Fig. 4.

Lineage tracing indicates lineage conversion of CD4⁺ T cells to CD4-CD8αβ⁺ T cells during an antigen-specific effector response. Genetic labeling of CD4⁺ T cells (a) Rosa26^{tdTomato+/−} Ox40-Cre^+/− (OxTom) mice were infected with 1 × 10⁵ cfu LCMV Armstrong by i.p. injection. Eight days post-infection, splenic lymphocytes were harvested and analyzed for tdTomato (tdTom) expression among LCMV-specific CD4-CD8αβ⁺ T cells (Live/DeadhuCLIP87-101-I-A^b− LCMVgp66-77-I-A^b+ CD44^hi TCRβ⁺ CD4⁻, marked in brown) and effector CD4⁺ T cells (Live/Dead⁻ huCLIP87-101-I-A^b− LCMVgp66-77-I-A^b+ CD44^hi TCRβ⁺ CD4⁺, marked in green). b, c Naïve CD4⁺ T cells (TCRβ⁺ CD44^lo tdTom⁻ CD4⁺) and naïve CD8⁺ T cells (TCRβ⁺ CD44^lo tdTom⁻ CD8⁺) were FACS-sorted from the splenic lymphocytes of OxTom mice. b 1 × 10⁶ FACS-sorted CD4⁺ or CD8⁺ OxTom T cells were adoptively transferred to TCRα^−/− recipient mice by i.v. injection, and recipient mice were infected with 1 × 10⁵ cfu LCMV Armstrong by i.p. injection 24 h post-adoptive transfer. c Eight days post-infection, splenic lymphocytes were harvested and analyzed for tdTom expression among effector (CD44^hi) cells (middle panel). CD8αβ expression was assessed among CD44^hi tdTom⁺ cells (bottom panel). n = 5 from 2 independent experiments for (a), and n = 2 from 1 experiment for (b) and (c). Statistical significance was determined by the Mann–Whitney U test; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant. Adoptive transfer of serially-enriched CD4⁺ T cells (d) Naïve CD4⁺ T cells (TCRβ⁺ CD44⁻ CD4⁺) were FACS-sorted once (single sort; shown in black) or twice (double sort; shown in periwinkle) from the splenic lymphocytes of Rosa26^mT/mG (mT/mG) mice. Cells were enriched from the live donor population (Live-dead⁻ tdTom⁺; tdTom is a constitutive, ubiquitous label). 1 × 10⁶ naïve CD4⁺ T cells were adoptively transferred to TCRα^−/− recipient mice by i.v. injection, and recipient mice were infected with 1 × 10⁵ cfu LCMV Armstrong by i.p. injection. e Eight days post-infection, splenic lymphocytes were harvested and analyzed for CD4/CD8 expression among effector LCMV/MHCII-binding (CD44^hi TCRβ⁺ LCMVgp66-77-I-A^b+) live donor T cells. % CD4⁻ LCMVgp66-77-I-A^b+ cells among CD44^hi TCRβ⁺ live donor cells from single-sort vs. double-sort recipients is shown in (f). n = 4 from 1 experiment. Statistical significance was determined by the Mann–Whitney U test; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant