Phenotypic and cell cycle analysis of human peripheral blood mononuclear cells activated with interleukin-2 and/or...

The poor clinical response rates of cancer patients following immunotherapy with interleukin-2 (IL-2) and other cytokines has prompted attempts to enhance the response rate by using combinations of biological response modifiers. Peripheral blood mononuclear cells (PBMC) respond to interleukin-2 and mediate non-MHC-restricted cytotoxicity and cell proliferation. The addition of anti-human CD3 monoclonal antibody OKT3 has been reported to increase cytotoxicity by increasing the number of cells generated in response to the two stimuli; however, our results could not confirm this finding. In the present study we have investigated the proliferative capacity of individual populations of PBMC responding to IL-2 and OKT3 compared to either stimulus alone, in order to identify possible reasons for the failure of OKT3 to generate enhanced cytotoxic responses. PBMC were stained with monoclonal antibodies to surface antigens and propidium iodide in order to determine the phenotype of populations of PBMC progressing through the cell cycle. OKT3 alone caused an increase in PBMC progressing through the cell cycle, and addition of recombinant human IL-2 (rhIL-2) sustained this response. A higher percentage of CD56- or CD16-positive cells responded to the rhIL-2 alone, but the addition of OKT3 lowered the percentage of this phenotype and increased the number of CD3-positive T cells, which additionally demonstrated an increased CD25 expression. Comparison of the phenotypes progressing through the cell cycle in response to OKT3 plus rhIL-2 or rhIL-2 alone showed that a higher percentage of all populations responded to OKT3 plus rhIL-2, compared to IL-2 alone. Using flow cytometric cell sorting, those populations of cells with cytolytic activity were identified. Using flow cytometry, it was also possible to show minority cell populations (i.e., those representing less than 5% of the total number of cells) responding to individual stimuli and entering the cell cycle. This technique therefore offers distinct advantages over conventional proliferation assays.