An Experimental Study on the Effects of Sensitized-Donor Lymphocytes Infusion after Non-Myeloablative Allo-Stem Cell Transplantation.

Objectives :To explore the effects and mechanisms of the recipients chimerism, the rate of graft-versus-host disease (GVHD) and the immune state by infusion of donor lymphocytes (DLI) which were sensitized by the skin of recipients after non-myeloablative allogeneic stem cell transplantation (NSCT).

Methods :Female C57BL/6 mice (H-2^b, B6) as recipients received total-body irradiation (TBI) of 5.5 Gy (⁶⁰CoC-ray) on day 0 followed by allogeneic hematopoietic stem cells transplantation (allo-HSCT). The allo-grafts consisted of 2×10⁷ peripheral hematopoietic stem cells from mobilized male BALB/C (H-2^d) donor mice with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Two days after allo-HSCT, the recipient mice were given cyclophosphamide (Cy) (200mg/kg) intraperitoneally. Afterwards these recipient mice were infused 2×10⁶ sensitized or unsensitized-donor lymphocytes at the day of 28 after transplantation respectively. Recipients were observed clinical manifestation, phenotype, re-establishment of haematogenesis, histopathologic changes of internal organs suffered from GVHD and investigated donor chimerism by the semi-quantitate analyses of polymerase chain reaction (PCR). Immunologic reconstitution was evaluated through T-lymphocytes Subsets in peripheral blood detected by flow cytometer (FCM). Data was analyzed by SPSS 10.0 software and expressed as mean ± SD.

Results: The mice receiving sensitized-donor lymphocytes infusion did not suffer from GVHD and the phenotypic character of the recipient mice (black color) converted to that of the donor mice (white color), and to full-donor chimerism. It was found that the ratio of CD4⁺/CD8⁺ T lymphocytes of them decreased at the earlier period and increased after half month, but which were also lower than that of the normal value. mixed lymphocyte reaction (MLR) showed the specific hyporesponsiveness to donor mice. While various grades of aGVHD were observed in that of the unsensitized-DLI group and the mixed-chimerism were maintained, though it increased a little, and the ratio of CD4⁺/CD8⁺ T lymphocytes increased at first, then decreased to the normal level half month later. The effects were more significant in the group of sensitized-DLI than that of unsensitized-DLI. The result of MLR also showed hyporesponsiveness, but it exceeded that of the sensitized-DLI recipients. The effects were more significant in the group of sensitized-DLI than that of unsensitized-DLI (P<0.05). Conclusions Mixed-chimerism can be achieved using a nonmyeloablative TBI and CTX-based conditioning regimen combination with infusion of peripheral blood stem cells mobilized by rhG-CSF in fully mouse histocompatibility-2 complex (H-2) mismatched recipients, and donor chimerism can be promoted by administration of DLI. Furthermore, sensitized DLI converted mixed to comlete donor chimerism without GVHD. The ratio of CD4⁺/CD8⁺ and the reactiveness of MLR showed super dependability with the chimerism and the incidence of GVHD.