Proliferation, Expansion, Effector Differentiation and Survival of GVH-Reactive T Cells Following Delayed DLI to Mixed Chimeras.
Authors: Chakraverty Ronjon , Buchli Jennifer , Zhao Guiling , Sykes Megan 
Published: October 2019

Abstract
Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). DLI given immediately following lethal irradiation lead to severe GVHD. We examined the proliferation, expansion, differentiation and survival of GVH-reactive T cells following delayed DLI and compared the outcomes to those observed when identical DLI were administered early following lethal irradiation (TBI). MC recipients were prepared by TBI of BALB/c mice and reconstitution with mixed BALB/c and CD45.2 C57BL/6 (B6) T-cell depleted bone marrow (TCD BM). 10 weeks later, we transferred 1 x 107 CD45.1 B6 and 5 x 106 2C transgenic (tg) splenocytes. CD8+ T cells from 2C tg mice bear TCR specific for recipient class I MHC Ld. Polyclonal DLI-derived CD4+/CD8+ T cell responses were monitored by gating on CD45.1+ events and clonal 2C CD8+ T cell responses tracked using a clonotypic marker. For comparison, identical DLI together with TCD BM was administered to freshly irradiated BALB/c or B6 CD45.2 syngeneic recipients. MC recipients of delayed DLI developed a GVH reaction, as indicated by increases in donor chimerism, but no GVHD. In contrast, allogeneic recipients receiving DLI immediately following TBI developed lethal GVHD (median survival 32d vs. >100d post-delayed DLI, p<0.0001). By day 6, donor CD4+/CD8+ T cells had undergone almost equivalent proliferation as monitored by CFSE-dilution in MC and TBI allogeneic recipients. However, the kinetics and distribution of donor T cell expansion were distinct. Following delayed DLI, marked expansion of donor CD4+ cells (peak day 10) preceded expansion of CD8+ cells (peak day 13) in the spleen, with less accumulation in the lymph nodes, BM, liver and lung, and no accumulation in the gut. Histology revealed transient, mild lymphocytic infiltrates within the lung/liver but no evidence of colitis. In contrast, the kinetics of donor CD4+/CD8+ T cell expansion were more rapid in freshly irradiated recipients with CD4+/CD8+ responses peaking on day 4–7. The distribution was also different with major increases in donor CD4+/CD8+ numbers in the gut but less accumulation in the spleen. Histology confirmed severe colitis. The kinetics of proliferation, expansion and distribution of tg 2C CD8+ T cells showed a similar pattern to the polyclonal donor CD8+ T cell population. Following delayed DLI, 2C CD8+ T cells acquired a memory/activation phenotype (CD44hi, CD45RBlo, CD62Llo, CD49d+, CD27+) with similar kinetics to those observed in TBI mice developing GVHD. Despite the marked differences in trafficking to the gut, 2C CD8+ T cells in both groups of allogeneic recipients expressed equivalent levels of the gut homing receptor, α4β7. However, we also observed important differences: 1) IL-2Rαexpression was absent on 2C CD8+ T cells following delayed DLI, but was expressed at high levels (>50% by day +3) in freshly irradiated DLI recipients; 2) 2C CD8+ T cells showed greater reductions in expression of IL-7Rαfollowing delayed DLI; and 3) high rates of 2C CD8+ T cell apoptosis, as indicated by annexin V staining, were observed following delayed DLI with absolute numbers up to 6-fold greater than in TBI recipients. Thus, in contrast to responses in freshly irradiated mice that develop GVHD, GVH reactions induced by delayed DLI are characterized by delayed kinetics, a distinct distribution, marked apoptosis and reduced expression of cytokine receptors important for CD8+ T-cell survival.