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The Journal of Immunology, 2003, 170: 4127-4133.
Copyright © 2003 by The American Association of Immunologists

Regulation of T Cell Activation and Tolerance by Phospholipase C{gamma}-1-Dependent Integrin Avidity Modulation1

Andrew D. Wells2,3,*, Qing-Hua Liu2,{dagger}, Brian Hondowicz{dagger}, Jidong Zhang*, Laurence A. Turka* and Bruce D. Freedman3,{dagger}

* Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {dagger} Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ag receptor engagement without costimulation induces a tolerant state in CD4+ T cells termed anergy. Anergic CD4+ T cells are primarily characterized by the inability to produce IL-2, but the biochemical basis for this functional defect is not completely understood. We demonstrate that primary CD4+ T cells anergized by costimulatory blockade exhibit impaired TCR-coupled phospholipase C (PLC){gamma}-1 activation. This defect is associated with the marked reduction of multiple downstream signaling events required for IL-2 transcription, including mobilization of intracellular Ca2+ and activation of the mitogen-activated protein kinase cascade. We also found that primary anergic CD4+ T cells fail entirely to modulate their integrin binding avidity in response to TCR stimulation. Integrin avidity modulation is required for full T cell activation and effector function, and as we show in this study, is completely dependent upon PLC{gamma}-1 activity. Finally, analogs that mimic the actions of diacylglycerol and inositol 1,4,5-triphosphate, the immediate products of PLC{gamma}-1 activity, restored integrin avidity modulation and IL-2 production by anergic T cells. Thus, deficient coupling of PLC{gamma}-1 to the TCR appears to be a central biochemical defect that could potentially account for the failure of multiple functional responses in primary anergic CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD4+ T cells activated by specific Ag produce key cytokines and chemokines and express surface ligands that regulate diverse immunological activities, including cytotoxic activity of CD8+ lymphocytes and Ab production by B lymphocytes. Ag-activated CD4+ T cells also "home" to sites of inflammation where they produce proinflammatory cytokines and promote further leukocyte recruitment (1). In addition to signals from the Ag receptor, efficient responses of T cells require costimulation through receptors such as CD28 (2). An additional underlying requirement for the initiation and maintenance of effector functions of CD4+ T cells is the activation of {beta}1 and {beta}2 integrin receptors, which bind to ICAM on APCs or endothelial cells and to components of the extracellular matrix (3) during cell trafficking (4). The importance of these costimulatory mechanisms in the development of immune responses is demonstrated by the fact that disruption of the interactions between CD28 or integrins and their ligands can ameliorate autoimmunity (5, 6, 7) and inhibit organ transplant rejection in vivo (8, 9).

CD4+ T cells that receive Ag receptor signals in the absence of costimulation are rendered anergic. This hyporesponsive state is characterized by a reduced capacity to synthesize IL-2 and other effector cytokines upon subsequent engagement of the TCR (10). Although the molecular mechanism(s) behind the induction and maintenance of this tolerant state have not been completely defined, several biochemical defects have been associated with the reduced synthesis of IL-2 by anergic T cells, including defective coupling of the p42/44 extracellular-regulated kinase (ERK)4 and Jun-terminal kinase mitogen-activated protein kinase (MAPK) cascades to the TCR (11, 12, 13, 14, 15), resulting in an inability to assemble transcriptionally active Fos/Jun (AP-1) and NF-{kappa}B complexes on the IL-2 gene promoter (16). In this study we demonstrate that phospholipase C (PLC){gamma}-1 is a primary regulator of {beta} integrin activation during the initiation of effector responses of naive CD4+ lymphocytes and that a proximal defect in PLC{gamma}-1 activation could account for each of the functional defects of anergic CD4+ T cells, including diminished intracellular Ca2+ concentration ([Ca2+]i) signaling, reduced activation of the MAPK cascade, and decreased IL-2 production. These studies also demonstrate that the failure to up-regulate {beta} integrin avidity in response to TCR stimulation is a central feature of tolerant cells that is caused by failed TCR-induced activation of PLC{gamma}-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of primary effector and anergic CD4+ T cells

Pooled spleen and lymph node cells were cultured in vitro in medium (RPMI 1640 supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, and 2-ME) with 1 µg/ml anti-CD3 (145–2C11) plus either anti-CD28 (37.51, 1 µg/ml) or CTLA-4 Ig (10 µg/ml) for 3 days, followed by 1 day of rest in medium.

Measurement of IL-2 secretion by primed CD4+ T cells

CD4+ T cells were purified from primed cultures by negative selection on MACS columns (Miltyeni Biotec, Auburn, CA) using Abs against I-A/E, CD19, CD16/32, CD11b, and CD8 (BD PharMingen, San Diego, CA). Purified cells were restimulated for 24 h in 96-well plates coated with anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml). IL-2 secreted into the supernatant was measured by fluorescence-linked immunosorbent assay. IL-2 fluorescence-linked immunosorbent assay was performed by coating 5 µm latex beads (107/ml; Interfacial Dynamics, Portland, OR) with IL-2 capture Ab (2 µg/ml; BD PharMingen) in PBS for 2 h at 37°C. Beads were washed with PBS and blocked for 30 min at 37°C in culture medium. IL-2 capture beads (300,000/test) were incubated with 100 µl of culture supernatant or IL-2 standards (R&D Systems, Minneapolis, MN) for 2 h at 37°C and then washed in culture medium. Bead-bound IL-2 was detected by adding PE-conjugated anti-IL-2 (0.1 µg/test, BD PharMingen) for 30 min at 4°C, washing in PBS, and measuring fluorescence using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Fibronectin (FN) adhesion assay

Lymphocytes (0.5–1 x 106) from primed cultures were stained with PE-conjugated anti-CD4 Ab (0.1 µg/test; BD PharMingen) and allowed to settle for 5 min onto glass coverslips coated with 25 µg/ml FN (Life Technologies, Rockville, MD) in a warmed (37°C) microchamber on an inverted fluorescence microscope (Nikon, Melville, NY). PE-positive CD4+ T cells were visualized at x60 and enumerated. Unbound cells were removed from the visual field by gently perfusing the chamber with medium, and the bound CD4+ T cells were then enumerated. In some experiments, T cells were activated for 10 min before assessment of FN binding by cross-linking the TCR with biotinylated anti-CD3 (1 µg/ml) followed by streptavidin (1 µg/ml). This was followed by treatment with PMA (10 ng/ml; Sigma-Aldrich, St. Louis, MO) and the PLC{gamma} inhibitor U73122 or its inactive analog U73433 (1 µm; Calbiochem, La Jolla, CA). In some experiments, lymphocytes were pretreated with Ab against the {alpha}4 chain of very late antigen (VLA)-4 ({alpha}4{beta}1 integrin, 5 µg/ml; BD PharMingen) or the {alpha}L chain of LFA-1 ({alpha}L{beta}2 integrin, 5 µg/ml; BD PharMingen) before assessment of FN binding.

In vitro transmigration assay

Lymphocytes (3 x 105) from primed cultures were added in a volume of 100 µl to Trans-well 5 µm pore size tissue culture inserts (Costar, Cambridge, MA) coated with 25 µg/ml FN. The inserts were placed into 24-well plates containing 600 µl of culture medium and incubated at 37°C for 4 h. Lymphocytes that had migrated through the FN-coated filters were collected from the bottom wells and stained with fluorochrome-conjugated Abs to Thy1.2 (0.1 µg/test; BD PharMingen) and CD4. The number of migrated CD4+ T cells was determined using a FACSCalibur flow cytometer by comparing the number of Thy1.2+ CD4+ cells acquired per tube to a known number of latex beads (100,000) added to each tube at the time of acquisition. In some experiments lymphocytes were pretreated with Ab against VLA-4 before assessment of transmigration.

Flow cytometric measurement of integrin expression

Lymphocytes (0.5–1 x 106) from primed cultures were stained with PerCP-conjugated anti-CD4 Ab (BD PharMingen) and PE-conjugated Abs against either the {alpha}L chain of LFA-1 (CD18; BD PharMingen) or the {alpha}4 chain of VLA-4 (BD PharMingen) before subsequent analysis by flow cytometry.

Single-cell measurements of intracellular Ca2+

Lymphocytes were loaded with the cell-permeable calcium indicator fura 2-acetoxymethyl ester (3.0 µM; Molecular Probes, Eugene, OR) in RPMI for 15 min at 25°C. During the final 10 min of fura 2 loading, biotinylated anti-CD3 and anti-CD4 mAbs were added to lymphocyte suspensions. Cell suspensions were placed into the recording chamber on an inverted fluorescent microscope (Nikon) and allowed to adhere to poly-L-lysine (100 µg/ml; Sigma-Aldrich) treated coverslips for 5 min. Excess extracellular fura-2 acetoxymethyl and unbound Ab were washed from the microscope recording chamber with extracellular bath solution (155 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.4). Discrete bandwidth excitation light (340 ± 10 nm, 380 ± 10 nm) from a xenon source coupled to a computer-controlled monochromator (TILL; Applied Scientific Imaging, Eugene, OR) was delivered to the epifluorescence attachment of the microscope through a quartz fiber optic guide. Excitation light was directed through the fluorescent objective x100 (Nikon) via a dichroic mirror. The emitted fluorescence from fura 2-loaded cells was passed through a 470-nm long pass filter, and images were obtained with an intensified charge-coupled video camera (Model C2400-68; Hamamatsu, Ichinocho, Japan) connected to the side port of the inverted microscope. Four fluorescent video images were averaged and digitized (0.5 Hz) with a video frame grabber (Matrox; Dorval, Quebec, Canada) using Metafluor acquisition and analysis software (Universal Imaging, Downingtown, PA). Stored images were analyzed offline using the Metafluor package. Within cursor-defined areas of interest, paired 340/380 images were background subtracted, and the ratio was calculated. The absolute ratio values were exported into Microsoft Excel and converted to Ca2+ concentration (17). Calibration factors, Rmax (340:380 ratio obtained in presence of 5 µM ionomycin and 10 mM Ca2+), Rmin (340:380 ratio in presence of 5 µM ionomycin and excess EGTA), and F380max and F380min (minimum 510 nm emission with 380 nm excitation) were determined in situ.

Immunoblot analysis of ERK and PLC{gamma}-1 phosphorylation

T cells (106) from primed cultures were restimulated for the indicated time periods with latex beads coated with anti-CD3 and anti-CD4 Abs (5 µg/ml each) and immediately lysed in SDS loading buffer. The lysates were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and blocked overnight in TBS/Tween containing 10% nonfat dry milk. The membrane was probed with a phospho-specific mAb against PLC{gamma}-1 (1/1000; BioSource International, Camarillo, CA), and immunoreactive protein was visualized using an HRP-conjugated secondary antiserum (1/1000; Cell Signaling, Beverly, MA) and a chemiluminescent substrate (Pierce, Rockford, IL). The membrane was then stripped and probed sequentially with a phosphospecific rabbit antiserum against ERK (1/500; Santa Cruz Biotechnology, Santa Cruz, CA), a pan-ERK-specific mAb (1/500; Cell Signaling), and a pan-PLC{gamma}-1-specific mAb (1/500; BioSource International).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Interactions between B7 and the costimulatory receptor CD28 on T cells are necessary and sufficient to operate a "biochemical switch" controlling effector vs anergic cell fates (18). In these studies we chose to generate primary anergic CD4+ T cells by delivering a fully agonistic stimulus through the Ag receptor while blocking B7-mediated costimulation with CTLA-4 Ig (19). This well-defined system allows the generation of T cells that exhibit the central functional features of effector and anergic cells, both in vitro (20) and in vivo (21), and affords clear interpretation of results without the complexities inherent in many other models of T cell tolerance. Representative IL-2 levels produced upon TCR restimulation of effector (3.8 U/ml IL-2) and anergic CD4+ T cells (1.2 U/ml IL-2) confirm the expected distinct phenotypes of these cells.

Defective Ca2+ and MAPK signaling in tolerant T cells

Because IL-2 synthesis is central to the induction vs avoidance of anergy, we first focused on proximal TCR-coupled signaling pathways that regulate IL-2 transcription. Expression of the IL-2 gene in T cells normally requires the coordinated binding of the transcription factors NFAT, AP-1, and NF-{kappa}B to cis-elements in the IL-2 promoter (22). NFAT and NF-{kappa}B are regulated by the steady-state [Ca2+]i. However, both the efficiency and specificity of transcription factor activation is also modulated by dynamic [Ca2+]i changes (oscillations) in T cells (23). NF-{kappa}B activity is further controlled by TCR-coupled protein kinase C (PKC) activity (24, 25, 26) and CD28-coupled Akt activity (27), while the Fos/Jun dimer AP-1 relies to some extent on all three of these pathways (28, 29, 30, 31). Despite the established role for Ca2+ signaling in IL-2 transcription by primary lymphocytes, previous studies have suggested that [Ca2+]i is unaffected (14, 32, 33) or elevated (34) in anergic T cell clones. Therefore, we re-examined this question using single-cell methods to determine whether the dynamics of Ca2+ signaling are altered in primary anergic CD4+ T cells. As previously demonstrated (35), TCR engagement induced a rapid increase in [Ca2+]i in individual naive CD4+ T cells that reached a peak concentration (290 ± 29 nM) within ~2 min and decayed to a sustained (>10 min) steady-state level (137 ± 2 nM) (Fig. 1A). In effector CD4+ T cells, the peak [Ca2+]i elevation was slightly diminished (202 ± 42 nM); however, the sustained [Ca2+]i was not significantly different from that of the naive cells (Fig. 1B). In contrast, anergic CD4+ T cells exhibited a significantly smaller initial (129 ± 19 nM) and sustained (100 ± 4 nM) elevation in [Ca2+]i as compared with naive and effector CD4+ T cells (Fig. 1C), and the [Ca2+]i in anergic cells generally returned to basal levels within 10 min. Interestingly, previous studies that defined the dynamics of [Ca2+]i signaling required to activate the IL-2 promoter (23) would suggest that the relatively small elevations in [Ca2+]i exhibited by primary anergic CD4+ T cells in our model (Fig. 1D) are insufficient to activate transcription factors required for IL-2 production.



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  		FIGURE 1. Defect in TCR-coupled [Ca2+]i elevation in anergic CD4+ T cells. Naive (A), effector (B), and anergic (C) CD4+ T cells were subjected to co-cross-linking of CD3 and CD4, and [Ca2+]i was measured kinetically in individual CD4+ T cells (thin traces). The data shown are from one representative experiment. The thick trace in each plot represents the average of all the individual traces. D, Mean peak [Ca2+]i amplitude (± SEM) in naive, effector, and anergic cells from six experiments. E, Mean peak Tg (1 µM) induced calcium elevations in naive, effector, and anergic CD4+ T cells (± SEM).

 
The data above suggest that primary anergic CD4+ T cells inefficiently couple Ca2+ signaling pathways to the TCR. However, it is also possible that these results are caused by a more generalized problem with [Ca2+]i homeostasis in these cells. To test whether the diminished Ca2+ signaling in the anergic cells is caused by down-modulation of distal calcium regulatory elements, we tested the integrity of receptor-distal Ca2+ signaling components. To do this, we used the sarco(endo)plasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin (Tg) to mimic the effects of inositol 1,4,5-triphosphate (IP3), the physiological mediator of TCR-induced Ca2+ store release. Intracellular calcium store depletion by either IP3 or Tg triggers calcium release-activated calcium channels that are responsible for TCR-mediated Ca2+ influx. The use of Tg in this way maximally activates calcium release-activated calcium channels, and therefore triggers a maximal physiological Ca2+ response. Tg induced comparable elevations of intracellular Ca2+ in naive, effector, and anergized CD4+ T cells (Fig. 1E). The response of anergic CD4+ T cells to Tg was quantitatively comparable to that in naive cells and slightly increased as compared with effector cell responses (Fig. 1E). These data confirm that the primary anergic CD4+ T cells in our model are viable, that distal Ca2+ signaling pathways are intact in these cells, and that the defect in [Ca2+]i mobilization in these cells is caused by a more proximal signaling defect.

A second signal transduction pathway crucial for IL-2 transcription is the p21ras oncoprotein (Ras)/MAPK cascade (36). Previous studies have shown that anergized CD4+ T cell clones exhibit reduced MAPK activation in response to TCR engagement (11, 12, 13, 14). Therefore, we tested whether primary CD4+ T cells anergized by costimulatory blockade are likewise defective in coupling MAPK to the TCR. Whereas primary effector CD4+ T cells showed significant phosphorylation of ERK in response to TCR cross-linking (Fig. 2, lanes 1–4), primary anergic CD4+ T cells exhibited little or no ERK phosphorylation in response to this same stimulus (Fig. 2, lanes 5–8). Together with the observed inability to evoke significant Ca2+ signals (Fig. 1), this defect in TCR-coupled MAPK activation may explain the failure of primary anergic T cells to assemble the multiple transcriptional complexes required for efficient IL-2 synthesis.



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  		FIGURE 2. Defect in TCR-coupled ERK phosphorylation in anergic CD4+ T cells. Effector (lanes 1–4) and anergic (lanes 5–8) CD4+ T cells were subjected to co-cross-linking of CD3 and CD4 for the indicated time periods, and lysates were assessed for phosphorylated (upper panel) or total (lower panel) ERK by immunoblotting. One representative experiment of three is shown.

 
Defective integrin avidity modulation in tolerant T cells

The data above show that primary CD4+ T cells anergized by costimulatory blockade exhibit concomitant defects in at least two separate signal transduction pathways that are required for trans activation of the IL-2 gene. In addition to regulating IL-2 transcription, both intracellular Ca2+ (37, 38, 39) and Ras/MAPK (40, 41, 42) signaling have been shown to regulate the induction of integrin binding avidity and thereby regulate formation of an "immunological synapse" between T cells and Ag-bearing APCs (3), and interactions between T cells and adhesion molecules (ICAM, VCAM) expressed by vascular endothelium and in the extracellular matrix (4). Integrins expressed on the surface of naive T cells exist in an inactive state characterized by a very low avidity for integrin ligands (43). As a result, naive cells cannot bind to integrin ligands and migrate from the bloodstream into nonlymphoid tissues in vivo. Conversely, previously activated T cells can traffic into sites of inflammation, because the integrins expressed on the surface of these cells exist in an active state (44). Consequently, we next investigated whether a reduced capacity to activate integrin binding may also contribute to the hyporesponsiveness exhibited by anergic T cells.

Consistent with the paradigm described above, naive CD4+ T cells failed to adhere efficiently to the integrin ligand FN (Fig. 3A). However, primary effector cells did adhere efficiently to FN (Fig. 3A), and the majority of this binding could be inhibited by Abs against the {alpha}4 chain of the {alpha}4{beta}1 integrin VLA-4 (data not shown). Unlike effector cells, anergic CD4+ T cells did not bind efficiently to FN (Fig. 3A). Furthermore, the reduced adhesion of anergic CD4+ T cells corresponded to inefficient transmigration in response to FN as compared with effector CD4+ T cells in an in vitro assay of integrin-dependent migration (Fig. 3B). Both effector and anergic CD4+ T cells express high levels of the major integrins LFA-1 (Fig. 3C, top) and VLA-4 (Fig. 3C, bottom). Therefore, the relative inability of anergic cells to adhere to integrin ligands is not simply because of a lack of integrin expression.



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  		FIGURE 3. Anergic CD4+ T cells exhibit reduced integrin-mediated adhesion and FN-dependent migration. A, Effector CD4+ T cells (n = 6), anergic CD4+ T cells (n = 6), or unstimulated lymph node CD4+ T cells (naive; n = 5) were assessed for their capacity to bind to FN. Data from multiple experiments are shown plotted as the ratio of bound CD4+ cells to the total input CD4+ cells. B, Effector and anergic CD4+ T cells were assessed for their capacity to transmigrate through 5 µm filters coated with FN. The ratio of transmigrated CD4+ cells to the total input CD4+ cells for one representative experiment of three is shown. Addition of Abs against VLA-4 reduced transmigration by 50–75% in these assays (data not shown). C, Expression of the {beta}2 chain of LFA-1 and the {alpha}4 chain of VLA-4 on effector (solid black lines) vs anergic (solid gray lines) CD4+ T cells. Isotype control staining for each cell population is depicted by dotted lines. D, Signaling through the Ag receptor does not up-regulate adhesion of anergic CD4+ T cells to FN. Naive, effector, and anergic CD4+ T cells were subjected to medium (open bars), TCR cross-linking (hatched bars), or PMA stimulation (black bars) for 10 min before assessment of FN binding as in A. The mean ± SEM of four experiments is shown.

 
When a naive T cell is activated through the TCR, its capacity to adhere to integrin ligands is increased dramatically via a process termed avidity modulation (45, 46), which involves reorganization of the actin cytoskeleton and clustering of integrin receptors at the cell surface (47). However, the "inside-out" signaling events that mediate avidity modulation are not completely understood. To test whether TCR-coupled signaling pathways are capable of inducing integrin avidity modulation and reversion of the FN binding defect in anergic CD4+ T cells, we cross-linked the TCR on naive, effector, and anergic CD4+ T cells immediately before assessing FN binding capacity. As expected, signals through the TCR induced an ~6-fold increase in FN adhesion by naive CD4+ T cells (Fig. 3D, open vs hatched bars) but did not significantly affect the already high binding efficiency of effector cells. In contrast, CD3 cross-linking did not increase the avidity of anergic CD4+ T cells for FN. However, we did find that the diacylglycerol mimetic PMA, which bypasses proximal TCR-coupled signaling events to induce integrin avidity modulation in naive CD4+ T cells (45), restored FN binding by anergic cells (Fig. 3D, black bars). Thus, signaling pathways that control integrin avidity modulation are intact in anergic CD4+ T cells but are uncoupled from the TCR.

Defective PLC{gamma}-1 activation in tolerant T cells

However, the simplest explanation for the numerous distinct functional defects we observe in anergic CD4+ T cells is that they all reflect a single biochemical defect at a branchpoint that links intracellular Ca2+ mobilization, MAPK activation, IL-2 production, and integrin avidity modulation to the TCR. A prime candidate for such a nexus is the TCR-coupled enzyme PLC{gamma}-1. PLC{gamma}-1 hydrolyzes phosphatidyl inositol bisphosphate into the potent secondary messengers IP3 and diacylgycerol (48). IP3 induces the release of Ca2+ from intracellular stores (49), while diacylgycerol (and Ca2+) activates PKC and the Ras/Rap1 guanyl-releasing protein RasGRP (50, 51). These factors couple the MAPK cascade to the TCR and synergize with [Ca2+]i to activate several transcription factors required for IL-2 production. Finally, intracellular Ca2+, PKC, Ras and Rap-1 are all defined mediators of {beta}1 and {beta}2 integrin avidity modulation (37, 38, 39, 40, 41, 42), and PLC{gamma}-1 has been shown to be necessary for integrin activation in response to platelet-derived growth factor and epidermal growth factor in fibroblasts (52). Consequently, a defect in PLC{gamma}-1 activation could explain the constellation of downstream defects that we and others have identified in anergic CD4+ T cells.

PLC{gamma}-1 is tyrosine phosphorylated upon engagement of the TCR (53), and phosphorylation of PLC{gamma}-1 at tyrosine 783 is required for its functional activity (54, 55). To examine the extent of TCR-coupled PLC{gamma}-1 activation in effector vs anergic CD4+ T cells, we measured the phosphorylation of PLC{gamma}-1 in response to TCR engagement. Co-cross-linking of CD3 and CD4 on naive T cells using Ab-coated beads resulted, as expected, in strong phosphorylation of PLC{gamma}-1 on Tyr783, as measured by immunoblotting with a phospho-specific Ab (Fig. 4A). Whereas effector CD4+ T cells also showed strong and sustained phosphorylation of PLC{gamma}-1 following TCR engagement (Fig. 4, B, lanes 1–4, and C, black bars), TCR-mediated PLC{gamma}-1 phosphorylation in anergic CD4+ T cells was delayed, and at peak it reached a level only 30% of that in the effector cells (Fig. 4, B, lanes 5–8, and C, hatched bars). These data suggest that PLC{gamma}-1 is not coupled properly to the TCR in anergic cells. Importantly, the diacylglycerol analog PMA, which can reverse the inability of anergic T cell clones to produce IL-2 by directly activating signaling pathways immediately downstream of PLC{gamma}-1 (32), restored the FN binding capacity (Fig. 3D) and IL-2 production (data not shown) of primary anergic CD4+ T cells in our system. Thus, the defect responsible for failed integrin activation and IL-2 synthesis in anergic cells involves the generation of diacylglycerol.



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  		FIGURE 4. Defect in TCR-coupled PLC{gamma}-1 phosphorylation in anergic CD4+ T cells. A, Naive T cells were subjected to co-cross-linking of CD3 and CD4 for the indicated time periods, and lysates were assessed for Tyr783-phosphorylated PLC{gamma}-1 by immunoblotting. B, Effector (lanes 1–4) and anergic (lanes 5–8) CD4+ T cells were subjected to co-cross-linking of CD3 and CD4 for the indicated time periods, and lysates were assessed for Tyr783-phosphorylated (upper panel) or total (lower panel) PLC{gamma}-1 by immunoblotting. C, Quantification of phosphorylated PLC{gamma}-1 in the immunoblot shown in B. Band densities are plotted in arbitrary units where the density of the p-PLC{gamma}-1 signal at time zero is set to 1. One representative experiment of three is shown.

 
PLC{gamma}-1 regulates integrin avidity modulation in naive T cells

Given the stable defect in PLC{gamma}-1 activation, calcium signaling, and FN binding in anergic T cells, coupled with the ability of PMA to reverse defects in FN binding and IL-2 production by anergic cells, we asked whether PLC{gamma}-1 activation is central and sufficient to mediate integrin avidity modulation in naive T cells. To directly examine the role of PLC{gamma}-1 in integrin avidity modulation by the TCR, we used the PLC-specific inhibitor U73122. As shown in Fig. 5, TCR stimulation induced a significant increase in FN binding by naive CD4+ T cells that was completely inhibited by U731222, but not by the inactive analog U73433. Importantly, under conditions of PLC inhibition, we found that reconstitution of distal signaling pathways with PMA (10 ng/ml) and ionomycin (50 nM) restored FN binding capacity of naive cells to a level similar to that of untreated naive cells. Thus, PLC{gamma}-1 activation by the TCR is central to {beta} integrin avidity modulation in CD4+ T cells, and defective PLC{gamma}-1 activation in anergic cells can fully explain the defects exhibited by these tolerant cells.



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  		FIGURE 5. PLC{gamma} activity is required for TCR-mediated integrin adhesion. Inhibition of Ag receptor (CD3) mediated increases in FN binding by naive CD4+ T cells by the PLC inhibitor U73122 (1 µM), but not by the structurally similar but inactive analog U73433 (1 µM). In the presence of U73122, PMA (10 ng/ml) alone partially restored PLC-dependent FN binding, whereas the combination of PMA (10 ng/ml) and ionomycin (50 nM) resulted in a level of binding that was not significantly different from the level of CD3-induced binding in the absence of inhibitors. The mean ± SEM of three similar experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified major defects in multiple TCR-mediated signaling processes in primary anergic CD4+ T cells, including PLC{gamma}-1 phosphorylation, MAPK phosphorylation, intracellular Ca2+ mobilization, and integrin avidity modulation. The failure of anergic cells to adhere and migrate in response to integrin ligands, or to modulate their binding avidity in response to signals from the Ag receptor, has remarkable implications regarding their ability to participate in immune responses in vivo. These results suggest that T cells rendered anergic are compromised not only in their ability to proliferate, survive, and produce proinflammatory cytokines, but also in their capacity to interact productively with APC and to migrate into sites of inflammation. Despite high levels of LFA-1 expression, anergic CD4+ T cells do exhibit an ~50% decrease in staining compared with effector cells. Whereas it is formally possible that this decrease in {beta}2 integrin expression could contribute to reduced integrin binding activity, these levels are sufficient to mediate efficient binding following PMA and ionomycin-induced activation. Thus, it seems likely that the binding defect in anergic cells reflects an uncoupling of integrins from activation by inside out signaling pathways.

We also found that resting primary anergic CD4+ T cells maintain a basal intracellular Ca2+ level similar to that of normal naive T cells, but exhibit a strikingly limited capacity to elevate intracellular Ca2+ in response to TCR engagement. This observation contrasts with several previous reports that found no defect in intracellular Ca2+ mobilization in anergized T cell clones (14, 32, 33). We believe these disparate results may reflect fundamental differences in how intracellular Ca2+ signaling is regulated in primary T lymphocytes vs long-term T cell clones or lines. Our findings are also significant in the context of two previous studies that, in contrast to our data, noted an elevated steady-state level of intracellular Ca2+ in anergized lymphocytes. In one study, which relied on bulk measurements of [Ca2+]i, Gajewski et al. (34) foundthat a T cell clone rendered anergic by chronic stimulation on plate-bound anti-CD3 exhibited an elevated steady-state level of intracellular Ca2+ and was unable to further elevate [Ca2+]i upon restimulation through the TCR. A subsequent study by Healy et al. (56) revealed a similar elevated steady-state [Ca2+]i in anergic B lymphocytes generated by chronic Ag stimulation. However, using a single-cell approach similar to the one we use in this study, these investigators went on to demonstrate that asynchronous Ca2+ oscillations in individual, tolerant B cells were responsible for the increase in the mean steady-state [Ca2+]i, despite the fact that the oscillations decayed to resting levels between spikes. Moreover, following a period of Ag withdrawal, the Ca2+ oscillations in these tolerized B cells ceased, and the mean [Ca2+]i decayed to a level no different from that in naive cells. These results suggest that the apparent elevated basal [Ca2+]i in the T cell clones studied by Gajewski et al., as well as the inability to evoke any further change in [Ca2+]i in these cells, was most likely caused by a state of active, asynchronous Ca2+ oscillations within the population as a whole. These examples also emphasize that [Ca2+]i measurements based upon bulk cell populations cannot distinguish the complex [Ca2+]i dynamics and differential Ca2+ signaling patterns that may exist in normal vs anergic lymphocytes.

In these studies we have also accumulated significant evidence for the role of differential coupling of PLC{gamma}-1 to the TCR in the disparate function of the processes described above in effector vs anergic T cells. We show that TCR-mediated signaling for integrin activation in normal primary T lymphocytes is dependent upon PLC{gamma}-1 activity, and this integrin avidity modulation is defective in primary anergic T cells. However, our data indicate that calcium stores are normally filled, and that store-operated Ca2+ influx pathways can be normally activated in anergic T cells consistent with the interpretation that PLC{gamma}-1 activation, but not defects in more distal elements that regulate calcium mobilization, is the central defect in anergic cells. Further evidence in support of our interpretation is our observation that the combination of a diacylglycerol analog and calcium ionophore fully restore both integrin binding activity and IL-2 production by primary anergic T cells. Finally, we demonstrate that TCR-mediated phosphorylation of PLC{gamma}-1 on a specific tyrosine residue required for its activation is defective in primary anergic T cells. These data together localize the biochemical defect in primary anergic T cells to the generation of diacylglycerol and IP3, an activity mediated by PLC{gamma}-1, and establish a direct link between the products of PLC{gamma}-1 enzyme activity and the multiple downstream functions that are defective in primary anergic cells.

PLC{gamma}-1 activation is normally linked to the TCR through a scaffold comprised of Lck, {zeta}-associated protein-70, Src homology domain 2-containing leukocyte protein of 76 kDa, linker for activation of T cells, Vav, and the Tec family kinase Itk (57). Consequently, a defect in any of these upstream factors could explain the diminished phosphorylation of PLC{gamma}-1 in anergic CD4+ T cells. In fact, defective Lck and ZAP-70 activation has been documented in anergic T cell clones (15, 34, 58, 59). Our results do not identify which of these possible biochemical defect(s) is responsible for reduced PLC{gamma}-1 activation in tolerant CD4+ T cells, and a strict demonstration of a causal relationship between the PLC{gamma}-1 gene product itself and the anergic state awaits a genetic strategy to restore active PLC{gamma}-1 in the anergic T cells. However, our data do show that the immediate products of the activated PLC{gamma}-1 enzyme can influence the maintenance of the anergic state and that PLC{gamma}-1 serves as a single proximal regulator of Ca2+ signaling, MAPK activation, IL-2 production, and integrin avidity modulation in primary CD4+ T cells.


   Footnotes
 
1 B.D.F. was supported by National Institutes of Health Grants HD-01056 and AI-39678; L.A.T. was supported by National Institutes of Health Grants AI-37691, AI-41521, and AI-43620, the American Heart Association, and Juvenile Diabetes Foundation; A.D.W. was supported by National Institutes of Health Grants DK02771 and AI054643. Back

2 A.D.W., and Q.-H.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Bruce D. Freedman, Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Suite 389 E, Old Vet Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: bruce{at}vet.upenn.edu; or Dr. Andrew D. Wells at the current address: Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 802 Abramson Research Center, 3516 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: adwells{at}mail.med.upenn.edu Back

4 Abbreviations used in this paper: ERK, extracellular-regulated kinase; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; IP3, inositol 1,4,5-triphosphate; VLA, very late Ag; PKC, protein kinase C; Tg, thapsigargin; [Ca2+]i, intracellular Ca2+ concentration; Ras, p21ras oncoprotein; FN, fibronectin. Back

Received for publication November 20, 2002. Accepted for publication February 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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