Immune checkpoint inhibitors targeting the interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 induce tumor regression in a subset of non-small cell lung cancer patients. However, clinical response rates are less than 25%. Evaluation of combinations of immunotherapy with existing therapies requires appropriate pre-clinical animal models. In this study, murine lung cancer cells (CMT167 and LLC) were implanted either orthotopically in the lung or subcutaneously in syngeneic mice, and response to anti-PD-1/PD-L1 therapy was determined. Anti-PD-1/PD-L1 therapy inhibited CMT167 orthotopic lung tumors by 95%The same treatments inhibited CMT167 subcutaneous tumors by only 30%, and LLC orthotopic lung tumors by 35%. CMT167 subcutaneous tumors had more Foxp3+ CD4+ T cells and fewer PD-1+ CD4+ T cells compared to CMT167 orthotopic tumors. Flow cytometric analysis also demonstrated increased abundance of PD-L1high cells in the tumor microenvironment in CMT167 tumor-bearing lungs compared to CMT167 subcutaneous tumors or LLC tumor-bearing lungs. Silencing PD-L1 expression in CMT167 cells resulted in smaller orthotopic tumors that remained sensitive to anti-PD-L1 therapy, whereas implantation of CMT167 cells into PD-L1− mice blocked orthotopic tumor growth, indicating a role for PD-L1 in both the cancer cell and the microenvironment. These findings indicate that the response of cancer cells to immunotherapy will be determined by both intrinsic properties of the cancer cells and specific interactions with the microenvironment. Experimental models that accurately recapitulate the lung tumor microenvironment are useful for evaluation of immunotherapeutic agents.

Purpose: The source of glioblastoma (GBM)-associated immunosuppression remains multifactorial. We sought to clarify and therapeutically target myeloid cell-derived peripheral immunosuppression in patients with GBM.Experimental Design: Direct ex vivo T-cell function, serum Arginase I (ArgI) levels, and circulating myeloid lineage populations were compared between patients with GBM and normal donors or patients with other intracranial tumors. Immunofunctional assays were conducted using bulk and sorted cell populations to explore the potential transfer of myeloid cell-mediated immunosuppression and to identify a potential mechanism for these effects. ArgI-mediated immunosuppression was therapeutically targeted in vitro through pharmacologic inhibition or arginine supplementation.Results: We identified a significantly expanded population of circulating, degranulated neutrophils associated with elevated levels of serum ArgI and decreased T-cell CD3z expression within peripheral blood from patients with GBM. Sorted CD11b þ cells from patients with GBM were found to markedly suppress normal donor T-cell function in coculture, and media harvested from mitogen-stimulated GBM peripheral blood mononuclear cell (PBMC) or GBM-associated mixed lymphoid reactions showed ArgI levels that were significantly higher than controls. Critically, T-cell suppression in both settings could be completely reversed through pharmacologic ArgI inhibition or with arginine supplementation. Conclusions: These data indicate that peripheral cellular immunosuppression in patients with GBM is associated with neutrophil degranulation and elevated levels of circulating ArgI, and that T-cell function can be restored in these individuals by targeting ArgI. These data identify a novel pathway of GBM-mediated suppression of cellular immunity and offer a potential therapeutic window for improving antitumor immunity in affected patients. Clin Cancer Res; 17(22); 6992-7002. Ó2011 AACR.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3 mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ/TNFα/IL10 CD4 and CD8 T cells. Immunodepletion of CD4 but not CD8 T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway. This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. .

Macrophages represent an important component of the tumor microenvironment and play a complex role in cancer progression. These cells are characterized by a high degree of plasticity, and alter their phenotype in response to local environmental cues. While the M1/M2 classification of macrophages has been widely used, the complexity of macrophage phenotypes has not been well studied, particularly in lung cancer. In this study we employed an orthotopic immunocompetent model of lung adenocarcinoma in which murine lung cancer cells are directly implanted into the left lobe of syngeneic mice. Using multi-marker flow cytometry, we defined and recovered several distinct populations of monocytes/macrophages from tumors at different stages of progression. We used RNA-seq transcriptional profiling to define distinct features of each population and determine how they change during tumor progression. We defined an alveolar resident macrophage population that does not change in number and expresses multiple genes related to lipid metabolism and lipid signaling. We also defined a population of tumor-associated macrophages that increase dramatically with tumor, and selectively expresses a panel of chemokine genes. A third population, which resembles tumor-associated monocytes, expresses a large number of genes involved in matrix remodeling. By correlating transcriptional profiles with clinically prognostic genes, we show that specific monocyte/macrophage populations are enriched in genes that predict outcomes in lung adenocarcinoma, implicating these subpopulations as critical determinants of patient survival. Our data underscore the complexity of monocytes/macrophages in the tumor microenvironment, and suggest that distinct populations play specific roles in tumor progression.

Key Points Next-generation humanized mice differentially support human HSPC maintenance and myelopoiesis. MISTRG mice support long-term human HSPC maintenance demonstrated by quaternary transplantation and development of human tissue macrophages.

Eicosanoids, including prostaglandins, produced by cyclooxygenases (COX), and leukotrienes, produced by 5-lipoxygenase (5-LO) have been implicated in cancer progression. These molecules are produced by both cancer cells and the tumor microenvironment (TME). We previously reported that both COX and 5-LO metabolites increase during progression in an orthotopic immunocompetent model of lung cancer. While prostaglandins in the TME have been well studied, less is known regarding 5-LO products produced by the TME. We examined the role of 5-LO in the TME, using a model in which Lewis Lung Carcinoma cells are directly implanted into the lungs of syngeneic WT or 5-LO knockout (5-LO-KO) mice. Unexpectedly, primary tumor volume and liver metastases were increased in 5LO-KO mice. This was associated with an ablation of leukotriene production, consistent with production mainly mediated by the microenvironment. Increased tumor progression was partially reproduced in global LTC4 synthase knockout or mice transplanted with LTA4 hydrolase-deficient bone-marrow. Tumor bearing lungs of 5-LO-KO had decreased numbers of CD4 and CD8 T cells compared to WT controls, as well as fewer dendritic cells. This was associated with lower levels of CCL20 and CXL9, which have been implicated in dendritic and T cell recruitment. Depletion of CD8 cells increased tumor growth, and eliminated the differences between WT and 5-LO mice. These data reveal an anti-tumorigenic role for 5-LO products in the microenvironment during lung cancer progression through regulation of T cells, and suggest that caution should be used in targeting this pathway in lung cancer.

Transient suppression of peripheral immunity is a major source of complication for patients suffering from ischemic stroke. The release of Arginase I (ArgI) from activated neutrophils has recently been associated with T-cell dysfunction in a number of pathologies. However, this pathway has not been previously explored in ischemic stroke. Using the murine model of transient middle cerebral artery occlusion, we explored effects of stroke on peripheral T-cell function and evaluated the role of neutrophils and ArgI. Stimulation of splenic T cells from post-stroke animals with anti-CD3/CD28 resulted in decreased proliferation and interferon-γ production when compared with sham-surgery controls. Flow cytometric analysis of intrasplenic leukocytes exposed the presence of a transient population of activated neutrophils that correlated quantitatively with elevated ArgI levels in culture media. In vitro activation of purified resting neutrophils from unmanipulated controls confirmed the capacity for murine neutrophils to release ArgI from preformed granules. We observed decreased expression of the L-arg-sensitive CD3ζ on T cells, consistent with decreased functional activity. Critically, L-arg supplementation restored the functional response of post-stroke T cells to mitogenic stimulation. Together, these data outline a novel mechanism of reversible, neutrophil-mediated peripheral immunosuppression related to ArgI release following ischemic stroke.

Pre-clinical feasibility, safety, and efficacy testing of hematopoietic stem cell (HSC)-mediated gene therapy approaches is commonly performed in large animal models such as nonhuman primates (NHPs). Here we wished to determine whether mouse models would allow engraftment of NHP HSPCs which would enable more facile and less costly evaluation of promising strategies. In this study, we comprehensively tested two mouse strains for the engraftment of NHP CD34+ hematopoietic stem and progenitor cells. No engraftment of NHP HSPCs was observed in NSG mice, whereas the humanized MISTRG model did demonstrate dose-dependent multilineage engraftment of NHP cells in the peripheral blood, bone marrow, spleen, and thymus. Most importantly and closely mimicking the hematopoietic recovery of autologous stem cell transplants in the NHP, only HSC-enriched CD34+CD90+CD45RA– cell fractions did engraft and reconstituted the bone marrow stem cell niche in MISTRG mice. In summary, we here report the first “monkeynized” mouse xenograft model that closely recapitulates the autologous hematopoietic reconstitution in the NHP stem and progenitor cell transplantation and gene therapy model. Availability of this model has the potential to pre-evaluate novel HSC-mediated gene therapy approaches, inform studies in the NHP, and improve the overall outcome of large-animal experiments.