Investigations on autologous T-cells for adoptive immunotherapy of AIDS

Abstract

We report on the preclinical results of an immunotherapeutic approach of AIDS mediated by ex vivo propagated CD4+ and CD8+ T-cells. A mean yield of 6.23 x 10(9) lymphocytes, containing 1.82 x 10(9) CD4+, 3.23 x 10(9) CD8+ T-lymphocytes and 8.39 x 10(6) CD34+ peripheral blood progenitor cells (PBPC) were be obtained by continuous flow cytapheresis (CFC) in 15 asymptomatic HIV infected patients (CD4-count > 350/mm3). The CD4/CD8 ratio (mean: 0.53, SD: +/- 0.15) in the cell concentrates reflected the distribution of the circulating lymphocyte subsets in vivo. Absolute lymphocyte counts decreased at a mean of 404/microliter (25%) immediately after CFC but were replaced from the extravascular pool within one hour. Neither the CD4/CD8 ratio nor p24-antigen and neopterin levels did change significantly after cell separation. No alteration of the number of proviral DNA copies (1/10(3)-1/10(6)) could be detected in peripheral T-helper cells by semiquantitative PCR after lymphapheresis. Cells were cryopreserved in liquid nitrogen without substantial loss of viability or function. Ex vivo propagation of T-cells in a strictly autologous manner in the presence of PHA + IL-2 for 14d resulted in a 50-fold expansion rate (140-fold in healthy controls, p < 0.001). Viral replication could be controlled but not completely eliminated by cocultivation with autologous CD8+ T-lymphocytes as measured by limiting dilution nested PCR (NPCR). The expanded cells showed the typical phenotype of highly activated memory type T-lymphocytes (CD3+ CD45RO+ CD25+ HLA-DR+). The distribution of CD4+ and CD8+ T-cells did not reveal significant changes before and after culture indicating that both subsets were equally expanded. Functionally important membrane or intracellular epitopes which were found to be decreased in HIV infected subjects (CD7, CD55, CD59) before culture were reconstituted after ex vivo propagation of T-cells. The functional importance of the up-regulation of complement regulating epitopes (CD55, CD59) after culture could be proven by a significant inhibition of cytolysis of T-cells in the presence of autologous complement. The majority (75%) of expanded CD8+ T-cells stained positive with mAb TIA-1 which is directed to intracellular granules within cytotoxic T-cells. Furthermore, programmed cell death of expanded T-cells could be prevented by cocultivation with fibroblasts which are believed to secrete a cytokine pattern preventing activated T-cells from apoptosis after withdrawal of IL-2 and other stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)