Adoptive T cell immunotherapy using autologous lymphocytes is a viable treatment for patients with cancer and requires participation of Ag-specific CD4 and CD8 T cells. Here, we assessed the immunotherapeutic effects of autologous MUC1 peptide-stimulated CD4 + effector cells following adoptive transfer in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 + / Th1 effector cell generation, we show that three monthly treatment cycles of peripheral blood T cell restimulation and intraperitoneal re-infusion selectively modulated endogenous T cell-mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient survived long-term for nearly 16 months with recurrent disease and two patients expired within 3-5 months following final infusion. Although PBL from all patients showed elevated MUC1 cytolytic activity following therapy, such responses did not correlate with therapeutic efficacy. Long-term survivors showed elevated levels of systemic memory (CD45RO) and naïve (CD45RA) CD3/CD4/CD25 + T cells when compared to that of pre-treatment levels and similarly-treated short-term survivors. Such cells co-expressed different levels of Foxp3 and CTLA-4 that resulted in progressively lower systemic Foxp3/CTLA-4 memory T cell ratios that further correlated with disease-free survival. Lastly, these patients showed elevated levels of MUC1-specific T cells expressing the CCR5 and CCR1 chemokine receptors and the chemokine CCL4 associated with Th1 cell differentiation/memory. We suggest that effective Disclosures: This manuscript has not been published elsewhere and has not been submitted simultaneously for publication elsewhere.None of the authors have any potential financial conflict of interest related to this manuscript Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. immunotherapy with autologous MUC1-stimulated CD4 + effector cells induce differential levels of systemic "Ag-experienced" and "Ag-inexperienced" CD4/CD25 + TReg cell subpopulations that influence long-term tumor immunity in ovarian cancer patients. NIH Public Access

Adoptive T cell therapy for cancer patients optimally requires participation of CD4 T cells. In this phase I/II study, we assessed the therapeutic effects of adoptively transferred IL-10 and IFN-γ-producing CD4 effector cells in patients with recurrent ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that 3 monthly treatment cycles of autologous T cell restimulation and local intraperitoneal re-infusion modulated T cell mediated immune responses that were associated with enhanced patient survival. One patient remains disease free, another patient experienced prolonged survival for nearly 16 months with recurrent disease and two patients expired within 3–5 months following final infusion. Prolonged survivors showed elevated levels of systemic CD3+CD4+CD25+ and CD3+CD4+CD25− T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cell populations among these patients contained variable levels of “Inducible” Tr1 (CD4+CD25−FoxP3−IL-10+) and “Natural” (CD4+CD25+CD45RO+FoxP3+) TReg cell numbers and ratios that were associated with prolonged and/or disease-free survival. Moreover, peptide-restimulated T cells from these patients showed an elevation in both IFN-γ production, memory cell phenotype and select TNF family ligands associated with enhanced T cell survival and apoptosis-inducing activities. This suggests that intraperitoneally-administered Th1-like cells, producing elevated levels of IL-10, may require and/or induce differential levels of distinct systemic TReg subpopulations that influence, in part, long-term tumor immunity and enhanced memory/effector CD4-mediated therapeutic potentials. Furthermore, treatment efficacy and enhanced memory cell phenotype did not appear to be dependent on TReg cell numbers but upon ratios of “Inducible” and “Natural” TReg subpopulations.

The common γ chain receptor family of cytokines have been associated with T cell homeostasis, development and survival. In this study, we assessed the therapeutic effects of adoptively transferred CD4 T cells and their affects on endogenous expression of such cytokines in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that 3 monthly treatment cycles of autologous T cell restimulation and local intraperitoneal reinfusion resulted in diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient experienced prolonged survival for nearly 16 months with recurrent disease and two patients expired within 3-5 months following treatment. Prolonged survivors showed lower levels of systemic memory (CD8+CD45RO+)/naïve (CD8+CD45RA+) CD8 T cell ratios when compared to that of short-term survivors. Such subpopulations among prolonged survivors were predominantly FoxP3-NEG and at relatively greater levels when compared to that of corresponding pre-treatment conditions. Lastly, peptide-restimulated PBMCs from these patients showed variable gene expression levels of pre- and post-treatment IL-15, IL-21 and IL-9 that were further associated with prolonged and/or disease-free survival. This suggests that CD4 cell transfer may induce levels of endogenous cytokines associated with CD8 survival and/or homeostasis that may contribute to enhanced CD4 therapeutic potentials

Adoptive T cell therapy is a viable treatment for cancer patients and optimally requires participation of CD4 T cells. Here, we assessed the therapeutic effects of adoptively transferred autologous MUC1 peptide-stimulated CD4+ effector cells in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4+/Th1 effector cell generation, we show that 3 monthly treatment cycles of T cell restimulation and intraperitoneal re-infusion modulated T cell mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient survived long-term for nearly 18 months with recurrent disease and two patients expired within 3-5 months following final infusion. Long term survivors showed elevated levels of systemic CD3/CD4/CD25+ and CD3/CD4/CD25- T cells when compared to that of pre-treatment levels and similarly treated short-term survivors. Such cells co-expressed different levels of "Inducible" Tr1 (CD4/CD25-/FoxP3-/IL-10+) and "Natural" (CD4/CD25+/FoxP3+) TReg cell ratios that further correlated with disease-free survival. These patients showed elevated levels of Th1 cells and MUC1-specific T cells expressing chemokine receptors associated with T cell differentiation/memory. This suggests that MUC1-stimulated CD4 T cells induce differential levels and proportions of systemic TReg subpopulations that influence long term tumor immunity in such patients following treatment.

Adoptive T cell immunotherapy is a viable treatment for cancer patients and optimally requires participation of Ag specific CD4 T cells. Here, we assessed the therapeutic effects of adoptively transferred autologous MUC1 peptide stimulated CD4+ effector cells in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4+/Th1 effector cell generation, we show that 3 monthly treatment cycles of T cell restimulation and intraperitoneal re-infusion modulated T cell mediated immune responses that correlated with diminished serum CA125 tumor marker levels and enhanced patient survival. One patient survived long term (>5 yrs), another patient survived long term with recurrent disease (>5 yrs) and two patients expired within 100 days following final infusion. Long term survivors showed elevated levels of systemic memory (CD45RO) and naïve (CD45RA) CD3/CD4/CD25+ T cells. Such cells co-expressed different levels of Foxp3 and CTLA-4 that resulted in lower systemic Foxp3/CTLA-4 memory T cell ratios. Lastly, these patients showed elevated levels of MUC1 specific T cells expressing the CCR5 and CCR1 chemokine receptors associated with T cell differentiation and select migration. This data suggests that effective immunotherapy with autologous MUC1 peptide-stimulated CD4+ effector cells induce differential levels of systemic TReg cell subpopulations that influence long term tumor immunity in ovarian cancer patients following adoptive T cell transfer.