Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 5, 2012
Publication Date: December 2, 2012
Citation: Maggioli, M., Palmer, M.V., Vordermeier, H.M., Estes, D.M., Waters, W.R. 2012. Effector and memory T cell subsets in the response to bovine tuberculosis [abstract]. Conference of Research Workers in Animal Diseases. pg. 79. Technical Abstract: Long-term (i.e., 14d) cultured IFN-gamma ELISPOT assays of PBMC are used as a correlate of T cell central memory (Tcm) responses in cattle and humans. With bovine tuberculosis, vaccine-elicited Tcm responses correlate with protection against experimental Mycobacterium bovis infection. The objective of the present study was to characterize the relative contribution of Tcm, T effector memory (Tem), and T effector cells within short- (ex vivo) and long-term (cultured) antigen-specific assays by cattle (Holstein, 7 months of age, n = 8) in response to experimental M. bovis infection (10**4 cfu by aerosol). Peripheral blood mononuclear cells were stimulated with a cocktail of M. bovis purified protein derivative (PPDb, 5 µg/ml), rESAT-6:CFP-10 (1 µg/ml) and over-lapping peptide cocktails of Tb10.4 and Ag85A (1 µg/ml) for 13d with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6-CFP-10 (1 µg/ml), or PPDb (10 µg/ml) with addition of fresh autologous adherant cells for antigen presentation. After overnight stimulation, cells were analyzed for CD4, CD45RO, CD62L, CD44, and CCR7 (rat anti-human CD197) expression via flow cytometry. In response to rESAT-6:CFP-10 or PPDb, ~75% of CD4+, IFN-gamma+ cells expressed a Tcm phenotype (CCR7+, CD62Lhi, CD45RO+) and ~24% expressed a Tem phenotype (CCR7-, CD62Lmod, CD45RO+). The PBMC ex vivo response (i.e., short term, 20 hr stimulation) to rESAT-6:CFP-10 or PPDb consisted of ~56-59% T effector cells (CCR7-, CD62Llo, CD45RO-) and ~37-42% Tem cells. Only 3-4 % of the ex vivo response consisted of Tcm cells. Expression (mfi) of CD62L differed (p < 0.05) among Tcm (E:C / PPDb = 189 ± 47 / 141 ± 25), Tem (E:C / PPDb = 68 ± 14 / 53 ± 7) and T effector (E:C / PPDb = 13 ± 2 / 16 ± 4) cells. CD44 expression (mfi) on Tcm (E:C / PPDb: 71 ± 8 / 60 ± 9) exceeded (p < 0.02) that of Tem (E:C/PPDb = 25 ± 3 / 28 ± 4) and T effector (E:C / PPDb = 27 ± 3 / 20 ± 4) cells. These findings confirm that the 14d cultured ELISPOT assay to M. bovis specific antigens is primarily a measure of Tcm responses by cattle in response to M. bovis infection; whereas, ex vivo responses are primarily a measure of T effector responses.