PTC has been found to diminish experimental GVHD and is under investigation in clinical studies (Luznik Blood 2010). An important question is whether PTC affects dividing cells equivalently irrespective of whether they are driven by antigen, i.e. allo or lymphopenic conditions. Cyclophosphamide (cyc) is a DNA alkylating agent which causes death of dividing cells via double strand breaks. We reasoned that if alloantigen drives more rapid proliferation vs. cytokine driven proliferation, a threshold level of DNA damage may enable greater survival by donor non-antigen specific cells expanding under lymphopenic conditions. The difference in the rates of proliferation and the effect on donor cell survival after PTC were investigated in both syngeneic and MiHA mismatched transplants. To establish a basal rate of homeostatic proliferation by donor B6 T cells (H2b, CD45.2+), a syngeneic myeloablative transplant was performed with CFSE labeled donor CD4+ and CD8+ T cells using congenic B6 (H2b, CD45.1+) recipients. Based on the clinical dosing schedule, PTC was administered on days 3 and 4 at the lowest concentration found to ameliorate GVHD following MiHA allo-HCT in previous experiments. At 5 days post-syngeneic transplant we observed the greatest amount of proliferation via CFSE dilution in the mesenteric lymph nodes (MLN), followed by peripheral LN and the spleen. Notably, we did not observe a significant diminution in the numbers of total cells recovered from lymphoid tissues in PTC recipients and or in the percentage of cells in later cell generations as ascertained by CFSE. Interestingly, the dividing cells in the MLN displayed a more activated phenotype (CD44/CD62L expression) compared with donor cells in the other lymphoid compartments consistent with more rapid expansion. The same protocol was performed in the B6 (H2b, Ly9.2+)->BALB.B (H2b, Ly9.1+) MiHA disparate strain combination. In this MiHA disparate transplant, we observed more rapid expansion kinetics in the MLN vs. other tissues and detected a greater cell recovery vs. the group that received PTC. Cell recovery in groups that received PTC, as well as number of CFSE divisions, was comparable to that of the syngeneic model of homeostatic proliferation. This result leads us to the conclusion that at this lowest effective concentration of cyc, cells undergoing homeostatic proliferation are comparatively spared. In order to further test this hypothesis and rule out the possibility that homeostatic proliferation could be affected by an ongoing allogeneic response, we included CFSE labeled OT.1 transgenic CD8+ T cells as a donor population which undergoes homeostatic, i.e. non-host antigen reactive proliferation with polyclonal B6 donor T cells (B6, Thy1.1+) transplanted into MiHA disparate recipients. At 5 days post-transplant, polyclonal donor B6 T cells in later divisions (>4) were substantially diminished after PTC. However, the percentage of OT.1 T cells in later generations was not affected by PTC. Taken together, these results suggest that cells undergoing homeostatic proliferation do so at a rate which permits the repair of damage induced by 33mg/kg PTC on two successive days. To extend these findings, we asked whether cells undergoing lymphopenic expansion in a syngeneic system were in fact susceptible to PTC induced death. A single PTC dose of 66mg/kg resulted in a decrease in later (>2) CFSE generations of both OT-I and polyclonal CD8+ T cells undergoing lymphopenic expansion in the mesenteric LN.
We further employed CFSE labeled cells from Foxp3 FIR knock-in mice in these transplant models and followed the persistence of Tregs after syngeneic and allogeneic transplants. Results demonstrated that Tregs are required for successful PTC abrogation of GVHD in agreement with others (Luznik ASH 2010). Donor Tregs demonstrated more rapid proliferation in allogeneic transplants and were more susceptible to PTC deletion than CD4+FoxP3−cells. We therefore hypothesize that relatively few slowly proliferating non host allo-antigen specific Tregs are sufficient following PTC deletion of most anti-host alloreactive donor T cells to inhibit GVHD in myeloablative transplants and speculate that low numbers of Tregs transplanted after the administration of PTC should further improve clinical outcomes.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.