Search

4586 Background: Immune checkpoint blockade may play a role in the treatment of mUC. However, the role of CTLA4 blockade, the impact of chemotherapy on immune cell populations, and the optimal appr...

TPS3105 Background: Lymphomas are the 5th most incident cancer in the U.S. and indolent non-Hodgkin's lymphoma (iNHL) are incurable with standard therapy. We previously completed three trials of ‘i...

Tumour cells exist in a complex milieu of cellular and non-cellular components comprising fibroblasts, endothelial cells, immune cells and metabolites of cellular respiration. An elaborate interplay between these components and tumour cells exists with implications for immunological recognition of tumour cells. Tumours have been shown to alter their antigen and cytokine profiles, desensitise and impair immune defences, signal fibroblasts to facilitate metastasis, and take advantage of acidic and hypoxic conditions that impede normal cells. This paper aims to review the roles of the stroma, extracellular matrix and chemistry of the microenvironment on tumour growth, with particular emphasis on interactions with the immune system, and to highlight some of the novel therapeutic strategies that target the tumour microenvironment.

Purpose To characterize the number and functional status of CD4+CD25+ regulatory T cells (Tregs) in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC) treated with high-dose bolus interleukin-2 (IL-2). Patients and Methods Patients with MM or RCC treated with high-dose bolus IL-2 (600,000 IU/kg every 8 hours) at a single center provided pre- and post-treatment whole blood specimens. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, separated into cellular subsets, and analyzed by flow cytometry or used for in vitro proliferation assays. Results Between September 2003 and July 2005 57 patients were enrolled in the study with 48 patients available for analysis (45 MM, 12 RCC). Tregs were defined as CD4+CD25hi T cells, and this subset was significantly elevated in the cancer patients compared with normal donors (7.75% v 2.24%). The CD4+CD25hi T-cell pool in the patients constitutively expressed intracellular FoxP3, CTLA-4, and produced high amounts of IL-10. The Tregs were CCR7+ with 50% representing naïve and 50% central-memory T cells. The cells were functionally suppressive in mixed in vitro proliferation assays. Following IL-2 administration, the number and frequency of Tregs increased in patients with progressive disease but returned to normal levels in patients with objective clinical responses. Conclusion The number of Tregs, defined as CD4+CD25hi T cells is increased in patients with MM and RCC. High-dose IL-2 resulted in a significant decrease of Tregs in those patients achieving an objective clinical response to IL-2 therapy.

T suppressor and regulatory cells have been shown to play an important role in the maintenance of central and peripheral tolerance thereby preventing allograft rejection, autoimmunity and allergy. We have previously described a distinct population of antigen-specific CD8(+)CD28(-) T suppressor cells (T(S)). These CD8(+)CD28(-) T(S) cells can be generated in vitro after multiple rounds of stimulation of human peripheral blood mononuclear cells (PBMCs) with either allogenic- or xenogeneic-donor APCs. CD8(+)CD28(-) T(S) cells are FOXP3+, MHC class I-restricted and tolerize both professional antigen presenting cells, such as dendritic cells (DC) and nonprofessional APC such as endothelial cells (EC) by up-regulating the cell surface expression of inhibitory receptors immunoglobulin-like transcript (ILT)-3 and ILT4 and down-regulating the expression of costimulatory molecules such as CD58 and CD86. Tolerized ILT3(high), ILT4(high) APC anergize CD4(+) T(H) cells and can induce the generation of antigen-specific CD4(+)CD25(+) T regulatory cells (T(R)) cells and CD8(+)CD28(-) T(S) cells. In this review, we present our recent studies on the molecular characterization of these antigen specific T suppressor cells and tolerogenic APC.

Equine and rabbit antihuman thymocyte globulins (ATGs) have been used in renal transplantation for prevention and treatment of acute rejection. We now report that hyperacute and acute antibody-mediated rejection of renal allografts occurred in three newly transplanted patients who received ATG for induction therapy. Antibody studies performed using complement-dependent cytotoxicity, flow cytometry, enzyme-linked immunosorbent assay, and Luminex yielded negative results for antilymphocytic and antiendothelial cell antibodies in the pretransplant sera obtained from these patients. ATG treatment was initiated at the time of transplantation. One of the patients experienced hyperacute rejection and required transplant nephrectomy within 24 h of transplantation. The other two patients developed acute antibody-mediated rejection within 14 days after transplantation. None of the patients developed antihuman leukocyte antigen antibodies when humoral rejection occurred. However, xenoantibodies that strongly bound to human lymphocytes and, importantly, to activated endothelial cells, were identified in the sera obtained at the time of humoral rejection. Hence, our results strongly implicate ATG in the induction of antibody-mediated rejection of kidney allografts. Flow cytometry testing of ATG reactivity to endothelial cells may be useful in identifying and discarding the ATG lots containing xenoantibodies that can bind to activated endothelial cells of the transplant.

Endothelial cells have been shown to activate T cell responses to alloantigens, triggering transplant rejection. However, they may also play a role in tolerance induction. Using RT-PCR we show here that alloantigen specific CD8(+)CD28(-) T suppressor cells generated in vitro are FOXP3 positive and interact with human endothelial cells. This interaction results in the induction of inhibitory receptors and down-regulation of costimulatory and adhesion molecules, thus rendering endothelial cells tolerogenic. In turn, tolerized endothelial cells elicit the differentiation of CD8(+)CD28(-) FOXP3(+) T suppressor cells. Taken together our data demonstrate a functional and phenotypic overlap between tolerogenic dendritic cells and endothelial cells. Furthermore, alloantigen specific CD8(+)CD28(-) FOXP3(+) T cells, which trigger the upregulation of inhibitory receptors in endothelial cells, are present in the circulation of heart allograft recipients in quiescence as demonstrated by flow cytometry, RT-PCR and luciferase transcription assays. Their detection facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppressive therapy, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Modulation of endothelial cells in favor of promoting tolerance may be important for long-term survival of organ allografts.

Meeting abstracts Lymphomas are the 5th most common cancer in the U.S. and the most prevalent amongst these, low-grade B cell lymphomas are incurable with standard therapy. Previously, we completed three trials combining low-dose radiotherapy (XRT) with intratumoral administration of a TLR9 agonist

Purpose/Objectives: While viral antigens in HPV-related oropharyngeal cancer (HPVOPC) make it an attractive target for immunotherapy, understanding the immune effects of existing therapeutic approaches is essential for integrating immunotherapy into HPVOPC treatment. Preclinical data in several cancer models support an overall immunostimulatory effect of chemotherapy and radiation; however their effect on HPV-specific immunity on HPVOPC patients is unknown. We tested the hypotheses that platinum-based concomitant chemoradiation, with or without taxane-platinum-5FU (TPF) induction chemotherapy, induces a favorable profile of circulating immunocytes ad enhanced HPV-specific T cell responses in HPVOPC patients. Materials/Methods: Patients with stage II-IV HPVOPC treated with standard-of-care chemoradiation underwent serial blood sampling before, during, and after treatment for up to one year. Circulating immunocytes including effector CD4+ and CD8+ T cells and immunosuppressive regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) were profiled by flow cytometry. Expression levels of the negative costimulatory molecule PD-1 on T cells were also assessed by flow cytometry. HPV antigen-specific T cell responses in response to HPV16 E6 and E7 peptides were measured by luminex analysis of IFN-γ production. Results: Pre-treatment HPV-specific T cell responses were present in 12/18 patients, and an additional 4 patients acquired measureable responses following induction chemotherapy. Of the 16 patients who developed HPV-specific responses before completion of therapy, 10 lost these responses by 3 months post-treatment. The average level of IFN-g release by PBMC after stimulation with HPV peptides was non-significantly decreased at 3 weeks post-treatment, and significantly decreased at 3 and 6 months. Loss of pre-existing tumor-specific immune responses was associated with a striking (2-fold) decline in circulating CD4+ and CD8+ T cell numbers, a lesser decline in Treg (1.5-fold), marked elevation of MDSC (>2-fold), and decline in the CD8+:Treg and CD8+:MDSC ratios following chemoradiotherapy. PD-1 expression levels on total and CD45RO+ (memory) CD4+ T cells were elevated at 3 weeks after completion of chemoradiation, returning to baseline by 3 months. Conclusions: Contrary to our starting hypothesis, we found an overall immunosuppressive effect of chemoradiotherapy on immune responses in HPVOPC patients. Upregulation of PD-1 on CD4 T cells is a potential immunosuppressive mechanism amenable to targeted therapy with clinically available anti-PD-1 and anti-PD-L1 antibodies. Taken together, our results suggest that chemoradiation has profound effects on circulating immunocyte populations and the immune response to HPV+ OPC, and highlight the importance of further studies of the immune effects of standard-of-care treatment for HPVOPC. Citation Format: Andrew Sikora, Marshall Posner, Falguni Parikh, Seunghee Kim-Schulze, Vishal Gupta, Krzysztof Misiukiewicz, Alexis Patsias, Amelia Clark, Sangkon Oh, Dorothee Duluc. Cisplatin-based concurrent chemoradiotherapy antagonizes anti-tumor immunity in patients with HPV-positive oropharyngeal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2014-626

Background Oncolytic viruses represent a novel form of cancer immunotherapy. Vaccinia viruses encoding human T cell co-stimulatory molecules have demonstrated clinical activity in phase I clinical trials in patients with advanced melanoma. However, predictive biomarkers of therapeutic response have not yet been identified. Methods A customized microarray was performed to identify changes in peripheral blood mononuclear cell (PBMC) gene expression upon exposure to recombinant oncolytic vaccinia viruses. Up-regulated and down-regulated genes were identified and selected for further analysis using PBMC samples from normal donors and oncolytic virus-treated patients before and after viral injection. Quantitative PCR and flow cytometry of defined T cell subsets was performed to evaluate expression patterns and clinical correlations. Results The microarray identified 301 genes that were up-regulated and 960 genes that were down-regulated in T cells after exposure to oncolytic vaccinia virus. The B7.1 gene was highly up-regulated and the immunoglobulin-like transcript 2 (ILT2) gene was highly down-regulated by vaccinia-B7.1, which was consistent with the known inverse regulation of these two genes. We observed an inverse association between ILT2 expression in the tumor microenvironment and clinical response and further identified ILT2 as a marker of regulatory CD4+ and suppressor CD8+ T cell responses and whose down-regulation was predictive of therapeutic responses in patients treated with oncolytic virus immunotherapy. Conclusions ILT2 is a new putative biomarker of T cell and clinical response in patients treated with oncolytic vaccinia virus immunotherapy. Further confirmation of ILT2 as a biomarker requires prospective validation in a larger series of clinical trials.

Background Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. Methods and Results Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [ 3 H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2×10 4 to 8×10 4 receptors per cell and a K d of ≈5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. Conclusions These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.

2600 Background: The tumor microenvironment plays a critical role in mediating anti-tumor immunity following immunotherapy. To overcome the suppressive influence of the microenvironment, recombinant vaccinia and fowlpox viruses were designed to express B7.1 alone or a triad of costimulatory molecules, including B7.1, ICAM-1 and LFA-3 (TRICOM) for direct intra-lesional injection into tumors. The safety profile, immune response and therapeutic efficacy of the four poxviruses are reported herein. Methods: A series of phase I clinical trials were conducted in metastatic cancer patients who have measurable, superficial metastatic lesions (2 colorectal, 35 melanoma). Index tumors were injected with vaccinia virus expressing B7.1 (rV-B7.1, n = 12) or TRICOM (rV-TRICOM, n = 13) or fowlpox virus expressing B7.1 (rF-B7.1, n = 6) or TRICOM (rF-TRICOM, n = 6). Patients were evaluated for toxicity using NCI CTC and clinical response by standard RECIST criteria. Blood and tumor biopsies were collected for analysis of s...

3004 Background: The attenuated vaccinia virus MVA was engineered to deliver the tumor antigen 5T4 (MVA-5T4; TroVax). 5T4 is a glycoprotein expressed by nearly all RCCs. MVA-5T4 was tested in combi...

12500 Background: Metastatic renal cell carcinoma (RCC) is an immune responsive tumor. 5T4 is a tumor antigen expressed on the cell surface of clear cell and papillary RCC. We sought to evaluate the safety and immunologic effects of vaccination with modified vaccinia virus Ankara (MVA) expressing 5T4 followed by IL-2 in patients with advanced RCC. Methods: A single dose Phase I clinical trial was conducted in patients with metastatic RCC and who were eligible for high-dose IL-2. Vaccination was given every three weeks by intramuscular injection and was followed immediately by standard high-dose bolus IL-2 (600,000 IU/kg) after the second and third vaccinations. The regimen could be repeated if there was no evidence of disease progression. Peripheral blood and serum was collected before treatment and every three weeks for analysis. Routine blood counts, chemistry, and pituitary functions were monitored. Anti-5T4 antibody titers were determined by ELISA assay and 5T4-specific T cell responses were monitored by interferon-γ ELISPOT assay using overlapping peptides. Results: 25 patients with RCC are being sequentially enrolled. There have been no serious vaccine-related adverse events although typical Grade 3 IL-2 related toxicity has been seen in all patients. To date, all patients tested have developed an increase in 5T4-specific antibody titers following three initial vaccinations. T cell assays are currently in progress. Seven patients have completed the trial through at least one course of treatment (mean age 62.3 years). Of these 7, one patient who presented with a primary tumor in place and synchronous metastatic disease had an objective complete response of all metastatic disease and underwent a post-treatment nephrectomy; the primary tumor demonstrated highly necrotic tumor. Two additional patients have had stable disease and are receiving booster vaccinations every three months. Conclusions: These results demonstrate that local vaccination with an MVA virus expressing 5T4 administered in combination with high-dose IL-2 is safe and induces 5T4-specific antibodies in all patients. Additional T cell responses and clinical follow-up will be presented. The use of MVA-5T4 and IL-2 appears to be a promising approach for the treatment of advanced RCC. [Table: see text]

Estradiol Inhibits Pdgf-and Serum-Stimulated Map Kinase Activation in Vascular Smooth Muscle Cells † 215

Background Interleukin-2 (IL-2) induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC) but the antigen(s) responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA) encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity. Methods 25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg) after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses. Results There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12%) were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48%) had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261). All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs. Conclusion Vaccination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients. Trial registration number ISRCTN83977250