Research ArticleADAPTIVE IMMUNITY

Human thymopoiesis is influenced by a common genetic variant within the TCRA-TCRD locus

See allHide authors and affiliations

Science Translational Medicine  05 Sep 2018:
Vol. 10, Issue 457, eaao2966
DOI: 10.1126/scitranslmed.aao2966
  • Fig. 1 Thymic function associates with naïve T cell immune phenotypes.

    (A) βTRECs (blue) are episomal DNA generated during the TCRB recombination. sjTRECs (purple) derive from the deletion of the TCRD locus during TCRA locus recombination (shown in fig. S1B). DN, double negative; ISP, immature single positive; DP, double positive; SP, single positive. (B) Effect sizes of significant associations [adjusted P values (adj. P) < 0.05] between sjTRECs and immune cells and parameters measured by flow cytometry in 969 healthy individuals from the MI cohort. Effect sizes were estimated in a mixed model (see Supplementary Materials and Methods). MFI, mean fluorescence intensity. (C) Relationships between sjTRECs and the log10-transformed number of naïve CD4+ and CD8+ T cells, naïve Treg, and iNKT cells. Regression lines were fitted using linear regression. Adjusted P values were obtained using the mixed model and based on the Kenward-Rogers F test.

  • Fig. 2 Age and sex strongly affect thymic function in healthy donors.

    (A) sjTRECs as a function of age in 487 women (red) and 492 men (blue). (B) βTRECs as a function of age, in 264 women and 242 men donors with detectable amounts. (C) Number of intrathymic divisions as a function of age, in donors with detectable βTRECs. (D) sjTRECs as a function of sex in 487 women and 492 men. Regression lines were fitted using linear regression. P values were adjusted to control the false discovery rate at 5% and estimated on the basis of the Kenward-Rogers approximate F tests.

  • Fig. 3 Genome-wide association study reveals an impact of TCRA-TCRD genetic variation on thymic function.

    (A) Manhattan plot for genetic association with sjTRECs in the 969 donors of the MI cohort. Light and dark gray lines indicate the threshold for suggestive association (P = 1.0 × 10−5) and genome-wide significant association (P = 5.0 × 10−8), respectively. (B) Detailed view of the TCRA-TCRD locus. Primers (sjTREC-F/R) and probe (sjTREC-P) used to quantify sjTRECs are shown in red and cyan, respectively. (C) Fine mapping of the genetic association between the TCRA-TCRD locus and sjTRECs. Meta-analysis P values were obtained by combining array-based, probe-based, and imputed genotypes of the MI and MARTHA cohorts (table S2). Variants that are significantly associated at the genome-wide level are indicated in red. (D) Physical position of the four most strongly associated variants, relative to active transcription activity (26). The position of Dδ3 is indicated.

  • Fig. 4 Effect of TCRA-TCRD human genetic variation on thymic function in humanized immunodeficient mice.

    (A) Immunodeficient Balb/c Rag2−/−Il2rg−/−SirpaNOD (BRGS) mice were reconstituted with human CD34+ hematopoietic progenitors harvested from fetal livers with rs2204985 genotype AA (orange), GA (brown), or GG (purple). (B) Effects of rs2204985 genotypes on sjTRECs in all mice (AA, n = 19; GA, n = 58; GG, n = 15). (C) Effects of rs2204985 genotypes in immunophenotyped mice (AA, n = 5; GA, n = 31; GG, n = 13) on number of CD3+ thymocytes and (D) on thymocyte subsets at different developmental stages. Indicated P values correspond to the genotype effect in a linear model including genotype and mouse recipient sex.

  • Fig. 5 Effects of TCRA-TCRD human genetic variation on thymic TCR repertoire in humanized immunodeficient mice.

    The human TCRA-TCRD locus was sequenced using genomic DNA from 8 (3 males and 5 females) and 12 (4 males and 8 females) immunodeficient mice thymi grafted with rs2204985 AA (orange) and GG (purple) human fetal livers, respectively (table S3). (A) Effects of the donor genotype on V (left) and J (right) gene usage, among TCRα and TCRδ productive rearrangements. (B) Ratio of median percentage of V (left) or J (right) gene usage in GG-grafted mice, over that in AA-grafted mice. Gene segments used specifically by TCRδ are indicated in red, by TCRα in gray, and shared by both in cyan. Whiskers indicate bias-corrected and accelerated bootstrap 95% CIs. (C) Effect of genotypes on the percentage of TCRD specific J genes (TCRDJ01 to 04) among total TCRD and TCRA J genes used in productive rearrangements. (D) Effect of genotypes on the percentages of DV (left), DD (center), and DJ (right) genes usages among TCRD productive rearrangements. Genes are ordered according to their genomic location (see Fig. 3B). Blue asterisks indicate P < 0.05 obtained using nonparametric Mann-Whitney U test, adjusted for multiple testing using the false discovery rate as error rate.

  • Fig. 6 Combined effects of sex, age, and TCRA-TCRD genetic variation on human thymic function.

    sjTRECs as a function of age and rs2204985 genotypes in (A) the MI cohort (n = 969) and (B) the replication MARTHA cohort (n = 612). Regression lines were fitted using linear regression. P values were obtained with a mixed model of log10(sjTRECs), including rs2204985 genotypes as predictor; covariates were selected using a data-driven variable selection scheme; and correcting for population stratification was performed using the genetic relatedness matrix (GRM) as a random effect. Orange, brown, and purple indicate AA, GA, and GG genotype, respectively. (C) Proportions of variance of sjTRECs explained by age, sex, and TCRA-TCRD genetic variation in MI (left) and MARTHA (right) cohorts. The surface area and color of subrectangles indicate proportions attributed to specific predictors, as measured by the R2 of the regression model. (D) Difference between actual age and thymic age as a function of sex and rs2204985 variant. Thymic age is predicted from a regression model, where AA men are assumed as the baseline.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/457/eaao2966/DC1

    Composition of the MI Consortium

    Materials and Methods

    Fig. S1. Technical workflow of the study and validation of the TREC assay in the MI cohort.

    Fig. S2. Association of sjTRECs with immune cell counts and parameters.

    Fig. S3. Association of βTRECs with immune cell counts and parameters.

    Fig. S4. Association of sjTRECs with nonheritable factors.

    Fig. S5. Association of βTRECs with nonheritable factors.

    Fig. S6. Association of intrathymic division number with nonheritable factors.

    Fig. S7. Association of thymic function parameters with specific nonheritable factors.

    Fig. S8. Age and sex impact on sjTRECs in the MARTHA cohort.

    Fig. S9. Human immune system mice flow cytometry gating strategy.

    Fig. S10. Effect of the SNP rs2204985 polymorphism on the TCRD locus deletion alternative rearrangement δRec-Jα58.

    Fig. S11 Assessment of TCR repertoire overlap.

    Fig. S12. Annotated screenshot of Shiny Web application showing interface for visualization and prediction.

    Table S1. Demographic, medical, and lifestyle variables included in the MI study.

    Table S2. Statistics of association with sjTRECs for suggestive loci and replication at the TCRA-TCRAD locus.

    Table S3. TCRA-TCRD next-generation sequencing data.

    Table S4. Primers and probes used for sex determination and TREC quantification.

    Table S5. Primary human immune system mice data (Excel file).

    References (5460)

  • The PDF file includes:

    • Composition of the MI Consortium
    • Materials and Methods
    • Fig. S1. Technical workflow of the study and validation of the TREC assay in the MI cohort.
    • Fig. S2. Association of sjTRECs with immune cell counts and parameters.
    • Fig. S3. Association of βTRECs with immune cell counts and parameters.
    • Fig. S4. Association of sjTRECs with nonheritable factors.
    • Fig. S5. Association of βTRECs with nonheritable factors.
    • Fig. S6. Association of intrathymic division number with nonheritable factors.
    • Fig. S7. Association of thymic function parameters with specific nonheritable factors.
    • Fig. S8. Age and sex impact on sjTRECs in the MARTHA cohort.
    • Fig. S9. Human immune system mice flow cytometry gating strategy.
    • Fig. S10. Effect of the SNP rs2204985 polymorphism on the TCRD locus deletion alternative rearrangement δRec-Jα58.
    • Fig. S11 Assessment of TCR repertoire overlap.
    • Fig. S12. Annotated screenshot of Shiny Web application showing interface for visualization and prediction.
    • Table S1. Demographic, medical, and lifestyle variables included in the MI study.
    • Table S2. Statistics of association with sjTRECs for suggestive loci and replication at the TCRA-TCRAD locus.
    • Table S3. TCRA-TCRD next-generation sequencing data.
    • Table S4. Primers and probes used for sex determination and TREC quantification.
    • References (5460)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5. Primary human immune system mice data (Excel file).

Navigate This Article