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. 2024 Oct 15;13(10):903.
doi: 10.3390/pathogens13100903.

Assays for Assessing Mycobacterium avium Immunity and Evaluating the Effects of Therapeutics

Getahun Abate  1 , Krystal A Meza  1 , Chase G Colbert  1 , Christopher S Eickhoff  1
Affiliations

Assays for Assessing Mycobacterium avium Immunity and Evaluating the Effects of Therapeutics

Getahun Abate et al. Pathogens. .

Abstract

In Europe and North America, the prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing. Most pulmonary NTM infections are caused by the Mycobacterium avium complex (MAC). Sadly, the treatment of pulmonary MAC is suboptimal with failure rates ranging from 37% to 58%. Therefore, there is a need to develop new therapeutics. Developing new immunotherapies and studying their interaction with standard or new drugs requires reliable assays. Four different assays including CFSE-based flow cytometry, in vitro protection assays, IFN-γ ELISPOT, and murine infection models were optimized using a reference strain of MAC (ATCC 700898) to help with the development of immunotherapies for MAC. Expansion of proliferating and IFN-γ producing human T cells is optimal after 7 days of stimulation with MAC at a multiplicity of infection (MOI) of 0.1, achieving a stimulation index of 26.5 ± 11.6 (mean ± SE). The in vitro protection assay for MAC works best by co-culturing T cells expanded for 7 days with MAC (MOI 1)-infected autologous macrophages. Aerosol MAC infection of mice allows measurement of the effects of the BCG vaccine and clarithromycin. IFN-γ ELISPOT assays with live MAC (MOI 3) stimulation of splenocytes from mice immunized with BCG help identify differences between unimmunized mice and mice immunized with BCG. In conclusion, multiple assays are available for use to identify MAC-specific effector T cells, which will help in the development of new therapeutics or vaccines against pulmonary MAC.

Keywords: MAC; NTM; T cells; immunotherapy; macrophages; murine.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Typical FACS plot showing flow cytometric gating for proliferating and IFN-γ-producing T cells. (A) Gating strategy. (B) Proliferating and IFN-γ-producing T cells in PBMCs stimulated with BCG or M. avium compared to PBMCs rested in the medium. Similar results were obtained by gating SSC/FSC first followed by live-dead/CD3, CD3/γδ, CD4 or CD8, and then IFN-γ/CFSE.
Figure 2
Figure 2
Stimulation of PPD-positive PBMCs with MAC leads to significant expansion of CD4 and CD8 T cells. PBMCs from PPD-positive volunteers (n = 5) were stimulated with BCG or MAC WL for 7 days. Medium-rested (MR) PBMCs were used as negative controls. There was a significant expansion of CD4 (A) and CD8 (B) T cells with both BCG and MAC WL (*, p < 0.05, Mann–Whitney U test).
Figure 3
Figure 3
BCG vaccination in humans induces MAC cross-reactive T cells. Paired pre- and post-vaccination PBMCs from recently BCG-vaccinated volunteers living in the USA (n = 5) were used. PBMCs were labeled with CFSE and stimulated with different concentrations of MAC WL. Medium-rested PBMCs were used as negative controls. On day 7, cells were restimulated with PMA/ionomycin for 2 h, viable cells were counted, and cells were stained for surface and intracellular markers for the flow cytometry study. (A) Stimulation index of proliferating (CFSElo) and IFN-γ-producing CD4+ T cells. (B) Stimulation index of proliferating (CFSElo) and IFN-γ-producing CD4+ T cells. * p < 0.05 (Wilcoxon matched pairs test).
Figure 4
Figure 4
BCG-specific T cells cross-protect against MAC. Human monocytes from different volunteers were infected at different multiplicities of infection (MOI) overnight with MAC (ATCC 700898). Following infection, extracellular mycobacteria were washed away, and after various further incubation periods, macrophages were lysed and released mycobacteria by the 3H-uridine incorporation assay. MAC replicates inside macrophages, making them amenable to T cell effector functions. The results from a rapid 3H-uridine incorporation assay for MAC (A) were confirmed by CFU-plating of cultures at selected time points. (B) BCG-expanded T cells inhibit intracellular MAC potently as they inhibit intracellular BCG. PBMCs from BCG-vaccinated or latently individuals with TB infection were stimulated with the optimal concentration of live BCG in vitro for 7 days and co-cultured with autologous macrophages infected with either BCG (n = 8) or MAC. Residual mycobacteria were quantified 3 days after co-culture, and % inhibition was calculated by dividing the number of residual mycobacteria in the presence of BCG-stimulated PBMCs by the number of residual mycobacteria in co-cultures containing medium-rested PBMCs. BCG-expanded T cells inhibited intracellular MAV better than intracellular BCG (p < 0.01, Mann–Whitney U test).
Figure 5
Figure 5
BCG vaccination renders MAC reactive immunity. Three groups of C57BL/6 mice were used. The first group (n = 3) was kept without vaccination. The second group was vaccinated with BCG (10 × 106), intranasal (IN), and the third group was vaccinated with BCG, two doses 4 weeks apart. Mice were sacrificed 4 weeks after the last vaccination. Splenic cells were harvested and rested in the medium or stimulated overnight with live BCG and MAC, at a multiplicity of infection (MOI) of 3 in IFN-γ ELISPOT assays. Shown are mean ± SE from representative experiments expressed as IFN-γ spot-forming cells (SFCs) per million splenic cells. The number of IFN-γ SFCs following stimulation with BCG, MAC, and MAC-WL was significantly higher in BCG-vaccinated mice compared with unvaccinated mice (p < 0.05, Mann–Whitney U test). A second BCG vaccination did not significantly increase the number of mycobacteria-induced IFN-γ SFCs (p > 0.05).
Figure 6
Figure 6
Effects of the anti-MAC drug on MAC growth in the lungs. Mice were infected with MAC, and two weeks after infection, clarithromycin at a concentration ranging from 0.125 mg to 2 mg per 20 g was started. Clarithromycin was administered 5 days a week via gavage between weeks 2 and 6 post-infection. All mice were euthanized six weeks after infection, their lungs were homogenized, and CFUs were quantified by culturing on 7H10 media. All doses of clarithromycin used decreased lung CFUs significantly (p < 0.05).
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