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Abstracts J Immunother Volume 33, Number 8, October 2010 the loss of MHC class II expression (measured via HLA-DR) on CD14+ monocytes. In all malignancies measured to date, CD14+HLA-DRlo/neg monocytes are significantly elevated in peripheral blood of patients including glioblastoma patients (about 24 ± 20.0% of CD14+ monocytes were HLA-DRlo/neg; n = 24), lymphoma (27.8 ± 3.1%; n = 40), and prostate cancer (30.7 ± 15%; n = 22) compared to healthy donors (8.5 ± 8.0; n = 15). These percentages were converted into absolute cell counts and, in healthy donors, the absolute cell count of CD14+HLA-DRlo/neg monocytes ranged between 8 to 70 cells/mL with a median of 35 cells/mL compared to 134 cells/mL (range of 1 to 794 cells/mL) in GBM patients, 155 cells/mL (range of 25 to 591) and for lymphoma patients. These values have a large dynamic range in cancer patients, suggesting clinical and pathological heterogeneity within disease categories. CD14+HLA-DRlo/neg monocytes are functionally immunosuppressive as they inhibited T cell proliferation in an antigen independent manner. Most significantly, CD14+HLA- DRlo/neg monocytes were refractory in their ability to differentiate into mature dendritic cells that was independent of the maturation signal used. These findings have significant implications on the generation (both in vitro and in vivo) of DC for clinical use. Independently, others have shown CD14+HLA-DRlo/neg monocytes to be prognostic in the outcome of other immunosuppressive settings like sepsis. Our immunophenotyping analysis of a cohort of patients with/or at risk of acute lung injury with or without infection revealed that these patients had a median of 264 cells/mL (range of 31 to 1443; n = 29). Our data suggests that CD14+HLA- DRlo/neg cells may be a useful marker of immunosuppression in cancer patients as well as for patients at risk of infection. As such, the immunosuppressive functions of CD14+HLA-DRlo/neg monocytes should be considered during the development of novel biological therapies of cancer. Blockade of the PD-1 Pathway Improves Immunotherapy for Multiple Myeloma William H. Hallett*, William R. Drobyskiw, Bryon D. Johnson*. *Pediatrics; w Medicine, Medical Colllege of Wisconsin, Milwaukee, WI. Multiple Myeloma is an incurable plasma cell malignancy. Patients who fail conventional therapy are treated with high-dose therapy and hematopoietic stem cell transplantation (HSCT). This treatment typically reduces the tumor burden and can induce remission, but these patients frequently relapse from sites of chemotherapyresistant disease. We developed a combined immunotherapy approach that incorporates HSCT, adoptive transfer of T lymphocytes, and administration of a cell-based vaccine. This approach has been effective in a murine neuroblastoma model, but it confers no significant improvement in survival in mice with established myeloma (5T33 tumor line). Therefore, we sought to identify the mechanisms of immune evasion utilized by this myeloma. We observed high expression of B7-H1, the ligand for the inhibitory receptor PD-1, on 5T33 tumor cells. We also observed increased expression of PD-1 on both CD4 and CD8 T cells in tumor-bearing mice, and increased frequencies of PD- 1-expressing T cells were found only in lymphoid tissues containing tumor cells. Interestingly, we observed a similar pattern of expression when we evaluated clinical samples from multiple myeloma patients. We found increased B7-H1 expression on plasma cells in marrow samples from myeloma patients. Furthermore, we found that patient T cells displayed increased levels of PD-1. In our murine model, the increased expression of PD-1 on T cells correlated with tumor burden and was restricted to areas of local tumor accumulation. When PD-1 or B7-H1 blockade was used together with combined immunotherapy (HSCT, adoptive T cell transfer and vaccination) in tumor-bearing mice, we observed a significant improvement in survival (40% survival in treated mice vs. 0% survival in control mice). These data demonstrate an important role for the PD-1 pathway in suppressing immune responses to myeloma and suggest that blockade of this pathway may enhance immunotherapy for this disease. TLR Ligands Induce SOCS2 Gene Expression in Human Monocyte-Derived Dendritic cell via NF-kB Jin Hu*, DaoHua Lou*, Gunnar Norstedtw, Ann- Charlotte Wikströmz, Ola Winqvist*. *Department of Medicine; w Department of Molecular Medicine and Surgery; zDepartment of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden. The suppressor of cytokine signaling 2 (SOCS2) negatively regulates cytokine signaling, and STAT5b has been demonstrated as its regulatory transcription factor upon GH signaling. We previously reported that SOCS2 is induced by LPS stimulation in human DCs. Here we further investigate the transcriptional regulation of SOCS2 expression. Methods: Human DCs Generation: iDCs was generated by culturing monocytes with 50 ng/mL GM-CSF and 20 ng/mL IL-4 for 6 days. RT-PCR: Total RNA was extracted using TRIzol and reverse-transcribed for real-time PCR analysis. Bioinformatics: Genomic sequence of human SOCS2: UCSC. Prediction of transcription factor binding site (http://www.genomatix.de). Western blot: The cell lysates were submitted to SDS-PAGE, transferred to PVDF membranes for Western Blot analysis. Result: TLR ligands induce SOCS2 expression. iDCs were treated with various TLR ligands including Pam3CSK4(TLR1/2, 10 mg/mL), LPS(TLR4, 200 ng/mL), flagellin(TLR5, 1 mg/mL), poly- I:C(TLR3, 30 mg/mL), imiquimod(TLR7, 5 mg/mL), ssRNA40 (TLR8, 5 mg/mL) and CPG-ODN2336(TLR9, 5 mg/mL) for mRNA analysis. Real-time PCR result revealed that SOCS2 mRNA was induced by TLR 1/2, TLR 4, TLR 5, TLR 8 and TLR 9 signaling from 8 hours till 24 hours. No SOCS2 increase on TLR 3 and 7 stimulations. TLR 1/2 and 4 stimulation induced the strongest SOCS2 induction. The effect was moderate on TLR5, TLR8 and 9. NF-kB was predicted as transcription factors for human SOCS2 gene. STAT 3, 5 and 6, NF-kB, IRF 1, 3, 4 and 7 were found as putative SOCS2 transcriptional factors in human SOCS2 promoter region for TLR4 signaling. NF-kB is a transcription factor for human SOCS2 expression in TLR4 signaling. After inhibiting NF-kB pathway by pre-incubating iDCs 1 h with BAY11-7082 (10 mM), a IkBa phosphorylation inhibitor; BMS- 345541 (20 mM), a allosteric site-binding IKK-2 inhibitor, proteasome inhibitor MG-132 (20 mM) and protein synthesis inhibitor cyclohexamide (20 mg/mL), we stimulated the cells with LPS and extract whole protein for western blot analysis. The SOCS2 protein became significant after 8 hours and strong after 24 hours stimulation. The various NF-kB inhibitors inhibited SOCS2 expression demonstrating that NF-kB was necessary for the induction of SOCS2 transcription on TLR4 signaling. Discussion: SOCS2 expression is different after various TLR ligands stimulation in human DCs. Thus, SOCS2 expression is specific and restricted to certain TLRs. Inhibition of NF-kB translocation eliminated SOCS2 protein expression after TLR4 signaling in DCs. Thus, we here identify the transcriptional factor NF-kB as the major SOCS2 inducer after TLR signaling. Re-Programming of Tumor Derived T Regulatory Cells Mehmet O. Kilinc, Jamie L. Harden, Rachael B. Rowswell-Turner, Lauren P. Virtuoso. Microbiology and Immunology, State University of New York at Buffalo, Buffalo, NY. Sustained intratumoral IL-12 can restore cytotoxic function to tumor-associated CD8+ T effector/memory cells and induce the apoptotic death of Treg resulting in tumor regression. However, reversal of tumor immune suppression is transient and the tumors are rapidly re-infiltrated by Treg short-circuiting cytotoxic T cell activity. We predict that abrogation/delay of the Treg rebound will be critical to extending the antitumor effector window and achieving durable tumor regression. Based on recent reports demonstrating functional plasticity in CD4+ T cell subsets, we are testing strategies designed to re-program post-therapy Treg to T helper cells. One specific approach involves the local and 870 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts sustained delivery of siRNA and cytokines from controlled-release adjuvants. To determine whether Treg isolated from tumor draining lymph nodes (TDLN) could convert to Th17 cells, and establish the minimal cytokine requirement necessary for the phenotype switch, we performed in vitro culture assays. Foxp3 expression in tumor Treg cultured under Th17-polarizing conditions (TGF-B +IL-6) declined 1.5 fold. More importantly, the decrease of Foxp3 was paralleled with a similar fold increase in IL- 17 expressing cells (B1.8 fold) indicating that both changes may be occurring within the same subset. These findings support the idea of plasticity of mature Treg isolated from TDLN and provide a basis for the in vivo application of polarizing conditioning as part of an immunotherapeutic approach. Separately, sustained high level Foxp3 expression is required for maintenance of Treg phenotype and function. Since the Foxp3 stability is implicated in CD4 T cell plasticity, the transient knock down of Foxp3 expression in tumor Treg by siRNA may play a pivotal role in driving the conversion of tumor associated Treg to Th17. Our preliminary results in splenocytes confirmed the successful use of the gold nanoparticles (GNP) for siRNA delivery with no apparent toxicity and efficient suppression of gene activity for up to 8 days. We are currently testing these formulations in combination to determine whether the phenotype of post-therapy Treg can be modulated in vivo. A State of Dominant Tolerance is Rapidly Induced with Intravenous Dissemination but not with Subcutaneous Implantation in a Murine Leukemia Model Justin Kline*, Long Zhang*, Thomas F. Gajewski*w. *Medicine; w Pathology, University of Chicago, Chicago, IL. While significant progress has been made in the identification of immune evasion mechanisms utilized by solid cancers, negative regulatory pathways employed by hematological malignancies have been under-explored. C1498 is an aggressive acute myelogenous leukemia (AML) cell line from C57BL/6 mice which we have transduced to express the model SIY peptide antigen. This model enables monitoring of endogenous CD8 + T cell responses by specific tetramer and ELISPOT analyses, and also allows use of adoptively transferred 2C TCR Tg T cells to track specific responses. When implanted subcutaneously (SC) into syngeneic mice, C1498.SIY cells induced a robust functional CD8 + T cell response as measured by SIY/K b tetramer analysis and SIY-specific IFN-g ELISPOT. In contrast, when injected intravenously (IV), a minimal SIY-specific immune response was detected. Interestingly, when C1498.SIY cells were injected IV 5 days prior to a subsequent SC C1498.SIY challenge, the SIY-specific T cell response was severely blunted, suggesting that IV tumor dissemination induces a state of dominant tolerance. This observation was unique to the IV setting, as a prior SC C1498.SIY challenge did not blunt but rather augmented the immune response to a subsequent SC C1498.SIY challenge. Tolerance induced by IV C1498.SIY appeared to be largely antigen-specific, as it did not significantly inhibit immune responses generated against OVA-expressing tumors. In order to dissect the mechanism(s) of tolerance induced by IV C1498.SIY, we employed adoptive transfer of CFSE-labeled 2C TCR Tg T cells. While similar numbers of 2C T cells had seen antigen as measured by expression of CD44, 2C T cells with IV tumor underwent significantly fewer cell divisions and were less functional upon restimulation compared with 2C T cells with SC tumor. Interestingly, when C1498.SIY cells were administered IV first, prior to SC tumor implantation, CFSE dilution of transferred 2C T cells was severely blunted and associated with minimal accumulation of divided cells, consistent with cell death. Collectively, these results suggest that IV C1498.SIY may induce anergy and/or deletion of tumor-specific CD8 + T cells, the final mechanism of which is currently being elucidated. Our results have implications for the future development of immunotherapy for patients with established disseminated leukemia in whom a dense state of tumor antigen-specific T cell tolerance may exist. MHC Class II-Dependent Strain-Specific Differences in Phenotype and Function of Tumor-Infiltrating Myeloid Cells are Associated with Differential Outcome of TRAMP-PSA Tumor Growth in HLA-DR2b Transgenic Mouse Model Elena N. Klyushnenkova*w, Surmeet Singhw, Richard B. Alexander*w. *VA Maryland Health Care System; w Surgery, University of Maryland, Baltimore, MD. We have recently demonstrated that MHC class II alleles can differentially affect anti-tumor immune responses and the outcome of tumor growth in HLA-DR2b transgenic mice (J Immunol. 2009;182:1242–46). The presence of a ‘‘permissive’’ HLA-DR2b allele in our model was associated with the development of strong antigen-specific antibody responses, suppression of CD8 T cell responses and enhanced growth of TRAMP tumor cells engineered to express prostate-specific antigen (PSA). In contrast, mice bearing a ‘‘non-permissive’’ I-A b allele that did not support CD4 T cell responses to PSA, developed strong CD8 T cell responses in the absence of antibody responses, and rejected PSA-expressing tumors. We also analyzed a composition of the tumor-infiltrating leukocytes in DR2b+ and DR2b- mice bearing TRAMP-PSA tumors by flow cytometry. In DR2b+ mice, CD4 T cells, including CD25+foxp3+ regulatory T cells, were accumulated in larger number compared to DR2b-mice. The analysis of tumor-infiltrating myeloid cells also revealed significant stain-specific differences, particularly in the phenotype and activation status of CD11b+Ly6C low cells. Ly6C low cells accumulated in the tumors of DR2b+ mice had a different phenotype compared to DR2bmice, and expressed higher levels of activation markers, most notably CD40. Although the function of CD11b+Ly6C low cells is poorly characterized, they are regarded as ‘‘stationary’’ monocytes with a potential to differentiate into CD8a-dendritic cells and cross-tolerize T cells through the programmed death ligand (PDL)- 1. Due to their differential ability to process antigen, these cells were also reported to favor CD4 T cell activation. Up-regulation of CD40 on these cells observed in our experiments may serve as an indicator of their cross-talk with CD4 T cells at the tumor site, which is consistent with a ‘‘permissive’’ status of the HLA-DR2b allele. In contrast, tumor-infiltrating Ly6C low cells from DR2bmice expressed low levels of CD40, which could indicate lack of a cross-talk between CD4 T cell and APC consistent with a ‘‘nonpermissive’’ status of I-A b allele. Based on our observations, we propose that a failure to reject TRAMP-PSA tumors in DR2b+ mice can be partially due to the significant presence of ‘‘stationary’’ tolerogenic Ly6C low myeloid cells that could preferentially interact with tumor-infiltrating CD4 T cells in an MHC class II-restricted Ag-specific manner, thus preventing somehow the effective terminal CTL differentiation. Supported by a grant from the US Department of Veterans Affairs and a pilot grant from the Baltimore Research and Education Foundation. Human Myeloid Suppressor Cell Induction and Functional Analysis Melissa G. Lechner, Alan L. Epstein. USC Keck School of Medicine, Los Angeles, CA. Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid suppressor cells (MSC). MSC suppress anti-tumor T-effector responses through arginine depletion, reactive oxygen and nitrogen species production, and regulatory T cell expansion. In cancer patients and experimental tumor models, increased MSC correlates with more aggressive disease and poor prognosis. This study examined the ability of 100 human solid tumor cell lines to induce MSC using a novel in vitro tumor co-culture method. Newly induced MSC were characterized for suppression, morphology, phenotype, and gene expression. A group of 17 MSC-inducing cancer cell lines, as well as 6 non-MSC control cancer cell lines, was then evaluated for the production of putative MSC-promoting factors (TGFb, IL-1b, IL-4, IL-6, IL-10, GM-CSF, M-CSF, IDO, FLT3L, c-kit L, TNFa, COX2, and VEGF) by ELISA and quantitative RT-PCR r 2010 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 871
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