Aberrant T-cell exhaustion in severe combined immunodeficiency survivors with poor T-cell reconstitution after transplantation

Abstract

Background: Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes.

Objective: We hypothesized that CD4⁺ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients.

Methods: We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT.

Results: Compared to post-HCT SCID patients with normal CD4⁺ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA⁺/CCR7⁺ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8⁺ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8⁺ T cells was inversely correlated with CD4⁺ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4⁺ T cells showed a transcriptional signature of exhaustion.

Conclusions: Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.

Keywords: Conditioning chemotherapy; T-cell exhaustion; hematopoietic cell transplantation (HCT); immune reconstitution; severe combined immunodeficiency (SCID).

Figure 1.. Low CD4+ counts post-HCT were correlated with few naïve T cells, reduced recent thymic emigrants and restricted T-cell diversity.

(A) Patients were separated into those with normal (n=37) and low (n=24) absolute CD3⁺ T-cell counts (left panel) and those with normal (n=32) and low (n=29) absolute CD4⁺ T-cell counts (right panel). (B-C) Frequency of CD8⁺ (B) and CD4⁺ (C) T-cell subsets in normal CD4⁺ (n=32) and low CD4⁺ (n=29) groups. T_CM=central memory T cells (CD45RA⁻/CCR7⁺); T_Naive=naïve T cells (CD45RA⁺/CCR7⁺); T_EM=effector memory T cells (CD45RA⁻/CCR7⁺); T_EMRA=effector memory RA T cells (CD45RA⁺/CCR7⁻). (D) Recent thymic emigrant (RTE) CD4⁺ T-cell (CD45RA⁺ CD31⁺/CD4⁺) frequency (left panel) and counts (right panel) among the normal CD4⁺ (n=29) and low CD4⁺ (n=27) groups. (E) T-cell receptor excision circles (TRECs) in normal CD4⁺ (n=28) and low CD4⁺ (n=27) groups. (F) Number of polyclonal Vβ T-Cell Receptor peaks in spectratype analysis of normal CD4⁺ (n=30) and low CD4⁺ (n=27) patients. Error bars indicate mean±SEM. Statistical significance was assessed by Wilcoxon-Mann-Whitney test. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001.

Figure 2.. Poor T-cell reconstitution was associated with increased expression of inhibitory receptors (IR) and an activated T-cell state, even within naive cells.

(A) Frequency of the indicated IRs and (B) activation markers on CD8⁺ T cells in healthy controls (n=6) and patients with normal (n=20–32) or low (n=12–29) CD4⁺ T cells. (C) Expression of intracellular granzyme B and perforin in CD8⁺ T cells from patients with normal (n=27) or low (n=24) CD4⁺ T cells. (D) Frequency of the indicated IRs and (E) activation markers on CD4⁺ T cells in healthy controls (n=6) and patients with normal (n=20–32) or low CD4⁺ T cells (n=13–29). (F) Expression of the indicated IRs and (G) activation markers on CD8⁺ T_Naive cells in patients with normal (n=17–32) or low (n=12–29) CD4⁺ T cells. Error bars indicate mean±SEM. Statistical significance was assessed by Kruskal-Wallis test (A, B, D, E) or Wilcoxon-Mann-Whitney (C, F, G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3.. Exhaustion score was inversely correlated to quality of T-cell reconstitution.

(A) Z score of the indicated IR on CD8⁺ T cells in patients with normal (n=32) and low (n = 29) CD4⁺ T cells. (B) Exhaustion scores of patients with normal (n=32) and low (n = 29) CD4⁺ T cells. The exhaustion score is the sum of the Z scores of PD1, 2B4 and CD160 IR on total CD8⁺ T cells (left panel) or naive CD8⁺ T cells (right panel). (C-F) Correlations between the exhaustion score on CD8⁺ T cells and the number of (C) CD4⁺ T cells (n=61), (D) CD45RA⁺ CD31⁺/CD4⁺ T cells (n=56), (E) T-cell receptor excision circles (TRECs) (n = 55) and (F) Polyclonal Vβ T-cell receptor peaks by spectratyping (n=57). Error bars indicate mean±SEM. Statistical significance was assessed by Wilcoxon-Mann-Whitney (A-B). *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001. Correlation was assessed by Pearson correlation coefficient (C-F). Coefficient R² and P-values are shown.

Figure 4.. Elevated exhaustion score was associated with lack of HCT conditioning regimen and low donor myeloid engraftment.

(A) Exhaustion score of CD8⁺ T cells in RIC/MAC (n=20) vs. non-conditioned (None/IS; n=41) patients. (B) Exhaustion score on total CD8⁺ T cells according to SCID genotypes: IL2RG/JAK3 (n=31), RAG1/RAG2 (n=8), DCLRE1C (n=5), IL7R/CD3/CD45 (n=5), and others (n=12). (C) CD8⁺ T-cell exhaustion score according to graft type: MRD (n=11), MORD (n=3), MUD (n=8), MMRD (n=31), and UCB (n=7). (D) Exhaustion score according to HLA compatibility: Match (n=24), Mismatch (n=22) and Haploidentical (n=15). (E) Exhaustion score in patients with donor (n=11, more than 80% of myeloid cells of donor origin) or recipient (n=26, less than 5% of myeloid cells of donor origin) myeloid chimerism. Error bars indicate mean±SEM. Statistical significance was assessed by Wilcoxon-Mann-Whitney (A), Kruskal-Wallis (B-D) or Student t test (E). *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001. cGvHD: chronic Graft-versus-host disease; DCLRE1C, DNA cross-link repair 1C; IL2RG, Interleukin-2 receptor gamma chain; IL7R, Interleukin-7 receptor alpha chain; IS: immunosuppression; JAK3, Janus kinase 3; MAC: myeloablative conditioning; MMRD, mismatched related donor; MORD, matched other related donor; MRD, matched related donor; MUD, phenotypic matched unrelated donor; RAG, Recombinase activating gene; RIC: reduced-intensity conditioning; UCB, unrelated umbilical cord blood.

Figure 5.. Unconditioned SCID patients with low CD4⁺ counts had skewed T-cell differentiation and high exhaustion scores that correlated with their poor T-cell reconstitution.

(A) CD4⁺ cell number in non-conditioned (None/IS) patients with normal (n=13) vs. low (n=28) CD4⁺ T cells. (B-C) Percentage of CD8⁺ (B) and CD4⁺ (C) T-cell subsets in None/IS patients with normal (n=13) vs. low (n=28) CD4⁺ T cells. T_CM=central memory T cells (CD45RA^−/CCR7⁺); T_Naive=Naive T cells (CD45RA⁺/CCR7⁺); T_EM=effector memory T cells (CD45RA⁻/CCR7⁻); TEMRA=effector memory RA T cells (CD45RA⁺/CCR7⁻). (D-E) Global exhaustion score in None/IS patients with normal (n=13) vs. low (n=28) CD4⁺ T cells. The exhaustion score is the sum of the Z scores of PD1, 2B4 and CD160 IRs on CD8⁺ (D) or naive CD8⁺ (E) T cells. (F) Correlation between the exhaustion score on CD8⁺ T cells and the time after HCT (months) in NONE/IS patients (n=41; upper panel) and RIC/MAC patients (n=20; lower panel). (G) Exhaustion score in None/IS patients with normal (n=13) vs. low (n=28) CD4⁺ T cells on CD8⁺ T cells at 0–5, 5–15 and more than 15 years after HCT (Y, years). (H) Correlation between the exhaustion score on CD8⁺ T cells and the time after HCT (months) in NONE/IS patients with low CD4⁺ counts (n=28; upper panel) and normal CD4⁺ counts (n=13; lower panel). Error bars indicate mean±SEM. Statistical significance was assessed by Wilcoxon-Mann-Whitney test (A-F). *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001. Correlation was assessed by Pearson correlation coefficient (F, H). Coefficient R2 and P-values are shown.

Figure 6.. Unconditioned SCID patients with low CD4⁺ counts showed a transcriptional signature of exhaustion.

(A) Volcano plot of genes differently expressed (DE) between CD8⁺ T cells from individuals with normal vs. low CD4⁺ T cells following unconditioned HCT. Labels indicate DE (False Discovery Rate (FDR)<10%) known to be associated with T-cell exhaustion. (B) Gene set enrichment analyses showing that genes known to be up-regulated in exhausted T cells were enriched among up-regulated genes in individuals with low CD4⁺ T-cell counts relative to individuals with normal counts. Genes on the X-axis were ranked from the most up-regulated to the most downregulated in patients with low vs. normal CD4⁺ T cells. (C) Heatmap showing expression levels for exhaustion-associated genes that were differentially expressed between patients with normal vs. low CD4⁺ T cells.