Preclinical studies using immobilized OKT3 to activate human T cells for adoptive immunotherapy: optimal conditions for the proliferation and induction of non-MHC-restricted cytotoxicity.

In order to obtain large numbers of T cells for adoptive immunotherapy after bone marrow transplantation (BMT), we optimized conditions for long-term proliferation of T cells that exhibit non-MHC-restricted cytotoxicity using immobilized anti-CD3 (OKT3) activation and culture in IL-2. Proliferation and cytotoxicity directed at Daudi, K562, and B cell lines were used to determine (1) the optimal concentration of IL-2 and the optimal time of exposure to immobilized OKT3 for maintaining growth and cytotoxicity, (2) the starting populations that can be used, (3) the T cell subsets that mediate cytotoxicity, and (4) the optimal medium and concentration of serum for maintaining growth and cytotoxicity. Peripheral blood lymphocytes (PBL) activated with OKT3 would proliferate and mediate cytotoxicity at IL-2 doses as low as 30 IU/ml. Increasing the IL-2 concentrations beyond 600 IU/ml did not augment the proliferative or cytotoxic responses of PBL. A 24-hr incubation on OKT3 was sufficient to activate PBL. Increasing the incubation time on OKT3 from 24 to 72 hr did not significantly enhance cytotoxicity. Comparisons between PBL and purified T cells (E-rosette) indicated that either cell population could be activated with OKT3 in the presence of IL-2 to proliferate and mediate non-MHC-restricted cytotoxicity. Purified populations of CD4+ or CD8+ T cells demonstrated equivalent proliferation and cytotoxicity when activated using IL-2 and OKT3. With equal concentrations of human or fetal bovine serum, RPMI 1640 and X-Vivo 10 were comparable for supporting proliferation and cytotoxicity. These conditions are being used to activate and expand T cells for clinical trials that involve infusing activated T cells into recipients after autologous BMT.

Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation.

We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft-versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.