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. 1998 Sep 1;95(18):10797-802.
doi: 10.1073/pnas.95.18.10797.

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

A Geluk  1 , V Taneja, K E van Meijgaarden, E Zanelli, C Abou-Zeid, J E Thole, R R de Vries, C S David, T H Ottenhoff
Affiliations

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

A Geluk et al. Proc Natl Acad Sci U S A. .

Abstract

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4(+) T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20. DR3.Ab0 mice, immunized with bacillus Calmette-Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1-20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guérin but not to p1-20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

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Figures

Figure 1
Figure 1
(A) Proliferative T cell responses of LNCs from HLA-DR3.Ab0 mice immunized with BCG (10 μg), hsp65 (10 μg), p1–20 (100 μg), or PBS, in IFA. Proliferation is indicated in delta cpm (mean cpm in experimental wells − mean cpm in control wells without Ag). (B) Percentage inhibition of proliferative T cell responses by mAb specific for HLA-DR, mouse CD4, and mouse CD8, H2-Eα, and H2-Eβ LNCs of BCG-immunized mice were challenged in vitro with BCG, hsp65, or p1–20 in the presence of indicated mAb. (C) IFN-γ production by DR3-restricted LNCs from DR3.Ab0 mice immunized with BCG, hsp65, or p1–20. The in vitro challenge is indicated in the key.
Figure 2
Figure 2
(A) T cell reactivity of hsp65-immunized DR3.Ab0 to a complete set of hsp65-derived 20-mer peptides containing 10 amino acids overlap. (B) T cell reactivity of hsp65-immunized DQ8.Ab0 to a complete set of hsp65-derived, 20-mer peptides containing 10 amino acids overlap. Amino acid positions are indicated on the x-axis.
Figure 3
Figure 3
T cell recognition of specific peptide epitopes by DR3.Ab0 (A), DQ8.Ab0 (B), and DR3/DQ8.Ab0 (C) mice, after immunization with hsp65 (10 μg) in IFA. The in vitro challenge is given in the keys.
Figure 4
Figure 4
Reactivity of Ag85-immunized DR3.Ab0 mice to 85B-derived, 20-mer peptides containing 10 amino acids overlap (A). In vitro proliferation of LNCs from DR3.Ab0 mice immunized with BCG (10 μg), Ag85 (10 μg), or p51–70 (100 μg) in IFA (B).
Figure 5
Figure 5
Reactivity of M. tuberculosis-induced, 85B-reactive human T cell lines, derived from three DR3+ and one DR3 − (DR1/DR7) individuals. T cell proliferation induced by M. tuberculosis, Ag85, or peptide epitopes restricted by DR3 (p51–70) or DR1 (p71–90) are shown. The highly crossreactive M. leprae peptide was used, which differs only at two residues from the M. tuberculosis peptide but is crossrecognized. T cell reactivity is indicated as stimulation index (SI). Background values were <300 cpm.
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