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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2009 April 15.
Published in final edited form as:
PMCID: PMC2657555
NIHMSID: NIHMS96894

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

Abstract

The Src family kinase, Fyn, is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells, and have thymocytes and T cells with compromised responses following antibody cross-linking of their TCRs. Here we have addressed the role of Fyn in peptide/MHC class II-induced CD4+ T cell responses. In Fyn-deficient mice, CD4+ T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naïve and regulatory DO11.10+CD4+ T cells in the periphery were comparable with their wild type counterparts. Conjugation with Ovap323-339 loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10+Fyn-deficient CD4+ T cells was virtually indistinguishable from the response of DO11.10+ wild type CD4+ T cells. Proliferation of Fyn-deficient T cells was not more dependent on co-stimulation through CD28. In addition, we have found that differentiation, in vitro or in vivo, of transgenic CD4+ Fyn-deficient T cells into IL-4 secreting effector cells was unimpaired, and under certain conditions DO11.10+Fyn-deficient CD4+ T cells were more potent cytokine-producing cells than DO11.10+ wild type CD4+ T cells. These data demonstrate that ablation of Fyn expression does not alter most antigen-driven CD4+ T cell responses with the exception of cytokine production, which under some circumstances, is enhanced in Fyn-deficient CD4+ T cells.

Keywords: Fyn kinase, CD4+ T cell activation, IL-4, IFNγ

Introduction

Recognition of antigen by the T cell antigen receptor (TCR) initiates numerous coordinated signaling pathways that ultimately control T cell behavior. TCR signaling is initiated by phosphorylation of the immunoreceptor-tyrosine based activation motifs (ITAMs) present in the ζ-chain and CD3 chains of the TCR by the Src-family kinases, Lck and Fyn (1, 2). Phosphorylated ITAMs recruit a second type of intracellular tyrosine kinase, ZAP-70, leading to its activation. Subsequent signaling reactions are initiated by active ZAP-70, Lck and/or Fyn (1). Lck is associated with the co-receptors CD4 and CD8, which contribute to the activation of conventional α/β T cells by binding to major histocompatibility complex (MHC) molecules stabilizing the interaction between the TCR and antigen-bound MHC molecules, and by recruiting Lck to the TCR which promotes TCR-mediated signaling (3, 4). In contrast, Fyn associates with the TCR ζ-chain at low stochiometery, which may nonetheless allow it to initiate TCR signaling through the phosphorylation of ITAMs within the ζ-chain (5, 6). A variety of studies have suggested that Fyn also plays a positive role in TCR signaling at subsequent steps through its phosphorylation of SLAP-130/Fyn-binding protein (FYB) (7), Vav (8), WASp (Wiskott-Aldrich syndrome protein) (9) and Pyk-2 (10), all of which promote cytoskeletal re-organization and the formation of the immunological synapse between T cells and antigen-presenting cells (APC) (7, 9). Indeed, two studies utilizing TCR transgenic Fyn-deficient T cells have suggested that in the absence of Fyn, T cell interactions with APCs are weaker (79). In addition, Fyn can phosphorylate PAG (phosphoprotein associated with glycolipid-enriched membranes) also called Cbp (Csk-binding protein), which acts to downregulate TCR signaling through its recruitment to the membrane of Csk, a kinase that inhibits both Fyn and Lck through phosphorylation of negative regulatory tyrosines near their C-termini (11, 12). Thus, Fyn kinase has been implicated in both the propagation and inhibition of TCR-mediated signaling.

Fyn kinase is also involved in the signaling pathway downstream of the CD2-related cell adhesion molecule SLAM (signaling lymphocyte activation molecule), which promotes T cell differentiation to a T helper type 2 (Th2) phenotype (13). SLAM associates with the adaptor molecule SAP (signaling lymphocyte activation molecule (SLAM)-associated protein), which in turn associates with Fyn and in part signals through it (1315). The formation of the SLAM/SAP/Fyn complex has been shown to promote T cell secretion of IL-4 (13). Defects in SAP, which is encoded on the X-chromosome, lead to the disease X-linked lymphoproliferative (XLP) syndrome in which there are defects in the control of Epstein-Barr virus infection and in the generation of germinal centers (1618). Thus, Fyn kinase is thought to be involved in T cell activation and in differentiation to cytokine-producing effector T cells downstream of both the TCR and SLAM.

An important approach for understanding the unique roles of Fyn in T cell function is the analysis of T cells from Fyn-deficient mice. In the periphery of these mice, conventional α/β T cells are present at normal frequency and number (19, 20), but NKT cells are greatly reduced in number (21, 22). This defect may result from failed positive selection of CD1d-dependent NKT cell precursors in the thymus (21, 22), and/or because of the absence of a Fyn-dependent SAP-mediated signal required for the development of these cells (23, 24). In contrast, development of conventional α/β T cells in the thymus appears to be normal in fyn−/− mice, with the exception of impaired thymocyte deletion in response to certain super-antigens, including Mls-1a (19). Fyn-deficient thymocytes and mature peripheral T cells exhibit striking defects in their in vitro proliferative response to stimulation with anti-CD3 antibodies and have substantially decreased anti-CD3-induced signaling reactions including intracellular calcium mobilization and tyrosine phosphorylation of proteins (19, 20, 25). These observations indicate that Fyn plays an important role in TCR signaling in situations where the co-receptors CD4 and CD8 do not participate and therefore, Lck is not efficiently recruited to the TCR. But whether or not Fyn plays a unique role in TCR signaling in response to peptide-MHC complexes presented by APCs is less clear. Stimulation of AD10 TCR-transgenic Fyn-deficient CD4+T cells with antigen and APCs in vitro results in relatively normal phosphorylation of signaling components, calcium mobilization, dephosphorylation and nuclear translocation of NF-AT, and IL-2 production (25). In contrast, other studies have reported defects in the interactions of Fyn-deficient T cells with APCs (79).

To address the role of Fyn in the physiological response of CD4+ α/β T cells following stimulation of the TCR with peptide in the context of MHC molecules, we bred the fyn-mutation to the BALB/c genetic background and also introduced a transgene encoding the DO11.10 TCR, which recognizes a peptide from ovalbumin when presented by the BALB/c class-II MHC molecule, I-Ad (26). Here we report that DO11.10 TCR-transgenic Fyn-deficient T cells developed normally, and that their association with APCs, their proliferation in response to antigen stimulation, and their need for co-stimulation from B7-1 and B7-2 were all similar to Fyn-expressing (wild type) DO11.10 TCR-transgenic T cells. Moreover, Fyn-deficient CD4+ T cells did not have a defect in their ability to differentiate into IL-4 secreting effector cells following simulation with antigen in vitro, or in response to infection with Nippostrongylus brasiliensis. Under certain conditions, however, DO11.10+ Fyn-deficient CD4+ T cells were more potent producers of effector cytokines than their wild type counterparts. Combined, these data demonstrate that antigen-driven activation and differentiation to Th2 effector cells is unimpaired in the absence Fyn kinase, and reveals a previously unappreciated level of redundancy, with respect to Fyn, in antigen-induced CD4+ T cell responses.

Materials and Methods

Mice

BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, MN), or Charles River Laboratory (Wilmington, MA) or bred within our colony. BALB/c Fyn-deficient mice were generated by crossing mice expressing the inactivated Fyn-allele, originally described by Soriano et al., (19), on a mixed C57BL6/129S7/Sv background to BALB/c mice for 6–9 generations (G6-G9) and then to homozygosity for the inactivated Fyn-allele. DO11.10 TCR transgenic BALB/c mice, which express a transgenic TCR that recognizes a peptide from chicken egg ovalbumin (Ovap323-339) in association with I-Ad (26), were obtained from Dr A. Abbas (University of California, San Francisco - UCSF). To generate DO11.10 TCR-transgenic Fyn-deficient mice G6 Fyn-deficient mice were crossed to the DO11.10 TCR-transgenic mice and were bred to homozygosity for the inactivated Fyn-allele. Once generated, the DO11.10 TCR-transgenic Fyn-deficient mice were maintained within our colony. BALB/c B7-1 and -2 double knockout mice (27) were a gift from Dr A. Abbas (UCSF).

The genotype of the DO11.10 TCR-transgenic and Fyn-deficient mice used in these experiments was confirmed by PCR, using DNA samples prepared from murine tail sections, and primers and PCR conditions as described by Jackson Laboratories. The genotype of B7-1 and B7-2 deficient animals was confirmed by the lack of expression of B7 molecules by flow cytometry. All experimental mice were used at 8–13 weeks of age and for each experiment mice were matched for sex and age (within 2 weeks), and animals were housed in a specific pathogen free animal facility at The University of California (San Francisco, CA) under conditions that meet institutional animal care and use committee (IACUC) and NIH guidelines.

Antibodies and Reagents

Anti-CD3ε (145-2C11), anti-CD28 (37.51), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD69 (H1.2FE), anti-CD25 (2A3), anti-CD5 (53-7.3), anti-CD62L (MEL-14), anti-CD16/CD32 (2.4G2), anti-B7-1 (16-10A1), anti-B7-2 (24F), anti-SiglecF (E50-2440), anti-CD11b (M1/70), anti-Gr1 (RB6-865), anti-Ter119 (TER-119), anti-NK1.1 (PK136), anti-B220 (RA3-6B2), anti-IL-4 (11B11) anti-IFNγ (XMG1.2), anti-murine IgE (R35-72) and anti-murine IgE (R35-118) and isotype controls for rat IgG2a,κ (R35-95), mouse IgG1,κ (MOPC-31C), Armenian hamster IgG1,κ (A19-3) unconjugated or conjugated to biotin, fluorescein isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), PE-Cy7, PerCp-Cy5.5 as needed were purchased from Becton Dickinson PharMingen (San Diego, CA). Anti-CD4-Alexa700 was purchased from BioLegend (San Diego, CA). Anti-DO11.10-APC or -PE (KJ1-26) and Anti-CD4-APC-Alexa750 were purchased from Caltag/Invitrogen (Carlsbad, CA). Anti-FoxP3-FITC (FJK-16s) was purchased from eBioscience (San Diego, CA).

A peptide from chicken ovalbumin, Ovap323-339, was synthesized and purified by Genmed Synthesis (South San Francisco, CA). Phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A were purchased from Sigma-Aldrich (St. Louis, MO). The intracellular dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), Indo-1 AM, orange-fluorescent tetramethylrhodamine (CMTMR) and 7-hydroxy-9H I,3-dichloro-9,9-dimethylacridin-2-one (DDAO) were purchased from Molecular Probes, Invitrogen (Carlsbad, CA).

Flow Cytometry and staining with CFSE

A single cell suspension was prepared from thymus or spleen for analysis or purification by flow cytometry. In each case, prior to staining, binding to Fc receptors was inhibited by incubating with an anti-CD16/CD32 antibody. Cells were then stained with different combinations of antibodies as indicated. All surface staining was done for 30 min on ice in Ca2+- and Mg2+-free phosphate buffered saline (PBS) containing 1% FCS with 0.5% sodium azide for analysis, or without sodium azide for cell sorting. For analysis, data were acquired using a fluorescence-activated cell sorter (FACS) Calibur with Cell Quest software or LSR II (Becton Dickinson. San Diego, CA). For purification, a MoFlo high performance cell sorter was used (Dakocytomation. Fort Collins, CO).

For intracellular staining for FoxP3, thymocytes or splenocytes were treated with an anti-CD16/CD32 antibody and stained with anti-CD4-PECy7, anti-CD8-PE and anti-DO11.10-APC, or with anti-CD4-PECy7, anti-CD25-PE and anti-DO11.10-APC, respectively. Cells were then fixed, permeabilized and stained intracellularly with anti-FoxP3-FITC overnight at 4°C using the FITC anti-mouse/rat Foxp3 Staining set from eBioscience (#71-5775 San Diego, CA), as per the manufacturer’s instructions. To assess cytokine production by intracellular staining for cytokines, lymph nodes cells were surface stained with anti-CD8-FITC and anti-CD4-PECy7 and subsequently fixed, permeabilized and stained intracellularly with anti-IL-4-APC and anti-IFNγ-PE using a Cytofix/Cytoperm kit from Becton Dickinson (#554714) as per the manufacturer’s instructions.

To assess proliferation by flow cytometry, purified CD4+ or naïve DO11.10+ T cells (3×107 cells/ml) were labeled with 3μM CFSE in PBS/0.5% FCS in a 37°C water bath for 8 mins. Excess CSFE was removed by the addition of fresh RPMI-1640 containing 10% FCS.

Purification of CD4+ T cells

CD4+ T cells were prepared from the lymph nodes and spleen of mice by negative selection using a CD4+ T cell isolation kit from Miltenyi Biotec (Auburn, CA), and an autoMACS (Miltenyi Biotec), as per the manufacturer’s instructions. The purity and activation state of the purified T cells was assessed by flow cytometry. Cells from non-TCR-transgenic mice were prepared for analysis by staining with anti-CD4-APC, anti-CD69-FITC or anti-CD62L-FITC, whereas samples from TCR-transgenic animals were stained with DO11.10-APC, anti-CD4-PECy7, and anti-CD69-FITC or anti-CD62L-FITC. FACS analysis revealed purity to be in the range of 90–95% CD4+ T cells, which predominantly expressed high levels of CD62L and were negative for CD69 (data not shown). 80–85% of CD4+ T cells prepared from DO11.10 TCR-transgenic wild type or Fyn-deficient mice expressed the DO11.10 TCR transgene (data not shown).

Naïve DO11.10+ T cells were prepared from the lymph nodes and spleen of DO11.10 TCR-transgenic wild type mice or DO11.10 TCR-transgenic Fyn-deficient mice. To prepare the spleen samples, a single cell suspension was made, the red blood cells were lyzed, and the B220+ cells were removed through the use of anti-B220 Dynal beads (Invitrogen, Carlsbad, CA), as per the manufacturer’s instructions. The B220-depleted splenocytes and lymph node cells were then stained with anti-DO11.10-APC, anti-CD25-PE and anti-CD62L-FITC and sorted for live DO11.10+, CD25 and CD62Lhi lymphocytes. The typical purity was within the range of 89–95% (data not shown).

In vitro CD4+ T cell stimulation

For stimulation with plate-bound anti-CD3, 96-well flat bottom tissue culture plates (Corning Costar, Lowell, MA.) were coated with various concentrations of anti-CD3ε in PBS at 4°C overnight. Purified CD4+ T cells from wild type BALB/c mice or G9 Fyn-deficient mice were plated at a starting cell density of 1×105 well in 200μl of RPMI-1640 (UCSF Cell culture Facility) supplemented with 10% FCS (Gibco Invitrogen, Carlsbad, CA.), 1mM Hepes, 1mM non-essential amino acids, 1mM pyruvate, 1mM L-glutamine and 1mM penicillin and streptomycin (all of which were obtained from the UCSF Cell culture Facility) and 5×10−5M 2-mercaptoethanol. Where indicated, 10ng/ml IL-2 (BD Biosciences, San Jose, CA.) or 2μg/ml of an anti-CD28 antibody was included in the cultures. CD4+ T cells were cultured at 37 °C and 5% CO2 for 48 to 72 hrs. Replicate wells were prepared for proliferation and cytokine production. To assess changes in cell surface marker expression cells were stimulated with 3μg/ml plate-bound anti-CD3, harvested at 17–42hrs, stained with anti-CD4-Alexa700, anti-CD25-APC and anti-CD69-PE and the data acquired using an LSRII. Proliferation was assessed by addition of 1μCi [methyl-3H]-Thymidine (3H-TdR. Amersham, Little Chalfont, UK) incorporation for the last 8 hrs of culture. The level of 3H-TdR incorporated was measured with liquid scintillation using a Trilux scintillation counter (Wallac, San Francisco, CA).

To assess antigen-induced proliferation, CFSE-labeled Fyn-deficient naïve DO11.10+ T cells or CFSE-labeled wild type naïve DO11.10+ T cells were cultured for 40–96 hrs in 200μl RPMI-1640 medium, supplemented as described, at 2.5×104 cells/well in 96-well U-bottom plates (Corning Costar) with syngeneic sex-matched splenocytes from BALB/c mice, or B7-1 and B7-2-double knockout BALB/c mice that had been previously treated with mitomycin C (50μg/ml per 25×106 cells/ml for 30 min at 37°C) at a cell density of 2.5×105/well and various concentrations of Ovap323-339. Proliferation of unlabeled naïve DO11.10+ T cells was also assessed by 3H-TdR incorporation during the last 8 hrs of culture.

For in vitro cytokine production, naïve DO11.10+ T cells were cultured in supplemented RPMI-1640 medium at a starting cell density of 2.5×105 cells/ml with 1μg Ovap323-339/ml and 2.5×106 mitomycin C-treated splenocytes/ml in 24-well flat bottom plates (Corning Costar). After 96 hrs, the cells were harvested, and the dead cells were removed by centrifugation over lympholyte-M density gradient (Cedarlane Laboratories, Hornby, Ontario, Canada) at 2000rpm for 20mins at room temperature. Live cells were harvested and restimulated at 2.5×104/well with 1μg/ml Ovap323-339 and 2.5×105 mitomycin C-treated splenocytes in 96-well U-bottom plates for 18 hrs. The supernatant from each well was harvested and assessed for the presence of IL-4 and IFNγ by ELISA using BD OptEIA kits for IL-4 and IFNγ (PharMingen, San Diego, CA), as per the manufacturer’s instructions.

Assessment of calcium mobilization

Splenocytes and lymph node cells from either wild type or Fyn-deficient mice were pooled and loaded with Indo-1 AM in RPMI 1640 supplemented with 1% BSA (Sigma) and 20mM HEPES for an hour at room temperature. Cells were washed and then stained with anti-Gr1-FITC, anti-Ter119-FITC, anti-NK1.1-FITC, anti-CD11b-FITC, anti-B220-FITC, and anti-CD8α-PE-Cy7, with or without the addition of anti-CD3-biotin, for 30 min on ice. Prior to analysis, 2 μg/ml propidium iodine (Sigma), and in some instances anti-CD4-biotin, was added and the cells warmed to 37°C for 3 minutes after which time calcium mobilization was measured by flow cytometry using an LSRII. Baseline readings were taken for 30 seconds, after which time 25 ug/mL streptavidin (Pierce – ImmunoPure Streptavidin) was added to the cells and measurements continued. The median intracellular calcium concentration was determined by the ratio of fluorescence 405nm emission to 530nm emission over time. Exclusion of PI+FITC+PE-Cy7+ cells from the analysis allowed us to measure the calcium mobilization in CD4+ T cells.

Adoptive transfer and in vivo T cell stimulation

CD4+ T cells from DO11.10 TCR-transgenic mice were purified, the frequency of DO11.10 TCR expressing cells determined, and the cells CFSE-labeled. 2.5–5×106 CFSE-labeled CD4+ T cells, containing an identical number of DO11.10+ cells, were transferred into sex-matched recipient BALB/c mice by tail vein injection. Across all experiments the number of transferred DO11.10+ T cells was within the range 2–4×106 cells/recipient mouse. The following day, recipient mice were immunized sub-cutaneously (sc) with four 50μl injections of 1mg/ml Ovap323-339 in complete Freud’s adjuvant (CFA). Three days after recipient mice were immunized with antigen, draining (brachial and inguinal) and non-draining (cervical) lymph nodes were harvested from the recipient mice. Lymph node cells were stained with anti-DO11.10-APC and the level of CFSE dilution assessed by flow cytometry. To assess cytokine production, draining and non-draining lymph nodes were harvested five days after immunization, the frequency of DO11.10+ cells ascertained by flow cytometry, and lymph node cells containing 1–1.5×104 DO11.10+ T cells/well were restimulated over night with 1μg/ml Ovap323-339 in U-bottom plates. The following day the supernatants were harvested and assessed for the presence of cytokines by ELISA.

Nippostrongylus brasiliensis infection and assessment of inflammation in the lungs

Third-stage N. brasiliensis larvae (L3) were isolated from the cultured feces of infected rats. Wild type BALB\c mice and G9-fyn−/− BALB\c mice were infected with 500 larvae by sc injection and given antibiotic water (2g/L neomycin sulfate and 0.1g/L polymixinB) for 5 days. Infected mice were sacrificed after 10 days, and the lungs perfused with 10mL PBS. Lungs were harvested, crushed and passed through 70mm filters to generate single cell suspensions, prior to staining. Cells were then incubated with 2.4G2, stained with the appropriate antibody cocktail (28) and labeled with the vital dye DAPI (Roche). Counting beads (Caltag, Invitrogen) were used to calculate total cell numbers. Data was acquired on a BD LSRII and analyzed using FlowJo software. To assess cytokine production, mediastinal and mesenteric lymph nodes were harvested, a single cell suspension prepared and 5×106 cells/ml were cultured for 4 hrs in supplemented RPMI-1640 containing 100pg/ml PMA and 1ng/ml ionomycin, 10μg/ml brefeldin A was added for the last 2 hrs of culture. Cells were assessed for the presence of intracellular IL-4 and IFNγ by flow cytometry. The concentration of IgE in the serum of infected mice was assessed by ELISA using the monoclonal antibody (mAb) R35-72 as a capture antibody, and the biotinylated mAb R35-118 for detection. The bound IgE was visualized using streptavidin-alkaline phosphatase.

Assessment of T cell/APC conjugate stability

CFSE-labeled DO11.10+CD4+T cells (5×105) and CMTMR-labeled A20 B cells (5×105) were combined and incubated in RPMI-1640 containing 10% FCS 1mM L-glutamine, 1mM penicillin and streptomycin, and 5×10−5M 2-mercaptoethanol, at 37°C. After 45 minutes, the samples were agitated and 5×105 DDAO-labeled A20 B cells were added. Ninety minutes later, samples were agitated again, fixed with 1% para-formadlehyde, and analyzed by flow cytometry. The percentage of T cell/APC conjugates was determined by gating on CFSE positive cells, and assessing the frequency of CSFE+ cells that were also positive for CMTMR or DDAO.

Results

Fyn-deficient CD4+ T cells fail to proliferate, produce effector cytokines or mobilize calcium in response to stimulation with anti-CD3 antibodies

The role of Fyn tyrosine kinase in TCR signaling has been studied by examining the response of Fyn-deficient thymocytes or T cells to anti-TCR antibodies, super-antigen, or antigen presented by APCs (13, 19, 20, 25, 29, 30). These studies were done with T cells isolated from fyn−/− mice on a mixed background (129/sv x C57BL/6) or partially backcrossed onto the C57BL/6 background. In our initial experiments, we examined the anti-CD3-induced response of CD4+ T cells from fyn−/− BALB/c mice and compared it that of CD4+ T cells from wild type BALB/c mice. Stimulation with plate-bound anti-CD3 antibody induced strong proliferation of wild type, but not of Fyn-deficient, CD4+ T cells (Figure 1A). Similar results were obtained with Fyn-deficient T cells on inbred C57BL/6 background (data not shown). These data are similar to what has been has previously been reported using T cells isolated from less inbred Fyn-deficient mice (13, 25). Stimulation with plate-bound anti-CD3 in combination with IL-2 or anti-CD28 induced a low level of proliferation in Fyn-deficient CD4+ T cells, but their response was always much less than that obtained from wild type controls (Figure 1B and C). Cytokine production following anti-CD3 stimulation was also assessed, and low levels of IL-4 and IFNγ were detected in the supernatants of wild type CD4+ T cells following stimulation with plate-bound anti-CD3 alone, or in combination with IL-2 or anti-CD28, (Figure 1D and E). In contrast, neither IL-4 nor IFNγ were present in the supernatants of Fyn-deficient CD4+ T cells that had been stimulated under identical conditions (Figure 1D and E). The absence of cytokine-production from Fyn-deficient CD4+ T cells was not surprising given that these cells did not proliferate and therefore presumably could not differentiate into cytokine producing effector T cells (31). Moreover the difference between cytokine production of CD4+ T cells from wild type or Fyn-deficient mice was not due to contaminating NKT cells as the method used to purify CD4+ T cells specifically removed NKT cells with an anti-CD49b antibody. Stimulation with plate-bound anti-CD3 also induced activation-associated changes in the expression of several cell surface molecules, including CD69 and CD25. We compared the expression of these molecules on Fyn-deficient CD4+ T cells with that of wild type controls following anti-CD3 stimulation for 17–42 hr. Although fyn−/− CD4+ T cells did up-regulate CD69 and CD25, their response of was always less than that observed for wild type CD4+ T cells irrespective of the length of stimulation (Figure 1F and G and data not shown). These data suggest that Fyn-deficient CD4+ T cells receive a weak TCR signal from plate-bound anti-CD3 that is insufficient to fully activate these cells. To compare TCR signal strength in wild type and fyn−/− CD4+ T cells we measured calcium mobilization following stimulation with anti-CD3-biotin and strepavidin, or with the combination of anti-CD3-biotin, anti-CD4-biotin, and streptavidin. Following stimulation with anti-CD3-biotin and strepavidin Fyn-deficient CD4+ T cells had a weak calcium signaling response when compared to wild type CD4+ T cells from BALB/c mice (Figure 1H). However, when fyn−/− CD4+ T cells were stimulated with co-crosslinked anti-CD3 and anti-CD4, the calcium response was much more similar to that obtained for wild type CD4+ T cells (Figure 1I). Similar results have previously been described using T cells from Fyn-deficient mice on a mixed or partially inbreed C57BL/6 background (19, 20, 25, 32). These data show that cross-linking of CD3 alone induced a weak TCR signal in Fyn-deficient CD4+ T cells, which was insufficient to induce proliferation or cytokine production, but adequate to produce a modest change in the expression of cell surface molecules.

Figure 1
Fyn-deficient T cells proliferate poorly, fail to produce cytokines, modestly up-regulate of activation markers and weakly flux calcium in response to stimulation with anti-CD3 antibodies

Characterization of T cell development in DO11.10+ Fyn-deficient mice

We wanted to assess how fyn−/− CD4+ T cells respond to antigen in a more physiological context in which co-receptors and adhesion molecules participate in the T cell responses. Therefore mice deficient in Fyn kinase were bred to DO11.10 TCR-transgenic mice, which express a TCR that recognizes a peptide from chicken ovalbumin (Ovap323-339) in association with the I-Ad MHC class II molecule (26). Initially, the development of CD4+ T cells in the thymus of DO11.10+ Fyn-deficient mice was examined to determine if development of CD4+ T cells with the DO11.10 TCR was altered in the absence of Fyn kinase. Thymic cell number (data not shown) and the frequency of double negative, double positive and CD4 single positive thymocyte sub-populations were comparable in DO11.10+ wild type mice and DO11.10+ Fyn-deficient mice (Figure 2B and C). Similarly, the expression of CD4, CD8, the DO11.10 TCR, CD3 and the maturation markers CD5 and CD69 were equivalent between the two strains (Figure 2B–G and data not shown). In addition, intracellular staining for the expression of FoxP3 was used to assess the presence of regulatory CD4+ T cells in the thymus. The frequency of FoxP3+ cells and level of FoxP3 expression were equivalent in DO11.10+CD4+CD8 wild type thymocytes and DO11.10+CD4+CD8 fyn−/− thymocytes (mean 0.5% +/− S.D. 0.16 n=3 and mean 0.4% +/− S.D. 0.18 n=4, respectively, and data not shown). These results indicate that CD4+ T cell development, positive selection and the formation of antigen-specific regulatory T cells was unaffected by the absence of Fyn kinase.

Figure 2
Normal development of DO11.10 TCR-transgenic CD4+ T cells in the absence of Fyn kinase

The effect of Fyn-deficiency on the peripheral T cell population in DO11.10 TCR-transgenic mice

In the spleen, the number and frequency of total and transgenic TCR-expressing CD4+ T cells was similar in DO11.10+ wild type mice and DO11.10+ Fyn-deficient mice (Figure 3A, B and data not shown). The expression on these cells of various cell surface molecules, including DO11.10 TCR, CD4, CD3, CD28, CD69, CD25, CD62L, and CD44, was assessed, and in all cases the level of expression was almost identical between the two strains of mice (Figure 3C–F, and data not shown). Particularly noteworthy, the expression levels of a range of activation markers seen on DO11.10 TCR-transgenic and non-transgenic Fyn-deficient CD4+ T cells were consistent with a naïve T cell phenotype (Figure 3 and data not shown). Similarly, we found that splenic CD4+ T cells from non-transgenic Fyn-deficient BALB/c mice or Fyn-deficient C57BL/6 mice predominately had a resting T cell phenotype that was similar to that of wild type control T cells (data not shown). Thus, our results do not support the hypothesis that the loss of signaling from Fyn kinase results in an activated T cell phenotype, in contrast to the results of an earlier study with Fyn-deficient mice that had been partially backcrossed onto a C57BL/6 background (11). Finally, the frequency of splenic DO11.10+CD4+ regulatory T cells determined by assessing the expression of FoxP3 was found to be very similar between DO11.10+ wild type mice (12.5% +/− 1.7 S.D. n=3) and DO11.10+ Fyn-deficient mice (9.0% +/− 1.2 S.D. n=3). These results were not dissimilar to the frequency of FoxP3+CD4+ splenic T cells observed in BALB/c mice (14.2% +/− 1.9 S.D. n=3). Combined, these data indicate that the DO11.10 TCR-transgenic CD4+ T cells in Fyn-deficient mice resemble their wild type counterparts in cell number, in the level of spontaneous activation, and in the frequency of FoxP3+ regulatory T cells.

Figure 3
Peripheral DO11.10 TCR-transgenic Fyn-deficient CD4+ T cells have a naïve phenotype

Antigen-induced proliferation and cytokine production of DO11.10+ Fyn-deficient CD4+T cells in vitro

The normal development and phenotype of DO11.10 TCR-transgenic CD4+ T cells from Fyn-deficient mice made it possible to compare their in vitro and in vivo activation to that of their wild type counterparts. To assess antigen-induced proliferation in vitro naïve DO11.10+ wild type and DO11.10+ Fyn-deficient T cells were purified by fluorescence-activated cell sorting for DO11.10+CD62LhiCD25 T cells, CFSE labeled and stimulated with various doses of Ovap323-339 in the presence of mitomycin C-treated splenocytes as a source of APCs. After 40h or 60h in culture, the TCR-transgenic T cells were harvested, and the dilution of CFSE was assessed by flow cytometry. Following 40h in culture, cell division was not observed from either DO11.10+ wild type T cells or DO11.10+ Fyn-deficient T cells that had been stimulated with a low dose (30ng/ml) of Ovap323-339 (Figure 4A). However, higher doses of the ovalbumin peptide (100 and 1000ng/ml) induced one or two cell divisions from a similar proportion of the DO11.10+ wild type T cells and the DO11.10+ fyn−/− T cells (Figure 4B and C). By 60h, all of the different Ovap323-339 concentrations had stimulated 3 or 4 rounds of cell division in both DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells (Figure 4D–F). Similar numbers of DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells had completed each division for each concentration of Ovap323-339 and length of time in culture. Incorporation of 3H-TdR was also equivalent (Figure 4M). Thus, under these in vitro conditions, proliferation of DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells was virtually identical, irrespective of antigen dose or length of stimulation.

Figure 4
Fyn-deficient DO11.10 TCR-transgenic CD4+ T cells proliferate normally in response to antigen

Although we did not observe any decrease in the in vitro response to antigen of DO11.10 TCR-transgenic Fyn-deficient T cells, it was possible that the response of DO11.10+ Fyn-deficient T cells to antigen was more dependent on co-stimulation through CD28 than is the response of wild type DO11.10+ CD4+ T cells. To test this possibility, the proliferation experiments were repeated using splenocytes from B7-1 and B7-2 deficient mice as APCs. In the absence of co-stimulation provided by CD28, the proliferation of DO11.10+ wild type and DO11.10+ Fyn-deficient T cells was diminished when compared to stimulation with antigen and B7-expressing splenocytes (Figure 4G–M), however the Fyn-deficiency did not affect the proliferation of DO11.10+ T cells (Figure 4G–L). The similarities of the proliferative responses of wild type and Fyn-deficient T cells stimulated with Ovap323-339 in the presence and absence of co-stimulation from B7-1 and B7-2, was confirmed by 3H-thymidine incorporation (Figure 4M). In addition, changes to the level of expression of CD69 and CD25 in response to stimulation with Ovap323-339 and APCs were assessed and found to be similar for both wild type and Fyn-deficient DO11.10+CD4+ T cells, although the response was diminished when B7-deficient splenocytes were used as APCs (data not shown). These results indicate that a deficiency in Fyn kinase did not alter the antigen-specific activation or proliferation of CD4+ T cells in vitro. In addition, these experiments revealed that the proliferative response of Fyn-deficient T cells was not more dependent on co-stimulation through B7-1 and/or B7-2 when compared to the response of their wild type counterparts.

Cytokine production following in vitro priming and restimulation of DO11.10+ Fyn-deficient T cells and DO11.10+ wild type T cells was also assessed. In contrast to the proliferative response, marked differences were observed for cytokine production from DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells. Supernatants from DO11.10+ Fyn-deficient T cell cultures consistently contained approximately two-fold more IL-4 than those from identical cultures containing DO11.10+ wild type T cells (Figure 5A). In contrast, the supernatants from cultures containing wild type T cells typically had more IFNγ than did those from cultures containing DO11.10+ Fyn-deficient T cells (Figure 5B). For both IL-4 and IFNγ the difference in cytokine production was significant, although to a lesser degree for IFNγ. Thus, antigen-specific in vitro activation of DO11.10+ Fyn-deficient T cells resulted in the secretion of more IL-4 and less IFNγ compared to the corresponding activation of DO11.10+ wild type T cells.

Figure 5
DO11.10 TCR-transgenic Fyn-deficient CD4+ T cells more readily differentiate into IL-4 producing cells following in vitro activation with antigen

Activation of DO11.10+ Fyn-deficient CD4+ T cells with antigen in vivo

Next the DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells were stimulated in vivo with antigen in adjuvant. CD4+ T cells were purified from DO11.10+ wild type mice and DO11.10+ Fyn-deficient mice and the frequency of DO11.10 positive cells and the expression of activation markers determined by flow cytometry. Almost all of the purified CD4+ T cells had a naïve phenotype similar to that illustrated in Figure 3. Purified T cells were labeled with CFSE and transferred into naïve, sex-matched recipients. Recipient mice were subsequently immunized sc with 200μg of Ovap323-339 emulsified in CFA. Three days after immunization, proliferation of the transferred cells was assessed. As observed for antigen stimulation in vitro, the in vivo proliferative responses of DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells induced by immunization with Ovap323-339 in CFA were similar (Figure 6A). In response to immunization with Ovap323-339 in CFA many of the DO11.10+ wild type T cells and DO11.10+ Fyn-deficient T cells present in the draining lymph nodes underwent division with the majority of cells present in divisions 6 and 7 after 3 days (Figure 6A). In the non-draining lymph nodes, proliferation could also be observed, however the response was greatly reduced (data not shown). Immunization with Ovap323-339 in CFA did not induce proliferation of CFSE-labeled DO11.10CD4+ T cells from wild or Fyn-deficient mice (data not shown).

Figure 6
In vivo activation with Ovap323-339 in CFA of DO11.10+CD4+ wild type T cells and DO11.10+CD4+ Fyn-deficient T cells reveals similar proliferation but enhanced cytokine production in the absence of Fyn

Cytokine production following immunization was also examined. Five days after the recipient mice had been immunized, they were sacrificed, and the lymph nodes harvested. Draining lymph node cells were restimulated in vitro with 1μg/ml Ovap323-339 for 18 hrs and the supernatants from these cultures were subsequently assessed for the presence of IL-4 and IFNγ. IFNγ was detectable in the supernatant from DO11.10+ wild type T cells or DO11.10+ Fyn-deficient T cells that had been primed in vivo with peptide in CFA (Figure 6B). Interestingly, the supernatants from cultures containing DO11.10+ Fyn-deficient T cells contained more IFNγ than did the supernatants from their wild type counterparts (Figure 6B). Neither DO11.10+ wild type nor DO11.10+ Fyn-deficient T cells from mice that had been primed with Ovap323-339 in CFA produced detectable levels of IL-4 (data not shown), consistent with previous work demonstrating that CFA immunization promotes Th1 cytokine production (33, 34). These results, and those illustrated in Figure 5, indicate that under certain conditions DO11.10+ Fyn-deficient T cells are better cytokine producers than their wild type counterparts (Figures 5 and and66).

Infection with Nippostrongylus brasiliensis elicits a strong Th2 response in both Fyn-deficient mice and wild type mice

To assess the ability of Fyn-deficient T cells to differentiate into IL-4 secreting cells in vivo we utilized the N. brasiliensis infection model, which induces CD4+ T cell differentiation to Th2 effector cells, and inflammation in the lungs characteristic of a Th2 response (35). Cytokine production of CD4+ T cells was assessed 10 days after infection. T cells from the draining lymph nodes of the lung (mediastinal) and gastrointestinal tract (mesenteric) were restimulated in vitro with PMA and ionomycin and cytokine synthesis was assessed by intracellular cytokine staining and flow cytometry. The frequency of IL-4 producing T cells seen in both infected wild type mice and infected fyn−/− mice was higher than the percentage of IFNγ producing cells, consistent with a primary Th2 response (Figure 7A–D). There was no difference in the magnitude of the response between infected wild type mice and infected G9 fyn−/− mice (Figure 7A–D). We also assessed the concentration of serum IgE, an antibody whose production is dependent on IL-4 (36), in mice that had been infected by N. brasiliensis. The sera of both infected wild type mice and infected fyn−/− mice contained increased concentrations of IgE (Figure 7E), whereas IgE could not be detected in the sera of non-infected controls, or of infected rag−/− mice (data not shown). While the mean serum IgE concentration from infected Fyn-deficient mice was less than that from infected wild type mice, this difference was not significant (student T test p=0.3). In addition, both wild type mice and G9 fyn−/− mice were able to completely clear the N. brasiliensis infection, as worms were undetectable in the small intestine of both strains of mice 10 days post infection (data not shown). Finally, we observed a similar increase in the total number of cells infiltrating the lungs, and specifically in the numbers of eosinophils and CD4+ T cells infiltrating the lungs in infected wild type mice and infected fyn−/− mice (Figure 7F–H). These results clearly indicate that Fyn-deficiency does not alter CD4+T cell recruitment, differentiation into Th2 effector cells or helper cell function necessary for IgE production, during the course of an in vivo response.

Figure 7
Nippostrongylus brasiliensis infection elicits a Th2 response in both Fyn-deficient mice and wild type mice

Fyn-deficiency does not alter the formation or stability of DO11.10+ T cell/APC conjugates

The limited effects on the in vivo responses of Fyn-deficient T cells was quite surprising given the literature indicating that Fyn participates in the cytoskeleton rearrangements involved in the immunological synapse through its phosphorylation of Vav, Wasp and Pyk-2 (7, 9). Therefore, we wanted to assess whether the deficiency in Fyn affected the formation or stability of DO11.10+ T cell/APC conjugates. To do this, we compared the frequency of conjugates formed between CFSE-labeled DO11.10+ wild type T cells or DO11.10+ Fyn-deficient T cells and Ovap323-339 loaded APCs that had been stained with different fluorochromes. We employed a competitive assay measuring the frequency of conjugates between DO11.10 T cells and two populations of APCs that were added to the T cells at different times and/or were loaded with different concentrations of Ovap323-339.

In this assay, the frequency of conjugates and the ratio of conjugates with the two different APCs provide an indication of the stability of the T cell/APC conjugate. In the absence of peptide only around 8–10% of DO11.10+ wild type T cells or DO11.10+ Fyn-deficient T cells formed conjugates with the APC added first (APC1) and fewer formed new conjugates with the APC added second (APC2) (Figure 8). Loading APC1 with 1μg/ml Ovap323-339 increased the percentage of T cell/APC conjugates by approximately 3 fold, with little difference being observed in the frequency of conjugates with DO11.10+ wild type T cells or with DO11.10+ fyn−/− T cells (Figure 8), indicating that DO11.10+ Fyn-deficient T cells are as capable as DO11.10+ wild type T cells in forming T cell/APC conjugates. When both APCs were loaded with 1μg/ml Ovap323-339, some DO11.10+ T cells that had previously formed conjugates with APC1 switched APCs and formed new conjugates with APC2. In addition, the total percentage of T cells forming conjugates with APCs was increased (Figure 8). When APC2 was loaded with a higher concentration of Ovap323-339 than APC1, more T cells formed conjugates with APC2, and fewer remained conjugated to APC1, however no difference was observed between the responses of DO11.10+ Fyn-deficient T cells or of DO11.10+ wild type T cells (Figure 8). These data indicate that the formation and stability of T cell/APC conjugates was unaffected by the absence of Fyn kinase and correspond to the observed minimal effects of Fyn-deficiency on T cell responses to antigen in vitro and in vivo.

Figure 8
Fyn-deficiency does not alter the stability of DO11.10+ T cell/APC conjugates

Discussion

Previous studies have suggested that Fyn is involved in several important T signaling pathways including: the initiation and feedback inhibition of TCR-mediated signaling (5, 6, 8, 37, 38), signaling events downstream of the TCR leading to cytoskeletal rearrangement and the formation of stable T cell/APC conjugates (79), and signaling of the SLAM/SAP complex, which promotes differentiation to Th2 effector cells (1315). Indeed, following stimulation with soluble or plate-bound anti-CD3 Fyn-deficient CD4+ T cells have a major defect in proliferation, differentiation into cytokine producing effector cells and TCR signaling when compared to wild type T cells (Figure 1 and 19, 20, 25). Therefore, we set out to assess whether Fyn-deficient CD4+ T cells had a defect in their response to respond to antigen in the context of MHC-II. We found that the development and peripheral expansion of DO11.10+ TCR transgenic CD4+ T cells was unaffected by a deficiency in Fyn kinase. The presence of a large number of naïve resting antigen-specific T cells in the DO11.10+ Fyn-deficient mice gave us the opportunity to compare the responses of wild type and Fyn-deficient CD4+ T cells in detail. Surprisingly, antigen-induced proliferation of DO11.10+ Fyn-deficient T cells was comparable to that of DO11.10+ wild type T cells in vitro and in vivo, even in the absence of co-stimulation. Nor did we did detect a difference in the stability of DO11.10+ T cell conjugation with Ovap323-339-loaded APCs between wild type and Fyn-deficient T cells. Interestingly, the absence of Fyn did not cause a defect in Th2 cytokine production, as has previously been reported in Fyn-deficient T cells and SAP-deficient T cells (13, 29). Thus, Fyn was not essential for the function of SAP in CD4+ T cells. Surprisingly, DO11.10+Fyn-deficient T cells did produce higher levels of effector cytokines than DO11.10+ wild type T cells following antigen-induced activation, under certain conditions, in vitro or in vivo.

Our assessment of positive selection in the thymus of DO11.10+ Fyn-deficient mice showed that it was unaffected by the absence of signaling though Fyn (Figure 2 and data not shown). Similarly, positive selection was found to be normal in TCR transgenic Fyn-deficient mice that expressed either the CD8-selecting H-Y TCR or 2C TCR (30). Given the importance of TCR recognition of self-peptide-MHC complexes at low avidity for positive selection of conventional CD4 or CD8 T cells (39), these results suggest that TCR signaling in response to such ligands is not greatly affected in thymocytes by the absence of Fyn kinase. This is in contrast to the defect observed in Fyn-deficient mice in NKT cell development, which is dependent on the recognition of CD1d molecules bound to lipid antigens (21, 22, 40). One difference between the role of Fyn in thymic development of conventional α/β T cells and NKT cells may reflect the involvement of the CD4 and CD8 co-receptors in the former process and their association with Lck. Recruitment of CD4 or CD8, and thereby Lck, to the TCR efficiently provides Src-family kinase function to initiate TCR signaling in α/β T cells (Figure 1 and 25). The role of Fyn may be more important in NKT cell development in part because these cells do not utilize CD4 or CD8 as co-receptors. Alternatively, Fyn has been shown to promote NKT cell development following ligation of SLAM family receptors (24). Fyn-deficient mice have also been reported to have a defect in thymic negative selection induced by the super-antigen, Mls-1a (19). However, this does not seem to be a general defect, as negative selection of Fyn-deficient conventional α/β thymocytes in response to a second super-antigen, Staphylococcal enterotoxin A, is normal (19), as is the negative selection of HY TCR-transgenic CD8+ thymocytes in Fyn-deficient male mice (30).

In the periphery, the vast majority of CD4+ T cells in DO11.10+ Fyn-deficient mice had a naïve phenotype (Figure 3), this is in contrast to previously published results of Yasuda et al., (2002) who found evidence for an activated phenotype of CD4+ and CD8+ T cells from Fyn-deficient mice (11). However, we have not seen spontaneous activation of T cells from non-transgenic Fyn-deficient BALB/c mice or from highly inbred non-transgenic Fyn-deficient C57BL/6 mice (data not shown). Therefore, the DO11.10+ TCR-transgenic Fyn-deficient T cells appear to be typical in retaining the naïve resting phenotype in the periphery.

Proliferation of peripheral DO11.10+ Fyn-deficient T cells in response to antigen in the context of MHC was almost indistinguishable from the response of DO11.10+ wild type T cells (Figures 4 and and6).6). The similarity of the in vitro and in vivo proliferative responses of DO11.10+ T cells was surprising given the numerous signaling defects that have been described in Fyn-deficient T cells (8, 10, 19, 20, 25, 41). Our data for antigen-driven proliferation of Fyn-deficient DO11+CD4+ T cells was similar to that obtained for Fyn-deficient TCR transgenic CD8+ T cells that express the 2C TCR (30). However, assessment of antigen-induced CD8+ T cell proliferation from two other Fyn-deficient TCR transgenic mouse strains has produced conflicting results. Utting et al., (1998) showed that following stimulation with antigen Fyn-deficient HY+CD8+ T cells proliferate less well than their wild type counterparts (30). In contrast, Filby et al., (2007) recently showed that antigen-induced proliferation of Fyn-deficient F5 TCR transgenic CD8+ T cells was more vigorous than their wild type counterparts (41). Given these conflicting results it is difficult to form a coherent theory as to the role of Fyn in antigen-induced proliferation of CD8+ T cells, although one possibility is that these data are a reflection of differences in the affinity of TCRs for their respective antigens. The difference between CD4+ and CD8+ T cells could also relate to the fact that CD4 binds more effectively to Lck than does CD8 (2, 42). For this reason CD8+ T cells may be more sensitive to signaling defects that result from Fyn-deficiency, whereas the greater amount of Lck recruited to CD4 may make Fyn dispensable for TCR signaling in CD4+ T cells. Irrespective of the role of Fyn in CD8+ T cell proliferation, our data clearly shows that Fyn-deficient CD4+ T cells proliferate normally in response to antigen under a variety of conditions.

Several studies have shown that Fyn plays a positive role in cytoskeletal remodeling and the formation of stable T cell/APC conjugates (79). When stimulated with antigen, AD10+CD4+ Fyn-deficient T cells do not effectively phosphorylate Vav (8, 25), which is required for actin polarization and re-organization promoting the formation of the immunological synapse (43, 44). Correspondingly, AD10+CD4+ fyn−/− T cells were reported to form less stable conjugates with antigen-loaded APCs than wild type AD10+CD4+ T cells (8). In similar experiments, Badour et al., (2004) found that OT-II+CD4+ Fyn-deficient T cells formed poor immunological synapses with ovalbumin-presenting APCs, and provided evidence that this was due to a failure to phosphorylate WASp resulting in impaired actin polymerization and cytoskeletal rearrangement. More recently, OT-I+CD8+ Fyn-deficient T cells were shown to have a defect in the reorganization of the microtubule-organizing center (MTOC) when stimulated with antigen and APCs (7). Reorganization of the MTOC involves Pyk2 and Vav, both of which are phosphorylated by Fyn (8, 10). In spite of the reported defects in cytoskeletal reorganization, both AD10+CD4+ Fyn-deficient T cells and OT-I+CD8+ Fyn-deficient T cells produced IL-2 at levels similar to that seen in wild type controls following antigen stimulation. Moreover, we found that DO11.10+CD4+Fyn-deficient T cells did not have a defect in the formation of conjugates with Ovap323-339 loaded APCs (Figure 8). The ability of DO11.10+Fyn-deficient T cells to form stable T cell/APC conjugates is consistent with our observations that their antigen-induced proliferation was normal (Figures 4 and and66).

Although DO11.10+ Fyn-deficient T cells proliferated normally in response to antigenic stimulation, we did observed a moderate increase in antigen-induced effector cytokine production in some circumstances (Figures 5 and and6).6). Similarly, Filby et al., (2007) have reported that Fyn-deficient F5 TCR transgenic CD8+ T cells produced more IL-2 following antigen stimulation when compared to wild type F5+CD8+ T cells (41). However, in our experiments Fyn-deficient CD4+ T cell were not always hyper-responsive with respect to cytokine production, as following infection with N. brasiliensis G9 Fyn-deficient mice had a comparable cytokine response to that of wild type BALB/c mice (Figure 7). In any case, our data clearly show that Fyn-deficient T cells are capable of differentiating into Th1 or Th2 effector T cells, and that under certain conditions Fyn-deficient T cells are more potent producers of effector cytokines that their wild type counter-parts (Figures 57).

Previous work has shown that Fyn can play a positive role in Th2 differentiation and IL-4 production through its interaction with SAP, a key signaling adapter associated with several CD2-like adhesion molecules including SLAM (13, 29). Following the homotypic interaction between SLAM molecules, SAP binds to SLAM, and SAP-associated Fyn phosphorylates SLAM, initiating an intracellular signaling pathway that promotes IL-4 production (13). Anti-CD3 stimulation of SAP-deficient T cells or SAPR78A T cells, which have a mutation that prevents SAP’s interaction with Fyn, results in the induction of mRNA for T-bet but not GATA-3, the key transcription factors regulating Th1 and Th2 differentiation, respectively, whereas under the same conditions both T-bet and GATA-3 are produced in wild type T cells (13, 29). These results suggested that one of the downstream events following the formation of the SLAM/SAP/Fyn complex is the increased expression of GATA-3, a transcription factor that promotes the production of Th2 cytokines (45, 46). While, data from Cannons et al (2004) and Davidson et al (2004) suggested that signaling through Fyn can promote CD4+ T cells to differentiate to IL-4-secreting T cells, our data clearly showed that signaling through Fyn was not obligatory for IL-4 production or Th2 immune responses (Figure 5 and and7).7). Surprisingly, we found that under certain conditions Fyn-deficient DO11+CD4+ T cells produced more IL-4 than wild type CD4+ T cells (Figure 5). In agreement with our data, Fyn-deficient CD4+ T cell have previously been shown to be more potent producers of IL-4 following in vitro stimulation with anti-CD3 in the presence of APCs, even in the absence of costimulation though CD28 (47). Further in a murine model of airway allergy in which mice were sensitized to, and subsequently challenged with, aerosolized ovalbumin Th2-dependent eosinophil infiltration of the lung and the levels of IL-4 and IL-5 in the bronchiolar lavage were higher in mice deficient in the hematopoietic form of Fyn, FynT, than in wild type control mice (48). Combined, these data show that under different conditions naïve Fyn-deficient T cells can differentiate into IL-4-producing effector cells secreting as much, or more cytokine, than wild type T cells.

This study clarifies the effect of a deficiency in Fyn kinase on antigen-induced CD4+ T cells activation. Through the use of TCR-transgenic Fyn-deficient T cells we have directly assessed the effect of a Fyn-deficiency on antigen-induced activation in vitro and in vivo. Our results clearly show that Fyn-deficient T cells formed stable conjugates with antigen loaded APCs; that antigen-induced proliferation was normal, even in the absence of co-stimulation; and with respect to antigen-induced cytokine production, Fyn-deficient CD4+ T cells were as good as, or under certain conditions better, cytokine producers than wild type CD4+ T cells. In addition, we did not observe a defect in IL-4 production by Fyn-deficient CD4+ T cells, demonstrating that the signaling via SLAM/SAP that promotes T cell differentiation to Th2 phenotype can proceed via a pathway that is independent of Fyn kinase. Taken together, these data reveal a previously unappreciated level of redundancy in T cell signaling that allows conventional α/β CD4+ T cells to respond well following antigen recognition in the absence of Fyn kinase.

Acknowledgments

The authors wish to acknowledge Shuwei Jiang for assistance with purification of cells by flow cytometry on the MoFlo, Barbara Sheer for help in preparing mouse tissues, Julia Lyandres for typing the TCR-transgenic and Fyn-deficient mice, members of Dr Abbas’s laboratory for technical advice and reagents and Mark Lanett for his assistance.

This work was supported by The Joseph Marino Research Award from the Crohn’s & Colitis Foundation of America (for A.A.M), NIH grants PO1 AI-35297 (for A.L.D), AI-26918 (for R.M.L) and RO1 AI-052116 (for M.F.K).

Abbreviations

Ovap323-339
chicken ovalbumin peptide 323-339
CFSE
carboxyfluorescein diacetate succinimidyl ester
CFA
complete Freud’s adjuvant
3H-TdR
[methyl-3H]-Thymidine
CMTMR
orange-fluorescent tetramethylrhodamine
DDAO
7-hydroxy-9H I,3-dichloro-9,9-dimethylacridin-2-one
SLAM
signaling lymphocyte activation molecule
SAP
SLAM associated protein

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