Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing

Abstract

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

Figure 1

Prolonged T lymphopenia and relative expansion of B cells and NK cells after CBT. T cells (CD3⁺CD4⁺ and CD3⁺CD8⁺), B cells (CD19⁺), and NK cells (CD16⁺ and/or CD56⁺) were prospectively measured by multiparameter flow cytometry on fresh samples. At baseline and after CBT, CD4⁺ and CD8⁺ T cells were relatively reduced, whereas early B-cell and NK-cell recovery was evident. Surviving CB transplant recipients demonstrated rebound of CD4⁺ and CD8⁺ T cells at later intervals after transplantation. Horizontal lines depict normal values for healthy stem cell transplant donor previously studied by Storek et al. Error bars represent SEM.

Figure 2

Functional CD4⁺ and CD8⁺ T-cell recovery is delayed in CB transplant recipients. We used a cytokine flow cytometric assay wherein peripheral blood mononuclear cells are stimulated with a superantigen (SEB) or an overlapping pentadecapeptide mixture derived from the CMV pp65 protein. We compared SEB-stimulated and CMV-specific CD4⁺ (A) and CD8⁺ (B) T cells at approximately post-CBT day 100 to those seen in other allogeneic PBSC or marrow SCT recipients previously assessed at the same interval using identical methods. In all cases, CB transplant recipients exhibited reduced functional responses measured by the proportion of CD69⁺ IFNγ⁺ T cells after stimulation, relative to allogeneic SC transplantation controls, though differences in CMV-specific T-cell responses were greater in the CD4⁺ subset (vs the CD8⁺ subset). These data demonstrate that a significant subset of CB transplant recipients developed positive CMV-specific T-cell responses from adoptively transferred naive cells by day 100 (CD4⁺: 44% and CD8⁺: 50%). Results are shown only for those patients surviving to day +100, who were assessable (eg, CMV-seropositive), and who had sufficient events in the relevant gate to draw firm conclusions regarding functional frequencies. Error bars represent SEM.

Figure 3

Thymic regeneration failure after CBT. The frequency of recent thymic emigrants in peripheral blood was quantitated using a real-time PCR assay measuring T-cell receptor (TCR) δ excision circles produced during TCR-α rearrangement. (A) Longitudinal assessment of thymic function in CB transplant recipients (n = 26, median age = 33) demonstrates reduced thymic function in baseline and post-CBT thymopoietic recovery that was measurable in only 2 subjects and significant and sustained in only 1 recipient. The horizontal line depicts the number of TRECs present in a group of healthy control subjects who were age-matched (n = 18, median age = 34). (B) Thymic regenerative failure in CB transplant recipients relative to autologous (auto) and allogeneic (allo) SCT recipients. Median values of TRECs in CBT were dramatically reduced relative to other prospectively analyzed SCT recipients, despite a significantly reduced median age of the CBT population (median age 33 vs 43 for Colorado allogeneic SCT recipients and 50 for Colorado autologous SCT recipients). Values for CB transplant recipients were significantly lower than other groups at all time points (P < .01).

Figure 4

Late memory skewing of CD4⁺ and CD8⁺ T cells after CBT. The relative proportions of CD4⁺ and CD8⁺ T cells in 4 distinct maturation stages (from least mature to more mature: CD45RA⁺CCR7⁺, CD45RA⁻CCR7⁺, CD45RA⁻CCR7⁻, CD45RA⁺CCR7⁻) were assessed using 6-color flow cytometry. Relative proportions of naive and memory T cells in CB transplant recipients (n = 32) were compared with healthy SCT donors (n = 100). CB transplant recipients had, at baseline and after transplantation, relatively reduced proportions of naive and central memory CD4⁺ and CD8⁺ T cells and greater proportions of effector and late-effector memory (ie, CCR7⁻) T cells. Differences relative to healthy donors were statistically significant at all time points (P < .05).

Figure 5

Preservation of CCR7⁺ CD4⁺ T cells at day 30 is associated with improved survival after CBT. We examined actuarial survival in patients stratified by the median ratio of CCR7⁺ to CCR7⁻CD4⁺ T cells at post-CBT day 30. Patients above the median ratio had significantly improved survival after CBT (median survival 240 days vs 700 days, P = .013).