Itk, a member of the Tec family of tyrosine kinases, is critical for TCR signaling, leading to the activation of phospholipase Cγ1. Early biochemical studies performed in tumor cell lines also implicated Itk in CD28 signaling. These data were complemented by functional studies on primary Itk−/− T cells that suggested a negative role for Itk in CD28 signaling. In this report, we describe a thorough analysis of CD28-mediated responses in T cells lacking Itk. Using purified naive CD4+ T cells from Itk−/− mice, we examine a range of responses dependent on CD28 costimulation. We also analyze Akt and glycogen synthase kinase-3β phosphorylation in response to stimulation of CD28 alone. Overall, these experiments demonstrate that CD28 signaling, as well as CD28-mediated costimulation of TCR signaling, function efficiently in the absence of Itk. These findings indicate that Itk is not essential for CD28 signaling in primary naive CD4+ T cells.
Two signals are required for the optimal activation of naive T cells, one from the TCR and the second from a costimulatory receptor. On naive T cells, the CD28 receptor provides the primary costimulatory signal following interaction with CD80 or CD86 on APCs (for review, see Refs. 1 and 2). Functionally, CD28 costimulation enhances the survival, cell cycle progression, and cytokine production by activated T cells. Although tremendous effort has been directed at elucidating the signaling pathway(s) initiated by CD28 stimulation, the detailed mechanism by which CD28 costimulation operates has not yet been determined, in part due to the difficulty of distinguishing the TCR- vs the CD28-mediated signals in primary T cells.
The Tec family tyrosine kinase, Itk, has been previously implicated in CD28 signaling. Although Itk is primarily associated with TCR signaling (3, 4, 5), a number of biochemical studies have demonstrated an interaction between Itk and CD28. Specifically, Itk coimmunoprecipitates with CD28 from Jurkat tumor cells and, in addition, is tyrosine phosphorylated in response to CD28 cross-linking (6, 7). In vitro studies using recombinant proteins indicate that Itk binding to CD28 depends on the activity of the Src-family tyrosine kinase, p56lck (Lck) (8, 9). Structure-function analysis of CD28 additionally demonstrated that the Src homology 3 domain of Itk binds to proline-rich sequences in the CD28-cytoplasmic tail, an interaction that has been suggested to enhance Itk kinase activity (7). Finally, Itk has been shown to phosphorylate all four tyrosine residues of the CD28-cytoplasmic tail in in vitro kinase assays (10), providing additional evidence for a positive role of Itk in CD28 signaling.
To date, only a single study has addressed the role of Itk in CD28 signaling in primary T cells. Surprisingly, this study concluded that Itk is a negative regulator of CD28 signaling. This latter conclusion was based on the finding that CD4+ T cells from Itk−/− mice showed enhanced proliferative responses to CD28 costimulatory signals compared with cells from wild-type (WT)3 mice (11). One complication of this initial study is the fact that Itk−/− mice have a greatly increased population of previously activated/memory CD4+ T cells compared with controls, potentially skewing the responses of these cells to TCR plus CD28 stimulation, independently of a role for Itk in CD28 signaling. Based on this concern, we chose to readdress the role of Itk in CD28 signaling using a panel of assays that assess CD28 signaling in the presence, as well as the absence, of TCR stimulation. Overall, our data demonstrate that Itk is not a negative regulator of CD28 costimulatory activity; in contrast, to the best of our knowledge, all aspects of CD28 signaling are intact in the absence of Itk.
Materials and Methods
The Itk−/− mouse line (12) was backcrossed to C57BL/10 for >10 generations. Where indicated, Itk−/− mice were crossed to 5C.C7 TCR-transgenic Rag2−/− mice (Taconic Farms). All mice used were 6–12 wk of age and maintained in a specific, pathogen-free facility, following review and approval by the Institutional Animal Care and Use Committee.
Preparation of naive CD4+ T cells
CD4+ T cells were purified from lymph nodes and spleens as described previously (13). CD4+CD44low (naive) T cells were sorted on a BD Biosciences FACSVantage cell sorter. Purified populations were >95% CD4+CD44low cells. For some experiments, naive CD4+ T cells (CD62Lhigh) were purified on an AutoMACS following labeling with anti-CD4-FITC (BD Pharmingen), anti-FITC MultiSort kit (Miltenyi Biotec), and anti-CD62L-magnetic microbeads (Miltenyi Biotec). This purification strategy yielded 90–97% pure CD4+CD44low T cells. The CD4+ T cells from Rag2−/−5C.C7TgxItk−/− and Rag2−/−5C.C7TgxItk+/− mice are consistently >95% CD44low.
T cell stimulations
Purified naive CD4+ T cells were stimulated with surfactant-free, sulfate-charged, 4.9-μm white polystyrene latex beads (Interfacial Dynamics) coated with the following combinations of mAbs: anti-CD3ε (145-2C11; BD Pharmingen) at 0.5 μg/ml plus a hamster IgG isotype control (eBiosciences) at 4.5 μg/ml; anti-CD3ε at 0.5 μg/ml plus anti-CD28 (37.51; eBiosciences) at 4.5 μg/ml; anti-CD28 at 5 μg/ml; or a hamster IgG isotype control at 5 μg/ml. For Ab coating, latex beads were incubated at 1 × 107/ml with the indicated combinations of Abs at 37°C for 1.5 h with rotation. For biochemical assays, naive CD4+ T cells were stimulated with anti-CD28 Ab-coated plates.
To stimulate naive CD4+ T cells, 1 × 105 purified cells were incubated with an equal number of Ab-coated latex beads. Where indicated, PMA (Sigma-Aldrich) was added at various concentrations. For stimulation of Rag2−/− 5C.C7Tg cells, 1 × 105 purified CD4+ T cells were incubated with varying concentrations of the moth cytochrome c (MCC) peptide (93-103) plus 1 × 105 mitomycin C-treated Chinese hamster ovary (CHO) cells expressing mouse MHC class II IEk (CHO-IEk; Ref. 14) or CHO cells expressing IEk and mouse CD80 (CHO-IEk/B7.1; Ref. 15). Proliferation was assessed by pulsing cells overnight with [3H]thymidine (NEN).
Western blot analysis
Following stimulation, cells were lysed as described previously (16). Forty micrograms of protein for each sample were transferred to polyvinylidene difluoride membranes and probed with Abs to phospho-Akt or phospho-glycogen synthase kinase-3β (GSK3β) (Cell Signaling Technology). After stripping, the membranes were reprobed with Abs to total Akt (Cell Signaling Technology), GSK3β (Santa Cruz Biotechnology), or the p85 subunit of PI3K (Upstate Biotechnology).
Quantitative real-time PCR
T cells were stimulated for 6 h, and RNA and cDNA were prepared as described previously (15). Real-time quantitative PCR was performed on an i-Cycler (Bio-Rad). Primer sequences are available upon request.
Preparation of nuclear extracts and detection of activated NF-κB (p65)
After stimulation, cells were harvested, and nuclear lysates were prepared. Five micrograms of nuclear protein were subjected to the NFκB (P65) functional ELISA using the BD TransFactor NF-κB p65 kit (BD Biosciences). Signals were analyzed on an EMax precision microplate reader (Molecular Devices).
Cells were stained with the indicated Abs 30 min at 4°C, washed, and analyzed on a BD FACSCalibur (BD Biosciences). Data were analyzed using CellQuest software (BD Immunocytometry Systems). The Abs used were anti-CD4-CyChrome (Cy), anti-CD4-PE, anti-CD69-FITC, anti-CD44-FITC, anti-CD44-Cy, anti-CD25-FITC, and anti-CD62L-PE (BD Pharmingen).
Results and Discussion
Increased numbers of memory phenotype CD4+ T cells in Itk−/− mice
Previous studies have documented that Itk−/− mice have a modest defect in positive selection, resulting in an ∼2-fold reduction in the total numbers of CD4+ T cells in the spleens and lymph nodes of Itk−/− mice compared with controls (12, 17, 18, 19). However, surprisingly, the population of CD4+ T cells in Itk−/− mice is highly enriched for cells with a previously activated or memory phenotype (CD4+CD44highCD62Llow). As shown in Fig. 1,A, we routinely find an ∼3-fold increase in the proportion of CD4+ T cells expressing high levels of CD44 in lymph nodes of Itk−/− mice compared with WT C57BL/10 mice. When CD4+CD44high T cells were analyzed for CD69 and CD25 expression, fewer cells from Itk−/− mice compared with controls expressed these early activation markers, suggesting that these cells have not been recently activated (Fig. 1 B). Overall, these data indicate that, although total T cell numbers are reduced in the Itk−/− mice (12, 17), the proportion of cells with a memory phenotype is actually increased.
Naive Itk−/− CD4+ T cells are not hyperresponsive to CD28 costimulation
A previous study described increased responsiveness of Itk−/− CD4+ T cells compared with WT CD4+ T cells following stimulation through CD28, leading to the conclusion that Itk is a negative regulator of CD28 signaling (11). To bypass the TCR-signaling defect intrinsic to Itk−/− T cells, these experiments used PMA plus anti-CD28 Ab as a stimulus. Consistent with these earlier data, we also observe that when total CD4+ T cells from Itk−/− mice or WT control mice are stimulated with PMA plus anti-CD28-coated beads, the response of the Itk−/− CD4+ T cells is significantly higher than that of the control cells (Fig. 1,C). However, because the CD4+ T cell population from Itk−/− mice contains an increased proportion of memory phenotype cells, we reasoned that the increased responsiveness of these cells might be attributable to this altered subset distribution. To test this possibility, we repeated this experiment using highly purified naive CD4+ CD44low T cells. As shown in Fig. 1, D and E, purified naive Itk−/− CD4+ T cells are not hyperresponsive to PMA plus anti-CD28 Ab stimulation and, under these conditions, respond comparably to WT naive CD4+ T cells.
These findings reopened the question of the role of Itk in CD28 signaling and function. Therefore, we proceeded to examine the responses of Itk−/− T cells to stimulation through the TCR plus CD28, using conditions in which T cell activation is dependent stringently on CD28 costimulation. For these experiments, purified Itk−/− and WT naive CD4+ T cells were stimulated with Ab-coated latex beads. When stimulated with beads coated with anti-CD28 Ab alone or anti-CD3 Ab alone, neither population of T cells exhibited any proliferative response. In contrast, when cells were stimulated with beads coated with a mixture of anti-CD3 plus anti-CD28 Abs (1:9 ratio), both populations of cells proliferated robustly. Although the response of the Itk−/− cells was reduced compared with that of the WT T cells, this response still represents an ∼300-fold enhancement over the response to anti-CD3 Ab alone. Based on these data, we conclude that CD28 costimulatory activity functions quite efficiently in naive Itk−/− CD4+ T cells.
To substantiate these findings using bona fide MHC/peptide stimulation in the presence or absence of B7.1 (CD80), we examined purified naive CD4+ T cells isolated from transgenic mice expressing the 5C.C7 TCR (5C.C7Tg). For these experiments, T cells from Rag2−/−5C.C7TgxItk−/− and Rag2−/−5C.C7TgxItk+/− mice were stimulated with CHO-IEk cells or CHO-IEk/B7.1 cells as APCs in the presence of varying concentrations of the MCC peptide. At each given peptide concentration, both Itk+/− as well as Itk−/− CD4+ T cells show a similar degree of increased responsiveness to stimulation with APCs expressing B7.1 compared with APCs that lack B7.1 (Fig. 2 B). These data confirm the conclusion that CD28 costimulation functions effectively in the absence of Itk.
CD28 costimulation enhances gene expression in the absence of Itk
One function of CD28 costimulation is to enhance gene expression induced by TCR signaling. Among the genes most dramatically affected by CD28 costimulation are those encoding the cytokine, IL-2, the survival factor, Bcl-xL, and the effector molecule, CD40L (20, 21, 22, 23). To assess whether CD28 costimulation leading to enhanced gene expression is functional in the absence of Itk, WT, and Itk−/−, naive CD4+ T cells were stimulated, and IL-2, Bcl-xL, and CD40L mRNA levels were measured by real-time quantitative PCR. As shown in Fig. 3, each of these genes exhibited enhanced mRNA levels following CD28 costimulation in both WT and Itk−/− T cells. Interestingly, the activation-induced increases in IL-2, Bcl-xL, and CD40L mRNA were abolished completely following addition of the PI3K inhibitor, LY294002 (Fig. 3). Taken together, these data demonstrate the effectiveness of CD28 costimulatory signals to enhance gene expression in the absence of Itk.
CD28 costimulation activates NFκB in the absence of Itk
The transcription factor, NF-κB, is an important target of the CD28 costimulatory pathway (24). To assess the ability of CD28 costimulation to activate NF-κB in the absence of Itk, we stimulated WT and Itk−/− naive CD4+ T cells with anti-CD3 Ab alone or in combination with anti-CD28 Ab. After 60 min, nuclear lysates were prepared from the cells, and levels of activated NF-κB were examined by ELISA. As can be seen in Fig. 4 A, anti-CD3 Ab stimulation is not sufficient to induce detectable NF-κB activation in either cell type, whereas anti-CD3 plus anti-CD28 Ab stimulation induced significant levels of activated NF-κB in both WT and Itk−/− T cells. These data confirm the ability of CD28 costimulatory signaling to function in the absence of Itk.
Stimulation of CD28 alone induces phosphorylation of Akt and GSK3β in the absence of Itk
Although signaling through CD28 alone does not lead to functional changes in T cells, several biochemical events can be detected following CD28 stimulation. One such signaling pathway is the activation of PI3K, leading to the phosphorylation and activation of the serine/threonine kinase, Akt, and the subsequent phosphorylation of GSK3β (25). To examine whether these events occurred normally in the absence of Itk, naive CD4+ T cells from WT and Itk−/− mice were stimulated with anti-CD28 Ab alone, and Akt and GSK3β phosphorylation were detected with phospho-Akt- and phospho-GSK3β-specific Abs. As shown in Fig. 4 B, Akt and GSK3β were both phosphorylated comparably in WT and Itk−/− CD4+ T cells. These data demonstrate that the CD28-PI3K-Akt-GSK3β signaling pathway is intact in the absence of Itk, indicating that Itk is not essential for CD28 signaling.
Using purified naive CD4+ T cells and defined stimulation conditions, we have examined in detail the requirement for Itk in CD28 signaling and in CD28-mediated costimulation. Because Itk is required for optimal TCR signaling, it is difficult to ascertain whether CD28 costimulatory activity is equally effective in the presence vs the absence of Itk. Nonetheless, our data definitively demonstrate that CD28-mediated costimulation functions efficiently in the absence of Itk and, to a first approximation, is as effective in Itk−/− T cells as in WT T cells. This conclusion is supported by our biochemical data showing that two measurable outcomes triggered by CD28 stimulation alone, namely the phosphorylation of Akt and GSK3β, are completely independent of Itk. It is possible that efficient CD28 signaling in Itk−/− T cells is due to compensation by another Tec-kinase family member, Rlk or Tec, also expressed in T cells, although examination of Rlk and Tec expression in Itk−/− T cells has not indicated any compensatory up-regulation of these additional Tec kinases (Ref. 12 and data not shown). Overall, these findings demonstrate that Itk is not essential for CD28 signaling or function in naive CD4+ T cells.
The authors have no financial conflict of interest.
We thank Jennifer Cannons, Regina Whitehead, and Sharlene Hubbard for technical assistance. We also thank Cynthia Chambers for helpful discussions and Yoko Kosaka, Andrew Miller, and Luana Atherly for critical reading of the manuscript.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grants AI37584 and CI00101 (to L.J.B.) and by University of Massachusetts Center for Diabetes and Endocrinology Research Grant DK32520.
Abbreviations used in this paper: WT, wild type; MCC, moth cytochrome c; CHO, Chinese hamster ovary; GSK3β, glycogen synthase kinase-3β.