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. 2019 Sep 6;10:2075. doi: 10.3389/fimmu.2019.02075

Figure 6.

Figure 6

Regulation of Ag-specific CD8 TRM responses by CD4 T cells via their effects on proliferation/survival, but not recruitment, of CD8 T cells in the lung. Mice were immunized i.m. with AdCh68Ag85A. At day 28 post-immunization, mice were administered i.n. with Ag85 complex (two doses 5 days apart) (Ag85 group). In a separate group of mice, CD4 T cells were depleted by i.p. injection of αCD4 mAb 1 day before and after RM-pull (Ag85/αCD4mAb group). BrdU was administered i.n. once daily for four consecutive days after the last dose of Ag85 complex proteins. Mice were sacrificed 5 days after the final administration of Ag85 complex, and mononuclear cells from BAL, lungs, MLN, and spleen were subjected to ex vivo stimulation with Ag85A CD8 peptide, and immunostaining for tetramer, BrdU, and Annexin V or intracellular cytokines. (A) Bar graph showing numbers of tetramer+ CD8 T cells in BAL, LPT, LV, MLN, and spleen. (B) Representative dotplots depicting frequencies of IFN-γ+ CD8 T cells. (C,D) Representative dotplots and bar graphs showing frequencies of BrdU+ (C) and Annexin V+Aqua+ (D) tetramer+ CD8 T cells out of total tetramer+ cells in BAL and LPT. (E) Experimental schema. Thy1.2 and Thy1.1 congenic mice were immunized i.m. with AdCh68Ag85A followed by RM-pull with Ag85 complex. CD8 T cells purified from Thy1.2 donor lungs were transferred i.v. into Th1.1 recipient mice post-Ag85 delivery and CD4 T cell depletion. BrdU was administered i.n. once after T cell transfer. Lung mononuclear cells were stained for tetramer, BrdU, and Annexin V 24 h post-T cell transfer. (F) Representative dotplots showing frequencies of Thy1.2 (donor) and Thy1.1 (recipient) and tetramer+ CD8 T cells out of total cells in LPT. Bar graph comparing numbers of Thy1.2 (donor) tetramer+ CD8 T cells in LPT between CD4 T cell-helped (Ag85) and -unhelped (Ag85/αCD4mAb) groups. (G) Bar graph comparing frequencies of BrdU+ or Annexin V+Aqua+ Thy1.2 (donor) tetramer+ CD8 T cells in LPT of CD4 T cell-helped (Ag85) and -unhelped (Ag85/αCD4mAb) groups. (H) Bar graph comparing numbers of Thy1.2 (donor) tetramer+ CD8 T cells in the spleen of CD4 T cell-helped (Ag85) and -unhelped (Ag85/αCD4mAb) groups. Data are presented as the mean ± S.E.M. of three mice/group, representative of two independent experiments. *P < 0.05, **P < 0.01 compared with Ag85 group.

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