Regulatory and Naïve T Cells in Unmanipulated Donor Grafts Are Not Associated with Acute Graft Vs Host Disease in Matched Sibling Transplants for AML

Acute graft-versus-host disease (aGVHD) occurs in approximately 20–50% of matched-related stem cell transplants (HCT). The presence of increased frequencies of CD4⁺CD25⁺ regulatory T cells (T_reg) in murine donor grafts has been shown to ameliorate aGVHD. In similar models, several groups have demonstrated that the transfer of equivalent numbers of naïve T cells (vs. various memory subsets) confers a relatively greater risk for aGVHD. To test the hypothesis that natural variations in donor graft naïve and T_reg content would be associated with the incidence of aGVHD in graft recipients, we conducted a detailed analysis of donor graft T cells within unmanipulated grafts of 70 SCT recipients transplanted for AML/MDS following the administration of a uniform fludarabine/busulfan conditioning regimen. Median follow up time was 38 months. 19% (n=13) of recipients experienced grades II–IV aGVHD (n=3 grades III–IV). Cryopreserved donor grafts samples were analyzed using 9-color flow cytometry (with a panel including a viability dye and MAbs recognizing CD4, CD8, CD25, CD127, CD45RA, CD27, Foxp3, and Ki-67). Tregs were defined as being CD4⁺CD25⁺CD127^loFoxp3⁺. CD45RA and CD27 were used to measure naïve (CD45RA⁺CD27⁺) and memory conventional CD4⁺ and CD8⁺ T cells. In addition, the proliferating fraction of all cells was defined by Ki-67 nuclear-antigen expression. We conducted analyses based on donor graft T cells assessed both continuously and by quartiles, with similar results. No significant associations were found between aGVHD incidence and total T_regs (HR 1.01, 95%CI 0.9–1.2, p 0.8), nor with proliferating T_regs (HR 1.2, 95%CI 0.2–5.9, p 0.8). Since murine models typically fix the dose of T_regs vs. conventional T cells (Tcon), we also analyzed GVHD risk by donor graft T_reg:Tcon ratio, finding no association (HR 1.03, 95%CI 0.8–2.1, p 0.3). Although patients in the highest T_reg:Tcon quartile received Treg doses known in vitro to induce suppression (>1:21.5), there was no reduction in aGVHD (HR 1.5 Quartile 4 vs others, 95%CI 0.5–4.9, p 0.5). Additionally, no significant association with aGVHD was found with total naïve T cells (HR 1, 95%CI 0.9–1.0, p 0.9), naïve CD4⁺ (HR 1, 95%CI 0.9–1.03, p 0.6), or naïve CD8⁺ cells (HR 0.98, 95%CI 0.9–1.03, p 0.6). Because naïve:memory cell ratios were also fixed in murine models demonstrating the importance of naïve T cells in GVHD, we also assessed naïve:memory T cell ratios within donor grafts with respect to recipient aGVHD. No reduction in aGVHD was observed considering naïve:memory ratios analyzed continuously (HR 1.1, 95%CI 0.3–4.2, p 0.8) or in the quartile receiving the lowest naïve:memory ratio (HR 0.5 Quartile 1 vs others, 95%CI 0.1–2.2, p 0.3). Our results demonstrate that donor graft T_reg and naïve T cell content, assessed in the context of unmanipulated PBSC transplantation in matched siblings, does not predict recipient aGVHD incidence. These data suggest that the therapeutic efficacy of T_reg enrichment and/ or naïve T cell depletion may require grafts to be manipulated to supraphysiologic levels for maximal therapeutic benefit.