A retrogenic mouse model for TB10-specific CD8+ T cells.

<p>(<b>a</b>) Frequency and amino acid sequences of CDR3α (top panel) and CDR3β (bottom panel) regions of TB10.4_4-11-specific CD8⁺ T cells from the lung of individual Vα2var mice infected with <i>M</i>. <i>tuberculosis</i>. Data were obtained by single-cell sorting and sequencing of 60–200 cells per mouse (n = 3). (<b>b</b>) Sequences of CDR3α (top panel) and CDR3β (bottom panel) amino acid sequences of the dominant clones of TB10.4_4-11-specific CD8⁺ T cells expanded in the lung of Vα2var mice infected with <i>M</i>. <i>tuberculosis</i>. Dashes are gaps introduced for purpose of sequence alignment. (<b>c</b>) VDJ rearrangement (CDR3β) of the dominant TB10.4_4-11-specific CD8⁺ clonotypes from the lung of Vα2var mice infected with <i>M</i>. <i>tuberculosis</i>, displaying Vβ (black), N (red), Dβ (blue), and Jβ (black) sequences. Boxed codon is the conserved arginine (Arg, “R”). (<b>d,e</b>) Flow-cytometry analysis of T cells from uninfected retrogenic mice expressing TCR3. (<b>d</b>) Represented are a dot plot of CD4 vs CD8 staining of CD3⁺ T cells (left panel), histograms of GFP expression within CD8⁺ T cells (middle panel), and a dot plot of Vα2 vs. Vβ11 staining in CD8⁺ T cells (right panel). Red, CD4⁺ T cells; blue, are colored in CD8⁺GFP^- T cells; green, CD8⁺GFP⁺ T cells. The numbers represent: the frequency of CD4⁺ vs. CD8⁺ T cells among gated CD3⁺ T cells (left); the frequency of CD3⁺CD8⁺ cells that express GFP (middle); and the frequency of CD3⁺CD8⁺GFP⁺ cells that are Vα2⁺Vβ11⁺ (right). <b>(e)</b> Tetramer staining of CD8⁺GFP^- T cells (dashed line) and CD8⁺GFP⁺ T cells (filled green histogram). The numbers represent the frequency of CD3⁺CD8⁺GFP⁺ cells that are stained by the tetramer. (<b>f</b>) IFNγ production by lung T cells from TCR3 retrogenic mice infected with <i>M</i>. <i>tuberculosis</i>. Lung cells were stimulated with TB10.4_4-11-peptide and analyzed by intracellular cytokine staining and flow cytometry. Represented are CD8⁺GFP⁺ T cells stimulated by TB10.4_4-11-peptide (filled green histogram) or left unstimulated (dashed line). The numbers represent the frequency of CD3⁺CD8⁺GFP⁺ cells that produce IFNγ after peptide stimulation. (<b>g</b>) Specific lysis of TB10.4_4-11-peptide pulsed EL4 targets by TCR3 retrogenic T cells at different effector to target ratios. Data are representative of >10 (d, e), >5 (f), and 3 (g) independent experiments.