WC1(+) gammadelta T cells indirectly regulate chemokine production during mycobacterium bovis infection in SCID-bo mice.

Previous studies have suggested an important role for WC1 (+)gammadelta T cells in the regulation of mycobacterial-induced inflammation in the spleen and liver of heterochimeric SCID-bovine (SCID-bo) mice. To examine the role of these cells, we investigated the levels of selected chemokines and IL12-p70 post-infection in reconstituted SCID-bo mice. Mice were treated with a monoclonal antibody specific for boWC1 to eliminate WC1-bearing cells. Isotype control treated or bovine gammadelta TCR-depleted mice were assayed in parallel. Following infection with Mycobacterium bovis, mice were examined post-infection for the expression of IL12-p70, IP-10, MIP-1alpha, lymphotactin and MIG by ELISA in plasma and from activated splenocytes. Treatment with the anti-bovine WC1 resulted in reduced serum plasma levels of IP-10, MCP-1, and IL-12p70 versus control mice. The potential of WC1 (+)gammadelta TCR-bearing cells to produce chemokines and cytokines was determined directly from peripheral blood of cattle. Our results indicate that these cells have a fairly restricted capability to produce the chemokines examined in SCID-bo mice, but may be a significant source of cytokines (IL-2, IL-10, IL-12, IL-15, and IFNgamma) and contribute to cytotoxicity through expression of FasL and perforin. In M.bovis-infected liver tissue, depletion of the WC1(+) subset was associated with increased numbers of CD3(+)T cells adjacent to venules and portal tracts. These results suggest that the WC1(+) subset in cattle may contribute to chemotaxis through indirect effects on chemokine levels. Further, activated WC1(+)gammadelta TCR(+) cells up-regulate cytokines with direct regulatory effects on T cell and macrophage function and express effector molecules with critical roles in cytotoxicity.

Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with Mycobacterium bovis BCG or M. bovisDeltaRD1.

The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.