Lamichhane, Purushottam

Background Ovarian cancer (OC), a highly lethal cancer in women, has a 48% 5-year overall survival rate. Prior studies link the presence of IL-17 and Th17 T cells in the tumor microenvironment to improved survival in OC patients. To determine if Th17-inducing vaccines are therapeutically effective in OC, we created a murine model of Th17-inducing dendritic cell (DC) (Th17-DC) vaccination generated by stimulating IL-15 while blocking p38 MAPK in bone marrow-derived DCs, followed by antigen pulsing. Methods ID8 tumor cells were injected intraperitoneally into mice. Mice were treated with Th17-DC or conventional DC (cDC) vaccine alone or with immune checkpoint blockade (ICB). Systemic immunity, tumor associated immunity, tumor size and survival were examined using a variety of experimental strategies. Results Th17-DC vaccines increased Th17 T cells in the tumor microenvironment, reshaped the myeloid microenvironment, and improved mouse survival compared with cDC vaccines. ICB had limited efficacy in OC, but Th17-inducing DC vaccination sensitized it to anti-PD-1 ICB, resulting in durable progression-free survival by overcoming IL-10-mediated resistance. Th17-DC vaccine efficacy, alone or with ICB, was mediated by CD4 T cells, but not CD8 T cells. Conclusions These findings emphasize using biologically relevant immune modifiers, like Th17-DC vaccines, in OC treatment to reshape the tumor microenvironment and enhance clinical responses to ICB therapy.

Abstract The PD-1:PD-L1 axis is a major immune regulatory pathway that blunts T cell activation in the tumor microenvironment. We recently identified that PD-1 is also upregulated on ovarian mouse tumor-infiltrating myeloid dendritic cells (TIDCs) that exhibit an immune suppressive phenotype. Extending those observations in this study, we now report that PD-1 is also expressed on human myeloid TIDCs of ovarian cancer patients, on which it suppresses the expression of co-stimulatory molecules (CD40 and CD80) as well as production of inflammatory cytokines. Mechanistic studies revealed that PD-1 on TIDCs is associated with Src homology domain-containing phosphatase-2 (SHP-2) and that PD-1-mediated blockade of NFkB-dependent cytokine release is SHP-2 dependent. PD-1 also inhibits NFkB-mediated antigen presentation which appears, however, to be independent of SHP-2. Thus, suppression of NFkB is critical in mediating PD-1-induced paralysis of ovarian tumor-associated immune TIDCs and is mediated through multiple signaling circuits that are both SHP-2 dependent and independent.

Abstract PD-1: PD-L1 is a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells; PD-1 has also been shown to be expressed on and thereby impact the functions of cells of innate arm such as dendritic cells (DCs) primarily at the tumor site but not in the periphery. We show here that one of the culprits in the tumor microenvironment responsible for enhancing PD-1 expression on the tumor-associated DCs is the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer. Based on inhibition and knockdown studies, we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Using murine model of ovarian carcinoma (ID8), we show that tumor-associated DCs express not only high levels of PD-1 on the surface but also have increased expression of STAT-3 and p-STAT3 compared to splenic DCs. Blockade of PD-1 on DCs led to increased production of IL-10 which correlated with a further increase in PD-1 expression. In in vivo studies, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ID8 ovarian tumor bearing mice; leading to a decrease in tumor mass and a significant increase in survival. These results show that IL-10 and PD-1 pathway intersect in DCs in theTME hence making them attractive targets for therapy of ovarian cancer.

Abstract Ligation of programmed cell death-1 (PD-1) in the tumor microenvironment is known to inhibit effective adaptive antitumor immunity. Blockade of PD-1 in humans has resulted in impressive, durable regression responses in select tumor types. However, durable responses have been elusive in ovarian cancer patients. PD-1 was recently shown to be expressed on and thereby impair the functions of tumor-infiltrating murine and human myeloid dendritic cells (TIDC) in ovarian cancer. In the present work, we characterize the regulation of PD-1 expression and the effects of PD-1 blockade on TIDC. Treatment of TIDC and bone marrow–derived dendritic cells (DC) with IL10 led to increased PD-1 expression. Both groups of DCs also responded to PD-1 blockade by increasing production of IL10. Similarly, treatment of ovarian tumor–bearing mice with PD-1 blocking antibody resulted in an increase in IL10 levels in both serum and ascites. While PD-1 blockade or IL10 neutralization as monotherapies were inefficient, combination of these two led to improved survival and delayed tumor growth; this was accompanied by augmented antitumor T- and B-cell responses and decreased infiltration of immunosuppressive MDSC. Taken together, our findings implicate compensatory release of IL10 as one of the adaptive resistance mechanisms that undermine the efficacy of anti–PD-1 (or anti–PD-L1) monotherapies and prompt further studies aimed at identifying such resistance mechanisms. Cancer Res; 77(23); 6667–78. ©2017 AACR.

Abstract PD-1 and PD-L1 form a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells, PD-1 has also been shown to be expressed on and hence impact the functions of cells of innate arm, such as dendritic cells (DCs), primarily at the tumor site but not in the periphery. Our aim in this study was to identify tumor-associated factor(s) responsible for PD-1 regulation on DCs and investigate how DCs in the tumor microenvironment respond to anti-PD-1-based immunotherapy. Both bone marrow-derived dendritic cells (BMDCs) as well as murine ovarian tumor ascites-derived DCs were used in the studies. Using in vitro cultures, we show that the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer, is potent regulator of PD-1 expression on DCs. Based on inhibition and siRNA knockdown studies; we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Treatment of DCs with IL-10 also led to an increase in expression of PD-L1 on surface of DCs as well as an increase in release of soluble PD-L1. Antibody-mediated blockade of PD-1 on DCs led to greatly increased production of IL-10 (> 4 fold) in cultures in vitro. Furthermore, treatment of tumor bearing mice with anti-PD-1 antibody led to a significant increase of IL-10 in ascites (> 6 fold) and periphery (> 4 fold). This compensatory release of IL-10 correlated with a further increase in PD-1 expression on DCs both in vitro and in vivo. In in vivo studies, while the blockade of PD-1 or IL-10 neutralization as monotherapy were ineffective, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ovarian tumor bearing mice; leading to a decrease in tumor growth and a significant increase in survival. These results show that the IL-10 and PD-1 pathways intersect with DCs in the tumor microenvironment. PD-1 blockade on DCs leads to compensatory release of IL-10 resulting in the maintenance of immunosuppression, and a combination regimen of PD-1 blockade and IL-10 neutralization has therapeutic benefit in ovarian tumors. These results serve as catalyst to explore the feasibility/efficacy of exploiting these targets in clinical settings for treatment of human ovarian cancer. Citation Format: Purushottam Lamichhane, Lavakumar Karyampudi, Barath Shreeder, James Krempski, Deborah Bahr, Matthew Block, Keith Knutson. Blockade of PD-1 signaling in tumor-associated dendritic cells results in compensatory IL-10 release maintaining immune suppression in ovarian cancer microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B66.

Abstract The PD-1:PD-L1 immune signaling axis mediates suppression of T-cell–dependent tumor immunity. PD-1 expression was recently found to be upregulated on tumor-infiltrating murine (CD11c+CD11b+CD8−CD209a+) and human (CD1c+CD19−) myeloid dendritic cells (TIDC), an innate immune cell type also implicated in immune escape. However, there is little knowledge concerning how PD-1 regulates innate immune cells. In this study, we examined the role of PD-1 in TIDCs derived from mice bearing ovarian tumors. Similar to lymphocytes, TIDC expression of PD-1 was associated with expression of the adapter protein SHP-2, which signals to NF-κB; however, in contrast to its role in lymphocytes, we found that expression of PD-1 in TIDC tonically paralyzed NF-κB activation. Further mechanistic investigations showed that PD-1 blocked NF-κB–dependent cytokine release in a SHP-2–dependent manner. Conversely, inhibition of NF-κB–mediated antigen presentation by PD-1 occurred independently of SHP-2. Collectively, our findings revealed that PD-1 acts in a distinct manner in innate immune cells compared with adaptive immune cells, prompting further investigations of the signaling pathways controlled by this central mediator of immune escape in cancer. Cancer Res; 76(2); 239–50. ©2015 AACR.

Abstract Purpose: It is well known that the immune system impacts the clinical course of ovarian cancer suggesting that immune-based approaches may effective in treating the disease. This has led to several clinical trials of novel treatments such as antibody, vaccine, and adoptive T cell therapy. PD-1:PD-L1 axis is a major immune regulatory pathway that blunts immune effectors in the tumor microenvironment. It is well known that high surface expression of PD-1 on tumor infiltrating T cells is a sign of their exhaustion. However, the complete mechanism behind the PD-1 regulation of T cell exhaustion is yet to be explored. Recently we identified that PD-1 is also expressed on ovarian tumor-infiltrating myeloid dendritic cells (DCs) that exhibit an immunosuppressive phenotype. Our goal in this study was to understand the mechanism by which PD-1 mediates the paralysis of ovarian cancer infiltrating DCs. Experimental procedures: PD-1+ DCs obtained from ID8 mouse model of ovarian cancer were used in this study. Data from this preclinical model was confirmed by using myeloid DCs obtained from ovarian cancer patient’s samples. Using standard flow cytometry, immunoassays (ELISA, multiplexed cytokine assay), PCR array, western blot and confocal microscopy, PD-1 mediated paralysis of ovarian cancer DCs was determined. Results: Our data shows that blockade of surface PD-1 on ovarian cancer infiltrating DCs results in their activation by, enhancing their surface co-stimulatory molecules expression, increasing their motility, enhancing their antigen presentation capacity, and increasing their production of immunostimulatory cytokines. Also, our results suggest that PD-1 expressed on tumor DCs mediates the suppression of NFkB which occurs through SHP-2 and IKK dependent mechanisms. Conclusion: Our study reveals that blockade of PD-1 on ovarian cancer infiltrating DCs results in the reversal of their paralysis to such an extent that it overrides their B7-H1 mediated suppression of T cells and this occurs through the reversal of PD-1 mediated tonic suppression of NFkB. Citation Format: Lavakumar Karyampudi James Krempski, Purushottam Lamichhane, Kimberly R. Kalli, Marshall D. Behrens, Doris M. Vargas, Lynn C Hartmann, Karen E Hedin, Ellen L. Goode, Keith L. Knutson. PD-1 mediated paralysis of ovarian cancer infiltrating dendritic cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1418.

Abstract Dendritic cells (DCs) play a pivotal role in the tumor microenvironment, which is known to affect disease progression in many human malignancies. Infiltration by mature, active DCs into the tumors confers an increase in immune activation and recruitment of disease-fighting immune effector cells and pathways. DCs are the preferential target of infiltrating T cells. However, tumor cells have means of suppressing DC function or of altering the tumor microenvironment in such a way that immune-suppressive DCs are recruited. Advances in understanding these changes have led to promising developments in cancer-therapeutic strategies targeting tumor-infiltrating DCs to subdue their immunosuppressive functions and enhance their immune-stimulatory capacity.

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.