Single-unit dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8+ T-cell response against the nonengrafted unit

Abstract

We investigated the potential role of an immune reaction in mediating the dominant engraftment of 1 cord blood unit in 14 patients who received a double-unit cord blood transplantation (CBT). In 10 patients, dominant engraftment of a single donor unit emerged by day 28 after CBT. In 9 of these 10 patients, a significant subset of CD8(+) CD45RO(+/-)CCR7(-) T cells, present in peripheral blood mononuclear cells and derived from the engrafting cord blood unit, produced interferon-gamma (IFN-gamma) in response to the nonengrafting unit. No significant population of IFN-gamma-secreting cells was detectable when posttransplantation peripheral blood mononuclear cells were stimulated against cells from the engrafted unit (P < .001) or from a random human leukocyte antigen disparate third party (P = .003). Three patients maintained persistent mixed chimerism after CBT, and no significant IFN-gamma-secreting cells were detected after similar stimulations in these patients (P < .005). Our data provide the first direct evidence in human double-unit CBT recipients that immune rejection mediated by effector CD8(+) T cells developing after CBT from naive precursors is responsible for the failure of 1 unit to engraft. Future investigations based on these findings may result in strategies to predict a dominant unit and enhance graft-versus-leukemia effect.

Figure 1

Conversion from naive to effector/effector memory T-cell phenotype after double-cord CBT. (A) CD8⁺ and CD4⁺ T cells in a representative unmanipulated fresh cord blood express a naive phenotype (CCR7⁺CD45RO⁻). (B) In a representative sample of day 14 posttransplantation PBMCs, CD8⁺ T cells express a primarily effector/effector memory phenotype (CCR7⁻CD45RO^+/−), and CD4⁺ T cells express primarily naive (CCR7⁺CD45RO⁻) and central memory (CCR7⁺CD45RO⁺) phenotypes.

Figure 2

Development of IFN-γ–secreting CD8⁺ T cells recognizing alloantigens expressed by the nonengrafting unit after double-unit CBT. (A) After a 5-hour stimulation, CD8⁺CD45RO^+/−CCR7⁻ IFN-γ–staining cells from PBMCs samples collected 14 to 28 days after transplantation are demonstrable against LCLs generated from the nonengrafting unit (i), but not the dominant unit (ii) or a random third party unit (iii). (B) IFN-γ–secreting CD8⁺ T cells are detectable among 9 of 10 patients establishing single donor dominance. (C) Alloreactive IFN-γ–secreting CD8⁺ T cells decline at later times after CBT. ■ represents time point at which the sample was drawn; solid lines, no administration of steroids for GVHD in the interval between sample assays; dotted lines, administration of steroids for GVHD in the interval between sample assays. Because 3 patients died before second PBMCs samples were collected, serial data for only 6 patients are shown.

Figure 3

Host CD8⁺ T cells reactive with cord blood alloantigens are present in 9 patients rejecting double CBT. Day 26 posttransplantation PBMCs were stimulated against LCLs generated from the transplanted cord units. (A) The majority of CD3⁺ cells are CD8⁺. (B) The CD8⁺ cells express primarily effector/effector memory phenotype (CD45RO^+/−CCR7⁻). (C) Host CD8⁺CD45RO^+/−CCR7⁻ cells stain positive for intracellular IFN-γ staining after stimulation with each of the transplanted cord unit.