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Subunit and Syk Kinase Replace the CD3
-Chain and ZAP-70 Kinase in the TCR Signaling Complex of Human Effector CD4 T Cells1
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* Departments of Surgery, and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201;
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Abstract |
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-related FcR
signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3
, but not CD3. The TCR/CD3/FcR complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases. |
Introduction |
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and CD3 subunits, followed by recruitment and activation of the ZAP-70 tyrosine kinase, mobilization, and activation of linker/adapter molecules and mitogen-activated protein kinases, culminating in nuclear gene transcription (for reviews, see Refs. 1 and 2). Following this initial activation, prolonged stimulation results in differentiation to effector T cells, the central mediators of the adaptive immune response that can also contribute to pathologies in autoimmune diseases, transplant rejection, and chronic viral infections (3, 4, 5). Because most studies of TCR signaling have focused on T cell lines, with fewer studies in primary T cells, it is not known how or whether TCR-mediated signaling changes as a result of activation and differentiation to an effector state.
We recently reported that human effector CD4 T cells exhibit alterations in the phosphorylation and expression of TCR-coupled signaling intermediates. In contrast to resting primary CD4 T cells, effector CD4 T cells exhibit increases in overall intracellular tyrosine phosphorylation, yet paradoxically they demonstrate a profound reduction in the expression of the TCR-coupled CD3 signaling subunit (6). This loss of a critical signaling intermediate in the presence of tyrosine phosphorylation events suggested that TCR-coupled signals may be transduced through an alternate signaling subunit in effector T cells. We considered the FcR signaling subunit, originally identified as a subunit of the high affinity IgE receptor (FcRI) and expressed in mouse NK-like T cells (7, 8) and intraepithelial 
T lymphocytes (9), as a likely candidate for replacing the signaling function of CD3 for two reasons. First, both CD3 and FcR are structurally and functionally homologous. They both contain intracytoplasmic immunoreceptor tyrosine-based activation motifs, associate with the TCR, and can functionally complement each other in vivo (7, 10, 11). Second, up-regulation of FcR expression is observed in peripheral T cells in patients with systemic lupus erythematosus (SLE) 3 (12, 13) and T cells that infiltrate tumor sites (14), which both bear striking phenotypic and biochemical similarities to in vitro-generated human effector T cells, including up-regulation of activation markers and decreases in CD3 expression (6, 13, 14).
In this study, we tested the hypothesis that FcR replaces the missing CD3-chain in the TCR/CD3 complex of human effector CD4 T cells. Indeed, we found a dramatic up-regulation of FcR protein in effector CD4 T cells that forms part of a new TCR/CD3/FcR signaling complex independent of CD3. In contrast to primary T cells, where the ZAP-70 kinase is recruited to TCR/CD3, effector cells recruit and phosphorylate the related p72syk (Syk) kinase to the TCR/CD3 complex, indicating differential signaling. These results demonstrate profound alterations in the molecular composition of the TCR signaling complex in differentiated effector cells and suggest that distinct signals are transduced through the TCR in naive and effector T cells.
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Materials and Methods |
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Heparinized peripheral venous blood was obtained from consenting healthy adult volunteers according to a protocol approved by the health use committee of Walter Reed Army Medical Center. Leukopacs from healthy volunteers were also purchased from BRT Laboratories (Baltimore, MD).
Antibodies
Anti-CD3 Ab (clone OKT3) was obtained from Ortho Biotech (Raritan, NJ), and IgM anti-CD3 (clone 2Ad2A2) was provided by Dr. R. Siliciano (The Johns Hopkins University, Baltimore, MD). Anti-FcR Ab directed against the peptide sequence CKHEKPPQ of FcR-chain was provided by Dr. J.-P. Kinet (Beth Israel Deaconess Medical Center, Boston, MA) and was also purchased from Upstate Biotechnology (Lake Placid, NY). Anti-actin Ab (clone I-19), anti-CD3 Ab (clone M-20), and anti-Syk Abs (clone 4D10 and agarose-conjugated clone LR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-TCRC
Ab (clone Jovi-1) was purchased from Ancell Corp. (Bayport, MN). Anti-ZAP-70 antisera was provided by Dr. R. Wange (National Institutes of Aging, Baltimore, MD). FITC-conjugated anti-CD14 (clone M5E2), anti-HLA-A,B,C (clone G46-2.6), FITC-coupled anti-CD16 (clone 3G8), and FITC-conjugated anti-CD25 (clone M-A251) were purchased from BD PharMingen (San Diego, CA). FITC-conjugated anti-CD3 (clone UCHT1) was purchased from Sigma-Aldrich (St. Louis, MO). Isotype control Abs and FITC- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary Abs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Isolation of CD4 T cell subsets and in vitro generation of effector CD4 T cells
CD4 T cells were purified from total PBMC using a CD4 T cell isolation kit (Miltenyi Biotec, Auburn, CA), followed by negative selection over a MACS magnetic column using the AutoMACS (Miltenyi Biotec) as previously described (6, 15), resulting in >95% purity. Naive (CD45RA) and memory (CD45RO) CD4 T cells were purified using anti-CD45RA- and anti-CD45RO-coupled MACS magnetic beads (Miltenyi Biotec), respectively, as previously described (6). For APCs, purified monocytes obtained by either elutriation or positive selection with anti-CD14 magnetic microbeads (Miltenyi Biotec) using AutoMACS were treated with 100 µg/ml mitomycin C (Roche, Indianapolis, IN) as previously described (6). Effector CD4 T cells were generated in vitro as described previously (6). Briefly, 106 CD4 T cells were cultured with 2 µg/ml soluble anti-CD3 (OKT3) Ab plus 2 x 106 autologous, mitomycin C-treated monocytes for 72–120 h at 37°C in complete RPMI medium (6). The resultant effector T cells were purified by centrifugation through Ficoll (LSM, ICN/Cappel, San Diego, CA), and residual monocytes were depleted using anti-CD14-coupled magnetic Dynabeads (Dynal Biotech, Lake Success, NY), resulting in 99% pure effector CD4 T cells (6).
Mixed lymphocyte reactions
Allogeneic effector CD4 T cells were generated by coculture of peripheral blood CD4 T cells (106/well) as responders with 2 x 106 mitomycin C-treated allogeneic monocytes in 24-well plates in complete RPMI medium for 5–7 days at 37°C. Effector cells were centrifuged through Ficoll, washed, and used directly for FACS analysis. For biochemical analysis, residual monocytes were depleted using anti-CD14 magnetic beads as described above, yielding 99% pure alloantigen-activated CD4 T cells.
Western blotting and immunoprecipitation experiments
For Western blot analyses, 107 cells in 100 µl of RPMI were untreated or activated by anti-CD3 IgM Ab for 2 min at 37°C, pelleted, and lysed in cold 1% Nonidet P-40 lysis buffer with protease/phosphatase inhibitors as previously described (6). Cells were lysed at 107 cells/35 µl of lysis buffer for Western blotting and 2.5–3.5 x 107 cells/150–200 µl of lysis buffer for immunoprecipitations. Lysates were resolved by 4–20% SDS-PAGE, gels were transferred to nitrocellulose, and blots were incubated with anti-CD3, anti-CD3, anti-ZAP-70, anti-FcR, and anti-actin Abs, followed by HRP-conjugated goat anti-mouse (Bio-Rad) or goat anti-rabbit (clone RG-96; Sigma-Aldrich) IgG as previously described (6). Bands were detected using ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) and were revealed with Hyperfilm ECL (Amersham Pharmacia Biotech). For anti-CD3, anti-ZAP-70, and anti-Syk immunoprecipitations, Nonidet P-40 cell lysates isolated as described above were precleared with 2 µg of rabbit IgG preadsorbed to protein A/Sepharose or protein A/Sepharose alone (Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°C, followed by Ab bound to protein A/Sepharose for 2 h at 4°C. Immunoprecipitates were washed and resuspended in sample buffer containing DTT before loading onto gels. For anti-TCRC immunoprecipitations, cells were lysed in digitonin lysis buffer consisting of 1% (w/v) digitonin, 150 mM NaCl, 10 mM HEPES (pH 7.5), 1 mM EDTA, 0.1% sodium azide, and 0.12% Triton X-100, freshly reconstituted with 10 µg/ml leupeptin, 100 µg/ml aprotinin, and 1 mM PMSF.
Flow cytometry
For analysis of cell surface phenotype, cells were washed and resuspended in stain buffer (PBS, 1% FCS, and 0.05% sodium azide) containing fluorescein-conjugated Abs for 30 min at 4°C. For intracellular staining, cells were fixed in 0.75% paraformaldehyde in PBS at 4°C, washed, and resuspended in permeabilization buffer (RPMI, 0.05% saponin, 10% FCS, 10 mM glycine, and 1 M HEPES, pH 7.4). Nonspecific binding was blocked with human IgG (Fc-block, Miltenyi Biotec). Cells were stained in permeabilization buffer, fixed with 1% paraformaldehyde, and analyzed using a FACSCalibur (BD Biosciences, San Jose, CA) with CellQuest software.
Confocal microscopy
Primary and effector CD4 T cells were adhered to poly-L-lysine-coated slides (Sigma-Aldrich) for 1 h on ice. For CD3 capping, cells were incubated with IgM anti-CD3 Ab for 10 min at 37°C, followed by fixation in 3.7% paraformaldehyde. Cells were washed three times with PBS (pH 7.4), blocked with Fc-block (Miltenyi Biotec), and surface-labeled with anti-CD3 and anti-TCR Ab, anti-TCR and anti-HLA Ab, or anti-CD3 and FITC-conjugated anti-CD16 Ab, followed by FITC- and TRITC-coupled secondary Abs. For CD3 and FcR cocapping analysis, cells surface-labeled with anti-CD3 and FITC-conjugated anti-mouse Ab were treated with permeabilization buffer (as described above), blocked with Fc block, and incubated on ice with anti-FcR Ab or isotype-matched control rabbit IgG. After washing, secondary anti-rabbit TRITC was added for 30 min. Stained cells were washed four times with PBS, air-dried, and mounted using Gel/Mount (Biomedia, Foster City, CA). Samples were analyzed with a laser scanning confocal fluorescence microscope (Axiovert 100 M scope; Zeiss, New York, NY) with 510 SP1 software (LSM, Jena, Germany).
RT-PCR
Total RNA was isolated using an RNeasy minikit (Qiagen, Valencia, CA) from 5 x 106 primary and effector CD4 T lymphocytes. Using 400 ng of total RNA, cDNA was synthesized with AMV reverse transcriptase (Promega, Madison, WI) and PCR-amplified for 28 cycles using FcR-chain-, Lyn-, ZAP-70-, and -actin-specific primers. Primers for PCR were synthesized by Sigma-Genosys (The Woodlands, TX): ZAP-70, 5'-GACGTGGCCATCAAGGTGCTGAAGCAG-3' and 5'-GCGCTGCTCCACGGTCAGGAAGTCG-3'; -actin, 5'-CATGGGTCAGAAGGATTCCT-3' and 5'-AGCTGGTAGCTCTTCTCCA-3'; FcR, 5'-GCCTCAGCTCTGCTATATCCTGGA-3' and 5'-GTTCTCCCTTCCCATATTTTAGCTG-3'; and Lyn, 5'-AACTAATGCCGACGTGATGACCG-3' and 5'-GATTCATTGCAATGGTCTCTGAAAC-3'. PCR products were electrophoresed on 1.2% SeaKem agarose gel (FMC BioProducts, Rockland, ME) and visualized with ethidium bromide.
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Results |
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expression in human effector CD4 T cells
We examined whether the FcR signaling molecule was expressed in human effector T cells in the context of decreased expression of CD3, CD3, and surface TCR proteins previously identified (6). We initially assessed FcR protein expression in Western blots of lysates derived from primary CD4 T cells purified directly from human peripheral blood and human effector T cells generated by activating primary CD4 T cells with anti-CD3 Ab in the presence of autologous monocytes (anti-CD3/monocytes). We previously reported that these in vitro-activated CD4 T cells displayed all the functional and phenotypic attributes of effector T cells, including up-regulation of activation/differentiation markers and production of high levels of IFN- (6). As shown in Fig. 1A, while primary CD4 cells (lane 1) express substantial levels of CD3 and CD3 proteins and lack FcR chain, effector T cells (lane 2) express high levels of FcR and low levels of CD3 and CD3 proteins (rows 3 and 4). ZAP-70 and actin expression remains unaltered in both cell types, and they served as controls for equal loading (Fig. 1A, rows 1 and 2). As expected, peripheral blood monocytes express FcR (lane 2 vs lane 5).
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expression in differentiated effector T cells, we measured FcR protein levels in effector CD4 T cells that were further stimulated with anti-CD3 and autologous monocytes or were rested in medium alone for an additional 48 h (Fig. 1A, lanes 3 and 4). While CD3 and CD3 expression remains low in stimulated effector T cells (Fig. 1A, lane 3), the expression of both these proteins increases in rested effector T cells (lane 4), as previously shown (6). By contrast, FcR expression persists in stimulated and rested effector T cells, albeit at lower levels (Fig. 1A, lanes 3 and 4, bottom row).
To ensure that the presence of FcR in effector CD4 T cell lysates was not due to the small number (<1.5%) of contaminating accessory cells (6), we examined FcR expression in primary and effector CD4 T cells by intracellular staining with anti-FcR in conjunction with surface staining for the T cell marker CD3, followed by FACS analysis (Fig. 1B). Although resting CD3+ primary T cells do not express FcR, 73.2% of CD3+ effector T cells express FcR protein (Fig. 1B). As controls, CD14+ PBMC were found to express high levels of FcR (Fig. 1B, right panel). These results demonstrate that up-regulation of FcR protein expression occurs specifically in CD3+ effector T cells and is not due to minute levels of accessory cell contamination.
We asked whether up-regulation of FcR expression in effector T cells occurred at the transcriptional level. Using RT-PCR analysis, we assessed the pattern of FcR transcript expression in resting and activated T cells. Fig. 1C (middle row) shows that resting CD4 T cells and CD4 T cells activated by anti-CD3 Ab alone do not express FcR transcripts (lanes 1 and 2). However, stimulation with both anti-CD3 and anti-CD28 results in slight up-regulation of FcR transcripts (Fig. 1C, lane 3), although the protein product is not visible (data not shown). Substantial up-regulation of FcR transcripts only occurred in purified T cells activated with anti-CD3/monocytes (Fig. 1C, lane 4). Actin transcripts (top row) served as the internal control, and PCR amplification of the Lyn kinase whose expression is specific to monocytes with minimal expression in T cells (16) was included to monitor possible contamination by FcR+ monocytic accessory cells. While monocytes express significant levels of Lyn transcripts, both resting and activated T cell preparations exhibit minimal Lyn transcript expression, further confirming the purity of the primary and effector T cell preparations. These results demonstrate that FcR expression is up-regulated in effector CD4 T cells at the transcriptional level, and the highest level of FcR expression is observed in T cells activated with anti-CD3/monocytes, as also assessed by Western blotting (Fig. 1A and data not shown).
Expression of FcR by Ag-activated effector cells
As shown above, we found up-regulation of FcR expression in human effector T cells generated in response to anti-CD3 stimulation, a noncognate TCR stimulus. We asked whether FcR expression was likewise up-regulated in effector T cells generated following antigenic stimulation. We thus activated purified human CD4 T cells for 5–7 days with alloantigen provided by monocytes from an unrelated donor and analyzed the resultant activated cells by flow cytometry and Western immunoblotting (Fig. 2). Fig. 2A shows representative FACS analysis of alloantigen-activated T cells stained with fluorescent Abs directed against CD3, CD25, and intracellular FcR. When gated for CD3+ T cells, 
7% of alloantigen-stimulated cells express the CD25 activated/effector cell marker compared with 0% of the parent CD4 T cells (Fig. 2A). Of the 7% CD25+ effector cells, more than half (56% of CD25+ T cells; 3.7% of total T cells) also expressed FcR (Fig. 2A, lower right panel, upper right quadrant), with an additional 2.7% FcR+CD3+CD25- T cells present in this alloantigen-activated T cell pool (upper left quadrant), compared with the total lack of FcR+ resting CD4 T cells (Fig. 2A, lower left panel). We also detected FcR expression in alloantigen-activated CD4 T cells by Western blot (Fig. 2B, lane 2), at a level lower than the amount of FcR expressed by anti-CD3-generated effector cells (lane 3), most likely due to the higher proportion of CD25+ cells generated by anti-CD3 stimulation (6). These results collectively demonstrate that Ag activation of primary CD4 T cells results in substantial up-regulation of FcR expression and that anti-CD3 generated effector cells represent a relevant model to explore FcR expression and signaling.
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/CD3 association and recruitment to a new TCR signaling complex
Because we identified up-regulation of FcR in effector cells in the context of decreased CD3 expression, we hypothesized that, like CD3, the FcR subunit associates with the TCR/CD3 complex. We used both cellular and biochemical approaches to address this hypothesis. On the cellular level we examined the expression and colocalization of CD3, TCR, and FcR on primary and effector CD4 T cells by confocal microscopy (Fig. 3). We found two striking differences in the TCR/CD3 complex expressed by effector vs resting CD4 T cells. First, the pattern of surface CD3 and TCR expression on effector CD4 T cells was distributed in discrete clusters, compared with the more uniform surface expression of TCR and CD3 on resting CD4 T cells (Fig. 3, rows 1–3), although both TCR and CD3 co-cap and co-localize on CD3-stimulated primary and effector CD4 T cells (rows 1–3). Second, effector, and not resting, CD4 T cells express the FcR subunit that is localized to the surface and co-caps with CD3 (Fig. 3, rows 4–6). To ensure that co-capping of TCR and FcR with CD3 was specific for TCR-associated proteins, we included a control demonstrating that MHC class I molecules (HLA-A, -B, and -C) do not co-cap with CD3 and the TCR in our assay (Fig. 3, rows 7–9). These results indicate that the surface distribution and composition of the TCR/CD3 complex differ in effector vs resting primary CD4 T cells.
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was not associated with CD3 via the CD16 IgG-binding FcR that is known to associate with the FcR subunit on macrophages (17), we used confocal microscopy to detect a potential association between CD3 and CD16. We did not observe CD16 expression or CD3-CD16 colocalization in resting and effector CD4 T cells (Fig. 3, rows 10 and 11), consistent with the lack of surface FcR (e.g., CD16, CD32, CD89, and CD64) by FACS analysis of these cell types (data not shown). These controls establish that FcR associates specifically to CD3 in the TCR/CD3 complex and does not associate via an FcR intermediate.
On the biochemical level, we analyzed the expression and association of TCR, CD3, and FcR subunit in resting and effector CD4 T cells by specific immunoprecipitation. We immunoprecipitated anti-TCR from lysates derived from resting and effector CD4 T cells and asked whether CD3, CD3, and/or FcR coprecipitated. As shown in Fig. 4, while CD3 coprecipitated with the TCR in resting and effector T cells (with lower levels of CD3 detected in effector cells, consistent with Western blots of whole effector cell lysates; Fig. 1A), CD3 coprecipitated with the TCR only in resting CD4 T cells and not in effector CD4 T cells (lane 1 vs lanes 2 and 3). By contrast, FcR coprecipitated with the TCR only in effector T cells and not in resting CD4 T cells (Fig. 4, lanes 2 and 3 vs lane 1). Thus, both the confocal and biochemical analyses provide compelling evidence that the TCR/CD3/CD3 complex in resting CD4 T cells changes to a TCR/CD3/FcR complex in effector T cells.
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Given the dramatic change in the composition of the proximal TCR signaling complex in effector T cells, we asked whether this alteration had an effect on the recruitment and/or activation of proximal kinases to TCR/CD3. Because Syk kinase is associated with FcR signaling in mast cells (18), whereas ZAP-70 kinase is associated with CD3 signaling in T cells (19), we hypothesized that the replacement of CD3 with FcR in effector cells may likewise result in differential recruitment of ZAP-70 and/or Syk. Indeed, we found that in resting CD4 T cells, CD3 coprecipitates with the CD3 signaling subunit and ZAP-70 kinase, but not with the FcR subunit or Syk kinase (Fig. 5A, lanes 1 and 2; and Fig. 5B, lanes 1 and 2). By contrast, CD3 in effector T cells coprecipitates with the FcR signaling subunit and Syk tyrosine kinase (Fig. 5A, lanes 3 and 4), but not with ZAP-70 kinase (Fig. 5B, lanes 3 and 4).
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FcR and Syk expression are specific to the effector T cell subset
To determine whether up-regulation of FcR and Syk was a unique feature of the effector T cell subset or was stably maintained following T cell activation and differentiation, we compared the expression of TCR-coupled components in naive, effector, and memory CD4 T cell subsets. We purified naive (CD45RA) and memory (CD45RO) CD4 T cell subsets from peripheral blood as previously described (6) and compared the expression of Syk, FcR, and other CD3 components to that in effector cells by Western blotting of whole cell lysates (Fig. 6). By this analysis we found that only effector cells exhibit a striking up-regulation of Syk and FcR and concomitant down-regulation of CD3 and CD3 (Fig. 6, lane 2), whereas both naive and memory CD4 T cells express comparable levels of CD3 and CD3 proteins and low levels of Syk and do not express the FcR subunit (Fig. 6, lanes 1 and 3). These results indicate that the changes in TCR signaling components identified here are specific to activated/effector T cells.
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/CD3/ZAP-70 complex, effector CD4 T cells express and transduce signals through an alternate TCR signaling complex that excludes CD3 and ZAP-70 and contains CD3, the FcR subunit, and the proximal Syk kinase (Fig. 7). This TCR/CD3/FcR complex is suggested by our ability to coimmunoprecipitate the TCR, CD3, and FcR with either anti-TCR or anti-CD3 Abs, and the complete colocalization of CD3 with surface TCR. The preferential phosphorylation of Syk rather than ZAP-70 in effector cells is similar to the biased phosphorylation of Syk over ZAP-70 we previously identified in mouse memory vs naive CD4 T cells (20), suggesting an integral role for Syk in previously activated T cells. Our findings reveal a novel role for both FcR and Syk in effector T cell signaling and suggest distinct signaling mechanisms for initiation vs perpetuation of T cell-mediated immune responses from naive and effector T cells, respectively.
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subunit and Syk kinase are critical mediators of signaling through the high affinity IgE receptor (FcRI) that triggers degranulation of mast cells and basophils (21). FcR is also the primary signaling molecule of the IgG-binding Fc receptors, FcRI (22, 23) and FcRIII (17). We found FcR in human effector T cells to be associated with the TCR/CD3 complex and did not detect the expression of any of the three types of human FcR (CD16, CD32, and CD64; Fig. 3 and data not shown). Taken together, our results and those from previous studies of mast cell and macrophage FcR signaling indicate that both FcR and Syk serve as signaling molecules in human immune effector cells of the innate and adaptive arms of the immune system. As we did not detect either CD3 down-regulation or FcR up-regulation in mouse effector T cells (S. Krishnan and D. L. Farber, unpublished observations), this signaling alteration appears specific to human T cells.
The FcR subunit may play structural, signaling, and/or functional roles in human effector cells. On the structural level, FcR is less efficient than CD3 in stabilizing surface TCR expression, as FcR+/CD3- T cells from CD3-deficient mice exhibit lower surface TCR expression than CD3+ T cells from wild-type mice (10, 11, 24). Human effector cells likewise exhibit a substantially lower level of surface TCR expression (6), consistent with the presence of FcR rather than CD3 in the TCR/CD3 complex.
The signaling potential of FcR has been most extensively studied in the context of its role as the primary signaling molecule through FcRI in mast cells. FcR associates with the Syk kinase in mast cells (25), and downstream signaling through FcRI couples to an alternative intermediate for calcium mobilization, namely sphingosine kinase (26). Our findings that Syk likewise associates with the TCR/CD3/FcR complex in effector cells suggests that downstream signaling in human effector T cells may resemble downstream mast cell signaling through FcRI. In addition, FcR has been shown to couple to discrete membrane microdomains (rafts) after mast cell activation (27). In effector T cells, we have detected FcR expression (but not CD3) in the raft fraction (data not shown), suggesting that one role of FcR in T cells may be to maintain the TCR and associated signaling molecules in membrane rafts for optimal activation.
FcR and/or Syk kinase may act to promote the survival and/or other distinct functions in effector T cells. FcR signaling in human monocytes has been shown to increase levels of the survival proteins Bcl-2 and Bcl-xL (28), suggesting that FcR may promote the survival of effector T cells that are otherwise prone to apoptosis (29). Transfection studies in T cell lines suggest that in the absence of CD3 and/or ZAP-70, FcR and/or Syk appear to be less efficient in promoting IL-2 production. For example, FcR-expressing CD3- T cell line transfectants produce lower levels of IL-2 than CD3+ transfectants (10), and human ZAP-70-/- T cells that express Syk produce lower levels of IL-2 than ZAP-70+ T cells (30, 31). Consistent with these previous results, the physiological switch in TCR signaling from CD3/ZAP-70 in naive T cells to FcR/Syk in effector T cells identified here correlates with a shift in cytokine profile from predominantly IL-2 by naive T cells to effector cytokine production by differentiated effector cells (6, 32). Determining the functional role of the novel signaling components in effector T cells is an important issue to address for future studies.
Our findings that Syk and/or FcR are up-regulated specifically in activated/effector T cells generated by stimulation with anti-CD3 or Ag and are not in resting naive or memory cells strongly suggest that these molecules can serve as both novel indicators for immune activation and targets for manipulation of ongoing immune responses. Up-regulation of FcR expression in T cells has also been shown to occur in vitro, in IL-2 cultured T cells (33), and in vivo in circulating T cells from individuals with SLE (12). Similar to effector CD4 T cells, SLE T cells exhibit altered tyrosine phosphorylation and association of the FcR-chain with CD3 and Syk (12). We propose that T cell-associated FcR or Syk expression can serve as a biomarker to assess immune activation for predicting autoimmune flare-ups and transplant rejection and to monitor vaccine efficacy. Developing therapies that specifically target effector cells will enable immunosuppression in autoimmune diseases and transplants without global T cell paralysis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Donna L. Farber, Department of Surgery, University of Maryland School of Medicine, MSTF Building, Room 400, 685 West Baltimore Street, Baltimore, MD 21201. E-mail: dfarber{at}smail.umaryland.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication November 25, 2002. Accepted for publication February 10, 2003.
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Y.-T. Juang, L. Sumibcay, M. Tolnay, Y. Wang, V. C. Kyttaris, and G. C. Tsokos Elf-1 Binds to GGAA Elements on the FcR{gamma} Promoter and Represses Its Expression J. Immunol., October 1, 2007; 179(7): 4884 - 4889. [Abstract] [Full Text] [PDF]
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 K. Tenbrock, Y.-T. Juang, V. C. Kyttaris, and G. C. Tsokos Altered signal transduction in SLE T cells Rheumatology, October 1, 2007; 46(10): 1525 - 1530. [Abstract] [Full Text] [PDF]
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E. Leeansyah, B. D. Wines, S. M. Crowe, and A. Jaworowski The Mechanism Underlying Defective Fc{gamma} Receptor-Mediated Phagocytosis by HIV-1-Infected Human Monocyte-Derived Macrophages J. Immunol., January 15, 2007; 178(2): 1096 - 1104. [Abstract] [Full Text] [PDF]
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 E. Urich, I. Gutcher, M. Prinz, and B. Becher Autoantibody-mediated demyelination depends on complement activation but not activatory Fc-receptors PNAS, December 5, 2006; 103(49): 18697 - 18702. [Abstract] [Full Text] [PDF]
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C. W. Thomson, W. A. Teft, W. Chen, B. P.-L. Lee, J. Madrenas, and L. Zhang FcR{gamma} Presence in TCR Complex of Double-Negative T Cells Is Critical for Their Regulatory Function J. Immunol., August 15, 2006; 177(4): 2250 - 2257. [Abstract] [Full Text] [PDF]
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B. D. Wines, H. M. Trist, P. A. Ramsland, and P. M. Hogarth A Common Site of the Fc Receptor {gamma} Subunit Interacts with the Unrelated Immunoreceptors Fc{alpha}RI and Fc{epsilon}RI J. Biol. Chem., June 23, 2006; 281(25): 17108 - 17113. [Abstract] [Full Text] [PDF]
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S. Krishnan, J. G. Kiang, C. U. Fisher, M. P. Nambiar, H. T. Nguyen, V. C. Kyttaris, B. Chowdhury, V. Rus, and G. C. Tsokos Increased Caspase-3 Expression and Activity Contribute to Reduced CD3{zeta} Expression in Systemic Lupus Erythematosus T Cells J. Immunol., September 1, 2005; 175(5): 3417 - 3423. [Abstract] [Full Text] [PDF]
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B. P.-L. Lee, E. Mansfield, S.-C. Hsieh, T. Hernandez-Boussard, W. Chen, C. W. Thomson, M. S. Ford, S. E. Bosinger, S. Der, Z.-x. Zhang, et al. Expression Profiling of Murine Double-Negative Regulatory T Cells Suggest Mechanisms for Prolonged Cardiac Allograft Survival J. Immunol., April 15, 2005; 174(8): 4535 - 4544. [Abstract] [Full Text] [PDF]
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A. Kikuchi-Maki, T. L. Catina, and K. S. Campbell Cutting Edge: KIR2DL4 Transduces Signals into Human NK Cells through Association with the Fc Receptor {gamma} Protein J. Immunol., April 1, 2005; 174(7): 3859 - 3863. [Abstract] [Full Text] [PDF]
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L. Krymskaya, W.-H. Lee, L. Zhong, and C.-P. Liu Polarized Development of Memory Cell-Like IFN-{gamma}-Producing Cells in the Absence of TCR {zeta}-Chain J. Immunol., February 1, 2005; 174(3): 1188 - 1195. [Abstract] [Full Text] [PDF]
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V. C. Kyttaris, Y.-T. Juang, K. Tenbrock, A. Weinstein, and G. C. Tsokos Cyclic Adenosine 5'-Monophosphate Response Element Modulator Is Responsible for the Decreased Expression of c-fos and Activator Protein-1 Binding in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., September 1, 2004; 173(5): 3557 - 3563. [Abstract] [Full Text] [PDF]
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M. Steinberg, O. Adjali, L. Swainson, P. Merida, V. D. Bartolo, L. Pelletier, N. Taylor, and N. Noraz T-cell receptor-induced phosphorylation of the {zeta} chain is efficiently promoted by ZAP-70 but not Syk Blood, August 1, 2004; 104(3): 760 - 767. [Abstract] [Full Text] [PDF]
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S. Krishnan, M. P. Nambiar, V. G. Warke, C. U. Fisher, J. Mitchell, N. Delaney, and G. C. Tsokos Alterations in Lipid Raft Composition and Dynamics Contribute to Abnormal T Cell Responses in Systemic Lupus Erythematosus J. Immunol., June 15, 2004; 172(12): 7821 - 7831. [Abstract] [Full Text] [PDF]
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A. E. Schade and A. D. Levine Cutting Edge: Extracellular Signal-Regulated Kinases 1/2 Function as Integrators of TCR Signal Strength J. Immunol., May 15, 2004; 172(10): 5828 - 5832. [Abstract] [Full Text] [PDF]
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L. Zhong, C.-H. Wu, W.-H. Lee, and C.-P. Liu {zeta}-Associated Protein of 70 kDa (ZAP-70), but Not Syk, Tyrosine Kinase Can Mediate Apoptosis of T Cells through the Fas/Fas Ligand, Caspase-8 and Caspase-3 Pathways J. Immunol., February 1, 2004; 172(3): 1472 - 1482. [Abstract] [Full Text] [PDF]
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S. Krishnan, D. L. Farber, and G. C. Tsokos T Cell Rewiring in Differentiation and Disease J. Immunol., October 1, 2003; 171(7): 3325 - 3331. [Full Text] [PDF]
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