Utilization of Phorbol Myristate Acetate as an Activation Probe for Primed Human T Lymphocytes

Goodwin, Joseph; Caplan, Barry; Babbitt, Bruce

doi:10.1097/00002371-199311000-00050

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“…The assay is based on the ability of activated, not resting, T cells to proliferate in response to the low dose (1 ng/ml) of PMA, the protein kinase C activator (3,4). When this assay was used to evaluate the effect of RBC on the levels of proliferative response of the PBMC in the secondary culture, there were no statistical differences of proliferation of the ex vivo-activated T cells between PBMC with and without RBC addition.…”

Section: Compositions Of Cd3mentioning

confidence: 99%

Effect of Contaminating Red Blood Cells on OKT3-Mediated Polyclonal Activation of Peripheral Blood Mononuclear Cells

Goodwin¹,

Zhang²,

Babbitt³

et al. 2002

Clin Vaccine Immunol

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Erythrocytes are typically present as impurities in the majority of peripheral blood mononuclear cell (PBMC) preparations. This study was undertaken to investigate the effects of contaminating red blood cells (RBC) on the ability of OKT3 to activate CD4 ؉ and CD8 ؉ T cells. Surprisingly, the levels of gamma interferon, tumor necrosis factor alpha, and interleukin-1␤ (IL-1␤) produced by PBMC upon stimulation by OKT3 were increased (P < 0.05) in a dose-dependent manner when increasing amounts of autologous RBC (RBC-to-PBMC ratios of 2:1, 10:1, and 50:1) were spiked into PBMC preparations. The OKT3-driven induction of the IL-2 receptor (CD25) and the proliferation of T lymphocytes in response to phorbol myristate acetate were not affected by the addition of RBC.Lymphocytes are among the most extensively studied cells of the hematopoietic system because of their central role in the generation of immune responses. Information provided from the study of T lymphocytes is important not only in understanding the basic concepts of immune function but also in enabling the development of lymphocyte-based adoptive immune therapies. Lymphocytes can be collected from the peripheral blood, lymphoid tissues, and certain internal organs. In most cases, lymphocytes are initially isolated from the peripheral blood compartment and purified by Ficoll density gradient centrifugation. However, regardless of the method used to isolate T cells or peripheral blood mononuclear cells (PBMC), there always exists a low level of contaminating red blood cells (RBC). In addition, when PBMC are isolated on a large scale, as with most ex vivo adoptive immunotherapy approaches, the level of contaminating RBC increases even further. It has been shown previously that lymphocytes in whole blood stimulated with mitogen produce more interleukin-2 (IL-2) than Ficoll-Hypaque-purified lymphocytes in culture (5). What remains unknown is the effect various levels of contaminating RBC have on the ability of well-characterized Tcell stimulants to activate lymphocytes under normal cell culture conditions.A unique form of outpatient adoptive immunotherapy referred to as autolymphocyte therapy (ALT) for the treatment of patients with metastatic renal cell carcinoma has been developed (6, 6a). Patients are infused monthly with ϳ10 9 T lymphocytes activated ex vivo in a conditioned medium containing a mixture of OKT3 (mouse monoclonal anti-CD3 antibody) and a broad panel of autologous cytokines. The cytokine mixture is generated by stimulation of patient PBMC ex vivo with 25 ng of OKT3/ml for 3 days during the first cycle of the therapy (8). During the secondary cycles, i.e., monthly, of therapy, patient PBMC are cultured with the autologous cytokine mixture from the first cycle of therapy for 5 days and then infused back into the patient.For this procedure, lymphoapheresis is performed for each cycle to collect large numbers of PBMC. The resulting apheresis cell products (ACP) are highly enriched in white blood cells and contain various amounts of RBC, platelets, ...

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Section: Compositions Of Cd3mentioning

confidence: 99%