INFLUENCE OF THE ADDITIONAL INJECTION OF HOST-TYPE BONE MARROW ON THE IMMUNE TOLERANCE OF MINOR ANTIGEN-MISMATCHED CHIMERAS

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Abstract
Background. It has previously been demonstrated that adding T cell-depleted (TCD) host bone marrow (BM) to an MHC-mismatched BM inoculum allows for induction of long-term stable chimeras without graft-versus-host disease (GVHD) even when non-TCD allogeneic BM was used. Aims. The present study was undertaken to investigate immune tolerance mechanisms in minor antigen-mismatched allogeneic BM chimeras when host-type BM was added to the BM inoculum. Methods. C3H (H2k, Thy 1.2, Mls 2a) recipients were conditioned with 9.5 gray (Gy) of total body irradiation. To exclude any interference with possible subclinical GVHD, 5×106 TCD AKR (H2k, Thy 1.1, Mls 1a) BM cells were injected with (syn + allo) or without (allo) 5×106 TCD C3H BM cells. Chimerism, clonal deletion, and T lymphocyte subsets were scored using FACS and anti-mouse Thy, Vβ6, Vβ3, CD3, CD4, or CD8 monoclonal antibodies. The stability of tolerance was studied by investigating mixed lymphocyte reaction and cytotoxic T cell induction in chimeras after immunization with host, donor, or third-party (BALB/c) splenocytes. Breaking of chimerism was attempted by injecting nontolerant 40×106 host-type splenocytes 2 months after BM transplantation. Cytokines and Vα14 mRNA were assayed using real time quantitative reverse transcriptase-polymerase chain reaction at 4 and 48 hr, respectively, after injection of nontolerant host-type splenocytes. Results. Both groups of mice became long-term stable mixed chimeras without any clinical sign of GVHD. Neither group was able to produce antihost nor anti-donor cytotoxic T cells, even after immunization. The addition of syngeneic BM to the allogeneic inoculum reduced the overall level of allogeneic chimerism (from ∼70% or ∼85% in peripheral blood lymphocytes and spleen, respectively, in allo chimeras versus ∼35% and ∼60% in syn + allo chimeras). Moreover, it resulted in complete clonal deletion of both host-reactive (Vβ3) and donor-reactive (Vβ6) lymphocytes in syn + allo chimeras in contrast to in allo chimeras, in which only donor-reactive lymphocytes were completely deleted. After nontolerant C3H splenocyte injection, high levels of interleukin 2 mRNA were produced and chimerism decreased in syn + allo chimeras. In contrast, in allo chimeras, this maneuver was followed by the production of higher levels of interleukin 4 and interferon-γ, and of Vα14 mRNA, as well as by the proliferation of CD3+CD4−CD8− (double-negative) T cells and by an increase of donor chimerism. Conclusion. The addition of host-type BM to the allogeneic inoculum has an influence on the level of chimerism, the extent of clonal deletion, and the reaction of chimeras after the injection of nontolerant host-type splenocytes. In the latter phenomenon, cytokine production and proliferation of Vα14+ CD3+CD4−CD8− (double-negative, natural killer T) lymphocytes may be involved.

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