Postthymic maturation influences the CD8 T cell response to antigen

Abstract

Complete T cell development requires postthymic maturation, and we investigated the influence of this ontological period on the CD8 T cell response to infection by comparing responses of mature CD8 T cells with those of recent thymic emigrants (RTEs). When activated with a noninflammatory stimulus or a bacterial or viral pathogen, CD8 RTEs generated a lower proportion of cytokine-producing effector cells and long-lived memory precursors compared with their mature counterparts. Although peripheral T cell maturation is complete within several weeks after thymic egress, RTE-derived memory cells continued to express inappropriate levels of memory cell markers and display an altered pattern of cytokine production, even 8 weeks after infection. When rechallenged, RTE-derived memory cells generated secondary effector cells that were phenotypically and functionally equivalent to those generated by their mature counterparts. The defects at the effector and memory stages were not associated with differences in the expression of T cell receptor-, costimulation-, or activation-associated cell surface markers yet were associated with lower Ly6C expression levels at the effector stage. This work demonstrates that the stage of postthymic maturation influences cell fate decisions and cytokine profiles of stimulated CD8 T cells, with repercussions that are apparent long after cells have progressed from the RTE compartment.

Fig. 1.

Compared with MN cells, RTEs produce a distinct effector response to a noninflammatory stimulus. (A) Experimental setup. (B) Representative plots of CD127 and KLRG1 expression by transferred donor cells and mean total number of donor cells and percentage of CD127⁺ KLRG1⁻ donor cells from 4 recipients. (C) Representative intracellular IL-2 and IFN-γ production by donor cells from recipient spleens after a 5-h peptide restimulation and mean percentage of IFN-γ⁺ and IFN-γ⁺ IL-2⁺ cells. Bars represent standard deviations from the mean. The indicated P values were calculated by using two-tailed Student's t tests with equal variance. T_x, thymectomized.

Fig. 2.

Compared with MN cells, RTEs generate fewer memory precursor cells in response to bacterial infection. (A) Experimental setup. (B) Representative sorting gates showing the strategy used to isolate OT-1 Tg RTEs and MN T cells from bead-depleted populations. Cells expressing CD4, NK1.1, CD11b, B220, or Ter-119 (dump⁺) were excluded, and dump⁻ cells were further selected for expression of CD62L (for both RTEs and MN T cells) and GFP (for RTEs). Approximately 3% of the brightest GFP⁺ cells were used as the source of RTEs, representing the newest emigrants. (C) Representative plots of CD127 and KLRG1 expression by transferred donor cells and mean total number of donor cells and percentage of CD127⁺ KLRG1⁻ donor cells from a total of 4–8 recipients. (D) Representative intracellular IL-2 and IFN-γ production by donor cells from recipient spleens after a 5-h peptide restimulation and mean percentage of IFN-γ⁺ and IFN-γ⁺ IL-2⁺ cells. Bars represent standard deviations from the mean. The indicated P values were calculated by using two-tailed Student's t tests with equal variance.

Fig. 3.

In response to a bacterial pathogen, a lower proportion of RTE-derived memory cells produce IL-2 than their MN-derived counterparts. The experiment was designed as in Fig. 2; however, mice were killed at day 60. (A) Representative plots of CD127 and KLRG1 expression by transferred donor cells and mean total number of donor cells and percentage of CD127⁺ KLRG1⁻ donor cells from a total of 4–6 recipients. (B) Representative intracellular IL-2 and IFN-γ production by donor cells from recipient spleens after a 5-h peptide restimulation and mean percentage of IFN-γ⁺ and IFN-γ⁺ IL-2⁺ cells. (C) Representative histograms of BrdU incorporation by donor cells and mean percentage of BrdU⁺ cells from a total of 6 recipients. Bars represent standard deviations from the mean. The indicated P values were calculated by using two-tailed Student's t tests with equal variance.

Fig. 4.

RTEs are not defective in their secondary effector response to a bacterial pathogen. The experiment was designed as in Fig. 2; however, mice were rechallenged with Lm-OVA on day 60 and killed 3 or 5 days later. (A) Representative plots of CD127 and KLRG1 expression by transferred donor cells and mean total number of donor cells and percentage of CD127⁺ KLRG1⁻ cells from a total of 3–6 recipients. (B) Representative intracellular IL-2 and IFN-γ production by transferred donor cells after a 5-h peptide restimulation and mean percentage of IFN-γ⁺ and IFN-γ⁺ IL-2⁺ cells. Bars represent standard deviations from the mean. n.s., not significant; P values calculated by using two-tailed Student's t tests with equal variance were all >0.5.

Fig. 5.

RTEs express appropriate levels of TCR-related, costimulatory, and activation markers after infection, apart from lower levels of Ly6C at the effector stage. Representative histograms of the indicated surface marker expression by uninfected (open histograms with gray line), MN-derived (open histograms with bold black line), and RTE-derived (filled gray histograms) OT-1 Tg CD8 T cells, analyzed 7 days after primary Lm-OVA infection. **, P < 0.05, two-tailed Student's t test with equal variance. Data are from 3 or more independent experiments for a total of 4–10 recipients.

Fig. 6.

RTEs are defective in their effector response to a viral pathogen. (A) Experimental setup. Note that both RTEs and MN T cells were sorted as dump⁻ CD62L^high and GFP⁻ (MN) or GFP⁺ (RTEs) from the same pool of donors. (B) Representative plots of CD127 and KLRG1 expression by transferred donor cells and mean total number of donor cells and percentage of CD127⁺ KLRG1⁻ cells from a total of 4–8 recipients. (C) Representative intracellular IL-2 and IFN-γ production by donor cells from recipient spleens after a 5-h peptide restimulation and mean percentage of IFN-γ⁺ and IFN-γ⁺ IL-2⁺ donor cells. Bars represent standard deviations from the mean. The indicated P values were calculated by using two-tailed Student's t tests with equal variance.