The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants

Abstract

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αβ T cell counts at birth persisted over time, with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Fig. 1.. Autosomal recessive pre-TCRα deficiency.

(A to B) Schematic representations of the isoform A (A) and B (B) proteins encoded by PTCRA. (C) Abundance of the indicated pre-TCRα isoforms in transcripts per million (TPM) across thymocyte developmental stages (DN1, DN2, DN3, ISP, DP early, DP late, and single-positive SP8 and SP4). The proportion of total PTCRA transcripts corresponding to isoform A in each thymocyte subset is indicated on the graph (dashed gray line). (D) Pedigree of the seven unrelated families displaying familial segregation of the mutant PTCRA alleles. The indicated mutant alleles, each with a unique color code, are labeled “M" in the pedigree. Individuals of unknown genotype are labeled “E?”. Asymptomatic individuals are annotated with a vertical bar. (E) MRI on axial sections at the level of the aortic arch: T1-weighted sequences after gadolinium injection (P5) and T2-weighted sequences (P6, P9, and controls), for P5, P6, P9, and age- and sex-matched controls. In patients, the thymic lodge, located between the sternum and the aortic arch (asterisk), appears empty (P5 and P6) or small (P9). By contrast, the thymus is clearly visible in controls, even after the onset of puberty (14-year-old girl).

Fig. 2.. Patient mutations and two mutants from gnomAD are loss-of-function or severely hypomorphic.

(A) Schematic representation of the artificial gene created to study the splicing between exons 1 and 2 of PTCRA. The two mutations tested are depicted. TSS: transcription start site. (B to D) HEK293T cells were transfected with an empty vector (EV) or with plasmids encoding the artificial gene with the WT or mutant PTCRA sequence described in A. (B) The RNA was subjected to RT-qPCR for PTCRA with a probe spanning the splice junction between exons 1 and 2. Data are displayed as 2^−ΔCt values after normalization relative to an endogenous control (ΔCt). The bar graphs show the mean ± SEM of three technical replicates, and are representative of three independent experiments. (C) Exon trapping. Bar graph showing the proportion of canonical or noncanonical PTCRA transcripts in the transfected HEK293T cells. (D) Total protein extracts were subjected to immunoblotting with an antibody against the DDK tag or GAPDH. Data representative of three independent experiments. (E) HEK293T cells were transfected with an empty plasmid or with a plasmid carrying a C-terminal DDK-tagged cDNA encoding the WT or the indicated variants of PTCRA isoform A. Total protein extracts were subjected to immunoblotting with an antibody against the DDK tag, pre-TCRα, or Vinculin. Data representative of four independent experiments. (F to K) TCRα–deficient Jurkat cells were transduced with an EV or with a plasmid encoding the WT isoform A, the WT isoform B or the indicated variant of pre-TCRα. The expression of TCRβ (F and G), CD3ζ (H and I) or CD69 (J and K) at the cell surface was evaluated by flow cytometry on the transduced cell lines. Data representative of three independent experiments. (F, H, and J) Histograms showing the mean fluorescence intensity (MFI) of cells transduced with the indicated PTCRA allele normalized against the MFI for EV. (G, I, and K) Representative flow cytometry histogram plot for the indicated PTCRA alleles. Cells transduced with the PTCRA alleles (black line, unshaded area) are compared to the cells transduced with the EV (shaded).

Fig. 3.. T cell immunophenotyping for patients with pre-TCRα deficiency

(A and B) CD3⁺ T cell counts as a function of age. The control range is represented by the gray area. (C) Thymic function was assessed in pre-TCRα–deficient patients (red and blue dots) and healthy local controls (black dots; n=101). The concentration of sjTRECs in the blood (sjTREC/10⁵ PBMCs) is presented as a function of age. (D) TCRαβ⁺ T cell counts as a function of age for naïve and memory T cells. (E) TCRγδ⁺ T cell counts as a function of age. The control range is represented by the gray area. (F) TCRγδ⁺ T cell counts as a function of age for naïve and memory T cells. (G) Frequency of TCRγδ⁺ T cells among total naïve (CD3⁺CD45RA⁺CCR7⁺) and memory (defined as non-naïve CD3⁺) T cells from patients (P3-P6 and P8) and controls (n=46). (H) Frequency of TCRγδ1⁺ and TCRγδ2⁺ T cells among naïve TCRγδ⁺ T cells from patients (P4, P8, and P9) and controls (n=8). (I) Cell counts as a function of age, for CD4⁺CD8⁻ T cells and CD4⁻CD8⁺ T cells. The control range is represented by the gray area. (J) Naïve αβ T cell counts as a function of age, for CD4⁺CD8⁻ T cells and CD4⁻CD8⁺ T cells. αβ T cells are defined here as CD3⁺TCRγδ⁻ cells. (K) Frequency of CD4⁻CD8⁻ cells in αβ (defined here as CD3⁺TCRγδ⁻) naïve (right) and memory (left) T cells from patients (P3-P6 and P8) and controls. (L to N) Phenotyping of individuals homozygous for the p.Asp51Ala mutation (D51A) and controls (n=12-18). (L) Frequency of TCRγδ⁺ T cells among total naïve and memory T cells. (M) TCRγδ⁺ T cell counts for naïve and memory T cells. (N) Frequency of CD4⁻CD8⁻ cells among αβ (defined here as CD3⁺TCRγδ⁻) naïve (right) and memory (left) T cells. (B, D, F, and J) P3 suffered from severe enteropathy and was on rituximab treatment. P7 received chemotherapy for lymphoma. These two patients are therefore depicted with triangles. (G, H, and K to N) C: controls; LOF: patients homozygous for LOF variants; and D51A: individuals homozygous for the p.Asp51Ala mutation.

Fig. 4.. Impaired generation of TCRαβ⁺ T cells in pre-TCRα–deficient ATOs.

In vitro T cell differentiation from positively selected peripheral blood CD34⁺ cells obtained from a healthy control, three patients with the p.Asp51Ala variant (D51A) and three patients with LOF PTCRA mutations (P1, P5, P6) after 5 weeks of culture in the ATO system. (A) Flow cytometry plots showing the expression of early and late T cell differentiation markers (CD7, CD5, CD1a, CD4, CD8β, TCRαβ, TCRγδ, and CD3) following gating on LIVE/DEAD⁻CD45⁺CD56⁻ cells. The data shown correspond to one control, one p.Asp51Ala patient and P1. (B) Plots of absolute counts/ATO for the various stages of T cell differentiation, for the cells isolated from the ATOs. (C) Bar graphs showing the ratio of absolute counts of TCRγδ⁺ cells to absolute counts of TCRαβ⁺ cells per ATO. C: controls, LOF: patients homozygous for LOF variants, D51A: individuals homozygous for the p.Asp51Ala variant.

Fig. 5.. Biases in the TRAD rearrangement repertoire indicate that TCRα chains are mostly generated by rearrangement of a TCRδ1 template in pre-TCRα–deficient individuals.

(A) Shannon’s entropy for TCRγ rearrangements in naïve and memory γδ T cells from controls (black; n=4) and pre-TCRα–deficient individuals (red; P1, P2, P4, and P8). (B) Shannon’s entropy for TCRα and TCRβ rearrangements in naïve and memory αβ T cells from controls (black; n=4) and pre-TCRα–deficient individuals (red; P1, P2, P4, P8, and P9). (C) Heatmap of paired gene rearrangements at the TRAD locus for naïve αβ T cells from four controls compared with five pre-TCRα–deficient individuals (P1, P2, P4, P8, and P9). The red color highlights V-J gene pairings overused in patients and the blue color highlights V-J gene pairings overused in controls. The TCRδ1 (TRDV1:TRDJ1) rearrangement is indicated with a black circle. (D) Fraction of TCRδ rearrangements in total productive TRAD rearrangements from sorted naïve and memory αβ T cells from controls (black; n=4) and pre-TCRα–deficient individuals (red; P1, P2, P4, P8, and P9). (E) Fraction of productive TCRδ rearrangements among total TCRδ rearrangements in naïve and memory αβ T cells from controls (black; n=4) and pre-TCRα–deficient individuals (red; P1, P2, P4, P8, and P9) (red). (F) Schematic representation of the TRAD locus before and after TCRδ1 rearrangement. (G) Percentage of productive TRA rearrangements involving TRAV24-41 in sorted naïve and memory αβ T cells from controls (black; n=4) and pre-TCRα–deficient individuals (red; P1, P2, P4, P8, P9). Unpaired t tests were used for comparisons in panels A, B, D, and E.

Fig. 6.. γδ⁺ thymocytes do not preferentially differentiate into αβ T cells in the absence of pre-TCRα, and TCRδ1 cannot act as a surrogate for pre-TCRα.

(A) Fraction of productive TCRγ among total TCRγ templates in sorted naïve and memory αβ (left) or γδ (right) T cells from controls (black, n=4) and pre-TCRα–deficient individuals (red, P1, P2, P4, P9). Unpaired t tests were used for all comparisons. (B) Fraction of expanded αβ T cell clones from two controls and two pre-TCRα–deficient individuals with a productive TRG rearrangement at the gDNA level. Of note ~90% of these clones were CD4⁺CD8⁻. (C) Heatmap of paired gene rearrangements of the TRB locus for naïve αβ T cells from controls compared with five pre-TCRα–deficient individuals (P1, P2, P4, P8). The red color highlights V-J gene pairings overused in patients and the blue color highlights V-J gene pairings overused in controls. (D) TCRαβ–deficient Jurkat cells were stably transduced with an empty plasmid or with a plasmid encoding TCRα, pre-TCRα, TCRδ1 or TCRγ. Each of the resulting cell lines was then cotransduced with another empty plasmid or with a plasmid encoding one of eight selected TCRβ chains. The expression of CD3 at the cell surface was evaluated by flow cytometry. Representative flow cytometry histogram plot for three independent experiments. Representative flow cytometry data are shown on the left. A recapitulative bar graph of the MFI for CD3 for each cell line is shown on the right.

Fig. 7.. Longitudinal studies of Ptcra^−/− mice.

(A) Schematic representation of thymocyte differentiation stages in mice. (B) Cell counts per thymus in Ptcra^−/− mice and WT mice aged 0-24 weeks, for the various thymocyte developmental stages. (C) Intracellular (IC) and membrane (Mb) expression of TCRβ for the indicated thymocyte subsets from Ptcra^−/− mice (bottom panel) and WT mice (upper panel). Cytometry data representative of 6 Ptcra^−/− mice and 6 WT mice are shown. (D) Counts of cells per microliter of blood or per spleen or per LN for Ptcra^−/− mice (red) and WT mice (black) aged 0-24 weeks for the indicated T cell subsets, including CD4-SP (CD3⁺TCRγδ⁻CD4⁺CD8⁻), CD8-SP (CD3⁺TCRγδ⁻CD4⁻CD8⁺), DN (CD3⁺TCR⁻γδ⁻CD4⁻CD8⁻), Treg (CD3⁺TCRγδ⁻CD4⁺CD8⁻CD25⁺), and γδ (CD3⁺TCRγδ⁻CD4⁺CD8⁻). (E) Representative flow cytometry plots of naïve and memory cell staining for CD4 and CD8 αβ T cells from the spleen and LNs of 12-week-old WT (black) and Ptcra^−/− mice (red). EM: effector memory; CM: central memory; N: Naïve. (F) Frequency of naïve cells among CD4-SP and CD8-SP αβ T cells, and of γδ T cells in the indicated tissue of Ptcra^−/− mice (red) and WT mice (black) from 0-24 weeks of age. (B, D and F) The data shown are the mean and standard deviation of 4 to 6 animals at each age.