Multiple Mycobacterium antigens induce interferon-gamma production from sarcoidosis peripheral blood mononuclear cells

Abstract

Studies of sarcoidosis immunology have noted oligoclonal T cell populations, suggesting cell-mediated immunity that is antigen-specific. Sarcoidosis immunology and pathology are most similar to mycobacterial infections. Mycobacterium tuberculosis infection in mice and humans reflects T helper 1 (Th1) immune responses to multiple cell wall and secreted antigens. We investigated if the oligoclonal immune response in individual sarcoidosis subjects could be elicited by multiple secreted mycobacterial antigens by performing ex vivo enzyme-linked immunospot assay (ELISPOT) on peripheral blood mononuclear cells (PBMC) from 30 sarcoidosis, 26 purified protein derivative negative (PPD-) control and 10 latent tuberculosis subjects (PPD+) to assess Th1 responses to mycobacterial superoxide dismutase A (sodA), catalase-peroxidase (katG) and early secreted antigenic target protein (ESAT-6). A significant difference was noted among the sarcoidosis and PPD- control subjects to ESAT-6 [12 of 30 versus one of 26 (P = 0.0014)], katG [nine of 30 versus none of 26 (P = 0.002)] and sodA [12 of 30 versus none of 26 (P = 0.002)]. There was no significant difference between sarcoidosis and PPD+ subjects. Twelve sarcoidosis subjects recognized two or more mycobacterial proteins, as well as multiple distinct epitopes within individual proteins. One sarcoidosis subject on whom we collected bronchoalveolar lavage (BAL) fluid and PBMC had no recognition of mycobacterial antigens using PBMC, but BAL fluid demonstrated strong Th1 immune responses to ESAT-6 and katG. Individual sarcoidosis subjects recognized not only multiple mycobacterial proteins, but multiple distinct peptides within a specific protein, thus demonstrating that multiple mycobacterial epitopes elicit the Th1 immune response observed. Immune responses by sarcoidosis T cells to mycobacterial proteins may have an important role in sarcoidosis pathogenesis.

Fig. 2

Analysis of the distribution of spot-forming cells/million peripheral blood mononuclear cells (PBMC) generated by stimulation with mycobacterial and Trypanosome brucei antigens. The bars represent the 25th, 50th and 75th percentile of each group of study participants. For the sarcoidosis subjects, the 25th and 50th percentile were the same. There was no statistically significant difference in the distribution of the T cell responses to any of the three mycobacterial antigens between the sarcoidosis and purified protein derivative positive (PPD⁺) controls, although there was between sarcoidosis and PPD⁻ controls. The PPD⁻ control that did recognize early secreted antigenic target protein (ESAT-6) antigens had a response quantitatively consistent with the sarcoidosis and PPD⁺ controls. Contrary to recognition of mycobacterial antigens, there was no distinction with regard to recognition of T. brucei whole cell wall lysate among sarcoidosis and PPD⁻ controls. There were too few PPD⁺ controls tested for recognition of T. brucei to compare to sarcoidosis.

Fig. 1

Sarcoidosis subjects recognize multiple mycobacterial antigens. Using enzyme-linked immunospot assay (ELISPOT) analysis, each of the study participants were tested for interferon-γ production after stimulation by catalase-peroxidase (katG), early secreted antigenic target protein (ESAT-6) and superoxide dismutase A (sodA) antigens. Sixteen of 30 sarcoidosis subjects had systemic responses to mycobacterial antigens which paralleled more closely the responses observed in purified protein derivative positive (PPD⁺) subjects, but was statistically significant from the response observed in PPD⁻ controls.

Fig. 3

Sarcoidosis subjects recognize numerous mycobacterial epitopes. Enzyme-linked immunospot assay (ELISPOT) analysis for 17 early secreted antigenic target protein (ESAT-6), two catalase-peroxidase (katG) and four superoxide dismutase A (sodA) peptides was performed on all 66 study participants. Among the sarcoidosis subjects who recognized mycobacterial proteins, not only was there recognition of multiple mycobacterial proteins, but they frequently recognized more than one epitope within a given protein. Among some of the peptides recognized there were amino acid overlap, but recognition of peptides which were distinct from each other was also observed with similar T cell frequencies.

Fig. 4

Flow cytometry of sarcoidosis bronchoalveolar fluid reveals T helper 1 (Th1) immune responses to early secreted antigenic target protein (ESAT-6) and catalase-peroxidase (katG) peptides. Intracellular cytokines staining for interleukin (IL)-2 and interferon (IFN)-γ was performed on bronchoalveolar lavage (BAL) fluid obtained from sarcoidosis 30, after stimulation with mycobacterial peptides from ESAT-6 and katG. Consistent with prior reports of sarcoidosis immunology, CD4⁺ T cells are increased in the sarcoidosis immune response. Both mycobacterial peptides were recognized by sarcoidosis CD4⁺ T cells, producing IL-2 and IFN-γ. In this subject (sarcoidosis 30), immune recognition was detected in the BAL fluid, a site of active sarcoidosis involvement but none in peripheral blood mononuclear cells.