Intratumoral T-cell receptor repertoire composition predicts overall survival in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and "True entropy." Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8+ effector memory and CD4+ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

Seasonal trends in lysogeny in an Appalachian oak-hickory forest soil.

Since 1989, investigations into viral ecology have revealed how bacteriophages can influence microbial dynamics within ecosystems at global scales. Most of the information we know about temperate phages, which can integrate themselves into the host genome and remain dormant via a process called lysogeny, has come from research in aquatic ecosystems. Soil environments remain under-studied, and more research is necessary to fully understand the range of impacts phage infections have on the soil bacteria they infect. The aims of this study were to compare the efficacy of different prophage-inducing agents and to elucidate potential temporal trends in lysogeny within a soil bacterial community. In addition to mitomycin C and acyl-homoserine lactones, our results indicated that halosulfuron methyl herbicides may also be potent inducing agents. In optimizing chemical induction assays, we determined that taking steps to reduce background virus particles and starve cells was critical in obtaining consistent results. A clear seasonal trend in inducible lysogeny was observed in an Appalachian oak-hickory forest soil. The average monthly air temperature was negatively correlated with inducible fraction and burst size, supporting the idea that lysogeny provides a mechanism for phage persistence when temperatures are low and host metabolism is slower. Furthermore, the inducible fraction was negatively correlated with both soil bacterial and soil viral abundance, supporting the idea that lysogeny provides a mechanism for temperate phage persistence when host density is lower. The present study is the first of its kind to reveal clear seasonal trends in inducible lysogeny in any soil.IMPORTANCELysogeny is a relationship in which certain viruses that infect bacteria (phages) may exist within their bacterial host cell as a segment of nucleic acid. In this state, the phage genome is protected from environmental damage and retains the potential to generate progeny particles in the future. It is thought that lysogeny provides a mechanism for long-term persistence for phages when host density is low or hosts are starved-two conditions likely to be found in soils. In the present study, we provide the first known evidence for a seasonal trend in lysogeny in a forest soil. Based on clear relationships observed between lysogeny, temperature, and soil microbial abundance, we find support for previous hypotheses regarding the factors governing lysogeny.

Tumor-associated fibrosis impairs immune surveillance and response to immune checkpoint blockade in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. Immune checkpoint blockade has improved survival for many patients with NSCLC, but most fail to obtain long-term benefit. Understanding the factors leading to reduced immune surveillance in NSCLC is critical in improving patient outcomes. Here, we show that human NSCLC harbors large amounts of fibrosis that correlates with reduced T cell infiltration. In murine NSCLC models, the induction of fibrosis led to increased lung cancer progression, impaired T cell immune surveillance, and failure of immune checkpoint blockade efficacy. Associated with these changes, we observed that fibrosis leads to numerically and functionally impaired dendritic cells and altered macrophage phenotypes that likely contribute to immunosuppression. Within cancer-associated fibroblasts, distinct changes within the Col13a1-expressing population suggest that these cells produce chemokines to recruit macrophages and regulatory T cells while limiting recruitment of dendritic cells and T cells. Targeting fibrosis through transforming growth factor-ß receptor signaling overcame the effects of fibrosis to enhance T cell responses and improved the efficacy of immune checkpoint blockade but only in the context of chemotherapy. Together, these data suggest that fibrosis in NSCLC leads to reduced immune surveillance and poor responsiveness to checkpoint blockade and highlight antifibrotic therapies as a candidate strategy to overcome immunotherapeutic resistance.

Systemic Alterations in Type-2 Conventional Dendritic Cells Lead to Impaired Tumor Immunity in Pancreatic Cancer.

Intratumoral T-cell dysfunction is a hallmark of pancreatic tumors, and efforts to improve dendritic cell (DC)-mediated T-cell activation may be critical in treating these immune therapy unresponsive tumors. Recent evidence indicates that mechanisms that induce dysfunction of type 1 conventional DCs (cDC1) in pancreatic adenocarcinomas (PDAC) are drivers of the lack of responsiveness to checkpoint immunotherapy. However, the impact of PDAC on systemic type 2 cDC2 development and function has not been well studied. Herein, we report the analysis of 3 cohorts, totaling 106 samples, of human blood and bone marrow (BM) from patients with PDAC for changes in cDCs. We found that circulating cDC2s and their progenitors were significantly decreased in the blood of patients with PDAC, and repressed numbers of cDC2s were associated with poor prognosis. Serum cytokine analyses identified IL6 as significantly elevated in patients with PDAC and negatively correlated with cDC numbers. In vitro, IL6 impaired the differentiation of cDC1s and cDC2s from BM progenitors. Single-cell RNA sequencing analysis of human cDC progenitors in the BM and blood of patients with PDAC showed an upregulation of the IL6/STAT3 pathway and a corresponding impairment of antigen processing and presentation. These results suggested that cDC2s were systemically suppressed by inflammatory cytokines, which was linked to impaired antitumor immunity.

Context-dependent activation of STING-interferon signaling by CD11b agonists enhances anti-tumor immunity.

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.

Stromal and therapy-induced macrophage proliferation promotes PDAC progression and susceptibility to innate immunotherapy.

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Background: Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. Methods: We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Results: Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Conclusions: Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

Stromal Reprogramming by FAK Inhibition Overcomes Radiation Resistance to Allow for Immune Priming and Response to Checkpoint Blockade.

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor-stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival. SIGNIFICANCE: Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711.

Neoadjuvant FOLFIRINOX Therapy Is Associated with Increased Effector T Cells and Reduced Suppressor Cells in Patients with Pancreatic Cancer.

PURPOSE: FOLFIRINOX has demonstrated promising results for patients with pancreatic ductal adenocarcinoma (PDAC). Chemotherapy-induced immunogenic cell death can prime antitumor immune responses. We therefore performed high-dimensional profiling of immune cell subsets in peripheral blood to evaluate the impact of FOLFIRINOX on the immune system. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells (PBMC) were obtained from treatment-naïve (n = 20) and FOLFIRINOX-treated patients (n = 19) with primary PDAC tumors at the time of resection. PBMCs were characterized by 36 markers using mass cytometry by time of flight (CyTOF). RESULTS: Compared with treatment-naïve patients, FOLFIRINOX-treated patients showed distinct immune profiles, including significantly decreased inflammatory monocytes and regulatory T cells (Treg), increased Th1 cells, and decreased Th2 cells. Notably, both monocytes and Treg expressed high levels of immune suppression-associated CD39, and the total CD39+ cell population was significantly lower in FOLFIRINOX-treated patients compared with untreated patients. Cellular alterations observed in responders to FOLFIRINOX included a significantly decreased frequency of Treg, an increased frequency of total CD8 T cells, and an increased frequency of CD27-Tbet+ effector/effector memory subsets of CD4 and CD8 T cells. CONCLUSIONS: Our study reveals that neoadjuvant chemotherapy with FOLFIRINOX enhances effector T cells and downregulates suppressor cells. These data indicate that FOLFIRINOX neoadjuvant therapy may improve immune therapy and clinical outcome in patients with PDAC.

Rethinking immune checkpoint blockade: 'Beyond the T cell'.

The clinical success of immune checkpoint inhibitors has highlighted the central role of the immune system in cancer control. Immune checkpoint inhibitors can reinvigorate anti-cancer immunity and are now the standard of care in a number of malignancies. However, research on immune checkpoint blockade has largely been framed with the central dogma that checkpoint therapies intrinsically target the T cell, triggering the tumoricidal potential of the adaptive immune system. Although T cells undoubtedly remain a critical piece of the story, mounting evidence, reviewed herein, indicates that much of the efficacy of checkpoint therapies may be attributable to the innate immune system. Emerging research suggests that T cell-directed checkpoint antibodies such as anti-programmed cell death protein-1 (PD-1) or programmed death-ligand-1 (PD-L1) can impact innate immunity by both direct and indirect pathways, which may ultimately shape clinical efficacy. However, the mechanisms and impacts of these activities have yet to be fully elucidated, and checkpoint therapies have potentially beneficial and detrimental effects on innate antitumor immunity. Further research into the role of innate subsets during checkpoint blockade may be critical for developing combination therapies to help overcome checkpoint resistance. The potential of checkpoint therapies to amplify innate antitumor immunity represents a promising new field that can be translated into innovative immunotherapies for patients fighting refractory malignancies.

Phenotypic and Functional Plasticity of CXCR6+ Peripheral Blood NK Cells.

Human NK cells are comprised of phenotypic subsets, whose potentially unique functions remain largely unexplored. C-X-C-motif-chemokine-receptor-6 (CXCR6) + NK cells have been identified as phenotypically immature tissue-resident NK cells in mice and humans. A small fraction of peripheral blood (PB)-NK cells also expresses CXCR6. However, prior reports about their phenotypic and functional plasticity are conflicting. In this study, we isolated, expanded, and phenotypically and functionally evaluated CXCR6+ and CXCR6- PB-NK cells, and contrasted results to bulk liver and spleen NK cells. We found that CXCR6+ and CXCR6- PB-NK cells preserved their distinct phenotypic profiles throughout 14 days of in vitro expansion ("day 14"), after which phenotypically immature CXCR6+ PB-NK cells became functionally equivalent to CXCR6- PB-NK cells. Despite a consistent reduction in CD16 expression and enhanced expression of the transcription factor Eomesodermin (Eomes), day 14 CXCR6+ PB-NK cells had superior antibody-dependent cellular cytotoxicity (ADCC) compared to CXCR6- PB-NK cells. Further, bulk liver NK cells responded to IL-15, but not IL-2 stimulation, with STAT-5 phosphorylation. In contrast, bulk splenic and PB-NK cells robustly responded to both cytokines. Our findings may allow for the selection of superior NK cell subsets for infusion products increasingly used to treat human diseases.

Dendritic Cell Paucity Leads to Dysfunctional Immune Surveillance in Pancreatic Cancer.

Here, we utilized spontaneous models of pancreatic and lung cancer to examine how neoantigenicity shapes tumor immunity and progression. As expected, neoantigen expression during lung adenocarcinoma development leads to T cell-mediated immunity and disease restraint. By contrast, neoantigen expression in pancreatic ductal adenocarcinoma (PDAC) results in exacerbation of a fibro-inflammatory microenvironment that drives disease progression and metastasis. Pathogenic TH17 responses are responsible for this neoantigen-induced tumor progression in PDAC. Underlying these divergent T cell responses in pancreas and lung cancer are differences in infiltrating conventional dendritic cells (cDCs). Overcoming cDC deficiency in early-stage PDAC leads to disease restraint, while restoration of cDC function in advanced PDAC restores tumor-restraining immunity and enhances responsiveness to radiation therapy.

Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies.

Although checkpoint immunotherapies have revolutionized the treatment of cancer, not all tumor types have seen substantial benefit. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in which very limited responses to immunotherapy have been observed. Extensive immunosuppressive myeloid cell infiltration in PDAC tissues has been postulated as a major mechanism of resistance to immunotherapy. Strategies concomitantly targeting monocyte or granulocyte trafficking or macrophage survival, in combination with checkpoint immunotherapies, have shown promise in preclinical studies, and these studies have transitioned into ongoing clinical trials for the treatment of pancreatic and other cancer types. However, compensatory actions by untargeted monocytes, granulocytes, and/or tissue resident macrophages may limit the therapeutic efficacy of such strategies. CD11b/CD18 is an integrin molecule that is highly expressed on the cell surface of these myeloid cell subsets and plays an important role in their trafficking and cellular functions in inflamed tissues. Here, we demonstrate that the partial activation of CD11b by a small-molecule agonist (ADH-503) leads to the repolarization of tumor-associated macrophages, reduction in the number of tumor-infiltrating immunosuppressive myeloid cells, and enhanced dendritic cell responses. These actions, in turn, improve antitumor T cell immunity and render checkpoint inhibitors effective in previously unresponsive PDAC models. These data demonstrate that molecular agonism of CD11b reprograms immunosuppressive myeloid cell responses and potentially bypasses the limitations of current clinical strategies to overcome resistance to immunotherapy.

Biallelic mutations in IRF8 impair human NK cell maturation and function.

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.