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The Journal of Immunology, 2007, 179, 3559 -3569
Copyright © 2007 by The American Association of Immunologists, Inc.
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Adhesion and Degranulation-Promoting Adapter Protein (ADAP) Positively Regulates T Cell Sensitivity to Antigen and T Cell Survival1

Kristen L. Mueller2,*, Molly S. Thomas*, Brandon J. Burbach*, Erik J. Peterson{dagger} and Yoji Shimizu3,*

* Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis, MN 55455; and {dagger} Department of Medicine, Center for Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The hemopoietic specific adapter protein ADAP (adhesion and degranulation-promoting adapter protein) positively regulates TCR-dependent, integrin-mediated adhesion and participates in signaling pathways downstream of the TCR that result in T cell activation. The specific role of ADAP in regulating Ag-dependent T cell interactions with APCs and T cell activation following Ag stimulation is not known. We used ADAP–/– DO11.10 T cells to demonstrate that ADAP promotes T cell conjugation to Ag-laden APCs. Complementary in vitro and in vivo approaches reveal that ADAP controls optimal T cell proliferation, cytokine production, and expression of the prosurvival protein Bcl-xL in response to limiting Ag doses. Furthermore, ADAP is critical for clonal expansion in vivo independent of Ag concentration under conditions of low clonal abundance. These results suggest that ADAP regulates T cell activation by promoting Ag-dependent T cell-APC interactions, resulting in enhanced T cell sensitivity to Ag, and by participating in prosurvival signaling pathways initiated by Ag stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antigen recognition by the TCR results in the induction of multiple signaling cascades that together result in T cell activation. Adapter proteins, which lack intrinsic enzymatic activity or direct effector function, are critical mediators of signaling pathways downstream of the TCR because they facilitate the formation of signaling complexes through their protein-protein and protein-lipid interaction domains. Acting as molecular scaffolds, adapter proteins allow effector proteins to come into close proximity to their substrates. Adhesion and degranulation-promoting adapter protein (ADAP)3 is a hemopoietic-specific adapter protein that was initially identified through its ability to interact with the Src family tyrosine kinase Fyn (1) and the adapter protein SLP-76 (2). Additional binding partners for ADAP include the adapter proteins SKAP55 (Src kinase-associated phosphoprotein of 55 kDa) and SKAP-HOM, actin regulatory proteins of the Ena/VASP (vasodilator-stimulated phosphoprotein) family, and the membrane-associated guanylate kinase (MAGUK) family member CARMA1 (caspase-recruitment domain (CARD)-MAGUK protein 1) (3, 4, 5, 6). Studies using ADAP-deficient mice demonstrated that ADAP is a positive regulator of T cell activation and integrin-mediated adhesion (7, 8). ADAP–/– T cells display impaired T cell proliferation, cytokine production, and induction of the activation markers CD25 and CD69 following Ab stimulation of the TCR and CD28. Despite these defects in T cell activation, ADAP–/– T cells exhibit normal calcium mobilization and phosphorylation of the signaling intermediates LAT (linker for activation of T cells), PLC-{gamma}1 (phospholipase C-{gamma}1), SLP-76 (Src homology 2 domain-containing leukocyte specific phosphoprotein of 76 kDa), Vav1, Lck, and Fyn (7, 8). Activities of PI3K, Fyn, Lck, ERK1/2, JNK, and p38 are intact as well, suggesting that ADAP acts downstream of many of the proximal signaling events that regulate T cell activation.

TCR signals necessary for full T cell activation and gain of effector function require a stable interaction with an Ag-expressing APC (9, 10, 11, 12). Stable contact between a T cell and an APC is mediated by the binding of the LFA-1 integrin, expressed on the T cell surface, to ICAM-1, expressed on the surface of the APC (13, 14, 15). An important functional outcome of LFA-1-dependent T cell-APC adhesion is a reduction of the Ag dose threshold required for T cell activation (16), thereby allowing T cells to mount productive immune responses to low Ag concentrations.

To facilitate T cell interactions with APCs, LFA-1 must first be activated (13, 17, 18). TCR stimulation generates intracellular signals that result in LFA-1 activation, which is characterized by the transient clustering of LFA-1 at the T cell-APC contact site. Together with changes in affinity mediated by conformational changes in the integrin, TCR-induced clustering of LFA-1 enhances the overall avidity of LFA-1 for ICAM-1. ADAP is a critical positive regulator of TCR-induced integrin activation (7, 8). ADAP–/– T cells exhibit impaired adhesion to purified LFA-1 and beta1 integrin ligands and do not cluster LFA-1 following anti-CD3 Ab stimulation (7, 8). In addition, overexpression of ADAP in a human T cell hybridoma results in enhanced Ag-dependent T cell conjugation and enrichment of ADAP in the immunological synapse, where it colocalizes with F-actin (19). These studies suggest that ADAP may regulate T cell activation by promoting stable interactions between T cells and APCs.

Although ADAP is necessary for changes in integrin activation downstream of the TCR, promotion of adhesion is unlikely to be the only mechanism by which ADAP regulates T cell activation. Purified ADAP–/– T cells stimulated with immobilized anti-CD3 and anti-CD28 Abs exhibit impaired proliferation (7, 8). Recent work has also demonstrated that ADAP is required for CD3- and CD28-dependent activation of NF-{kappa}B (6), a signal transduction pathway that regulates the expression of prosurvival proteins such as Bcl-xL (20, 21) following TCR stimulation. The regions of ADAP required for integrin activation are distinct from those necessary for NF-{kappa}B activation (6, 19, 22), suggesting the existence of multiple signaling pathways nucleated by ADAP that contribute to T cell activation.

In this study, we sought to elucidate the functional effects of ADAP deficiency on Ag-stimulated CD4 T cell activation both in vitro and during an in vivo immune response. We felt it was particularly important to investigate the role of ADAP in Ag-stimulated T cell activation because the duration of Ag exposure and Ag dose play important roles in determining the functional outcome of CD4 T cell activation and differentiation (16, 23, 24, 25, 26, 27). Our studies demonstrate that Ag-specific CD4 ADAP–/– T cells exhibit impaired conjugate formation with APCs expressing Ag. Using complementary in vitro and in vivo approaches, we also show that ADAP is critical for T cell activation in response to limiting doses of Ag and under conditions of low clonal abundance. Finally, ADAP is critical for Ag-induced expression of the prosurvival protein Bcl-xL. These studies suggest that ADAP regulates CD4 T cell activation by enhancing T cell sensitivity to Ag and by promoting T cell survival following TCR stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

ADAP–/– mice (7) were backcrossed onto the BALB/c background (>10 generations) and then bred to DO11.10 TCR transgenic mice (28) (The Jackson Laboratory). BALB/c mice (7–9 wk old) were purchased from Taconic or Charles River Laboratories. All mice were used between 6 and 12 wk of age. All experimental protocols involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (Minneapolis, MN).

Antibodies and reagents

The following Abs and reagents were purchased from eBioscience: function-blocking anti-CD11a mAb (clone M17/4), anti-B220 allophycocyanin, streptavidin-PE, streptavidin-allophycocyanin, anti-CD4-FITC, anti-CD8-FITC, anti-B220-FITC, anti-B220-PE-Cy5.5, anti-mouse IgG-FITC, anti-CD62L-PE, anti-IL-2-PE, rat IgG2b-PE isotype control, rat IgG2A-PE isotype control, anti-CD69-biotin, anti-CD25-biotin, anti-CD25-allophycocyanin, anti-CD69-PE, anti-CD4 Pacific Blue, and brefeldin A. The following Abs were purchased from BD Pharmingen: anti-I-Ad-FITC, anti-CD4-PE-Cy7, anti-CD11b-PerCP-Cy5.5, anti-CD62L-allophycocyanin-Cy7, and mAb KJ1-26-PE. Anti-Bcl-xL-PE and mouse IgG3-PE isotype control were purchased from Southern Biotech. Annexin V-allophycocyanin, CellTracker Orange 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR), and CFSE were purchased from Invitrogen/Molecular Probes. Ionomycin, PKH-26 reference beads, and LPS were purchased from Sigma-Aldrich and PMA was purchased from LC Laboratories. Anti-FITC microbeads, streptavidin microbeads, and LS columns were purchased from Miltenyi Biotec. The OVA323–339 peptide (OVAp; ISQAVHAAHAEINEAGR) has been reported previously (28) and was synthesized by Invitrogen Life Technologies.

Conjugate assays

Conjugate assays were performed as previously described (6). Briefly, 2 x 105 CD4+KJ1-26+ cells were incubated with 8 x 105 CellTracker Orange-labeled T cell-depleted splenocytes for 10 min at 37°C in 96-well round-bottom plates (Costar). Following conjugation, cells were fixed in 1% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min at room temperature. Cells were washed twice in PBS containing 2% calf serum and stained with mAb KJ1-26, anti-CD4, and anti-B220. Conjugates were analyzed by flow cytometry. A conjugate was defined as any KJ1-26+CD4+B220+ CellTracker Orange event.

In vitro cell cultures

CFSE-labeled control DO11.10 or ADAP–/– DO11.10 splenocytes (1 x 106) were cultured in 24-well plates (BD Biosciences) with OVAp or 10–50 ng/ml PMA and 200 ng/ml ionomycin in T cell medium (RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, streptomycin and 2-ME). Because of the reduced numbers of T cells found in ADAP–/– DO11.10 spleens (data not shown), ADAP–/– DO11.10 cultures initially contained 1 x 105 mAb KJ1-26+CD4+ cells while control cultures contained 2 x 105 mAb KJ1-26+CD4+ cells. Cells were harvested at 24, 48, or 72 h, labeled with mAb KJ1-26, anti-CD4, anti-B220, and anti-CD11b and analyzed by flow cytometry. An aliquot of PKH-26 reference beads was added to each FACS tube to enumerate the total number of cells in each sample as described previously (29). The number of DO11.10 T cells in each tube was then calculated by multiplying the total number of cells per FACS tube by the percentage of mAb KJ1-26+CD4+ cells in the sample. Supernatants were assessed for the presence of IL-2 at 48 h by ELISA (eBioscience) according to the manufacturer’s instructions.

CFSE labeling

Cells were resuspended at a concentration of 107 cells/ml in RPMI 1640 supplemented with 2% calf serum (RP2 medium) and incubated in a 37°C water bath for 10 min. CFSE (1 mM for in vitro cultures or 1.67 mM for in vivo assays) was added at a concentration of 1 µl/107 cells and cells were incubated for 10 min in a 37°C water bath. Cells were washed once in RP2 medium to stop the labeling reaction.

Intracellular staining

Intracellular staining methods for Bcl-xL in in vitro stimulated cells were adapted from a protocol described by Khoruts and colleagues (30). Briefly, cells were stained for surface markers, fixed with 2% PFA for 20 min at room temperature, washed twice with PBS, and permeabilized with 0.5% saponin/PBS supplemented with 25% FCS (superperm buffer). Cells were incubated with anti-Bcl-xL-PE (1/50) or the appropriate isotype control in 50 µl of superperm buffer for 30 min at room temperature, washed once with 0.5% saponin/PBS supplemented with 2% FCS (perm buffer), once with PBS, once with PBS supplemented with 2% FCS, and analyzed by flow cytometry. Intracellular IL-2 production for in vitro cultures was assessed following 48 h of stimulation. Brefeldin A was added during the last three hours of culture. Cells were stained for surface markers and intracellular staining for IL-2 was performed using fixation and permeabilization solutions according to the manufacturer’s instructions (eBioscience). For in vivo stimulated cells, the cells were washed twice in perm buffer and resuspended in 50 µl of superperm buffer containing mAb KJ1-26, anti-CD4, anti-B220, anti-CD11b, and anti-IL-2 or appropriate isotype controls. Cells were incubated for 30 min at room temperature, washed one time in perm buffer, resuspended in 50 µl of perm buffer containing 1.5 µl of streptavidin-allophycocyanin, and incubated for 30 min at room temperature. Cells were washed once in perm buffer, once with PBS, and once with PBS supplemented with 2% FCS and run on an LSR II flow cytometer (BD Biosciences).

Adoptive transfer and T cell activation in vivo

Adoptive transfer experiments were performed as previously described (31). A total of 1–3 x 106 CFSE-labeled mAb KJ1-26+CD4+ control or ADAP–/– T cells was transferred i.v. into BALB/c recipients. Twenty-four to 48 h later mice were challenged i.v. with a low (5–30 µg) or high (100–150 µg) dose of OVAp and 25 µg of LPS. On the appropriate day, spleens and lymph nodes were harvested, RBC were lysed, and single cell suspensions were analyzed by flow cytometry for the presence of donor T cells. Cells were labeled with anti-Fc receptor mAb (clone 2.4G2) followed by mAb KJ1-26-biotin and streptavidin, anti-B220, anti-CD11b, and anti-CD4. Non-T cells were identified as B220+ and CD11b+ and removed from further analysis. CD4+B220–CD11b– cells were analyzed for the presence of mAb KJ1-26+ cells.

Homing assays

Adoptive transfers of control DO11.10 or ADAP–/– DO11.10 T cells (3 x 106 cells/mouse) were performed as described above. Spleens or peripheral lymph nodes (inguinal, axillary, cervical, brachial, and mesenteric) were harvested 5 days following cell transfer and single cell suspensions were made and assessed for the presence of donor transgenic cells using the labeling strategy described above.

Low clonal precursor adoptive transfer and T cell activation in vivo

This protocol was adapted from Hataye et al. (32). Briefly, a volume containing 104 CFSE-labeled mAb KJ1-26+CD4+ADAP+/+ or ADAP–/– T cells was transferred i.v. into BALB/c recipients. Twenty-four to 48 h later, mice were challenged i.v. with 50 µg of OVAp and 25 µg LPS. Eight days later spleens and mesenteric lymph nodes were harvested and single cell suspensions were made. Cells were resuspended in a small volume of anti-Fc receptor mAb (clone 2.4G2) culture supernatant. Donor transgenic T cells were positively selected as previously described (32) using mAb KJ1-26-biotin and streptavidin magnetic beads. Purified T cells were spun down and resuspended in 100 µl of PBS supplemented with 2% calf serum. Cells (20 µl) were transferred into a FACS tube and 200 µl of PKH-26 counting beads was added to the tube. The remaining cell suspension was transferred to a FACS tube and labeled with mAb KJ1-26, anti-CD4, anti-B220, and anti-CD11b before flow cytometric analysis. The total number of cells per FACS tube was calculated as described above. The total number of cells recovered following purification was calculated as follows: ((number of cells in 20 µl) x (total volume of cells recovered in µl))/20 µl. The number of mAb KJ1-26+CD4+ donor cells per recipient was calculated by multiplying the total number of cells recovered by the percentage of mAb KJ1-26+CD4+ cells in the sample.

Short term in vivo stimulations

Short term in vivo stimulations were performed as previously described (33). Briefly, 6 h following i.v. challenge with OVAp and LPS, spleens were isolated, dissociated in 2% PFA, and incubated for 20 min at room temperature. Cells were washed twice in PBS and resuspended in anti-Fc receptor mAb (clone 2.4G2) culture supernatant and incubated for 15 min at 4°C. Aliquots (50 µl) were added to FACS tubes and intracellular staining was performed as described above.

Measurement of the average number of cell divisions

The average number of total cell divisions per input T cell was calculated as described previously (34).

Statistical analysis

All statistical analysis was performed using a Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
ADAP is a positive regulator of Ag-dependent T cell-APC conjugate formation

To assess the role of ADAP in regulating Ag-dependent T cell interactions with APCs, we bred ADAP–/– mice onto the DO11.10 TCR transgenic background and performed in vitro conjugate assays. Both control DO11.10 and ADAP–/– DO11.10 T cells expressed comparable levels of the DO11.10 clonotypic TCR and CD11a, the {alpha} subunit of the LFA-1 integrin (data not shown). DO11.10 T cells were incubated with OVAp-pulsed, T cell-depleted splenocytes and the percentage of T cells in conjugates with APCs was determined by flow cytometry. In the absence of Ag, both control ADAP+/+ and ADAP–/– DO11.10 T cells exhibited low levels of conjugate formation (Fig. 1). In the presence of Ag-pulsed APCs, ADAP–/– DO11.10 T cells displayed reduced conjugate formation compared with control DO11.10 T cells (Fig. 1). Although conjugate formation by ADAP–/– DO11.10 T cells was most impaired at lower Ag concentrations, significant decreases in conjugate formation were observed at all Ag concentrations tested (55% reduction at 0.3 µM OVAp compared with 20% reduction at 100 µM OVAp). Conjugate formation required the LFA-1 integrin, because the addition of an anti-LFA-1 function-blocking Ab largely inhibited conjugate formation (Fig. 1). These results demonstrate that ADAP positively regulates LFA-1 integrin-dependent T cell interactions with Ag-laden APCs in vitro.


Figure 1
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  		FIGURE 1. ADAP is critical for optimal conjugate formation of DO11.10 T cells with Ag-pulsed APCs. Conjugate formation of ADAP+/+ DO11.10 (gray bars) or ADAP–/– DO11.10 (open bars) T cells with OVAp-pulsed APCs was analyzed as described in Materials and Methods. In the indicated sample, cells were incubated in the presence of blocking Abs against the {alpha} subunit of LFA-1 while conjugating ({alpha}-CD11a). Data are presented as mean percentage of DO11.10 T cells in conjugates with CellTracker Orange+B220+ cells (±SD) from triplicate wells from one representative experiment of three independent experiments performed. *, p < 0.05; **, p < 0.002; and ***, p < 0.0009.

 
ADAP is critical for T cell proliferation and expression of CD25, CD69, and IL-2 following stimulation with suboptimal concentrations of Ag ex vivo

To characterize Ag-dependent proliferation of ADAP–/– T cells, we assessed the kinetics of T cell proliferation by tracking T cell numbers and CFSE dye dilution of ADAP–/– DO11.10 T cells stimulated over a range of OVAp concentrations. ADAP–/– DO11.10 T cells proliferated poorly in response to a low concentration of OVAp (0.2 µg/ml) (Fig. 2A). At 48 h, 14% of control DO11.10 T cells had undergone at least one round of cell division whereas only 3% of ADAP–/– DO11.10 T cells had begun to divide (Fig. 2B). At 72 h, the fold expansion of ADAP–/– DO11.10 T cells was reduced (Fig. 2A). Although both control DO11.10 T cells and ADAP–/– DO11.10 T cells had undergone several rounds of cell division, many fewer ADAP–/– DO11.10 T cells had divided (Fig. 2B). These results suggest that fewer T cells receive sufficient signals to initiate cell division in the absence of ADAP.


Figure 2
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  		FIGURE 2. ADAP positively regulates T cell proliferation and expression of CD25 and CD69 to low doses of Ag in vitro. Control DO11.10 or ADAP–/– DO11.10 splenocytes were stimulated with the indicated concentrations of OVAp (OVA) or PMA and ionomycin (P/I) and cells were harvested at 24, 48, and 72 h. A, Kinetics of ADAP–/– DO11.10 T cell proliferation. Graphs represent the fold increase in the absolute number of KJ1-26+CD4+ T cells compared with cells stimulated with medium only. Data represent the average of six individual wells. One representative experiment of four independent experiments is shown. B, Representative CFSE profiles of cells in A. Black line, Control DO11.10 T cells; shaded histogram, ADAP–/– DO11.10 T cells. Numbers in each panel represent the percentage of cells with diluted CFSE (top number, control DO11.10 T cells; bottom number, ADAP–/– DO11.10 T cells). C and D, Expression of CD25 and CD69. Data are presented as mean percentage of DO11.10 T cells expressing CD25 (C) or CD69 (D) (±SD) from five wells from one representative experiment of two independent experiments performed. *, p < 0.03; **, p < 0.004; ***, p ≤ 0.001.

 
Stimulation with an intermediate concentration of OVAp (2 µg/ml) resulted in only modest defects in ADAP–/– DO11.10 T cell proliferation. At both 48 and 72 h a larger percentage of ADAP–/– DO11.10 T cells remained CFSE-high compared with control DO11.10 T cells (48 h: 54% control DO11.10 T cells compared with 78% ADAP–/– DO11.10 T cells; 72 h: 14% control DO11.10 T cells compared with 33% ADAP–/– DO11.10 T cells), and at 72 h the fold expansion of ADAP–/– DO11.10 T cells was reduced (Fig. 2, A and B). At the highest concentration of OVAp tested (20 µg/ml), ADAP–/– DO11.10 T cells also exhibited a reduction in fold expansion at 72 h but underwent similar amounts of cell division as indicated by their CFSE dilution. Control and ADAP–/– DO11.10 T cells proliferated equivalently when stimulated with PMA and ionomycin (Fig. 2, A and B).

We also assessed expression of the early activation markers CD25 and CD69 on ADAP–/– DO11.10 T cells. Induction of CD25 and CD69 on ADAP–/– DO11.10 T cells was dramatically impaired at all time points examined when T cells were stimulated with a low Ag concentration (Figs. 2, C and D). Stimulation with increasing Ag concentrations largely rescued CD25 and CD69 expression on ADAP-deficient T cells. However, a small but statistically significant decrease in expression of both markers was observed on ADAP–/– DO11.10 T cells stimulated with a high OVAp concentration or with PMA and ionomycin (Fig. 2, C and D).

The initial characterization of ADAP-deficient T cells revealed a defect in IL-2 production following stimulation of CD3 and CD28 (7, 8). Thus, we examined the expression of IL-2 in Ag-stimulated ADAP–/– DO11.10 T cells. Compared with controls, ADAP–/– DO11.10 T cells exhibited impaired IL-2 expression under conditions of suboptimal Ag stimulation (Fig. 3, A and B). ADAP–/– DO11.10 T cells exhibited a reduction in IL-2 expression at both 24 (data not shown) and 48 h following stimulation with the lowest concentration of Ag tested (Fig. 3B). Stimulation with an intermediate Ag concentration resulted in a modest reduction in IL-2 expression by ADAP-deficient T cells, whereas the absence of ADAP had no effect on IL-2 expression when cells were stimulated with a high Ag concentration or PMA and ionomycin. Interestingly, a significant reduction in secreted IL-2 was observed at all Ag concentrations examined, with the greater reductions observed at lower Ag concentrations (Fig. 3C). The levels of secreted IL-2 under unstimulated conditions were below the limit of detection (data not shown). Together, these results suggest that ADAP is critical for T cell proliferation, the induction of early activation markers, and IL-2 production under conditions of limiting Ag stimulation.


Figure 3
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  		FIGURE 3. ADAP positively regulates Ag-induced expression of IL-2 in vitro. A, Representative histograms of IL-2 expression by KJ1-26+CD4+ T cells 48 h after no stimulation (U), stimulation with PMA/ionomycin (P/I), or the indicated concentrations of OVAp. Black line, IL-2; shaded histogram, isotype control. The percentage of cells positive for IL-2 was determined using the isotype control. B, Quantification of A. Gray bars, Control DO11.10 T cells; open bars, ADAP–/– DO11.10 T cells. Data are gated on KJ1-26+CD4+ cells and are presented as the mean percentage of IL-2+ cells (±SD). **, p < 0.06; and *, p < 0.02. Data presented are from one representative experiment of three independent experiments performed. C, IL-2 secretion as measured by ELISA at 48 h following stimulation with the indicated concentrations of OVAp. Data represent the average concentration of IL-2 from six samples (±SD). ***, p < 0.0001.

 
ADAP–/– T cells traffic normally through secondary lymphoid organs

Naive T cell entry into lymph nodes is a tightly regulated process that requires LFA-1-mediated T cell adhesion to ICAM-1-expressing high endothelial venules (35, 36, 37). Because ADAP–/– T cells display defective adhesion to ICAM-1 following TCR stimulation (7, 8), we investigated whether ADAP–/– T cells traffic normally into secondary lymphoid tissue. Equivalent numbers of resting control DO11.10 or ADAP–/– DO11.10 T cells were adoptively transferred into syngeneic recipients and the number of transferred cells in peripheral lymph nodes and spleen was assessed 5 days later. Similar numbers of mAb KJ1-26+CD4+ cells were present in both peripheral lymph nodes and spleen in mice that received control or ADAP–/– DO11.10 T cells (Fig. 4, A and B). Additionally, the average ratio of control to ADAP–/– T cells present in the spleen was 1.01 ± 0.5 (n = 16 control DO11.10 mice, n = 18 ADAP–/– DO11.10 mice; data not shown). These results suggest that ADAP is not required for naive T cell recirculation through secondary lymphoid tissue.


Figure 4
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  		FIGURE 4. ADAP does not regulate T cell recirculation through secondary lymphoid organs. Wild-type DO11.10 (gray bars) or ADAP–/– DO11.10 (open bars) T cells were transferred into syngeneic recipients 5 days before harvesting and the number of donor T cell numbers in the spleen and peripheral lymph nodes was determined. Data represent the mean number of KJ1-26+CD4+ cells (±SD) present in spleen (A) or peripheral lymph nodes (B). Data in A represent the mean of three independent experiments where a minimum of seven mice were used per condition. The data in B represent the mean cell number of two independent experiments where six mice were used per condition.

 
ADAP is critical for efficient clonal expansion to limiting concentrations of Ag

To determine the role of ADAP in regulating T cell proliferation in the context of an in vivo immune response, we adoptively transferred equivalent numbers (1–3 x 106) of control DO11.10 or ADAP–/– DO11.10 T cells into syngeneic recipients and challenged the mice i.v. with OVAp and LPS. The presence of transferred cells was assessed in the spleen 3 days following priming. Control DO11.10 T cells displayed dose-dependent clonal expansion following challenge with either a low or high dose of OVAp as measured by absolute cell numbers and CFSE dilution (Fig. 5, A and B). Proliferation of ADAP–/– DO11.10 T cells was also dose dependent, but the total number of ADAP–/– DO11.10 T cells was significantly reduced following challenge with a low dose of OVAp (Fig. 5A; p < 0.03). At this Ag concentration, ADAP–/– DO11.10 T cells underwent fewer rounds of cell division as measured by CFSE dilution (Fig. 5B) and the average number of cell divisions per ADAP–/– DO11.10 T cell was significantly decreased compared with the average number of cell divisions per control DO11.10 T cell (1.2 ± 0.04 vs 1.9 ± 0.01 divisions, respectively) (Fig. 5C). When challenged with a high dose of OVAp, control DO11.10 and ADAP–/– DO11.10 T cells exhibited similar cell numbers, CFSE dye dilution profiles, and average numbers of cell divisions (3.0 ± 0.16 vs 2.6 ± 0.16 divisions, respectively) per T cell (Fig. 5, A–C). To determine whether deficient clonal expansion of ADAP–/– DO11.10 T cells at low doses of Ag was the result of delayed kinetics, we assayed clonal expansion of ADAP–/– DO11.10 T cells at days 3, 5, and 7 following challenge with OVAp and LPS. Both control and ADAP–/– DO11.10 T cell numbers peaked at day 3 and then declined (Fig. 5D), suggesting that the reduction in ADAP–/– DO11.10 T cell numbers on day 3 following Ag challenge is not the result of these cells dividing with slower kinetics than control DO11.10 T cells. Together, these data demonstrate that ADAP is critical for optimal clonal expansion to a low Ag concentration but is dispensable for clonal expansion when a high Ag concentration is used.


Figure 5
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  		FIGURE 5. ADAP is required for optimal T cell clonal expansion to low doses of Ag in vivo under conditions of high clonal abundance. CFSE-labeled control or ADAP–/– DO11.10 T cells were transferred into syngeneic recipients. Mice were either left unchallenged (T.O., transfer only) or challenged 24–36 h after transfer i.v. with the indicated amounts of OVAp and LPS. A, The mean number of KJ1-26+CD4+ cells in the spleens of recipient mice 3 days following challenge (±SD) with OVAp or in mice left unchallenged (T.O.). *, p < 0.03. B, Representative CFSE dilution profiles on day 3 following challenge. Black line, Control DO11.10 T cells; shaded histogram, ADAP–/– DO11.10 T cells. C, The average number of cell divisions on day 3 following challenge. Gray bars, Control DO11.10 T cells; open bars, ADAP–/– DO11.10 T cells. ***, p < 0.0003. D, The kinetics of the clonal expansion of ADAP–/– DO11.10 T cells following challenge with 5 µg of OVAp and LPS. The mean number of KJ1-26+CD4+ cells in the spleen for each time point (±SD) is shown. **, p < 0.01. In A–C, 3 x 106 T cells were transferred into each recipient. In D, 1 x 106 T cells were transferred into each recipient. For all panels, one representative experiment of three independent experiments is shown where a minimum of three mice were used per condition.

 
ADAP-deficient T cells exhibit impaired clonal expansion when clonal abundance is low

Initial clonal precursor frequency affects the magnitude of clonal expansion by Ag-challenged naive T cells (32, 38). Because we observed a reduction in clonal expansion of ADAP–/– DO11.10 T cells following challenge with a suboptimal Ag dose under conditions of high clonal abundance (1–3 x 106 T cells), we next examined clonal expansion following a challenge of recipients receiving 10,000 control or ADAP–/– DO11.10 T cells with 50 µg of OVAp and LPS. At the peak of clonal expansion under these conditions (day 8), ADAP–/– DO11.10 T cells exhibited a profound defect in clonal expansion. Although control DO11.10 T cells expanded 500-fold, ADAP–/– DO11.10 T cells only expanded 4-fold (Fig. 6A). Although both populations diluted CFSE, indicating multiple rounds of cell division, control DO11.10 T cells diluted CFSE to a greater extent than ADAP–/– DO11.10 T cells (Fig. 6B). These results indicate that ADAP–/– DO11.10 T cells exhibit a profound defect in clonal expansion when the initial clonal precursor frequency is low and that the deficit in clonal expansion is at least partially the result of ADAP–/– DO11.10 T cells undergoing fewer rounds of cell division.


Figure 6
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  		FIGURE 6. ADAP regulates clonal expansion under conditions of low precursor frequency. Expansion of DO11.10 ADAP–/– T cells under conditions of low clonal abundance (10,000 transferred DO11.10 T cells per mouse). A, The mean number of KJ1-26+CD4+ cells detected on day 8 in unchallenged (T.O., transfer only) or OVAp and LPS challenged mice (OVA) (±SD). Gray bars, Control DO11.10 cells; open bars, ADAP–/– DO11.10 cells. *, p < 0.04. B, Representative CFSE profiles of KJ1-26+CD4+ cells from OVAp-challenged mice. Black line, Control DO11.10 T cells; shaded histogram, ADAP–/– DO11.10 T cells. For all panels, one representative experiment of three independent experiments is shown where a minimum of three mice were used per condition.

 
ADAP–/– DO11.10 T cells exhibit reduced IL-2 production in vivo

Because we observed defects in IL-2 expression following Ag stimulation in vitro and impaired clonal expansion of ADAP–/– DO11.10 T cells in vivo, we wanted to determine whether ADAP was also required for IL-2 secretion in vivo. To directly assess IL-2 production during an in vivo immune response, control DO11.10 or ADAP–/– DO11.10 T cells were adoptively transferred into syngeneic recipients and analyzed for the presence of intracellular IL-2 protein 6 h after OVAp/LPS challenge. Previous studies demonstrated maximum IL-2 production following i.v. challenge with OVAp and adjuvant at this time point (39). Expression of IL-2 by both control and ADAP–/– DO11.10 T cells was dose dependent (Fig. 7, A and B). Over multiple experiments, an average of 21% of control DO11.10 T cells expressed IL-2 compared with 14% of ADAP–/– DO11.10 T cells at 6 h postchallenge with a low dose of Ag (Fig. 7B). No differences in IL-2 expression were observed when mice were challenged with a high Ag dose (Fig. 7, A and B). These results demonstrate that ADAP is critical for IL-2 production by T cells that are stimulated with a limiting Ag dose.


Figure 7
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  		FIGURE 7. ADAP positively regulates IL-2 expression in vivo. Adoptive transfer experiments were performed as in Fig. 5, except that spleens were harvested 6 h after challenge and analyzed for intracellular IL-2 expression. A, Representative histograms from OVAp/LPS (OVA) or unchallenged mice (T.O., transfer only) are gated on KJ1-26+CD4+ cells and the percentage of IL-2+ cells is shown for each condition. B, Quantification of data from three combined experiments where six mice were used per condition; *, p < 0.05. Data represent the mean percentage of IL-2+ cells (±SD) per condition. Gray bars, ADAP+/+ DO11.10 cells; open bars: ADAP–/– DO11.10 cells.

 
Impaired clonal expansion of ADAP–/– T cells is not associated with deficient induction of CD69 and CD25

Because ADAP–/– T cells demonstrated defective expression of CD25 and CD69 following anti-CD3 Ab (7, 8) or Ag stimulation (Fig. 2, C and D), we examined expression of these early activation markers following Ag challenge in vivo. Surprisingly, we observed comparable increases in expression of both CD25 and CD69 on control DO11.10 and ADAP–/– DO11.10 T cells 24 h following challenge with either low-dose or high-dose OVAp and LPS (Fig. 8, A and B). This suggests that ADAP does not regulate the expression of these early activation markers in vivo.


Figure 8
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  		FIGURE 8. Expression of activation markers on DO11.10 ADAP–/– T cells in vivo. Adoptive transfer experiments were performed as in Fig. 5, except that spleens were harvested 24 h after challenge and analyzed for CD25 and CD69 expression. Representative histograms from OVAp/LPS (OVA) or unchallenged mice (T.O., transfer only) were gated on KJ1-26+CD4+ cells and the percentage of CD25+ cells (A) or CD69+ cells (B) is shown for each condition. One representative experiment of two independent experiments with a minimum of two mice per condition is shown. C and D, Adoptive transfer experiments were performed as in Fig. 5 except that spleens were harvested at the indicated time points and analyzed for CD62L expression. C, Representative histograms were gated on KJ1-26+CD4+ cells that had undergone at least four rounds of cell division as assessed by CFSE dilution. The percentage of CD62L+ cells is shown. D, Quantification of C. Gray bars, Control DO11.10 T cells; open bars, ADAP–/– DO11.10 T cells. **, p < 0.002. For C and D, data from one representative experiment of three independent experiments are shown where a minimum of three mice per condition were used.

 
Altered effector cell phenotype following Ag stimulation of ADAP–/– T cells

Because ADAP–/– DO11.10 T cells display deficient clonal expansion and cytokine production at low Ag doses, we were interested in determining whether the absence of ADAP affected the phenotype of effector T cells. Following activation, T cells decrease expression of several cell surface proteins, including CD62L (40). T cells express reduced amounts of CD62L over the course of several days following T cell activation, and this phenotype is associated with gain of effector function (40, 41). We assessed the expression of CD62L on control and ADAP–/– DO11.10 T cells on days 3, 5, and 7 following challenge with a suboptimal dose of OVAp and LPS. Because increased numbers of undivided ADAP–/– DO11.10 T cells or T cells that had undergone only a few rounds of cell division could account for differences in cell surface protein expression, only cells that had undergone four or more rounds of cell division were analyzed. OVAp-challenged control DO11.10 T cells had decreased expression of CD62L by day 3 and maintained this profile through day 7. Although a similar pattern of CD62L expression was observed on ADAP–/– DO11.10 T cells on day 3, fewer ADAP-deficient T cells maintained a CD62Llow profile on days 5 and 7 (Fig. 8, C and D). We also observed higher CD62L expression on OVAp-challenged ADAP–/– DO11.10 T cells in our low transfer model (data not shown). These results suggest that even if they undergo multiple rounds of cell division, ADAP–/– DO11.10 T cells do not receive the appropriate signals to fully decrease the expression of cell surface proteins associated with gain of effector function.

ADAP is required for Ag-induced expression of Bcl-xL

Recent studies indicate that ADAP has a previously unappreciated role in TCR signaling to NF-{kappa}B (6), which regulates TCR-mediated induction of prosurvival proteins such as Bcl-xL (20, 21). Thus, impaired clonal expansion of ADAP–/– DO11.10 T cells could also be due to a reduced ability of ADAP–/– T cells to survive following Ag challenge. To assess a role for ADAP in regulating Bcl-xL induction following Ag stimulation, we analyzed changes in Bcl-xL expression in control DO11.10 and ADAP–/– DO11.10 T cells following OVAp challenge (Fig. 9, A and B). In the absence of Ag, control DO11.10 T cells and ADAP–/– DO11.10 T cells expressed low levels of Bcl-xL. OVAp-stimulated control and ADAP–/– DO11.10 T cells both exhibited a bimodal pattern of expression of Bcl-xL that was dose dependent and increased over time (Fig. 9, A and B). Similar to our results with Ag-stimulated T cell proliferation, defects in Bcl-xL expression by ADAP–/– DO11.10 T cells were observed at the lowest Ag concentration. By 72 h, only 14% of ADAP–/– DO11.10 T cells stimulated with a low-dose OVAp expressed Bcl-xL compared with 70% of control DO11.10 T cells (Fig. 9A). Impaired induction of Bcl-xL by ADAP–/– DO11.10 T cells was also observed at 24 and 48 h after challenge with an intermediate or high dose of OVAp. By 72 h, control and ADAP–/– DO11.10 T cells stimulated with an intermediate or high dose of OVAp expressed similar levels of Bcl-xL (87 vs 82% for 1 µg/ml OVAp; 84 vs 85% for 10 µg/ml OVAp). These results indicate that ADAP is critical for Ag-induced Bcl-xL expression, particularly at limiting concentrations of Ag.


Figure 9
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  		FIGURE 9. ADAP positively regulates Ag-stimulated increases in Bcl-xL expression. Control DO11.10 or ADAP–/– DO11.10 splenocytes were cultured with the indicated concentrations of OVAp (OVA), PMA and ionomycin (P/I), or medium alone (U) for 24, 48, or 72 h. A, Representative histograms were gated on KJ1-26+CD4+ cells and the percentage of cells expressing Bcl-xL is indicated. Black line, Bcl-xL; shaded histogram, isotype control. B, Quantification of A. Data represent two combined independent experiments (±SD). C, CFSE by Bcl-xL profiles of KJ1-26+CD4+ cells stimulated with 0.1 mg/ml OVAp for the indicated periods of time. The percentage of cells in each quadrant is shown.

 
We found that expression of Bcl-xL correlated strongly with cell division (Fig. 9C). Although all control and ADAP–/– DO11.10 T cells remained CFSE-high at 24 h, a population of cells had gained expression of Bcl-xL. However, even before cell division started, a higher percentage of control DO11.10 T cells expressed Bcl-xL when compared with ADAP–/– DO11.10 T cells. Once cells began to divide, they expressed Bcl-xL at all time points examined. The lower expression of Bcl-xL on ADAP–/– DO11.10 T cells correlated with fewer T cells undergoing cell division. These results suggest that Bcl-xL expression is induced before cell division and is a good indicator of cells that have acquired the ability to divide.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we analyzed the role of the adapter protein ADAP in Ag-driven T cell activation. We demonstrated that ADAP is a positive regulator of Ag-dependent, LFA-1 integrin-mediated T cell conjugate formation with APCs. We also revealed that ADAP is critical for T cell expansion and cytokine expression in vitro and during an in vivo immune response to limiting concentrations of Ag or under conditions of low clonal abundance. Reduced expression of Bcl-xL following Ag stimulation of ADAP–/– T cells suggests that the observed defect in T cell expansion may be the result of reduced survival of ADAP–/– T cells. These data suggest that ADAP positively regulates T cell activation by at least two distinct mechanisms: 1) increasing T cell sensitivity to Ag by facilitating the formation of stable interactions between T cells and Ag-expressing APCs; and 2) enhancing T cell survival following Ag stimulation.

Our results showed that LFA-1-mediated, Ag-dependent T cell conjugate formation with Ag-pulsed APCs is impaired in the absence of ADAP (Fig. 1). These results are consistent with previous studies demonstrating that ADAP is critical for integrin-mediated adhesion and LFA-1-integrin clustering following TCR ligation (7, 8) and for Ag-dependent thymocyte adhesion to Ag-expressing APCs (42). Additionally, overexpression of ADAP enhances Ag-dependent T cell conjugate formation (19). Although we observed impaired conjugate formation between ADAP–/– T cells and APCs at all concentrations of Ag tested, the defect was most pronounced at low Ag doses. Similar findings have been reported with LFA-1–/– T cells (16). In these studies, the concentrations of Ag that resulted in diminished conjugate formation also induced impaired proliferation and cytokine production, demonstrating that LFA-1 promotes T cell activation by increasing T cell sensitivity to Ag. Our results suggest that ADAP facilitates T cell responses by promoting stable LFA-1-dependent T cell-APC interactions.

Our analysis of Ag-stimulated T cell proliferation, the induction of activation markers, and IL-2 production indicate that ADAP is particularly important when Ag concentrations are limiting. Although saturating concentrations of Ag produced equivalent expansion and cytokine production by wild-type and ADAP-deficient T cells, T cell proliferation and IL-2 expression was impaired in the absence of ADAP when a suboptimal Ag concentration was used (Figs. 2 and 3). The kinetics of ADAP–/– DO11.10 T cell proliferation suggest that the diminished proliferation of ADAP–/– T cells at the lowest Ag dose may be the result of a delay in the time necessary to commit to cell division. Additionally, T cells may cycle at a slower rate in the absence of ADAP because ADAP–/– DO11.10 T cells lagged by one round of cell division at 48 and 72 h when stimulated with the lowest Ag dose. Our results are similar to findings from several studies demonstrating that Ag dose and the duration of Ag exposure critically shape the CD4 T cell proliferative response. Lowering the Ag dose or shortening the length of Ag stimulation reduced the percentage of T cells that divide and the number of cell divisions that dividing T cells underwent and increased the time required for T cells to commit to cell division (24, 25, 26, 43). Taken together, these results suggest that by facilitating long-term interactions between T cells and Ag-expressing APCs, ADAP enhances T cell Ag sensitivity and lengthens the duration of signaling.

We investigated a role for ADAP during an in vivo immune response by using adoptive transfer of ADAP–/– DO11.10 donor T cells into syngeneic hosts and challenging with a high or low concentration of Ag and LPS. Similar to our studies using ex vivo cultured ADAP–/– DO11.10 T cells, we found that ADAP is critical for T cell clonal expansion following challenge with a low Ag dose but is dispensable when mice are immunized with a high dose of Ag. CFSE profiles of donor T cells challenged with a low Ag dose indicated that T cells underwent fewer rounds of cell division in the absence of ADAP, suggesting that the reduction in ADAP–/– DO11.10 T cell numbers at the peak of the response may be the result of ADAP–/– T cells requiring a longer period of stimulation to commence cell division coupled with a slower rate of cell division. Alternatively, reduced T cell numbers could also be the result of impaired T cell survival following stimulation with a low Ag dose. Our analysis of the kinetics of clonal expansion revealed that the reduced numbers of ADAP–/– DO11.10 T cells on day 3 are not the result of a profound delay in the kinetics of expansion. Under similar conditions, we also observed a defect in IL-2 expression in donor ADAP–/– DO11.10 T cells challenged with a low Ag dose, suggesting that early defects in IL-2 production may contribute to a reduced proliferative response to limiting Ag doses.

Similarities in in vivo clonal expansion profiles between ADAP–/– and CD18–/– DO11.10 T cells further support a role for ADAP in promoting T cell clonal expansion by enhancing T cell sensitivity to Ag. Although the effect of Ag dose on clonal expansion was not assessed in these studies, CD18–/– DO11.10 T cells exhibit impaired T cell clonal expansion following i.v. challenge with 50 µg of OVAp and LPS (36). CD18–/– DO11.10 T cells also exhibit impaired proliferation ex vivo in response to OVAp-pulsed APCs that is dependent on the Ag dose as well as impaired IL-2 expression following ex vivo restimulation (36). In contrast to our results, CD18–/– DO11.10 T cells exhibit impaired clonal expansion in vivo following stimulation with a relatively high Ag dose. This difference may trace to the fact that ADAP–/– T cells exhibit only a partial block in LFA-1 function (Fig. 1). The residual LFA-1 activity present in ADAP–/– DO11.10 T cells may allow these T cells to respond to higher Ag concentrations that may be disrupted in the total absence of LFA-1.

The most profound defect in clonal expansion of ADAP–/– DO11.10 T cells was observed under conditions of low clonal abundance. T cell expansion was diminished by two orders of magnitude when recipient mice received only 10,000 ADAP–/– DO11.10 T cells. The reduced clonal expansion of ADAP–/– DO11.10 T cells may be the result of fewer rounds of cell division because ADAP–/– DO11.10 T cells diluted CFSE to a lesser extent than control DO11.10 T cells. However, the CFSE profiles of ADAP–/– DO11.10 T cells do indicate that these cells underwent multiple rounds of cell division, suggesting that impaired cell survival could also account for the dramatically reduced numbers of ADAP–/– T cells observed at the peak of clonal expansion. The reduced potential for cell expansion exhibited by T cells at high clonal abundance is thought to be the result of intraclonal competition for resources (32). Thus, the differences in clonal expansion between control and ADAP–/– DO11.10 T cells may be partially masked when clonal frequency is high. When clonal abundance is low, competition for resources is removed and wild-type T cells can undergo much greater expansion. In contrast, the fold expansion of ADAP–/– DO11.10 T cells is unaffected by clonal abundance, suggesting that defective proliferation by ADAP–/– T cells cannot be overcome by greater access to growth factors or Ag-expressing APCs. Additionally, recent studies have revealed that the stability of Ag-dependent T cell-dendritic cell interactions is dependent on clonal abundance. Fewer stable T cell-dendritic cell contacts were observed under conditions of high clonal precursor frequency, and this correlated with less efficient T cell activation and proliferation on a per cell basis (44). Because ADAP is required for optimal T cell-APC interactions, more pronounced defects in T cell activation may be observed when the precursor frequency is low because ADAP–/– T cells are unable to form stable contacts with Ag-laden APCs.

Recently, we discovered a novel role for ADAP in regulating NF-{kappa}B activation downstream of the TCR and CD28 (6). Mutational analysis revealed that the regions of ADAP required for NF-{kappa}B activation are distinct from the regions required for LFA-1-mediated adhesion (6). Because NF-{kappa}B is an important regulator of cell survival in T cells (45), we were interested in determining a role for ADAP in T cell survival. We demonstrated that ADAP is required for Bcl-xL induction following stimulation with suboptimal Ag concentrations. Importantly, the concentrations of Ag where the most profound defects in Bcl-xL expression were observed were also the same concentrations at which we observed reduced proliferation of ADAP-deficient T cells. This suggests that diminished survival of ADAP–/– T cells may contribute to the proliferation defect of these cells.

ADAP acts downstream of protein kinase C{theta} (PKC{theta}) to activate NF-{kappa}B following TCR ligation by interacting with CARMA1 and facilitating the assembly of the CARMA1-Bcl10-Malt1 complex (6). Analysis of T cell function in PKC{theta}–/– and CARMA1–/– mice shows similarities to our results with ADAP–/– T cells. T cells from both ADAP–/– and PKC{theta}–/– mice exhibit reduced T cell proliferation and cytokine production following TCR and CD28 stimulation (7, 8, 46). Similar to ADAP–/– T cells, impaired T cell proliferation by PKC{theta}–/– T cells is correlated with reduced T cell survival and Bcl-xL expression (21, 47). This suggests that PKC{theta} and ADAP facilitate T cell activation by protecting T cells from apoptosis in a Bcl-xL- and NF-{kappa}B-dependent manner. Similar to ADAP–/– DO11.10 T cells, CARMA1–/– DO11.10 T cells also exhibited impaired proliferation to suboptimal Ag concentrations but normal proliferation to high Ag doses (48). Unlike ADAP, CARMA1 appears to be absolutely required for IL-2 secretion, as CARMA1–/– T cells do not secrete IL-2 in response to any concentration of Ag stimulation. Furthermore, CARMA1–/– T cells do not exhibit defects in Ag-dependent conjugate formation. Thus, ADAP may enhance Ag sensitivity by CARMA1-dependent regulation of NF-{kappa}B and via CARMA1-independent increases in T cell-APC conjugation.

In summary, we propose that ADAP functions to regulate T cell activation by two distinct mechanisms. First, ADAP enhances T cell sensitivity to Ag by promoting LFA-1-mediated T cell adhesion to Ag-expressing APCs. The stable T cell-APC interactions facilitated by ADAP allow T cells to mount more productive immune responses to lower Ag concentrations. This might be particularly relevant early in the T cell response, when both pathogen load and clonal precursor frequency is low. Second, ADAP facilitates T cell expansion through the induction of prosurvival signaling pathways, specifically the expression of Bcl-xL, that promote T cell survival following Ag stimulation. Thus, ADAP has multiple functions downstream of the TCR that promote optimal T cell responses to Ag.


   Acknowledgments
 
We thank S. Highfill and M. Schwartz for mouse genotyping and colony maintenance and M. Jenkins, J. Hataye, and members of the Shimizu laboratory for helpful discussions and advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grants R01AI038474 (to Y.S.), R01AI031126 (to Y.S.), and R01AI056016 (to E.J.P.), a grant from the Arthritis Foundation (to E.J.P.), an American Heart Association predoctoral fellowship (to K.L.M.), and National Institutes of Health Training Grant T32DE007288 (to M.S.T. and B.J.B.). Y.S. is supported in part by the Harry Kay Chair in Biomedical Research at the University of Minnesota. Back

2 Current address: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892. Back

3 Address correspondence and reprint requests to Dr. Yoji Shimizu, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, MMC 334/Room 6-112 NHH, 312 Church Street Southeast, Minneapolis, MN 55455. E-mail address: shimi002{at}umn.edu Back

4 Abbreviations used in this paper: ADAP, adhesion and degranulation-promoting adapter protein; CARMA1, caspase-recruitment domain (CARD)-membrane-associated guanylate kinase (MAGUK) protein 1; MAGUK, membrane-associated guanylate kinase; OVAp, OVA323–339 peptide; PFA, paraformaldehyde; PKC{theta}, protein kinase C{theta}; SLP-76, SH2 domain-containing leukocyte-specfic phosphoprotein of 76 kDa. Back

Received for publication November 2, 2006. Accepted for publication July 6, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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