Mapping of TH1 helper T-cell epitopes on major secreted mycobacterial antigen 85A in mice infected with live Mycobacterium bovis BCG.

TH1 cytokine secretion was examined in response to synthetic peptides of the 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis in seven different mouse strains infected with live M. bovis BCG. Twenty-eight overlapping 20-mer peptides covering the complete mature 295-amino-acid (AA) protein were synthesized. Significant interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion could be measured following in vitro stimulation of spleen cells with these peptides. H-2d haplotype mice reacted preferentially against the amino-terminal half of the protein, i.e., against peptide 5 (AA 41 to 60) and especially against peptide 11 (AA 101 to 120), which contained an I-Ed binding motif. H-2b haplotype mice, on the other hand, reacted against peptides from both amino- and carboxy-terminal halves of the protein, peptide 25 (AA 241 to 260) being the most potent stimulator of IL-2 and IFN-gamma production. (BALB/c x C57BL/6)F1 animals with the H-2d/b haplotype weakly recognized peptides specific for both parental lines. Finally, CBA/J (H-2k) and major histocompatibility complex class II mutant B6.C.bm12 mice, carrying a mutant I-A beta bm12 allele on an H-2b background, reacted only very weakly to the 85A peptides. Reactive T cells isolated from lungs of BCG-infected H-2b haplotype mice recognized the same epitopes as spleen cells, especially peptide 25. These data confirm previous findings regarding the powerful IL-2 and IFN-gamma-inducing properties of antigen 85 during infection with live M. bovis BCG.

The utility of the macrophage migration inhibition test for in vitro titration of tuberculins.

The purpose of this study was to explore the fundamental principle of the potency estimation of tuberculins, as applied in vitro by the macrophage migration inhibition test (MIT) under agarose. The MIT was performed using specifically sensitized mouse and guinea-pig peritoneal macrophages and serial dilutions of the analogous PPD (purified protein derivatives of tuberculin) tuberculins as antigen. Statistical studies performed included (a) the standard deviation of the mean migration areas, (b) the analysis of variance, (c) the regression analysis, (d) its corresponding linearity test and (e) the determination of the related correlation coefficient. It was shown for the first time and in both animal species under study that there is correspondence between the log dose-response relationship of the tuberculin PPD in MIT under agarose and the well known in tuberculin cutaneous reaction. The MIT may therefore successfully replace the in vivo titration of tuberculins.