Dendritic cells enhance the activity of human MUC1-stimulated mononuclear cells against breast cancer.

Dendritic cells (DCs) are among the most potent antigen-presenting cells (APCs), stimulating peripheral blood mononuclear cells (PBMCs) to generate antigen-specific cytotoxic T lymphocytes (CTLs). The objectives of this study were to determine if interleukin (IL)-4 is beneficial or detrimental for the generation of human DCs in vitro and to understand whether DCs generated in vitro in the presence or absence of IL-4 stimulate the killing of adenocarcinoma cells by CTLs in vivo. Mucin 1 (MUC1), a glycoprotein found on the surface of adenocarcinoma cells was used to load DCs. MUC1-loaded DCs generated in the absence of IL-4 were superior to their counterparts produced with IL-4 in stimulating PBMCs to kill human breast cancer MCF-7 cells in vitro. A corollary in vivo protection experiment was performed by injecting immunodeficient NOD-SCID mice with MCF-7 cells s.c. and MUC1-loaded CTLs, PBMCs, or DCs generated in the absence of IL-4, i.p. Mice that received CTLs and MUC1-loaded DCs on days 0, 2, 4, 9, 14 and 19 were completely protected against the development of MCF-7-derived tumors, while other schedules conferred lower protection. Therefore, tumor antigen-loaded DCs enhance the efficacy of adoptive CTL transfer, and should thus be considered for combinatorial immunotherapeutic regimens.

Cytotoxic T-lymphocyte immunotherapy for ovarian cancer: a pilot study.

The objective was to evaluate the toxicity and feasibility of intraperitoneal infusion of tumor-specific cytotoxic T lymphocytes (CTL) as therapy for recurrent ovarian cancer, and to determine if repetitive cycles of CTL generation and infusion measurably increases the host's ovarian cancer immune response. In this study, 7 subjects with recurrent ovarian cancer confined to the peritoneal cavity underwent up to 4 cycles, each cycle beginning with a leukapheresis for collection of precursor lymphocytes, which were stimulated in vitro with mucin 1, a tumor-specific antigen found commonly in ovarian cancer cells. The resulting new CTL for each cycle were reintroduced into the host by intraperitoneal infusion. Immunologic parameters (killer cells, cytokine production, memory T lymphocytes, and natural killer cells) were studied. Toxicity, CA-125, and survival data were also evaluated. The tumor marker CA-125 was nonstatistically significantly reduced after the first month of immunotherapy. However, after that it rose. Killer cells, cytokine production, and memory T lymphocytes increased after the first cycle of stimulation, but plateaued or reduced thereafter. The percent of natural killer cells inversely correlated with other immune parameters. Median survival was 11.5 months. One subject is free of disease since December, 2000. Multiple cycles, beyond 1 cycle, of T-cell stimulation followed by adoptive T-cell infusion, may not enhance the in vivo immune response.

Immunotherapy with IL-10- and IFN-γ-producing CD4 effector cells modulate "Natural" and "Inducible" CD4 TReg cell subpopulation levels: observations in four cases of patients with ovarian cancer.

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 three 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.

Number of treatment cycles influences development of cytotoxic T cells in metastatic breast cancer patients - a phase I/II study.

The influence of the number of apheresis-stimulation-infusion(s) cycles, and the time in culture before the infusion (one vs. two weeks), on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Two previously treated metastatic breast cancer patients with no evidence of disease, in complete remission (CR), were enrolled. Each apheretic peripheral blood mononuclear cell (PBMC) sample was stimulated twice with MUC-1 before infusion back into the patients. Killer T-cells responses against MUC-1-expressing MCF-7 (CTL), nonspecific natural killer (NK) and lymphokine-activated killer (LAK) target cell lines, as well as, cytokine production were measured before each infusion. Patients received 2 infusions per month for 4 months. There were no tumor recurrences or toxicity. CTL, NK and LAK cells, type 1 cytokine, gamma-interferon (G-INF), and CD4(+) and CD8(+) memory T-lymphocytes were initially generated, produced or induced, respectively, and then declined. The CTL, NK and LAK cells were only induced at the first infusion of the first month. Thus, maintaining PBMC in culture longer than the first infusion was of no benefit with regards to retaining functional killer T-cells. In conclusion, this study implies that one treatment is optimal.

Retention of immunogenicity produced by mucin 1 peptides with glycosylation site substitutions.

Mucin1 (MUC1) with altered glycosylation behaves as an antigen unique to adenocarcinomas (ADCs). As a step toward DNA vaccines, the goal of this work was to determine whether MUC1 peptides substituted with an asparagine at O-linked glycosylation sites, might expose MUC1 peptide backbone to serve as immunogens to generate cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells of patients with ADCs. Substitution of some or all tyrosine and serine residues by asparagine in MUC1 did not inhibit the generation of mucin-specific CTLs. This suggests that MUC1 tandem repeat altered sequences to prevent O-linked glycosylation may be useful as DNA vaccines with tumor specificity.

Tumor burden influences cytotoxic T cell development in metastatic breast cancer patients--a phase I/II study.

The influence of tumor burden on the generation of tumor antigen-specific cytotoxic T-lymphocytes (CTL) was investigated in a phase I/II clinical adoptive immunotherapy trial. Four previously treated metastatic breast cancer patients, two with macroscopic disease and two with no evidence of disease, in complete remission (CR), were enrolled. Each apheretic peripheral blood mononuclear cell (PBMC) sample was stimulated twice with MUC-1 before infusion back into the patients. CTL responses against MCF-7 cell line and cytokine production were measured before infusion. Patients received two monthly CTL infusions and were monitored for toxicity, tumor response as well as tumor marker levels. The CTL generated from patients with high tumor burdens had less cytokine production and lower cytotoxicity of MCF-7 than the CTL of patients in CR. The differences between the two groups were observed after the two MUC1 in vitro stimulations of the cells obtained in first apheresis. This difference increased after the two MUC1 stimulations of the cells obtained in the second apheresis. The cytotoxicity function was sustained from the first infusion to the second apheresis only for the patients in CR. This suggests that tumor burden had an inverse effect on the function of the generated CTL.

Autologous MUC1-specific Th1 effector cell immunotherapy induces differential levels of systemic TReg cell subpopulations that result in increased ovarian cancer patient survival.

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 immunotherapy with autologous MUC1-stimulated CD4(+) effector cells induces differential levels of systemic "Ag-experienced" and "Ag-inexperienced" CD4/CD25(+) TReg cell subpopulations that influence long-term tumor immunity in ovarian cancer patients.

MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

Adoptive immunotherapy of mucin1 expressing adenocarcinomas with mucin1 stimulated human peripheral blood mononuclear cells.

Mucin1 stimulated hematopoietic mononuclear cells (M1SHMC) from patients with breast cancer, adoptively transferred to non-obese diabetic, severe combined immunodeficient (NOD SCID) mice, extended survival in a therapy model of gross adenocarcinoma and prevented tumor growth in a model of minimal disease. M1SHMC exhibited specific lysis of a human breast adenocarcinoma cell line expressing mucin1, MCF-7 and produced interferon gamma. M1SHMC were injected intraperitoneally (IP) in NOD SCID mice after gross, palpable tumors appeared after MCF-7 were injected subcutaneously (SC). Survival was increased as compared to no M1SHMC controls. However tumors eventually regrew in all mice. To determine whether minimal disease (MD) could be controlled, NOD SCID were injected with MCF-7 cells, and on the same day, injected IP with M1SHMC. The M1SHMC injected mice were protected from tumor growth. These results imply that M1SHMC can prolong survival, but not cure NOD SCID mice bearing gross palpable adenocarcinomas. However in a minimal disease model tumor growth was prevented.