Thymic output, T-cell diversity, and T-cell function in long-term human SCID chimeras

Abstract

Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B, and sometimes NK-cell function. Nonablative human leukocyte antigen-identical or rigorously T cell-depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T-cell development in the recipients, leading to long-term survival. We reported previously that normal T-cell numbers, function, and repertoire developed by 3 to 4 months after transplantation in SCID patients, and the repertoire remained highly diverse for the first 10 years after BMT. The T-cell receptor diversity positively correlated with T-cell receptor excision circle levels, a reflection of thymic output. However, the fate of thymic function in SCID patients beyond 10 to 12 years after BMT remained to be determined. In this greater than 25-year follow-up study of 128 patients with 11 different molecular types of SCID after nonconditioned BMT, we provide evidence that T-cell function, thymic output, and T-cell clonal diversity are maintained long-term.

Figure 1

PBMC phenotype before and after BMT in SCID patients. Mean numbers (± SEM) of CD20 B, CD3 T, and CD16 NK cells were obtained by flow cytometric analysis of SCID PBMCs studied before (A; N = 151) and at the latest time after transplantation (B; N = 120). Results from 2 patients with Artemis deficiency (1 surviving), 1 with CD45 deficiency, 4 with SCID of unknown molecular type (1 surviving), and from the 5 patients who received transplants elsewhere for whom we do not have the original pretransplantation data, are not included in Figures 1 and 2. Values for normal infant (A) and pediatric to young adult (B) controls are shown for comparison. Using a conservative Bonferroni α level analysis for all of the pairwise comparisons, given the sample sizes and variances of the patients and control groups, differences in mean T-cell numbers (B) were found between γcDef, ADADef, IL7RαDef, RAG1/2Def, CD3Def, AutoRec, and the normal control range (P < .015); differences in mean B-cell numbers (B) were found between ADADef (P = .001) and RAG1/2Def (P < .001) and the normal control range; differences in mean NK-cell numbers (B) were found between γcDef (P < .001), ADADef (P < .001), IL7RαDef (P = .002), RAG1/2Def (P < .001), AutoRec (P = .001), and the normal control values.

Figure 2

PBMC function before and after BMT in SCID patients. [³H]Thymidine incorporation by proliferating lymphocytes in response to PHA, ConA, and PWM was evaluated before (A; N = 151) and at latest time studied after transplantation (B; N = 120). Values are mean responses in counts per minute (cpm; ± SEM). Using a conservative Bonferroni α level analysis for all of the pairwise comparisons, given the sample sizes and variances of the patients and control groups at the latest transplantation date (B), the proliferative response to PHA of γcDef, ADADef, RAG1/2Def SCIDs was different (P < .015) from that of normal controls. Mean proliferation to ConA stimulus was different from normal controls only for ADADef (P = .005), and for the response to PWM only the IL-7RαDef was different from normal control values (P < .001).

Figure 3

Analysis of T-cell function over time after BMT in all SCIDs. Proliferation values (cpm; N = 6299) to PHA (A), ConA (B), PWM (C), and Candida (D) stimulations of PBMCs from 128 patients over time. Graphs represent the change in the log₁₀ proliferation for PHA, ConA, and PWM stimulations, respectively, over time after the patient and transplantation effects were removed. Means for normal controls: PHA 187 073 cpm, SEM 2390 cpm; Con A 137 050 cpm, SEM 2289 cpm; PWM 92 191 cpm, SEM 2156 cpm; and Candida 48 793 cpm, SEM 2835 cpm.

Figure 4

Analysis of thymic function over time after BMT in all SCIDs. TREC values (N = 597) were observed over time in 115 patients for whom samples were available for analysis, each patient associated with 1 of 11 molecular types of SCID. (A) The combined results of all 115 patients. The points on the figure represent the predicted TREC value plus the residual after the patient and transplantation effects were removed. Lighter lines are then used to join points from the same patient before and 1 year after transplantation. The same analysis was performed for 15 IL7RαDef patients (B), 10 Jak3Def patients (C), and 57 γcDef patients (D); 95% confidence intervals were placed around the predicted TREC line over time for times more than 1 year after transplantation.

Figure 5

Thymic output correlates with TCR diversity. Shown are dot plots of the DKL values compared with TREC levels of samples taken from 36 SCID patients at multiple time points after BMT. DKL values from normal controls were < 0.08. ○ represents patients whose TREC value was less than 100; ●, patients whose TREC value was > 100.