Bone marrow CD34 þ cells generate fewer T cells in vitro with increasing age and following chemotherapy F. O FFNER , T. K ERRE , M. D E S MEDT AND J. P LUM Departments of Haematology and Immunology, University Hospital Ghent, Belgium Received 26 June 1998; accepted for publication 3 December 1998 Summary. To assess the influence of high-dose chemother- apy and age on the intrinsic capacity of stem cells to generate T cells, CD34 þ cells derived from bone marrow used in clinical transplantation were evaluated in an in vitro T-cell assay using a mouse thymic microenvironment. CD34 þ cells were sorted from healthy donor and autologous back-up bone marrow after density gradient centrifugation and depletion for CD1, 3, 4, 7, 8, 19 and glycophorin A using magnetic beads. CD34 þ cells were then introduced in day 14–15 fetal SCID mouse thymus lobes by incubation in hanging drops for 48 h. After transfer to gelfoam rafts they were cultured for variable time periods. The lobes were then homogenized in a tissue grinder for flow cytometric analysis gating on human cells. These were evaluated for CD4, CD8, CD3 and HLA-DR surface expression. 51 samples were analysed and three patterns of T-cell precursor development could be observed. In pattern A no human cells could be recovered, in pattern B maturation stopped at the CD4 þ CD8 ¹ CD3 ¹ pre-T-cell stage, and in pattern C matura- tion to double-positive CD4 þ CD8 þ thymocytes was reached. In 25 healthy donors under age 40 three showed pattern A, 12 pattern B and 10 pattern C, whereas in 16 healthy donors over the age 40 there were respectively four with A, seven with B and only five with C (P=0·01). In 10 patients who had previously received chemotherapy, none developed pattern C, five pattern B and five pattern A, in contrast to 15/41 pattern C, 19/41 pattern B and 7/41 pattern A in healthy donors. These data suggest an intrinsic loss of T-cell generation capacity from adult bone marrow stem cells in comparison to reports on stem cells of fetal origin. This loss correlated weakly with age, irrespective of thymic involution, and may be further reduced by prior chemotherapy. Keywords: thymopoiesis, bone marrow, stem cells, age, chemotherapy. Host defence following high-dose chemotherapy and stem cell reinfusion or transplantation depends on the recovery of granulocytes, monocytes and tissue macrophages, and on the reconstitution of immune competence of the lymphoid system. The recovery of granulocytes is early and can be accelerated by the growth factors or alternatively by the reinfusion of peripheral blood progenitors rather than bone marrow stem cells (reviewed in Lieschke & Burgess, 1992; To et al, 1997). Tissue macrophage reconstitution is slower and dependent on the target organ (Kennedy & Abkowitz, 1997). An important factor in the immune deficiency after high- dose chemotherapy is the late recovery of antigen-specific T- cell response that is pivotal in the induction of new antibody production and in the response to viral infections such as cytomegalovirus. T-cell reconstitution is generally defective for a long time, particularly in the allotransplant setting where graft-versus-host disease (GVHD) prophylaxis is given either as cyclosporin or by way of T-cell depletion (Lum, 1997; Keever et al, 1989; Parkman & Weinberg, 1997; Storek et al, 1997; Roux et al, 1996). The recovery of functional T-cell responses has been reported to vary on the stem cell source: responses to viral and alloantigens recover faster in patients receiving bone marrow grafts (Ottinger et al, 1996). The repopulation with T cells following stem cell reinfu- sion can theoretically proceed along four pathways (reviewed in Parkman & Weinbery, 1997; Mackall & Gress, 1997). First, T cells may be derived from expansion of postnaive donor T cells that are reinfused along with the stem cells (Mackall et al, 1996), and these would in theory transfer acquired donor immunity. Second, they may develop from the donor stem cells through a process of thymic education comparable to what happens in early childhood (Mackall & Gress, 1997). Third, they may be derived from stem cells through an extrathymic pathway (Dejbakhsh British Journal of Haematology , 1999, 104, 801–808 801  1999 Blackwell Science Ltd Correspondence: Dr F. Offner, Department of Haematology, 9K12IE, Universitair Ziekenhuis, 185 De Pintelaan, 9000 Gent, Belgium. e-mail: fritz.offner@rug.ac.be.