Comprehensive analysis of mycobacterium tuberculosis antigen-specific CD4+ T cell responses restricted by single HLA class II allotype in an individual

Abstract

Mycobacterium tuberculosis infection is generally asymptomatic as latent tuberculosis, but it is still known as the world's leading bacterial cause of death. The diagnosis of latent tuberculosis infection relies on the evidence of cellular immunity to mycobacterial antigens. Since the association between HLA class II and tuberculosis infection has been reported in several population groups, a detailed study on the CD4⁺ T cell response to major tuberculosis antigens is needed. To elucidate which HLA class II allotypes in an individual are preferentially used in tuberculosis, CD4⁺ T cells specific to TB10.4, Ag85b, ESAT-6, and CFP-10 of Mycobacterium tuberculosis antigens were analyzed comprehensively. A total of 33 healthy donors were analyzed by ex vivo and cultured ELISPOT using panels of artificial antigen-presenting cells expressing a single HLA class II allotype. The CD4⁺ T cell responses were increased by an average of 39-fold in cultured ELISPOT compared with ex vivo ELISPOT. In ex vivo and cultured ELISPOT, CD4⁺ T cell responses showed significantly higher by HLA-DR than those of HLA-DQ and HLA-DP locus. In cultured ELISPOT, 9 HLA-DR allotypes, 4 HLA-DQ allotypes, and 3 HLA-DP allotypes showed positive CD4⁺ T cell responses. Among ten donors with positive CD4⁺ T cell responses when tested for mixed Mycobacterium tuberculosis antigens, seven donors were positive for only a single allotype, and three were positive for two allotypes in an individual. However, only one allotype was used for a single antigen-specific response when a single tuberculosis antigen was used individually. These results on the distribution of HLA class II allotypes showing high CD4⁺ T-cell responses to Mycobacterium tuberculosis antigens and the intra-individual allotype dominance will provide valuable information for understanding the immunobiology and immunogenetics of tuberculosis, which can contribute to the development of more effective vaccines.

Keywords: CD4+ T cell; HLA class II allotype; Mycobacterium tuberculosis; allotype dominance; cultured ELISPOT; ex vivo ELISPOT; intra-individual dominance.

Figure 1

Schematic representation of ex vivo and cultured ELISPOT. (A) In vitro culture of antigen-specific CD4⁺ T cell for cultured ELISPOT. As the M. tuberculosis antigens, a mixture of peptide pools of TB10.4, Ag85b, ESAT-6, and CFP-10 was used. (B) The proliferation folds of CD4⁺ T cells during 14 days of culture. Error bars indicate the mean ± standard deviation. (C) Representative ex vivo ELISPOT and cultured ELISPOT of HD04 using aAPCs expressing a single HLA class II allotype pulsed without antigens (-) or with M. tuberculosis antigens (+). *Notation of the HLA allele.

Figure 2

Frequencies of CD4⁺ T cells specific for M. tuberculosis antigens restricted by HLA class II loci and correlation between ex vivo and cultured ELISPOT. (A) Ex vivo ELISPOT, (B) Cultured ELISPOT. Statistical analysis was performed using Wilcoxon test. *p <0.05, **p < 0.01, ***p < 0.001. Error bars present mean ± standard error of the mean. The frequencies M. tuberculosis-specific CD4⁺ T cells were adjusted to the frequency per 1×10⁶ CD4⁺ cells in peripheral blood to compare ex vivo and cultured ELISPOT. Correlation between ex vivo and cultured ELISPOT of HLA class II (C) which is the sum of the responses by all HLA class II allotypes, and of each HLA-DR (D), -DQ (E) and -DP (F) locus.

Figure 3

The frequencies of antigen-specific CD4 ⁺ T cells by allotypes. The frequencies of M. tuberculosis antigen-specific CD4 ⁺ T cells by HLA-DR (A), -DQ (B), and -DP (C) allotypes measured by ex vivo and cultured ELISPOT. The parentheses indicate the number of donors with the allotype. Error bars present mean ± standard error of the mean.

Figure 4

The distribution of CD4⁺ T cell responses by an HLA class II allotype within individuals. Colored bars indicate allotypes with positive responses higher than 100 SFCs per 5×10⁴ cells. Each color represents the corresponding allotype.

Figure 5

CD4⁺ T cell responses to each M. tuberculosis antigens TB10.4, Ag85b, ESAT-6, and CFP-10 by an HLA class II allotype. (A) Representative cultured ELISPOT to each antigen in HD04. CD4⁺ T cell responses by two allotypes (B) or an allotype (C) in a donor that showed a positive response were measured individually to TB10.4, Ag85b, ESAT-6, CFP-10, and a mixture of antigens. (D) Distribution of CD4⁺ T cell responses to each M. tuberculosis antigens. Statistical analysis was performed using Welch’s t-test. *p = 0.0373. Error bars present mean ± standard deviation.