Behrens, Marshall D.

Abstract Background: Ovarian cancer (OC) is commonly associated with ascites and predominantly remains in the peritoneal cavity. Soluble factors present in ascites such as growth factors and cytokines likely play a key role in local tumor behavior including stage, invasiveness and resectability and survival. Members of IL-12 family (IL-12p70, IL-23, IL-27 and IL-35) play a critical role in the regulation of inflammatory response. However, their role as local factors in ovarian cancer growth has not been well studied. We hypothesized that that the level of these important immunomodulatory and pro-inflammatory cytokines are associated with the clinical behavior of OC. Methods: Ascites fluid was collected and analyzed from women undergoing maximal debulking surgery for presumed OC, and relevant clinical endpoints were abstracted. Concentrations of IL-12 related cytokines (IL-23, IL-27, IL-35, and IL-12p70) were assessed by ELISA. Cytokines were also assessed on matching pre-surgery plasma samples. Cytokine concentrations between matched ascites and plasma samples were examined using scatter plots and Spearman correlations. Associations between ascites concentrations and clinical characteristics were assessed using Wilcoxon rank-sum tests. Associations between ascites levels with overall survival in newly diagnosed invasive OC patients were assessed by Cox proportional hazards models using a log2 transformation. Results: Ascites fluid was analyzed from 20 patients with primary epithelial OC including 18 serous histology, 19 patients with stage III or IV disease: 16 (80%) were optimally debulked. We observed that higher levels of IL-12p70 were associated with locally invasive disease including higher rates of sub-optimal debulking (p=0.0074) and stage IV disease (p=0.012). For other IL-12 family members we observed similar associations between higher cytokine levels and suboptimal disease and stage IV disease including IL-23 (p=0.73, p=0.17), IL-27 (p=0.041, p=0.018), and IL35 (p=0.12, p=0.11), respectively. In addition, increase in IL-23 was associated with worse overall survival (log2 HR = 1.85, p=0.031). Conclusions: Higher levels of IL-12 family of cytokines in ascitic fluid are associated with several surrogates of biological aggressiveness in OC including stage, resectability and survival. Given the importance of local tumor environment on clinical behavior, a detailed understanding of IL-12 pathway may help in developing novel therapeutic strategies to treat OC. Citation Format: Eati Basal, Mathew S. Block, Matthew J. Maurer, Krista Goergen, Courtney L. Erskine, Marshall Behrens, Kimberly R. Kalli, Keith Knutson, William Cliby. Levels of IL-12 family cytokines in ascitic fluid are associated with extent and resectability of ovarian cancer and correlate with overall survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2013-1434

Abstract Despite significant progress in the treatment of breast cancer particularly through the use of targeted therapy, relapse and chemo-resistance remain a major hindrance in the fight to minimize the burden of the disease. Accumulating evidence suggest that breast cancer stem cells (BCSCs), which show resistance to chemotherapy and radiation, drive tumor initiation and disease recurrence. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial to mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them in order to reverse EMT and attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL expression using AXL inhibitor MP470 (amuvatinib) or shRNA reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with expression of stem cell genes, regulation of metastases genes, increased tumorigenicity, and was important for BCSC invasion and migration. Inactivation of AXL also led to downregulation of NFκB pathway and reduced tumor formation in vivo. Together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anti-cancer therapies and to reduce breast cancer recurrence and metastases. Citation Format: Michael K. Asiedu, Francis Beauchamp-Perez, James N. Ingle, Marshall D. Behrens, Derek C. Radisky, Keith Knutson. AXL induces epithelial to mesenchymal transition and regulates the function of breast cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2013-LB-272

HLA‐DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA), while HLA‐DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4+ T‐cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen‐specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4+ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN‐γ. *0401 Tg mice harbor memory CD4+ T cells that differentiate into IL‐17+ cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg‐cell numbers in *0401 Tg mice, causes immune dysregulation.

Abstract Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.

Abstract To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8·CD4−/− mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8·CD8−/− mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8·CD8−/− and DQ8 mice produced rheumatoid factor. In addition, DQ8·CD8−/− mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4−/− mice. An increased frequency of CD3+ double-negative (DN) T cells was found in DQ8·CD8−/− compared with DQ8·CD4−/− and DQ8 mice. These CD3+ DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3+ and CD3+ DN T cells in DQ8·CD8−/− mice compared with DQ8·CD4−/− and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8+ T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.

Abstract To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2Aq) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by Aq. In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.

Abstract CD28 is required to achieve optimal T cell activation to an Ag. To determine the role CD28 costimulation plays in collagen-induced arthritis, we have generated DQ8 transgenic, CD28-deficient mice. DQ8 mice deficient for CD28 had comparable numbers of CD4 and CD8 T cells as DQ8.CD28+/+ mice. DQ8.CD28−/− mice develop collagen-induced arthritis with delayed onset and less severity than DQ8.CD28+/+ mice. T cells from DQ8.CD28−/− mice did not respond to type II collagen efficiently in vitro, although the response to DQ8-restricted peptides was similar to that in the parent mice. There was no functional defect in T cells as observed by proliferation with Con A. Cytokine analysis from in vitro study showed the production of high levels of the inflammatory cytokine, IFN-γ, in response to type II collagen. We observed an increase in CD4+CD28−NKG2D+ cells after immunization, suggesting an important role for cells bearing this receptor in the disease process. CD28−/− mice also have an increased number of DX5+ cells compared with CD28+/+ mice, which can lead to the production of high levels of IFN-γ. DQ8.CD28−/− mice had an increased number of cells bearing other costimulatory markers. Cells from DQ8.CD28−/− mice exhibited a lower proliferation rate and were resistant to activation-induced cell death compared with DQ8.CD28+/+ mice. This study supports the idea that CD28 plays a crucial role in the regulation of arthritis. However, in the absence of CD28 signaling, other costimulatory molecules can lead to the development of disease, thus indicating that the requirement for CD28 may not be absolute in the development of arthritis.

Abstract The human MHC class II genes are associated with genetic susceptibility to multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS of presumed autoimmune origin. These genes encode for proteins responsible for shaping immune response. The exact role of HLA-DQ and -DR genes in disease pathogenesis is not well-understood due to the high polymorphism, linkage disequilibrium, and heterogeneity of human populations. The advent of HLA class II-transgenic (Tg) mice has helped in answering some of these questions. Previously, using single-Tg mice (expressing the HLA-DR or -DQ gene), we showed that proteolipid protein (PLP)91–110 peptide induced classical experimental autoimmune encephalomyelitis only in DR3.Aβ° mice, suggesting that DR3 (DRB1*0301) is a disease susceptible gene in the context of PLP. Human population studies have suggested that HLA-DQ6 (DQB1*0601) may be a protective gene in MS. To test this disease protection in an experimental model, we generated double-Tg mice expressing both HLA-DR3 and -DQ6. Introduction of DQ6 onto DR3-Tg mice led to a decrease in disease incidence on immunization with PLP91–110 peptide indicating a dominant protective role of DQ6. This protective effect is due to high levels of IFN-γ produced by DQ6-restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis. Our study indicates that DQ6 modifies the PLP91–110-specific T cell response in DR3 through anti-inflammatory effects of IFN-γ, which is protective for experimental autoimmune encephalomyelitis. Thus, our double-Tg mouse provides a novel model in which to study epistatic interactions between HLA class II molecules in MS.

Abstract In humans, HLA-DR alleles sharing amino acids at the third hypervariable region with DRB1*0401(shared epitope) are associated with a predisposition to rheumatoid arthritis, whereas DRB1*0402 is not associated with such a predisposition. Both DRB1*0402 and DRB1*0401 occur in linkage with DQ8 (DQB1*0302). We have previously shown that transgenic (Tg) mice expressing HLA-DRB1*0401 develop collagen-induced arthritis. To delineate the role of “shared epitope” and gene complementation between DR and DQ in arthritis, we generated DRB1*0402, DRB1*0401.DQ8, and DRB1*0402.DQ8 Tg mice lacking endogenous class II molecules, AE°. DRB1*0402 mice are resistant to develop arthritis. In double-Tg mice, the DRB1*0401 gene contributes to the development of collagen-induced arthritis, whereas DRB1*0402 prevents the disease. Humoral response to type II collagen is not defective in resistant mice, although cellular response to type II collagen is lower in *0402 mice compared with *0401 mice. *0402 mice have lower numbers of T cells in thymus compared with *0401 mice, suggesting that the protective effect could be due to deletion of autoreactive T cells. Additionally, DRB1*0402 mice have a higher number of regulatory T cells and show increased activation-induced cell death, which might contribute toward protection. In DRB1*0401.DQ8 mice, activated CD4+ T cells express class II genes and can present DR4- and DQ8-restricted peptides in vitro, suggesting a role of class II+ CD4 T cells locally in the joints. The data suggest that polymorphism in DRB1 genes determines predisposition to develop arthritis by shaping the T cell repertoire in thymus and activating autoreactive or regulatory T cells.

Abstract Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer–bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1–negative tumors). Cancer Res; 74(11); 2974–85. ©2014 AACR.