Inhibitory mechanisms of cytotoxic T cell response by multidrug resistant Mycobacterium tuberculosis (Mtb) strain M

GEFFNER LAURA; JUAN BASILE; SABIO Y GARCÍA, CARMEN;; YOKOBORI NOEMÍ; BARRERA LUCÍA; RITACCO VIVIANA; SASIAIN MARÍA DEL CARMEN; DE LA BARRERA SILVIA

Vancouver

Simposio; Keystone Symposia 2011 - Tuberculosis: Immunology, Cell Biology and Novel Vaccination Strategies; 2011

Keystone Symposia Organization

In Argentina multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 90’s and disseminated to the community. Several MDR strains responsible for clustered MDR-TB cases have been isolated, including Muñiz strain belonging to Haarlem family (M) which has high infectivity/virulence. It is known that different Mycobacterium tuberculosis (Mtb) strains operate different immune evasion strategies for their survival in the host, which mainly depends on the virulence of the strain and the host immune responses. Previously, we have demonstrated that the MDR outbreak strain M induces weak CTL response, when compared with its related strain 410 (non prosperous), in healthy controls and TB patients. Thus, the aim of this work was to evaluate the mechanisms employed by M to hamper CTL response. Methods: Buffy coats’ PBMCs were cultured (5d) with M or 410 strains, and stained with PE-Cy5 anti-CD8. Mtb pulsed autologous macrophages (Mac) were stained with CFSE and mixed with CTLs (4h). Mac-CD8 conjugate formation was evaluated by flow cytometry. Intracellular expression of perforin, granzymeB, and granulysin, and surface expression of ICAM-1 and LFA-1 were evaluated in CD8+T cells. Results: M induced a lower %Perforin+CD8+T cells than 410, but similar %GranzymeB+ or Granulysin+CD8+T cells. Perforin MFI in CTL was similar between both strains, while GranzymeB and Granulysin were higher in M induced CTLs. Mac-CD8 conjugates were less frequently observed upon M than 410 stimulation. M and 410 induced similar LFA-1 MFI in CD8+T cells, while M induced less %ICAM-1+CD8+T cells than 410. M induced a lower CD69 expression than 410 in 18 h cultures, while CD25 expression in 5d cultures was not significantly different between those strains. Both strains induced a similar CD69 peak of expression (66h), thus M strain was not delaying T cell activation. PBMC preincubated with Mtb strains were activated with anti-CD3 with or not anti-CD28 and no inhibition was detected in terms of CD69 expression suggesting that low CD69 expression was not due to an inhibition of TCR activation by strain M. However, M-induced a reduced IL-2 expression in CD8+CD3+T, cytokine that is regulated upon CD69 signaling and controls upregulation of lytic molecules. Besides, blockade of CD69 with specific monoclonal antibody along 5d cultures inhibited CD107 expression in 410-stimulated CD8+ T cells resulting in a CD107 expression similar to that observed in M-stimulated cells.