Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)–signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)–signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok−/− thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

Visual Abstract

Functionally competent mature αβ T cells play a central role in the cell-mediated immune responses (14). Development of these cells in the thymus is an ordered process consisting of distinct differentiation stages defined by the expression of CD4 and CD8 coreceptors. Precursor thymocytes are CD4CD8 double negative (DN), which following pre-TCR–transduced signaling, differentiate into CD4+CD8+ double positive (DP) thymocytes. The DP thymocytes expressing a low level of TCRαβ receptor and the associated CD3 chains undergo thymic selection such that those expressing high-affinity TCR for self-peptide/self-MHC (pMHC) are negatively selected, whereas those expressing low-affinity TCR for pMHC are positively selected (57). Positively selected thymocytes further differentiate into MHC class II (MHCII)–specific CD4+ helper and MHC class I (MHCI)–specific CD8+ cytotoxic mature thymocytes that populate the peripheral lymphoid organs (810). How pMHC specificity of TCR/coreceptor translates into MHCII-specific CD4 helper and MHCI-specific CD8 cytotoxic lineage choice is not completely understood.

The CD4/CD8 binary lineage fate decision is strongly influenced by the duration and intensity of TCR signaling. A widely accepted kinetic signal strength model posits that positively selected DP thymocytes, irrespective of their MHC specificity, transcriptionally terminate Cd8 expression and become lineage uncommitted CD4+CD8lo thymocytes (8, 11, 12). Continued Cd4 transcription at this stage induces a sustained/stronger signal in MHCII-specific thymocytes, leading to an error-free CD4 helper lineage choice (13), whereas the downregulation of CD8 results in a disrupted/weaker signal in MHCI-specific thymocytes leading to CD8 cytotoxic lineage choice. Lck, a Src family tyrosine kinase essential for T cell development, is strongly associated with the cytoplasmic tail of CD4 than that of CD8 (14, 15). Thus, increased Lck activity due to continued CD4 expression then results in stronger TCR signaling in MHCII- than in MHCI-specific thymocytes (16, 17). Indeed, altered Lck activity is shown to direct positively selected thymocytes into alternate lineages (17, 18).

Induction of ThPOK (encoded by ZBTB7B, hereafter referred to as Thpok) in MHCII-signaled thymocytes is both necessary and sufficient for the CD4 helper lineage commitment (19). Similarly, Runx3 induction in MHCI-signaled thymocytes establishes a cytotoxic program in the CD8-committed thymocytes (12). ThPOK is proposed to suppress Runx3 expression and thereby impair the initiation of the cytotoxic program in MHCII-signaled thymocytes, leading to the CD4 helper lineage choice (1922). Accordingly, the loss and gain of ThPOK function results in the production of MHCII-specific CD8+ cytotoxic and MHCI-specific CD4+ helper T cells, respectively (23, 24). The Thpok silencer-mediated heritable epigenetic modifications control ThPOK expression in the signaled thymocytes and is suggested to play an important role in the CD4/CD8 lineage choice (25). These studies suggest that ThPOK induction during a temporal developmental window is critical for the CD4 helper lineage choice (25). Persistent TCR signaling in MHCII-specific thymocytes is proposed to reverse silencer-induced epigenetic modifications at the Thpok locus, leading to stable ThPOK expression, which then suppresses the cytotoxic program and thereby commits these cells into the CD4 helper lineage (21, 26). Based on these and other studies, it is proposed that persistent TCR signaling leading to ThPOK induction and the extent of this induction during a temporal lineage commitment window affects the CD4/CD8 lineage fate of positively selected thymocytes (22, 25, 27, 28). Although published data suggest that developmental constrain on the CD4 helper lineage commitment of MHCI-signaled thymocytes can be overcome by enforced ThPOK expression (23, 24), several questions remain to be addressed. For example, it is not clear why ThPOK induction in MHCI-signaled thymocytes lacking Tle proteins, which disrupt Runx3 function, or Runx1 and Runx3 or MAZR and Runx3 results in an incomplete CD8 to CD4 helper lineage redirection or the generation of “confused” DP mature T cells (2931). Further, the role of TCR signaling in ThPOK-induced CD4 helper lineage choice of MHCII- and MHCI-signaled thymocytes is not evaluated. Is suppression of the cytotoxic program in itself sufficient for establishing the CD4 helper lineage? Thus, it remains to be investigated if the CD4 helper lineage choice, irrespective of MHC specificity, requires the same level of ThPOK or if it is also influenced by TCR signaling in MHCI- versus MHCII-specific thymocytes.

In the present investigation, we show that the efficiency of CD4 helper lineage commitment of MHCI-signaled thymocytes is proportional to ThPOK dose. Further, a ThPOK dose that induced the partial CD8 to CD4 helper lineage redirection of MHCI-signaled thymocytes expressing monoclonal or polyclonal TCRs completely restored the CD4 helper lineage commitment of MHCII-signaled thymocytes expressing monoclonal or polyclonal TCRs in ThPOK-deficient mice. Importantly, this differential ThPOK-induced CD4 helper lineage commitment correlated, at least in part, with TCR signal strength as augmenting TCR signaling significantly enhanced the CD4 helper lineage choice of MHCI-signaled thymocytes; however, it was still significantly less efficient than the CD4 helper lineage choice of MHCII-signaled cells. Together, our results provide crucial insights into the mechanism of ThPOK-induced CD4 helper lineage choice of thymocytes specific for disparate MHC and a critical role for TCR signaling in this process.

MHCI-restricted OTI+Rag−/− (chicken OVA Ag-specific) and P14+TCRα−/− (lymphocytic choriomeningitis virus GP33–) transgenic mice were obtained from Taconic Biosciences or Nathalie Labrecque (Centre de Recherche Hopital Maisonneuve-Rosemont). MHCII-specific OTII+Rag−/− mice were from The Jackson Laboratory. MHCII−/− and Nur77-GFP mice (32) were obtained from The Jackson Laboratory. All TCR transgenic mice were in Rag-deficient background unless mentioned otherwise. ThPOK transgenic mice were generated by cloning the genomic DNA encompassing the two coding exons flanking an intron into human CD2 expression vector. The following primers were used for cloning the ThPOK transgene: forward primer 5′-GGCGGAATTCCCAGGGAAGCAGAAGATGGGGAGCCCCGAGGA-3′ and reverse primer 5′-GCCCTTCCCCGGGCTTTTAAGAGGACTCCATGGCACC-3′ (ThPOK sequence is underlined, and ThPOK start codon in the forward primer is in bold letters). The PCR product was digested with EcoRI and XmaI restriction enzymes, and agarose gel purified and cloned into the EcoRI and XmaI cut hCD2 expression vector. The cloned DNA insert was sequenced to ensure fidelity of the ThPOK coding sequence. DNA was digested to release the insert from the vector backbone, and agarose gel purified DNA devoid of the vector backbone was injected into the fertilized mouse eggs. Three independent founder lines were established, and all of them showed an increased frequency of CD4+ mature T cells and severely reduced number of CD8+ mature T cells in the lymphoid organs. ThPOK-deficient mice were generated in the laboratory or acquired from Dan Littman (New York University). Constitutively active Lck transgenic mouse line, dLGF, is described elsewhere (17) and was obtained from Paul Jolicoeur (33). Mice were genotyped by peripheral blood analysis and/or PCR of genomic DNA isolated from tail snippets. Lymphoid organs harvested from 5–7-wk-old mice were analyzed. Any mice that showed signs of ThPOK-induced thymic leukemia (34), usually observed in >12-wk-old mice, were excluded from the analysis. All mice were housed under specific pathogen-free conditions at the Centre de Recherche Hopital Maisonneuve-Rosemont. Animal care was approved by the Institutional Animal Care Committee in accordance with the Canadian Committee on Animal Care.

A total of 1 × 106 thymocytes or RBC-depleted spleen cells or stimulated T cells were incubated with a combination of fluorescently labeled Abs to CD4 (GK1.5), CD8 (53-6.7), TCRβ (H57-957), CD5 (53-7.3), CD69 (H1.2F3), CD24 (M1/69), CD44 (IM7), CD62L (MEL-14), NK1.1 (PK136), CD154 (MR1), IFN-γ (XMG1.2), IL-4 (11B11), Vα2 (B20.1), Vβ5 (MR9-4), ThPOK (D9V5T) or donkey anti-rabbit secondary Ab (Poly4064), phospho-Src (pY418; clone K98-37), and phospho-CD3z (pY142, clone 3ZBR4S) and analyzed by flow cytometry using LSRFortessa X-20 (BD Biosciences) or LSR II (BD Biosciences). Abs were obtained from eBioscience, BioLegend, or Cell Signaling Technology. For ThPOK staining, the human Foxp3 staining kit (eBioscience) was used for cell fixation and permeabilization using the manufacturer’s protocol. Data were analyzed using FlowJo software (Tree Star). Gating strategy involving TCR transgenic mice is shown in Fig. 1. Unless mentioned otherwise, this flow cytometry gating strategy was used for the analysis of all the thymic and splenic T cells described in the manuscript.

Various thymic or splenic T cell subsets were FACS purified, and total RNA was isolated using TRIzol (Invitrogen). cDNA were synthesized using a commercial kit (Bio-Rad Laboratories). Quantitative PCR (QPCR) for Thpok, Runx3d, Socs1, Nur77, Perforin, St8sia6, St3gal2, Cxxc5, and endogenous Thpok was performed in triplicate using SYBER green dye (Bio-Rad Laboratories) or EvaGreen (Abcam). Amplification of housekeeping gene Hprt served as an internal control. QPCR data were analyzed by Applied Biosystems software ABI 7500 v2.0.5. Data were normalized to Hprt expression in each population. Relative expression values were calculated using ΔΔ cycle threshold method. Ratio of gene-specific values to housekeeping gene for wild-type (WT) or OTI reference subset was treated as one. Data are presented as an average of triplicate values and SD. The following QPCR primers were obtained from the Integrated DNA Technologies or designed in our laboratory: total Thpok, 5′-TGTCACAAGATAATCCACGGG-3′ and 5′-GGTCGTAGCTATGCAGGAAG-3′; Runx3d, 5′-CGACATGGCTTCCAACAG-3′ and 5′-CGGCGGAGTAGTTCTCATC-3′; Socs1, 5′-CAGAAAAATGAAGCCAGAGACC-3′ and 5′-ATTCCACTCCTACCTCTCCAT-3′; Nur77, 5′-CCATGTGCTCCTTCAGACAG-3′ and 5′-GCTCTGGTCCTCATCACTG-3′; Perforin, 5′-GTACAACTTTAATAGCGACACAGTA-3′ and 5′-AGTCAAGGTGGAGTGGAGGT-3′; endogenous Thpok 5′-CCTCAGCGTTCAGGAGAAGAT-3′ and 5′-GCTGCTGTGGTCTGGGAAT-3′ (sequence unique for endogenous Thpok is underlined); St8sia6, 5′-CCACCTCGTAGCTCATGTTAG-3′ and 5′-CGGCAAGCAGAAGAATATGAC-3′; St3gal2, 5′-GGTGTTGTGTGACTTGAATTGG-3′ and 5′-GTTTGACAGCCACTTTGACG-3′; Cxxc5, 5′-ATCACTGAAACCACCGGAAG-3′ and 5′-TTGTAGGAACCGAAAGACTGG-3′; Hprt, 5′-CCTCATGGACTGATTATGGACAG-3′ and 5′-TCAGCAAAGAACTTATAGCCCC-3′; and Thpok transgene copy number, 5′-TTGAGGCTGTGGTGGTGGCAGT-3′ and 5′-GGTGAGGAAGAAGAGGAGGA-3′.

Mature T cell subsets from spleen of OTI (CD8+) and OTI mice expressing specific ThPOK transgene (CD4+, CD8+, and DN) mice were purified and cultured in the presence of irradiated (2500 rad) BL/6 splenocytes pulsed with cognate OVA peptide (SIINFEKL) for 5–7 d. Purified mature T cell subsets from WT (CD4+ and CD8+) and ThPOK-H+MHCII−/− (CD4+, CD8+, and DN) mice were stimulated with irradiated splenocytes obtained from BALB/c mice. In some cases, purified T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (in suspension). The stimulated cells were stained with a combination of CD69, CD154, CD4, CD8, and TCRβ-specific Abs and analyzed by flow cytometry. For cytokine staining, the activated T cells were restimulated with PMA plus ionomycin in the presence of brefeldin for 4 h, surface stained, fixed in 2% paraformaldehyde, permeabilized, washed and stained with anti–IL-4 and anti–IFN-γ Abs, and analyzed by flow cytometry.

FACS-sorted thymic subsets were cultured in 96-well flat-bottom plates at a concentration of 1 × 106 cells/ml. Cultures were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% (vol/vol) FBS, l-glutamine (2 mM), 2-ME (50 μM), streptomycin (100 mg/ml), penicillin (10 U/ml), and IL-7 (1 ng/ml). After 2 d of culture, cells were collected and analyzed by flow cytometry.

For luciferase reporter assay, the promoter sequence of Actin and Nur77 was cloned into the EcoRV and HindIII cut pGL4.17 vector (Promega). The promoter sequence was amplified using genomic DNA and the following primers: Nur7 promoter, 5′-TCGCCGGTCGACTCGATATCAGGAGATGGAGTTCGATGGCCC-3′ and 5′-GTCGCCTCTAGATCAAGCTTACCAAGCACCTTGCAGACCCTTC-3′; and Actin promoter, 5′-GGGGTGGCCGGTACCAGAGACACTAGCTAACGGCCC-3′ and 5′-GGGCCCGGGAAGCTTCTGGTGGCGGGTGTGGACCGG-3′.

The promoter-reporter DNA was cotransfected with either a ThPOK-YFP or control YFP plasmid (pMSCV) using the lipofectamine 2000 (Invitrogen) at a ratio of 3:1 (promoter-reporter to YFP) in 293T HEK cells. Twenty-four hours after transfection, an equal number of YFP-expressing cells were seeded in a 96 flat-bottom plate. Socs1 promoter-driven luciferase plasmid was used as a positive control (kind gift of Hyun Park, National Institutes of Health). Luciferase activity was measured 48 h after transfection using the Luciferase Assay System (Promega).

Statistical analysis was performed using GraphPad Software or Microsoft Excel software. Data are displayed as a mean with SD error bar. Unpaired two-tailed Student t test was used for determining the statistical significance when thymic and splenic T cell subsets from different mice were compared. For experiments involving a comparison of T cell subsets isolated from the same mouse, a paired Student t test was used for evaluating the statistical significance. A p value ≤ 0.05 was considered statistically significant (*p ≤ 0.05, **p ≤ 0.005, and ***p ≤ 0.0005).

To investigate if ThPOK-mediated suppression of the cytotoxic program in MHCI-signaled thymocytes is in itself sufficient for inducing the CD4 helper lineage program and the role of TCR signaling and MHC specificity in this process, we generated three independent ThPOK founder lines (ThPOK-H, ThPOK-163, and ThPOK-611) in which ThPOK expression is driven by human CD2 promoter/enhancer cassette (35). All the progenies of three ThPOK founders showed, in agreement with the previously published reports (23, 24), increased and decreased frequencies of CD4+ and CD8+ mature T cells, respectively, in the lymphoid organs (Supplemental Fig. 1). Although CD8+ mature T cells in the spleen (TCR+) and thymus (CD24CD69TCR+) of ThPOK-611+ and ThPOK-163+ mice were almost completely absent, we consistently detected a small number of CD8+ mature T cells in the lymphoid organs of ThPOK-H+ mice (Supplemental Fig. 1A, 1B). Accordingly, compared with WT control, CD4/CD8 ratio of mature T cells increased by ∼20-fold in ThPOK-H+ mice and >100-fold in ThPOK-163+ and ThPOK-611+ mice (Supplemental Fig. 1C).

To investigate the basis of differential CD4/CD8 phenotype of the three transgenic mice, we analyzed ThPOK protein expression by intracellular staining. In WT mice, the basal ThPOK staining observed in preselection DP thymocytes increased as signaled thymocytes matured into CD4+CD8lo and CD4+ thymocytes (Supplemental Fig. 1D). This ThPOK-specific staining pattern in WT thymic subsets is in agreement with ThPOK induction in MHCII- but not MHCI-signaled thymocytes and its continued expression in CD4+ mature T cells (23). Importantly, compared with WT control, significantly higher ThPOK expression was observed in all the thymic subsets, including the preselection DP thymocytes from the three ThPOK transgenic mice (Supplemental Fig. 1E, 1G), which correlated with the observed CD4/CD8 phenotype in the thymus of these mice. Interestingly, ThPOK levels in DP thymocytes showed a hierarchical pattern with that in ThPOK-611 > ThPOK-163 > ThPOK-H; DP thymocytes from ThPOK-611+ mice showed a significantly higher ThPOK level compared with ThPOK-H+ DP thymocytes (Supplemental Fig. 1G). Similar to the thymic subsets, significantly higher ThPOK expressed was observed in the splenic CD4+ mature T cells from the three transgenic mice compared with WT CD4+ mature T cells (Supplemental Fig. 1F, 1G). The differential ThPOK staining in thymocytes from the three transgenic mice was not correlated with transgene copy number (Supplemental Fig. 1H). Interestingly, variegated ThPOK expression observed in the preselection DP thymocytes was lost as the signaled thymocytes matured as judged by largely uniform ThPOK staining in CD4+CD8lo and CD4+ thymocytes and CD4+ splenic T cells (Supplemental Fig. 1E, 1F) from the three ThPOK transgenic mice; a small number of CD4+CD8lo and CD4+ thymocytes and mature T cells, particularly from ThPOK-H+ and ThPOK-163+ mice, showed a slightly lower ThPOK staining. At present, the reason for this change in ThPOK expression pattern in DP thymocytes versus mature T cells from these mice is not clear. Irrespective, we consistently observed ∼1.5–2-fold more ThPOK expression in CD4+ mature T cells from the spleen of three transgenic mice compared with that in CD4+ mature T cells from the spleen of WT mice.

To evaluate the impact of differential ThPOK levels on the CD8 to CD4 helper lineage redirection, we bred the three ThPOK transgenic lines to mice expressing MHCI-restricted OTI-TCR (Vβ5+Vα2+; all mice Rag−/−). In these mice, intrathymic signaling in MHCI-specific thymocytes does not induce endogenous ThPOK expression and thus allows us to study the role of transgenic ThPOK expression in the CD8 to CD4 helper lineage redirection. Indeed, we observed a ThPOK dose-dependent impact on the CD8 to CD4 helper lineage redirection in OTI mice expressing each ThPOK transgene as judged by the hierarchical pattern of the CD4+ mature T cell frequency in the thymus and spleen of these mice (Fig. 1A, 1B) with that in OTI+ThPOK-611+ > OTI+ThPOK-163+ > OTI+ThPOK-H+ mice. Thus, there were only 16% Vα2+CD4+ mature T cells in the spleen of OTI+ThPOK-H+ mice, whereas it was 44% in OTI+ThPOK-163+ and 68% in OTI+ThPOK-611+ mice compared with <1% in OTI control mice (Fig. 1A). An increase in the CD4+ mature T cell frequency observed in the spleen was also observed in the thymus of these mice, indicating an efficient lineage redirection in OTI+ThPOK-611+ mice compared with partial lineage redirection in OTI+ThPOK-163+ and OTI+ThPOK-H+ mice (Fig. 1B). As expected, the frequency of CD8+ mature T cells in the thymus and spleen showed an opposing pattern (Fig. 1A, 1B), resulting in the CD4/CD8 ratio in OTI+ThPOK-611+ mice significantly higher than that in OTI+ThPOK-163+ or OTI+ThPOK-H+ mice (Fig. 1C). We also noticed a significant number of DN mature T cells in OTI+ThPOK-H+ and OTI+ThPOK-163+ mice; as many as 25–40% of total splenic T cells were DN in these mice (Fig. 1A). The DN mature T cells in OTI+ThPOK-H+ mice did not express NK1.1 and were CD62LhiCD44lo, indicating that they were not innate or memory T cells (Supplemental Fig. 2A) (36, 37).

FIGURE 1.

The ThPOK dose impacts the CD8 to CD4 helper lineage redirection. To assess the impact of individual ThPOK transgene on the CD8 to CD4 helper lineage redirection, each ThPOK transgenic line was introduced into OTI+Rag−/− mice and T cells were analyzed by flow cytometry. (A) The frequency of mature T cells (TCRβ+Vα2+) and CD4/CD8 profiles of splenic T cells in OTI mice expressing the indicated ThPOK transgene are shown. (B) CD4/CD8 and CD69/TCR profile of total thymocytes, CD69/CD24 profile of TCR+ thymocytes, and CD4/CD8 profile of mature thymocytes (CD69-CD24-TCR+) from the indicated strain of mice are shown. (C) The CD4/CD8 ratio for Vα2+ T cells from the spleen (left) and mature thymocytes (right) for the indicated strain of mice is shown. (D) ThPOK protein levels in DP, CD4+8lo, mature CD4+, and CD8+ thymocytes (CD69CD24TCR+; top histograms), and splenic T cell subsets (bottom histograms) from the indicated strain of mice are shown. Numbers in histograms represent the mean fluorescence intensity (MFI) values. (E) Compilations of ThPOK MFI for the indicated thymic and splenic T cell subsets from the indicated mice are shown. For DP thymocytes, ThPOK MFI is relative to that in DP thymocytes from ThPOK-H. For all other thymic subsets, ThPOK MFI is relative to the corresponding WT thymic subset. ThPOK MFI for splenic subsets is relative to that in splenic CD4+ mature T cells from WT mice. ThPOK MFI for CD4+ mature T cells from OTI mice and DN mature T cells from OTI and WT mice are ND because of the lack of a substantial number of these cells. (F) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in the indicated mice are shown. Also shown are the total splenocytes and the frequency and number of splenic T cells and T cell subsets from OTI mice expressing or not the indicated ThPOK transgene (n > 12). Data are representative examples of four or more independent experiments (A, B and D). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005. n/d, not determined.

FIGURE 1.

The ThPOK dose impacts the CD8 to CD4 helper lineage redirection. To assess the impact of individual ThPOK transgene on the CD8 to CD4 helper lineage redirection, each ThPOK transgenic line was introduced into OTI+Rag−/− mice and T cells were analyzed by flow cytometry. (A) The frequency of mature T cells (TCRβ+Vα2+) and CD4/CD8 profiles of splenic T cells in OTI mice expressing the indicated ThPOK transgene are shown. (B) CD4/CD8 and CD69/TCR profile of total thymocytes, CD69/CD24 profile of TCR+ thymocytes, and CD4/CD8 profile of mature thymocytes (CD69-CD24-TCR+) from the indicated strain of mice are shown. (C) The CD4/CD8 ratio for Vα2+ T cells from the spleen (left) and mature thymocytes (right) for the indicated strain of mice is shown. (D) ThPOK protein levels in DP, CD4+8lo, mature CD4+, and CD8+ thymocytes (CD69CD24TCR+; top histograms), and splenic T cell subsets (bottom histograms) from the indicated strain of mice are shown. Numbers in histograms represent the mean fluorescence intensity (MFI) values. (E) Compilations of ThPOK MFI for the indicated thymic and splenic T cell subsets from the indicated mice are shown. For DP thymocytes, ThPOK MFI is relative to that in DP thymocytes from ThPOK-H. For all other thymic subsets, ThPOK MFI is relative to the corresponding WT thymic subset. ThPOK MFI for splenic subsets is relative to that in splenic CD4+ mature T cells from WT mice. ThPOK MFI for CD4+ mature T cells from OTI mice and DN mature T cells from OTI and WT mice are ND because of the lack of a substantial number of these cells. (F) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in the indicated mice are shown. Also shown are the total splenocytes and the frequency and number of splenic T cells and T cell subsets from OTI mice expressing or not the indicated ThPOK transgene (n > 12). Data are representative examples of four or more independent experiments (A, B and D). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005. n/d, not determined.

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Similar to non-TCR transgenic background, ThPOK-specific staining in DP thymocytes was hierarchical with that in OTI+ThPOK-611+ > OTI+ThPOK163+ > OTI+ThPOK-H+ cells and was substantially higher compared with similar subsets from OTI control (Fig. 1D, 1E). Similar to the analysis of ThPOK transgenic mice with WT background, variegated ThPOK expression observed in DP thymocytes was lost in a majority of the redirected CD4+ mature T cells from the three ThPOK transgenic mice expressing OTI-TCR and was ∼1.5–2-fold higher compared with ThPOK expression in CD4+ mature T cells from WT mice (Fig. 1D, 1E). Interestingly, DN and CD8+ mature T cells from the spleen of OTI+ThPOK-H+ or OTI+ThPOK-163+ mice continued to express a significant amount of ThPOK compared with ThPOK levels in CD4+ mature T cells from WT mice (Fig. 1D, bottom panels, Fig. 1E). Although total thymocytes in OTI mice expressing or not individual ThPOK transgene were comparable, the frequency and number of selected thymocytes were reduced in ThPOK-expressing mice likely because of impaired Runx3 expression (Fig. 1F; Refs. 11, 38, 39) see below)). As expected, the frequency and number of CD4+ single-positive thymocytes was significantly increased, whereas that of CD8+ single-positive thymocytes decreased in all three ThPOK transgenic OTI lines compared with control. Similarly, the frequency and number of TCR+ splenic cells was reduced in ThPOK-expressing OTI mice compared with control, likely reflecting a reduced thymic maturation and survival/expansion of the redirected T cells in the periphery (Fig. 1F; Refs. 23, 24). Nevertheless, the frequency and cell number compilation data show a significant increase in CD4+ and/or DN mature T cells and a decrease in CD8+ mature T cells in all OTI mice expressing individual ThPOK transgene compared with control (Fig. 1F). Additionally, the DN mature T cells appeared to be mostly derived from CD4+ thymocytes in OTI+ThPOK-H+ mice (Supplemental Fig. 2B).

To rule out the possibility that the ThPOK-H–mediated partial CD8 to CD4 helper lineage redirection was not specific to the OTI model, we introduced the ThPOK-H transgene into mice expressing MHCI-specific monoclonal TCR (P14-TCR) or polyclonal TCRs (MHCII−/−). Similar to OTI+ThPOK-H+ mice, P14+ThPOK-H+ mice also showed partial CD8 to CD4 helper lineage redirection as judged by the presence of CD4+, DN, and CD8+ mature T cells in the spleen of these mice (Supplemental Fig. 3A). Importantly, the introduction of ThPOK-H transgene in MHCII−/− mice also resulted in the partial CD8 to CD4 helper lineage redirection (Fig. 2A, 2B) and a significant increase in the CD4/CD8 ratio in the thymus and spleen of ThPOK-H+MHCII−/− mice compared with MHCII−/− mice (Fig. 2C). Similar to the OTI model, we noted a decrease in thymic selection and mature splenic T cell frequency and number in ThPOK-H+MHCII−/− mice (Fig. 2D). In the thymus of ThPOK-H+MHCII−/− mice, the frequency and number of CD4+ thymocytes was significantly increased, whereas that of CD8+ thymocytes was significantly decreased compared with control mice (Fig. 2D). In the spleen, we observed a similar pattern except that the number of CD4+ mature T cells were only slightly higher in ThPOK-H+MHCII−/− mice compared with control mice likely due to their differentiation into DN mature T cells in ThPOK-H+MHCII−/− mice (Fig. 2B, 2D). Together, the generation of a small number of CD4+ mature T cells and the presence of a substantial number of DN mature T cells with impaired cytotoxic function but lack of activation of helper function (see below) suggests that ThPOK-H induces partial CD8 to CD4 helper lineage redirection of thymocytes expressing MHCI-specific monoclonal or polyclonal TCRs.

FIGURE 2.

ThPOK induces partial CD8 to CD4 helper lineage redirection in MHCII−/− mice. The CD4/CD8 profiles of TCR+ thymocytes (A) and splenocytes (B) from MHCII−/− (left) and ThPOK-H+MHCII−/− (right) mice are shown. (C) The CD4/CD8 ratio in the thymus and spleen of MHCII−/− (black bars) and ThPOK-H+MHCII−/− (white bars) mice is shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (TCR+) in MHCII−/− expressing or not ThPOK-H are shown. Also shown are the total splenocytes and the frequency and number of splenic T cells and T cell subsets from MHCII−/− mice expressing or not ThPOK-H transgene (n > 8). Data are representative of six or more independent experiments (A and B). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 2.

ThPOK induces partial CD8 to CD4 helper lineage redirection in MHCII−/− mice. The CD4/CD8 profiles of TCR+ thymocytes (A) and splenocytes (B) from MHCII−/− (left) and ThPOK-H+MHCII−/− (right) mice are shown. (C) The CD4/CD8 ratio in the thymus and spleen of MHCII−/− (black bars) and ThPOK-H+MHCII−/− (white bars) mice is shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (TCR+) in MHCII−/− expressing or not ThPOK-H are shown. Also shown are the total splenocytes and the frequency and number of splenic T cells and T cell subsets from MHCII−/− mice expressing or not ThPOK-H transgene (n > 8). Data are representative of six or more independent experiments (A and B). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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To determine the basis for the presence of CD4+, CD8+, and DN mature T cells in OTI+ThPOK-H+ mice, we assessed Thpok, Runx3d, Socs1, and Nur77 levels in each T cell subset purified from the same mouse. Socs1 is positively regulated by ThPOK (21) and Nur77, which influences CD8+ mature T cell development by modulating Runx3 expression (40), is suggested to be preferentially expressed in CD4+ mature T cells (41). QPCR and flow cytometric analysis of mature T cells from OTI+ThPOK-H+ mice showed graded ThPOK expression levels with that in CD4+ > DN > CD8+ mature T cells (Fig. 3A), which is in agreement with staining data (Fig. 1D, 1E). In agreement with ThPOK expression analysis, the expression of Runx3 from a distal promoter (Runx3d) was completely abolished in CD4+ and DN mature T cells and reduced in CD8+ mature T cells from OTI+ThPOK-H+ mice compared with CD8+ mature T cells from OTI mice (Fig. 3B). Similarly, Socs1 was mostly expressed in CD4+ mature T cells (Fig. 3C), whereas Nur77 expression was directly proportional to ThPOK levels in the three mature T cell subsets from OTI+ThPOK-H+ mice (Fig. 3D). Nur77 expression was significantly higher in CD4+ mature T cells (p = 0.0032) but not in DN or CD8+ mature T cells from OTI+ThPOK-H+ mice compared with CD8+ mature T cells from OTI mice. Comparable CD5 levels [a surrogate marker for TCR signal strength (42)] in CD4+ and CD8+ mature T cells (Supplemental Fig. 3B) from OTI+ThPOK-H+ and OTI control suggest that the differential Nur77 expression observed in mature T cells may be due to differential ThPOK expression (32). We then evaluated Nur77-GFP reporter expression in DP thymocytes (to exclude the influence of intrathymic signaling on Nur77 expression) from OTI+ThPOK-H+ and control mice. Comparable CD5 levels but higher GFP expression was detected in DP thymocytes from OTI+ThPOK-H+ mice compared with OTI control mice expressing Nur77-GFP reporter (Fig. 3E); however, an increase in Nur77-GFP expression in the presence of transgenic ThPOK did not appear to be significant. In cell transfection studies, we did not observe any increase in Nur77 promoter-driven luciferase expression in the presence of ThPOK (Supplemental Fig. 3C). These data suggest that ThPOK may not be involved in regulating Nur77 expression.

FIGURE 3.

ThPOK modulates lineage-specific gene expression in T cell subsets. CD4+, CD8+, and DN mature T cells from the spleen of OTI+ThPOK-H+ mice were isolated and the expression levels of Thpok (A, left), Runx3d (B), Socs1 (C), and Nur77 (D) were evaluated by QPCR and were compared with that in CD8+ mature T cells from OTI mice (normalized to Hprt expression). Data depicts the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD8+ mature T cells from OTI mice. (A, right) ThPOK protein levels in the indicated splenic T cell subsets from OTI+ThPOK-H+ or OTI+ mice are shown. Also shown is ThPOK mean fluorescence intensity (MFI) compilation for the indicated T cell subsets (relative to WT CD4+ mature T cells). (E) MFI of CD5 and Nur77-GFP expression in DP thymocytes from OTI+ThPOK-H+, OTI, and WT mice are compared (left) and compiled (right; relative to OT1). (F) shows perforin expression levels by QPCR in CD4+, DN, and CD8+ mature T cells from the spleen of OTI+ThPOK-H+ mice compared with CD8+ mature T cells from OTI control mice (normalized to Hprt expression). (G) Purified T cell subsets from OTI mice expressing or not ThPOK-H were activated for 5–7 d in the presence of irradiated splenocytes from BL/6 mice pulsed with OTI peptide (SIINFEKL) and then restimulated with PMA/ionomycin in the presence of brefeldin for the analysis of IFN-γ expression. (H) CD154 expression in the cognate-peptide activated indicated T cell subsets from OTI+ and OTI+ThPOK-H+ mice is shown. Data are representative of two to six independent experiments. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 3.

ThPOK modulates lineage-specific gene expression in T cell subsets. CD4+, CD8+, and DN mature T cells from the spleen of OTI+ThPOK-H+ mice were isolated and the expression levels of Thpok (A, left), Runx3d (B), Socs1 (C), and Nur77 (D) were evaluated by QPCR and were compared with that in CD8+ mature T cells from OTI mice (normalized to Hprt expression). Data depicts the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD8+ mature T cells from OTI mice. (A, right) ThPOK protein levels in the indicated splenic T cell subsets from OTI+ThPOK-H+ or OTI+ mice are shown. Also shown is ThPOK mean fluorescence intensity (MFI) compilation for the indicated T cell subsets (relative to WT CD4+ mature T cells). (E) MFI of CD5 and Nur77-GFP expression in DP thymocytes from OTI+ThPOK-H+, OTI, and WT mice are compared (left) and compiled (right; relative to OT1). (F) shows perforin expression levels by QPCR in CD4+, DN, and CD8+ mature T cells from the spleen of OTI+ThPOK-H+ mice compared with CD8+ mature T cells from OTI control mice (normalized to Hprt expression). (G) Purified T cell subsets from OTI mice expressing or not ThPOK-H were activated for 5–7 d in the presence of irradiated splenocytes from BL/6 mice pulsed with OTI peptide (SIINFEKL) and then restimulated with PMA/ionomycin in the presence of brefeldin for the analysis of IFN-γ expression. (H) CD154 expression in the cognate-peptide activated indicated T cell subsets from OTI+ and OTI+ThPOK-H+ mice is shown. Data are representative of two to six independent experiments. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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Because ThPOK is proposed to suppress the cytotoxic program in mature T cells (28), we wondered about the functionality of the three T cell subsets, particularly CD8+ and DN mature T cells that expressed a significant amount of ThPOK and reduced levels of Runx3. To this end, we evaluated the expression of genes involved in cytotoxic and helper function in these T cell subsets. In agreement with Runx3 and ThPOK expression analysis, we observed, compared with CD8+ mature T cells from OTI mice, almost complete ablation of perforin and severely reduced IFN-γ expression in CD4+ and DN mature T cells (Fig. 3F, 3G). Interestingly, perforin and IFN-γ expression was significantly reduced in CD8+ mature T cells isolated from OTI+ThPOK-H+ mice as well (Fig. 3F, 3G). Upon activation, CD4+, but not DN or CD8+, mature T cells from OTI+ThPOK-H+ mice upregulated CD154, a CD4 helper lineage marker (Fig. 3H). This was also observed in mature T cells subsets isolated from ThPOK-H+MHCII−/− mice; activated CD4+ mature T cells from ThPOK-H+MHCII−/− mice expressed CD154 and IL-4 (Supplemental Fig. 3D, 3E), whereas DN and CD8+ mature T cells from ThPOK-H+MHCII−/− mice continued to express IFN-γ with DN mature T cells expressing lower amounts. Together, these data suggest that a ThPOK level sufficient to suppress the cytotoxic program does not activate the helper program (phenotype of DN mature T cells) and that a higher amount of ThPOK is required to redirect MHCI-signaled thymocytes into the CD4 helper lineage.

ThPOK is proposed to form a positive autoregulatory loop (27). Hence, we wondered if the transgenic ThPOK induced the expression of endogenous ThPOK in the signaled thymocytes and whether this contributed to the CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice. To address this, we first evaluated, using specific QPCR primers, the endogenous ThPOK expression in various T cell subsets from OTI+ThPOK-H+ mice. Indeed, we observed a significant increase in endogenous ThPOK expression in the splenic CD4+ mature T cells of OTI+ThPOK-H+ mice (Fig. 4A). To assess if this endogenous ThPOK induction played any role in the CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice, we analyzed CD4/CD8 phenotype of OTI+ThPOK-H+ mice expressing endogenous ThPOK or not. Surprisingly, we did not observe any significant changes in the frequency of CD4+ and CD8+ thymocytes in OTI+ThPOK-H+Thpok−/− mice compared with OTI+ThPOK-H+Thpok+/+ control (Fig. 4B). A slight decrease in CD4+ and increase in CD8+ mature T cell frequency in the spleen of OTI+ThPOK-H+Thpok−/− mice compared with OTI+ThPOK-H+Thpok+/+ mice was noticed; however, it did not result in any significant change in the CD4/CD8 ratio in these mice [0.41 ± 0.09 in OTI+ThPOK-H+Thpok+/+ versus 0.28 ± 0.035 in OTI+ThPOK-H+Thpok−/− mice; (Fig. 4C; relative to OTI control)]. The frequency/number of various thymic and splenic T cell subsets in OTI+ThPOK-H+ mice were comparable irrespective of the presence or absence of endogenous ThPOK (Fig. 4D; some mice were in Rag+/− background, which did not affect the CD4/CD8 phenotype). These data suggest that endogenous ThPOK played an insignificant role in the CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice.

FIGURE 4.

Insignificant contribution of endogenous ThPOK in the CD8 to CD4 helper lineage redirection by ThPOK-H. (A) To assess endogenous ThPOK levels, various T cell subsets from OTI+THPOK-H+ mice were purified for QPCR analysis. Data show endogenous ThPOK levels in the indicated splenic T cell subsets from OTI+ThPOK-H+ mice compared with that in CD8+ mature T cells from OTI mice (normalized to Hprt expression). Data depicts the average of triplicate values with SD and are expressed as fold increase over endogenous ThPOK levels in control CD8+ mature T cells from OTI mice. To determine the impact of endogenous ThPOK expression on ThPOK-H–mediated CD4 helper lineage choice OTI+ThPOK-H+Thpok−/− mice were generated. (B) shows the CD4/CD8 profiles of the spleen and thymus isolated from OTI+, OTI+ThPOK-H+Thpok+/+, and OTI+ThPOK-H+Thpok−/− mice. (C) The CD4/CD8 ratio in the spleen of indicated mice relative to OTI is shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in the indicated mice are shown (n > 6). Also shown are total splenocytes and the frequency and number of splenic T cells and T cell subsets in these mice. Data are representative of three or more independent experiments (A and B). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 4.

Insignificant contribution of endogenous ThPOK in the CD8 to CD4 helper lineage redirection by ThPOK-H. (A) To assess endogenous ThPOK levels, various T cell subsets from OTI+THPOK-H+ mice were purified for QPCR analysis. Data show endogenous ThPOK levels in the indicated splenic T cell subsets from OTI+ThPOK-H+ mice compared with that in CD8+ mature T cells from OTI mice (normalized to Hprt expression). Data depicts the average of triplicate values with SD and are expressed as fold increase over endogenous ThPOK levels in control CD8+ mature T cells from OTI mice. To determine the impact of endogenous ThPOK expression on ThPOK-H–mediated CD4 helper lineage choice OTI+ThPOK-H+Thpok−/− mice were generated. (B) shows the CD4/CD8 profiles of the spleen and thymus isolated from OTI+, OTI+ThPOK-H+Thpok+/+, and OTI+ThPOK-H+Thpok−/− mice. (C) The CD4/CD8 ratio in the spleen of indicated mice relative to OTI is shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in the indicated mice are shown (n > 6). Also shown are total splenocytes and the frequency and number of splenic T cells and T cell subsets in these mice. Data are representative of three or more independent experiments (A and B). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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The presence of a large number of DN and CD8+ mature T cells in ThPOK-H+ mice expressing MHCI-specific TCR (OTI, P14, or MHCII−/−), despite expressing ∼1.5–2-fold more transgenic ThPOK compared with endogenous ThPOK levels in CD4+ mature T cells from WT mice, suggests that differential amount of ThPOK may be required for the CD4 helper lineage choice of MHCI- and MHCII-specific thymocytes. Alternately, the observed phenotype of OTI+ThPOK-H+ mice could be because of the ThPOK-H transgene–specific effect. To address these questions, we evaluated the impact of ThPOK-H on the rescue of CD4 helper lineage development of MHCII-specific thymocytes in ThPOK-deficient mice. To this end, we generated OTII+Thpok−/− mice expressing or not ThPOK-H transgene. In OTII+Thpok+/+ mice, >95% of the Vα2+ mature T cells are CD4+, which are directed into CD8 lineage in the absence of ThPOK (Fig. 5A). Indeed, the introduction of ThPOK-H transgene into OTII+Thpok−/− mice completely rescued CD4 helper lineage commitment; >95% of Vα2+ mature T cells were CD4+ in the thymus and spleen of OTII+ThPOK-H+Thpok−/− mice, which was similar to that in OTII+Thpok+/+ mice (Fig. 5A, 5B). ThPOK-specific staining of the mature CD4+ thymocytes and splenic T cells showed a ∼2-fold higher expression compared with endogenous ThPOK expression in control CD4+ mature T cells from OTII+Thpok+/+ mice (Fig. 5C). The frequency and absolute cell numbers in the thymus and spleen of OTII+ThPOK-H+Thpok−/− mice were comparable to that in littermate control OTII+ThPOK-H+Thpok+/+ mice (Fig. 5D).

FIGURE 5.

ThPOK-H completely rescues CD4 helper lineage development in OTII+Thpok−/− mice. To evaluate the ability of ThPOK-H transgene to rescue CD4 helper lineage development of MHCII-specific thymocytes, the transgene was introduced into OTII+Thpok−/− mice. (A) shows the CD4/CD8 profile of mature thymocytes (CD24CD69TCR+), and (B) shows the CD4/CD8 phenotype of splenic T cells from the indicated mice. (C) ThPOK protein levels in CD4+ mature T cells from the thymus and spleen of OTII+Thpok+/+ (shaded histogram) and OTII+ThPOK-H+Thpok−/− (open histogram) mice are shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in OTII+ThPOK-H+Thpok−/− and littermate OTII+ThPOK-H+Thpok+/+ (all Rag−/−) control mice are shown. Also shown are total splenocytes and the frequency and number of splenic T cells and T cell subsets from the same mice (n > 4). Data are representative of three or more independent experiments (A–C).

FIGURE 5.

ThPOK-H completely rescues CD4 helper lineage development in OTII+Thpok−/− mice. To evaluate the ability of ThPOK-H transgene to rescue CD4 helper lineage development of MHCII-specific thymocytes, the transgene was introduced into OTII+Thpok−/− mice. (A) shows the CD4/CD8 profile of mature thymocytes (CD24CD69TCR+), and (B) shows the CD4/CD8 phenotype of splenic T cells from the indicated mice. (C) ThPOK protein levels in CD4+ mature T cells from the thymus and spleen of OTII+Thpok+/+ (shaded histogram) and OTII+ThPOK-H+Thpok−/− (open histogram) mice are shown. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) in OTII+ThPOK-H+Thpok−/− and littermate OTII+ThPOK-H+Thpok+/+ (all Rag−/−) control mice are shown. Also shown are total splenocytes and the frequency and number of splenic T cells and T cell subsets from the same mice (n > 4). Data are representative of three or more independent experiments (A–C).

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To further support the observation that the same level of ThPOK differentially influences the CD4 helper lineage choice of MHCI- and MHCII-specific thymocytes, we introduced each of the three ThPOK transgene into Thpok−/− mice expressing polyclonal TCR repertoire. In Thpok−/− mice, positively selected MHCII-specific thymocytes are redirected into the CD8 lineage, and thus, the peripheral CD8+ mature T cell population consists of MHCI- and MHCII-specific T cells. Indeed, each of the ThPOK transgene rescued CD4 helper development and impaired CD8 cytotoxic development in Thpok−/− mice (Supplemental Fig. 4A–C). Together, these data strongly suggest that, compared with CD4 helper lineage choice of MHCII-specific thymocytes, an efficient CD8 to CD4 helper lineage redirection of MHCI-specific thymocytes requires a higher amount of ThPOK, and the partial CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice is unlikely because of the ThPOK-H transgene–specific effect.

The partial CD8 to CD4 helper lineage redirection of MHCI-signaled thymocytes but complete rescue of CD4 helper lineage choice of MHCII-signaled thymocytes prompted us to ask if differential TCR signaling played a role in the ThPOK-H–mediated CD4 helper lineage choice of MHCI- and MHCII-specific thymocytes. We considered the possibility that weak TCR signaling in MHCI-specific thymocytes, compared with that in MHCII-specific thymocytes, may be responsible for an inefficient CD4 helper lineage choice of MHCI-specific thymocytes expressing ThPOK-H transgene. If so, we reasoned that increasing the TCR signal strength may enhance the efficiency of ThPOK-H–mediated CD4 helper lineage choice of MHCI-specific thymocytes. To test this notion, we introduced constitutively active Lck encoding transgene, dLGF, (16, 17, 33) into OTI+ThPOK-H+ mice. We bred mice to obtain OTI+dLGF+ThPOK-H+ triple transgenic mice with a Rag−/− background and analyzed CD4/CD8 development in these mice. As reported previously (16), increased TCR signaling due to constitutively active Lck led to an increase in the frequency of Vα2+CD4+ and a decrease in the frequency of Vα2+CD8+ mature T cells in the thymus and spleen of OTI+dLGF+ mice compared with control mice (Fig. 6A). Importantly, analysis of mature T cells in the thymus and spleen of OTI+dLGF+ThPOK-H+ mice showed a significant increase in the frequency and number of Vα2+CD4+ T cells, whereas that of Vα2+CD8+ mature mature T cells was significantly reduced compared with OTI+ThPOK-H+ mice (Fig. 6A, 6B). About 70% of mature T cells were CD4+ in the spleen and thymus of triple transgenic mice, resulting in a significantly higher CD4/CD8 ratio compared with OTI+ThPOK-H+ or OTI+dLGF+ mice (Fig. 6C). Of note, the frequency and absolute number of DN mature T cells were also reduced in the spleen of triple transgenic mice compared with OTI+ThPOK-H+ mice but was still higher compared with OTI+dLGF+ mice (Fig. 6A, 6B).

To ascertain that the efficient CD8 to CD4 helper lineage redirection was not specific to the introduction of dLGF transgene into OTI+ThPOK-H+ mice, we analyzed the CD4/CD8 phenotype of OTI+dLGF+ThPOK-163+ mice as well. Indeed, an increase in the CD4+ and a decrease in the CD8+ mature T cell frequency was observed in the thymus and spleen of OTI+dLGF+ThPOK-163+ mice compared with OTI+ThPOK-163+ mice (Supplemental Fig. 4D). We then evaluated the expression of St8sia6 and St3gal2, the helper lineage-associated genes (31, 41), and Cxxc5, a ThPOK target gene that negatively regulates CD154 and is highly expressed in CD8+ mature T cells (43). Indeed, QPCR analysis showed a significantly elevated expression of St8sia6 and St3gal2 and a decreased expression of Cxxc5 in CD4+ mature T cells from the triple transgenic mice, which was similar to the expression of these genes in CD4+ mature T cells but opposite to their expression in CD8+ mature T cells from WT mice (Fig. 6D). The DN mature T cells from the triple transgenic mice showed a significantly lower St8sia6 and St3gal2 expression compared with CD4+ mature T cells from the same mice. Interestingly, DN mature T cells, which upon activation failed to upregulate CD154, expressed very little Cxxc5, suggesting a possible complex regulation of CD154 expression in the activated CD4+ mature T cells (43). Upregulation of St8sia6 and St3gal2 and suppression of Cxxc5 was also observed in the redirected CD4+ mature T cells isolated from OTI+ThPOK-163+ and OTI+ThPOK-611+ mice as well (Supplemental Fig. 4E). Together, these data strongly suggest that an elevated TCR signal strength and transgenic ThPOK act synergistically in redirecting MHCI-signaled thymocytes into the CD4+ helper T cell lineage.

FIGURE 6.

Augmenting TCR signal strength enhances ThPOK-induced CD8 to CD4 helper lineage redirection. The role of increased TCR signal strength in promoting CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice was investigated by introducing dLGF transgene into OTI+ThPOK-H+ mice. (A) shows a representative example of the CD4/CD8 profile of mature thymocytes (left) and splenic T cells (right) from the indicated mice. (B) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) as well as splenic T cells and subsets from the indicated mice are shown (n > 6). (C) The CD4/CD8 ratio of mature thymic and splenic T cells in the indicated mice is shown. (D) Mature T cells from the spleen of WT (CD4+ and CD8+), OT1+dLGF+ (CD4+), and OTI+dLGF+ThPOK-H+ (CD4+ and DN) mice were isolated, and the expression of St8sia6, St3gal2, and Cxxc5 was evaluated by QPCR. Data depict the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD4+ mature T cells from WT mice (normalized to Hprt expression). Data are representative of >6 independent experiments (A) and two experiments (D). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 6.

Augmenting TCR signal strength enhances ThPOK-induced CD8 to CD4 helper lineage redirection. The role of increased TCR signal strength in promoting CD8 to CD4 helper lineage redirection in OTI+ThPOK-H+ mice was investigated by introducing dLGF transgene into OTI+ThPOK-H+ mice. (A) shows a representative example of the CD4/CD8 profile of mature thymocytes (left) and splenic T cells (right) from the indicated mice. (B) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) as well as splenic T cells and subsets from the indicated mice are shown (n > 6). (C) The CD4/CD8 ratio of mature thymic and splenic T cells in the indicated mice is shown. (D) Mature T cells from the spleen of WT (CD4+ and CD8+), OT1+dLGF+ (CD4+), and OTI+dLGF+ThPOK-H+ (CD4+ and DN) mice were isolated, and the expression of St8sia6, St3gal2, and Cxxc5 was evaluated by QPCR. Data depict the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD4+ mature T cells from WT mice (normalized to Hprt expression). Data are representative of >6 independent experiments (A) and two experiments (D). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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An efficient CD8 to CD4 helper lineage redirection of MHCI-specific thymocytes in the presence of augmented TCR signaling and ∼2-fold more transgenic ThPOK protein (compared with endogenously expressed ThPOK in WT CD4+ mature T cells) in OTI+dLGF+ThPOK-H+ and OTI+dLGF+ThPOK-163+ mice could be because of two overlapping possibilities: augmented TCR signaling 1) induces endogenous ThPOK that contributes to this lineage redirection or 2) plays a role in the CD4 helper lineage choice of MHCI-specific thymocytes that is independent of ThPOK. To investigate these possibilities, we first measured endogenous ThPOK levels in the positively selected thymocytes from OTI+dLGF+ mice. As expected, we detected a significant ThPOK induction in the CD4+CD8lo thymocytes from OTI+dLGF+ mice compared with OTI control (Fig. 7A); ThPOK induction was essential for the generation of CD4+ mature T cells in these mice as indicated by the absence of these cells in the thymus and spleen of OTI+dLGF+Thpok−/− mice (Fig. 7B). Thus, it was conceivable that the induction of endogenous ThPOK due to increased TCR signal strength substantially contributed to the increased frequency of CD4+ mature T cells in OTI+dLGF+ThPOK-H+ mice. Therefore, to evaluate the relative contribution of the two sources of ThPOK (transgenic and endogenous) in the CD4 helper lineage choice in triple transgenic mice, we ablated ThPOK expression in these mice (all mice Rag−/−). We predicted that if endogenous ThPOK induced by augmented TCR signaling primarily contributed to the increased frequency of CD4+ mature T cells in OTI+dLGF+ThPOK-H+ mice, then ablating endogenous ThPOK in these mice would result in a CD4 frequency that would be lower compared with the CD4 frequency in OTI+dLGF+ThPOK-H+ThPOK+/+ mice, but it would be comparable to the CD4 frequency in OTI+ThPOK-H+Thpok−/− mice (Fig. 4B). Indeed, ablating the endogenous ThPOK expression resulted in a small but significant decrease (p < 0.02) in the splenic CD4+ mature T cell frequency in OTI+dLGF+ThPOK-H+Thpok−/− mice compared with ThPOK-sufficient control mice; >50% of mature T cells were still CD4+ in the thymus and spleen of OTI+dLGF+ThPOK-H+Thpok−/− mice compared with >60% in triple transgenic ThPOK-sufficient mice (Fig. 7C, 7D). Importantly, despite expressing only the transgene-encoded ThPOK, the CD4+ mature T cell frequency (52%) in the spleen of OTI+dLGF+ThPOK-H+Thpok−/− mice was still significantly higher than the CD4+ mature T cell frequency (20%) observed in the spleen of OTI+ThPOK-H+Thpok−/− mice (Fig. 7C, 7D; p < 0.0001). The frequency and number of DN and CD8+ splenic T cell subsets were comparable in triple transgenic mice expressing endogenous ThPOK or not (Fig. 7D).

To ascertain the observed differential CD4 frequency in OTI+dLGF+ThPOK-H+Thpok−/− mice, we analyzed the CD4/CD8 phenotype of OTI+dLGF+ThPOK-163+Thpok−/− mice as well. Indeed, we observed only a small decrease in the splenic CD4+ mature T cell frequency in OTI+dLGF+ThPOK-163+Thpok−/− mice (48%) compared with OTI+dLGF+ThPOK-163+Thpok+/+ mice (54%), but it was higher compared with that in OTI+ThPOK-163+Thpok−/− mice (29%; Supplemental Fig. 4D). Of note, ThPOK expression analysis showed a slightly higher frequency of ThPOKlo DP thymocytes from OTI+dLGF+ThPOK-H+Thpok−/− mice compared OTI+ThPOK-H+Thpok−/− control, suggesting a possible influence of augmented TCR signaling on the transgenic ThPOK levels in DP thymocytes (Fig. 7E). Significantly higher phospho-Src staining in DP and CD4+CD8lo thymocytes confirmed augmented TCR signaling in OTI+dLGF+ mice expressing ThPOK-H or not compared with OTI or WT control (Fig. 7F, 7G). We also observed elevated, albeit insignificant, phospho-CD3ζ levels in DP and CD4+CD8lo thymocytes from these mice compared with OTI control [it was significantly higher compared with similar subsets from WT mice (Fig. 7F, 7G)]. The increased pSrc and pCD3ζ staining observed in DP thymocytes in OTI mice expressing dLGF transgene became less pronounced in CD4+CD8lo thymocytes, reflecting a possible impact of intrathymic signaling and/or the limit of sensitivity of phospho-specific Ab staining. We then evaluated the expression of CD4 helper lineage genes in purified T cells from the triple transgenic Thpok−/− mice. Indeed, the expression pattern of St8sia6, St3gal2, and Cxxc5 in CD4+ mature T cells from OTI+dLGF+ThPOK-H+Thpok−/− mice was similar to that in CD4+ mature T cells from WT mice, which is upregulation of St8sia6 and St3gal2 and downregulation of Cxxc5 (Fig. 7H). Collectively, these data strongly suggest that augmenting TCR signal strength in MHCI-specific thymocytes significantly promotes the ThPOK-induced CD8 to CD4 helper lineage redirection. These data also suggest that TCR signaling plays a role in CD4 helper lineage choice that may be independent of ThPOK.

FIGURE 7.

Evaluating the contribution of endogenous and transgenic ThPOK in CD4 helper lineage choice in OTI+dLGF+ThPOK-H+ mice. (A) The CD4+CD8lo thymocytes were purified from OTI+dLGF+ and control OTI mice, and endogenous ThPOK expression was analyzed by QPCR. A representative example shows the average of triplicate values ± SD and are expressed as relative fold increase over preselection DP thymocytes from OTI control mice. (B) shows the CD4/CD8 profiles of the mature thymocytes (CD69CD24Vα2+) and splenic T cells from OTI+dLGF+ mice expressing or not endogenous ThPOK. (C) The contribution of endogenous and transgenic ThPOK in the CD4 helper lineage choice of MHCI-specific thymocytes with augmented TCR signaling was evaluated by assessing the CD4/CD8 phenotype of the mature thymocytes (top) and splenic T cells (bottom) isolated from the indicated mice. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) as well as splenic T cells and subsets from the indicated mice are shown (n > 6). (E) ThPOK-specific staining in DP thymocytes and CD4+ mature thymocytes and splenic T cells from the indicated mice is shown. (F) Histograms show phospho-Src (left panels) and phospho-CD3ζ (right panels) expression levels in DP and CD4+CD8lo thymocytes from the indicated mice. (G) The compilation of mean fluorescence intensity (MFI) data for pSrc and pCD3ζ for DP and CD4+CD8lo subsets from the indicated mice are shown and are expressed relative to MFI values in WT subsets. (H) The expression of St8sia6, St3gal2, and Cxxc5 was evaluated by QPCR in mature T cell subsets purified from the spleen of WT (CD4+ and CD8+) and OTI+dLGF+ThPOK-H+Thpok−/− (CD4+ and DN) mice. Data depict the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD4+ mature T cells from WT mice (normalized to Hprt expression). Data are representative of two to six independent experiments (A–C, E, F, and H). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 7.

Evaluating the contribution of endogenous and transgenic ThPOK in CD4 helper lineage choice in OTI+dLGF+ThPOK-H+ mice. (A) The CD4+CD8lo thymocytes were purified from OTI+dLGF+ and control OTI mice, and endogenous ThPOK expression was analyzed by QPCR. A representative example shows the average of triplicate values ± SD and are expressed as relative fold increase over preselection DP thymocytes from OTI control mice. (B) shows the CD4/CD8 profiles of the mature thymocytes (CD69CD24Vα2+) and splenic T cells from OTI+dLGF+ mice expressing or not endogenous ThPOK. (C) The contribution of endogenous and transgenic ThPOK in the CD4 helper lineage choice of MHCI-specific thymocytes with augmented TCR signaling was evaluated by assessing the CD4/CD8 phenotype of the mature thymocytes (top) and splenic T cells (bottom) isolated from the indicated mice. (D) The frequency and absolute number of TCR+ and CD4+CD8lo subsets in total thymocytes and CD4+ and CD8+ mature thymocytes (CD24CD69TCR+) as well as splenic T cells and subsets from the indicated mice are shown (n > 6). (E) ThPOK-specific staining in DP thymocytes and CD4+ mature thymocytes and splenic T cells from the indicated mice is shown. (F) Histograms show phospho-Src (left panels) and phospho-CD3ζ (right panels) expression levels in DP and CD4+CD8lo thymocytes from the indicated mice. (G) The compilation of mean fluorescence intensity (MFI) data for pSrc and pCD3ζ for DP and CD4+CD8lo subsets from the indicated mice are shown and are expressed relative to MFI values in WT subsets. (H) The expression of St8sia6, St3gal2, and Cxxc5 was evaluated by QPCR in mature T cell subsets purified from the spleen of WT (CD4+ and CD8+) and OTI+dLGF+ThPOK-H+Thpok−/− (CD4+ and DN) mice. Data depict the average of triplicate values with SD and are expressed as fold increase over the expression of individual genes in control CD4+ mature T cells from WT mice (normalized to Hprt expression). Data are representative of two to six independent experiments (A–C, E, F, and H). *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

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The CD4+ mature T cell frequency in OTI+dLGF+ThPOK-H+Thpok−/− mice, although higher than the CD4+ mature T cell frequency in OTI+ThPOK-H+Thpok−/− mice, was still significantly lower than the CD4+ mature T cell frequency in OTII+ThPOK-H+Thpok−/− mice (Fig. 8A; p < 0.0005). CD4+ mature T cells in all these three mouse strains expressed the same amount of ThPOK but received differential intrathymic signaling (MHCI-induced signaling, MHCI-induced signaling combined with augmented TCR signaling, and MHCII-induced signaling). Thus, it was possible that TCR signaling in MHCI-specific CD4+ mature T cells from OTI+dLGF+ThPOK-H+ mice, although elevated compared with that in MHCI-specific CD4+ mature T cells from OTI+ThPOK-H+ mice, may still be lower than that in MHCII-specific CD4+ mature T cells from OTII mice. Therefore, we compared CD5 levels in the thymic and splenic T cells from OTI mice expressing or not dLGF with that from OTII mice to assess their TCR signal strength. In each experiment, we calculated CD5 levels in the thymocytes and mature T cell subsets from various mice relative to CD5 levels in the relevant thymic subsets or CD8+ mature T cells from OTI mice. As expected, DP and CD4+CD8lo thymocytes from OTI mice expressing dLGF transgene showed significantly higher CD5 levels compared with the OTII subset (Fig. 8B). Interestingly, CD5 levels in CD4+ thymocytes from the two mice were quite comparable (Fig. 8C), likely reflecting stronger intrathymic signaling transduced in OTII+ thymocytes, but became significantly higher in the CD4+ splenic T cells from OT1+dLGF+ mice compared with that from OTII mice (Fig. 8D). In OTI+dLGF+ThPOK-H+ mice, we observed a similar trend in CD5 expression levels in DP, CD4+CD8lo thymocytes (Fig. 8B, 8C), and CD4+ splenic T cell (Fig. 8D) subsets compared with similar subsets from OTII mice. Of note, CD5 levels were lower in CD4+ mature thymocytes and splenic T cells from OTI+dLGF+ThPOK-H+ mice compared with CD5 levels in similar subsets from OTI+dLGF+ mice. As well, CD5 levels in DN and/or CD8+ mature T cells from OTI+dLGF+ or OTI+ThPOK-H+ mice were lower compared with that in CD8+ mature T cells from OTI mice. An analysis of CD5 levels in the splenic CD4+ mature T cells from OTI+dLGF+ThPOK-163+ mice also showed a similar trend. CD5 levels in CD4+ mature T cells from OTI+dLGF+ThPOK-163+ mice were significantly higher compared with that in CD4+ mature T cells from OTII mice, and they were slightly lower compared with CD5 levels in CD4+ mature T cells from OTI+dLGF+ mice (Supplemental Fig. 4F). It is unclear whether differential intrathymic signaling, which influences CD5 levels and correlates with mature T cell function (44, 45), may be responsible for the altered CD5 levels in CD4+ or CD8+ mature T cells in the presence of transgenic ThPOK in OTI+dLGF+ mice. Nevertheless, these data support the notion that TCR signaling in MHCI-specific OTI+ thymocytes expressing dLGF transgene is significantly higher than that in MHCII-specific OTII+ thymocytes. Collectively, our in-depth analysis of CD4/CD8 lineage choice of MHCI-specific thymocytes with or without augmented TCR signaling and of MHCII-specific thymocytes in the presence of the same amount of ThPOK strongly suggest that ThPOK-induced CD4 helper lineage choice of developing thymocytes is critically influenced by quantitative as well as differential TCR signaling.

FIGURE 8.

Comparison of CD4+ mature T cell frequency and TCR signal strength in MHCI- and MHCII-specific thymic and splenic T cells from control mice and ThPOK-H mice expressing or not dLGF transgene. (A) The efficiency of ThPOK-H–mediated CD4 helper lineage choice of thymocytes with differential TCR signaling was evaluated by comparing the CD4+ splenic T cell frequencies in the indicated mice. To assess relative TCR signal strength, CD5 levels in various thymic subsets and mature T cells from OTI+, OTI+dLGF+, OTI+dLGF+ThPOK-H+, and OTII+ mice were compared. (B) CD5 levels in DP and CD4+CD8lo thymocytes from the indicated mice were normalized to CD5 levels in the relevant thymic subsets from OTI mice. CD5 levels in the mature CD4+ and CD8+ thymocytes (C) and splenocytes (D) were normalized to CD5 levels in mature CD8+ thymocytes and splenocytes from OTI mice. Each symbol represents one mouse. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

FIGURE 8.

Comparison of CD4+ mature T cell frequency and TCR signal strength in MHCI- and MHCII-specific thymic and splenic T cells from control mice and ThPOK-H mice expressing or not dLGF transgene. (A) The efficiency of ThPOK-H–mediated CD4 helper lineage choice of thymocytes with differential TCR signaling was evaluated by comparing the CD4+ splenic T cell frequencies in the indicated mice. To assess relative TCR signal strength, CD5 levels in various thymic subsets and mature T cells from OTI+, OTI+dLGF+, OTI+dLGF+ThPOK-H+, and OTII+ mice were compared. (B) CD5 levels in DP and CD4+CD8lo thymocytes from the indicated mice were normalized to CD5 levels in the relevant thymic subsets from OTI mice. CD5 levels in the mature CD4+ and CD8+ thymocytes (C) and splenocytes (D) were normalized to CD5 levels in mature CD8+ thymocytes and splenocytes from OTI mice. Each symbol represents one mouse. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

Close modal

In the present manuscript, we have investigated the impact of ThPOK levels on the CD4 helper lineage choice of MHCI- and MHCII-specific thymocytes and the role of TCR signaling in it. Specifically, we have evaluated the impact of ThPOK levels on the CD4 helper lineage choice of thymocytes with differential TCR signaling. Our data strongly suggest that MHCI-restricted thymocytes require a higher level of ThPOK in preselection thymocytes for an efficient CD8 to CD4 helper lineage redirection (ThPOK-611 mice), whereas relatively low/moderate levels (still higher than ThPOK levels in WT CD4+ mature T cells) result in the partial CD8 to CD4 helper lineage redirection (ThPOK-H and ThPOK-163 mice). The lower frequency of mature T cells observed in the spleen of OTI+ThPOK-163+ and OTI+ThPOK-611+ mice likely reflects a reduced thymic maturation and/or the effect of mismatched coreceptor expression (CD4+ mature T cells) or lack of coreceptor expression (DN mature T cells) on survival and/or homeostatic expansion of the redirected T cells (23, 24). A consequence of partial lineage redirection is that a substantial number of CD8+ and DN mature T cells are detected in OTI+ThPOK-H+ or OTI+ThPOK-163+ mice. It is interesting that the DN mature T cells in OTI+ThPOK-H+ mice fail to maintain CD4 expression despite almost complete suppression of Runx3 expression indicating a complex regulation of Cd4 expression requiring sustained TCR signaling in developing thymocytes (46). In vitro differentiation culture data supports such a notion; disrupting intrathymic TCR signaling in an in vitro culture of purified CD4+ thymic subsets from OTI+ThPOK-H+ mice results in the loss of CD4 expression in a significant number of cells, leading to the generation of DN mature T cells. Importantly, DN and CD8+ mature T cells, particularly the former, continued to express a substantial amount of ThPOK that compromised the cytotoxic function but still failed to activate the helper program in these cells. A simple explanation would be that activation of the helper program in MHCI-signaled thymocytes requires a higher amount of ThPOK than that required for suppression of the cytotoxic program (phenotype of DN mature T cells). However, it was paradoxical that the ThPOK level in DN mature T cells from OTI+ThPOK-H+ or OTI+ThPOK-163+ mice was significantly higher compared with endogenous ThPOK levels in MHCII-restricted CD4+ mature T cells from WT mice and yet failed to redirect them into the CD4 helper lineage. The inability of ThPOK-H to induce an efficient CD8 to CD4 helper lineage redirection is unlikely because of the variegated expression as the same ThPOK-H transgene completely rescued CD4 helper development in Thpok−/− mice expressing or not OTII-TCR.

We propose two mutually nonexclusive possibilities that may explain the ability of the same amount of transgenic ThPOK to completely rescue the CD4 helper development in Thpok−/− or OTII+Thpok−/− mice but induce an inefficient CD8 to CD4 helper lineage redirection of MHCI-specific thymocytes (in OTI+, P14+, or MHCII−/− mice). It is possible that genes responsible for activating the helper program in MHCI-specific thymocytes, because of weak or shorter duration of TCR signaling, are epigenetically modified in such a way that they are inaccessible or accessible for a shorter time for ThPOK-mediated regulation, and in such a case, a significantly higher amount of ThPOK (than the one required for CD4 helper lineage choice of MHCII-specific thymocytes) would be required to override this constrain on CD4 helper lineage choice of MHCI-specific thymocytes. A significantly higher CD4+ mature T cell frequency in OTI+dGLF+ThPOK-H+Thpok−/− mice compared with OTI+ThPOK-H+Thpok−/− mice, both expressing the same amount of ThPOK but differing in their TCR signal strength, strongly suggests that TCR signal strength plays a critical role in establishing the ThPOK-mediated CD4 helper lineage choice. We propose that augmented TCR signal strength, although critical for ThPOK induction, promotes the CD4 helper lineage choice by extending the window of lineage choice during which the target gene loci are accessible readily or for a longer time for ThPOK-mediated modulation. Any MHCI-signaled thymocytes expressing ThPOK at levels comparable to that induced in MHCII-signaled thymocytes but remaining outside this temporal lineage commitment window will differentiate into CD8+ mature T cells with a compromised cytotoxic function. Such a possibility is supported by the observation that although ThPOK induction in MHCI-signaled thymocytes, because of a compound deficiency of Runx1 and Runx3 or Tle1/3/4 or MAZR and Runx3, upregulates helper lineage genes including Cd4, it fails to completely suppress the expression of cytotoxic lineage genes including Cd8, resulting in the generation of a large number of CD4+CD8+ mature T cells of undefined functional potential (2931). Compromised cytotoxic function, but failure to upregulate CD4 or secrete IL-4 following retroviral-mediated ThPOK expression in the peripheral CD8+ mature T cells, also supports such a notion (47). These data are in agreement with the signal strength model of CD4/CD8 lineage commitment; irrespective of MHC specificity, stronger TCR signaling may alter the chromatin structure such that not only the CD4 helper lineage–specifying genes such as Gata3, Tox, or c-Myb are induced (4851) but the accessibility of the target gene loci by ThPOK is enhanced as well, leading to the suppression of the CD8 cytotoxic lineage choice and imprinting of the CD4 helper lineage choice in these cells. These data also suggest that stronger TCR signaling may be sufficient for the CD4 helper lineage commitment even in the absence of ThPOK, provided those critical for the CD8 cytotoxic lineage commitment are suppressed (22).

Although strong TCR signaling is critical for the CD4 helper lineage choice, the MHC specificity of developing thymocytes appears to play an equally important role in the process. Comparing the CD4 helper development of MHCI-specific thymocytes in OTI+dLGF+ThPOK-H+Thpok−/− and MHCII-specific thymocytes in OTII+ThPOK-H+Thpok−/− mice provides some insight into this issue. A significantly higher frequency of CD4+ mature T cells in OTII+ThPOK-H+Thpok−/− mice (MHCII-specific) compared with OTI+dLGF+ThPOK-H+Thpok−/− mice (MHCI-specific), both expressing the same amount of ThPOK, cannot simply be explained by the TCR signal strength model as thymic subsets, and CD4+ mature T cells from OTII+ mice show a significantly lower TCR signaling compared with that in similar subsets from OTI+dLGF+ mice expressing or not ThPOK transgene. Our data then suggest that TCR signaling in MHCI- and MHCII-specific thymocytes are likely to be different not only quantitatively but qualitatively as well, and introducing constitutively active Lck in MHCI-specific thymocytes mimics the quantitative aspect. We propose that continuous TCR signaling in the positively selected MHCII-specific thymocytes not only results in a stronger TCR signal that keeps the lineage commitment window “open” for a longer time but also induces the expression of CD4 helper lineage–establishing genes whose continued expression likely requires ThPOK.

Based on these data, we propose a model that links TCR signaling to the CD4/CD8 lineage choice of MHCI- and MHCII-signaled thymocytes. We propose that qualitatively distinct and stronger TCR signaling opens the window of lineage commitment during which the CD4 helper lineage–specifying genes are induced in MHCII-signaled thymocytes. It is conceivable that during the CD4 helper lineage specification phase, Gata3 induced by TCR signaling functions, for instance, as a “pioneer” transcription factor that remodels the chromatin landscape, which then facilitates the ability of other transcription factors to access the target gene loci in association with or independently of the pioneering factor (5254). The persistent TCR signaling, along with Gata3 expression, would then initiate ThPOK induction (25, 55) in MHCII-signaled thymocytes, which collectively play a role in the CD4 helper lineage commitment and maintenance. The induction of the helper program in MHCI-signaled thymocytes but the inability to sustain it (56) may be due to inadequate chromatin alterations, leading to the insufficient induction of ThPOK and/or its residency at the target gene loci. In such a case, very high ThPOK expression would be necessary for the efficient redirection of the MHCI-signaled thymocytes into the CD4 helper lineage.

In conclusion, considerably different efficiency of CD4 helper lineage choice in three different mouse models expressing the same amount of ThPOK but different modes of TCR signaling (OTI+ThPOK-H+Thpok−/−, OTI+dLGF+ThPOK-H+Thpok−/−, and OTII+ThPOK-H+Thpok−/−) provides a critical insight into the mechanism of the CD4 helper lineage choice of developing thymocytes. Our data link stronger TCR signaling to ThPOK induction and strongly suggest that the CD4 helper lineage choice by a defined amount of ThPOK is critically influenced by the TCR signal strength and MHC specificity of developing thymocytes during a temporal window of lineage commitment.

We thank Martine Dupuis for cell sorting, Marie-Philippe Boucher and Karel Prudhomme for animal care, Paul Jolicoeur for dLGF mice, Qinzhang Zhu (I.R.C.M.) for the generation of ThPOK transgenic mice, and Claude Perreault, Heather Melichar, and Jean-Philippe Bastien for critical comments.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DN

double negative

DP

double positive

MHCI

MHC class I

MHCII

MHC class II

pMHC

self-peptide/self-MHC

QPCR

quantitative PCR

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data