Donor lymphocyte count and thymic activity predict lymphocyte recovery and outcomes after matched-sibling hematopoietic stem cell transplant

Abstract

Delayed immune recovery is a characteristic feature of allogeneic hematopoietic stem cell transplantation in adult recipients. Although recipient thymic T-cell neogenesis contributes to T-cell regeneration after transplantation, thymic recovery in the transplant recipient decreases with increasing age, and is diminished by intensive preconditioning regimens and graft-versus-host disease. In adult recipients, most events that determine transplant success or failure occur during the period when the majority of circulating T cells is derived from the donor's post thymic T-cell repertoire. As a result, the make-up of the donor lymphocyte compartment may strongly influence immune recovery and transplant outcomes. The aim of this study was to examine donor lymphocyte counts in a series of patients undergoing an allogeneic hematopoietic stem cell transplant to identify the potential contribution of donor regulatory and conventional T lymphocyte populations to immune recovery and transplant outcomes. We examined donor lymphocyte subset counts in relation to post-transplant lymphocyte recovery and transplant events in 220 consecutive myeloablative, T-cell-depleted, HLA-identical sibling hematopoietic stem cell transplant recipients with hematologic malignancies. In a multivariate analysis, absolute numbers of donor CD4(+) recent thymic emigrants were associated with overall survival (P=0.032). The donors' absolute lymphocyte count and thymic production of regulatory T cells were both associated with extensive chronic graft-versus-host disease (P=0.002 and P=0.022, respectively). In conclusion, these results identify donor immune characteristics that are associated with lymphocyte recovery, extensive chronic graft-versus-host disease, and survival in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using peripheral blood samples drawn from donors and patients enrolled in the ClinicalTrials.gov-registered trials NCT00001623, NCT00001873, NCT00353860, NCT00066300, NCT00079391, and NCT00398346.

Figures

Figure 1.

Flow cytometric analysis of CD4+ recent thymic emigrants (RTE) and regulatory T cells (Treg). Donor peripheral blood mononuclear cells were stained, acquired, and analyzed as outlined in the Design and Methods section. To identify CD4 subsets, live T cells were discriminated from dead cells, monocytes, and B cells in a dump (CD14+ and CD19+ cells, and dead cells staining brightly with ViViD) versus a CD3 bivariate plot. Next, singlets were identified in a forward scatter-area (FSC-A) versus –height (FSC-H) plot, and intact cells in a forward scatter-area versus side scatter-area (SSC-A) plot. CD4 cells were next gated on a CD45RA versus a CD4 plot, which also served to eliminate fluorochrome, in this instance, TxPE, aggregates. Within the CD4 population we identified (i) rRTE as cells co-expressing both CD31 and CD45RA, and (ii) natural and induced Treg (nTreg and iTreg, respectively) and conventional T cells (Tconv) in a FOXP3 versus Helios plot. Within the Treg and Tconv subsets, RTE were identified again using a CD31 versus CD45RA bivariate plot.

Figure 2.

Lymphocyte reconstitution and cumulative incidence of extensive chronic GvHD. The reconstitution of peripheral blood lymphocytes was assessed at 30 days, 100 days, and 1 year after HSCT. Patients were categorized into two groups on the basis of donor ALC. Group A (solid line) consists of patients whose donors had more than the median ALC (>2,110 cells/μL), and group B (dashed line) consists of patients whose donors had less than the median ALC. (A) Mean (+ SEM) ALC is shown for the donors, and for the patients at 30 days, 100 days, and 1 year after HSCT. (B) Patients whose donors had more than the median ALC had a low cumulative incidence of extensive chronic GvHD. (C) The reconstitution of natural Treg (nTreg, Foxp3+Helios+) was assessed at 1 year after HSCT and compared to the mean donor nTreg count. (D) Cumulative incidence of extensive chronic GvHD by donor nTreg count in a matched analysis performed on donor and 1-year patient samples from 55 donor/patient pairs. P values were calculated using a two-sided t-test or a log-rank test.

Figure 3.

Thymic production of nTreg and risk of extensive chronic GvHD. Patients whose donors had a higher thymic production of nTreg (RTE nTreg count/percent CD4 RTE) had a lower cumulative incidence of extensive chronic GvHD. The P value between the two groups was calculated using a log-rank test.

Figure 4.

Donor CD4+ recent thymic emigrant (RTE) counts and overall survival. Patients whose donors had higher CD4+ RTE counts had a better overall survival. The P value between the two groups was calculated using a log-rank test.