Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens.

Intratumoral B cell responses are associated with more favorable clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). However, the antigens driving these B cell responses are largely unknown. We sought to discover these antigens by using single-cell RNA sequencing (scRNA-Seq) and immunoglobulin (Ig) sequencing of tumor-infiltrating immune cells from 7 primary PDAC samples. We identified activated T and B cell responses and evidence of germinal center reactions. Ig sequencing identified plasma cell (PC) clones expressing isotype-switched and hypermutated Igs, suggesting the occurrence of T cell-dependent B cell responses. We assessed the reactivity of 41 recombinant antibodies that represented the products of 235 PCs and 12 B cells toward multiple cell lines and PDAC tissues and observed frequent staining of intracellular self-antigens. Three of these antigens were identified: the filamentous actin (F-actin), the nucleic protein RuvB like AAA ATPase 2 (RUVBL2), and the mitochondrial protein heat shock protein family D (Hsp60) member 1 (HSPD1). Antibody titers against F-actin and HSPD1 were substantially elevated in the plasma of patients with PDAC compared with healthy donors. Thus, PCs in PDAC produce autoantibodies reacting with intracellular self-antigens, which may result from promotion of preexisting, autoreactive B cell responses. These observations indicate the chronic inflammatory microenvironment of PDAC can support the adaptive immune response.

T cell-Mediated Development of Stromal Fibroblasts with an Immune-Enhancing Chemokine Profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune-suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

T cell-mediated development of stromal fibroblasts with an immune-enhancing chemokine profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAFs) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA-sequencing of CAFs from mouse pancreatic adenocarcinomas identified a sub-population of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8+ T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

AMD3100/Plerixafor overcomes immune inhibition by the CXCL12-KRT19 coating on pancreatic and colorectal cancer cells.

A recent Phase 1 clinical study of the immunological effects of inhibiting the chemokine receptor, CXCR4, in patients with pancreatic ductal adenocarcinoma or colorectal cancer suggests that stimulation of CXCR4 on immune cells suppresses the intratumoural immune reaction. Here, we discuss how CXCR4 mediates this response, and how cancer cells elicit it.

CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response.

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Cancer cell CCR2 orchestrates suppression of the adaptive immune response.

C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2-/- cancer cells did not induce immune suppression in Batf3-/- mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response.

Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.

Embryonic FAP+ lymphoid tissue organizer cells generate the reticular network of adult lymph nodes.

The induction of adaptive immunity is dependent on the structural organization of LNs, which is in turn governed by the stromal cells that underpin LN architecture. Using a novel fate-mapping mouse model, we trace the developmental origin of mesenchymal LN stromal cells (mLNSCs) to a previously undescribed embryonic fibroblast activation protein-α (FAP)+ progenitor. FAP+ cells of the LN anlagen express lymphotoxin ß receptor (LTßR) and vascular cell adhesion molecule (VCAM), but not intercellular adhesion molecule (ICAM), suggesting they are early mesenchymal lymphoid tissue organizer (mLTo) cells. Clonal labeling shows that FAP+ progenitors locally differentiate into mLNSCs. This process is also coopted in nonlymphoid tissues in response to infection to facilitate the development of tertiary lymphoid structures, thereby mimicking the process of LN ontogeny in response to infection.

Immunofibroblasts are pivotal drivers of tertiary lymphoid structure formation and local pathology.

Resident fibroblasts at sites of infection, chronic inflammation, or cancer undergo phenotypic and functional changes to support leukocyte migration and, in some cases, aggregation into tertiary lymphoid structures (TLS). The molecular programming that shapes these changes and the functional requirements of this population in TLS development are unclear. Here, we demonstrate that external triggers at mucosal sites are able to induce the progressive differentiation of a population of podoplanin (pdpn)-positive stromal cells into a network of immunofibroblasts that are able to support the earliest phases of TLS establishment. This program of events, that precedes lymphocyte infiltration in the tissue, is mediated by paracrine and autocrine signals mainly regulated by IL13. This initial fibroblast network is expanded and stabilized, once lymphocytes are recruited, by the local production of the cytokines IL22 and lymphotoxin. Interfering with this regulated program of events or depleting the immunofibroblasts in vivo results in abrogation of local pathology, demonstrating the functional role of immunofibroblasts in supporting TLS maintenance in the tissue and suggesting novel therapeutic targets in TLS-associated diseases.

Distinct fibroblast subsets drive inflammation and damage in arthritis.

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice.

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3ß1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients.

Stromal Cells in the Tumor Microenvironment.

The tumor microenvironment comprises a mass of heterogeneous cell types, including immune cells, endothelial cells, and fibroblasts, alongside cancer cells. It is increasingly becoming clear that the development of this support niche is critical to the continued uncontrolled growth of the cancer. The tumor microenvironment contributes to the maintenance of cancer stemness and also directly promotes angiogenesis, invasion, metastasis, and chronic inflammation. In this chapter, we describe on the role of fibroblasts, specifically termed cancer-associated fibroblasts (CAFs), in the promotion and maintenance of cancers. CAFs have a multitude of effects on the growth and maintenance of cancer, and here we focus on their roles in modulating immune cells and responses; CAFs both inhibit immune cell access to the tumor microenvironment and inhibit their functions within the tumor. Finally, we describe the potential modulation of CAF function as an adjunct to bolster the effectiveness of cancer immunotherapies.

Unresolved endoplasmic reticulum stress engenders immune-resistant, latent pancreatic cancer metastases.

The majority of patients with pancreatic ductal adenocarcinoma (PDA) develop metastatic disease after resection of their primary tumor. We found that livers from patients and mice with PDA harbor single disseminated cancer cells (DCCs) lacking expression of cytokeratin 19 (CK19) and major histocompatibility complex class I (MHCI). We created a mouse model to determine how these DCCs develop. Intraportal injection of immunogenic PDA cells into preimmunized mice seeded livers only with single, nonreplicating DCCs that were CK19- and MHCI- The DCCs exhibited an endoplasmic reticulum (ER) stress response but paradoxically lacked both inositol-requiring enzyme 1α activation and expression of the spliced form of transcription factor XBP1 (XBP1s). Inducible expression of XBP1s in DCCs, in combination with T cell depletion, stimulated the outgrowth of macrometastatic lesions that expressed CK19 and MHCI. Thus, unresolved ER stress enables DCCs to escape immunity and establish latent metastases.

Fenofibrate prevents skeletal muscle loss in mice with lung cancer.

The cancer anorexia cachexia syndrome is a systemic metabolic disorder characterized by the catabolism of stored nutrients in skeletal muscle and adipose tissue that is particularly prevalent in nonsmall cell lung cancer (NSCLC). Loss of skeletal muscle results in functional impairments and increased mortality. The aim of the present study was to characterize the changes in systemic metabolism in a genetically engineered mouse model of NSCLC. We show that a portion of these animals develop loss of skeletal muscle, loss of adipose tissue, and increased inflammatory markers mirroring the human cachexia syndrome. Using noncachexic and fasted animals as controls, we report a unique cachexia metabolite phenotype that includes the loss of peroxisome proliferator-activated receptor-α (PPARα) -dependent ketone production by the liver. In this setting, glucocorticoid levels rise and correlate with skeletal muscle degradation and hepatic markers of gluconeogenesis. Restoring ketone production using the PPARα agonist, fenofibrate, prevents the loss of skeletal muscle mass and body weight. These results demonstrate how targeting hepatic metabolism can prevent muscle wasting in lung cancer, and provide evidence for a therapeutic strategy.

Cancer Immunotherapy Trials Underutilize Immune Response Monitoring.

Immune-related radiological and biomarker monitoring in cancer immunotherapy trials permits interrogation of efficacy and reasons for therapeutic failure. We report the results from a cross-sectional analysis of response monitoring in 685 T-cell checkpoint-targeted cancer immunotherapy trials in solid malignancies, as registered on the U.S. National Institutes of Health trial registry by October 2016. Immune-related radiological response criteria were registered for only 25% of clinical trials. Only 38% of trials registered an exploratory immunological biomarker, and registration of immunological biomarkers has decreased over the last 15 years. We suggest that increasing the utilization of immune-related response monitoring across cancer immunotherapy trials will improve analysis of outcomes and facilitate translational efforts to extend the benefit of immunotherapy to a greater proportion of patients with cancer.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Connecting the Metabolic and Immune Responses to Cancer.

Separate research fields have advanced our understanding of, on the one hand, cancer immunology and, on the other hand, cachexia, the fatal tumor-induced wasting syndrome. A link between the host's immune and metabolic responses to cancer remained unexplored. Emerging work in preclinical models of colorectal and pancreatic cancer has unveiled tumor-induced reprogramming of liver metabolism in cachexia that leads to suppression of antitumor immunity and failure of immunotherapy. As research efforts in metabolism and immunology in cancer are rapidly expanding, it is timely to discuss the metabolic and immunological determinants of the cancer-host interaction. We also present the hypothesis that the convergence of host metabolism and antitumor immunity may offer a platform for biomarker-driven investigations of new combination therapies.

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity.

In patients with cancer, the wasting syndrome, cachexia, is associated with caloric deficiency. Here, we describe tumor-induced alterations of the host metabolic response to caloric deficiency that cause intratumoral immune suppression. In pre-cachectic mice with transplanted colorectal cancer or autochthonous pancreatic ductal adenocarcinoma (PDA), we find that IL-6 reduces the hepatic ketogenic potential through suppression of PPARalpha, the transcriptional master regulator of ketogenesis. When these mice are challenged with caloric deficiency, the resulting relative hypoketonemia triggers a marked rise in glucocorticoid levels. Multiple intratumoral immune pathways are suppressed by this hormonal stress response. Moreover, administering corticosterone to elevate plasma corticosterone to a level that is lower than that occurring in cachectic mice abolishes the response of mouse PDA to an immunotherapy that has advanced to clinical trials. Therefore, tumor-induced IL-6 impairs the ketogenic response to reduced caloric intake, resulting in a systemic metabolic stress response that blocks anti-cancer immunotherapy.