OR.85. Leukocyte Responses during Recombinant Human Interleukin-21 Treatment of Patients with Stage IV Melanoma

Abstracts

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42 MAPKs. Moreover, our data suggests that PLD plays an important role in the activation of NFAT and NF-κB transcription factors, in T cell proliferation and in the production of cytokines, IL-2 and IFN-γ. Overall, our results point to a pivotal role for PLD in the intracellular signaling cascade activated by TCR and in Tcell functional responses. Thus, our results suggest that PLD might be a suitable target for the treatment of inflammatory and autoimmune diseases and for prophylaxis of transplant rejection. doi:10.1016/j.clim.2008.03.091

Immuno-oncology Sunday, June 8 2:45 pm–4:45 pm OR.85. Leukocyte Responses during Recombinant Human Interleukin-21 Treatment of Patients with Stage IV Melanoma Kresten Skak,1 Klaus Stensgaard Frederiksen,1 Jenny Lau,2 Ulrik Mouritzen,3 Grant McArthur,4 Ian Davis,5 Birte K. Skrumsager,5 Mary Sartor,2 Rune Overgaard,3 Dorthe Lundsgaard,1 Lasse Tengbjerg Hansen.3 1Novo Nordisk A/S, Maaloev, Denmark; 2Westmead Hospital, Sydney, NSW, Australia; 3Novo Nordisk A/S, Bagsvaerd, Denmark; 4Cancer Trials Australia, Melbourne, VIC, Australia; 5Austin Health, Heidelberg, VIC, Australia Interleukin-21 is a class I cytokine with effects on several leukocyte subsets. Leukocyte responses were monitored in an open-label phase 2a trial of intravenous recombinant human IL21 (rIL-21) treatment of patients with stage IV melanoma administered as 3-9 cycles of daily dosing for 5 days followed by 9 days of rest. Peripheral blood leukocyte responses were monitored by flow cytometry, effector molecule qRT-PCR, and serum sCD25 (sIL-2Rα) analysis. On Day 4 of treatment, a marked drop in number of NK-cells and both CD4+ and CD8+ Tcells was observed. Between dosing, a rebound effect beyond baseline levels was observed. CD8+ T-cells and NK-cells had increased surface expression of CD25 (IL-2Rα), CD69, the lymph-node homing marker CD62L and increased mRNA expression of the effector genes, IFN-γ, granzyme B, and perforin. In accordance with immune activation, increased serum levels of sCD25 were observed as early as 24-hours postdosing. No changes in activation markers on CD4+ T-cells were observed; this suggests that separate mechanisms drive the rIL21-mediated leukocyte redistribution and activation of effector functions in CD8+ T-cells and NK-cells. In contrast to lymphocytes, increases in numbers of monocytes and plasma cells were observed. In particular, a 6-fold increase was observed in the FcγRIII+ monocyte subset, which was associated with increased expression of the ADCC-mediating FcγRIII (CD16). These data demonstrate that administration of rIL-21 to melanoma patients induces a variety of leukocyte responses related to effector functions that warrant further investigations in relation to anti-tumour effects of this cytokine. doi:10.1016/j.clim.2008.03.092

OR.86. Accumulation of Tumor Infiltrating FOXP3+ CD4+ Regulatory T Cells in Tumors and Not in Peripheral Blood in Patients with Melanoma Mojgan Ahmadzadeh,1 Aloisio Felipe-Silva,2 Maria Merino,2 Steven Rosenberg.1 1NCI/NIH, Bethesda, MD; 2NCI, Bethesda, MD Studies of regulatory T cells in human tumors have been difficult and functional studies have been limited due to low cell viability and purity. Using a novel functional assay, we examined tumor infiltrating T cells in the single cell suspensions of enzymatically digested tumors to enumerate regulatory T cells. We observed more than four-fold increase in the frequency of FOXP3+ CD4+ T cells in tumors compared to peripheral blood of patients and healthy donors. To distinguish between activated and regulatory Tcells, we stimulated tumor infiltrating Tcells in the tumor digests with PMA and ionomycin for 6 hours followed by intracellular cytokine and FOXP3 costaining. Our findings revealed that, similar to regulatory T cells in peripheral blood of healthy individuals, tumor infiltrating FOXP3+ CD4+ T cells were unable to produce IL-2 and IFN-g in contrast to FOXP3- T cells. Using the combination of functional and phenotypic parameters, we concluded that tumor infiltrating FOXP3+ CD4+ T cells exhibited the characteristics of regulatory T cells. We then utilized FOXP3 expression as a marker to distinguish regulatory T cells in the intratumoral and peritumoral sections of 18 melanoma tumors using immunohistochemical analysis. We found that there was a significantly higher percentage of FOXP3+CD4+ regulatory T cells intratumorally than peritumorally. These findings reveal selective accumulation of regulatory T cells in the close proximity of tumors and it further highlights that the frequency of regulatory T cells in the peripheral blood are not representative of the tumor microenvironment. doi:10.1016/j.clim.2008.03.093

OR.87. Optimizing Anti-tumor Immunity: Reversing Systemic iNKT Cell Immunoregulatory Defects in Murine Prostate Cancer Michael Nowak, Simo Arredouani, Martin Sanda, Steven Balk, Mark Exley. Beth Israel Deaconess Medical Center, Boston, MA Breaking tolerance to self-antigens represents a promising treatment approach, especially for non-critical organ cancers. Invariant natural killer T cells (iNKT) recognize glycolipids presented on CD1 molecules by dendritic and other cell types, thereby regulating immune reactions. Importantly, in multiples types of model and human cancer, we and others have found both numerical as well as functional iNKT defects. In particular, decreased iNKT numbers were accompanied by reversible functional defects: decreased IFN-γ production by iNKT in advanced prostate cancer, resulting in a detrimental Th2 cytokine profile of iNKT in such patients. We analyzed numerical and functional iNKT defects in the murine TRAMP prostate cancer model. In this model, probasin-directed expression of SV40 T antigen drives the sequential development of prostatic intraepithelial