Journal: Infection and Immunity

Article Title: Identification and Evaluation of Novel Protective Antigens for the Development of a Candidate Tuberculosis Subunit Vaccine

doi: 10.1128/IAI.00014-18

Figure Lengend Snippet: Characterization of the phenotype of antigen-specific T cells in the lung parenchyma and vasculature of ChAdOx1.PPE15-vaccinated mice. (a) BALB/c and C57BL/6 mice were primed with BCG and boosted 10 weeks later with either i.n. or i.d. ChAdOx1.PPE15. At 4 weeks after the last vaccination, the animals were challenged with 50 to 100 CFU of aerosolized M. tuberculosis Erdman. Each dot represents one animal, and the lines represent the median value for each group ( n = 8 mice). The Kruskal-Wallis test followed by Dunn's multiple-comparison test was used to test for significant differences. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Tetramer staining was combined with intravascular staining to discriminate the lung parenchyma from lung vasculature cells. (b) Representative plots of I-A(b) PPE15 1–15 tetramer and intravascular staining of CD4 + T cells. (c) Number of tetramer-positive (tet + ) and intravascular staining-negative CD45 − CD4 + (left) and intravascular staining-positive CD45 + CD4 + (right) T cells. (d) Number of tetramer-positive and intravascular staining-negative CD4 + T cells that are CXCR3 + and KLRG1 − (left) and tetramer-positive and intravascular staining-positive CD4 + T cells that are CX3CR1 + KLRG1 + (right). (e to g) The same as for panels b to d, respectively, but for CD8 + T cells using the H-2D b PPE15 192–200 tetramer. (h and i) Number of tetramer-positive and intravascular staining-negative CD4 + (h) or CD8 + (i) T cells that were CXCR3 + PD1 + . *, P < 0.05. BC15i.n. and BC15i.d., BCG-ChAdOx1.PPE15 administered by the i.n. and i.d. routes, respectively.

Article Snippet: Cells were stained with the I-A(b) tetramer at 4°C for 30 min, after which the cells were washed twice with PBS and then surface stained at 4°C with KLRG1, CXCR3, CX3CR1 (all from BioLegend, UK), and PD1 (eBioscience).

Techniques: Comparison, Staining