Minor Histocompatibility Antigen-Specific CD8+ Memory T Cells in Hematopoietic Compartments Survive the Immediate Non-Ablative and Ablative TBI Milieu.

Minor histocompatibility (MiHA) antigens induce CD8⁺ T-cell responses that mediate resistance to bone marrow engraftment in an MHC-identical, MiHA-disparate marrow transplant model, in which recipients are sensitized to donor antigens (BALB.B à B6_BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6_BALB.B). We initially sought to determine the distribution and partially characterize the phenotype of H60-specific CD8⁺ cells in the blood, spleen and marrow compartments of B6 mice primed with 3 x 10⁷ BALB.B lymphoid cells and ≥3 weeks later boosted with 2 x 10⁷ cells. An H60 tetramer (LTFNYRNL/H2-K^b) conjugated to PE was used to detect H60-specific CD8+ cells in these compartments. Eight days following the second immunization, the mean frequency of circulating H60-specific cells was 12.2% ± SE 0.88 of CD8+ cells (range: 5.6 – 20.5%). The frequency of splenic H60-specific CD8⁺ cells was equivalent to that of circulating antigen-specific cells, thus peripheral blood levels of H60-specific CD8⁺ cells appear to be representative of those resident in the spleen. Interestingly, in the marrow compartment, the frequency of H-60⁺ cells amongst the CD8 T cell population was higher compared to peripheral blood and spleen levels, suggesting that H60-specific CD8⁺ cells in this compartment may comprise both migrant cells from the periphery and resident cells in the marrow elicited during priming. In both BM and spleen, >90% of CD8⁺ H60⁺ cells expressed the memory phenotype(CD44⁺, Ly6C⁺) and as expected, did not express early activation markers (CD25, CD69). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8⁺ cell must first survive the immediate post-BMT milieu in the hemopoietic compartments. To examine this question, B6_BALB.B mice irradiated at 3.0, 6.0 and 9.0 Gy were analyzed 24 hours later for the presence of H60-specific CD8⁺ cells in the spleen and marrow compartments. Although there was an expected irradiation dose-dependent decrease in absolute numbers of CD8⁺ H60⁺ cells in the two compartments, there was a dose-dependent increase in percent of CD8⁺ T cells expressing the H60 TCR in both compartments. This observation indicates enhanced survival of these antigen-specific CD8⁺ memory T cells post-conditioning. Preliminary results indicate that 24h post-BMT into 9.0 Gy TBI recipients, there was BrDU uptake in marrow and splenic CD8⁺H60⁺ T cells in B6_BALB.B transplanted with 1 x 10⁷ BALB.B BM-TCD. Approximately 80% of CD8⁺H60⁺ T cells in the marrow compartment of primed recipients of BALB.B cells exhibited proliferation by BrDU uptake. Thus, donor MiHA-disparate marrow grafts elicit antigen-driven proliferation early post-BMT by CD8⁺ memory T cells in both compartments consistent with the potential importance of these cells in mediating resistance against progenitor engraftment across these MiHA differences.