Short telomere syndromes cause a primary T cell immunodeficiency

Abstract

The mechanisms that drive T cell aging are not understood. We report that children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell-aging phenotypes seen in adults 5 decades older, including depleted naive T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting that newborn screening may identify individuals with germline telomere maintenance defects. Telomerase-null mice with short TL showed defects throughout T cell development, including increased apoptosis of stimulated thymocytes, their intrathymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells upregulated DNA damage and intrinsic apoptosis pathways, while older adult T cells upregulated extrinsic apoptosis pathways and programmed cell death 1 (PD-1) expression. T cells from mice with short TL also showed an active DNA-damage response, in contrast with old WT mice, despite their shared propensity to apoptosis. Our data suggest there are TL-dependent and TL-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.

Keywords: Aging; Genetics; Telomeres.

Figure 1. T cell primary immunodeficiency and its complications in telomerase mutation carriers.

(A) Telogram showing total lymphocyte TL measured by flow cytometry and FISH of mutation carriers relative to a nomogram of healthy controls. Those who developed opportunistic infections are noted. One TERT mutation carrier (patient 4, Table 1) did not have TL measured, so only 27 of 28 patients studied are plotted. (B and C) Images showing vesicular rash characteristic of VZV reaction (patients 3 and 5 in Table 1, respectively). (D) Brain MRI showing evidence of enhancing periventricular flare (marked by arrows) in a 19-year-old who died from fatal CMV encephalitis (Table 1, patient 4). (E) Chest CT scan image from a patient who developed concurrent P. jiroveci pneumonia that was complicated secondarily by CMV pneumonitis; the latter was treatment refractory and ultimately fatal. (F) Proportion of telomerase mutation carriers with lymphocyte count abnormalities (defined as at least 2 SD below the age-adjusted mean). Low CD4 counts and low IgM levels were the most common anomalies. Data are derived from 17 patients, including 7 from Table 1 for whom the full immune evaluation was available.

Figure 2. Telomerase mutation carriers have premature skewing of T cell subsets and decreased TRECs.

(A) Telogram showing the age-adjusted lymphocyte TL for each individual falling in 1 of 3 groups studied. (B) Difference in TL from the age-adjusted median for cases shown in A. YC and OA groups cluster around the age-adjusted median, whereas ST patients are at or below the first percentile. (C) Representative flow plots of peripheral CD4⁺ T cells from YC and ST subjects. (D) Percentage of naive CD4⁺ T cells, defined as CD3⁺CD4⁺CD45RA⁺CCR7⁺. (E) Representative flow plots from YC and ST cases showing naive CD8⁺ T cells (CD3⁺CD8⁺CD45RA⁺CCR7⁺) and terminally differentiated CD8⁺ TEMRAs, defined as CD3⁺CD8⁺CD45RA⁺CCR7^neg. (F) Percentage of CD8 naive and TEMRA populations as defined in E. For C–F, n = 5 YC, 2 male/3 female; n = 6 ST, 2 male/4 female; and n = 5 OA, 3 male/2 female. (G) Quantification of RTEs defined as CD4⁺CD45RA⁺CD31⁺. n = 6 YC, 2 male/4 female; n = 6 ST, 3 male/3 female; n = 4 OA, 2 male/2 female. (H) TRECs measured by quantitative PCR in telomerase mutation carriers. Data from each of the 10 patients (5 male/5 female) are graphed relative to an age-adjusted nomogram with the fifth percentile shown. The normal range was derived from 254 controls. For 9 patients, the mutated gene is shown, and 1 patient had classic features of dyskeratosis congenita (DC). In an infant with DKC1 mutation, TREC levels were undetectable. Error bars represent SEM. *P < 0.05; **P< 0.01; ***P < 0.001, Student’s t test, 2-sided.

Figure 3. T cell–intrinsic apoptosis contributes to T cell lymphopenia.

(A) Schematic for congenic transplant of bone marrow hematopoietic stem progenitor cells (HSPCs), defined as lineage-negative population. Cells were transplanted into WT or fourth-generation telomerase RNA-null mice (mTR^–/–G4). Donor-derived T cell fractions were assessed. (B and C) Quantification of donor-derived CD4⁺ and CD8⁺ T cells in thymuses and peripheral blood at 4 and 8 weeks after transplantation for WT and mTR^–/–G4 recipient mice (n = 6 recipients were studied, 3 male/3 female for each genotype at each time point). (D) Thymocyte apoptosis rates in CD3^negCD4^–CD8^– (DN) populations 1, 2, 3, and 4, defined by their cell-surface markers, as shown. (E) Apoptotic fraction of CD3^loCD4⁺CD8⁺ (DP) thymocytes in newborn. (F and G) Apoptotic fraction of CD3^hiCD4⁺ and CD3^hiCD8⁺ thymocytes. For D–G, the apoptotic fraction was quantified as the total annexin V⁺ population in newborn mice (1 week old, n = 6/group, sex not determined because of young age). (H–J) Peripheral blood absolute CD4⁺, CD8⁺ T cell counts and the CD4/CD8 ratio, respectively. For H–J, n = 10 WT, 7 male/3 female, n = 9 mTR^–/–G4, 4 male/5 female, 6–16 weeks). (K) Peripheral T cell apoptosis quantified at 48 hours as total annexin V⁺ PI^neg plus annexin V⁺ PI^lo populations for splenocytes stimulated with CD3/CD28. Total T cells were isolated from 10 WT (7 female/3 male) and 9 mTR^–/–G4 (4 female/3 male), 20–25 weeks; 7 old WT (5 female/2 male), 55–67 weeks. (L) Apoptosis quantified at 48 hours (as in K) for T cells isolated from peripheral blood of YC (n = 13, 8 male/5 female), ST (n = 9, 3 male/6 female), and OA (n = 5, 3 male/2 female). The analysis for K and L included total T cells. Error bars represent SEM. *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 4. The TCR repertoire is restricted in telomerase mutation carriers.

(A) Expression of 24 Vβ proteins by flow cytometry for YC, ST, and OA relative to 85 controls. Each column represents data from a single individual, with gray representing percentage of Vβ-expressing T cells within 1 SD from the mean and the colors representing greater deviation from means derived from controls. The degree of deviation is noted in the key. Data are shown for CD3⁺, CD4⁺, and CD8⁺ T cells and are summarized in the bottom 2 rows. (B–D) Bar graphs show mean number of Vβ families deviating 1–2, or more than 2, SD for CD3⁺, CD4⁺, and CD8⁺ T cells, respectively. n = 6 YC, 2 male/4 female; n = 7 ST, 2 male/5 female; n = 5 OA, 3 male/2 female. Error bars in B–D represent SEM. (E) T cell diversity as measured by the mean unique sequences per T cell determined by deep sequencing of the CDR3 of the TCR-β gene on sorted CD8⁺ T cells (n = 4/group, 2 male/2 female for each). (F) Unique productive sequences per CD8⁺ T cell for data generated for E. Whiskers in E and F mark the minimum and maximum values. (G) Pielou’s J index, a calculation of evenness of Vβ usage where 1 represents an even distribution and 0 represents complete dominance of 1 Vβ. (H) Dot plot of total usage of the 20 most frequently utilized Vβ genes in YC compared with ST subjects shows a higher usage in ST patients. Inset shows summed usage frequency of the top 5 most frequently used Vβ genes; the increased usage of highly utilized genes is reflected in a decreased usage of the subsequent Vβ genes (i.e., those ranked 6 to 20). *P < 0.05; **P < 0.01, Mann-Whitney U test.

Figure 5. Distinct pathways to apoptosis in short telomere and OA T cells.

(A) Heatmap and dendrogram of gene expression showing the mean subtracted expression values on a log₂ scale. For each of 12 samples, YC, ST, and OA groups (2 male/2 female/group), the log₂ expression value was subtracted from the mean log₂ expression value of the entire cohort. The dendrogram showing relatedness of the samples is above, and relatedness of the gene transcripts is to the left. The differential change in gene expression is shown as positive and negative change on color scale indicated in key. (B) Venn diagram shows 4 of 20 nonoverlapping upregulated pathways in IPA involved in apoptosis. (C and D) CD95 expression in CD4⁺ and CD8⁺ T cells, respectively. (E and F) PD-1 expression in CD4⁺ and CD8⁺ T cells, respectively. For C–F, n = 5 YC, 2 male/3 female; n = 6 ST, 2 male/4 female; and n = 5 OA, 3 male/2 female. (G and H) Kap1 and p-Kap1 levels on protein from isolated mouse T splenocytes. p-Kap1 and actin were detected first. Then the blot was stripped and reblotted with Kap1 antibody. Protein from irradiated splenocytes is a positive control. (H) Shown are quantification data from 3 independent Western blots from a total of 11 mice: WT (30 weeks, 1 male/3 female), mTR^–/–G4 (30–33 weeks, 4 female), and old WT mice (50–73 weeks, 3 female). (I) qRT-PCR from unstimulated T splenocytes. Each data point represents an independent experiment with ages similar to those in H. (J) Model of T cell–aging mechanisms showing differences in immunophenotype and T cell apoptosis program in young ST T cells and OA with normal TL. Older individuals with short telomeres are predicted to have extrinsic and intrinsic apoptotic mechanisms contributing. Error bars represent SEM. *P < 0.05; **P < 0.01, Student’s t test.