The immunoregulatory effects of merocyanine 540 on in vitro human T- and B-lymphocyte functions.

YE-SENSITIZED photoirradiation is a relatively reD cent addition to the arsenal of techniques used for the elimination of tumor cells from autologous bone marrow (BM) grafts.’ One photosensitizer, merocyanine 540 (MC 540), has been characterized extensively in preclinical and is now being used in a phase I clinical trial for extracorporeal purging of autologous marrow grafts in patients with acute lymphocytic or nonlymphocytic leukemia, Hodgkin’s or non-Hodgkin’s lymphoma, or stage IV neuroblastoma.’o~” The preclinical evaluation of MC 540 has shown that MC 540 has a low acute systemic toxicity” and low mutagenic potential.” It preferentially photosensitizes normal erythroid progenitor cells, leukemiabymphoma cell^,'^*^^-^ and neuroblastoma cell^.'^^ By contrast, pluripotent hematopoietic progenitor cells as measured by day 12 colony-forming unit-spleen (CFU-S) or CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) are quite resistant to MC 540-sensitized ph~toirradiation~.’~ and substantial reductions of tumor cell concentrations can be achieved without causing excessive damage to the graft’s capacity to rescue lethally irradiated host~.’~~~~~~’~ In all of the reported animal experiments, survival was the end-point and the exact kinetics of hematopoietic and lymphopoietic reconstitution were not determined. The kinetics of the hematopoietic and lymphopoietic recovery may be influenced by T cells and accessory cells (ie, cells other than the pluripotent hematopoietic stem cell) and it is conceivable that the damage done to T cells and accessory cells by a given purging procedure may affect clinical transplantation. This report addresses the effects of MC 540-sensitized photoirradiation on the functions of T and B cells. Specifically, we ask whether treatment with MC 540sensitized photoirradiation: (1) modulates T-cell proliferative responses to mitogens; (2) alters the ability of T cells to respond to alloantigens; (3) modulates T-cell helper activity; and (4) affects the ability of B cells to produce polyclonal Ig. In this study, we show that treatment with MC 540 and white light is a potent inhibitor of T- and greater than 90%. In polyclonal lg synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited EpsteinBarr virus-stimulated lg synthesis. These data show that Tand B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions. 0 1991 by The American Society of Hematology. B-lymphocyte functions at doses of dye and light comparable with or lower than those used in clinical trials. MATERIALS AND METHODS Viability after dye-mediatedphotoiradiation (DMP). Viability of the lymphocytes used for these assays was checked before culture and at 2, 6, and 24 hours after MC 540 DMP using trypan blue exclusion. Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-hypaque density gradient centrifugation (F/H) of whole heparinized blood. T (E+) and non-T (E-) subpopulations were separated by rosetting with 2-aminoethylisothiuronium bromide hydrobromide-modified sheep red blood cells (RBC) as described.” The T-cell populations were greater than 95% E+ cells and the non-T-cell populations contained less than 5% E+ cells by rosetting. Unless otherwise mentioned, the initial lymphocytes placed into culture were viable based on trypan blue exclusion after treatment. RPMI 1640 supplemented with penicillinstreptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum (FBS; Hyclone, Logan, UT) was used for culture except for the mixed lymphocyte cultures. Human AI3 serum (10%) was used in the mixed lymphocyte cultures. ’H-thymidine incorporation after Lymphocyte separation.