Fig 1.
Chart of the extended H2 genomic region in novel recombinant congenic strains.
Chromosome segments transferred from I/St (Н2j) mice onto the B6 (Н2b) genetic background are shown in grey. Markers (all—from MGI-Mouse Genome Database (http://www.jax.org), are shown in the left column, followed by their genomic positions in mega base pairs (Mb). Gene symbols and locations (according to the Ensembl Genome assembly GRCm38.p2 (http://www.ensembl.org) are shown in the right column. All strains bearing the Н2j-originated genetic material highlighted by the red border displayed a TB-susceptible phenotype (S, bottom), other strains were TB-resistant (R, bottom).
Fig 2.
Survival curves of mice infected with M. tuberculosis.
Survival of parental B6 and I/St and recombinant congenic mice (males) following aerosol challenge with ~500 CFU of M. tuberculosis H37Rv. Recombinant strains B6.I-9.3.19.8, B6.I-9.5.7, B6.I-9.5, B6.I-9.3, B6.I-249.1.15.46 and B6.I-249.1.15 all displayed similar (P > 0.1) intermediate mean survival time (MST) compared to hyper-susceptible I/St and relatively resistant B6 mice (P < 0.0001, log-rank test), which reflects the input of the intra-H2 QTL in susceptibility. All recombinant strains were tested in 3–10 independent experiments (total N = 20–70 animals). Summary of 3–5 experiments is displayed (Kaplan-Meier survival analysis).
Table 1.
Allelic polymorphisms in the coding parts of the protein-encoding genes*.
Fig 3.
Genotypes and TB phenotypes of new recombinant mouse strains B6.I-100 and B6.I-139.
A–The genome chart for B6.I-100 and B6.I-139 recombinant strains. Chromosome segments transferred from I/St onto B6 genetic background are shown in grey. B–survival curves of B6.I-100, B6.I-139 and parental strains mice after aerosol challenge with ~500 CFU of M. tuberculosis. MST ± SEM (days): B6 = 249 ± 10; I/St = 63 ± 11; B6.I-100 = 152 ± 13; B6.I-139 = 233 ± 14; B6.I-249.1.15.46 = 153 ± 11. Recombinant strains were tested in 3–5 independent experiments (total N = 20–40 males). Summary of 3 experiments is displayed (Kaplan-Meier survival analysis). C–CFU counts in infected lungs at days 28 and 70 post-challenge (4 mice per group, **P < 0.05, ANOVA). The representative results of one out of 2 independent experiments are present. D–genes and their positions in the D17Mit21 – H2-Ea interval according to Ensembl. Red borders–location of the candidate gene.
Fig 4.
The differences in H2-Ab1 AA sequences between B6.I-100 and B6.I-139 mice.
Protein structure alignment of H2-Ab1molecules. Gene annotations are from UniProt Domain structure http://www.uniprot.org: 1–27 –signal peptide; 28–122 - β1 polymorphic domain; 123–216 - β2 conservative domain; 217–226 –connecting peptide (CP); 227–247 –transmembrane domain (TM); 248–265 –cytoplasmic domain (CD). H2b –H2j AA substitutions are highlighted.
Fig 5.
Possession of the H2-Ab1 j-like alleles results in a more severe TB infectious course.
A–Representative tuberculous lung lesions at day 35 post-infection. Hematoxylin and eosin staining, magnification x100. Arrows show granulomatous structures. B–TNF-α and IL-6 production in infected lungs at day 60 post-challenge. Whole-lung homogenates from individual mice (3 per group) were assessed in the ELISA format. The results are expressed as mean ± SD from two independent experiments (total N = 6), P < 0.05 for B6.I-100 and B6.I-139, ANOVA. C–The number of lung IFN-γ-producing CD4+ T cells was assessed by intracellular staining for IFN-γ at 35 days post-challenge. After culturing with mycobacterial sonicate, the lung cell population gated for CD3 expression was analyzed as displayed. Results of one of two similar experiments (total N = 6) are shown, with statistics for 3 individual mice per group provided in quadrants. In controls (cells from normal mice with mycobacterial sonicate, or cells from infected mice without antigen in culture) the per cent of IFN-γ-producing lung CD4+ T cells never exceed 0.1. P < 0.05 for B6.I-100 and B6.I-139, ANOVA.
Fig 6.
CD4+ T cells recognize mycobacterial antigens in the context of H2-A molecule.
Mycobacterial antigens were presented by APC expressing different gene combinations and alleles of Class II molecules (see legend) to: (A) polyclonal I/St T cell line, or to highly purified immune lymph node CD4+ T cells (pooled from 2–3 mice) from I/St (B), B6.I-100 (C) and B6.I.139 mice (D). Representative data from one out of two similar independent experiments are presented, results are expressed as mean ± SEM of triplicate cultures. Y-axis: Δ cpm (counts per minute) = mean cpm of antigen-stimulated wells—the mean cpm of non-stimulated wells. Stimulation index (SI) mean cpm of antigen-stimulated wells/ mean cpm of non-stimulated wells. (E)–CD4+ T-cells from (B6 x B6.I-9.3.19.8) F1 mice stimulate bacteriostatic activity of B6 and F1, but not of B6.I-9.3.19.8 macrophages (mph). Results are present as [3H]-uracil uptake from one out of two experiments provided similar results (CPM ± SEM for triplicates, P < 0.001, ANOVA). See Materials and Methods for details.
Fig 7.
Molecular model of H2-Aj molecule in comparison to H2-Ab.
Top view of structural overlay of the peptide-binding domains of H2-Ab (blue) and H2-Aj (red) alleles, bound to CLIP peptide (green). α- alpha and β–beta chains. 1 –α- subunit 310 helix, 2- β subunit region with two AA (P65E66) deletions in j-haplotype (A). Comparison of the H-bond network between H2-Ab (B) and H2-Aj (C) molecules containing CLIP peptide backbone (P-1-P10). MHC Class II conserved residues that contribute to the peptide- MHC hydrogen-bonding network are shown in stick representation. Dashed lines indicate conservative hydrogen bonds with the exception of Ab74 and Kb71 (marked red) in the H2-Aj molecule. D -Comparison of pocket structures of the MHC- binding groove between H2-Ab (blue) and H2-Aj (red). The CLIP peptide backbone is shown in green, P1, P4, P6, P7 and P9 pockets in grey. AA substitutions in the α-chain contribute mostly to the differences in the P1 structure, AA substitutions in the β-chain determine differences in P4, P6, P7 and P9 pockets (E). Potentially most important substitutions are marked and their side chains shown.