Collagen gel blocks encapsulating cancer cells in microfluidics were treated with various concentrations of oxaliplatin (OXP). Then, DCs were attached on the side of the collagen gel blocks, and migration of DCs within the 3D gels was quantitatively analyzed. Interactions between OXP-treated cancer cells and DCs were observed by high-resolution time-lapse imaging. Active infiltration of DCs was predominantly observed when OXP was administrated, indicating OXP-treated cancer cells release factors promoting DC motility. The highest frequency of DC recruitment was detected at a moderate OXP concentration, suggesting that optimizing the dosage of cytotoxic chemotherapy is crucial in order to improve immunogenic cell death (ICD). High-resolution video microscopy revealed that DCs employ trogocytosis to disassemble dying/dead cancer cells and acquire antigens, as opposed to phagocytosing the entire cancer cells. Microfluidic chip-based observations may provide new insights for the design of new therapeutic strategies to combine chemotherapy and immunotherapy.

mRNA vaccines are recognized as potent tools for immunization against viral diseases and cancer. However, the lack of a vaccine adjuvant limits the efficacy of these treatments. Here, we used cGAS mRNA, which encodes the DNA innate immune sensor, complexed with lipid nanoparticles (LNPs) to boost the immune response. By introducing specific mutations in human cGAS mRNA (hcGASK187N/L195R), we significantly enhanced cGAS activity, resulting in a more potent and sustained STING-mediated interferon (IFN) response. cGAS mRNA-LNPs exhibited stimulatory effects on maturation, antigen engulfment and antigen presentation by antigen-presenting cells (APCs) both in vitro and in vivo. Moreover, the hcGASK187N/L195R mRNA-LNP combination has shown a robust adjuvant effect, amplifying the potency of mRNA and protein vaccines by inducing strong humoral and cell-mediated responses. Remarkably, the hcGASK187N/L195R mRNA-LNP complex, either alone or in combination with antigens, demonstrated exceptional efficacy in eliciting antitumor immunity. In addition to its immune-boosting properties, hcGASK187N/L195R mRNA-LNP exerted synergistic antitumor effects with IFNγ directly on tumor cells, further promoting tumor restriction. In conclusion, we developed a cGAS-mRNA-based immunostimulatory adjuvant compatible with various vaccine forms to boost the adaptive immune response and cancer immunotherapies.

Agonistic anti-CD40 with anti-PD-1 can elicit objective responses in a small number of patients with pancreatic ductal adenocarcinoma (PDA). Better understanding of their individual effects on the PDA tumor microenvironment will help inform new strategies to further improve outcomes. Herein, we map tumor-specific CD8+ T-cell differentiation following agonistic anti-CD40 and/or anti-PDL1 in PDA. Rare Tcf1+Slamf6+ CD8+ T cells (TSTEM) are shown to seed memory precursors that transition into a continuum of exhausted and effector T cells. In tumors, anti-PDL1 drove the clonal expansion of Gzmk+ progenitor exhausted (CD8+ T cells, whereas anti-CD40 promoted CD4+ T-cell clonal expansion and accumulation of CD8+ TTSTEM. Cloning the most frequent intratumoral T-cell receptors (TCRs) revealed identical neoepitope specificity, yet the top TCRs from anti-PDL1  anti-CD40 cohorts lacked tetramer binding suggesting lower affinity. Anti-CD40 + anti-PDL1 markedly drove the clonal hyperexpansion of a unique exhausted T-cell (TEX) subset in spleen. TEX were enriched for IL2R, and provision of IL-15 complex (IL-15C) mitigated systemic and intratumoral T-cell exhaustion when combined with anti-CD40 + anti-PDL1, resulting in enhanced antitumor effects, prolongation of animal survival, and resistance to orthotopic tumor rechallenge. Mechanistically, while anti-CD40 + anti-PDL1 mitigated Tox, IL-15C + anti-CD40 + anti-PDL1 increased T-bet thereby conferring a higher T-bet:Tox ratio in tumor-specific CD8+ T cells. Collectively, agonistic anti-CD40 and anti-PDL1 drove systemic and intratumoral CD8+ T-cell clonal expansion and acquisition of exhaustion features. Provision of IL-15C altered the trajectory of T-cell differentiation induced by immunotherapy, resulting in PDA eradication and long-lived antitumor memory T cells.

Microvascular proliferation (MVP) is a disease-defining hallmark of glioblastoma (GBM) and other World Health Organization (WHO) grade 4 gliomas. MVP also serves as a poor prognostic marker in various solid tumors. Despite its clinical significance, the mechanisms and biological consequences of MVP are controversial and remain unclear. In this study, we performed single cell RNA-sequencing (scRNA-seq) on paired CD45-CD105+ vascular/perivascular stromal cells (PVSCs) and CD45+CD105± immune cells from 16 primary glioma patient samples, both with and without MVP. This analysis revealed the presence of developmentally-related mesenchymal stem cells (MSCs) alongside cancer-associated fibroblasts (CAFs), pericytes, fibromyocytes, and smooth muscle cells within the CD45-CD105+ compartment. RNA velocity analysis identified PDGFRB as a putative driver gene guiding MSCs toward more mature PVSCs in the context of MVP. Signaling network analysis and digital spatial profiling uncovered interactions between PDGFRB+ PVSCs and immunosuppressive myeloid cell subsets enriched in the perivascular niche, suggesting targetable receptor-ligand interactions. Additionally, a gene signature of MVP-associated PVSCs from gliomas predicted worse prognosis in multiple other solid tumors. This study provides a transcriptomic cell atlas of PVSCs and immune cells in glioma, helping to refine the biological model of MVP which has traditionally focused on endothelial cells.

The systemic immunological effects of combining anti-CTLA4 therapy with PD-(L)1 blockade remain incompletely characterized, despite the widespread use of this combination in treating various solid tumors across multiple stages of disease. Herein, we investigated the additive impact of anti-CTLA4 on peripheral immune signatures in patients undergoing PD-(L)1 blockade, using blood samples from a cohort of patients receiving checkpoint inhibitor therapy for advanced solid tumors. We performed in-parallel analysis of peripheral blood mononuclear cells (PBMC) using Cytometry by Time-of-Flight (CyTOF) and plasma cytokines using Luminex immunoassay. Our study cohort included 104 patients, 54 who received anti-PD(L)1 alone and 50 who received anti-PD(L)1 in combination with anti-CTLA4. As compared to single-agent anti-PD(L)1, combination therapy was associated with a greater expansion of CD4+ T helper cell subsets, including Th17 (adjusted p=0.04) and regulatory T cells (Treg) (adjusted p=0.02), after multivariable and multiple testing adjustment. In patients receiving anti-CTLA4, examination of functional marker expression within the Th17 subset revealed an increase in expression of the Th1-related transcription factor TBET (p=0.003). Assessment of the peripheral cytokine signatures showed an increase in Th1-associated cytokines (p=0.002) in recipients of combination anti-PD(L)1 and anti-CTLA4, particularly the IFN-inducible cytokines MIG (adjusted p=0.05) and IP-10 (adjusted p=0.05). Our results confirm prior reports that anti-CTLA4 therapy is associated with augmentation of Th17 cell subsets, and they also show that anti-CTLA4 may reshape CD4+ T-cell responses through Th17-to-Th1 plasticity, revealing a potential mechanism for enhanced antitumor immunity with broader implications immune modulation in immunotherapy.

Immunosuppressive myeloid cells are critical obstacles to T cell-centered immune checkpoint blockade therapies, which have been successful in treating a fraction of cancer patients. How tumor cells interact with myeloid cells to regulate immune responses and tumor development is unclear. Here, we report that certain membrane tyrosine kinase Eph receptors, including EphA7 and EphB1, specifically bind the immune inhibitory receptors leukocyte immunoglobin like receptor family B 5 (LILRB5) and leukocyte immunoglobin like receptor family B 2 (LILRB2). These Eph receptors induce LILRB5-mediated signaling activation, and LILRB5 also activates Eph receptor signaling. Activation of LILRB5 promoted immunosuppressive marker expression and inhibited activating marker expression on myeloid cells from cancer patients in vitro. Upon myeloid cell-specific expression of LILRB5 in transgenic mice, the interaction between the Eph receptor on tumor cells and LILRB5 on myeloid cells led to increased tumor growth, increased immunosuppressive myeloid cells, and decreased frequencies of functional T cells compared to control mice. Eph-induced LILRB5 signaling and functions were reversed by LILRB5 blockade. In sum, certain Eph receptors functionally interact with the myeloid checkpoint receptor LILRB5 resulting in bi-directional signaling, and LILRB5 plays an important role in supporting immunosuppressive myeloid cells and sustaining tumor development.

Immune checkpoint blockade (ICB) has revolutionized the therapeutic landscape across various cancer types. However, the emergence of resistance to ICB therapy limits its clinical application. Therefore, it is necessary to better understand immune-resistance mechanisms that could be targeted by actionable drugs, and important to identify predictive markers for selecting patients. Here, by analyzing transcriptomic data from patients treated with PD-1 blockade and tumor models refractory to anti-PD-1 therapy, we identified WEE1 as a resistance factor conferring cancer stem cell (CSC)-like properties as well as immune-refractory phenotypes to tumor cells. WEE1 is transcriptionally upregulated by stemness factor NANOG and predominantly localized in the cytoplasm, not the nucleus, following AKT-dependent S642 phosphorylation in immune-refractory tumor cells. Mechanistically, cytoplasmic WEE1 drove AKT hyperactivation via the HSP90A/TCL1A/AKT auto-amplification loop andupregulated the expression of refractory factors such as CYCLIN A for hyperproliferation and MCL-1 for resistance to T cell killing. Of note, CXCL10 was downregulated, resulting in insufficient T cell infiltration. The NANOG/WEE1/AKT axis was also conserved in various human cancers. Importantly, targeting WEE1 with a clinically relevant inhibitor sensitized NANOG+ immune-refractory tumors to ICB, reinvigorating antitumor immunity by disrupting the HSP90A/TCL1A/AKT loop. Thus, our findings demonstrate the oncogenic role of cytoplasmic WEE1 in immune-refractoriness and CSC-like properties of tumor cells through AKT hyperactivation and provide a rationale for combining a WEE1 inhibitor to control anti-PD-1 therapy-refractory tumors.

The tumor microenvironment (TME) in solid tumors contains myeloid cells that modulate local immune activity. STING signaling activation in these myeloid cells enhances local type I interferon (IFN) production, inducing an innate immune response that mobilizes adaptive immunity and reprograms immunosuppressive myeloid populations to drive antitumor immunity. Here, we generated TAK-500, an immune cell directed antibody drug conjugate (iADC), to deliver a STING agonist to CCR2+ human cells and drive enhanced antitumor activity relative to non-targeted STING agonists. Preclinically, TAK-500 triggered dose-dependent innate immune activation in vitro. In addition, a murine TAK-500 iADC surrogate enhanced innate and adaptive immune responses both in vitro and in murine tumor models. Spatially resolved analysis of CCR2 and immune cell markers in the TME of >1,000 primary human tumors showed the CCR2 protein was predominantly expressed in intratumoral myeloid cells. Collectively, these data highlight the clinical potential of delivering a STING agonist to CCR2+ cells.

Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients who have ovarian tumors infiltrated with high frequencies of T cells are associated with a greater survival probability. However, therapies to revitalize tumor-associated T cells, such as PD-L1/PD-1 or CTLA4 blockade, are ineffective for the treatment of ovarian cancer. In this study, we demonstrate that for ovarian cancer, Toll-Like Receptor 5 (TLR5) signaling, for which the only known ligand is bacterial flagellin, governed failure of PD-L1 and CTLA4 blockade. Mechanistically, chronic TLR5 signaling on CD11c+ cells in vivo and in vitro impaired the differentiation of functional IL-12-producing XCR1+CD103+ conventional type 1 dendritic cells (cDC1), biasing CD11c+ precursor cells toward myeloid subsets expressing high levels of PD-L1. This culminated in impaired activation of CD8+ T cells, reducing CD8+ T-cell function and ability to persist within the ovarian tumor microenvironment. Expansion of cDC1s in situ using FLT3L in combination with PD-L1 blockade achieved significant survival benefit in TLR5 knockout mice bearing ovarian tumors, whereas no benefit was observed in the presence of TLR5 signaling. Thus, we have identified a host-intrinsic mechanism leading to the failure of PD-L1 blockade for ovarian cancer, demonstrating that chronic TLR5 signaling on CD11c+ cells is a barrier limiting the efficacy of checkpoint therapy.

Traditional anti-cancer therapies induce tumor cell death and subsequent release of Damage Associated Molecular Patterns (DAMPs) that activate the innate inflammatory response. Paradoxically, after treatment, macrophages often adopt a pro-wound healing, rather than pro-inflammatory, phenotype and contribute to cancer progression. We found that in areas proximal to DAMP release, tumor cells upregulate the expression of Pros1. Tumor-secreted Pros1 binds to the macrophage Mer receptor, consequently limiting responsiveness to DAMPs by preventing Toll Like Receptor (TLR) signal transduction. Pharmacological inhibition of PTP1b signaling downstream of Mer rescued the pro-inflammatory response, even in the presence of Pros1. Combining PTP inhibition with traditional therapeutics, like chemo- or radiotherapy, rescued the innate immune response to DAMPs, increased immune infiltration, and resulted in a 40-90% reduction in tumor growth in multiple treatment refractory preclinical models. Our findings suggest using PTP1b inhibitors may be a tumor agnostic means of improving the efficacy of some of the most widely used anti-cancer therapeutic agents.

Immune checkpoint inhibitors (ICIs) have changed the treatment paradigm for many cancers but have not shown benefit in prostate cancer (PCa). Chronic inflammation contributes to the immunosuppressive prostate tumor microenvironment (TME) and is associated with poor response to ICIs. The primary source of inflammatory cytokine production is the inflammasome. Here, we identify PIM kinases as regulators of inflammasome activation in tumor-associated macrophages (TAMs). Analysis of clinical data from a cohort of treatment naïve, hormone-responsive PCa patients revealed that tumors from patients with high PIM1/2/3 displayed an immunosuppressive TME characterized by high inflammation and a high density of repressive immune cells, most notably TAMs. Macrophage-specific knockout of PIM reduced tumor growth in syngeneic models of PCa. Transcriptional analyses indicated that eliminating PIM from macrophages enhanced the adaptive immune response and increased cytotoxic immune cells. Combined treatment with PIM inhibitors and ICIs synergistically reduced tumor growth. Immune profiling revealed that PIM inhibitors sensitized PCa tumors to ICIs by increasing tumor suppressive TAMs and increasing the activation of cytotoxic T cells. Our data implicate macrophage PIM as a driver of inflammation that limits ICI potency and provide preclinical evidence that PIM inhibitors are an effective strategy to improve the ICI efficacy in PCa.

Denosumab, a RANKL inhibitor, is primarily used to prevent osteoclastogenesis in the treatment of conditions such as osteoporosis, bone metastasis, and giant cell tumour of bone (GCTB). RANKL also plays an important role in immunity by activating NF-κB and its target genes, including the osteopontin-coding gene SPP1 (also known as OPN), which is linked to CXCL9:SPP1 macrophage polarization and prognosis. In this study, we explored an additional role of denosumab in enhancing antitumour immunity in patients. Single-cell RNA sequencing was performed on nine human GCTB samples, including six untreated and three treated only with denosumab, to exclude confounding treatment factors linked with bone metastasis samples. We further analysed paired pre- and post-denosumab treated samples from a cohort of nine GCTB patients and conducted a pan-cancer analysis of 34 distinct types of cancers. Our single-cell analysis of GCTB resulted in a comprehensive cell atlas revealing an antitumour role of denosumab in inhibiting SPP1 expression and augmenting active cytotoxic T cell abundance. Furthermore, we validated this immunomodulatory role of denosumab using the paired GCTB samples. Finally, the pan-cancer analysis supported a negative correlation between SPP1 and CD8A levels, with the CD8A:SPP1 ratio correlating with overall survival in 14 cancer types, which was superior to either CD8A or SPP1 alone. Our research provides clinical evidence that denosumab improves antitumour immunity by decreasing SPP1 expression and enhancing cytotoxic T cell activity, serving as a milestone in the development of innovative use of denosumab and offering potential benefits to patients with elevated levels of SPP1.

Cervical tumors are usually treated using surgery, chemotherapy, and radiotherapy, and would benefit from immunotherapies. However, the immune microenvironment in cervical cancer remains poorly described. Tertiary lymphoid structures (TLS) were recently described as markers for better immunotherapy response and overall better prognosis in cancer patients. We evaluated the cervical tumor immune microenvironment, specifically focusing on TLS, using combined high-throughput phenotyping, soluble factor concentration dosage in the TME and spatial interaction analyses. We found that TLS presence was associated with a more inflammatory soluble microenvironment, with the presence of B cells as well as more activated macrophages and dendritic cells (DCs). Furthermore, this myeloid cell activation was associated with expression of immune checkpoints, such as PD-L1 and CD40, and proximity of activated conventional type 2 DCs (DC2) to CD8+ T cells, indicating better immune interactions and tumor control. Finally, we associated TLS presence, greater B cell density, and activated DC density with improved progression-free survival, substantiating TLS presence as a potential prognostic marker. Our results provide evidence that TLS presence denotes cell activation and immunotherapy target expression.