Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles

Abstract

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA-CD62L+/RA+CD62L- were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA-DR+ and CD8+CD45RA-CD62L+/RA+CD62L- T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.

Fig. 1

Analysis of TRECs (log₁₀) in CD4⁺ and CD8⁺ T cells in CVID (i) and in healthy individuals (ii). PBMC were isolated from heparinized venus blood of CVID patients and normal donors by Ficoll-Isopaque (Lymphoprep-Nycomed, Oslo, Norway) gradient centrifugation. CD4⁺ and CD8⁺ T cells were separated using magnetic beads coated with anti-CD4 and anti-CD8 MoAbs. Real time PCR analysis was performed on CD4⁺ and CD8⁺ T cells with TREC specific primers to detect recent thymic emigrants, and on the GAPDH to standardize for DNA content. TREC levels in CD4⁺ (a) and in CD8⁺ (b) T cells were significantly reduced in CVID patients when compared with age-matched healthy individuals.

Fig. 2

Distribution of TRECs in CD4⁺ and CD8⁺ T cells according to age in CVID patients (a) and in healthy subjects (b). CD4⁺ and CD8⁺ T cells were isolated in 17 CVID patients and 15 normal subjects ranging in age from 24 to 61 years, as described in Materials and methods. TREC levels were quantified using RT-PCR and values (copies/100 ngDNA) were transformed to log₁₀. The Pearson's correlation coefficient (r) and the regression line are indicated in the figures.

Fig. 3

Correlation between percentage of naive (CD45RA⁺CD62L⁺) CD4⁺ T cells and TREC levels in CD4⁺ T cells (log₁₀). The data indicate that there is a direct correlation between naive CD4⁺ phenotype and TREC levels in our cohort. Pearson's correlation coefficient (r) and P-value are indicated within the figure.

Fig. 4

Correlation between TREC levels (log₁₀) in activated (HLA-DR⁺) and memory (CD45RA⁺CD62L⁻ + CD45RA⁻CD62L⁺) CD8⁺ T cells (log₁₀). Activated CD8⁺ T cells (a) are correlated inversely with TRECs as well as memory (b) CD8⁺ T cells. Pearson's correlation coefficients (r) and P-values are indicated within the figure.