Requirements for Eomesodermin and PLZF in the development of innate-like CD8⁺ T cells

Abstract

Conventional and non-conventional T cell development occurs in the thymus. Non-conventional thymocytes that bear characteristics typically associated with innate immune cells are termed innate-like lymphocytes (ILLs). Mice harboring a tyrosine to phenylalanine mutation in the adaptor protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) at residue 145 (Y145F mice) develop an expanded population of CD8⁺CD122⁺CD44⁺ ILLs, typified by expression of the T-box transcription factor Eomesodermin (Eomes). Y145F mice also have an expanded population of γδ T cells that produce copious amounts of IL-4 via a mechanism that is dependent on the BTB-ZF transcription factor promyelocytic leukemia zinc finger (PLZF). Using mice with T cell specific deletion of Eomes, we demonstrate that this transcription factor is required for CD8⁺ ILL development in Y145F as well as WT mice. Moreover, we show that PLZF and IL-4 are also required for the generation of this ILL population. Together, these data shed light on the cell-intrinsic and cell-extrinsic factors that drive CD8⁺ ILL differentiation.

Introduction

The T cell compartment largely consists of conventional CD4⁺ and CD8⁺ T cells. These cells develop in the thymus and exit to peripheral lymphoid organs as naïve T cells. Additional populations of thymically-derived T cells have also been identified, including those with regulatory or innate-like functions. T cells constituting this latter subset are termed innate-like lymphocytes (ILLs), because they acquire effector function during development, prior to peripheral activation. Other characteristics of ILLs include specificity for non-classical MHC molecules, selection on hematopoietic cells, and expression of molecules typically associated with activated or memory T cells, such as CD44, CD122 (the β chain of the IL-15 and IL-2 receptors), and NK1.1 (reviewed in (1)). NKT cells and a variety of CD8⁺ populations including H2-M3 specific T cells, CD8αα⁺ T cells, and mucosal-invariant T cells (MAIT), have been classified as ILLs (1). ILLs have been found to participate in host defense against bacteria and viruses, and additional studies have suggested that NKT cells can aid in anti-tumor responses (2-5). In some contexts, however, ILL participation during an immune response may be disadvantageous. Several reports implicate these cells in asthma and autoimmunity, while others have linked them to negative outcomes following infection with particular organisms (5, 6).

Several transcription factors regulate thymocyte commitment to distinct T cell lineages. GATA3 and Th-POK regulate CD4⁺ T cell differentiation, Runt domain transcription factors (Runx) are critical for CD8⁺ T lineage commitment (7, 8), and Foxp3 is required for the development of CD4⁺ T regulatory cells (9, 10). Transcriptional control of non-conventional T cell development has also been studied, but our knowledge is limited largely to the NKT lineage. NKT cells are dependent on several transcription factors including, but not limited to, promyelocytic leukemia zinc finger (PLZF), Runx1, ETS1, ELF4, c-myc, RelA, and T-bet (reviewed in (11)). Transcription factors that regulate CD8⁺ ILL development are less well defined.

Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is an adaptor protein critical for thymocyte development due to its role in TCR signal transduction (12). Through its protein-protein interaction domains, SLP-76 nucleates a signaling complex containing linker of activation of T cells (LAT), growth factor receptor-bound protein 2 (GRB2)-related adapter downstream of Shc (GADS), phospholipase C (PLC)-γ1, and the Tec family tyrosine kinase IL-2-inducible T cell kinase (Itk) (13). We recently provided evidence that mutation of tyrosine 145 of SLP-76 (Y145F) results in altered thymocyte selection and in development of a population of non-conventional CD8 single-positive (SP) thymocytes (14). In addition to exhibiting such ILL hallmarks as enhanced expression of CD44 and CD122, Y145F CD8⁺ ILLs express elevated mRNA levels of Eomesodermin (Eomes)(14), a T-box transcription factor that regulates differentiation during embryogenesis and directs fate and function of effector and memory T cells (14-17). Y145F mice also have an expanded population of γδ T cells that express high levels of promyelocytic leukemia zinc finger (PLZF), a member of BTB-ZF family of transcription factors (18). These PLZF⁺ γδ T cells produce copious amounts of IL-4 (18), which has been implicated in driving Eomes expression in CD8SP thymocytes (19).

Here we find that Eomes is essential for CD8⁺ ILL development in Y145F as well as wild-type (WT) mice. We also observe that, in the presence of Y145F-derived bone marrow, WT thymocytes differentiate into Eomes⁺CD8⁺ ILLs, indicating that non-cell-autonomous factors direct CD8⁺ ILL development. Development of this population is also dependent upon PLZF and IL-4, as Y145F CD8+ ILLs do not develop in the absence of PLZF and are significantly diminished upon administration of a blocking antibody to IL-4. Taken together, our findings highlight key cell-intrinsic and cell-extrinsic factors that control the development of CD8⁺ ILLs.

Materials and Methods

Mice

Y145F, PLZF^−/− and Cd4-cre Eomes^F/F mice have been described (13, 14, 20, 21). Animals were housed at the University of Pennsylvania or Sloan-Kettering and experiments were performed in accordance with protocols approved by their Institutional Animal Care and Use Committees.

Flow cytometry and Real-time PCR

Cells were stained and analyzed as described (13, 18). Antibodies to the following proteins were from BD Pharmingen unless otherwise noted: CD3 Alexa Fluor 700, CD4 PerCP-Cy5.5, PE-Cy7 (Biolegend), or APC-eFluor 780 (eBioscience), CD8a FITC, APC, Alexa Fluor 700 (eBioscience), Pacific Blue (Biolegend), or PE-Cy7 (eBioscience), CD44 FITC, CD45.1 PE-Cy7 (eBioscience), CD45.2 FITC (eBioscience), CD122 PE or biotin, streptavidin PE-Texas Red, CD124 PE, TCRγδ PE or APC (eBioscience), CXCR3 PE, Eomes AF647 (eBioscience), mouse anti-mouse PLZF (Santa Cruz) followed by anti-mouse IgG1 FITC, IFN-γ Pacific Blue (eBioscience), IL-4 PE-Cy7 (eBioscience). Aqua LIVEDEAD (Invitrogen) was used to gate out dead cells. Real-time PCR was performed as described (14).

Ex vivo stimulation of thymocytes

Thymocytes were isolated and stimulated directly ex vivo at 37°C for 5 hours with 50 ng/mL PMA and 500 ng/mL ionomycin in the presence of 1 μg/mL Brefeldin A. Cells were then assayed for cytokine production by intracellular staining and flow cytometric analysis.

Bone Marrow Chimeras

C57BL/6-SJL (CD45.1) mice were irradiated with 950 rads and injected i.v. with a 1:1 mixture of T cell-depleted (Magnetic bead depletion, Qiagen) bone marrow (BM) from C57BL/6-SJL (CD45.1) mice and WT C57BL/6 (CD45.2) or Y145F (CD45.2) mice. Recipients were reconstituted with 4×10⁶ cells. Recipients were analyzed 9-10 weeks post transplantation.

In Vivo IL-4 Blockade

Y145F mice (2-3 weeks of age) were injected intra-peritoneally with either PBS or 1mg/mouse of neutralizing anti-IL-4 antibody (11B11) a total of three times over the course of two weeks.

Statistical Analysis

Prism (GraphPad Software) was used for statistical analysis. Student's t-test was used to assess statistical significance. For linear-regression analysis, P values indicate the significance of slope being non-zero, and r² represents the goodness of fit.

Results

SLP-76 Y145F mice generate CD8⁺ ILLs

Mice bearing a tyrosine to phenylalanine mutation at residue 145 of SLP-76 (Y145F) have defective positive and negative selection of conventional T cells (14). Additionally, these mice have a diminished CD4:CD8 ratio, owing to a greater than 2-fold increase in the absolute number of CD8SP thymocytes, accompanied by a more modest decrease in CD4SP T cell numbers (Fig. 1A). Further analysis of the CD8SP population revealed that this compartment is largely composed of CD122⁺ CD44⁺ cells (ref (14) and Fig. 1B). Compared to WT CD8SP thymocytes, Y145F CD8SP cells express elevated levels of Eomes protein and perforin mRNA, a direct target of Eomes (16). Moreover, Y145F CD8SP thymocytes produce IFN-γ following direct ex vivo stimulation, another hallmark of Eomes⁺ lymphocytes (Fig. 1C). Taken together, these data indicate that the majority of CD8SP thymocytes from Y145F mice are ILLs.

FIGURE 1.

CD8⁺ ILLs develop in Y145F mice.

A, Flow cytometric analysis of WT and Y145F thymi. Plots are gated on live thymocytes. The bar graph shows the absolute number of CD4SP and CD8SP thymocytes in WT and Y145F mice.

B, Flow cytometric analysis for CD44 and CD122 expression on WT and Y145F CD8SP thymocytes. The total number of CD8SP CD122⁺CD44⁺ thymocytes from WT and Y145F mice are shown in the bar graph (n>8).

C, Flow cytometric analysis for Eomes expression in WT and Y145F CD8SP thymocytes (n=8). Quantitative real-time reverse-transcription PCR for perforin mRNA in purified WT and Y145F CD8SP CD69⁺ thymocytes is shown in the bar graph (n=3). The right FACS plots show intracellular detection of IFN-γ in WT and Y145F thymocytes stimulated with PMA/Ionomycin (n=8).

CD8⁺ ILL fate in Y145F mice is conferred by cell-extrinsic factors

Development of conventional CD8⁺ thymocytes depends on both cell-intrinsic transcriptional programs and cell-extrinsic factors, such as cytokines (22). To determine whether CD8⁺ ILL development in Y145F mice is driven by cell-intrinsic or cell-extrinsic factors, mixed bone marrow (BM) chimeras were generated in which WT (CD45.1) and Y145F (CD45.2) BM were mixed and transplanted into lethally-irradiated WT (CD45.1) hosts. WT (CD45.1):WT (CD45.2) mixed BM chimeras served as controls. Percent chimerism was defined as the percent CD45.2⁺CD4⁻CD8⁻ (double negative, DN) thymocytes of total DN thymocytes. When mixed with Y145F donor BM, the percent of Eomes⁺ CD8SP thymocytes derived from WT BM was equivalent to the percent derived from Y145F donor BM (Fig. 2A and B). Thus, Y145F-derived cells are capable of directing WT thymocytes to adopt an ILL phenotype. The extent of CD8⁺ ILL development in the mixed BM chimeras positively correlated with the relative contribution of Y145F cells (Fig. 2C). These data are consistent with a model in which CD8⁺ ILL development in Y145F mice is driven by cell-extrinsic factors.

FIGURE 2.

Cell-extrinsic factors govern CD8⁺ ILL development, but cell-intrinsic factors control enhanced PLZF⁺ γδ T cell generation in Y145F mice. Analysis of mixed bone marrow chimeras (n=7).

A, Eomes expression in WT (CD45.1⁺) or WT (CD45.2⁺) CD8SP thymocytes (left panels) and in WT (CD45.1⁺) or Y145F (CD45.2⁺) CD8SP thymocytes (right panels).

B, Percent Eomes⁺ of WT (CD45.1⁺) or Y145F (CD45.2⁺) CD8SP thymocytes from all chimeras.

C, Linear regression analysis of the percent Eomes⁺ of CD8SP thymocytes in WT- or Y145F-derived populations as a function of the percent Y145F chimerism—defined as percent of CD45.2⁺ DN thymocytes. r² values describe the linear relationship (goodness of fit) between the percent Y145F chimerism and CD8⁺ ILL development. P values indicate whether the slope of the regression line is significantly different from zero.

D, PLZF and γδ TCR expression in WT (CD45.1⁺) or WT (CD45.2⁺) thymocytes (left panels) and in WT (CD45.1⁺) or Y145F (CD45.2⁺) thymocytes (right panels). E, Percent PLZF⁺ γδ⁺ of total WT (CD45.1⁺) or Y145F (CD45.2⁺) thymocytes from all chimeras.

F, Linear regression analysis of the percent PLZF⁺ γδ⁺ thymocytes in WT- or Y145F-derived populations as a function of the percent Y145F chimerism, as defined in C.

One explanation for our observation that Y145F thymocytes induce the ILL fate in WT CD8⁺ thymocytes in a dose-dependent fashion could be the presence of a cytokine-producing “inducer” cell that is more abundant among Y145F than WT thymic cells. IL-4 has been previously found to positively regulate Eomes expression (23). Eomes⁺ CD8SP cells from Y145F mice express high levels of CXCR3 and IL-4 receptor (supplemental Fig. 1), consistent with data implicating IL-4 signaling in upregulation of these receptors (19, 24). Additionally, Y145F mice contain an expanded population of γδ T cells that produce IL-4 in a PLZF-dependent manner (18, 25). Thus, we analyzed WT:Y145F chimeras for the presence and origin of PLZF⁺ γδ⁺ cells. In these mice, a greater frequency of Y145F-derived precursors developed into PLZF⁺ γδ⁺ thymocytes compared to WT-derived precursors (Fig. 2D and E). Further, the degree of Y145F chimerism did not affect the percent of Y145F- or WT-derived PLZF⁺ γδ T cells within the thymus (Fig. 2F). Unlike induction of Eomes and commitment to the CD8⁺ ILL lineage, enhanced development of PLZF⁺ γδ T cells among Y145F thymocytes appears to be driven by cell-intrinsic factors.

PLZF and IL-4 direct the development of CD8⁺ ILLs

To determine if PLZF⁺ cells play a role in establishing the CD8⁺ ILL fate in Y145F mice, PLZF^−/−Y145F mice were generated. In PLZF^−/−Y145F mice, the frequency and number (data not shown) of CD8SP thymocytes expressing Eomes, CD44 and CD122, and capable of rapid IFN-γ production was reduced to WT levels (Fig. 3A and B). Analysis of the function of γδ T cells in PLZF^−/−Y145F mice revealed a dramatic reduction in IFN-γ and IL-4 production (Fig. 3C). These data are consistent with reports showing PLZF induces innate-like characteristics, including the ability to produce cytokines, within γδ⁺ subsets (18, 25). These data demonstrate that PLZF is essential for the development of CD8⁺ ILLs in Y145F mice.

FIGURE 3.

CD8⁺ ILL development in Y145F mice is dependent on PLZF.

A, Eomes expression in CD8SP thymocytes from the indicated genotypes (n=4 mice per genotype for A-C).

B, IFN-γ in CD8SP thymocytes from indicated genotypes, stimulated in vitro with PMA/ionomycin.

C, IL-4 and IFN-γ production in TCRγδ⁺ thymocytes from indicated genotypes, stimulated in vitro with PMA/ionomycin.

Next, we directly addressed whether IL-4 regulates CD8⁺ ILL development in Y145F mice. To this end we dampened IL-4 signaling in vivo by treating mice with neutralizing antibodies against IL-4. Compared to Y145F mice injected with PBS, Y145F mice receiving anti-IL-4 antibody treatment exhibited an increase in the thymic CD4:CD8 ratio (Fig. 4A). IL-4 blockade also resulted in a decreased percentage of CD8SP thymocytes expressing classic markers of ILLs, including CD44, CD122, and Eomes (Fig. 4B and C). Taken together, these data are consistent with a model in which IL-4, likely derived from a PLZF⁺ T cell population, induces Eomes⁺CD8⁺ ILL development. This model is consistent with recent reports that show IL-4 signaling and PLZF are required for CD8⁺ ILL development in other model systems (25, 26).

FIGURE 4.

IL-4 blockade diminishes the CD8⁺ ILL compartment in Y145F mice.

A, Flow cytometric analysis of thymi from Y145F animals treated with PBS or with blocking anti-IL-4 antibody (n=4 mice per group for A-C).

B, Flow cytometric analysis for CD44 and CD122 expression on CD8SP thymocytes from indicated mice.

C, Flow cytometric analysis for Eomes expression on CD8SP thymocytes from indicated mice.

Eomes is required for CD8⁺ ILL development in Y145F and WT mice

Given the role of Eomes in directing cell fates (16, 17), Eomes^F/FCd4-Cre⁺ mice were bred to Y145F mice to test whether ILL development in Y145F mice is dependent on Eomes. Deletion of Eomes in the T cell compartment of Y145F mice restored the percent and number of Y145F CD8SP thymocytes to levels seen in WT mice (Fig. 5A and supplemental Fig. 2A). The CD4:CD8 ratio in Y145F Eomes^F/FCd4-Cre⁺ mice was increased compared to Y145F mice, although it was not restored to WT levels (supplemental Fig. 2B). Failure of Y145F Eomes^F/FCd4-Cre⁺ mice to achieve a completely normalized CD4:CD8 ratio is likely due to an Eomes-independent effect of the Y145F mutation on the selection of conventional CD4SP thymocytes.

FIGURE 5.

Eomesodermin is required for CD8⁺ ILL development in Y145F mice.

A, Flow cytometric analysis of thymi from indicated genotypes. Plots gated on live thymocytes (n=3-10 mice per genotype for A-C).

B, CD44 and CD122 expression on CD8SP thymocytes from indicated genotypes.

C, IFN-γ in thymocytes of indicated genotypes stimulated with PMA/ionomycin.

Within the CD8SP compartment, it was the selective loss of CD8⁺ CD122⁺CD44⁺ cells that accounted for the decrease in the total number of CD8SP cells in Y145F Eomes^F/FCd4-Cre⁺ mice (Fig. 5B and supplemental Fig. 2C). Additionally, ILL generation was sensitive to Eomes hemizygosity, as Eomes^F/+Y145FCd4-Cre⁺ mice have an intermediate phenotype with respect to CD8⁺ ILL numbers (supplemental Fig. 2A-C). The loss of CD8⁺ ILLs correlated with a loss of IFN-γ-producing CD8SP thymocytes in the absence of Eomes (Fig. 5C). These data support a requirement for Eomes in CD8⁺ ILL development in Y145F mice.

While CD8⁺ ILLs are clearly apparent and dependent on Eomes in Y145F mice, it remained to be seen if this population represented an Eomes-dependent population found normally in the thymus of WT mice. To address this issue, we examined thymi of WT and Eomes^F/FCd4-Cre⁺ mice for the presence of CD8⁺ ILLs. We found a significant decrease in the percentage of CD8⁺CD122⁺CD44⁺ ILLs in Eomes^F/FCd4-Cre⁺ thymi, relative to WT thymi (Fig. 6A and B). The percent of IFN-γ-producing CD8⁺ cells was also significantly decreased in Eomes-deficient thymocytes, compared to their WT counterparts (Fig. 6C). Taken together, these data are consistent with an essential role for Eomes in the development of CD8SP cells with phenotypic and functional properties of ILLs in WT mice.

FIGURE 6.

Eomes regulates CD8⁺ ILL development in wild-type mice.

A, Flow cytometric analysis of thymi from WT and Eomes^F/FCd4-Cre⁺ mice (n=6 mice per group for A-C).

B, Percent CD122⁺CD44⁺ cells of total CD8SP thymocytes from indicated genotypes.

C, Percent IFNγ-producing cells of total CD8SP thymocytes from indicated genotypes after stimulation with PMA/Iono. Expression of IFN-γ assessed by flow cytometric analysis.

Discussion

In this report, we have used a model of altered T cell signaling to highlight a population of CD8⁺ ILLs found normally in WT mice. We show that development of the CD8⁺ ILLs present in Y145F mice require Eomesodermin and PLZF and propose that IL-4 may serve as the CD8⁺ ILL-extrinsic factor linking the function of these two transcription factors. We previously demonstrated that mutation of Y145 of SLP-76 drives the enhanced development of a population of γδ T cells that express high levels of PLZF, and that PLZF is responsible for IL-4 production by these cells (18). We show here that IL-4 is required for Eomes upregulation and development of the CD8⁺ ILL phenotype. These results are consistent with the extrinsic nature of CD8⁺ ILL generation (Fig. 2) and the reported role of IL-4 in Eomes regulation (23). Our findings are also in agreement with recent reports suggesting that IL-4 production by PLZF⁺ non-conventional T cells results in elevated numbers of CD8+ ILLs in the setting of Id3 or KLF2 deletion (26, 27). Additionally, in an independent study, Eomes was shown to be required for IL-4-mediated upregulation of CXCR3 in bystander CD8SP thymocytes developing in the context of Klf2 deficiency, suggesting a cell-intrinsic requirement for Eomes during CD8⁺ ILL development (19). We find that Eomes and PLZF expression are mutually exclusive in WT and Y145F thymocytes and that PLZF expression is maintained in Eomes^F/FCd4-Cre⁺ mice (supplemental Fig. 3). Preliminary data also indicate that PLZF expression is not altered in Y145F Eomes^F/F Cre⁺ mice, consistent with a CD8⁺ ILL intrinsic role for Eomes (S.M.G., unpublished observations). However, it remains to be formally tested whether Eomes is required for the development or survival of an IL-4-producing “inducer cell”. Similarly, while we have not observed expression of PLZF in CD8⁺ ILLs in Y145F mice (data not shown), we have not formally ruled out the possibility that a precursor of the CD8⁺ ILL expresses and is dependent upon PLZF.

Our proposed mechanism of ILL development in Y145F mice—a pathway intrinsically dependent on Eomes and extrinsically driven by a PLZF dependent IL-4 producing population—may be more universal, as similar findings have been observed in Itk^−/− and conditionally Cbp^−/− mice, which also develop a preponderance of CD8⁺ ILLs (26, 28-30). The link between the SLP-76 mutant- and Itk deficient-phenotypes may rest in the functional relationship between these two molecules, as SLP-76 Y145 is a binding site for Itk, and this tyrosine is critical for inducible Itk activation following TCR ligation (31). In addition to Y145F and Itk^−/− mice having increased numbers of CD8⁺ ILLs, the frequency of PLZF⁺ γδ thymocytes capable of producing IL-4 is increased in both mouse strains (14, 18, 32, 33). Our studies have demonstrated that the loss of PLZF or blockade of IL-4 signaling in Y145F mice impairs CD8⁺ ILL development. Taken together, these data are consistent with the possibility that PLZF⁺ γδ cells serve as the source of IL-4 that drives the ILL phenotype, though formal testing of the hypothesis is required. Moreover, how mutation of the SLP-76/Itk signaling axis influences PLZF⁺ γδ T cell development remains to be elucidated.

Eomes regulates several of the cell surface phenotypes and functions that define CD8⁺ ILLs, such as CD122 and perforin expression, as well as IFN-γ production (16, 34). One prediction for these studies was that deletion of Eomes in Y145F mice would not alter the number of CD8SP thymocytes, only their phenotype. However, we found that Eomes deficiency resulted in not only the loss of CD8SP thymocytes with an innate-like phenotype, but also a decrease in the absolute number of CD8SP cells. This result is similar to the impact IL-15 deficiency has on CD8⁺ ILL numbers in Itk^−/− mice (28). IL-15 signaling is dependent on CD122, thus it is possible that Eomes regulates the survival of CD8⁺ ILLs through its regulation of CD122 expression. Alternatively, other Eomes-regulated genes may control the differentiation and/or survival of this innate-like population.

Innate-like lymphocytes contribute both positively and negatively to the outcome of immune responses. CD8⁺ ILLs generated in Itk^-/- mice have been shown to contribute to the low bacterial burden observed in these mice following Listeria infection (35). How the CD8⁺ ILL population from Y145F mice and other gene deletion models influences additional immune responses is unknown. Studies focused on this question and how Eomes contributes to these outcomes may provide insight into how the immune response can be manipulated to enhance its effectiveness. Additionally, understanding Eomes and PLZF regulation during thymocyte development will aid in our understanding of conventional and non-conventional T cell development.

Abbreviations

ILL

Innate-like lymphocyte

BM

bone marrow

Eomes

Eomesodermin

PLZF

Promyelocytic leukemia zinc finger

SLP-76

Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa

SP

single positive

DN

CD4⁻CD8⁻ double negative

WT

wild type