Expression of the transcription factor cKrox in peripheral CD8 T cells reveals substantial postthymic plasticity in CD4-CD8 lineage differentiation

Abstract

Most T cells belong to either of two lineages defined by the mutually exclusive expression of CD4 and CD8 coreceptors: CD4 T cells are major histocompatibility complex (MHC) II restricted and have helper function, whereas CD8 T cells are MHC I restricted and have cytotoxic function. The divergence between these two lineages occurs during intrathymic selection and is thought to be irreversible in mature T cells. It is, however, unclear whether the CD4-CD8 differentiation of postthymic T cells retains some level of plasticity or is stably maintained by mechanisms distinct from those that set lineage choice in the thymus. To address this issue, we examined if coreceptor or effector gene expression in mature CD8 T cells remains sensitive to the zinc finger transcription factor cKrox, which promotes CD4 and inhibits CD8 differentiation when expressed in thymocytes. We show that cKrox transduction into CD8 T cells inhibits their expression of CD8 and cytotoxic effector genes and impairs their cytotoxic activity, and that it promotes expression of helper-specific genes, although not of CD4 itself. These observations reveal a persistent degree of plasticity in CD4-CD8 differentiation in mature T cells.

Figure 1.

cKrox inhibits CD8 transcription in mature CD8 T cells. (A) CD4-depleted splenocytes transduced with either control (left) or cKrox (right) supernatants were analyzed for GFP and for CD4 and CD8 surface expression by flow cytometry. Two parameter dot plots gated on all live cells are shown. The mean fluorescence intensity of CD8 staining was 3,735 in control- and 867 in cKrox-transduced cells. Results are representative of more than five experiments. (B) T cells from E8(I)-hCD2 reporter mice were transduced with either control or cKrox retrovirus and analyzed by flow cytometry for GFP, hCD2, CD4, and CD8 expression. Two parameter dot plots gated on CD8⁺ cells are shown. Results are representative of three experiments performed on three distinct founder-derived lines, all with CD8-specific hCD2 expression.

Figure 2.

cKrox represses cytotoxic effector gene expression and function in CD8 T cells. (A) Expression of perforin, granzyme B (Gran B), and IFN-γ genes was analyzed by RT-PCR in sorted GFP⁺ CD8 T cells 3 d after transduction with control or cKrox retrovirus. Results are presented relative to gene expression in control-transduced cells (arbitrarily set to 100 for each gene). (B) Analyses of granzyme B protein expression by intracellular staining and flow cytometry are depicted by overlaid histograms showing staining of cKrox- or control-transduced CD8⁺ T cells or of control-transduced CD4 T cells. (C) cKrox impairs CD8 cell cytotoxicity. P14 TCR T cells transduced with either control or cKrox retroviruses were sorted for GFP expression and assessed for cytotoxicity against LCMV gp33-loaded EL-4 targets labeled with the DiD Vybrant Dye (see Fig. S3 for gating strategy). Graphs plot the percentage of targets lysed (7-AAD⁺) by infected (GFP⁺) or uninfected (GFP⁻) effectors against effector/target ratios. (D) CD4-depleted splenocytes transduced with either control (left) or cKrox (right) retrovirus were restimulated for 4 h by PMA and ionomycin, and analyzed for GFP and IFN-γ expression by intracellular cytokine staining and flow cytometry. Numbers indicate the percentage of GFP⁻ or GFP⁺ cells that stained for intracellular IFN-γ. Results in each panel are representative of three or more experiments.

Figure 3.

cKrox-induced Eomes repression mediates in part its reduction of IFN-γ production. (A) cKrox represses Eomes but not T-bet expression. Sorted GFP⁺CD8⁺ cells were analyzed as in Fig. 2 A for Eomes and T-bet gene expression. (B) cKrox- or control-transduced CD4-depleted splenocytes were analyzed by flow cytometry for CD122 and CD127 surface expression. Two parameter dot plots are gated on all live cells. Numbers represent mean fluorescence intensity of CD122 or CD127 expression in GFP⁺ cells. Results are representative of three experiments. (C and D) CD4-depleted splenocytes were cotransduced with Eomes-NGFR and cKrox-GFP viruses. 3 d later, flow cytometric analysis of CD8, NGFR, and GFP expression distinguishes CD8 cells infected with either the Eomes or cKrox virus, with both viruses (both), and uninfected CD8 cells (none), as depicted in Fig. S4 A. (C) Analyses of IFN-γ production by intracellular cytokine staining are shown as single-parameter histograms gated on each population. Numbers represent the percent of cells producing IFN-γ. (D) Perforin gene expression was analyzed by RT-PCR on sorted cells from each population and is expressed relative to uninfected cells (set to 1 arbitrarily). Results in each panel are representative of two separate transductions.

Figure 4.

cKrox promotes expression of helper-specific genes by CD8 T cells. (A) Lymph node cells transduced with cKrox or control retrovirus were analyzed by intracellular staining and flow cytometry for IL-2 production. Overlaid histograms gated on CD8⁺GFP⁺ (left) or CD4⁺GFP⁺ (right) cells show IL-2 expression after restimulation with PMA and ionomycin (filled) or without restimulation (dotted line). (B) mRNA expression of Gata3 and IL-4 was analyzed by RT-PCR in cKrox- or control-transduced CD8 cells and in untransduced CD4 cells, all activated in type 2 conditions. Results are relative to control-transduced CD8 cells (arbitrarily set to 1). Results in each panel are representative of three experiments.