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

Abstract

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 B6BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6BALB.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-Kb) 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, B6BALB.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 B6BALB.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.