Introduction: Transplantation of haematopoietic stem cell (HSCT) from human leucocyte antigen (HLA)-disparate parental donors presents an approach for the treatment of patients lacking an HLA-matched donor, but little is known about differences in immune reconstitution as compared to conventional BMT. We prospectively compared paediatric recipients of positively selected CD34+ peripheral blood stem cells from unrelated or HLA-mismatched donors vs. recipients of unmanipulated bone marrow from matched sibling donors.
Patients: Immune reconstitution was studied in 20 children (20 transplants [5 sibling, vs. 8 UD, 7 haplo]; median age was 9,6 years; range 7m-26y; 7 female, 13 male). Blood samples were drawn before allo-HSCT, on days 14 (take), 30, 60, 100, 200 and 365 and >15months after SCT.
Methods: We analyzed lymphocyte subpopulations using flow cytometry. Cytokines (IFNg, IL2, TNFa, IL4, IL5, IL10) were determined by FACS after in vitro stimulation with PMA, ionomycin and brefeldin for 24h. Additionally, we measured IL2, IL7, IL13, IL15, IFNg and TGFb in unstimulated sera by ELISA. Further more, we studied TREC values and did spectratype analysis by real time-PCR and gel electrophoresis.
Results: After allo-HSCT, NK-cells were the cells, which regenerated first. In T-cells, decreased absolute counts could be detected as well as an inverted CD4-CD8-ratio during the first year after SCT. In CD4+ T-cells, the memory phenotype (CD45RO+) predominated. Sibling recipients show a faster T-cell regeneration than recipients of UD or mismatched family donors (MM).
As to cytokine levels in unstimulated sera, IFNg levels remained stable, while we saw high level of IL4 shortly after allo-HSCT; IL4 levels decreased during the first year after SCT. TGFb showed increased levels post-transplantation for up to two years. In stimulated T-cells, we measured a rise in Th2-cytokines (IL4, IL5, IL10) until d60 and a decrease of IFNg/TNFa. Th2-cytokines returned to pre-transplantation levels until d200, whereas Th1-cytokines rise to higher level than before transplantation. We could find that IL2 was produced predominantly by CD4+ / IFNg+ cells and by CD8+cells. Patients who received T-cell depleted stem cell preparations show lower IFNg and TNFa values and higher Th2-cytokine levels than patients who received bone marrow.
TREC counts appear earlier in sib HSCT (median d60) than in MM-transplant (median d200). As to spectratype values, we found a distribution of the 24Vb gene families of 4,18% per Vb-family with clonal expansion, especially in the first months after HSCT, of BV6b>BV23>BV1>BV16.
Disclosure: No relevant conflicts of interest to declare.