T Cell Factor-1 and β-Catenin Control the Development of Memory-like CD8 Thymocytes

Archna Sharma, Qinghua Chen, Trang Nguyen, Qing Yu and Jyoti Misra Sen
J Immunol April 15, 2012, 188 (8) 3859-3868; DOI: https://doi.org/10.4049/jimmunol.1103729

Abstract

Innate memory-like CD8 thymocytes develop and acquire effector function during maturation in the absence of encounter with Ags. In this study, we demonstrate that enhanced function of transcription factors T cell factor (TCF)-1 and β-catenin regulate the frequency of promyelocytic leukemia zinc finger (PLZF)-expressing, IL-4–producing thymocytes that promote the generation of eomesodermin-expressing memory-like CD8 thymocytes in trans. In contrast, TCF1-deficient mice do not have PLZF-expressing thymocytes and eomesodermin-expressing memory-like CD8 thymocytes. Generation of TCF1 and β-catenin–dependent memory-like CD8 thymocytes is non–cell-intrinsic and requires the expression of IL-4 and IL-4R. CD8 memory-like thymocytes migrate to the peripheral lymphoid organs, and the memory-like CD8 T cells rapidly produce IFN-γ. Thus, TCF1 and β-catenin regulate the generation of PLZF-expressing thymocytes and thereby facilitate the generation of memory-like CD8 T cells in the thymus.

In addition to conventional αβ T cells, recent studies have demonstrated the presence of subsets of thymocytes that acquire effector functions in the thymus as a result of the maturation process. A subset of CD8 single-positive (SP) mature thymocytes, referred to as “memory-like” or “innate-like,” has been described in mice with mutations in Krüppel-like factor 2 (KLF2), inducible T cell kinase, CREB binding protein, inhibitor of DNA binding 3, and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa genes (1–9). Nonmutant BALB/c mice also show an increase in the frequency of CD8 memory-like cells (1, 8, 10). Memory-like CD8 thymocytes share characteristics with conventional memory CD8 T cells, including higher expression of surface markers CD44 and CD122, intracellular eomesodermin (Eomes), and rapid production of cytokines after TCR activation (5). In addition to a memory-like phenotype, memory-like CD8 thymocytes respond to TCR signals by rapidly producing IFN-γ and therefore appear competent to provide immune protection (8, 11). Generation of memory-like CD8 thymocytes is dependent on thymocytes that express transcription factor promyelocytic leukemia zinc finger (PLZF) (1). PLZF-expressing thymocytes are expanded in the thymuses of KLF2, inducible T cell kinase, and inhibitor of DNA binding 3 gene-deficient mice and produce IL-4, which is required for the generation of memory-like CD8 thymocytes (1, 8, 9). However, the mechanisms that regulate the increase in the accumulation of PLZF-expressing and IL-4–producing thymocytes and consequent increase in memory-like CD8 thymocytes remain to be fully understood.

TCF1 is highly expressed in thymocytes and critically regulates T cell development at multiple developmental stages (12, 13). Despite this, we and others have demonstrated that normal naive T cells develop in TCF1-deficient mice and migrate to populate the spleen and the lymph nodes (13–15). Early events in TCR-activated TCF1-deficient T cells are also comparable to control T cells (16, 17). TCF1-deficient CD4 T cells fail to make IL-4 due to lack of GATA-3 expression and produce enhanced IFN-γ and IL-17 because TCF1 directly negatively regulates IFN-γ and IL-17 genes (16, 17). TCF1 also plays a definitive role in the generation and maintenance of conventional Ag-driven CD8 memory T cells (18–22). Whereas TCF1 is highly and constitutively expressed in thymocytes, expression of β-catenin is intrathymically regulated in a developmentally sensitive manner, suggesting that signal-dependent expression of β-catenin might be limiting during thymocyte development (15, 23, 24). We have previously shown that enforced expression of β-catenin from the proximal Lck promoter results in an increase in memory-like CD8SP thymocytes (25). However, transgenic expression of β-catenin from the CD4 promoter failed to induce the generation of memory-like CD8SP thymocytes (26). The reason for this difference in phenotype of the two mouse strains that expressed ectopic β-catenin in thymocytes might be explained based on two hypotheses. First, the level of transgene expression might be different. Second, the stage of thymocyte development at which expression of transgenic β-catenin is initiated plays an important role in regulating the generation of memory-like CD8SP thymocytes. These studies outline the scope of the role played by TCF1 and cofactors β- and γ-catenin in T cell function during an immune response.

In this study, we provide a mechanism by which TCF1 and β-catenin–dependent gene expression regulates the generation of memory-like mature CD8 thymocytes. Enforced expression of transgenic β-catenin, from the Lck promoter, in TCF1-sufficient mice (β-CAT-Tg) increases the generation of CD4+αβTCR+ thymocytes that express PLZF and produce IL-4. Enhanced IL-4 production by PLZF-expressing thymocytes induces the generation of Eomes-expressing memory-like CD8 thymocytes. Deletion of IL-4 or IL-4R expression abrogates memory-like CD8 thymocytes but not PLZF-expressing CD4 thymocytes, demonstrating that IL-4R–dependent signals are required for the generation of CD8 memory-like cells. In contrast, PLZF-expressing CD4 thymocytes and Eomes-expressing memory-like CD8 thymocytes are absent in TCF1-deficient mice, which demonstrates the requirement for TCF1-dependent gene expression. Finally, we show that the thymic CD8 memory-like cells migrate to the periphery and rapidly produce IFN-γ ex vivo.

Materials and Methods

Animals

β-CAT-Tg mice, expressing β-catenin in thymocytes and T cells from the proximal Lck promoter, have been described previously (25). TCF1-deficient mice (12) were provided by H. Clevers (Hubrect Institute, Utrecht, The Netherlands). RAG2-deficient and IL-4–deficient mice were purchased from The Jackson Laboratory. IL-4Rα–deficient mice (27) were provided by Z. Zhu (The Johns Hopkins Asthma and Allergy Center, Baltimore, MD). IL-4–deficient and IL-4Rα–deficient mice were bred with β-CAT-Tg mice to generate IL-4 knockout (KO)-β-CAT-Tg and IL-4RαKO-β-CAT-Tg mice. All mice were on C57BL/6 genetic background. Age-matched littermate controls or C57BL/6 mice were used in all experiments. All mice were bred and maintained in an animal facility at the National Institute on Aging according to National Institutes of Health regulations and were in compliance with the guidelines of National Institute on Aging animal resources facility, which operates under the regulatory requirements of the U.S. Department of Agriculture and Association for Assessment and Accreditation of Laboratory Animal Care.

Abs and flow cytometry

Cells were harvested, stained, and analyzed on a FACSCalibur (Becton Dickinson). Dead cells were excluded by forward light scatter or forward light scatter plus propidium iodide. All data were acquired and are presented on a log scale. The following Abs conjugated to FITC, PE, PerCP-Cy5.5, or allophycocyanin (all from BD Biosciences) were used for staining: anti-CD4 (GK1.5), anti-CD8α (53-6.7), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD122 (TM-β1), anti–IL-4Rα (mIL4R-M1), anti–TCR-β (H57-597), anti-TCRγδ (GL3), anti-STAT6 (pY641) (J71-773.58.11), anti-integrin β7-chain (M293), anti-CD103 (M290), Vβ2 TCR (B20.6), Vβ3 TCR (KJ25), Vβ4 TCR (KT4), Vβ5.1/5.2 TCR (MR9-4), Vβ6 TCR (RR4-7), Vβ8.1/8.2 TCR (MR5-2), Vβ9 TCR (MR10-2), and Vβ14 TCR (14-2). FITC- or PE-conjugated anti–IL-7Rα (A7R34), anti-CXCR3 (CXCR3-173), anti–IFN-γ (XMG1.2), anti–IL-4 (11B11), anti-Eomes (Dan11mag), and anti–T-bet (eBio4B10) Abs were purchased from eBioscience. PE-conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 were obtained from the tetramer facility of the National Institutes of Health. Purified anti-CD3 (145-2C11) and anti-CD28 (37.51) were from BD Pharmingen. PLZF staining was done as described (28). In brief, cell surfaces were stained with Abs, and then cells were fixed with the Foxp3 staining buffer set (eBioscience). Permeable cells were incubated with Ab to PLZF (D-9; Santa Cruz Biotechnology) followed by anti-mouse Ig G1 (A85-1; BD Biosciences) alone or with anti–IL-4 (BVD4-1D11) where appropriate. For Eomes staining and T-bet staining, cells were stained for 1 h with anti-Eomes (Dan11mag) and anti–T-bet (eBio4B10) with a Foxp3 staining buffer set (eBioscience).

In vitro cytokine production

For in vitro cytokine production, 1 × 106 cells/ml were incubated for 5 h in RPMI 1640 medium plus 10% (v/v) FCS with or without PMA (50 ng/ml) and ionomycin (1.5 μM; Sigma-Aldrich). GolgiStop (BD Pharmingen) was added for the final 3 h. Cells were then harvested and stained for intracellular cytokines with a BD Cytofix/Cytoperm kit.

Cell sorting and quantitative real-time RT-PCR

Cell suspensions of total thymocytes were surface stained with anti-CD4 and anti-CD8 followed by electronic cell sorting on a DakoCytomation MoFlo. Total mRNA was reverse transcribed using poly (dT) and SuperScript III reverse transcriptase (Invitrogen). SYBR Green quantitative real-time RT-PCR was performed using PCR Master Mix (Applied Biosystems) for 40 cycles with annealing and extension at 60°C. Primer sequences will be provided upon request. The expression of target gene was determined relative to GAPDH and calculated as 2−(Ct target gene − Ct GAPDH).

Mixed bone marrow chimera

Bone marrow (BM) was isolated from femurs and tibias of β-CAT-Tg and IL-4RαKO-β-CAT-Tg mice. T cells were depleted and β-CAT-Tg BM cells were mixed with IL-4RαKO-β-CAT-Tg BM cells at a 1:1 ratio and injected i.v. into sublethally irradiated Rag2-deficient mice. Chimeric mice were sacrificed and analyzed 16–18 wk after transplant.

Retroviral infection

CD8+ T cells were purified using a mouse CD8+ T cell isolation kit (Miltenyi Biotec). Purified CD8+ T cells were activated by plate-bound anti-CD3 and -CD28 Abs for 2 d and infected with murine stem cell virus (MSCV)-based retroviruses that express human CD8 as a marker (MSCV-Vector) or coexpress stabilized mouse β-catenin (MSCV-β-CAT). Human CD8+ populations were electronically sorted and analyzed for mRNA.

Stimulation of CD8 T cells with cytokines

Electronically sorted CD122−CD44lo naive CD8 T cells from C57BL/6 mice were treated with 10 ng/ml IL-2, 50 ng/ml IL-4, 5 ng/ml IL-7, 100 ng/ml IL-15, or the combination of cytokines as indicated for 20 h. For in vitro CD8 T cell differentiation, lymph node cells were stimulated with 1 μg/ml soluble anti-CD3 for 3 d and then rested in cultures that contained 5 ng/ml IL-7 and 10 ng/ml IL-4 for another 6 d. CD8 T cells were purified by positive selection using biotin-anti-CD8 and anti-biotin magnetic beads (Miltenyi Biotec). All cytokines were purchased from R&D Systems.

Preparation of small intestine intraepithelial lymphocytes

Intraepithelial lymphocytes (IEL) from small intestines of control and β-CAT-Tg mice were prepared according to a modification of a previously published procedure (29). Briefly, small intestine was removed and carefully cleaned from its mesentery and fecal contents flushed out from small intestine. The intestinal tissue was cut longitudinally and then into 1-cm pieces and shaken three times at 200 rpm for 20 min at 37°C in RPMI 1640 media with 5 mM EDTA and 0.145 mg/ml DTT. The cells in suspension were collected and centrifuged in a discontinuous 40/70% Percoll (GE Healthcare Bio-Sciences, Uppsala, Sweden) gradient at 1600 rpm for 20 min. Cells from the 40/70% interface were collected, washed, and resuspended in complete RPMI 1640 media supplemented with 10% FBS.

Statistics

Statistical significance was determined by the Student t test.

Results

TCF1 and β-catenin regulate memory-like CD8 T cell generation in the thymus

We have noted that enforced expression of the stabilized form of β-catenin from the proximal Lck promoter in β-CAT-Tg mice results in an increase of TCR-βhi CD8SP thymocytes that exhibit a memory phenotype (25) (Fig. 1A). In contrast, we note (Fig. 1A), as has been reported before (12), that TCF1-deficient CD8SP thymocytes contain a high representation of immature SP cells and a lower proportion of TCR-βhi mature CD8 thymocytes compared with control. Further analysis of the TCR-βhi CD8SP population revealed that most β-CAT-Tg CD8SP thymocytes expressed high levels of Eomes (Fig. 1B). In contrast, most CD8SP TCF1-deficient thymocytes showed very low Eomes expression (Fig. 1B). We note that TCF1-deficient mice showed a significantly small population (0.087 ± 0.016 × 106 compared with control mice, 0.15 ± 0.012 × 106; p = 0.004) of TCR-βhi CD8 thymocytes that expressed intermediate levels of Eomes. An increase in the relative abundance of Eomes mRNA in β-CAT-Tg CD8 memory-like cells compared with control CD8SP thymocytes (data not shown) showed that Eomes expression is regulated at the mRNA level. These data are congruent with impaired TCF1-dependent regulation of Eomes expression in conventional Ag-driven CD8 memory cells (21). β-CAT-Tg CD8SP thymocytes also expressed higher levels of IL-4Rα–chain compared with control or TCF1-deficient CD8SP thymocytes (Fig. 1B). We examined expression of other surface memory markers and found that β-CAT-Tg CD8SP thymocytes expressed high levels of memory markers, including CD44, CD122, and CXCR3 (Fig. 1C). Finally, ex vivo β-CAT-Tg CD8 memory-like cells produced higher levels of IFN-γ compared with control CD8SP thymocytes (Fig. 1D). Taken together, these data demonstrate that TCF1 and β-CAT-Tg regulate the generation of Eomes-expressing CD8SP thymocytes that acquire memory-like phenotype and function during their development in the thymus.

FIGURE 1.
FIGURE 1.

Memory-like CD8 T cell development in the thymus is regulated by β-catenin and TCF1. (A) Flow cytometric analysis of surface CD4/CD8 expression on total thymocytes and TCR-β expression on the gated CD8SP population from control, β-CAT-Tg, and TCF1-deficient mice. Numbers show the percentage of cells. Data are representative of eight independent analyses. The graph in the upper right panel shows total thymic cellularity, and the graph in the lower right panel shows absolute numbers of TCR-βhi CD8SP thymocytes from control, β-CAT-Tg, and TCF1-deficient mice. (B) Flow cytometric analysis of Eomes and IL-4Rα expression on the gated TCR-βhi CD8SP thymocytes from control, β-CAT-Tg, and TCF1-deficient mice. Data are representative of six independent analyses. (C) Flow cytometric analysis of cell surface memory marker expression on the gated CD8SP thymocytes from control and β-CAT-Tg mice. Data are representative of eight independent analyses. (D) Flow cytometric analysis is shown of intracellular IFN-γ expression on gated CD8SP thymocytes after 5 h PMA plus ionomycin stimulation of total thymocytes from control and β-CAT-Tg mice. Data are representative of three independent analyses with a total of five to six mice in each group.

TCF1 and β-catenin regulate the generation of PLZF-expressing thymocytes

Several recent studies have indicated that generation of memory-like CD8 thymocytes is dependent on the presence of PLZF-expressing thymocytes (1, 8, 9). To determine whether generation of TCF1 and β-catenin–dependent memory-like CD8SP thymocytes was regulated by PLZF-expressing thymocytes, we assayed for PLZF-expressing thymocytes in β-CAT-Tg and TCF1-deficient mice. Costaining with PLZF and TCR-β–chain showed that β-CAT-Tg thymocytes have a significantly (p = 5.5 × 10−6) higher percentage and number of TCR-β+ cells that also express PLZF compared with control thymocytes (Fig. 2A). In contrast, the frequency and number of TCR-β+ thymocytes expressing PLZF was significantly decreased (p = 1.8 × 10−14) in TCF1-deficient mice (close to none) compared with control mice (Fig. 2A). These data show that TCF1 and β-catenin regulate the generation of PLZF-expressing TCR-β+ thymocytes. Because major PLZF-expressing thymocytes are known to be NKT cells, defined by their binding to CD1d tetramers loaded with the glycolipids, we stained thymocytes with CD1d tetramers loaded with glycolipid α-galactosylceramide analog PBS-57 and PLZF. We found that β-CAT-Tg thymocytes had a significantly (p = 7.9 × 10−5) higher percentage and number of PLZF-expressing CD1d-PBS-57 tetramer binding NKT cells compared with control thymocytes (Fig. 2B). These data suggest that there is an increase in PLZF-expressing NKT cells in β-CAT-Tg mice. Further characterization of PLZF-expressing thymocytes in β-CAT-Tg mice showed that they express CD4 but not CD8 (Fig. 2C). Finally, the relatively higher abundance of PLZF mRNA in CD4SP but not CD8SP thymocytes showed that β-catenin expression enhanced PLZF expression in CD4SP but not CD8SP thymocytes (Fig. 2D). Taken together, these data show that TCF1 and β-catenin promote the generation of PLZF-expressing thymocytes and support the hypothesis that the increase in memory-like CD8 thymocytes is along the principles outlined in other mutant mice that show an increase in this population of thymocytes.

FIGURE 2.
FIGURE 2.

TCR-β+ and CD1dPBS-57+ PLZF-expressing thymic population is increased in β-CAT-Tg mice. (A) Flow cytometric analysis of expression of PLZF and TCR-β on total thymocytes from control, β-CAT-Tg, and TCF1-deficient mice. Numbers adjacent to outlined areas (upper panels) and graphs (lower panels) show percentage and absolute numbers of PLZF+TCR-β+ cells as indicated. Data are representative of five independent analyses with a total of 8–10 mice per group. (B) Flow cytometric analysis of expression of PLZF and CD1dPBS-57 on total thymocytes from control and β-CAT-Tg mice. Numbers adjacent to outlined areas (left panels) and graphs (right panels) show percentage and absolute numbers of PLZF+CD1dPBS-57+ cells as indicated. Data are representative of five independent analyses with a total of 8–10 mice per group. (C) Flow cytometric analysis of expression of PLZF on gated CD4 and CD8SP populations of total thymocytes from control and β-CAT-Tg mice. Numbers adjacent to outlined areas show percentage of PLZF+ CD4SP and PLZF+ CD8SP thymocytes as indicated. Data are representative of five independent analyses. (D) Real-time PCR analysis for Zbtb16 mRNA in purified CD4SP and CD8SP thymocytes, presented relative to Hprt. Data are from four independent samples.

PLZF-expressing β-CAT-Tg thymocytes produce IL-4

PLZF-expressing thymocytes have been shown to produce IL-4 (1, 8, 9). To determine whether PLZF-expressing β-CAT-Tg thymocytes produce IL-4, we stimulated total thymocytes with PMA and ionomycin and measured IL-4 production by intracellular staining followed by flow cytometric analysis. We found that β-CAT-Tg thymocytes contained 5- to 10-fold higher percentage of PLZF+ IL-4–producing cells after 5 h in vitro stimulation compared with control thymocytes (Fig. 3A). Double staining with CD4 and intracellular IL-4 Abs confirmed that β-CAT-Tg CD4SP thymocytes contained a higher percentage of IL-4–producing cells as compared with their control counterparts (Fig. 3B). Thus, a subset of CAT-Tg TCR-β+CD4+ thymocytes express PLZF and produce IL-4 in β-CAT-Tg mice. In contrast, β-CAT-Tg CD8SP thymocytes did not show enhanced IL-4 production compared with control CD8SP thymocytes (Fig. 3B). Accordingly, relative increase in Il4 transcripts in β-CAT-Tg CD4SP thymocytes compared with control CD4SP thymocytes showed regulation at the transcription level (Fig. 3C). Parenthetically, KLF2 expression in β-CAT-Tg CD4 or CD8SP thymocytes is comparable to control thymocytes (data not shown), suggesting that the effect of TCF1 and β-catenin–dependent regulation may not go through KLF2. Thus, CAT-Tg TCR-β+PLZF+ CD4SP thymocytes produce IL-4 resulting from increased transcription. We propose that β-catenin expression in developing thymocytes enhances the generation of PLZF-expressing thymocytes that overproduce IL-4 and promote the generation of memory-like CD8 thymocytes.

FIGURE 3.
FIGURE 3.

β-CAT-Tg PLZF+ CD4SP thymocytes overproduce IL-4. (A) Flow cytometric analysis of intracellular expression of IL-4 and PLZF in control and β-CAT-Tg thymocytes stimulated for 5 h with PMA and ionomycin. Numbers adjacent to outlined areas show percentage of IL-4+PLZF+ cells. Data are representative of four independent analyses. (B) Flow cytometric analysis of intracellular IL-4 expression on gated CD4 and CD8SP thymocytes after 5 h PMA plus ionomycin stimulation of total thymocytes from control and β-CAT-Tg mice. Numbers adjacent to outlined areas show percentage of IL-4+ CD4SP and IL-4+ CD8SP thymocytes as indicated. Data are representative of five independent analyses. (C) Real-time PCR analysis for Il4 mRNA in purified CD4SP and CD8SP thymocytes, presented relative to Actb. Data are from four independent samples.

IL-4/IL-4Rα expression is required for generation of β-CAT-Tg memory-like CD8SP thymocytes

To demonstrate that IL-4 production by PLZF+ cells in β-CAT-Tg mice was required for the generation of memory-like CD8 thymocytes, we generated IL-4KO-β-CAT-Tg and IL-4RαKO-β-CAT-Tg mice by breeding β-CAT-Tg mice with IL-4KO and IL-4RαKO mice, respectively. Analysis of thymocytes from these mice showed that in contrast to β-CAT-Tg CD8SP thymocytes, IL-4KO-β-CAT-Tg (Fig. 4A) and IL-4RαKO-β-CAT-Tg (Fig. 4B) CD8SP thymocytes were CD44loCD62Lhi, similar to control, IL-4KO, and IL-4RαKO CD8SP thymocytes. Additionally, cell surface expression of memory-like phenotype markers CD122 and CXCR3 also showed that IL-4KO-β-CAT-Tg and IL-4RαKO-β-CAT-Tg CD8SP thymocytes fail to acquire the memory-like phenotype noted in β-CAT-Tg CD8SP thymocytes (Fig. 4C). Increased expression of Eomes noted in β-CAT-Tg CD8SP thymocytes was also abolished in IL-4KO-β-CAT-Tg and IL-4RαKO-β-CAT-Tg CD8SP thymocytes (Fig. 4C). Thus, expression of IL-4 and IL-4Rα is required to promote generation of CD8SP thymocytes with a memory-like phenotype in β-CAT-Tg mice. We confirmed that β-CAT-Tg, IL-4KO-β-CAT-Tg, and IL-4RαKO-β-CAT-Tg thymocytes express comparable levels of transgenic β-catenin protein (data not shown). Finally, in contrast to memory-like β-CAT-Tg CD8SP thymocytes, which rapidly produce IFN-γ ex vivo, IL-4–deficient β-CAT-Tg CD8SP thymocytes fail to produce IFN-γ (Fig. 4D). These data demonstrate that IL-4 is also required for the memory-like function of β-CAT-Tg CD8SP thymocytes. We conclude that the generation of memory-like CD8SP phenotype in β-CAT-Tg thymus is dependent on IL-4 produced by PLZF-expressing thymocytes in a cell-extrinsic manner.

FIGURE 4.
FIGURE 4.

The memory-like CD8 phenotype in β-CAT-Tg mice is dependent on IL-4 and IL-4Rα. (A) Flow cytometric analysis of the cell surface memory markers CD44 and CD62L on the gated CD8SP population of total thymocytes from control, β-CAT-Tg, IL-4KO, and IL-4KO-β-CAT-Tg mice. Numbers show the percentage of cells. Data are representative of four independent analyses. (B) Flow cytometric analysis of the cell surface memory markers CD44 and CD62L on the gated CD8SP population of total thymocytes from control, β-CAT-Tg, IL-4RαKO, and IL-4RαKO-β-CAT-Tg mice. Numbers show the percentage of cells. Data are representative of four independent analyses. (C) Flow cytometric analysis of the surface CD122, CXCR3, and intracellular Eomes expression in the gated CD8SP thymocytes from control, β-CAT-Tg, IL-4KO, IL-4RαKO, IL-4KO-β-CAT-Tg, and IL-4RαKO-β-CAT-Tg mice. Data are representative of four independent analyses. (D) Flow cytometric analysis is shown of intracellular IFN-γ expression on gated CD8SP thymocytes after 5 h PMA plus ionomycin stimulation of total thymocytes from control, β-CAT-Tg, and IL-4KO-β-CAT-Tg mice. Data are representative of four independent analyses.

β-CAT-Tg CD8SP thymocytes experience higher IL-4/IL-4Rα signals and require IL-4Rα expression on the cell surface

Next, we demonstrate that increased IL-4R signaling regulates memory-like phenotype of CAT-Tg CD8SP thymocytes in a cell-intrinsic manner. To determine whether β-CAT-Tg memory-like CD8SP thymocytes, which express higher levels of IL-4Rα (Fig. 1B), receive higher IL-4/IL-4Rα signaling we examined STAT6 phosphorylation levels. Transient stimulation with IL-4 resulted in a measurable increase in pSTAT6 levels in control and IL-4KO CD8SP thymocytes but not in IL-4Rα–deficient CD8SP thymocytes (Fig. 5A). Next, we assayed pSTAT6 levels in freshly isolated β-CAT-Tg CD8SP thymocytes. We found that ex vivo β-CAT-Tg CD8SP thymocytes showed significantly increased levels of pSTAT6 compared with control CD8SP thymocytes without stimulation with IL-4 in vitro (Fig. 5B). The increase in pSTAT6 levels in β-CAT-Tg CD8SP thymocytes was abolished in thymocytes from IL-4–deficient or IL-4Rα–deficient β-CAT-Tg mice (Fig. 5B). These data show that β-CAT-Tg CD8SP thymocytes receive higher IL-4/IL-4Rα signals in vivo.

FIGURE 5.
FIGURE 5.

Enhanced IL-4 signaling and IL-4Rα expression regulate β-CAT-Tg CD8 memory-like phenotype. (A) Flow cytometric analysis of intracellular pSTAT6 in gated CD8SP thymocytes from ex vivo control, IL-4KO, and IL-4RαKO total thymocytes transiently stimulated with IL-4 for 20 min. Data are representative of two independent analyses. (B) Flow cytometric analysis of intracellular pSTAT6 on gated CD8SP thymocytes from ex vivo total thymocytes from control, β-CAT-Tg, IL-4KO-β-CAT-Tg, and IL-4RαKO-β-CAT-Tg mice. Data are representative of two independent analyses. Graph on right side represents pSTAT6 levels as mean fluorescence intensity (MFI). (C) Flow cytometric analysis of the cell surface memory markers on the gated IL-4Rα− and IL-4Rα+ CD8SP population of total thymocytes from β-CAT-Tg and IL-4RαKO-β-CAT-Tg 1:1 bone marrow chimera. Data are representative of two independent analyses with a total of five chimera mice. (D) Flow cytometric analysis of Eomes expression on the gated IL-4Rα− and IL-4Rα+ CD8SP thymocytes from β-CAT-Tg and IL-4RαKO-β-CAT-Tg 1:1 bone marrow chimera. Data are representative of two independent analyses with a total of five chimera mice.

To further substantiate the requirement of IL-4Rα expression on CAT-Tg CD8SP thymocytes for the generation of memory-like CD8SP thymocytes, we generated mixed BM chimeras in RAG2-deficient mice using a mix of equal numbers of BM cells from IL-4Rα–sufficient β-CAT-Tg and IL-4Rα–deficient β-CAT-Tg mice. After 16 wk we assayed for the memory phenotype of CD8SP thymocytes. We found that only IL-4Rα–expressing but not IL-4Rα–deficient β-CAT-Tg CD8SP thymocytes showed upregulation of surface memory markers CD44, CD122, CXCR3 (Fig. 5C), and intracellular Eomes (Fig. 5D). We conclude that memory-like CD8 T cell generation in β-CAT-Tg mice is dependent on IL-4Rα expression on the CD8SP thymocytes. These data further substantiate the notion that development of memory-like CD8SP thymocytes in β-CAT-Tg mice is not cell-autonomous and requires signals from the IL-4Rα in addition to enhanced β-catenin expression in a TCF1-sufficient background.

CD8 memory-like thymocytes migrate to the periphery and are functional

The phenotypic description of β-CAT-Tg CD8 T cells in the periphery showed the same memory-like phenotype as the CD8SP thymocytes with high levels of memory markers CD44 and CD62L (Fig. 6A). CD62L and β7 integrin regulate the entry of mature T cells into peripheral lymphoid tissues (30, 31). CAT-Tg memory-like CD8 T cells in the lymph nodes express high levels of other surface memory markers and CD62L (L-selectin) and lower levels of CD103 and β7 integrin (Fig. 6B). β-Cat-Tg CD4 and CD8 T cells represent a TCR repertoire comparable to control mice, which shows that T cells in β-CAT-Tg mice are polyclonal (data not shown). Intracellular staining showed that β-CAT-Tg memory-like CD8 T cells expressed high levels of Eomes compared with control CD8 T cells, whereas expression of T-bet was comparable to control (Fig. 6C). Abundance of Eomes and T-bet mRNA expression using real-time RT-PCR showed that Eomes mRNA expression was markedly higher, whereas T-bet mRNA level was moderately lower, in peripheral β-CAT-Tg CD8 T cells than in control CD8 T cells (Fig. 6D). Additionally, β-CAT-Tg CD8 T cells expressed higher levels of mRNA for Prf1 (perforin) and Prdm1 (Blimp-1) (Fig. 6D). These observations are consistent with the fact that Eomes is a critical regulator of perforin expression in CD8 T cells (32). To determine whether memory-like CD8 T cells were functional, we stimulated freshly isolated ex vivo CD8 T cells with PMA and ionomycin to assess IFN-γ production. We found that a greater percentage of β-CAT-Tg CD8 T cells produced IFN-γ as compared with control CD8 T cells (Fig. 6E). These data suggest that β-CAT-Tg memory-like CD8 T cells rapidly produce IFN-γ upon stimulation. We also studied the phenotype of CD8 T cells in the small intestine of β-CAT-Tg mice and found that the β-CAT-Tg IEL CD8 T cells showed increased CD44 (but not CD62L) expression compared with control, suggesting an increase in memory phenotype (Fig. 6F). These data show that the memory-like CD8SP thymocytes generated in the thymus of CAT-Tg mice, in the absence of antigenic stimulation, migrate to the peripheral lymphoid organs and are functional.

FIGURE 6.
FIGURE 6.

Peripheral CD8 T cells in β-CAT-Tg also have memory-like features and function. (A) Flow cytometric analysis of the cell surface memory markers CD44 and CD62L on the gated CD8 T population of lymph node cells from control and β-CAT-Tg mice; numbers show the percentage of cells. Data are representative of 10 independent analyses. (B) Flow cytometric analysis of the cell surface memory markers CD122, IL-7Rα, IL-4Rα, CXCR3, CD103, and β7 expression on the gated CD8 T population of lymph node cells from control and β-CAT-Tg mice. Data are representative of 10 independent analyses. (C) Flow cytometric analysis of Eomes and T-bet expression on the gated CD8 T population of lymph node cells from control and β-CAT-Tg mice. Data are representative of six independent analyses. (D) Real-time PCR analysis for mRNA abundance of Eomes, Tbx21, Prf1, and Prdm-1 in purified ex vivo CD8 T cells from lymph nodes of control and β-CAT-Tg mice, presented relative to Actb. Data are from four independent samples. (E) Flow cytometric analysis is shown of intracellular IFN-γ expression on gated CD8 T cells after 5 h PMA plus ionomycin stimulation of lymph node cells from control and β-CAT-Tg mice. Data are representative of four independent analyses. (F) Flow cytometric analysis of the expression of cell surface memory markers CD44 and CD62L on the gated CD8 T population of IEL from small intestines of control and β-CAT-Tg mice. Numbers show the percentage of cells.

TCF1 and β-catenin–dependent CD8 memory-like T cells require IL-4 and IL-4Rα expression

To determine whether memory-like CD8 T cells found in the peripheral compartment of β-CAT-Tg mice required IL-4 signaling, we compared the CD8 T cells in β-CAT-Tg mice with CD8 T cells from IL-4KO-β-CAT-Tg and IL-4RαKO-β-CAT-Tg mice. We found that memory-like CD8 T cells were absent from β-catenin–expressing mice that lacked IL-4 or IL-4R (Fig. 7A, 7B). To confirm the requirement for the expression of IL-4R on the memory-like CD8 T cells, we analyzed peripheral CD8 T cells from mixed BM chimeric mice described above. We found that when IL-4R expression was deleted, CD8 T cell memory-like phenotype was absent (Fig. 7C). Taken together, these data show that IL-4α expression is required for TCF1 and β-catenin–induced memory-like CD8 T cells.

FIGURE 7.
FIGURE 7.

IL-4 or IL-4Rα–chain deficiency results in loss of memory-like phenotype of β-CAT-Tg CD8 T cells. (A) Flow cytometric analysis of the cell surface memory markers and Eomes expression by the gated CD8T cells from lymph nodes from control, β-CAT-Tg, IL-4KO, and IL-4KO-β-CAT-Tg mice; CD44 and CD62L expression (upper panel) and CD122, CXCR3, and Eomes expression (lower panel) are shown. Numbers show the percentage of cells. Data are representative of four independent experiments. (B) Flow cytometric analysis of the cell surface memory markers and Eomes expression by the gated CD8T cells from lymph nodes from control, β-CAT-Tg, IL-4RαKO, and IL-4RαKO-β-CAT-Tg mice; CD44 and CD62L expression (upper panel) and CD122, CXCR3, and Eomes expression (lower panel) are shown. Numbers show the percentage of cells. Data are representative of four independent experiments. (C) Flow cytometric analysis of the cell surface memory markers on and Eomes expression in the gated IL-4Rα− and IL-4Rα+ CD8 T population of splenocytes from β-CAT-Tg and IL-4RαKO-β-CAT-Tg 1:1 bone marrow chimera. Data are representative of two independent analyses with a total of five chimera mice.

Finally, we wanted to determine whether overexpression of β-catenin or cytokine-dependent signaling promoted memory-like features in CD8 T cells. We expressed β-catenin using a retroviral expression system in control naive CD8 T cells. We found that memory-like features were not induced upon expression of β-catenin in naive CD8 T cells from control mice (Fig. 8A). Instead, we found that treatment of naive CD8 T cells with IL-4, but not other cytokines, promoted memory-like features (Fig. 8B). Thus IL-4R–dependent signaling regulated the generation of memory-like CD8 T cells in β-CAT-Tg mice. Interestingly, activated TCF1-deficient CD8T cells treated with IL-4 failed to induce expression of Eomes, perforin, IL-4Rα and CD122, whereas expression of T-bet remained unchanged (Fig. 8C). These data support the notion that TCF1 expression is also required in a cell-intrinsic manner for the generation of CD8 memory-like cells. We propose that TCF1-dependent cell-intrinsic aspects support the development of IL-4–dependent CD8 memory-like features.

FIGURE 8.
FIGURE 8.

IL-4 signals induce memory-like gene expression in naive CD8 T cells. (A) CD8 T cells were infected with control MSCV-human CD8 vector or MSCV-β-CAT-human-CD8 construct. Human CD8+-infected CD8 T cells were sorted and the relative abundance of mRNA for transcription factors and effector molecules was analyzed by real-time RT-PCR. Data are presented relative to Actb and are from six independent samples. (B) CD122−CD44lo sorted naive CD8 T cells were treated with various cytokines and the relative abundance of mRNA for transcription factors and effector molecules was analyzed by real time RT-PCR. Data are presented relative to Actb and are from four to six independent samples. (C) Lymph node cells from control and TCF1-deficient mice were stimulated with anti-CD3 for 3 d and then rested in culture that contained IL-7 and IL-4 for 6 d. After this in vitro stimulation, CD8 T cells were purified and transcription factors and effector molecules analyzed by real-time RT-PCR. Data are presented relative to Actb and are from three independent samples.

Discussion

In this study, we demonstrate that enforced expression of β-catenin in a TCF1-sufficient background facilitates the generation of PLZF-expressing CD4+TCR-αβ+ thymocytes, which in turn induce the generation of memory-like CD8SP thymocytes. We show that expression of both IL-4 and IL-4Rα is required for the generation of TCF1 and β-catenin–dependent memory-like CD8SP thymocytes. Finally, we demonstrate that CD8 memory-like thymocytes migrate to the periphery and rapidly produce high levels of IFN-γ ex vivo, suggesting that they are functional. In light of the observation that TCF1 is highly expressed in most thymocytes, we propose that expression of β-catenin is limiting in the development of PLZF-expressing thymocytes that promote the generation of CD8 memory-like thymocytes.

We have previously reported that transgenic expression of β-catenin from the proximal Lck promoter in a TCF1-sufficient background results in increased numbers of CD8SP thymocytes that acquire a memory-like phenotype in the thymus (25). However, when β-catenin was overexpressed using the CD4 promoter, a similar phenotype was not noted (26). Data in the present study provide an explanation for this discrepancy. One major difference between expression from the proximal Lck promoter and the CD4 promoter is the timing of gene expression during thymocyte development. Proximal Lck promoter initiates gene expression as early as the DN2 stage, whereas CD4 promoter-dependent expression is first noted in post-β selection DN4/pre-DP thymocytes. We propose that timing of β-catenin stabilization during T cell development is essential to facilitate the development of PLZF-expressing thymocytes and thereby to promote the generation of memory-like CD8 thymocytes. Signals that stabilize β-catenin at these developmental stages remain to be defined. However, two possible candidates that regulate β-catenin expression might be considered. First, in light of previous reports from our laboratory that TCR (33) and pre-TCR (34) signals stabilize β-catenin, one might consider that the strength of lineage decision signals transmitted through the TCR might stabilize β-catenin to different extents, allowing for PLZF expression in a subset of thymocytes. The second possibility is the involvement of Wnt-dependent signals for β-catenin stabilization in a developmentally sensitive manner. Further research will be useful in resolving these issues.

TCF1 and β-catenin have been shown to be required for the generation of long-lived Ag-driven CD8 memory T cells (18–22). In the absence of TCF1, Ag-driven memory CD8 T cells expressed low Eomes and failed to provide adequate long-term memory, whereas double-transgenic mice overexpressing TCF1 and β-catenin show enhanced generation of memory CD8 T cells. One difference between Ag-driven CD8 memory cells and CD8 memory-like cells generated in the thymus is that the former are clonal whereas the latter are polyclonal. One common feature is TCF1 and β-catenin–dependent expression of Eomes, which in turn regulates several aspects of CD8 memory cells. Future studies will be required to determine the extent of functional overlap between these cell populations.

The memory features in bystander CD8SP thymocytes in various gene knockout models have been shown to be dependent on the PLZF+ thymocyte population (1, 8–10, 35). PLZF-expressing thymocytes were shown to produce IL-4, which was essential for the development of the memory-like CD8SP thymocytes in the absence of encounter with Ag (1, 8, 9). In the present studies, using mice that have enforced expression of β-catenin in IL-4–sufficient and –deficient background, we show that IL-4 is required for the generation of TCF1 and β-catenin–induced memory-like CD8SP thymocytes. Experiments with mixed bone marrow chimeras using a mix of equal numbers of bone marrow cells from IL-4Rα–sufficient β-CAT-Tg and IL-4Rα–deficient β-CAT-Tg mice in RAG2-deficient mice showed that in the same background only IL-4Rα–chain-expressing β-CAT-Tg CD8 thymocytes acquire the memory-like phenotype. Thus, IL-4 signals are required for the generation of memory-like CD8SP thymocytes generated when expression of β-catenin is enforced at the appropriate developmental stage.

Thus, data provided in this study show that β-catenin induction is limiting for the generation of PLZF-expressing thymocytes, which in turn promote the generation of CD8 memory-like thymocytes that migrate to the periphery and rapidly produce IFN-γ.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the National Institute on Aging animal facility for maintaining animals, S. Luo and team for genotyping, the tetramer facility of the National Institutes of Health for providing PE-conjugated mouse CD1d tetramers loaded with glycolipid PBS-57, H. Clevers (Hubrecht Institute, Utrecht, The Netherlands) for providing TCF1-deficient mice, and Z. Zhu (The Johns Hopkins Asthma and Allergy Center, Baltimore, MD) for providing IL-4Rα–deficient mice. We are grateful to Drs. Alycia Williams and Mark Soloski for help with isolation of IELs.

Footnotes

  • This work was supported by the Intramural Research Program of the National Institute on Aging at the National Institutes of Health.

  • Abbreviations used in this article:

    BM
    bone marrow
    β-CAT-Tg
    β-catenin-transgenic (mice)
    Eomes
    eomesodermin
    IEL
    intraepithelial lymphocyte
    KLF2
    Krüppel-like factor 2
    KO
    knockout
    MSCV
    murine stem cell virus
    PLZF
    promyelocytic leukemia zinc finger
    SP
    single-positive
    TCF
    T cell factor.

  • Received December 21, 2011.
  • Accepted February 12, 2012.

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