TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Tumor-Infiltrating Myeloid Cells Confer De Novo Resistance to PD-L1 Blockade through EMT-Stromal and Tgfß-Dependent Mechanisms.

SIGNIFICANCE: Most patients with bladder cancer do not respond to ICB targeting of the PD-L1 signaling axis. Our modeling applied a de novo resistance signature to show that tumor-infiltrating myeloid cells promote poor treatment response in a TGFß-dependent mechanism.

TIM-3 and TIGIT are possible immune checkpoint targets in patients with bladder cancer.

The resurgence of immunotherapy as an effective anticancer strategy has been coupled with more mature understandings of the underlying immune pathways and the development of novel immune checkpoint targets. The clinical development of antibodies first directed against cytotoxic T-lymphocyte-associated antigen 4, and later against program death 1, achieved durable disease control in a subset of patients across a large number of tumor types. Previous work demonstrates that targeting the programmed death 1 pathway alone does not result in complete restoration of T cell function and in some cancers, targeting this axis does not restore T cell function at all, suggesting a need to identify other molecules and inhibitory pathways that are involved in T cell exhaustion. In a comprehensive immune profiling study of patients with bladder cancer, we demonstrate T-cell immunoglobulin domain and mucin domain-containing molecule and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain as possible targets as perhaps monotherapy or in combination with other immune checkpoint inhibitors.

Myeloid Cell-associated Resistance to PD-1/PD-L1 Blockade in Urothelial Cancer Revealed Through Bulk and Single-cell RNA Sequencing.

PURPOSE: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer. EXPERIMENTAL DESIGN: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures. RESULTS: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer. CONCLUSIONS: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139.

Harnessing Natural Killer Cell Function for Genitourinary Cancers.

Natural killer (NK) cells are potently cytolytic innate lymphocytes involved in the immune surveillance of tumors and virally infected cells. Although much progress has been made in manipulating the ability of T cells to recognize and eliminate tumors, a comprehensive understanding of NK-cell infiltration into solid tumors, and their amenability to immunomodulation, remains incomplete. This article discusses recent studies showing that urologic tumors are infiltrated by NK cells and that these NK cells are often dysfunctional, but that strategies interfering with inhibitory axes have significant potential to alleviate this dysfunction.

Fibroblast Growth Factor Receptor 3 Alterations and Response to PD-1/PD-L1 Blockade in Patients with Metastatic Urothelial Cancer.

Prior studies have demonstrated that fibroblast receptor 3 (FGFR3)-mutant urothelial cancers (UCs) are associated with decreased T-cell infiltration. As FGFR3 mutations are enriched in luminal-like UC and luminal-like UC has been shown to be relatively less responsive to PD-1/PD-L1 inhibition (checkpoint inhibition [CPI]), these data have led to the speculation that FGFR3 mutations may be causally related to poor T-cell infiltration and that UC patients harboring FGFR3 mutations may be suboptimal candidates for CPI. Using data derived from two clinical trials exploring CPI in metastatic UC, we demonstrate no statistically significant difference in response rates in patients with FGFR3-mutant versus wild-type UC. We present hypothesis-generating data, suggesting that similar response rates may be explained by a "balancing out" of previously identified independent positive and negative predictors of CPI sensitivity; that is, compared with FGFR3 wild-type UC, FGFR3-mutant UC is associated with a similar tumor mutational burden, lower T-cell infiltration, but also lower stromal/transforming growth factor beta (TGF-ß) signals. Based on our findings, FGFR3 mutation status is not a biomarker of resistance to CPI. Indeed, the single-agent activity of both FGFR3 inhibitors and CPI in FGFR3-mutant UC, and potential non-cross resistance provide a strong pragmatic rationale for combination approaches. PATIENT SUMMARY: In this report, we examined the impact of a mutated gene found in a subset of urothelial cancers on response to treatment with immunotherapy. We found that patients with tumors harboring mutations in the gene FGFR3 respond to immunotherapy similarly to patients without such mutations.

Immune phenotype of peripheral blood mononuclear cells in patients with high-risk non-muscle invasive bladder cancer.

PURPOSE: To explore the immune phenotype of peripheral blood mononuclear cells (PBMC) in patients with high-risk non-muscle invasive bladder cancer (NMIBC). METHODS: We prospectively collected blood samples from patients with high-risk NMIBC treated at our institution. PBMC were analyzed by flow cytometry to determine the frequency of T cells and NK cells and the expression of immunoregulatory molecules (Tim-3, TIGIT and PD-1). PBMC from healthy donors (HD) were included for comparison, and associations with response to BCG were investigated. RESULTS: A total of 38 patients were included, 19 BCG responders and 19 BCG refractory. Compared to 16 PBMC from HD, the frequency of total NK cells was significantly higher in patients with NMIBC [15.2% (IQR: 11.4, 22.2) vs. 5.72% (IQR: 4.84, 9.79); p = 0.05], whereas the frequency of T cells was not statistically different. Both Tim-3 and TIGIT expressions were significantly higher in NMIBC compared to HD, particularly in NK cells [13.8% (11.0; 22.4) vs. 5.56% (4.20; 10.2) and 34.9% (18.9; 53.5) vs. 1.82% (0.63; 5.16), respectively; p < 0.001]. Overall, the expression of PD-1 in all cell types was low in both NMIBC patients and HD. The immune phenotype was not significantly different before and after initiation of BCG. However, the proportion of CD8+ T cells before BCG was significantly higher in responders. CONCLUSION: The immune phenotype of PBMC from patients with high-risk NMIBC was significantly different from HD, regardless of the presence of disease or the initiation of BCG. Peripheral CD8+ T cells could play a role in response to BCG.

Intestinal Monocyte-Derived Macrophages Control Commensal-Specific Th17 Responses.

Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Dendritic cells (DCs) and macrophages (Mfs) integrate microbial signals and direct adaptive immunity. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. Th17 cells are critical for mucosal immune protection and at steady state are induced by commensal bacteria, such as segmented filamentous bacteria (SFB). Here, we examined the roles of mucosal DCs and Mfs in Th17 induction by SFB in vivo. We show that Mfs, and not conventional CD103(+) DCs, are essential for the generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

Escaping Negative Selection: ILC You in the Gut.

How commensal-specific T cells are controlled in the periphery is poorly understood. In a recent issue of Science, Hepworth et al. (2015) show that ILC3s induce apoptosis of microbiota-specific CD4 T cells in a form of extrathymic negative selection.

Induction of Th17 cells by segmented filamentous bacteria in the murine intestine.

Segmented filamentous bacteria (SFB) are Gram-positive, anaerobic, spore-forming commensals that reside in the gut of many animal species. Described more than forty years ago, SFB have recently gained interest due to their unique ability to modulate the host immune system through induction of IgA and Th17 cells. Here, we describe a collection of methods to detect and quantify SFB and SFB adhesion in intestinal mucosa, as well as SFB-specific CD4 T cells in the lamina propria. In addition, we describe methods for purification of SFB from fecal material of SFB-monoassociated gnotobiotic mice. Using these methods we examine the kinetics of SFB colonization and Th17 cell induction. We also show that SFB colonize unevenly the intestinal mucosa and that SFB adherence occurs predominantly in the terminal ileum and correlates with an increased proportion of SFB-specific Th17 cells.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen.

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

Altered glycosylation of MUC1 influences its association with CIN85: the role of this novel complex in cancer cell invasion and migration.

MUC1 is a transmembrane glycoprotein abnormally expressed in human adenocarcinomas. The extracellular domain of MUC1 contains a variable number of tandem repeats (VNTR) region that is extensively O-glycosylated in normal epithelia and underglycosylated in tumor cells. This change in posttranslational modification of MUC1 leads to changes in its normal functions including, we hypothesized, its interaction with other molecules. We identified CIN85, an adaptor protein involved in multiple cellular processes including signal transduction, cytoskeletal remodeling and cancer cell invasion, as one of several proteins that associate with MUC1 in tumor cells. CIN85 associates with both the cytosolic tail and the extracellular VNTR of MUC1. Co-immunoprecipitation and confocal immunofluorescence confirmed that MUC1 and CIN85 co-localize primarily at the plasma membrane but the complex can be found also in the cytosol and on the cytoskeleton. MUC1 and CIN85 are both over-expressed in early as well as advanced clinical stages of breast cancer and co-localize on invadopodia-like structures implicated in cell invasion. siRNA-mediated silencing of CIN85 and/or MUC1 revealed that MUC1 enhances CIN85-dependent breast cancer cell migration and invasion in vitro. However, ectopic expression of MUC1 enhances the motility induced by CIN85. When tested in vivo in a tumor metastasis model of B16 melanoma, mice injected with CIN85-depleted melanoma cells exhibited few or no lung metastasis and, similarly to the in vitro results, overexpression of MUC1 recovered the shCIN85-reduced metastatic process. Our findings implicate this newly identified CIN85/MUC1 complex associated with invadopodia-related molecules in promoting the invasive and metastatic potential of breast cancer.

Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic dendritic cells that are marked by differential expression of pancreatic enzymes.

Dendritic cells (DC) elicit immunity to pathogens and tumors while simultaneously preserving tolerance to self. Efficacious cancer vaccines have been a challenge because they are based on tumor Ags, some of which are self-Ags and thus subject to self-tolerance. One such Ag is the tumor-associated mucin MUC1. Preclinical testing of MUC1 vaccines revealed existence of peripheral tolerance to MUC1 that compromises their efficacy. To identify mechanisms that act early postvaccination and might predict vaccine outcome, we immunized human MUC1 transgenic mice (MUC1.Tg) i.v. with a MUC1 peptide vaccine against which they generate weak immunity and wild-type (WT) mice that respond strongly to the same peptide. We analyzed differences in splenic DC phenotype and function between the two mouse strains at 24 and 72 h postvaccination and also performed unbiased total gene expression analysis of the spleen. Compared to WT, MUC1.Tg spleens had significantly fewer DC, and they exhibited significantly lower expression of costimulatory molecules, decreased motility, and preferential priming of Ag-specific Foxp3(+) regulatory T cells. This tolerogenic DC phenotype and function was marked by a new putative biomarker revealed by the microarray: a cohort of pancreatic enzymes (trypsin, carboxypeptidase, elastase, and others) not previously reported in DC. These enzymes were strongly upregulated in the splenic DC from vaccinated WT mice and suppressed in the splenic DC of vaccinated MUC1.Tg mice. Suppression of the enzymes was dependent on regulatory T cells and on signaling through the IL-10R and correlated with global downregulation of DC immunostimulatory phenotype and function.

Vaccines based on abnormal self-antigens as tumor-associated antigens: immune regulation.

Abnormal expression of "self" antigens on tumors compared with normal cells provides opportunities and challenges for development of cancer vaccines. We review recent work in pre-clinical transgenic mouse models and in clinical trials that has elucidated multiple regulatory mechanisms that interfere with the induction of effective immunity. We discuss these as being either part of the normal function of the immune system or being driven by the tumor microenvironment. Collectively this work shows that it is possible to design vaccines based on tumor-associated antigens and elicit effective immunity against abnormal expression of these antigens on tumors without causing autoimmunity.

Biological activities of cytokine-neutralizing hyaluronic acid-antibody conjugates.

Wound healing represents a highly regulated, orchestrated response of cells recruited to sites of injury. High molecular weight hyaluronic acid was conjugated with monoclonal antibodies to the cytokine interleukin-1beta to create a matrix-forming polymer capable of modifying healing. Using gel electrophoresis and fluorescence immunosorbent assays, we determined a degree of antibody functionalization per hyaluronic acid chain of 13.6+/-1.6%. The biological activity of the conjugate in vitro, measured using a nuclear factor-kappaB translocation assay in activated THP-1 monocytes, was comparable in inhibiting cytokine signaling to a similar level as the unconjugated antibody. Incisional wound studies in Sprague-Dawley rats indicates that viscous hyaluronic acid solutions were immunologically active, but covalent functionalization with antibodies against tumor necrosis factor-alpha and interleukin-1beta resulted in significant reductions in the inflammatory response. Covalent attachment of cytokine-neutralizing antibodies to matrix-forming polymers could lead to the development of materials capable of locally regulating wound healing and inflammatory responses in the setting of tissue regeneration.

Estrogen receptor beta functions through nongenomic mechanisms in lung cancer cells.

Recent studies have shown that estrogens promote the growth of lung cancer cells and may potentially be responsible for increased susceptibility to lung cancer in women. These observations raise the possibility of using antiestrogens in treating and preventing lung cancer. However, it is not clear how estrogen receptors (ERs) modulate the growth of non-small cell lung cancer (NSCLC) cells. Our Western blotting and real-time PCR analysis showed that NSCLC cells expressed ERbeta, but not ERalpha. In addition, ERbeta-specific ligands, but not ERalpha-specific ligands, promoted the growth of lung cancer cells. Furthermore, knockdown of ERbeta by short hairpin RNA constructs resulted in loss of estrogen-dependent growth of lung cancer cells. Interestingly, endogenous ERbeta failed to transcriptionally activate estrogen response element (ERE)-luciferase constructs in NSCLC cells, suggesting a lack of genomic function. Upon further investigation, ERbeta was found to be in the cytoplasm in all lung cancer cells and failed to translocate to the nucleus in the presence of estrogen, as observed by biochemical, ArrayScan, and confocal microscopy experiments. Nonetheless, estrogen caused rapid activation of cAMP, Akt, and MAPK signaling pathways in lung cancer cells. Immunohistochemical analysis of lung tumor biopsies showed strong ERbeta staining in the cytoplasm, whereas no staining was observed for ERalpha. In conclusion, our results suggest that that proliferative effects of estrogen in lung cancer cells is mediated primarily, if not exclusively, by the nongenomic action of ERbeta.