Suppression Of Antitumor Immune Responses Research Articles

Correlation of PDL1 tumor expression and treatment outcomes in patients with renal cell carcinoma (RCC) receiving tyrosine kinase inhibitors: COMPARZ study analysis.

416 Background: The interaction of PDL1 (B7H1) with its receptor PD-1 on activated T cells contributes to suppression of antitumor immune responses. Tumor PDL1 expression has been associated with poor outcomes in RCC but has not been investigated as a biomarker of response in RCC patients receiving standard vascular endothelial growth factor (VEGF)-targeted therapy. Methods: Formalin-fixed paraffin-embedded tumor samples were collected at baseline from consenting patients enrolled in COMPARZ—a phase lll clinical trial comparing pazopanib and sunitinib as first-line interventions for metastatic RCC (Motzer et al, NEJM 2013). PDL1 expression was evaluated using the anti-PDL1 mouseIgG1 (clone 5H1; Thompson) on the Leica automated immunohistochemistry platform. Additional dual PDL1/CD68 staining was performed on tumor associated macrophages (TAMs). Tumor PDL1 expression was quantified by an H-score (HS). PDL1+ TAMs were assessed semiquantitatively. In addition, intra-tumor CD8+ T cells were quantified morphometrically. The association between PDL1 HS, CD8+T cell counts, and survival was investigated using Kaplan-Meier analysis. Results: HS data were available from 453 of 1110 patients. 64% of patients had negative (HS = 0) PDL1 expression (HS range 0-290), but PDL1 expression was associated with tumours containing higher numbers of infiltrating macrophages. Peripheral CD8+ T cells in the invasive margin surrounding the tumor were also observed. Patients with HS >50 (n = 61, 13%) had significantly shorter overall survival (OS) in both pazopanib (19.7 vs 31.6 mo) and sunitinib (15.3 vs 27.7 mo) arms. In both arms, patients with HS >50 with intratumoral CD8+T cell counts >300 had the shortest OS. Results were similar for progression-free survival and persisted on multivariate analysis. Conclusions: This is the largest report to show that tumors’PDL1 expression and CD8+ T cell counts are associated with treatment outcome in metastatic RCC patients. Increased PDL1, or increased PDL1 plus tumor CD8+ T cell counts, were associated with shorter OS. These findings may have major implications for future trial designs that involve PD-1 inhibitors.

Effects of Adjuvant Chemoradiotherapy on the Frequency and Function of Regulatory T Cells in Patients with Head and Neck Cancer

Regulatory T cells (Treg) accumulate in tumor tissues and the peripheral blood of cancer patients and may persist after therapies. This cross-sectional study examines effects of adjuvant chemoradiotherapy (CRT) on Treg numbers and function in head and neck squamous cell carcinoma (HNSCC) patients. The frequency and absolute numbers of CD4(+), ATP-hydrolyzing CD4(+)CD39(+) and CD8(+) T cells, and expression levels of CD39, CD25, TGF-β-associated LAP and GARP on Treg were measured by flow cytometry in 40 healthy donors (NC) and 71 HNSCC patients [29 untreated with active disease (AD); 22 treated with surgery; 20 treated with CRT]. All treated subjects had no evident disease (NED) at the time of phlebotomy. In an additional cohort of 40 subjects with AD (n = 15), NED (n = 10), and NC (n = 15), in vitro sensitivity of CD4(+) T-cell subsets to cisplatin and activation-induced cell death (AICD) was tested in Annexin V-binding assays. CRT decreased the frequency of circulating CD4(+) T cells (P < 0.002) but increased that of CD4(+)CD39(+) Treg (P ≤ 0.001) compared with untreated or surgery-only patients. Treg frequency remained elevated for >3 years. CRT increased surface expression of LAP, GARP, and CD39 on Treg. In vitro Treg were resistant to AICD or cisplatin but conventional CD4(+) T cells (Tconv) were not. CRT-induced Treg from AD or NC subjects upregulated prosurvival proteins whereas Tconv upregulated proapoptotic Bax. Highly suppressive, cisplatin-resistant Treg increase in frequency and persist after CRT and could be responsible for suppression of antitumor immune responses and recurrence in HNSCC.

B cell regulation of anti-tumor immune response

Our laboratory has been investigating the role of B cells on tumor immunity. We have studied the immune response in mice that are genetically lacking in B cells (BCDM) using a variety of syngeneic mouse tumors and compared immune responses in BCDM with those seen in wild type (WT) immunocompetent mice (ICM). A variety of murine tumors are rejected or inhibited in their growth in BCDM, compared with ICM, including the EL4 thymoma, and the MC38 colon carcinoma in C57BL/6 mice, as well as the EMT-6 breast carcinoma in BALB/c mice. In all three murine models, tumors show reduced growth in BCDM which is accompanied by increased T cell and NK cell infiltration, and a more vigorous Th1 cytokine response, and increased cytolytic T cell response in the absence of B cells. Reconstitution of the mice with B cells results in augmented tumor growth due to a diminished anti-tumor immune response and in reduction in CD8+ T cell and NK cell infiltration. Studies involving BCR transgenic mice indicated that B cells inhibit anti-tumor T cell responses through antigen non-specific mechanisms. More recent studies using the EMT-6 model demonstrated that both the number and function of Treg cells in ICM was increased relative to that seen in BCDM. Increased expansion of Treg cells was evident following EMT-6 implantation in ICM relative to that seen in non-tumor-bearing mice or BCDM. The percentage and number of Tregs in spleen, tumor draining lymph nodes, and the tumor bed are increased in ICM compared with BCDM. Treg functional capacity as measured by suppression assays appears to be reduced in BCDM compared with ICM. In contrast to other described types of B regulatory activity, adoptive transfer of B cells can rescue tumor growth independently of the ability of B cells to secrete IL-10, and also independently of MHC-II expression. In experiments using the MC38 adenocarcinoma model, BCDM reconstituted with WT B cells support tumor growth while tumor growth continues to be inhibited in BCDM reconstituted with OX40L(-/-) B cells. This suggests that interaction between OX40 on T cells and OX40-ligand on B cells may be important in modulating anti-tumor immune response. Ongoing experiments in the laboratory indicate that B cells migrate to the site of tumor and acquire expression of immunosuppressive ligands and/or cytokines that contribute to the inhibition of anti-tumor immune response. Significant infiltration of human tumors by Treg cells as well as B cells suggests that observations made in murine systems may be applicable to human tumors as well.

Regulatory T cell subsets in human cancer: are they regulating for or against tumor progression?

Regulatory T cells (Treg) play a key role in maintaining the balance of immune responses in human health and in disease. Treg come in many flavors and can utilize a variety of mechanisms to modulate immune responses. In cancer, inducible (i) or adaptive Treg expand, accumulate in tissues and the peripheral blood of patients, and represent a functionally prominent component of CD4+ T lymphocytes. Phenotypically and functionally, iTreg are distinct from natural (n) Treg. A subset of iTreg expressing ectonucleotidases, CD39 and CD73, is able to hydrolyze ATP to 5'-AMP and adenosine (ADO) and thus mediate suppression of those immune cells which express ADO receptors. iTeg can also produce prostaglandin E2 (PGE2). These iTreg, expanding in response to tumor antigens and cytokines such as TGF-β or IL-10, are presumably responsible for the suppression of anti-tumor immune responses and for successful tumor escape. On the other hand, in cancers associated with prominent inflammatory infiltrates, e.g., colorectal carcinoma or certain types of breast cancer, iTreg down-regulate excessive inflammation by producing ADO and/or PGE2 and protect the host from tissue injury and tumor development. Thus, iTreg utilizing the same adenosine pathway play a key but dual role in cancer, and their plasticity is controlled and driven by the microenvironment. Thus, monitoring for the frequency and functions of iTreg rather than nTreg is important in cancer. In addition, elimination of iTreg by various available strategies prior to immunotherapies may not be beneficial in all cases and needs to be undertaken with caution.

Abstract A252: A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers.

Abstract Introduction: Within the tumor microenvironment, CSF1R signaling is thought to play an important role in recruitment and differentiation of tumor-associated macrophages and osteoclasts, promoting disease progression through suppression of anti-tumor immune response, promotion of angiogenesis, tumor cell metastasis and tumor-induced osteolysis. ARRY-382 is a potent, highly selective, oral inhibitor of CSF1R. This Phase 1 dose-escalation study was designed to determine the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of ARRY-382 in patients (pts) with advanced or metastatic cancers refractory to standard treatment. Methods: Pts received ARRY-382 orally once daily (QD) in 28-day cycles. Study design included an initial accelerated titration phase, followed by 3+3 dose escalation and an expansion phase. Safety was assessed by adverse events (AEs), ECGs, clinical laboratory tests, physical exams and vital signs. PK parameters were estimated based on serial plasma PK samples. Levels of pERK in pt monocytes stimulated with CSF1 ex vivo, circulating CD14dim/CD16+ nonclassical monocytes (NCM) and CSF1 were evaluated as markers of CSF1R inhibition. Urinary collagen type 1 cross-linked N[[Unable to Display Character: &amp;#8209;]]telopeptide (NTX) was evaluated as a marker of bone turnover. Results: Twenty-six pts received ARRY-382 at doses ranging from 25 to 500 mg QD. The MTD of ARRY-382 was 400 mg QD. Dose-limiting toxicities were increased creatine kinase (CK), increased AST and pyrexia (1 pt each, all Grade 3). The most common drug-related AEs were fatigue (42%), increased CK (27%), nausea (23%), decreased appetite (15%) and vomiting (12%). Grade 3/4 AEs in &amp;gt;1 pt were increased CK (23%), pneumonia (15%), anemia (8%) and vomiting (8%). Increases in ARRY-382 exposure were approximately dose proportional. At the MTD, a Cmax of 3.06 µg/mL was achieved with a Ctrough &amp;gt; 1 µg/mL after repeated dosing. Doses ≥ 200 mg QD resulted in &amp;gt; 80% mean reduction from Baseline in monocyte pERK, consistent with exposures above the ARRY-382 IC50 value. The PD activity of ARRY-382 was consistent with CSF1R inhibition. Reductions in NCM were observed at doses ≥ 200 mg QD, with a 96% mean decrease from Baseline observed at the MTD. Reductions in urinary NTX were observed at doses ≥ 50 mg QD. Five of 7 pts with elevated urinary NTX at Baseline experienced reductions to within the normal range in the first cycle, including 3 pts with bone metastases. Increases in CSF1 appeared dose-dependent with ∼28-fold maximal increase from Baseline observed at the MTD in the first cycle. No objective responses were observed, although 4 pts (15%) experienced stable disease which lasted &amp;gt; 3 months for 2 pts. Conclusions: ARRY-382 was generally well tolerated in pts with advanced or metastatic cancers. Exposure and PD activity of ARRY[[Unable to Display Character: &amp;#8209;]]382 indicate that CSF1R was significantly inhibited at tolerated doses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A252. Citation Format: Johanna C. Bendell, Anthony W. Tolcher, Suzanne F. Jones, Muralidhar Beeram, Jeffrey R. Infante, Paul Larsen, Kevin Rasor, Jennifer E. Garrus, Jinfang Li, P. Louann Cable, Christine Eberhardt, Jennifer Schreiber, Selena Rush, Kenneth W. Wood, Emma Barrett, Amita Patnaik. A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A252.

A perylene derivative regulates HIF-1α and Stat3 signaling pathways

It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.

HLA Class II tetramers reveal tissue-specific regulatory T cells that suppress T-cell responses in breast carcinoma patients

Regulatory T cells (Tregs) play an important role in controlling antitumor T-cell responses and hence represent a considerable obstacle for cancer immunotherapy. The abundance of specific Treg populations in cancer patients has been poorly analyzed so far. Here, we demonstrate that in breast cancer patients, Tregs often control spontaneous effector memory T-cell responses against mammaglobin, a common breast tissue-associated antigen that is overexpressed by breast carcinoma. Using functional assays, we identified a HLA-DRB1*04:01- and HLA-DRB1*07:01-restricted epitope of mammaglobin (mam34–48) that was frequently recognized by Tregs isolated from breast cancer patients. Using mam34–48-labeled HLA Class II tetramers, we quantified mammaglobin-specific Tregs and CD4+ conventional T (Tcon) cells in breast carcinoma patients as well as in healthy individuals. Both mammaglobin-specific Tregs and Tcon cells were expanded in breast cancer patients, each constituting approximately 0.2% of their respective cell subpopulations. Conversely, mammaglobin-specific Tregs and CD4+ Tcon cells were rare in healthy individuals (0.07%). Thus, we provide here for the first time evidence supporting the expansion of breast tissue-specific Tregs and CD4+ Tcon cells in breast cancer patients. In addition, we substantiate the potential implications of breast tissue-specific Tregs in the suppression of antitumor immune responses in breast cancer patients. The HLA Class II tetramers used in this study may constitute a valuable tool to elucidate the role of antigen-specific Tregs in breast cancer immunity and to monitor breast cancer-specific CD4+ T cells.

Comment on “Myeloid-Derived Suppressor Cells Suppress Antitumor Immune Responses through IDO Expression and Correlate with Lymph Node Metastasis in Patients with Breast Cancer”

In a recent issue of The Journal of Immunology , Yu and colleagues elegantly describe that IDO is a crucial player for myeloid-derived suppressor cell (MDSC)-mediated suppression of anti-tumor immune responses ([1][1]). Hence, they depicted that IDO expression was upregulated in MDSC isolated from

Correlation of PDL1 tumor expression and outcomes in renal cell carcinoma (RCC) patients (pts) treated with pazopanib (paz).

3021 Background: The interaction between PDL1 (B7H1) and its receptor PD-1 on activated T cells plays an important role in the inhibition of T-cell responses and contributes to suppression of antitumor immune responses. Tumor PDL1 expression has been associated with poor outcomes in RCC. This study investigates the correlation between PDL1 tumor expression and outcomes in RCC pts treated with paz. Methods: Using IHC, we retrospectively analyzed baseline FFPE tumor samples for PDL1 from 2 paz RCC studies: a single arm phase II trial and randomized placebo (pbo)-controlled phase III study. PDL1 expression was analyzed by MedTox Laboratories using the anti-PDL1 MouseIgG1 clone 5H1 (Thompson) on the Leica automated IHC platform. Additional dual PDL1/CD68 staining was carried out to delineate tumor and macrophage PDL1 expression. Tumor PDL1 expression was quantified by H-Score (HS) and PDL1+ macrophages were assessed semi-quantitatively. Association between PDL1H scores and PFS was investigated by Kaplan-Meier analysis using optimal cutoff of PDL1tumor HS (minimum p value, log rank test). Results: The optimal cut-point of PD-L1 tumor HS, relative to PFS, was identified as HS &gt; 3. In the phase II study (46 available samples out of 225), HS range was 0-150 and most samples had negative (HS = 0, n = 34, 74%) or low (HS 1-3, n=4, 9%) PDL1 expression. Pts with HS &gt; 3 (n = 8, 17%) had significantly shorter PFS (2.6 mo) than those with HS ≤ 3 (12 mo; p = .0005). In the phase III study (N = 160 available samples: paz, 113 of 290; pbo, 47 of 145), HS range was 0-280. Most patients had negative (n = 122/160, 76%) or low (n = 9/160, 6%) PD-L1 expression, with 18% (29/160) having HS &gt; 3. Pbo-arm pts with HS &gt; 3 (n = 6/47, 13%) had shorter PFS (2.3 vs 5.5 mo p = .0207). Paz-arm pts with HS &gt; 3 (n = 23/113, 20%) trended toward shorter PFS (7.3 vs 11 mo, p = .1405). Conclusions: PDL1 appears to be a prognostic marker with PDL1 HS &gt; 3 associated with shorter PFS. Limitations of the study include the retrospective nature of the analysis with limited pt samples available, low or negative PDL1 expression in the vast majority of pts, and use of archival samples that may not accurately reflect PDL1 status at study entry. Additional results (tumor volume, OS) will be presented.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production

BackgroundCTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma.MethodsCTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student’s t-test.ResultsAll melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts.ConclusionsOur studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

Interleukin 21–Induced Granzyme B–Expressing B Cells Infiltrate Tumors and Regulate T Cells

The pathogenic impact of tumor-infiltrating B cells is unresolved at present, however, some studies suggest that they may have immune regulatory potential. Here, we report that the microenvironment of various solid tumors includes B cells that express granzyme B (GrB, GZMB), where these B cells can be found adjacent to interleukin (IL)-21-secreting regulatory T cells (Treg) that contribute to immune tolerance of tumor antigens. Because Tregs and plasmacytoid dendritic cells are known to modulate T-effector cells by a GrB-dependent mechanism, we hypothesized that a similar process may operate to modulate regulatory B cells (Breg). IL-21 induced outgrowth of B cells expressing high levels of GrB, which thereby limited T-cell proliferation by a GrB-dependent degradation of the T-cell receptor ζ-chain. Mechanistic investigations into how IL-21 induced GrB expression in B cells to confer Breg function revealed a CD19(+)CD38(+)CD1d(+)IgM(+)CD147(+) expression signature, along with expression of additional key regulatory molecules including IL-10, CD25, and indoleamine-2,3-dioxygenase. Notably, induction of GrB by IL-21 integrated signals mediated by surface immunoglobulin M (B-cell receptor) and Toll-like receptors, each of which were enhanced with expression of the B-cell marker CD5. Our findings show for the first time that IL-21 induces GrB(+) human Bregs. They also establish the existence of human B cells with a regulatory phenotype in solid tumor infiltrates, where they may contribute to the suppression of antitumor immune responses. Together, these findings may stimulate novel diagnostic and cell therapeutic approaches to better manage human cancer as well as autoimmune and graft-versus-host pathologies.

Abstract 4945: The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy.

Abstract The dendritic cell (DC) -based vaccine immunotherapy has been a promising cancer immunotherapy, but has been insufficient to eradicate the tumor in patients with advanced cancer. This can result from the complicated tumor microenvironment (TME) that is implicated in suppression of anti-tumor immune responses. Several immune cell types in TME, such as Tregs, myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs), have been reported to regulate anti-tumor immune responses negatively. Cancer-associated fibroblasts (CAFs) are also primary stromal cells in TME, and contribute to tumor growth and metastases through the secretion of TGF-β and stromal cell-derived factor-1 (SDF-1). We considered that TME-targeted strategies should be innovated for the development of the potent cancer immunotherapy. On the basis of these viewpoints, we focused on the role of CAFs in TME, and hypothesized that inhibition of CAFs would lead to improvement of systemic anti-tumor immune responses and enhancement of the potency of the DCs-based vaccine immunotherapy. In this study, we applied tranilast in order to inhibit CAFs, the anti-fibrotic and -allergic agent that is used clinically and has been shown to inhibit fibroblast in the scar tissue. In in vitro studies, we examined effects of tranilast on CAFs that were isolated from established EG7 (mouse lymphoma cells) tumors. As results, tranilast was able to suppress the proliferation of CAFs, and decrease the production of SDF-1 as well as TGF-β from CAFs. Regarding the effect on Tregs, tranilast was able to decrease the induction of them from spleen cells of normal mice. Based on these results, we confirmed that tranilast could inhibit the function of CAFs. Next, we examined the association between inhibition of CAFs and anti-tumor immune responses in tumor-bearing mouse model. C57BL/6 mice bearing EG7 were administered tranilast into the established tumor in combination with tumor antigen-loaded DCs vaccination, and were evaluated anti-tumor immune responses. As results, the population of CAFs was decreased by targeting them, leading to lower expressions of TGF-β as well as SDF-1 in TME. Inhibition of CAFs in TME resulted in the decreased distributions of Tregs in TME and tumor-draining lymph nodes (TDLs). On the induction of effector cells, antigen-specific CD8+ cells producing IFN-γ were significantly increased in TDLs and spleen through inhibition of CAFs in TME. In these mice, systemic antigen-specific cytotoxic responses were augmented, leading to suppression of tumor growth as compared with mice in control groups. These results demonstrate that CAFs are associated with immune suppression, and inhibition of CAFs functions in TME can augment systemic anti-tumor immune responses. Our mouse models provide a new rationale with TME-targeted strategies for enhancing the potency of the DCs-based vaccine immunotherapy. Citation Format: Yasuhiko Ohshio, Ryosuke Kaku, Keiko Ishida, Masayuki Hashimoto, Shoji Kitamura, Koji Teramoto, Jun Hanaoka, Noriaki Tezuka. The cancer-associated fibroblasts-targeted strategycan augment the potency of the dendritic cell-based vaccine immunotherapy. [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 4945. doi:10.1158/1538-7445.AM2013-4945

Abstract 1539: Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo.

Abstract Macrophages promote tumor growth by stimulating tumor-associated angiogenesis, cancer-cell invasion, migration, and intravasation, as well as suppression of antitumor immune responses. We previously defined the macrophages which were harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated macrophages (EMϕ)” and demonstrated EMϕ promoted tumor growth and metastasis in orthotopic mouse models. The aim of this study is to elucidate the mechanism of tumor promotion by EMϕ. Transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with the human pancreatic cancer cell line XPA1 stably expressing RFP in the cytoplasm and GFP in the nucleus. GFP-expressing macrophages from the GFP mice with the dual-color pancreatic tumor were harvested and defined as EMϕ. Macrophages were also harvested from transgenic GFP mice without tumors and identified as “naïve macrophages (NMϕ)”. EMϕ and NMϕ were compared for their ability to enhance cancer-cell proliferation. Using a skin-flap-window imaging model, the mitotic index of the cancer cells significantly increased after injection of EMϕ compared to NMϕ (p = 0.001). Next, we cultured cancer cells in 3-D on Gelfoam® and found that the cancer cells cultured with EMϕ proliferated to a greater extent compared to those cultured with NMϕ (p = 0.014). We also found that the cancer cells cultured in the conditioned medium harvested from EMϕ had enhanced proliferated compared to cancer cells cultured in the conditioned medium from NMϕ (p = 0.016). These results suggest that EMϕ secrete factors which increase cancer cell growth and can serve as a novel therapeutic target for pancreatic cancer. Citation Format: Yukihiko Hiroshima, Masashi Momiyama, Mohamed Hassanein, Ali Maawy, Rhiana Menen, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo. [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 1539. doi:10.1158/1538-7445.AM2013-1539

Evaluation of Immunological Changes in Patients Treated With SBRT and Radiation Therapy -- A Prospective Cohort Study

Evaluation of Immunological Changes in Patients Treated With SBRT and Radiation Therapy -- A Prospective Cohort Study

Shaping the glioma immune microenvironment through tryptophan metabolism.

The metabolism of the essential amino acid tryptophan is a key microenvironmental factor shaping the immunobiology of many tumor types. The current concept suggests that in the tumor microenvironment, tryptophan is metabolized by specialized dioxygenases, chiefly indoleamine-2,3-dioxygenase (IDO), which is expressed by tumor cells and antigen-presenting cells. High IDO activity leads to the depletion of tryptophan from the local microenvironment, while immediate tryptophan metabolites, particularly kynurenine, accumulate to high micromolar levels. Both the depletion of tryptophan and the accumulation of kynurenine lead to profound suppression of T-cell responses. Orally active IDO inhibitors are currently being explored in clinical trials for their efficacy in enhancing antitumor immune responses. Recent evidence points at alternative routes of tryptophan catabolism via tryptophan-2,3-dioxygenase, which is particularly expressed in malignant gliomas resulting in the production of high amounts of kynurenine. Tryptophan-2,3-dioxygenase-derived kynurenine in turn leads to the promotion of glioma growth and invasiveness and the suppression of antitumor immune responses by binding to the aryl hydrocarbon receptor expressed in glioma cells and glioma-infiltrating T cells. These new data open up novel therapeutic approaches to alleviate glioma-mediated immunosuppression. This review summarizes the current view on the relevance of tryptophan metabolism as an important immunosuppressive, proinvasive and growth-promoting metabolic pathway in malignant glioma.

Myeloid-Derived Suppressor Cells and anti-tumor T cells: a complex relationship

Myeloid-Derived Suppressor Cells (MDSC) are immature myeloid cells that are potent inhibitors of immune cell function and which accumulate under conditions of inflammation, especially cancer. MDSC are suggested to promote the growth of cancer by both enhancement of tumor angiogenesis and metastasis and also inhibition of antitumor immune responses. The presence of deficient and/or defective antitumor adaptive and innate immune responses, coincident with accumulation of MDSC in lymphoid organs and tumor parenchyma, supports the notion of a causal relationship. The potent ability of MDSC to inhibit several components and phases of immune response highlights the likelihood that targeting the inhibitory functions of MDSC may maximize the therapeutic potential of antitumor immunotherapy. In order to guide the rational development of immunotherapeutic strategies that incorporate inhibition of MDSC activity and enzymatic functions, thorough understanding of the role of MDSC in antitumor immune responses is required. In this manuscript we review the multifaceted inhibitory functions of MDSC and consider the role of MDSC-induced inhibition of antitumor T cell effector phase.Support for this research is from NIH R01 CA108573.

The expression, function, and clinical relevance of B7 family members in cancer.

The modulation and suppression of anti-tumor immune responses is a characteristic feature of tumor cells to escape immune surveillance. Members of the B7 family are involved in this process, since the level of activation of the anti-tumor immune response depends on the balance between co-stimulatory and co-inhibitory signals. Some molecules are often overexpressed in tumors, which has been associated with the pathogenesis and progression of malignancies as well as their immunological and non-immunological functions. The B7 homologs play a key role in the maintenance of self-tolerance and the regulation of both innate and adaptive immunity in tumor-bearing hosts. Furthermore, the blockade of negative signals mediated by the interaction of co-inhibitory ligands and counter-receptors of the B7 family is currently being studied as a potential immunotherapeutic strategy for the treatment of cancer in humans.

Analysis of regulatory T cells in patients of NSCLC with malignant pleural effusion.

e17511 Background: Enrichment of regulatory T (Treg) cells at the affected anatomic site in cancer may suppress the anti-tumor immune response influencing the cancer progression. Understanding of the clinical relevance of Treg mediated suppression of anti-tumor immune response and mechanisms underlying their preferential trafficking to the affected anatomic site is still limited. The aim of this study was to enumerate the frequencies of Treg cells in malignant pleural effusion and peripheral blood of patients with advanced NSCLC and it’s trend after treatment. Methods: Treg frequencies were evaluated in pleural effusion and peripheral blood of the patients with advanced NSCLC (n=27) using flowcytometry and compared with peripheral blood of age and sex matched healthy controls (n=15) and tubercular pleural effusions (n=10). The Treg cells were characterized as CD4+CD25+Foxp3+ T cells gated on CD4+CD25+ T cells. We assessed the effect of treatment response on Treg frequency. We have also looked for the expression of chemokine receptors CCR4 and CCR6 on the Tregs in pleural effusion and peripheral blood of the patients. Results: Compared to healthy controls, frequency of CD4+CD25+Foxp3+ Tregs was significantly increased in peripheral blood of patients with NSCLC (p=0.0036). In pleural effusion of patients, Treg frequency was higher than their corresponding peripheral blood (p=0.025). As compared to tubercular pleural effusion Treg frequency was higher in malignant effusion (p&lt;0.0001). We had 12 patients who completed treatment and in whom response evaluation was available. Treg frequency reduced at the time of response (PR or SD) and increased again at disease progression. Surface expression of CCR4 and CCR6 was higher on Treg cells as compared to non Treg CD4 cells among the patients (p=0.0001; p=0.001 respectively). However, there was no difference in expression of these chemokine receptors on Tregs in pleural fluid and peripheral blood. Conclusions: Tregs are increased in patients of NSCLC, both at disease site and in systemic circulation. This increase may be chemokine receptors mediated. Treg frequency changes with treatment and response. Modulation of Tregs may have therapeutic implication in the management of advanced NSCLC.

Immunotherapy of Cancer: Reprogramming Tumor-Immune Crosstalk

The advancement of cancer immunotherapy faces barriers which limit its efficacy. These include weak immunogenicity of the tumor, as well as immunosuppressive mechanisms which prevent effective antitumor immune responses. Recent studies suggest that aberrant expression of cancer testis antigens (CTAs) can generate robust antitumor immune responses, which implicates CTAs as potential targets for immunotherapy. However, the heterogeneity of tumor cells in the presence and quantity of CTA expression results in tumor escape from CTA-specific immune responses. Thus, the ability to modulate the tumor cell epigenome to homogenously induce expression of such antigens will likely render the tumor more immunogenic. Additionally, emerging studies suggest that suppression of antitumor immune responses may be overcome by reprogramming innate and adaptive immune cells. Therefore, this paper discusses recent studies which address barriers to successful cancer immunotherapy and proposes a strategy of modulation of tumor-immune cell crosstalk to improve responses in carcinoma patients.

Regulatory T-cell-mediated inhibition of antitumor immune responses is associated with clinical outcome in patients with liver metastasis from colorectal cancer.

Adaptive regulatory T cells (Tregs) contribute to an immunosuppressive microenvironment in colorectal cancer (CRC). Here, we examined whether the level of Treg-mediated inhibition of antitumor immune responses in patients with metastatic CRC (metCRC) selected for liver resection is associated with clinical outcome. Preoperatively and at follow-ups, we did flow-based phenotyping, examined antitumor immunity using peptides from carcinoembryonic antigen (CEA) protein in the presence or absence of CD4(+)CD25(+)CD127(dim/-) cells (Tregs) and determined cytokine and PGE(2) levels in patient blood samples. At 18 months post-surgery, 8 patients were disease free (7 alive and 1 dead of unrelated cause) and 10 had experienced disease recurrence (7 alive and 3 dead of metCRC). Prior to surgery, the patients demonstrated Treg-mediated suppression of TNFα and IFNγ expression that could be perturbed through the PGE(2)/cAMP pathway and the immune suppression was significantly higher in the group that later developed disease recurrence (P = 0.046). Furthermore, the post-surgery plasma PGE(2) levels were related to the clinical outcome (PGE(2) levels of 280 ± 47 vs. 704 ± 153 pg/ml (mean ± SEM) for disease free and recurrent disease, respectively). T-cell phenotyping revealed higher frequencies of COX-2(+) cells in the patients with recurrent disease. These findings support the notion that the level of Treg-mediated suppression of adaptive antitumor immune responses at the time of surgery may influence later clinical outcome of metCRC and provide valuable prognostic information.