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The Journal of Immunology, 2000, 164: 6105-6112.
Copyright © 2000 by The American Association of Immunologists

Altered Composition of the Immunological Synapse in an Anergic, Age-Dependent Memory T Cell Subset1

Michael D. Eisenbraun*,{ddagger}, Ami Tamir{dagger} and Richard A. Miller2,*,{dagger},{ddagger},§

* Cellular and Molecular Biology Graduate Program and {dagger} Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI 48109; and {ddagger} Institute of Gerontology and § Geriatrics Center and Veterans Affairs Medical Center, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In young mice, memory CD4 T lymphocytes with high P-glycoprotein activity (P-gphigh) are unresponsive to TCR stimulation in vitro but can be activated by PMA plus ionomycin. The proportion of these hyporesponsive cells increases considerably with age. The earliest events in T cell activation were studied in P-gphigh and P-gplow CD4 memory cells at the single-cell level using confocal immunofluorescence methods. Recruitment of both linker for activation of T cells (LAT) and protein kinase C-{theta} to the immunological synapse, i.e., the site of T cell interaction with stimulator cells, was greatly impaired in P-gphigh cells from both young and old mice. Translocation of NF-AT to the nucleus, CD69 expression, and proliferative capacity were also diminished to a similar extent in P-gphigh cells under the same activation conditions. In contrast, movement of c-Cbl to the synapse region occurred in a high proportion of CD4 memory T cells regardless of P-gp subset or age. Moreover, although P-gplow cells frequently recruited both c-Cbl and LAT to the APC synapse, cells in the less responsive P-gphigh subset frequently relocated c-Cbl, but not LAT, to the interface region. In some systems, c-Cbl can act as a negative regulator of receptor-dependent tyrosine kinases, and alterations of c-Cbl to LAT ratios in the P-gphigh subset may thus contribute to the hyporesponsiveness of this age-dependent, anergic memory cell population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T lymphocytes expressing the P-glycoprotein (P-gp)3 transporter are normally present in both humans and mice and can be identified based on their ability to extrude the fluorescent dye rhodamine-123 (R-123) (1, 2). Although P-gp is known to confer multidrug resistance to tumor cells (3), its physiological function in normal T cells remains unresolved (4). In mice, the proportion of T cells with high P-gp activity (P-gphigh phenotype), increases significantly with age (5), particularly within the set of CD4 memory (CD4 M) cells marked by high level expression of CD44 and low expression of CD45RB and CD62L. Moreover, P-gphigh CD4 M T cells exhibit characteristics that are consistent with those of anergic T cells (6), including poor proliferation and low production of cytokines including IL-2, IL-4, IL-5, IL-10, and (in young mice) IFN-{gamma} (7, 8). P-gphigh CD4 T cells also show diminished ability to produce intracellular Ca2+ signals in responses to anti-CD3 stimulation (9). The age-dependent accumulation of these hypofunctional P-gphigh CD4 M T cells could contribute to diminished protective immunity in old age, but the molecular basis for their poor responses is not yet clear. Studies of knockout mice (4) have shown that P-gp itself neither promotes nor impairs production of IL-2, IL-4, IL-5, IL-10, or IFN-{gamma}, T cell proliferation, or generation and function of cytotoxic effector cells. However, the ability of PMA plus ionomycin to induce proliferation of P-gphigh T cells (8) suggests that signaling from the TCR might be inhibited in this cell subset.

TCR engagement initiates a series of well-studied biochemical events (10), including the redistribution of membrane, cytoskeletal, and cytosolic proteins (11). One example is that T cell activation leads to the accumulation of glycolipid-enriched microdomains (GEMs) at the "immunological synapse," i.e., the point of contact between the T cell membrane and the Ag-bearing APC or antigenic surface (12, 13). GEM aggregation is believed to be important for concentrating and organizing components involved in TCR signaling and simultaneously excluding those with negative regulatory roles (14, 15, 16, 17). The adapter protein linker for activation of T cells (LAT), which through its constitutive palmitoylation is confined primarily to GEMs, is one of the components brought to the site of TCR engagement by this relocalization process (18, 19). LAT plays an essential role in the transmission of signals from the TCR complex to other key signaling proteins (20, 21, 22), including phospholipase C{gamma}1, phosphatidylinositol 3-kinase, and Grb2, which then in combination initiate downstream events such as the activation of Ras, influx of Ca2+, and translocation of NF-AT to the nucleus.

The recruitment of the T cell-specific form of protein kinase C (PKC), i.e., PKC-{theta}, from the cytosol to the immunological synapse is thought to play a critical role in this activation process, in that translocation of PKC-{theta} in TCR-transgene-bearing T cells is stimulated by APCs bearing agonist peptides, but not by APCs bearing closely related antagonist or nonagonist peptides (23). Recruitment of PKC-{theta} to the membrane is necessary for its activation and involvement in signaling (24). Our group has previously used immunofluorescent methods to show, at the single-cell level, a decline with age in the proportion of CD4+ and CD8+ T cells that mobilize PKC-{theta} to the T cell/APC interface in responses triggered by anti-CD3 hybridoma cells (25).

The accumulation of inhibitory molecules at the T cell/APC interface is also likely to influence the outcome of the interaction. The proto-oncoprotein c-Cbl, which can associate with LAT and the protein tyrosine kinases (PTKs) Fyn, Syk, and ZAP-70 during activation, has recently been shown to act as a negative regulator of PTK activity (reviewed in Ref. 26) and to enhance ubiquitination of activated receptors after stimulation (27, 28, 29). Additionally, c-Cbl was reported to be constitutively associated with Fyn and to have a role in attenuating signals from the TCR in anergic human T cell clones (30). Differences in responsiveness between the P-gphigh and P-gplow CD4 M T cell subsets might thus in principle reflect alterations in the balance of positive and negative regulators at the immunological synapse. Therefore, we have used immunofluorescence methods to examine activation-induced relocalization of LAT, PKC-{theta}, and c-Cbl in P-gp subsets of the CD4 M population freshly isolated from young and old mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Five-month-old and 15- to 18-mo-old CB6F1 male mice were obtained from the National Institute on Aging-funded colonies at Charles River Laboratories (Wilmington, MA) and Harlan (Indianapolis, IN). Mice were housed under specific pathogen-free conditions at the University of Michigan for at least 1 mo before use, until animals were between the ages of 6 and 8 mo ("young") or between 18 and 22 mo ("old"). Only mice without any visible signs of splenomegaly, skin lesions, or tumors were used in experiments.

Reagents

Abs. Primary detection of intracellular proteins was performed using rabbit polyclonal antisera specific for mouse CD3{epsilon} (Dako, Carpinteria, CA), LAT (Upstate Biotechnology, Waltham, MA), or PKC-{theta} (Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal antiserum to c-Cbl (Santa Cruz Biotechnology), or a mouse mAb specific for NF-AT1 (Santa Cruz Biotechnology). Secondary detection was performed using either a Texas Red-conjugated goat anti-mouse (Jackson ImmunoResearch Labs, West Grove, PA), goat anti-rabbit (Jackson ImmunoResearch Labs), or donkey anti-goat polyclonal (Polysciences, Warrington, PA) Abs. For c-Cbl and LAT double staining, a fluorescein-conjugated donkey anti-rabbit polyclonal Ab (Jackson ImmunoResearch Labs) was used in combination with the Texas Red-conjugated anti-goat polyclonal Ab for secondary detection. F-actin was stained using Alexa 488-labeled phalloidin (Molecular Probes, Eugene, OR).

Cell lines. B cell hybridomas expressing monoclonal hamster Abs specific for murine CD3{epsilon} (clone 145-2C11 (2C11)) or DNP (clone UC8) (American Type Culture Collection, Manassas, VA) were maintained in DMEM with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids, and 5 x 10-5 M 2-ME at 37°C and 10% CO2. Flow cytometric analyses confirmed that both types of hybridoma cells not only express membrane-bound Ig but also display significant surface levels of the costimulatory ligands B7 and ICAM-1 (M.D.E. and R.A.M., unpublished data).

Cell preparation

CD4 M T lymphocytes were purified from splenic cell suspensions by negative immunomagnetic depletion as previously described, with one minor modification (8). In brief, B cell-depleted splenic T cells were incubated with optimal concentrations of anti-CD62L (MEL-14) and anti-CD8 (53-6.7) purified ascites for 40 min on ice. Cells were then washed once and depleted of CD62Lhigh (naive) and CD8+ T cells using BioMag goat anti-rat IgG (PerSeptive Biosystems, Framingham, MA) according to the supplier’s instructions. The purity of recovered CD3+ T cells was >90% CD44high and CD4+ as assessed by flow cytometry. Cells were then immediately stained with 5 µM R-123 as previously described (5) and were sorted into R-123dim (P-gphigh) and R-123bright (P-gplow) subsets using an Epics Elite ESP flow sorter (Coulter, Miami, FL). Recovered cells were washed twice with RP-FC (RPMI 1640, 10% FCS, 5 x 10-5 M 2-ME, 2 mM L-glutamine, and antibiotics), resuspended at 4 x 106 cells/ml in RP-FC, and then incubated at 37°C for 15–30 min.

For each T cell stimulation experiment, 2C11 and UC8 cells in log-phase growth were harvested, washed two times with prewarmed (37°C) HBSS, and then resuspended at 3 x 106 cells/ml in prewarmed RP-FC before use. When used for CD69 expression analyses or proliferation assays, hybridoma cells were additionally gamma-irradiated with ~4000 rad in a cesium irradiator before being combined with sorted T cells.

Slide preparation and microscopy

P-gphigh or P-gplow CD4 M T cells were combined with equal volumes of 2C11 or UC8 hybridoma cells to achieve a 4:3 cell ratio, respectively, using cells resuspended at the concentrations described above. In some cases, where indicated, ionomycin was also added to a final concentration of 1 µM. Cell mixtures were incubated at 37°C for 15 min and then gently resuspended and spread (50 µl/slide) onto prewarmed poly-L-lysine-coated slides (Sigma, St. Louis, MO). Slides were incubated for another 15 min at 37°C to promote cell attachment, fixed in freshly prepared 3.7% formaldehyde/PBS for 20 min, and then finally washed three times in PBS. Slides designated for immunofluorescence detection of LAT, PKC-{theta}, c-Cbl, or NF-AT were further permeabilized with 0.1% (or 0.2%, for NF-AT slides only) Triton X-100/PBS for 5 min before three washes in PBS. All slides were placed in blocking solution (1% BSA/0.1% NaN3/PBS) and stored at 4°C for at least 24 h. Slides were then stained with appropriate primary Abs diluted in blocking solution (anti-CD3{epsilon}, anti-c-Cbl, anti-PKC-{theta}, and anti-NF-AT, each used at 10 µg/ml; anti-LAT used at 20 µg/ml; phalloidin used at 1:1000) for 2 h at room temperature in a humidified chamber. With the exception of phalloidin-stained samples, slides were washed three times in PBS and a final time in blocking solution and then were counterstained for 90 min in the dark as above using appropriate secondary Abs diluted 1:200 in blocking solution. All slides were washed four times in PBS before mounting coverslips with ProLong antifade reagent (Molecular Probes). After drying overnight at room temperature, slides were coded for blind analysis and stored at 4°C protected from light. Single- and two-color confocal analyses were performed at x100 magnification on a Nikon Diaphot microscope equipped with a Bio-Rad MRC 600 confocal laser imaging system (Bio-Rad, Hercules, CA). Single-color fluorescence analysis (NF-AT only) was performed at x100 magnification on a Zeiss Axioskop microscope equipped with a low-light CCD camera (MicroImage Video Systems, Boyertown, PA). Randomly selected T cell-hybridoma cell conjugates were analyzed by confocal or fluorescence microscopy only if the following criteria were first met under phase contrast light microscopy conditions: 1) tightly formed cell-to-cell contact, 2) T cell in contact with only one hybridoma cell, 3) both cells in the same relative z-axis plane, and 4) no significant overlap of cell membranes at contact interface. One hundred acceptable conjugates on each slide (50 on double-stained slides) were visualized by confocal or fluorescence microscopy and scored as either positive or negative for substantial translocation and accumulation of the studied protein(s) at the interface membrane or in the nucleus (NF-AT only) of each selected T cell. For studies of protein clustering at the membrane synapse, T cells were scored as positive if 75% or more of the fluorescent signal was confined to the ~25% of the membrane juxtaposed to the stimulator cell. For NF-AT, cells were scored as positive if they exhibited at least a 50% decline in cytoplasmic fluorescence, compared with unconjugated cells, with a reciprocal increase in nuclear fluorescence.

Analysis of CD69 expression

P-gphigh or P-gplow CD4 M T cells (2.5 x 104/well) were transferred alone or with equal numbers of 2C11 or UC8 cells into 96-well round-bottom plates and then were cultured for 6 h at 37°C and 5% CO2. Cells were collected, washed once with cold PBS, and then stained with a combination of PE-labeled anti-CD4 (PharMingen, San Diego, CA) and either anti-CD69 or isotype-matched FITC-labeled Abs (PharMingen). Stained and fixed cells were analyzed on a FACScan with Lysis II software (Becton Dickinson, Mountain View, CA). A total of 10,000 events were acquired for each sample using a light scatter gate to exclude dead cells and hybridoma cells. Percentages of CD69+ cells were determined for CD4-gated memory T cells using WinMDI analysis software (Joseph Trotter, Scripps Institute, San Diego, CA).

Proliferation assay

P-gphigh or P-gplow CD4 M T cells (5 x 104/well) were transferred alone or with equal numbers of 2C11, UC8, or cells into 96-well flat-bottom plates and then were cultured for 72 h at 37°C and 5% CO2. Cells were pulsed with 0.5 µCi/well [3H]thymidine during the last 6 h of culture. Plates were then harvested and processed for liquid scintillation counting to determine levels of thymidine incorporation.

Statistical analyses

Statistical comparisons between results for each test group from the various protein redistribution analyses and cellular response assays were performed by two-way ANOVA, using age and P-gp subset as the two independent factors before a posthoc least significant difference test for planned comparisons. All values reported in the text represent mean averages ± SEM. The n values indicate the number of individual mice tested except where noted otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CD69 and proliferative capacity are reflective of LAT, PKC-{theta}, and NF-AT redistribution

Previous work (7, 8) has shown that P-gphigh cells in the memory CD4 subset proliferate poorly in vitro, and produce only low levels of cytokines, in responses to plate-immobilized anti-CD3 Abs when compared with cells of the P-gplow subset. To see whether the responses to live anti-CD3{epsilon} hybridoma (2C11) cells were also subset-dependent, we examined the capacity of stimulated P-gphigh and P-gplow CD4 M T cells from young and old mice to progress through early and late phases of the first cell cycle after coculture with 2C11 cells. Surface expression of CD69 at 6 h was used to assess the fraction of T cells that were able to advance into an early state of activation (31). The data revealed (Fig. 1GoA) that a majority of P-gplow cells from young and old mice expressed CD69 in response to 2C11 stimulation. The proportion of P-gphigh cells expressing CD69 was more than 2-fold lower (p < 0.001 at both ages). UC8 hybridoma cells, used as a negative control, only elicited CD69 expression on fewer than 5% of cells from either subset or age group in these conditions (not shown). Proliferation induced by 2C11 hybridoma cells was also evaluated at 72 h of culture (Fig. 1GoB) and revealed that P-gplow cells from young and old mice responded more strongly (p < 0.001) than P-gphigh cells, which is consistent with previous studies (7, 8). In addition, P-gplow cells from old mice were significantly (p < 0.001) less responsive than P-gplow cells from young donors; this age effect, which cannot be attributed to the change with age in the number of CD44high M T cells, is also consistent with previous work using purified anti-CD3 Abs (7, 8).



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  		FIGURE 1. CD69 expression and proliferation responses of P-gphigh and P-gplow CD4 M T cells in response to 2C11 cells. P-gp subsets purified from CD4 M T cells from young or old mice were combined with equal numbers of gamma-irradiated 2C11 hybridoma cells to measure induced CD69 expression and proliferative capacity. A, Surface expression of CD69 was analyzed by flow cytometry after a 6-h culture period. Results presented for each cell type are represented as mean percentages ± SEM of CD69+ cells for n = 2 independent experiments. Fewer than 5% of T cells incubated alone or with UC8 cells (negative control; anti-DNP) were CD69+ (data not shown). *, Significant (p < 0.05) difference between subsets at each age. B, Proliferative capacity evaluated at the end of a 72-h culture period by [3H]thymidine incorporation. Results presented as mean cpm ± SEM (n = 4 independent experiments). Average counts for 2C11 or T cells alone, or for T cells cocultured with UC8 control stimulator cells, were <1000 cpm (data not shown). Statistical comparisons between groups were performed by two-way ANOVA using a posthoc least significant difference test. Symbols indicate a significant difference between subsets in each age group (**, p < 0.001) or between young and old P-gplow cells.

 
Activation-induced redistribution of LAT and PKC-{theta} is impaired in P-gphigh CD4 M T cells and declines with age

To test the idea that the hyporesponsiveness of the P-gphigh subset of CD4 M T cells might reflect alterations in the early stages of signal transduction, we used immunofluorescence assays to follow the redistribution of LAT and PKC-{theta}, two molecules previously shown to play critical roles in T cell activation (20, 21, 22, 32), in T cells allowed to form conjugates in coculture with 2C11 stimulators. Each T cell/2C11 conjugate was scored as positive or negative for LAT (or, in parallel experiments, PKC-{theta}) relocalization to the synapse region, using coded slides to conceal age and subset information from the investigator. Fig. 2Go, A and B, shows examples of positive and negative relocalization responses for LAT and for PKC-{theta}, respectively. In previous work (25) and in other pilot studies not shown, the proportion of conjugates showing relocalization of both proteins increased between 15 and 30 min and was constant thereafter at least through 60 min after the initiation of the coculture. Previous work from our laboratory also found no effect of age on the proportion of CD4+ cells that formed conjugates with 2C11 stimulators (25) and, although not formally quantitated in the current study, similar conjugate numbers were observed regardless of age and P-gp phenotype for CD4 M cells. One hundred conjugates were scored for each subset from each donor mouse. The results for four to six mice tested in this way are shown in Fig. 2Go (C and D). Control samples were analyzed in which species-matched normal antiserum was substituted for the specific anti-LAT or anti-PKC-{theta} polyclonal Abs to estimate the "false positive" rate; these background rates are indicated by dashed lines on each graph. We found that LAT clustered at the synapse in about half of the P-gplow cells from young mice after conjugation with 2C11 cells. LAT relocalization also occurred in 40% of P-gplow cells from old mice, a proportion that is slightly, but significantly (p < 0.01), lower than that seen for cells from young donors. In contrast, fewer than 22% of P-gphigh cells from young or old donors exhibited LAT relocalization; this is significantly (p < 0.001) lower than in the corresponding P-gplow subset at each age. Indeed, the P-gplow subset from old donors is indistinguishable from the false-positive level estimated using nonspecific Ab in place of the anti-LAT reagent. In control experiments using UC8 cells in place of 2C11, very few conjugates were present, and among these the frequency of cells showing LAT relocalization was indistinguishable from background levels determined for control-stained samples (data not shown).



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  		FIGURE 2. Single-cell analysis of LAT and PKC-{theta} redistribution in stimulated P-gphigh and P-gplow CD4 M T cells. Subsets of CD4 M T cells with high (P-gphigh) or low (P-gplow) R-123 efflux activity from young or old CB6F1 mice were combined for 30 min with 2C11 cells and then stained by immunofluorescence to localize LAT or PKC-{theta} using confocal microscopy. A and B, A set of six images (a-f) is shown for each indicated protein. The three left panels in each set (a, c, and e) show Nomarski images of representative cell conjugates (T cells are the smaller cells in each image). The three right panels in each set (b, d, and f) show the corresponding confocal fluorescence images. In each set, b and d show examples in which the indicated protein had accumulated at the interface between the T cell and the 2C11 stimulator, whereas panel f shows an example in which redistribution of the protein had not occurred. Each confocal image is representative of the focal z-axis center plane of the conjugated T cell. C and D, The proportion of conjugated T cells showing relocalization of LAT (C) or PKC-{theta} (D) to the T cell/APC interface, shown as mean percentage ± SEM for n = 4–6 mice examined for each subset. One hundred conjugates were scored on coded slides for each subset for each mouse. Dashed lines represent the mean percentage of "false positives" estimated using species-matched normal antisera in place of the primary detection polyclonal Abs. Symbols indicate significant differences between subsets in mice of the same age (*, p < 0.001) or between age groups for the same P-gp subsets ({dagger}, p < 0.005).

 
P-gp subsets of CD4 M T cells were also examined for localization of PKC-{theta}, whose activation is thought to depend on interaction between phosphorylated LAT and phospholipase C{gamma}1 for production of diacylglycerol (21). PKC-{theta} is a key component in the TCR signaling cascade that leads to an up-regulation of AP-1 (32) and has been shown by immunofluorescent methods to translocate to the immune synapse of TCR-transgenic T cells only in response to agonist peptides and not in response to antagonist peptides (23). Fig. 2GoB shows examples of positive and negative responses for PKC-{theta} in T cells conjugated with 2C11 stimulators. The results of a series of such experiments, shown in Fig. 2GoD, were very similar to those seen for LAT. The fraction of cells showing PKC-{theta} localization was over 2-fold lower in the P-gphigh subset than in the P-gplow subset for each age group (p < 0.001). In addition, comparisons between different donor ages revealed a significant decline with age in PKC-{theta} relocalization for both the P-gphigh and P-gplow subsets (p < 0.001). The accumulation with age of P-gphigh cells within the CD4 memory population, together with the age-dependent decline in PKC-{theta} relocalization within each P-gp subset, thus both contribute to the decline with age in PKC-{theta} redistribution previously noted in CD4 cells of aged mice (25).

To see whether impaired redistribution of LAT and PKC-{theta} in P-gphigh CD4 M T cells was the result of upstream alterations in TCR movement or in F-actin assembly at the immunological synapse, we examined the frequency of 2C11-induced CD3{epsilon} clustering and actin capping in cells from each P-gp subset by confocal microscopy. Fig. 3Go, A and B, shows examples of positive and negative CD3{epsilon} clustering (detected using a polyclonal Ab that recognizes the cytoplasmic domain of CD3{epsilon}) and F-actin capping (detected using an actin-specific, fluorochrome-labeled bicyclic peptide (33)). 2C11 stimulators induced accumulation of CD3{epsilon} at the synapse region in the majority of T cells (76–79%), regardless of the P-gp subset or donor age examined (Fig. 3GoC). We note that this dramatic redistribution of CD3{epsilon} is not seen in conjugates formed between TCR-transgenic T cells and peptide-bearing lymphoma cell stimulators (Ref. 16); and A. Tamir, M. D. Eisenbraun, G. G. Garcia, and R. A. Miller, manuscript in preparation), but it presumably reflects the high affinity of the 2C11 Ab for the CD3{epsilon} molecule (34). The F-actin analysis (Fig. 3GoD) gave similar results for young mice, with 70–80% of cells in either subset showing F-actin clustering in the synapse region. F-actin clustering in the subsets from aged donors was slightly but significantly lower compared with that of young T cells (p < 0.05 for both subsets), but again there was no difference between P-gplow and P-gphigh cells. These data thus indicate that the differences between P-gphigh and P-gplow cells in LAT and PKC-{theta} relocalization cannot be attributed to alterations in either CD3{epsilon} translocation to or F-actin assembly at the synapse region. Similarly, we found no effects of age on the total amount of LAT per CD4 cell (A. Tamir, M. D. Eisenbraun, G. G. Garcia, and R. A. Miller, manuscript in preparation) or on the levels or functional activity of PKC-{theta} per CD4 or CD8 cell (25). In addition, flow cytometric analyses of both P-gphigh and P-gplow subsets of CD4 M T cells failed to show any discernable differences in the surface expression levels of TCR-ß, CD2, CD28, or LFA-1 that might have contributed to variations in T cell activation (data not shown).



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  		FIGURE 3. Single-cell analysis of CD3{epsilon} and F-actin localization in stimulated P-gphigh and P-gplow CD4 M T cells. Conjugates of CD4 M T cell subsets and 2C11 cells were prepared and analyzed by immunofluorescence confocal microscopy as described in Fig. 2Go. A and B, A set of six images (a-f) is shown for each indicated protein. The three left panels in each set (a, c, and e) show Nomarski images, and the right panels (b, d, and f) show the corresponding confocal fluorescence images. Panels b and d show examples in which the indicated protein had accumulated at the interface, whereas panel f shows an example in which redistribution of the protein had not occurred. C and D, The proportion (mean ± SEM; n = 4–5 mice; 100 counted cells per sample) of conjugated T cells showing a concentration of CD3{epsilon} (C) or F-actin (D) at the interface. Dashed lines represent the mean percentage of "false positives" estimated using species-matched normal antisera in place of the anti-CD3{epsilon} rabbit polyclonal Ab. *, Significant difference (p < 0.05) between young and old mice for each subset.

 
Nuclear translocation of NF-AT is also impaired in P-gphigh CD4 M T cells

Aging leads to a decline in the proportion of CD4 T cells that exhibit intracellular changes in Ca2+ concentration (35, 36), reflecting at least in part the relative unresponsiveness of cells in the P-gphigh pool (9). Increases in cytosolic Ca2+ after TCR stimulation appear to depend upon LAT (21, 22). This mobilization of Ca2+ triggers a number of other important signaling events, including the calcineurin-dependent activation of NF-AT and its subsequent translocation to the nucleus (37). Therefore, we examined the ability of P-gphigh and P-gplow CD4 M T cells from young and old mice to relocalize NF-AT from cytoplasm to nucleus after stimulation either by 2C11 cells or, as a positive control, by the receptor-independent ionophore ionomycin. Fig. 4GoA shows examples of positive and negative responses using the NF-AT mAb for immunofluorescence microscopy. For the P-gplow cells, stimulation by 2C11 cells (Fig. 4GoB) induced NF-AT migration to the nucleus in 52% (young mice) or 43% (old mice), but in much smaller proportions (17% and 14%, respectively; p < 0.001) of P-gphigh cells from donors of either age. There was no significant effect of donor age on NF-AT translocation in either subset. When 1 µM ionomycin was included along with 2C11 cells, NF-AT translocation was induced in a large majority of cells (~80%) regardless of the P-gp subset or donor age (Fig. 4GoC). This suggested that NF-AT translocation was impaired in P-gphigh cells, not because of changes in NF-AT or in proteins required for response to Ca2+ influx, but because of defects in the events required to initiate calcium influx.



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  		FIGURE 4. Single-cell analysis of NF-AT translocation in stimulated P-gphigh and P-gplow CD4 M T cells. Conjugates of CD4 M T cell subsets and 2C11 cells were analyzed by immunofluorescence microscopy to detect T cells with nuclear-localized NF-AT. A, Representative results. Three of the panels (a, b, and c) show examples of conjugates (T cells are indicated by "T" in each image) in which NF-AT has translocated to the T cell nucleus (indicated by arrow) after activation by 2C11 cells alone (a and b) or in the presence of added ionomycin (c). Panel d shows an example in which NF-AT translocation was not induced. B and C, Proportion of conjugated T cells of each type showing NF-AT localization to the nucleus in response to 2C11 activation alone (B) or 2C11 cells plus ionomycin (C); other details are as in Figs. 2Go and 3Go. *, Significant (p < 0.001) differences between subsets for each age.

 
Interface clustering of c-Cbl occurs more frequently than LAT in P-gphigh CD4 M T cells

c-Cbl, like LAT, interacts with TCR-associated proteins in activated (26) and, in some cases, resting T cells (30) and is thought to regulate T cell activation by inhibiting tyrosine kinases and possibly also by serving as an E3 ubiquitin ligase that promotes the internalization and degradation of TCRs (26). To investigate whether c-Cbl plays a role in impairing responses of P-gphigh CD4 memory T cells to TCR stimulation, we analyzed its cellular localization in P-gphigh and P-gplow CD4 M T cells from young and old mice after activation with 2C11 cells. Fig. 5GoA shows examples of positive and negative responses in the c-Cbl system, and Fig. 5GoB summarizes the results of P-gp subset comparisons from four young and five old mice tested in parallel. A high proportion of P-gplow cells from young and old mice (52 ± 9.8%, n = 4, and 45 ± 6.6%, n = 5, respectively) exhibited localization of c-Cbl to the synapse area after contact with 2C11 stimulators. Interestingly, c-Cbl clustering was also triggered in more than half of the P-gphigh cells (58 and 67% for young and old donors, respectively), a striking contrast to the much lower levels of LAT and PKC-{theta} relocalization determined for this same population (Fig. 2Go). For old animals the percentage of P-gphigh cells with clustered c-Cbl was significantly higher than for the P-gplow subset (p = 0.03).



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  		FIGURE 5. Single-cell analysis of c-Cbl localization in stimulated P-gphigh and P-gplow CD4 M T cells. Conjugates of CD4 M T cell subsets and 2C11 cells were analyzed as described above. A, Representative images. Panels a, c, and e show Nomarski images, and panels b, d, and f show the corresponding confocal fluorescence images. Panels b and d show examples in which c-Cbl had accumulated at the synapse, and panel f shows an example where c-Cbl remains evenly distributed in the T cell. B, The proportion of conjugated T cells showing c-Cbl relocalization to the synapse; details are as in Fig. 2Go. *, Significant difference (p < 0.05) between P-gphigh and P-gplow cells from old mice.

 
Studies of anergic T cell clones (30) have suggested that c-Cbl may play a role in inhibiting their activation, consistent with the idea that preferential relocalization of c-Cbl in P-gphigh memory T cells might contribute to the hyporesponsiveness of this subset. To explore this possibility further, we simultaneously stained slides with Abs to c-Cbl and LAT. Fig. 6Go shows examples of T cells in which contact with 2C11 led to relocalization of LAT, c-Cbl, both, or neither, and Table IGo summarizes the proportions of cells in these categories determined in a series of comparisons using CD4 M subsets from three young and four old animals. The results of this analysis (Table IGo) revealed that both donor age and memory T cell subset influenced c-Cbl and LAT localization after activation. In P-gplow cells from mice of either age group, the majority of those cells showing translocation of either LAT or c-Cbl showed translocation of both molecules in the same cell. In contrast, P-gphigh cells showing relocalization of c-Cbl alone were about three times more frequent than those in which c-Cbl clustering occurred together with LAT migration. These subset differences were statistically significant (p < 0.001) for cells of both donor ages. In addition there were two effects of age per se: 1) a significant, >3-fold decline with age in the proportion of both P-gphigh and P-gplow cells that showed relocalization of LAT alone (p < 0.005), and 2) a corresponding increase with age in the proportion of cells in each subset that showed rearrangement of both c-Cbl and LAT, which reached statistical significance for the P-gphigh subset only (p = 0.05). These results are consistent with a model that attributes the anergic state of the P-gphigh CD4 M cell population to increased concentrations of c-Cbl at the immunological synapse.



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  		FIGURE 6. Two-color visualization of c-Cbl and LAT localization in stimulated P-gphigh and P-gplow CD4 M T cells: representative images. Left panels in each set show LAT localization detected by a FITC-conjugated secondary Ab, whereas right panels show c-Cbl localization detected by a Texas Red-conjugated secondary Ab. Each pair of images shows an example of one of the four possible localization patterns: a, LAT but not c-Cbl localized at the 2C11 interface (indicated by arrow); b, c-Cbl but not LAT localized at the interface; c, both proteins colocalized at the interface; and d, neither protein localized at the interface.

 

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  		Table I. Two-color analysis of c-Cbl and LAT localization in P-gphigh and P-gplow CD4 M T cells1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
P-gphigh CD4 M T cells resemble anergic T cells in their poor proliferative and cytokine responses (6). We have previously shown that T cells from P-gp-deficient mice (mdr1a knockout strain) are functionally indistinguishable from controls in tests of cytokine production, proliferation, and cytotoxicity (4), suggesting that the hyporesponsiveness of P-gphigh cells may reflect a multifaceted effect of cell differentiation rather than some effect of P-gp per se. Consistent with this model, other studies have shown that P-gphigh CD4 M T cells express elevated levels of the Ca2+ binding protein sorcin (9) and decreased levels of the P-gp-related TAP1 transporter (38). Based on the previous observation that stimulation with PMA + ionomycin could restore proliferative capacity to P-gphigh CD4 M T cells (8), the current study was undertaken to address the possibility that impaired TCR signaling might contribute to the unresponsiveness of this memory cell subset.

We found that activation-induced migration and clustering of LAT and PKC-{theta}, two key proteins that act early in TCR-initiated signaling pathways, occurred far less frequently in P-gphigh than in P-gplow CD4 M T cells. P-gphigh cells did initiate responses to the anti-CD3 stimulus, though, as indicated by relocalization of both CD3{epsilon} and F-actin cap formation. Translocation of NF-AT from the cytoplasm to the nucleus (a downstream event that relies on LAT (19, 22)) was also found to occur at a much lower frequency in CD4 M T cells with a P-gphigh phenotype. However, translocation of NF-AT could readily be induced in P-gphigh cells when ionomycin was added, suggesting that the defect in NF-AT movement was secondary to upstream signaling events in these cells. Our data suggest that intracellular signals are not generated or transmitted efficiently from the TCR complex to early acting components like LAT and PKC-{theta} in P-gphigh CD4 M T cells.

Several possibilities that might account for this lack of signaling include decreased activity/expression of PTKs involved in initiating signals from the TCR (i.e., Fyn, Lck, or ZAP-70), increased phosphatase activity, or negative regulation by one or more signaling components. A recent report from Boussiotis et al. (30) has shown that c-Cbl is constitutively associated with Fyn in anergic human T cells and that it prevented IL-2 synthesis and proliferation after TCR stimulation through its involvement in activating Rap1. c-Cbl is also known to become associated with PTKs and LAT during productive T cell activation, where it appears to act both as an inhibitor of PTK function (39, 40) and as an E3 ubiquitin ligase (27, 28, 29) to terminate responses to signaling. Our immunofluorescent analysis showed that c-Cbl, like LAT and PKC-{theta}, is preferentially concentrated in the synapse region in stimulated P-gplow CD4 M T cells. However, the P-gphigh subset included many cells in which the interface region contained c-Cbl alone, rather than both c-Cbl together with LAT. These findings suggest a model in which c-Cbl may be constitutively associated with the TCR in P-gphigh CD4 M T cells and acts to inhibit early steps in the activation process, including the recruitment of LAT into the synapse region. Future experiments using biochemical methods will help to ascertain whether c-Cbl in P-gphigh CD4 M T cells is constitutively bound to TCR complexes or if it is triggered to translocate to the APC interface by a mechanism that also inhibits the recruitment of LAT.

Our argument that these changes are relevant to age-related alterations in T cell activation is not based on contrasts between young and old cells, which are fairly small, but instead on the change with age in the relative proportions of P-gphigh and P-gplow memory T cells. This paper, like others published previously (7, 8, 9), documents large differences (often 4-fold or more, as in Fig. 1GoB) between P-gphigh and P-gplow T cells. Because aging leads to a 3-fold increase in the proportion of CD4 memory T cells in the P-gphigh subset (5), the poor function of P-gphigh CD4 memory cells could be a significant factor in age-related decline in CD4 memory cell responses. Because the cellular composition of the immune system varies with age, studies that use unfractionated populations of T cells, or even of pools enriched for CD4+ or CD8+ cells, from young and old animals to analyze the effects of age on the biochemistry of TCR signaling cannot discriminate the effects of aging on cell responsiveness from the effects of altered subset composition. In the present system, separation of CD4+ T cells into functionally distinct subpopulations of memory cells provided the opportunity both to characterize activation defects in P-gphigh CD4 M T cells and also to identify age-related alterations in the activation properties of P-gphigh and P-gplow CD4 M T cells that occur independent of changes in their proportions.

Comparisons of the P-gplow subset of CD4 M T cells from young and old mice (the subset that is preferentially responsive in tests of proliferation and cytokine production) revealed a significant decline with age in the proportion of these cells that could undergo LAT and PKC-{theta} redistribution. Assembly of F-actin, on the other hand, decreased slightly but to a similar extent in both P-gplow and P-gphigh cells with age. With regards to both LAT and PKC-{theta}, this age effect seems not to result from changes in protein expression, because Western blot analyses detected a change in neither LAT (A. Tamir, M. D. Eisenbraun, G. G. Garcia, and R. A. Miller, manuscript in preparation) nor PKC-{theta} (25) protein levels with age in CD4+ T cells. These results were also consistent with previous studies that have reported decreases with age in the activation-induced redistribution of PKC-{theta} in CD4+ T cells (25) and polymerization of F-actin in total T cells (41). The redistribution of CD3{epsilon} seen in 2C11 conjugates (but not in conjugates of TCR transgenic T cells to peptide-loaded APC (16)) seems unlikely to be typical of immune responses to natural Ags in vivo but was in any case insensitive to age or subset in this system.

The data on c-Cbl relocalization provide a possible clue to the basis for hyporesponsiveness in the P-gphigh subset, suggesting that preferential association of c-Cbl with one or more of the components of the TCR complex might interfere with recruitment of LAT (and possibly PKC-{theta} or other key elements) into the synapse. Such an effect might involve steric hindrance by competition for binding sites potentially accessible to c-Cbl or other coupling factors or might involve c-Cbl-dependent activation of enzymatic machinery. Although it is not known whether c-Cbl protein levels differ between P-gp subsets, Western blot experiments have shown that c-Cbl levels do not change with age in CD4+ cells (G. G. Garcia and R. A. Miller, unpublished data), making it unlikely that an alteration in c-Cbl levels can account for the observed shift in the ratio of c-Cbl to LAT at the synapses of conjugated P-gphigh cells. The immunofluorescence approach has the potential to allow molecular analysis of aging effects on signal transduction even in T cell subsets which, like the P-gphigh and P-gplow subsets of the CD4 M population, are difficult to obtain in quantities sufficient for standard biochemical analyses.


   Acknowledgments
 
We thank Dr. Jill Macoska (University of Michigan) for generously providing the Zeiss fluorescent microscope and digital camera system and Dr. Gonzalo Garcia (University of Michigan) for his advice and critical review of this work.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AG09801 and AG08808. M.D.E. was also supported by the University of Michigan Institute of Gerontology from National Institutes of Health Training Grants AI07413 and AG00114. Back

2 Address correspondence and reprint requests to Dr. Richard A. Miller, 5316 Cancer Center and Geriatrics Center Building, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940. Back

3 Abbreviations used in this paper: P-gp, P-glycoprotein; R-123, rhodamine-123; CD4 M, CD4 memory; GEM, glycolipid-enriched microdomain; LAT, linker for activation of T cells; PKC, protein kinase C; PTK, protein tyrosine kinase; 2C11, clone 145-2C11. Back

Received for publication December 15, 1999. Accepted for publication March 23, 2000.


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