Journal: Infection and Immunity

Article Title: Interleukin-21 Induces Short-Lived Effector CD8 + T Cells but Does Not Inhibit Their Exhaustion after Mycobacterium bovis BCG Infection in Mice

doi: 10.1128/IAI.00147-18

Figure Lengend Snippet: Kinetics of numbers of Ag-specific effector CD8+ T cells in IL-21R−/− mice following BCG infection. (A and B) The absolute number of OVA-specific CD44+ CD62L− CD8+ T cells, CD44+ CD62L+ CD8+ T cells, short-lived effector cells (SLECs), or early effector cells (EECs) was calculated by multiplying the total number of lung MNCs or spleen cells by the percentage of each subset. On the indicated days after infection with 2 × 106 CFU of rBCG-OVA, the lung MNCs and spleen cells were stained either with anti-CD8, anti-CD44, and anti-CD62L MAbs and OVA257–264 H-2Kb tetramers or with anti-CD8, anti-KLRG1, and anti-CD127 MAbs and OVA257–264 H-2Kb tetramers. (C) The mean fluorescence intensities (MFI) of T-bet, Blimp-1, Bcl-6, and Eomes in OVA257–264-specific CD8+ T cells in the lung were calculated on days 14 and 21 after rBCG-OVA infection. For intracellular expression of T-bet, Blimp-1, Bcl-6, and Eomes by OVA257–264-specific CD8+ T cells, lung MNCs were surface stained with an anti-CD8 MAb and OVA257–264 H-2Kb tetramers and were intracellularly stained with each MAb. (D) The absolute number of OVA257–264-specific exhausted CD8+ T cells was calculated by multiplying the total number of lung MNCs by the percentage of the PD-1high KLRG1low subset. On the indicated days after infection with 2 × 106 CFU of rBCG-OVA, lung MNCs were stained with anti-CD8, anti-KLRG1, and anti-PD-1 MAbs and OVA257–264 H-2Kb tetramers. Data from one experiment representative of three separate experiments are shown. Each value shown is the mean ± standard deviation for four mice of each group. Statistically significant differences between IL-21R−/− mice and WT mice are indicated (*, P < 0.05; **, P < 0.01). N.S., not significant.

Article Snippet: I-A/I-E-V500 (M5.144.15.2), allophycocyanin- and Cy.7-conjugated CD8α (RM53-6.7), CD44-V450 (IM7), phycoerythrin (PE)- and Cy.7-conjugated CD127 (A7R34), Alexa Fluor 488-conjugated Bcl-6 (K112-91), Alexa Fluor 647-conjugated Blimp-1 (5E7), and PE- and Cy.7-conjugated streptavidin antibodies were purchased from BD Biosciences (San Jose, CA).

Techniques: Infection, Staining, Fluorescence, Expressing, Standard Deviation

Journal: Infection and Immunity

Article Title: Interleukin-21 Induces Short-Lived Effector CD8 + T Cells but Does Not Inhibit Their Exhaustion after Mycobacterium bovis BCG Infection in Mice

doi: 10.1128/IAI.00147-18

Figure Lengend Snippet: IL-21R signaling for differentiation of Ag-specific CD8+ T cells in adoptive transfer experiments following BCG infection. (A) Naïve CD8+ T cells purified from WT (Ly5.1/5.1) and IL-21R−/− (Ly5.1/5.2) OT-I mice were transferred i.v. into WT mice (Ly5.2/5.2), which were inoculated i.t. with 2 × 106 CFU rBCG-OVA on the next day. Two weeks after infection, lungs were harvested. (B) Percentages of OT-I cells in the lung after rBCG-OVA infection. (C) The absolute number of OT-I cells expressing Ly5.1/5.1 or Ly5.1/5.2 was calculated by multiplying the total number of lung MNCs or mediastinal lymph node (MLN) cells by the percentage of each subset. On day 14 after infection, the lung MNCs and MLNs were stained with anti-CD8, anti-Ly5.1, and anti-Ly5.2 MAbs and OVA257–264 H-2Kb tetramers. (D) Numbers in the dot plot show the percentage of each region in a gated population. A representative fluorescence-activated cell sorter profile is shown. On day 14 after infection, lung MNCs were stained with anti-CD8, anti-Ly5.1, anti-Ly5.2, anti-CD127, and anti-KLRG1 MAbs and OVA257–264 H-2Kb tetramers. (E) The absolute number of SLECs or EECs was calculated by multiplying the total number of lung MNCs by the percentage of each subset. (F) Histogram overlays of CFSE on CD8+ T cells. Shown are levels for naïve CD8+ T cells before transfer (filled histogram), WT OT-I CD8+ T cells after infection (shaded histogram), and IL-21R−/− OT-I CD8+ T cells after infection (open histogram). The MFI of CFSE in WT or IL-21R−/− OT-I CD8+ T cells was calculated on day 14 after rBCG-OVA infection. To examine the division of OT-I cells in vivo, the cells were first labeled with 1 μM CFSE and then injected intravenously into sex-matched WT mice, which were subsequently injected with 2 × 106 CFU rBCG-OVA. On day 14 after infection, lung MNCs were stained with anti-CD8, anti-Ly5.1, and anti-Ly5.2 MAbs. Data from one experiment representative of two separate experiments are shown. Each value shown is the mean ± standard deviation for four mice. Statistically significant differences between IL-21R−/− OT-I CD8+ T cells and WT OT-I CD8+ T cells are indicated (*, P < 0.05; **, P < 0.01).

Article Snippet: I-A/I-E-V500 (M5.144.15.2), allophycocyanin- and Cy.7-conjugated CD8α (RM53-6.7), CD44-V450 (IM7), phycoerythrin (PE)- and Cy.7-conjugated CD127 (A7R34), Alexa Fluor 488-conjugated Bcl-6 (K112-91), Alexa Fluor 647-conjugated Blimp-1 (5E7), and PE- and Cy.7-conjugated streptavidin antibodies were purchased from BD Biosciences (San Jose, CA).

Techniques: Adoptive Transfer Assay, Infection, Purification, Expressing, Staining, Fluorescence, In Vivo, Labeling, Injection, Standard Deviation

Journal: Infection and Immunity

Article Title: Interleukin-21 Induces Short-Lived Effector CD8 + T Cells but Does Not Inhibit Their Exhaustion after Mycobacterium bovis BCG Infection in Mice

doi: 10.1128/IAI.00147-18

Figure Lengend Snippet: Kinetics of Ag-specific CD8+ T cells following rBCG–Ag85B–IL-21 inoculation. The lung MNCs were harvested on the indicated days, at an early stage after rBCG–Ag85B–IL-21 or rBCG–Ag85B inoculation. (A) The numbers of bacteria recovered from the lungs of infected mice on the indicated days were determined. (B) The absolute numbers of TB10.4- or Mtb32a-specific CD8+ T cells were determined by staining with an anti-CD8 MAb and TB10.4 or an Mtb32a MHC class I tetramer. (C to E) The absolute numbers of TB10.4- or Mtb32a-specific IFN-γ+ CD44+ CD8+ T cells were calculated by multiplying the total number of lung MNCs by the percentage of IFN-γ+ CD44+ CD8+ T cells stimulated with the TB10.4 or Mtb32a peptide. The lung MNCs were stained with an anti-CD8, anti-KLRG1, anti-CD127, or anti-PD-1 MAb and TB10.4 or Mtb32a MHC class I tetramers. Data from one experiment representative of three separate experiments are shown. Each value shown is the mean ± standard deviation for four mice of each group. Asterisks indicate statistically significant differences between rBCG–Ag85B–IL-21-infected mice and rBCG–Ag85B-infected mice (*, P < 0.05; **, P < 0.01). N.S., not significant.

Article Snippet: I-A/I-E-V500 (M5.144.15.2), allophycocyanin- and Cy.7-conjugated CD8α (RM53-6.7), CD44-V450 (IM7), phycoerythrin (PE)- and Cy.7-conjugated CD127 (A7R34), Alexa Fluor 488-conjugated Bcl-6 (K112-91), Alexa Fluor 647-conjugated Blimp-1 (5E7), and PE- and Cy.7-conjugated streptavidin antibodies were purchased from BD Biosciences (San Jose, CA).

Techniques: Infection, Staining, Standard Deviation