The Protein Tyrosine Kinase Tec Regulates a CD44highCD62L− Th17 Subset

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44highCD62L− Th17 subset in mice deficient for the protein tyrosine kinase Tec. CD44highCD62L− Tec−/− CD4+ T cells produced enhanced IL-17 upon activation, showed increased expression levels of IL-23R and RORγt, and IL-23–mediated expansion of Tec−/− CD4+ T cells led to an increased production of IL-17. Tec−/− mice immunized with heat-killed Streptococcus pneumoniae displayed increased IL-17 expression levels in the lung postinfection with S. pneumoniae, and this correlated with enhanced pneumococcal clearance and reduced lung inflammation compared with Tec+/+ mice. Moreover, naive Tec−/− OT-II CD4+ T cells produced higher levels of IL-17 when cultured with OVA peptide-loaded bone marrow-derived dendritic cells that have been previously activated with heat-killed S. pneumoniae. Taken together, our data indicated a critical role for Tec in T cell-intrinsic signaling pathways that regulate the in vivo generation of CD44highCD62L− effector/memory Th17 populations.

M embers of the Tec kinase family (Tec, Btk, Itk, Rlk, and Bmx) play an important role during lymphocyte development and activation, and mutations in Tec family kinases are linked with immunodeficiencies in humans and mice (1,2). Three members of the Tec kinase family are expressed in T cells: Tec, Itk, and Rlk. Itk 2/2 mice have defects in T cell development, affecting in particular conventional T cells, thus leading to increased numbers of CD4 + and CD8 + T cells with a CD44 high CD62L 2 effector/memory phenotype that rapidly se-crete high levels of effector cytokines such as IFN-g after PMA/ ionomycin stimulation and thus were described as innate-like T cells. Moreover, TCR-mediated activation and proliferation of naive T cells as well as their polarization into Th2 effector T cells are impaired in the absence of Itk. Additionally, Itk is also important in the activation of b 1 integrins and the regulation of the actin cytoskeleton upon activation. Several studies implied a role for Rlk in Th1 cell polarization, although Rlk 2/2 mice had only a mild T cell signaling defect. The analysis of Itk 2/2 Rlk 2/2 T cells, however, revealed that Rlk can compensate for loss of Itk in TCR-mediated signaling events. Taken together, these studies showed that Itk and Rlk play an important role in the development and function of T cells (for reviews, see Refs. [3][4][5][6]. To address the role of Tec in lymphocytes, we analyzed previously generated Tec 2/2 mice (7). Initial studies with Tec 2/2 animals focused on B cells, and it was shown that Tec regulates B cell development in the absence of Btk. With respect to T cells, studies using a murine T cell hybridoma line have shown that Tec is activated in response to TCR/CD28 stimulation (8,9). CD28 engagement leads to recruitment of Tec to the cytoplasmic tail of CD28, and overexpression of Tec activates both Il2 and Il4 promoters (10), However, T cell proliferation and IL-2 production in response to TCR/CD28 stimulation were normal in the absence of Tec (7), although another study showed that downmodulation of Tec kinase by antisense oligonucleotide treatment in primary murine splenocytes impaired Il2 gene induction (11). Recently, it was reported that Tec expression is upregulated in effector T cells, in particular Th2 cells (12). Taken together, the function of Tec in primary T lymphocytes is poorly understood.
In this study, we performed a comprehensive analysis of the helper T cell lineages in Tec 2/2 mice. Il23r and Rorc expression were enhanced in CD44 high CD62L -Tec 2/2 CD4 + T cells, and IL-23mediated expansion of Tec 2/2 CD4 + T cells led to increased production of IL-17. Ex vivo, PMA/ionomycin-stimulated Tec 2/2 splenocytes contained an increased population of IL-17-producing CD44 high CD4 + T cells, whereas the percentage of IFN-g-or IL-4-producing CD44 high CD4 + T cells was similar between Tec +/+ and Tec 2/2 mice. This indicated elevated Th17 effector/memory subsets in Tec-deficient mice. Upon TCR-mediated activation, CD44 high CD62L 2 Tec +/+ CD4 + T cells strongly upregulated Tec expression, and CD44 high CD62L 2 Tec 2/2 CD4 + T cells produced a larger amount of IL-17A (referred to herein as  and showed increased levels of Il17f and Il21. Moreover, Tec 2/2 mice immunized with heat-killed Streptococcus pneumoniae expressed larger amounts of Il17 mRNA in the lung after intranasal infection, and this correlated with enhanced pneumococcal clearance and reduced CFU/lung in Tec 2/2 mice. Because IL-17-expressing CD4 + T cells are important for an efficient clearance of S. pneumoniae (13,14), these data suggested increased IL-17-producing CD4 + T cells in Tec-deficient mice upon immunization. Finally, naive Tec 2/2 OT-II CD4 + T cells produced higher levels of IL-17 when stimulated with OVA peptide-loaded bone marrow-derived dendritic cells (BMDCs) that had been previously activated with heat-killed S. pneumoniae. Thus, our data suggest a critical role for Tec in T cell-intrinsic signaling pathways that regulate the in vivo generation of a CD44 high CD62L 2 Th17 effector subset.

Materials and Methods
Mice Animals used in this study were 6-12 wk of age unless otherwise indicated. The animals were bred and maintained in the animal facility of the Medical University of Vienna, and all animal experiments were approved by the animal committee of the Medical University of Vienna and by Federal Ministry for Science and Research. Tec 2/2 mice were previously described (7) and backcrossed to C57BL/6 for at least 10 generations. OT-II mice (15) expressing a TCR specific for OVA 323-339 in the context of I-A b were obtained from Dr. Dagmar Stoiber-Sakaguchi. CD45.1 + congenic mice were obtained from the European Mouse Mutant Archive (Département de Cryopréservation, Distribution, Typage et Archivage Animal, Centre National de la Recherche Scientifique, Orleans, France).

Purification of CD4 + T cells
Pooled cell suspensions of lymph nodes and spleens were incubated with biotinylated anti-CD8a, anti-CD11b, anti-CD11c, anti-CD45R, anti-Ly-6G, anti-Ter-119, and anti-NK1.1 Abs in PBS/2% FBS. The CD4 + T cells were then purified by negative depletion using streptavidin beads (BD Biosciences) according to the manufacturer's instructions. The purity of the cells was assessed by flow cytometry and was routinely .96%. Where indicated, CD4 + T cells were further sorted into CD44 low CD62L + (naive) and CD44 high CD62L 2 populations on a FACSAria cytometer (BD Biosciences), and the purity of the populations was routinely .99% for naive cells and .97% for CD44 high CD62L 2 cells.

Repeated Th17 polarization
Naive CD4 + T cells were stimulated with 1 mg/ml anti-CD3ε and 3 mg/ml anti-CD28 in the presence of 1 ng/ml TGF-b1 and 20 ng/ml IL-6. Cells were split 1:2 after 48 h, transferred into new 48-well plates without stimulation, and were further cultured for 3 d without additional cytokines. On days 5 and 10, the cells were restimulated with 0.3 mg/ml anti-CD3ε in the presence of 1 ng/ml TGF-b1 and 20 ng/ml IL-6 and cultured for another 5 d. IL-17 production on days 5, 10, and 15 was assessed by PMA/ ionomycin stimulation in the presence of GolgiStop (BD Biosciences) for 4 h and subsequent intracellular cytokine staining.

RT-PCR and quantitative real-time PCR analysis
Total RNA was extracted (TRIzol; Sigma-Aldrich) and converted into cDNA using random primers (Fermentas, Burlington, Ontario, Canada) and SuperScript II reverse transcriptase (Invitrogen). cDNA was prepared from in vitro-activated CD4 + T cells after 6 d in culture, restimulated for 8 h with 0.3 mg/ml plate-bound anti-CD3ε, and analyzed by RT-PCR. In addition, samples obtained and treated as indicated in the figure legends were subjected to quantitative real-time PCR (qRT-PCR) analysis on an Applied Biosystems Prism 7300 sequence detection system under standard conditions (Applied Biosystems, Foster City, CA). Genes analyzed were detected using the commercially available SYBR Green assay (Applied Biosystems). Il23r mRNA was quantified using a TaqMan gene expression assay (Applied Biosystems), according to the manufacturer's instructions. The following primer sequences were used for RT-PCR and qRT-PCR (designated with an asterisk): Hprt1 (17)
S. pneumoniae infections S. pneumoniae serotype 3 (ATCC 6303) were grown to midlogarithmic phase in Todd-Hewitt broth. Mice were immunized on days 0, 21, and 35 FIGURE 1. Activated Tec 2/2 CD4 + T cells display Th17 features. A, IL-17, IFN-g, and IL-4 expression levels in supernatants of activated total (naive and CD44 high ) Tec +/+ and Tec 2/2 CD4 + T cells measured by ELISA. CD4 + T cells were activated with anti-CD3ε plus anti-CD28 in the presence of rhIL-2 and restimulated on day 5 with anti-CD3ε (+). 2, Unstimulated controls. Data are representative of five independent experiments. B, Intracellular expression of IFN-g and IL-17 in Tec +/+ and Tec 2/2 CD4 + T cells cultivated as described in A. Cells were restimulated on day 5 with PMA/ionomycin. Data are representative of four independent experiments. C, RT-PCR analysis shows Il17f and Il23r expression in Tec +/+ and Tec 2/2 CD4 + T cells cultivated as described in A and restimulated with anti-CD3ε. Hprt1 expression was used as loading control. Data are representative of two independent experiments. D, CD4 + T cells were activated as described in A and restimulated with IL-23 for the indicated time periods. The phosphorylation status and expression of STAT3 was assessed by immunoblot analysis. Data are representative of three independent experiments. A-D, Data shown were generated from the same batch of CD4 + T cells. with 10 8 heat-killed S. pneumoniae in 200 ml PBS i.p. Four weeks later, mice were anesthetized with isoflurane (Baxter, Deerfield, IL), and 8 3 10 5 CFU S. pneumoniae (in 50 ml sterile saline) was inoculated intranasally. Forty-eight hours postinfection, mice were anesthetized with ketamine (Pfizer, New York, NY) and sacrificed by bleeding out via heart puncture. Whole lungs were harvested and homogenized in sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, OK), after which CFU/lung were determined from serial dilutions, plated on blood agar plates, and incubated at 37˚C for 16 h before colonies were counted. Lung homogenate (80 ml) was added to 350 ml RLT buffer (Qiagen, Hilden, Germany) for RNA extraction using a Qiagen RNeasy kit according to the manufacturer's instructions, which included a DNase step.

Histology
Lungs for histology were harvested 48 h after S. pneumoniae infection, fixed in 10% formalin, and embedded in paraffin. Four-micrometer sections were stained with H&E and analyzed by a pathologist who was blinded for groups. To score lung inflammation and damage, the entire lung surface was analyzed with respect to the following parameters: interstitial inflammation, edema, endotheliitis, bronchitis, pleuritis, and thrombi formation. Each parameter was graded on a scale of 0-4 as fol lows: 0, absent; 1, mild; 2, moderate; 3, moderately severe; and 4, severe. The inflammation score was expressed as the sum of the scores for each parameter, with the maximum being 24. FIGURE 2. Increased Th17 gene expression profile in Tec 2/2 CD44 high CD62L 2 CD4 + T cells upon activation. A-D, Naive and CD44 high CD62L 2 CD4 + T cells of Tec +/+ and Tec 2/2 mice were activated with anti-CD3ε plus anti-CD28 and restimulated on day 5 with anti-CD3ε. Subsequently, different parameters were investigated without (2) and with (+) anti-CD3ε restimulation. A, Expression of Tec was assessed by immunoblot analysis. Phospholipase Cg1 (PLCg1) was used as loading control. Data are representative of two independent experiments. B, IL-17 secretion was assessed by ELISA. Data are representative of five independent experiments. C, Summary showing IL-4, IFN-g, and IL-17 levels in CD44 high CD62L 2 CD4 + T cells as determined by ELISA. For each experiment, Tec +/+ cytokine levels were set as one, and the diagram shows relative cytokine levels. Each symbol represents one experiment. D, Expression of Il17f, Il21, Rorc, and Il23r in CD44 high CD62L 2 CD4 + T cells was assessed by qRT-PCR and normalized to Hprt1 expression. Expression levels in unstimulated Tec +/+ CD44 high CD62L 2 CD4 + T cells were set as one, and relative expression levels to Tec +/+ cells are shown. Data are representative of two independent experiments. E, Naive CD4 + T cells (CD45.1 + ) were cultured with either Tec +/+ or Tec 2/2 CD44 high CD62L 2 CD4 + T cells. The "cocultures" were activated with anti-CD3ε plus anti-CD28 in the presence of rhIL-2. On day 5, cells were restimulated with IL-23 (left panel) or IL-6 (right panel). The phosphorylation of STAT3 in gated CD45.2 + cells was assessed by intracellular pSTAT3 detection. F, Naive Tec +/+ or Tec 2/2 CD4 + T cells (CD45.2 + ) were cocultured with CD44 high CD62L 2 CD4 + T cells (CD45.1 + ) as described in E. The cocultures were restimulated on day 5 with IL-23 (left panel) or IL-6 (right panel). E and F, Data are representative of two independent experiments. ., Tec-specific band; p, nonspecific background band.

Induction of experimental autoimmune encephalomyelitis
Mice were injected s.c. with 100 ml of an emulsion consisting of heat-killed Mycobacterium tuberculosis-supplemented (strain H37Ra) CFA (Difco, Detroit, MI) and 50 mg myelin oligodendrocyte glycoprotein peptide (aa . Mice received 200 ng pertussis toxin (Sigma-Aldrich) i.p. on the day of immunization and 2 d later. Disease progression was assessed daily, and clinical scores were assigned according to the following criteria: 0, unaffected; 1, flaccid tail; 2, impaired righting reflex and/or gait; 3, partial hind limb paralysis; 4, total hind limb paralysis; 5, total hind limb paralysis with partial forelimb paralysis; 6, death.

Induction and evaluation of allergic airway inflammation and mucus production
Mice were immunized with 25 mg OVA (Sigma-Aldrich) or PBS in 2 mg alum i.p. on day 0 and day 5. Mice were challenged 1 wk later with nebulized 1% OVA in PBS with an ultrasonic nebulizer for 60 min twice daily on days 12 and 13. Forty-eight hours after the last aerosol challenge, the tracheas were cannulated and the lungs were lavaged with PBS. Total leukocytes from bronchoalveolar lavage fluid were counted with a hemocytometer. Cytospin slides were stained with May-Grünwald-Giemsa to determine the cell differential.

Statistical analysis
All data are expressed as the mean 6 SD (except otherwise indicated). Statistical analysis was performed by using a Student t test. The p values are indicated in the figures.

Activated Tec 2/2 CD4 + T cells display Th17 features
Flow cytometric analysis of bone marrow cells or splenocytes for the expression of B220, IgD, IgM, Ter-119, CD61, CD11b, and Gr1 from Tec-deficient mice did not reveal any differences compared with wild-type mice (7). Furthermore, thymocytes and peripheral T cell numbers as well as CD4/CD8 subset distribution are normal in Tec 2/2 mice (7). Moreover, there was no difference between Tec +/+ and Tec 2/2 CD4 + T cells in the proliferative capacity and production of IL-2 in response to anti-CD3ε with or without anti-CD28 stimulation (Supplemental Fig. 1A, 1B). Additionally, Ca 2+ flux and the overall tyrosine phosphorylation patterns were similar in Tec +/+ and Tec 2/2 T cells (Supplemental Fig. 1C, 1D), indicating normal ex vivo activation upon TCR stimulation. Next, we investigated whether cytokine production is altered upon restimulation of Tec 2/2 CD4 + T cells. Therefore, purified total CD4 + T cells (i.e., both naive CD44 low CD62L + and CD44 high CD62L 2 memory-like populations) were activated with anti-CD3ε plus anti-CD28, rested, and restimulated at day 6 with anti-CD3ε. Under these conditions, Tec 2/2 CD4 + T cells produced much higher levels of IL-17 compared with Tec +/+ CD4 + T cells (Fig. 1A). In contrast, IL-4 and IFN-g secretion was not altered in the absence of Tec (Fig. 1A). Intracellular cytokine staining of Tec 2/2 CD4 + T cells revealed the presence of an IL-17-producing subset that was distinct from IFN-g-producing cells (Fig. 1B). Additionally, Tec 2/2 effector CD4 + T cells expressed also higher levels of other Th17-related genes such as Il17f and Il23r (for a review, see Ref. 21) upon TCR restimulation (Fig. 1C).
IL-23R signaling activates STAT3, and IL-23-activated STAT3 binds to the Il17 gene locus and induces IL-17 expression (22). Therefore, we investigated whether elevated expression of the Il23r in Tec 2/2 effector CD4 + T cells correlates with increased STAT3 phosphorylation. Tec +/+ CD4 + T cells that were previously activated under nonpolarizing conditions showed low levels of STAT3 phosphorylation (pSTAT3) upon IL-23 stimulation, whereas Tec 2/2 CD4 + T cells displayed much higher levels of pSTAT3 (Fig. 1D). These data suggest that increased IL-23R expression in the absence of Tec leads to enhanced pSTAT3 and thus increased IL-17 production upon activation of total CD4 + T cells.
Enhanced Th17 effector subsets in Tec 2/2 mice Next we wanted to determine whether there is already a difference in IL-17 production between Tec +/+ and Tec 2/2 naive and CD44 high CD62L 2 CD4 + T cells under homeostatic conditions. Tec +/+ and Tec 2/2 mice displayed a similar CD44/CD62L expression profile on peripheral CD4 + T cells, although there was a mild reduction of the naive CD44 low CD62L + subsets and a corresponding small increase in CD44 high CD62L 2 effector-like CD4 + T cells (Fig. 3A). Ex vivo PMA/ionomycin stimulation of splenocytes revealed an increase in IL-17-producing cells within the CD44 high CD4 + T cells in Tec 2/2 mice (Fig. 3B); however, there was no increase in IFN-g-producing cells within the CD44 high subset (Fig. 3C). Enhanced IL-17 was also observed when either invariant NKT cells or gdT cells were excluded from the CD4 + T cell gate (Supplemental Fig. 2A, 2B), indicating that the increase in IL-17 + cells is not due to differences in innate-like and/or nonconventional T cell subsets. Moreover, the increased IL-17-producing population was only observed in splenic CD4 + T cells but not in CD4SP thymocytes from 4-and 8-wk-old mice (Supplemental Fig. 2C, Fig. 3B, respectively), suggesting that this subset does not represent a distinct helper sublineage that originates already during thymocyte development.
It has been shown that Il23r and Rorc are expressed ex vivo in CD44 high CD62L 2 CD4 + T cells, but not in naive CD4 + T cells (23,24). The observation that Il23r and Rorc expression is increased upon restimulation in Tec 2/2 CD44 high CD62L 2 CD4 + T cells prompted us to test whether in sorted naive and CD44 high CD62L 2 CD4 + T cells the Il23r and Rorc expression differs already between Tec +/+ and Tec 2/2 mice. Purified Tec 2/2 CD44 high CD62L 2 CD4 + T cells displayed increased Il23r and Rorc ex-  . CD44 high CD62L 2 CD4 + T cells display Th17 features. A, Ex vivo expression analysis of Il23r and Rorc by qRT-PCR in Tec +/+ and Tec 2/2 CD44 high CD62L 2 CD4 + T cells. Expression in each subset was normalized to Hprt1 expression. For Il23r and Rorc expression, expression levels in Tec +/+ CD44 high CD62L 2 CD4 + T cells were set as one, and relative expression levels to Tec +/+ cells are shown. Data are representative of three independent experiments. B, Total Tec +/+ and Tec 2/2 CD4 + T cells were stimulated with anti-CD3ε plus anti-CD28 in the presence of IL-23, anti-IL-4, and anti-IFN-g. Cells were restimulated on day 5 with anti-CD3ε, and IL-17 levels in the supernatant were determined by ELISA. Each symbol represents one mouse.
pression levels compared with Tec +/+ CD44 high CD62L 2 CD4 + T cells, whereas Il23r and Rorc expression was not detectable in the naive subsets of both genotypes (Fig. 4A).
Tec-deficient mice immunized with heat-killed S. pneumoniae display enhanced pneumococcal clearance postinfection Th17 cells are important for an efficient clearance of S. pneumoniae colonization in the lung (13,14). To investigate whether the increased IL-17-producing CD4 + T cell population with a Th17 signature in Tec 2/2 mice leads to a better protection against S. pneumoniae, Tec +/+ and Tec 2/2 mice were intranasally infected and CFU/lung and lung IL-17 expression were determined 48 h postinfection (Fig. 5A). Under these conditions, similar CFU/lung were detected in Tec +/+ and Tec 2/2 mice (Fig. 5B) and there was no difference in the expression of IL-17 (Fig. 5C).
To study whether S. pneumoniae-specific effector/memory Th17 responses in Tec 2/2 mice are enhanced, Tec +/+ and Tec 2/2 mice were immunized i.p. with heat-killed S. pneumoniae followed by two booster injections at days 21 and 35. Twenty-eight days after the last injection, mice were infected intranasally and CFU/ lung were determined 2 d postinfection (Fig. 5A). Tec 2/2 mice showed decreased levels of CFU/lung compared with immunized Tec +/+ mice (Fig. 5B), as well as reduced signs of inflammation (Fig. 5D, 5E), indicating a better bacterial clearance in the lung. The enhanced clearance of S. pneumoniae correlated with increased Il17 expression levels in Tec 2/2 lung homogenates (Fig. 5C).

T cell-intrinsic alterations in the absence of Tec
Since Tec-deficient CD44 + CD62L 2 CD4 + T cells produced enhanced levels of IL-17, we tested whether the in vitro generation of Th17 cells is altered in the absence of Tec. Th17 cells generated from naive Tec 2/2 CD4 + T cells in the presence of TGF-b1 plus IL-6 (27) produced similar levels of IL-17 compared with Tec +/+ cells (Fig. 6A, upper panels, and Fig. 6B), indicating that Tec kinase does not regulate the in vitro differentiation of Th17 cells via TGF-b1 and IL-6. Tec was also not required for the development of FoxP3-expressing inducible regulatory T cells generated from naive CD4 + T cells in the presence of TGF-b1 (Fig.  6A, lower panels).
Because immunization with heat-killed S. pneumoniae prior to infection resulted in enhanced Il17 expression in the lung of Tec 2/2 mice, we tested whether naive Tec 2/2 CD4 + T cells might show enhanced IL-17 production if stimulated with dendritic cells that have been activated with heat-killed S. pneumoniae. For this, Tec +/+ and Tec 2/2 mice were intercrossed with OT-II transgenic mice, which express TCRs (V b 5, V a 2) restricted to MHC class II. Naive Tec +/+ and Tec 2/2 OT-II CD4 + T cells were isolated and incubated with OVA peptide-loaded Tec +/+ or Tec 2/2 BMDCs that have been previously activated with heat-killed S. pneumoniae. Tec 2/2 OT-II CD4 + T cells showed enhanced IL-17 production compared with Tec +/+ OT-II CD4 + T cells when incubated with heat-killed S. pneumoniae-activated Tec +/+ BMDCs (Fig. 6C). A similar increase in IL-17 production was also observed when naive Tec 2/2 OT-II CD4 + T cells were stimulated with OVA peptide-loaded Tec 2/2 BMDCs that have been previously activated with heatkilled S. pneumoniae (Fig. 6C). Taken together, these data strongly suggest a T cell-intrinsic defect in the absence of Tec.

Discussion
In this study, we investigated the role of the nonreceptor protein tyrosine kinase Tec in CD4 + helper T cells. Our study revealed that Tec had no major role during T cell development and for anti-CD3ε with or without anti-CD28-induced proliferation. Although studies suggested that Tec is involved in the activation of the IL-4 promoter (10) and that Tec is upregulated in Th2 cells (12), we did not observe any alteration in the Th2 (or in Th1) cytokine pattern of Tec 2/2 CD4 + T cells in vitro (Supplemental Fig. 3). Moreover, there was a similar eosinophil infiltration in the lung of Tec +/+ and Tec 2/2 mice in a Th2-driven allergic airway inflammation model (Supplemental Fig. 4). This is in contrast to Itk, which plays an important role in these processes (4,5). This indicates a different utilization of Tec family kinases in the T cell lineage. Whether other Tec family kinases compensate for the loss of Tec in Th2dependent processes remains to be determined; however, there was no compensatory upregulation of Itk or Rlk in the absence of Tec under homeostatic conditions (Supplemental Fig. 5).
An important finding of our study is the identification of an essential role for Tec in the regulation of a CD44 high CD62L 2 Th17 subset. CD44 high CD62L 2 CD4 + T cells are slightly increased in Tec-deficient mice, and they produced more IL-17 upon FIGURE 6. Ag-specific activation reveals T cell-intrinsic defects in Tecdeficient CD4 + T cells. A, Tec +/+ and Tec 2/2 naive CD4 + T cells were stimulated under Th17 or inducible regulatory T cell conditions. Cells were restimulated on day 4 with PMA/ionomycin. Intracellular expression of IL-17 and FoxP3 in Tec +/+ and Tec 2/2 Th17 (upper panel) and regulatory T (lower panel) cells is differentiated from naive CD4 + T cells. Numbers in the dot plots indicate the percentage of cells in the respective quadrants. Data shown are representative of eight independent experiments for TGF-b1 plus IL-6 Th17 differentiation cultures, and of two independent experiments for TGF-b1-induced regulatory T cell differentiation. B, Diagram represents the summary of all Th17 differentiations as described in A. C, Naive Tec +/+ and Tec 2/2 OT-II CD4 + T cells were isolated and incubated for 5 d with OVA peptide-loaded Tec +/+ or Tec 2/2 BMDCs that have been previously activated with heat-killed S. pneumoniae (S.p.) or that have not been activated (con). IL-17 cytokine levels in the supernatants of the T/DC cocultures were determined by ELISA. Data show the summary of three independent experiments. activation compared with Tec +/+ mice. Because Th1 or Th2 memory cells were not increased, this indicated a general bias toward a Th17 memory in Tec 2/2 mice. We consider it unlikely that this CD44 high subset represents an innate-like lineage with immediate effector function similar to the increase in innate-like T cells observed in Itk-deficient mice (29)(30)(31)(32)(33) or that a natural Th17 population as described recently (34) was expanded in the absence of Tec. The increase in IL-17-producing CD44 high CD4 + T cells upon PMA/ionomycin stimulation was restricted to peripheral CD4 + T cells and not observed in CD4SP thymocytes. This argues against a distinct lineage already generated in the thymus. Also, we did not observe alterations in invariant NKT and gdT cell subsets. Thus, our results rather indicate that conventional Th17 subsets are enhanced in Tec 2/2 mice and that TCR stimulation leads to an expansion of these Th17 effector/memory subsets. First, we observed that ex vivo-isolated CD44 high CD62L 2 CD4 + T cells expressed increased levels of Il23r and Rorc in the absence of Tec. Second, the addition of IL-23, known to induce the expansion of preformed effector Th17 cells (23,27,28,35), led to increased IL-17 levels in Tec 2/2 CD4 + T cell cultures. Third, upon activation, Tec 2/2 CD44 high CD62L 2 CD4 + T cells expressed also higher levels of Il17f and Il21, which is indicative of a Th17 signature. Additionally, lungs of Tec 2/2 mice displayed reduced inflammation and enhanced IL-17 expression compared with Tec +/+ mice after S. pneumoniae infection, but only if mice were immunized with heat-killed S. pneumoniae before the infection. This suggests that there might be also enhanced Th17 memory development in Tec-deficient mice during an immune response, leading to a protective Th17 response upon rechallenge. Taken together, these data indicate that Tec deficiency enhances the in vivo generation of a CD44 high CD62L 2 Th17 effector/ memory subset.
One question that arises is why a CD44 high CD62L 2 CD4 + T cells subset with Th17 characteristics is enhanced in the absence of Tec. In vitro, Th17 differentiation from naive CD4 + T cells was shown to occur upon anti-CD3ε/CD28 stimulation in the presence of IL-6 plus TGF-b1 (27); however, other cytokines were shown to contribute to the differentiation and homeostatic maintenance of Th17 cells (19,36,37). Tec 2/2 Th17 cells (as well as transgenic OT-II Tec 2/2 Th17 cells; data not shown) generated with IL-6 plus TGF-b1 did not display differences in the extent of IL-17 production even after repetitive stimulation (Supplemental Fig. 6). However, naive Tec 2/2 OT-II CD4 + T cells produced more IL-17 compared with Tec +/+ OT-II CD4 + T cells when stimulated with OVA peptide-loaded BMDCs that were matured/activated with heat-killed S. pneumoniae, an extracellular bacterium that elicits Th17 responses in vivo (13,14). This indicates that naive Tec 2/2 CD4 + T cells, dependent on the activation conditions, have the potential to produce more IL-17 than do their Tec +/+ counterparts. Moreover, this suggests that the increase in Th17 effector T cells in vivo might be in part due to T cell-intrinsic defects. The strong upregulation of Tec expression in activated CD44 high CD4 + T cells further suggests a T cell-intrinsic role for Tec downstream of TCR signaling. However, preliminary results showed that Tec 2/2 BMDCs produced increased levels of TGF-b1 even without activation, and after activation with heat-killed S. pneumoniae they also produced increased levels of IL-6, IL-1b, IL-23, and TNF-a, all cytokines that favor Th17 responses (Supplemental Fig. 7A). Moreover, supernatants from heat-killed S. pneumoniae-treated Tec 2/2 BMDCs induced more IL-17 production in wild-type T cells compared with Tec +/+ BMDC supernatants (Supplemental Fig. 7B). It is therefore likely that T cell-intrinsic alterations together with T cell-extrinsic changes lead to the enhanced Th17 CD44 high CD62L 2 subset in Tec 2/2 mice, and that the enhanced Th17 subsets under homeostatic conditions might have arisen from an altered response against commensal bacteria in the absence of Tec (21,38).
Tec 2/2 CD44 high CD62L 2 CD4 + T cells expressed higher amounts of Il17a and Il17f. Thus, the role of Tec in Th17 cells is different to the function of Itk. Whereas Itk selectively regulates the expression of Il17a but not of Il17f in an NFAT-dependent manner (39), Tec negatively regulates Th17 signature genes such as Il17a, Il17f, Rorc, Il21, and Il23r. However, Tec 2/2 mice showed a similar incidence and clinical score compared with Tec +/+ mice in an experimental autoimmune encephalomyelitis model (Supplemental Fig. 8). In contrast, Tec-deficient mice displayed enhanced pneumococcal clearance and IL-17 expression in the lung after immunization and infection. Since IL-17 production is beneficial for immunity against a growing number of extracellular pathogens, including S. pneumoniae (13,14,40), it is tempting to speculate that Tec might be a potential target to increase host defense against these pathogens without raising the risk for Th17-mediated autoimmunity.
Taken together, our study showed that Tec deficiency leads to the enhanced in vivo generation of Th17 effector/memory subsets. Additional studies aiming to further characterize the cellular and molecular pathways controlled by Tec in CD4 + T cells and other hematopoietic cells are required to fully understand the role of Tec in the generation and function of Th17 cells.