Generation of MUC1‐stimulated Mononuclear Cells using Optimizied Conditions

Abstract

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)‐stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF‐7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony‐stimulating factor, gamma‐interferon and interleukin‐10) was 45 × 10−8 m (1 μg/ml); except for tumour necrosis factor (TNF)‐alpha which was 22 × 10−8 m (0.5 μg/ml). The unwashed MC were superior to washing them with Ficoll–Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 × 10−8 m (1 μg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.

Costimulation with anti-cluster of differentiation 3 and anti-cluster of differentiation 28 reduces the activity of mucin 1-stimulated human mononuclear cells

“…Whilst costimulation with anti-CD3/CD28 antibody beads may be utilized for the activation of lymphocytes (17), the results of the present study suggested that costimulated M1SHMCs, whilst exhibiting higher rates of proliferation, possess a reduced ability to kill cancer cells, and thus this method of treatment may not be advisable following antigen activation of lymphocytes under the conditions used here. We have previously shown that continued stimulation of CTL rendered them anergic (9). In support of this, constitutive proliferating CAR T cells showed inferior antitumor effect (18).…”

“…The following day, the tube was placed into the Cell culture conditions. Procedures were performed as previously described (9). Mononuclear cells were not human leukocyte antigen typed, as our previous studies (10)(11)(12) and another literature study (13) identified that cytotoxicity by M1SHMC may be non-major histocompatibility complex-restricted.…”

Number of Treatment Cycles Influences Development of Cytotoxic T Cells in Metastatic Breast Cancer Patients – A Phase I/II Study

“…CTL stimulation and ex vivo expansion were performed as we have detailed elsewhere (Wright, 2008) and describe briefly next.…”

“…The maximum target control wells had 2% Triton X-100 (Sigma, St. Louis, MO, USA) added instead of effector cells to lyse the target cells. The assays were incubated for 18 hours, which we found to be superior to 4 hours (Wright, 2008), at 37°C and 5% CO 2 and were performed in triplicate. The plates were centrifuged and one-half of the 100μl supernatants were harvested.…”

“…Immunophenotyping was performed by immunofluorescent flow cytometry (BD Pharmingen Inc., San Diego, CA, USA) per manufacturer's instructions (Wright, 2008). % lysis (mean experimental cpm -mean spontaneous cpm) (mea = n n maximum cpm -mean spontaneous cpm) × 100…”