Innate Immunosurveillance Research Articles

Abstract A54: Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma

Abstract Macrophage infiltration into solid malignancies most often portends a poor prognosis. In pancreatic ductal adenocarcinoma (PDAC), macrophages dominate the leukocyte infiltrate with effector T lymphocytes remaining scarce. Strategies to restore productive immunosurveillance in PDAC have largely focused on inducing anti-tumor T cell immunity. However, we have previously shown in patients and a mouse model of PDAC that immune modulation with a CD40 agonist can induce macrophages to facilitate tumor regression. Thus, strategies that restore innate immunosurveillance may hold promise in the treatment of PDAC. To further investigate the role of macrophages as targets for anti-tumor immunotherapy, we utilized the KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model of PDAC. We previously reported a role for liposomal targeted macrophages in facilitating tumor regression induced with CD40 therapy. However, unexpectedly we found by immunohistochemistry that macrophages targeted by liposomes do not infiltrate PDAC tumors in response to CD40 therapy. This finding suggested a role for additional leukocyte subsets in mediating the anti-tumor effect. Using immunohistochemistry, we found that systemic CD40 activation induced the infiltration of Ly6C+ myeloid cells into KPC tumors. Based on this finding, we hypothesized that macrophages may regulate the infiltration of Ly6Chi inflammatory monocytes, which are the critical effector myeloid cells mobilized in the peripheral blood by CD40 agonist therapy. In support of this hypothesis, we found that tumor regression induced with a CD40 agonist was abrogated by treatment with the anti-Ly6C/Ly6G RB6-8C5 depleting antibody. Flow cytometric analysis of peripheral blood revealed that RB6-8C5 and liposomes target distinct myeloid subsets. Whereas RB6-8C5 was found to deplete Ly6ChiCCR2+ inflammatory monocytes, CEL depleted Ly6Cneg myeloid cells. Our findings suggest that CD40-induced tumor regression in PDAC is mediated by inflammatory monocytes which infiltrate tumor lesions under the regulation of extra-tumoral macrophages. Citation Format: Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. Ly6Chi inflammatory monocytes mediate tumor regression in a mouse model of spontaneously arising pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A54.

Association of variants in genes encoding for macrophage-related functions with clinical outcome in patients with locoregional gastric cancer

Nuclear factor-kappaB (NF-κB) and CCL2/CCR2 chemokine axis play a central role in tumor progression such as stimulation of angiogenesis, acceleration of tumor invasion and migration, and suppression of innate immunosurveillance in the macrophage-related functions. There have been few reports regarding association of the macrophage function-related genes with the clinical outcome in gastric cancer. We hypothesized that variants in genes encoding for NF-κB and CCL2/CCR2 axis may predict prognosis in gastric cancer and tested whether the functional single-nucleotide polymorphisms (SNPs) will be associated with clinical outcome in patients with gastric cancer across two independent groups. This study enrolled two cohorts which consisted of 160 Japanese patients and 104 US patients with locoregional gastric cancer. Genomic DNA was analyzed for association of 11 SNPs in NFKB1, RELA, CCL2, and CCR2 with clinical outcome using PCR-based direct DNA sequencing. The univariable analysis showed four SNPs had significant association with clinical outcome in the Japanese cohort, NFKB1 rs230510 remained significant upon multivariable analysis. The patients with the A allele of the NFKB1 rs230510 had significantly longer overall survival (OS) compared with those with the T/T genotype in both the Japanese and US cohort in the univariable analysis. In contrast, genotypes with the T allele of CCL2 rs4586 were significantly associated with shorter OS compared with the C/C genotype in the US cohort [hazard ratio (HR) 2.43; P = 0.015] but longer OS in the Japanese cohort (HR 0.58; P = 0.021), resulting in the statistically significant opposite impact on OS (P = 0.001). Our study provides the first evidence that the NFKB1 rs230510 and CCL2 rs4586 are significantly associated with the clinical outcome in patients with locoregional gastric cancer. These results also suggest that the genetic predisposition of the host may dictate the immune-related component of the tumor for progression in gastric cancer.

Stress-related and homeostatic cytokines regulate Vγ9Vδ2 T-cell surveillance of mevalonate metabolism

The potentially oncogenic mevalonate pathway provides building blocks for protein prenylation and induces cell proliferation and as such is an important therapeutic target. Among mevalonate metabolites, only isopentenyl pyrophosphate (IPP) has been considered to be an immunologically relevant antigen for primate-specific, innate-like Vγ9Vδ2 T cells with antitumor potential. We show here that Vγ9Vδ2 T cells pretreated with the stress-related, inflammasome-dependent cytokine interleukin 18 (IL-18) were potently activated not only by IPP but also by all downstream isoprenoid pyrophosphates that exhibit combined features of antigens and cell-extrinsic metabolic cues. Vγ9Vδ2 T cells induced this way effectively proliferated even under severe lymphopenic conditions and the antioxidant N-acetylcysteine significantly improved reconstitution of γδ T cells predominantly with a central memory phenotype. The homeostatic cytokine IL-15 induced the differentiation of effector cells in an antigen-independent fashion, which rapidly produced abundant interferon γ (IFNγ) upon antigen re-encounter. IL-15 induced effector γδ T cells displayed increased levels of the cytotoxic lymphocyte-associated proteins CD56, CD96, CD161 and perforin. In response to stimulation with isoprenoid pyrophosphates, these effector cells upregulated surface expression of CD107a and exhibited strong cytotoxicity against tumor cells in vitro. Our data clarify understanding of innate immunosurveillance mechanisms and will facilitate the controlled generation of robust Vγ9Vδ2 T cell subsets for effective cancer immunotherapy.

Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

KISS1 induction of metastatic dormancy: A therapeutic opportunity?

HER2+ breast cancer patients are presented with either synchronous (S-BM), latent (Lat), or metachronous (M-BM) brain metastases. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Here, employing brain metastatic models, we show that metabolic diversity and plasticity within brain-tropic cells determine metastatic fitness. Lactate secreted by aggressive metastatic cells or lactate supplementation to mice bearing Lat cells limits innate immunosurveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immunosurveillance, oxidize glutamine, and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched M-BM brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Inhibiting xCT function attenuates residual disease and recurrence in these preclinical models.

Protective Immunosurveillance and Therapeutic Antitumor Activity of γδ T Cells Demonstrated in a Mouse Model of Prostate Cancer

In contrast to Ag-specific alphabeta T cells, gammadelta T cells can kill malignantly transformed cells in a manner that does not require the recognition of tumor-specific Ags. Although such observations have contributed to the emerging view that gammadelta T cells provide protective innate immunosurveillance against certain malignancies, particularly those of epithelial origin, they also provide a rationale for developing novel clinical approaches to exploit the innate antitumor properties of gammadelta T cells for the treatment of cancer. Using TRAMP, a transgenic mouse model of prostate cancer, proof-of-concept studies were performed to first establish that gammadelta T cells can indeed provide protective immunosurveillance against spontaneously arising mouse prostate cancer. TRAMP mice, which predictably develop prostate adenocarcinoma, were backcrossed with gammadelta T cell-deficient mice (TCRdelta(-/-) mice) yielding TRAMP x TCRdelta(-/-) mice, a proportion of which developed more extensive disease compared with control TRAMP mice. By extension, these findings were then used as a rationale for developing an adoptive immunotherapy model for treating prostate cancer. Using TRAMP-C2 cells derived from TRAMP mice (C57BL/6 genetic background), disease was first established in otherwise healthy wild-type C57BL/6 mice. In models of localized and disseminated disease, tumor-bearing mice treated i.v. with supraphysiological numbers of syngeneic gammadelta T cells (C57BL/6-derived) developed measurably less disease compared with untreated mice. Disease-bearing mice treated i.v. with gammadelta T cells also displayed superior survival compared with untreated mice. These findings provide a biological rationale for clinical trials designed to adoptively transfer ex vivo expanded autologous gammadelta T cells for the treatment of prostate cancer.

Intrinsic sensor of oncogenic transformation induces a signal for innate immunosurveillance.

Multiple cell-autonomous mechanisms exist in complex metazoans to resist oncogenic transformation, including a variety of tumor- suppressor pathways that control cell proliferation and apoptosis. In vertebrates, additional mechanisms of tumor resistance could potentially rely on cancer cell elimination by specialized cytotoxic leukocytes, such as natural killer (NK) cells. Such mechanisms would require that cancer cells be reliably distinguished from normal cells. The ligands for NKG2D, an activating NK cell receptor, are expressed on many tumor cell lines and at least some primary human tumors. However, it is unknown whether their expression is induced as a direct result of oncogenic transformation in vivo. We provide evidence that NKG2D ligands are induced on spontaneously arising tumors in a murine model of lymphomagenesis and that c-Myc is involved in their regulation. Expression of NKG2D ligands is induced at an early, distinct stage of tumorigenesis upon acquisition of genetic lesions unique to cancer cells, potentially defining a critical step in carcinogenesis. This finding suggests that the regulation of NKG2D ligands depends on a mechanism for intrinsic sensing of oncogenic transformation.