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Keywords:

  • minor histocompatibility antigen;
  • central memory T cell;
  • bone marrow

It has been shown that minor histocompatibility antigens (mHAgs) can function as targets for the graft-versus-leukaemia effect (Goulmy, 1997) following human leucocyte antigen (HLA)-identical allogeneic haematopoietic cell transplantation (HCT) and donor lymphocyte infusion (Marijt et al, 2003). We previously identified two haematopoietic-specific mHAgs, ACC-1 and ACC-2 (Akatsuka et al, 2003), and demonstrated that T cells specific for ACC-1 were detected in the peripheral blood (PB) up to 7 month postHCT in a patient from whom the original ACC-1-specific cytotoxic T cell (CTL) clone had been generated (Nishida et al, 2004). As it has recently been proposed that bone marrow (BM) can function as a secondary lymphoid organ and contribute to long-term T cell memory for pathogens and malignant disease (reviewed in Di Rosa & Pabst, 2005), this study was conducted to investigate whether T cells specific for mHAgs could feasibly be generated from BM, rather than PB, long after HCT.

Another patient who received an HLA-identical, ACC-1-disparate HCT for chronic myelomonocytic leukaemia was identified. At 14 months postHCT, she had complete donor chimerism in PB and remained disease free. After informed consent, we examined the phenotype and proliferative capacity of ACC-1-specific T cells in mononuclear cells (MCs) obtained from the patients’ PB and BM. Three-colour flow cytometry detected a 3·5-fold higher percentage of ACC-1-specific T cells among the CD8+ population in BM than PB (0·72% vs. 0·21%), with a trend of more CD62L+ cells in the BM (Fig 1A, upper panels). After CD8+ cell selection by immunomagnetic beads, tetramer+ cells were further stained with antibodies against CD28, CDw197 (CCR7), CD44 (H-CAM, as a marker for memory T cells) and CD49d (VLA4α, a receptor for vascular cellular adhesion molecule-1 expressed on BM stromal cells). BM tetramer+ cells contained a higher percentage of CCR7+ cells than PB (83% vs. 50%), and a threefold higher percentage of CD28+ cells were detected in the BM tetramer+ CCR7+ population (47% vs. 16%, Fig 1A lower panel). More than 97% of the cells were CD44+ and CD49d+ in both PB and BM (data not shown). These findings indicate that the vast majority of tetramer+ cells were memory T cells that possessed the property of being able to migrate to BM, and that most tetramer+ cells in BM also expressed CCR7, a lymph node homing receptor, as a central memory T (TCM) cell marker. It is of note that more than half of BM ACC-1-specific CTL expressed CD28, an important costimulatory molecule for effective CD8+ T cell responses that interacts with CD80/86 expressed by antigen-presenting cells to stimulate interleukin-2 production (Topp et al, 2003).

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Figure 1.  Characterisation of CD8+ T cells specific for the ACC-1 minor histocompatibility antigen in peripheral blood (PB) and bone marrow (BM) at 14 months following human leucocyte antigen (HLA)-identical allogeneic haematopoietic cell transplantation. (A) Mononuclear cells (MC) were isolated from PB and BM and stained with fluorescence-conjugated monoclonal antibodies and HLA-A24/ACC-1 tetramer (Nishida et al, 2004). In lower panels, CD8+ MC were first sorted and then stained as above. Percentages shown are of the gated T cells as indicated. (B) Remaining MC were stimulated with 0·1 μmol/l ACC-1 peptide (DYLQYVLQI) directly added to cell suspension on day 0 and 7 in RPMI 1640 medium supplemented with 6% pooled human serum. On day 14, donor-derived OKT3-activated CD4+ blasts, pulsed with the same concentration of peptide, were added as antigen-presenting cells. Interleukin-2 (10 U/ml) was added on days 1 and 4 after the second and third stimulations. Growing T cells were stained as above. (C) Cytolytic activity of the T cell line generated from BM in the right panel of Fig 1B is shown.

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Next we tested whether these tetramer+ cells could be expanded by ACC-1 peptide stimulation. Peripheral blood mononuclear cells (PBMC) or bone marrow mononuclear cells (BMMC) from the same aliquot (c. × 106) were stimulated twice in the presence of 0·1 μmol/l (predetermined concentration) of ACC-1 peptide alone, and once with ACC-1-pulsed (0·1 μmol/l), donor-derived antigen-presenting cells. Staining with tetramer was done 21 d after the first stimulation. As shown in Fig 1B, stimulated BMMC produced a large population of tetramer+ cells, whereas PBMC did not yield any significant tetramer+ population (confirmed in an independent experiment, data not shown). The T cell line induced from the BMMC was cytotoxic to recipient B-lymphoblastoid cells (Fig 1C). The reason for the poor induction of tetramer+ cells from the PBMC at 14 month postHCT is most probably a result of their reduced capacity to proliferate in vitro long after HCT, as PBMC harvested on days 94 and 180 postHCT readily generated 37% and 18% of tetramer+ populations respectively (data not shown). These observations suggest that BM may be a superior source of mHAg-specific TCM that could possibly be expanded and used for immunotherapy, although it is to be determined whether our finding is a general phenomenon not only in other HCT cases, but also in other mHAgs.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. References

The authors thank Dr W. Ho for critically reading the manuscript and Keiko Nishida for tetramer production. This study was supported by Grants-in-Aid for Scientific Research and Scientific Research on Priority Areas, from the Ministry of Education, Culture, Science, Sports and Technology, Japan.

References

  1. Top of page
  2. Acknowledgements
  3. References
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  • Di Rosa, F. & Pabst, R. (2005) The bone marrow: a nest for migratory memory T cells. Trends in Immunology, 26, 360366.
  • Goulmy, E. (1997) Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Immunological Reviews, 157, 125140.
  • Marijt, W.A., Heemskerk, M.H., Kloosterboer, F.M., Goulmy, E., Kester, M.G., Van Der Hoorn, M.A., Van Luxemburg-Heys, S.A., Hoogeboom, M., Mutis, T., Drijfhout, J.W., Van Rood, J.J., Willemze, R. & Falkenburg, J.H. (2003) Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proceedings of the National Academy of Sciences of the United States of America, 100, 27422747.
  • Nishida, T., Akatsuka, Y., Morishima, Y., Hamajima, N., Tsujimura, K., Kuzushima, K., Kodera, Y. & Takahashi, T. (2004) Clinical relevance of a newly identified HLA-A24-restricted minor histocompatibility antigen epitope derived from BCL2A1, ACC-1, in patients receiving HLA genotypically matched unrelated bone marrow transplant. British Journal of Haematology, 124, 629635.
  • Topp, M.S., Riddell, S.R., Akatsuka, Y., Jensen, M.C., Blattman, J.N. & Greenberg, P.D. (2003) Restoration of CD28 expression in CD28 CD8+ memory effector T cells reconstitutes antigen-induced IL-2 production. Journal of Experimental Medicine, 198, 947955.