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The Journal of Immunology, 2002, 168: 118-126.
Copyright © 2002 by The American Association of Immunologists

Ly-6A.2 Expression Regulates Antigen-Specific CD4+ T Cell Proliferation and Cytokine Production1

S. Christine Henderson, Mihir M. Kamdar and Anil Bamezai2

Department of Cellular Biology, University of Georgia, Athens, GA 30602


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ly-6 proteins appear to serve cell adhesion and cell signaling function, but the precise role of Ly-6A.2 in CD4+ T lymphocytes is still unclear. Overexpression of Ly-6A.2 in T lymphocytes has allowed us to analyze the influence of elevated Ly-6A.2 expression on T cell function. In this study we report reduced proliferation of CD4+ T cells overexpressing Ly-6A.2 in response to a peptide Ag. Moreover, the Ly-6A.2-overexpressing CD4+ cells generated elevated levels of IL-4, a key factor that propels the differentiation of naive CD4+ T cells into Th2 subset. The hyporesponsiveness of Ly-6A.2 transgenic CD4+ T cells is dependent on the interaction of Ly-6A.2 T cells with the APCs and can be reversed by blocking the interaction between Ly-6A.2 and a recently reported candidate ligand. Overexpression of Ly-6A.2 in CD4+ T cells reduced their Ca2+ responses to TCR stimulation, therefore suggesting effects of Ly-6A.2 signaling on membrane proximal activation events. In contrast to the observed Ag-specific hyporesponsiveness, the Ly-6A.2 transgenic CD4+ T cells produced IL-4 independent of the interactions between Ly-6A.2 and the candidate Ly-6A.2 ligand. Our results suggest that 1) interaction of Ly-6A.2 with a candidate ligand regulates clonal expansion of CD4+ Th cells in response to an Ag (these results also provide further functional evidence for presence of Ly-6A.2 ligand on APC); and 2) Ly-6A.2 expression on CD4+ T cells promotes production of IL-4, a Th2 differentiation factor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growth and differentiation of T cells following an encounter with an Ag are critical for proper immune function. The specificity of the clonal expansion and differentiation of lymphocytes are determined by the interaction of TCR with its ligand, a peptide-MHC complex expressed on the APCs. Clonal expansion of CD4+ T cells is tightly regulated, as unchecked growth can cause toxic effector functions and unbalanced immune responses to foreign Ags. Although the mechanism of regulation of T cell growth remains largely unknown, previous studies suggest that surface expression of CTLA-4, Fas/APO-1, IL-2R proteins may regulate CD4+ T cell growth (1, 2, 3). Mice lacking expression of CTLA-4, Fas, or IL-2R show lymphoproliferation and abnormal accumulation of activated T cells, suggesting their importance in regulating homeostasis of either naive and/or cycling T cells (4, 5, 6, 7). Moreover, single gene mutations of Cbl, SLP, HPK1, and Csk in mice are known to cause hyperproliferation of T cells and therefore suggest a role of these kinases in inhibiting T cell stimulation (8, 9, 10, 11). The cell surface proteins that regulate these intracellular kinases have not been identified. Many of these regulatory molecules or growth inhibitory pathways may function in different T cell subsets or at different stages of clonal expansion or even work synergistically. Mechanisms underlying these intricate regulations of clonal expansion following an encounter with foreign Ag remains unclear.

Stimulation of CD4+ T cells with Ag initiates production of a variety of cytokines including IL-2, IFN-{gamma}, and IL-4. Some of these cytokines (e.g., IL-2) play a central role in clonal expansion, whereas other cytokines produced influence differentiation of naive CD4+ T cells into either Th1 (IFN-{gamma}) or Th2 (IL-4) subset (reviewed in Refs. 12 and 13). Therefore, understanding the influence of accessory proteins on naive or activated CD4+ T cells in generation of cytokines (type and quantity) that direct T cell differentiation is critical, especially when cytokine-induced differentiation can determine the kind of immune response generated against a pathogen or allergen (reviewed in Refs. 14, 15, 16, 17). The role of accessory molecules expressed on naive or activated T cells in generation of cytokines that in turn influence T cell differentiation remains unresolved.

The mouse Ly-6 locus encodes a family of GPI-anchored, developmentally regulated cell surface proteins (reviewed in Refs. 18 and 19). Members of Ly-6 gene family are excellent markers of different lineages of hemopoietic origin, including lymphocytes (20, 21, 22, 23, 24, 25), monocytes (26, 27), bone marrow cells (20, 27, 28), and granulocytes (29). There are shared motifs among the mouse Ly-6 proteins, including 8–10 conserved cysteine residues which are also found in human CD59, epidermal growth factor, urokinase plasminogen activator receptor, squid Sgp-2, SP-10 (sperm Ag) (reviewed in Ref. 30), snake neurotoxins/cytotoxins (29), and Caenorhabditis elegans odr-2 (31). All these proteins from different species have been grouped together into Ly-6 supergene family based on their limited amino acid similarity and the presence of conserved cysteine residues. Published reports have suggested role of Ly-6 protein in T cell signaling (32, 33) and cell adhesion (34, 35, 36). Surface expression of Ly-6A.2 is important for immunoresponsiveness of both T-T hybridomas (37) and normal T cells (38). Interestingly, surface expression of TCR/CD3 expression on T-T hybridomas is important for stimulation through the Ly-6 protein (39, 40). Moreover, ectopic expression of Ly-6A.2 transgene on CD4+CD8+ thymocytes promotes maturation of CD4+ (not CD8+) T cells in the thymus in the absence of TCR-MHC interaction (41). These data suggest that Ly-6A.2 expression influences cell growth and differentiation that are dependent or independent of signaling through the Ag receptor. Contrary to some published reports, the CD4+ T cells from Ly-6A mutant mice show a modestly higher proliferation in response to anti-CD3 Ab than their controls (42). Moreover, Abs against Ly-6A.2 inhibit anti-CD3-induced IL-2 production by T-T hybridoma (43). The role of Ly-6A.2 expression in Ag-specific response of primary CD4+ T cells remains untested and the mechanism by which Ly-6A.2 expression may augment or inhibit the TCR-induced activation is unknown.

Ly-6A.2 and other members of the Ly-6 gene family, including E48 protein and Ly-6C, participate in cell-cell adhesion (35, 36). We recently reported biochemical characterization of a candidate ligand that binds Ly-6A.2 (44). The Ly-6A.2 candidate ligand is expressed on the majority of B cells and macrophages (Refs. 34 and 44 and our unpublished data). Moreover, a ligand for Ly-6 day (Ly-6dL), another member of the Ly-6 gene family, was recently identified that shows expression in almost all mouse tissues analyzed (45). Ly-6dL shows similarity to epidermal growth factor domain of mouse notch (motch-1) protein, known for its role in directing cell fate decisions in a variety of cell types during development (reviewed in Ref. 46). These observations raise the possibility that Ly-6 proteins may mediate cell function by binding to a ligand, but the consequences of these Ly-6-ligand interactions are unknown.

To examine the role of Ly-6A.2 expression on the function of CD4+ T cells, we bred the Ly-6A.2 transgenic (Tg)3 mice with DO11 TCR-Tg mice. In this study we report that the overexpression of Ly-6A.2 on CD4+ T cells inhibits responses initiated by the TCR in the presence of peptide Ag presented on APCs. Surprisingly, the same Tg CD4+ T cells are hyperresponsive to a combination of anti-TCR/CD3 and CD28 Abs, in the absence of APC. Our Ab against a candidate Ly-6A.2 ligand reversed the Ag-specific hyporesponsiveness. These observations suggest that Ly-6A.2 expression exerts both inhibitory and activating roles depending on how T cells are stimulated. Interaction of Ly-6A.2 with a candidate Ly-6A.2 ligand negatively regulates T cell proliferation. Moreover, Ly-6A.2 Tg DO11 CD4+ T cell primary cultures produce large amounts of IL-4 in response to the OVA323–339 peptide, suggesting that Ly-6A.2 expression may participate in differentiation of CD4+ T cells into Th2 subset.


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

Ly-6A.2 Tghigh and Tglow mice (4–6 wk of age) used in this study have been previously reported (47). The Ly-6A.2 Tg mice were bred to the DO11 TCR-Tg mice (48) (generous gift from Dr. D. Loh) and MRL-lpr/lpr (The Jackson Laboratory, Bar Harbor, ME) for testing the role of Ly-6A.2 expression in Ag-specific responses. Heterozygous Ly-6A.2 Tghigh and Tglow mice were bred with homozygous DO11 TCR-Tg mice and screened for the presence of Ly-6A.2 Tg sequence as described before (47). The heterozygous Ly-6A.2 Tg+ DO11 TCR double Tg mice were used for experiments, and littermates from the same breeding that expressed only DO11 TCR transgene served as controls. Expression of DO11 TCR transgene was confirmed by staining with KJ1-26, anti-DO11 TCR Ab (49) (generous gift from Dr. P. Marrack). Ly-6A.2 Tghigh and Ly-6A.2 Tglow mice were bred with MRL-lpr/lpr mice and backcrossed mice of seven and eight generations were used in the experiments. Following each backcross the mice expressing the mutated Fas gene were screened using forward (5'-GTAAATAATTGTGCTTCGTCAG-3') and reverse (5'-TAGAAAGCTGCACGGGTGTG-3') primers yielding mutant fas (lpr/lpr) of 212 bp and a wild-type (+/+) 184-bp PCR fragment. Seven- or eight-generation backcrossed mice on MRL-lpr/lpr background that only expressed Ly-6A.2 transgene and possessed mutations of fas gene on both chromosomes (yielding only a 212-bp PCR fragment) were used for our experiments. Mice with Fas mutations on both chromosomes (Fas-/-) or one of two chromosomes (Fas+/-) that did not express the Ly-6A.2 transgene and were generated in the same breeding served as controls.

Cell preparation

CD4+ T cells from Ly-6A.2 Tg or non-Tg mice were prepared from the lymph nodes. Lymph node cells were incubated with 100 µl of anti-CD8 (3.155) and anti-MHC class II (M5/114) Abs for 30 min at 4°C. Samples were washed three times with the PBS supplemented with 0.1% BSA. Following the washing step, cells were incubated with Dynal beads M-450 coupled with sheep anti-mouse IgG Ab, as per the manufacturer’s instructions (Dynal Biotech, Oslo, Norway) for 45 min at 4°C. Depletion of contaminating cells was achieved by magnetic separation, and purity of CD4+ cells ranged from 85 to 95%.

Flow cytometry

A total of 1 x 106 lymph node or purified CD4+ cells were incubated with anti-CD4-PE, anti-CD8-FITC, anti-Ly-6A/E (D7) (BD PharMingen, San Diego, CA), and anti-DO11 TCR (KJ1-26) (49) Abs followed by appropriate fluorochrome-conjugated second step reagents. Cells were analyzed on an EPICS Elite Analyzer flow cytometer (Beckman Coulter, Fullerton, CA).

ELISA for detection of anti-DNA Abs and cytokines

For detection of anti-DNA Abs, the microtiter wells were coated with poly-L-lysine (25 µg/ml) for 24 h at 4°C. Excess of poly-L-lysine was removed by washing with 0.1 M TBS containing 0.1% Tween 20 before coating of dsDNA at 5 µg/ml for 2 h at room temperature (RT). Sera from mice were analyzed at 1/10, 1/100, and 1/1000 dilution by incubating for 60 min at RT. The presence of anti-dsDNA Abs was detected by incubation with protein G-alkaline phosphatase at 1/4000 dilution for 1 h at RT and the assay was developed in the presence of the substrate, p-nitrophenyl phosphate.

For cytokine ELISA, microtiter wells (Costar, Cambridge MA) were coated with appropriate capture Ab in 0.1 M Na2HPO4 binding buffer (pH 9.0) overnight at 4°C. Plates were washed five times with PBS plus 0.05% Tween 20 and blocked with 100 ml of 1% BSA in PBS for 30 min at RT. After five washes, cytokine standards and samples diluted in blocking buffer/Tween 20 were added to wells for overnight incubation at 4°C. Plates were washed six times before adding appropriate capture Ab for a 1-h incubation at RT. Incubation was followed by six washes after which 100 µl of streptavidin-HRP conjugate (Vector Laboratories, Burlingame, CA) was added at a 1/2000 concentration. Plates were incubated for 30 min at RT and washed eight times before addition of the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO). The assay was read at 405 nm by an ELISA reader.

Ab immobilization

Microculture wells (Costar) were coated with either purified anti-CD3 (145-2C11), anti-CD28 (37N) (50), or anti-DO11 TCR (KJ1-26) for 2 h at 37°C in carbonate-bicarbonate buffer, pH 9.6. Unbound Abs were removed by washing with 0.1 M PBS, pH 7.4, five times before the addition of the purified CD4+ T cells.

Ag stimulation and cell culture

A total of 1 x 105 purified CD4+ T cells were cocultured with 5 x 105 irradiated APCs in RPMI 1640-based culture medium (Irvine Scientific, Santa Ana, CA). The cultures were conducted in the presence of either stimulating chicken OVA (cOVA)323–339 (SQAVHAAHAEINEAGRE) or nonstimulating cOVA324–334 (QAVHAAHAEIN) peptides (synthesized at Molecular Genetics Instrumentation Facility, University of Georgia, Athens, GA). The precise culture condition is listed in the appropriate figures. Cultures were pulsed after 72 h with 1 µCi of [3H]thymidine for the last 20 h of culture.

Calcium responses

CD4+ T cells purified from the lymph nodes of BALB/c mice were loaded with Indo-1 AM (Molecular Probes, Eugene, OR) at 2 µg/ml final concentration. The cell loading was conducted at a cell concentration of 2 x 106 cells/ml in loading medium consisting of HBSS with 1% BSA for 30 min at 37°C. Following incubation, cells were centrifuged for 8 min at 1000 rpm, and pellet was resuspended in cell loading medium to a final concentration of 2 x 106 cells/ml. Cells were then stored at 22°C and protected from light until analysis. Indo-1 AM-loaded cells were warmed to 37°C for 5–10 min before analysis. Agonists were added at varying time points and the cells were analyzed on an EPICS 753 Flow Cytometer (Coulter, Hialeah, FL) at 37°C in cell loading medium. Anti-CD3 (145-2C11) and control Ab, anti-H-2Kk (10.2.16), were used at a concentration of 10 µg/ml. Rabbit anti-mouse IgG and ionomycin were used at final concentrations of 25 and 2 µg/ml, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ly-6A.2-overexpressing CD4+ T cells are hyporesponsive to cOVA323–339 peptide presented by splenic APC

Previous studies have suggested both activating and inhibitory effects of Ly-6A.2 expression on CD4+ T cell responses. Moreover, the role of Ly-6A.2 expression on Ag-specific T cell activation of normal T cells is unclear. To clarify these issues and to fully understand the role of Ly-6A.2 expression in Ag-specific responses we bred the Ly-6A.2high Tg mice to DO11 TCR-Tg mice. CD4+ T cells were isolated from the lymph nodes of Tg mice and tested for their responsiveness to cOVA323–339 peptide in the presence of irradiated syngeneic APC. Ly-6A.2 Tghigh DO11 TCR+CD4+ T cells proliferated 7- to 8-fold lower than CD4+ T cells from Ly-6A.2 Tg- DO11 TCR+ control littermates (Fig. 1GoA). Hyporesponsiveness of Ly-6A.2 TghighCD4+ T cells was observed at 1 µM (Fig. 1GoA) and lower (0.5 and 0.25 µM) cOVA323–339 peptide concentration (data not shown). DO11 TCR-Tg CD4+ T cells did not respond to cOVA324–334 control peptide as expected (Fig. 1GoA). Ly-6A.2 Tg+CD4+ T cells also showed reduced responses to antiCD3 mAb (Fig. 1GoB). Similar levels of hyporesponsiveness of Ly-6A.2 TghighCD4+ T cells were observed when mitomycin-treated splenic cells were used to present the cOVA peptide (data not shown).



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  		FIGURE 1. Ag-specific responsiveness of the purified Ly-6A.2 Tg CD4+ T cells. Variable numbers of purified CD4+ T cells from Ly-6A.2 Tg- ({square}) or Ly-6A.2 Tghigh ({blacksquare} and {blacktriangleup}) mice were cocultured with 5 x 105 irradiated syngeneic spleen cells. A, The cultures were conducted in the presence of either cOVA324–334 (control, {blacktriangleup}) or cOVA323–339 (specific peptide, {square} and {blacksquare}) at a 1-µM concentration. B, Cultures were also set up with anti-CD3 (145-2C11; 1/100 dilution of culture supernatant), cOVA323–339, or cOVA324–334 (1 µM), or a combination of PMA (20 ng/ml) and ionomycin (0.125 µg/ml). C, Additional cultures with a combination of anti-Ly-6A/E plus 20 ng/ml of PMA were also conducted. Cells were harvested after 72 h of culture. The data are expressed as arithmetic mean counts per minute of incorporated [3H]thymidine incorporated. Error bars indicate SD. T cells from either Tg- or Tghigh mice did not proliferate when cultured with OVA323–329 in the absence of the irradiated syngeneic APC (<350 cpm). A representative experiment of at least five independent experiments is shown.

 
To examine whether the reduced proliferation in Ly-6A.2 Tg+ DO11 Tg+CD4+ T cells was not due to a general unresponsive (refractory) status of these cells, we tested the ability of the Ly-6A.2 Tghigh DO11 Tg CD4+ T cells to proliferate in the presence of PMA and calcium ionophore, which induce receptor-independent signaling in T cells. CD4+ T cells from Ly-6A.2 Tghigh mice were fully responsive to activation by PMA and calcium ionophore (Fig. 1GoB). Moreover, cross-linking of Ly-6A.2 with an anti-Ly-6A.2 Ab induced higher proliferation in the CD4+ Ly-6A.2 Tg T cells than the CD4+ T cells from non-Tg mice (Fig. 1GoC). Higher proliferation was observed in cultures with 1.25 x 105 cells as compared with cultures with 0.6 x 105 cells as expected. These results indicate that the hyporesponsiveness observed in CD4+Ly-6 Tg+ T cells is OVA323–339 peptide-APC specific.

High expression of the Ly-6A.2 transgene ameliorates the lymphoproliferative disorder in lpr/lpr mice

Lymphoadenopathy and autoimmunity in the MRL-lpr/lpr mice are consequences of spontaneous mutation in the Fas gene (5). T lymphocytes lacking the expression of Fas protein undergo activation-mediated cell death after recognizing an unidentified autoantigen bound to an appropriate MHC class II molecule (51). Interaction of the TCR with the self-MHC proteins is critical for initiation of this autoimmune phenotype (52, 53). We sought to examine whether the overexpression of the Ly-6A.2 on T cells from the lpr mice might inhibit autoreactivity through the TCR and reverse lymphoproliferative disorder in these mice. For this experiment the Ly-6A.2 Tghigh mice were backcrossed to the MRL-lpr/lpr mice, and Ly-6A.2 Tghigh lpr/lpr and Ly-6A.2 Tg- lpr/lpr mice were analyzed. The Tg- MRL-lpr/lpr mice develop large lymph nodes at ~16–18 wk of age as expected. Interestingly, the expression of high levels of Ly-6A.2 in the MRL-lpr/lpr mice results in lymph nodes of normal cellularity (Fig. 2GoA). Consistent with the previous observation (54), the MRL-lpr/lpr mice have an abnormal subset of CD4-CD8-Thy-1+ T cells in the lymph node (Fig. 2GoB). The abnormal subset is not observed in the Ly-6A.2 Tghigh MRL-lpr/lpr and normal mice (Fig. 2GoB). Moreover, relative amounts of anti-DNA Ab (Ab titers), which is a signature of the autoimmune phenotype, were significantly decreased in the lpr/lpr mice overexpressing Ly-6A.2 (Fig. 2GoD). These results suggest that the overexpression of Ly-6A.2 in the MRL-lpr/lpr mice suppresses the proliferation of T cells in the lymph node and reverses the autoimmune phenotype. Consistent with the above observation is the finding that CD4+ T cells from Ly-6A.2 Tghigh MRL-lpr/lpr mice do not proliferate in the presence of syngeneic APC (Fig. 2GoC). These results indicate that the overexpression of Ly-6A.2 suppresses lymphoproliferation and autoimmune phenotype in MRL-lpr/lpr mice.



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  		FIGURE 2. Ly-6A.2 transgene expression ameliorates lymphoadenopathy in MRL-lpr/lpr mice. A, Cellularity of lymph nodes from normal, Ly-6A.2 Tg-/+ lpr, Ly-6A.2 Tg- lpr/lpr, or Ly-6A.2 Tghigh lpr/lpr mice were analyzed. The total number of cells obtained from four lymph nodes per mouse is shown, and at least five mice from each group were analyzed. B, Lymph node cells were stained with anti-CD4-PE and anti-CD8 FITC. CD4+CD8-, CD4-CD8+, and CD4-CD8- subsets were gated, and the number of cells present in the gates (n = 3 experiments) are shown. C, Lymph node cells (5 x 105) were cultured with 5 x 105 irradiated syngeneic splenic APC for 6 or 8 days followed by pulsing with [3H]thymidine prior to the last 6–8 h of the culture. The data are expressed as the arithmetic mean counts per minute of [3H]thymidine incorporated ± SD (n = 3 experiments). D, Anti-dsDNA Ab titers (1/10 to 1/103) were analyzed from the sera of Ly-6A.2 Tg- lpr/lpr ({square}) or Ly-6A.2 Tg+ lpr/lpr ({blacksquare}) mice by ELISA as described in Materials and Methods. The pooled normal mouse serum was used as control ({circ}).

 
Reduced proliferation of Ly-6A.2 Tg CD4+ T cells in response to peptide Ag requires interaction with APCs

The finding that Ly-6A.2 overexpression decreased peptide-stimulated but not the PMA plus calcium ionophore responses might suggest that the Ly-6A.2 affects signaling through the TCR or that a Ly-6A.2 ligand on the APC mediates this inhibition. To distinguish between these two possibilities, purified CD4+ T cells were cultured with anti-TCR plus anti-CD28 Abs bound to the microtiter wells, in the absence of APCs. Previous experiments have demonstrated that cross-linking of TCR and CD28 with the Abs directed against them was necessary and sufficient to activate naive T cells (50). Fig. 3GoB shows that CD4+ T cells from DO11 TCR x Ly-6A.2 double Tg mice proliferate in response to the stimulation through the TCR and costimulatory molecule. Indeed, much higher proliferation was observed with DO11 x Ly-6A.2 double Tg T cells in comparison to the responses of CD4+ T cells from DO11 TCR single Tg mice. Similar results were obtained with a combination of plate-bound anti-DO11 TCR (KJ1-26) and anti-CD28 (Fig. 3GoB) or anti-TCR (H57) and anti-CD28 (data not shown). Taken together these data suggest that CD4+ T cells overexpressing Ly-6A.2 are capable of proliferating well (even more than the non-Tg CD4+ T cells) in response to anti-TCR stimulation in the absence of APC. The Ag-specific hyporesponsiveness of Ly-6A.2 Tg CD4+ T cells occurs by a non-cell-autonomous mechanism that requires interactions with the APC.



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  		FIGURE 3. Responsiveness of CD4+ T cells from DO11 TCR Ly-6A.2 double Tg mice through Ag receptor and costimulatory molecule in the absence of APCs. Purified CD4+ T cells (1 x 105) from the Ly-6A.2 Tg- (open symbols) or the Ly-6A.2 Tghigh (closed symbols) mice were cultured in wells coated with different concentrations of anti-CD3 (145-2C11) (A) alone (circles) or in combination with anti-CD28 (37N) at 10 µg/ml concentration (diamonds); or anti-TCR (KJ1-26) (B) alone (circles) or in combination with 10 µg/ml of anti-CD28 (37N) (diamonds). Cells were pulsed and harvested after 48 h of culture. The data are expressed as arithmetic mean counts per minute of [3H]thymidine incorporated ± SD. A representative experiment of five experiments is shown.

 
Reduced Ca2+ responses in CD4+ T cells overexpressing Ly-6A.2

To characterize the mechanism underlying hyporesponsiveness we first sought to test the effects of Ly-6A.2 overexpression on Ca2+ response of these cells after loading with a calcium-sensitive dye, Indo-1 (55), followed by cross-linking of the TCR/CD3 complex. As calcium binds to Indo-1, the peak emission wavelength shifts from 500 to 400 nm. This event is quantified in a flow fluorocytometer and data are displayed as a ratio of emission fluorescence at 395 and 510 nm as function of time. Lower Ca2+ responses were observed in Ly-6A.2 Tg CD4+ T cells than in non-Tg CD4+ T cell controls (Fig. 4GoB). Both the mean intensity of Ca2+ flux (Fig. 4GoB) as well as the total number of Ly-6A.2 Tg+CD4+ T cells responding (data not shown) to TCR/CD3 stimulation were significantly reduced. The lack of any Ca2+ response with the control Ab indicates the specificity of the responses observed (Fig. 4GoA). These results suggest that Ly-6A.2 expression affects early signaling events.



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  		FIGURE 4. Overexpression of Ly-6A.2 on CD4+ T cells inhibits calcium responses mediated through the TCR complex. Purified CD4+ T cells from either Ly-6Tg- (dashed line) or Ly-6Tg+ (solid line) were loaded with Indo-1 and exposed to isotype control (anti-H-2Kk) (A) at 10 µg/ml, or anti-CD3 (B) followed by rabbit anti-mouse IgG (cross-reactive with hamster IgG). Mean calcium response (ratio of 395:510 nm) by the stimulated cells is shown and total calcium present in the cells is evaluated by addition of ionomycin at a 2 µg/ml concentration. One representative experiment of three experiments is shown.

 
Cytokine production by Ly-6A.2 Tg CD4+ T cells

Impaired anti-TCR-mediated Ca2+ responses and proliferation of Ly-6A.2 Tg CD4+ T cells suggest a decrease in the production of cytokines that are key regulators of T cell growth. Therefore, we sought to quantitate the production of IL-2 (Fig. 5GoA), IFN-{gamma} (Fig. 5GoB), and IL-4 (Fig. 5GoC). These results demonstrate that the Ly-6A.2 Tg and non-Tg T cells produce IL-2, a key growth factor. Comparable amounts of IL-2 were produced by CD4+ T cells from the non-Ly-6A.2 Tg and the Ly-6A.2 Tghigh mice on days 1 and 2 postactivation, but significantly reduced IL-2 was detected in day-3 cultures with the Ly-6A.2 Tg cells (Fig. 5GoA). In contrast, the Ly-6A.2 Tg CD4+ T cells produced comparable or even more IFN-{gamma} than their non-Tg controls (Fig. 5GoB). To our surprise the CD4+ T cells from Ly-6A.2 Tg mice generated more IL-4 in response to the OVA323–339 peptide than their non-Tg controls (Fig. 5GoC). High amounts of IL-4 were detected in day-2 and -3 cultures, suggesting that Ly-6A.2 transgene expression promotes generation of IL-4 that is undetectable in the primary cultures in which CD4+ T cells were stimulated through the Ag receptor (our detection sensitivity, 62.5 pg/ml). Taken together these results suggest that Ly-6A.2 expression influences CD4+ T cell growth by inhibiting full production of IL-2. Ly-6A.2 expression may also influence the differentiation of naive T cells by producing elevated levels of IL-4.



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  		FIGURE 5. Ly-6A.2 Tg CD4+ T cells produce elevated levels of IL-4 in response to OVA323–339 presented by splenic APC. Purified CD4+ T cells (1 x 105) from either non-Tg ({diamond}) or Ly-6A.2 Tghigh ({diamondsuit}) were stimulated with cOVA323–339 peptide as described in Fig. 1Go and Materials and Methods. IL-2 (A), IFN-{gamma} (B), and IL-4 (C) were quantitated in supernatants from day-1 to -3 cultures by ELISA as described in Materials and Methods. Each data point is expressed as arithmetic mean value ± SD (n = 2). A representative experiment of three experiments is shown.

 
The elevated production of IL-4 by Ly-6A.2 Tg CD4+ T cells in response to stimulation through the Ag receptor is independent of APC

To test whether the elevated production of IL-4 was dependent on interaction of the CD4+ T cell with APCs or reflected intrinsic ability of these cells to produce IL-4 in response to TCR stimulation we stimulated CD4+ cells with anti-CD3 and anti-CD28 in the absence of APC. Plate-bound anti-CD3 Ab alone or in combination with plate-bound anti-CD28 induce large amounts of IL-4 in DO11 x Ly-6A.2 double Tg CD4+ T cells and not in the DO11 TCR single Tg controls (Fig. 6GoA). Similar observations were made when anti-DO11 TCR Ab was used either alone or in combination with anti-CD28 (Fig. 6GoB). These observations strongly suggest that the ability of IL-4 production by Ly-6A.2 Tg T cells is induced by a cell-autonomous mechanism that does not require interaction with APC.



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  		FIGURE 6. Ly-6A.2 Tg CD4+ T cells produce elevated levels of IL-4 by a cell-autonomous mechanism in the absence of APC. IL-4 was quantitated in supernatants from day-3 cultures of either Ly-6Tg- (open symbols) or Ly-6A.2 Tg (closed symbols) mice stimulated with either anti-CD3 alone (diamonds) or a combination of anti-CD3 and anti-CD28 (circles) in the absence of APC by ELISA as described in Materials and Methods. Each data point is expressed as arithmetic mean value ± SD (n = 2). A representative experiment of three experiments is shown.

 
Role of Ly-6A.2-ligand interactions in Ag-specific inhibition of proliferation and IL-4 production by Ly-6A.2 Tg CD4+ T cells

We have generated mAb against the Ly-6A.2 ligand. This Ab recognizes a 66-kDa protein expressed in the majority of professional APCs in the spleen and blocks the binding of a candidate ligand expressing cells to Ly-6A.2-overexpressing Chinese hamster ovary cells (44). We sought to test whether the anti-Ly-6A.2 ligand Ab reversed the peptide-specific hyporesponsiveness that was observed with Ly-6A.2 Tg CD4+ T cells. Fig. 7GoA shows that an Ab against the candidate Ly-6A.2 ligand (9AB2), but not the hamster control Ab (9E3), reverses this inhibition. These results strongly suggest that inhibition of CD4+ T cells to peptide Ag may be mediated through the interaction of the overexpressed Ly-6A.2 with a candidate Ly-6A.2 ligand expressed on the APC. In contrast, the presence of anti-Ly-6A.2 ligand Ab did not alter the production of IL-4 in response to OVA323–339 peptide (Fig. 7GoB). These later results are consistent with our observation that IL-4 production is independent of APC (Fig. 6Go). Taken together these data suggest that hyporesponsiveness and elevated production of IL-4 show differential dependence on ligand interaction.



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  		FIGURE 7. Role of the candidate Ly-6A.2 ligand in OVA323–339 peptide-specific CD4+ T cell proliferation and IL-4 production. A, Purified CD4+ T cells from the Ly-6A.2 Tg- (open bars) or Ly-6A.2 Tghigh (hatched bars) mice were cocultured with 5 x 105 irradiated syngeneic spleen cells. These cultures were conducted in the presence of either cOVA324–334 (control) or cOVA323–339 (specific-peptide) at a 1 µM concentration either in the absence or in the presence of anti-ligand (9AB2, 1/4 dilution of supernatant) or control hamster (9E3 at 1/4 dilution of supernatant) Ab. Cells were harvested after 72 h of culture. The data are expressed as the arithmetic mean counts per minute of [3H]thymidine incorporated ± SD (n = 2). A representative experiment of four independent experiments is shown. B, IL-4 production was quantitated in the supernatants of these day-3 cultures with the OVA323–339 peptide as described in Materials and Methods. A representative experiment of three experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous reports suggest that mouse Ly-6 proteins have both cell adhesion (34) and cell signaling (32, 33) function. Although the ligands that might bind Ly-6 proteins have been proposed (44, 45), the functional consequence of their interaction with Ly-6 proteins remains unclear. We demonstrate that Ly-6A.2 expression negatively regulates Ag-specific CD4+ T cell responses by interacting with a candidate ligand. Ly-6A.2 Tg CD4+ T cells show lower Ca2+ fluxes and IL-2 production than CD4+ T cells from non-Tg controls in response to TCR stimulation. To our surprise the primary cultures of Ly-6A.2 Tg CD4+ T cells generated IL-4 in response to peptide stimulation that was independent of interaction with the candidate Ly-6A.2 ligand. These data suggest that Ly-6A.2 expression regulates T cell proliferation and possibly differentiation of CD4+ T cells after encountering an Ag. These data also provide functional evidence of a candidate ligand expressed on APC that is recognized by our recently reported hamster anti-ligand Ab.

Our previous reports suggest the presence of Ly-6A.2 ligand on the majority of B cells and macrophages (34, 44). APC-dependent inhibition of CD4+ Tg T cell responses to peptide Ag is consistent with these published data, because splenic APC is comprised of B cells and macrophages. Suppression of lymphoproliferative disorder and autoimmune symptoms in the Ly-6A.2 Tg+ lpr/lpr mice that are normally observed in MRL-lpr/lpr mice in the absence of Ly-6A.2 transgene supports these in vitro observations. The inhibitory responses are proportional to the level of Ly-6A.2 transgene expression, inasmuch as 10-fold lower transgene expression on peripheral CD4+ T cells (from Ly-6A.2 Tglow mice) showed reduced proliferation in response to OVA323–339 peptide (ranging from 0 to 40% inhibition compared with the wild-type controls) (our unpublished observations). Consistent with this finding is the observation that Ly-6A.2low lpr/lpr mice did not show reduced lymphoproliferative disorder (our unpublished observation). These results corroborate a previously published report that shows a modest hyperresponsiveness of T cells lacking expression of Ly-6A.2 knockout mice in response to anti-CD3 stimulation (35). Taken together these observations demonstrate that Ly-6A.2 overexpression inhibits Ag-specific responses of CD4+ T cells that are primarily mediated by interaction of Ly-6A.2 with its candidate ligand.

Marked peptide-specific hyporesponsiveness and amelioration of the lymphoproliferative disorder in lpr/lpr mice was only observed with high levels of Ly-6A.2 transgene expression. The expression of Ly-6A.2 on naive CD4+ T cells from Tghigh mice is 100- to 200-fold higher than the expression of the endogenous levels that could be considered as nonphysiological. However, endogenous Ly-6A.2 on non-Tg T cells is inducible and achieves similar levels of expression as on Tg T cells when activated through the Ag receptor or stimulated with IFN-{gamma} alone (Refs. 56 and 57 and our unpublished data). Therefore, we favor the physiologic relevance of our Tg model. We postulate that these high levels are achieved physiologically during the immune response against a foreign Ag and that up-regulated Ly-6A.2-ligand interactions exert their inhibitory affect after sufficient clonal expansion and effector functions have been generated by the CD4+ Th cells. Future in vitro and in vivo experiments will directly address this issue.

Analyses of cell lines and normal T cells suggest either an activating (38, 39, 58) or an inhibitory (42, 43) role of the Ly-6A protein. It is unclear why the expression of Ly-6A.2 has these opposing affects. Our data may provide some insights into these apparently contradictory observations. Our results suggest that one candidate Ly-6A.2 ligand expressed on splenic APC can inhibit TCR-mediated responses in CD4+ T cells by interacting with Ly-6A.2 (Figs. 1Go and 2Go), whereas the same Ly-6A.2 Tg CD4+ T cell shows hyperresponses to TCR signaling (an opposite outcome) in the absence of Ly-6A.2-ligand interactions (Fig. 3Go). These data do not rule out the possibility that hyperresponsiveness may also be induced by interaction with another Ly-6 ligand. Interaction of Ly-6A.2 with a recently reported ligand for Ly-6d (45), another member of Ly-6 gene family, has not been examined. Taken together our results suggest that hypo- or hyperproliferation of Ly-6A.2 in Ly-6A.2 Tg CD4+ T cells may depend on how these cells are stimulated.

We have used anti-candidate ligand Ab to block Ly-6A.2-ligand interactions, and it remains a possibility that these experimental systems are nonphysiologic, even though the Abs have provided insights into the functions of a number of other reported receptor-ligand interactions. Therefore, the use of soluble Ly-6A.2 protein to block interactions of Ly-6A.2 with its ligand should provide insights into the physiologic role of Ly-6A.2. Unfortunately, the Ly-6A.2-IgG (dimer) is unable to recognize the candidate ligand on splenic APCs and does not inhibit Ly-6A.2-dependent cell-cell adhesion, rendering this reagent unusable to address this issue. Whether the absence of Ly-6A.2-IgG1 binding to the candidate binding is due to its low affinity is not known. Future studies with a highly multimerized form of Ly-6A.2 may address this issue. In results obtained with two independent experimental systems, our overexpression (Tg) and Ly-6A knockout mice (42) are consistent with each other, and Ab directed against the candidate ligand reversed the Ag-specific hyporesponsiveness (Fig. 7Go); therefore, we favor the interpretation that interaction of Ly-6A.2 with the candidate ligand mediates inhibition of CD4+ T cell clonal expansion.

The Ag-stimulated T cells double every 4.5 h and therefore have a potential to generate 1 x 1012 cells in 1 wk. This profound proliferation compounded with limited space available in the lymphoid compartment may potentiate the toxic effects and autoimmune consequences; therefore, these processes are under tight regulation. Up-regulation of CTLA-4 and Fas on activated CD4+ T cells is known to negatively regulate T cell proliferation (1, 2). IL-2 has been recognized as a T cell growth factor, but recent observations also suggest its importance in propriocidal regulation of T cell growth by inducing apoptosis in cycling T cells (reviewed in Ref. 3). These results strongly suggest that the expression of CTLA-4, Fas, and IL-2R on T cells exerts their role in regulation of homeostasis of naive or Ag-stimulated T cells. A number of published studies have suggested a growth inhibitory role of GPI-anchored proteins (including Ly-6) (reviewed in Ref. 59). Our results suggest that interaction of Ly-6A.2 with the candidate ligand inhibits T cell proliferation and therefore regulates clonal expansion of T cells following their encounter with a foreign Ag. These later observations are consistent with the expression pattern of Ly-6A.2. Naive CD4+ T cells express low levels of Ly-6A.2 protein that is profoundly increased (100- to 200-fold) upon T cell activation and by treatment with type I and type II IFNs (Refs. 46 and 47 and our unpublished observations). We propose that Ly-6A.2-ligand interactions do not affect the initiation of T cell proliferation but instead down-regulate their proliferation when high-level expression of endogenous Ly-6A.2 is achieved following T cell activation. These results may suggest that the regulation of T cell proliferation may occur once the proliferating T cells have performed their effector function. Further studies are needed to determine the precise stage at which Ly-6A.2 expression contributes to T cell proliferation.

To begin to understand the mechanism of Ag-specific inhibitory responses of CD4+ T cells overexpressing Ly-6A.2 protein, we focused on early signaling events. We report that Ca2+ responses are significantly affected. Additional experiments are needed to precisely determine the mechanism of reduced Ca2+ fluxes in Ly-6A.2 Tg cells. We suspected that reduced initial signaling in T cells would affect IL-2 production. To our surprise, the production of IL-2 on days 1 and 2 of T cell response was unaltered. Significant effects were observed in the production of this growth factor on day 3 of the culture. It is possible that production of IL-2 is reduced on days 1 and 2 of the culture, but our assays were unable to detect these differences. These results suggest that overall IL-2 production is significantly diminished, therefore reducing the clonal expansion of T cells. Reduced production of IL-2 was observed on day 3 but not days 1 and 2 of the culture, suggesting that regulation of T cell proliferation by Ly-6A.2 may occur at a later stage where normal expression is highly up-regulated (typically 48 h after the initial culture with Ag). Alternatively, the Ly-6A.2-ligand interactions alter signaling through the IL-2R and therefore inhibit growth of T cells regardless of IL-2 production, as has been previously observed with Abs against GPI-anchored proteins (reviewed in Ref. 59). Further experimentation is needed to completely and precisely address this question.

How cell proteins expressed on naive or activated T cells participate in T cell differentiation is not entirely clear. A number of factors influence development of Th1 and Th2 effector T cells, including relative concentration of cytokines present. IL-4 is a key regulator of differentiation of naive CD4+ T cells into Th2 subset, whereas IL-12 and IFN-{gamma} promote differentiation into Th1 effector cell. The Th1 and Th2 differentiation factors are derived from varied sources, including macrophages and dendritic cells (IL-12), NK cells, and T cells (IFN-{gamma}). T cells are known to generate initial bursts of IL-4, perhaps not in large enough amounts to be detectable in the primary cultures (60). In addition, the nature, dose, and route of administration of the Ag, as well as the nature of interacting APC, influence the generation of differentiation factors (reviewed in Ref. 61). Some recent reports suggest that interaction of CD28 with B7-2 (62, 63, 64, 65), CD4 with MHC class II (66, 67), and OX-40 with OX-40 ligand (68, 69) promote differentiation to Th2 but not to Th1 cells. In contrast, interaction of LFA-1 with ICAM-1 or -2 (70, 71), CD28 with B7-1 (62, 72), and CD40 with CD40L (73) promote Th1 differentiation. Moreover, naive T cells lacking expression of CTLA-4 differentiate into Th2 subset, suggesting a role of CTLA-4 in the Th1 differentiation pathway (74). The mechanisms by which the accessory proteins on T cells and their interaction with their ligands regulate differentiation of naive CD4+ T cell is unknown, but it is possible that different T cell surface proteins may influence production of different cytokines that in turn regulate differentiation. It was surprising that the Ly-6A.2 Tg CD4+ T cells generated large amounts of IL-4 in response to appropriate peptide ligand in the primary cultures. IL-4 was not detected on day 1 of the culture; however, >3.5 ng/ml of IL-4 was detected on days 2 and 3 of culture. These observations raise the possibility that elevated cell surface expression of Ly-6A.2 may regulate differentiation of naive T cell differentiation into the Th2 type by increasing IL-4 concentrations during primary stimulation. More studies need to be conducted to determine the cell-autonomous mechanism underlying IL-4 production and establishing the influence of Ly-6A.2 expression in differentiation of naive CD4+ T cells into Th2 phenotype.

Several members of the Ly-6 supergene family appear to participate in regulating important functions in other tissues by yet unidentified mechanisms. A human Ly-6 protein inhibits osteoclast formation and bone resorption (75); mutation in odr-2 gene that encodes a Ly-6-related protein causes defect in the ability to chemotax to odorants that are recognized by the two AWC olfactory neurons in C. elegans (31); and murine lynx1 that is expressed on hippocampus, cortex, and cerebellum modulates nicotinic acetylcholine receptors in mammalian brain (76). Moreover, mutation in secreted Ly-6/uPAR-related protein, another member of human Ly-6 superfamily, causes a rare autosomal recessive skin disorder, mal de Meleda, characterized by transgressive palmoplanter keratoderma, keratotic skin lesions, and perioral erythema (77). Our results suggest that one way Ly-6 proteins modulate signaling and mediate their function is by interacting with their ligand(s).


   Acknowledgments
 
We thank Dr. Dennis Loh for making the DO11 TCR-Tg mice available and Andrea English for excellent technical support. We also thank Dr. Ken Rock for reagents and cell lines.


   Footnotes
 
1 This work was supported by Research Project Grant RPG-97-089-01-CIM from the American Cancer Society (to A.B.). Back

2 Address correspondence and reprint requests to Dr. Anil Bamezai, Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602. E-mail address: abamezai{at}cb.uga.edu Back

3 Abbreviations used in this paper: Tg, transgenic; RT, room temperature; cOVA, chicken OVA. Back

Received for publication June 21, 2001. Accepted for publication October 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, J. M. Green, C. B. Thompson, J. A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405.[Medline]
  2. Russell, J. H., B. Rush, C. Weaver, R. Wang. 1993. Mature T cells of autoimmune lpr/lpr mice have a defect in antigen-stimulated suicide. Proc. Natl. Acad. Sci. USA 90:4409.[Abstract/Free Full Text]
  3. Lenardo, M., K. M. Chan, F. Hornung, H. McFarland, R. Siegel, J. Wang, L. Zheng. 1999. Mature T lymphocyte apoptosis: immune regulation in a dynamic and unpredictable antigenic environment. Annu. Rev. Immunol. 17:221.[Medline]
  4. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270:985.[Abstract/Free Full Text]
  5. Watanabe-Fukinaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  6. Willerford, D. M., J. Chen, J. A. Ferry, L. Davidson, A. Ma, F. W. Alt. 1995. Interleukin-2 receptor {alpha} chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
  7. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P. S. Ohashi, H. Griesser, et al 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor {beta}. Science 268:1472.[Abstract/Free Full Text]
  8. Joazeiro, C. A., S. S. Wing, H. Huang, J. D. Leverson, T. Hunter, Y. C. Liu. 1999. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286:309.[Abstract/Free Full Text]
  9. Liou, J., F. Kiefer, A. Dang, A. Hashimoto, M. H. Cobb, T. Kurosaki, A. Weiss. 2000. HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1. Immunity 12:399.[Medline]
  10. Sosinowski, T., A. Pandey, V. M. Dixit, A. Weiss. 2000. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling. J. Exp. Med. 191:463.[Abstract/Free Full Text]
  11. Saito, T.. 1998. Negative regulation of T cell activation. Curr. Opin. Immunol. 10:313.[Medline]
  12. O’Garra, A., N. Arai. 2000. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell Biol. 10:542.[Medline]
  13. Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, M. Afkarian, T. L. Murphy. 2000. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18:451.[Medline]
  14. Wills-Karp, M.. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17:255.[Medline]
  15. Parronchi, P., E. Maggi, S. Romagnani. 1999. Redirecting Th2 responses in allergy. Curr. Top. Microbiol. Immunol. 238:27.[Medline]
  16. Locksley, R. M., D. J. Fowell, K. Shinkai, A. E. Wakil, D. Lacy, M. Bix. 1998. Development of CD4+ effector T cells and susceptibility to infectious diseases. Adv. Exp. Med. Biol. 452:45.[Medline]
  17. Morel, P. A., T. B. Oriss. 1998. Crossregulation between Th1 and Th2 cells. Crit. Rev. Immunol. 18:275.[Medline]
  18. Rock, K. L., H. Reiser, A. Bamezai, J. McGrew, B. Benacerraf. 1989. The Ly-6 locus: a multigene family encoding phosphatidyl-inositol-anchored membrane proteins concerned with T cell activation. Immunol. Rev. 111:195.[Medline]
  19. Gumley, T. P., I. F. McKenzie, M. S. Sandrin. 1995. Tissue expression, structure and function of the murine Ly-6 family of molecules. Immunol. Cell Biol. 73:277.[Medline]
  20. Kimura, S., N. Tada, Y. Liu-Lam, U. Hammerling. 1984. Studies of the mouse Ly-6 alloantigen system. II. Complexities of the Ly-6 region. Immunogenetics 20:47.[Medline]
  21. Lanki, D. W., E. I. Ma, W. Havran, F. Fitch. 1984. Cell surface structures involved in T cell activation. Immunol. Rev. 81:65.[Medline]
  22. Leo, O., M. Foo, D. M. Segal, E. Shevach, J. A. Bluestone. 1987. Activation of murine T lymphocytes with monoclonal antibodies: detection on Lyt-2+ cells of an antigen not associated with the TCR complex but involved in T cell activation. J. Immunol. 139:1214.[Abstract]
  23. Schlueter, A. J., T. R. Malek, C. N. Hostetler, P. A. Smith, P. deVries, T. J. Waldschmidt. 1997. Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression. J. Immunol. 158:4211.[Abstract]
  24. MacNeil, I., J. Kennedy, D. I. Goldfrey, N. A. Jenkins, C. Mineo, D. J. Gilbert, N. G. Copeland, R. L. Boyd, A. Zlotnik. 1993. Isolation of a cDNA encoding thymic shared antigen-1: a new member of the Ly-6 family with a possible role in T cell development. J. Immunol. 151:6913.[Abstract]
  25. Pflugh, D. L., S. E. Maher, A. L. Bothwell. 2000. Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression. J. Immunol. 165:313.[Abstract/Free Full Text]
  26. Jutila, M. A., F. G. Kroese, K. L. Jutila, A. M. Stall, S. Fiering, L. A. Herzenberg, E. L. Berg, E. C. Butcher. 1988. Ly-6C is a monocyte/macrophage and endothelial cell differentiation antigen regulated by interferon-{gamma}. Eur. J. Immunol. 18:1819.[Medline]
  27. Patterson, J. M., M. H. Johnson, D. B. Zimonjic, T. A. Graubert. 2000. Characterization of Ly-6M, a novel member of the Ly-6 family of hematopoietic proteins. Blood 95:3125.[Abstract/Free Full Text]
  28. Houlden, B. A., P. M. Hogarth, I. F. McKenzie. 1986. Interrelationships of the "Ly-6 complex" antigens. Immunogenetics 23:226.[Medline]
  29. Fleming, T. J., C. O’hUigin, T. R. Malek. 1993. Characterization of two novel Ly-6 genes: protein sequence and potential structural similarity to {alpha}-bungarotoxin and other neurotoxins. J. Immunol. 150:5379.[Abstract]
  30. Palfree, R. G.. 1996. Ly-6-domain proteins: new insights and new members: a C-terminal Ly-6 domain in sperm acrosomal protein SP-10. Tissue Antigens 48:71.[Medline]
  31. Chou, J. H., C. I. Bargmann, P. Sengupta. 2001. The Caenorhabditis elegans odr-2 gene encodes a novel Ly-6-related protein required for olfaction. Genetics 157:211.[Abstract/Free Full Text]
  32. Rock, K. L., E. T. H. Yeh, C. F. Gramm, S. I. Haber, H. Reiser, B. Benacerraf. 1986. TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes. J. Exp. Med. 163:315.[Abstract/Free Full Text]
  33. Malek, T. R., G. Ortega, R. A. Chan, R. A. Kroczek, E. M. Shevach. 1986. Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies. J. Exp. Med. 164:709.[Abstract/Free Full Text]
  34. Bamezai, A., K. L. Rock. 1995. Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells. Proc. Natl. Acad. Sci. USA 92:4294.[Abstract/Free Full Text]
  35. Brakenhoff, R. H., M. Gerretsen, E. M. Knippels, M. van Dijk, H. van Essen, D. O. Weghuis, R. J. Sinke, G. B. Snow, G. A. van Dongen. 1995. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion. J. Cell Biol. 129:1677.[Abstract/Free Full Text]
  36. Classon, B. J., R. L. Boyd. 1998. Thymic-shared antigen-1 (TSA-1): a lymphostromal cell membrane Ly-6 superfamily molecule with a putative role in cellular adhesion. Dev. Immunol. 6:149.[Medline]
  37. Yeh, E. T. H., H. Reiser, A. Bamezai, K. L. Rock. 1988. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell 52:665.[Medline]
  38. Flood, P. M., J. P. Dougherty, Y. Ron. 1990. Inhibition of Ly-6A antigen expression prevents T cell activation. J. Exp. Med. 172:115.[Abstract/Free Full Text]
  39. Bamezai, A., H. Reiser, K. L. Rock. 1988. T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP. J. Immunol. 141:1423.[Abstract]
  40. Sussman, J. J., T. Saito, E. M. Shevach, R. N. Germain, J. D. Ashwell. 1988. Thy-1- and Ly-6-mediated lymphokine production and growth inhibition of a T cell hybridoma require coexpression of the T cell antigen receptor complex. J. Immunol. 140:2520.[Abstract]
  41. Henderson, S. C., A. Berezovskaya, A. English, D. Palliser, K. L. Rock, A. Bamezai. 1998. CD4+ T cells mature in the absence of MHC class I and II expression in Ly-6A.2 transgenic mice. J. Immunol. 161:175.[Abstract/Free Full Text]
  42. Stanford, W. L., S. Haque, R. Alexander, X. M. Liu, A. M. Latour, H. R. Snodgrass, B. H. Koller, P. M. Flood. 1997. Altered proliferative response by T lymphocytes of Ly-6A (Sca-1) null mice. J. Exp. Med. 186:705.[Abstract/Free Full Text]
  43. Codias, E. K., J. E. Rutter, T. J. Fleming, T. R. Malek. 1990. Down-regulation of IL-2 production by activation of T cells through Ly-6A/E. J. Immunol. 145:1407.[Abstract]
  44. English, A., R. Kosoy, R. Pawlinski, A. Bamezai. 2000. A monoclonal antibody against the 66-kDa protein expressed in mouse spleen and thymus inhibits Ly-6A.2-dependent cell-cell adhesion. J. Immunol. 165:3763.[Abstract/Free Full Text]
  45. Apostolopoulos, J., I. F. McKenzie, M. S. Sandrin. 2000. Ly6d-L, a ligand for mouse Ly6d. Immunity 12:223.[Medline]
  46. Robey, E.. 1999. Regulation of T cell fate by Notch. Annu. Rev. Immunol. 17:283.[Medline]
  47. Bamezai, A., D. Palliser, A. Berezovskaya, J. McGrew, K. Higgins, E. Lacy, K. L. Rock. 1995. Regulated expression of Ly-6A.2 is important for T cell development. J. Immunol. 154:4233.[Abstract]
  48. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  49. Haskins, K., R. Kubo, J. White, M. Pigeon, J. Kappler, P. Marrack. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J. Exp. Med. 157:1149.[Abstract/Free Full Text]
  50. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature 356:607.[Medline]
  51. Freed, J. H., A. Marrs, J. VanderWall, P. L. Cohen, R. A. Eisenberg. 2000. MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus. J. Immunol. 164:4697.[Abstract/Free Full Text]
  52. Jevnikar, A. M., M. J. Grusby, L. H. Glimcher. 1994. Prevention of nephritis in major histocompatibility complex class II-deficient MRL-lpr mice. J. Exp. Med. 179:1137.[Abstract/Free Full Text]
  53. Christianson, G. J., R. L. Blankenburg, T. M. D. Marshak-Rothstein, D. C. Roopenian. 1996. {beta}2-Microglobulin dependence of the lupus-like autoimmune syndrome of MRL-lpr mice. J. Immunol. 156:4932.[Abstract]
  54. Morse, H. C. 3d, W. F. Davidson, R. A. Yetter, E. D. Murphy, J. B. Roths, R. L. Coffman. 1982. Abnormalities induced by the mutant gene Ipr: expansion of a unique lymphocyte subset. J. Immunol. 129:2612.[Abstract]
  55. Grynkiewicz, G., M. Poenie, R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440.[Abstract/Free Full Text]
  56. Dumont, F. J., R. Dijkmans, R. G. E. Palfree, R. D. Bolz, L. Coker. 1987. Selective upregulation of interferon-{gamma} of surface molecules of the Ly-6/E complex in resting T cells: the Ly-6A/E and TAP antigens are preferentially enhanced. Eur. J. Immunol. 17:1183.[Medline]
  57. Dumont, F. J., R. D. Boltz. 1987. The augmentation of surface Ly-6A/E molecules in activated T cells is mediated by endogenous interferon-{gamma}. J. Immunol. 139:4088.[Abstract]
  58. Lee, S. K., B. Su, S. E. Maher, A. L. Bothwell. 1994. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity. EMBO J. 13:2167.[Medline]
  59. Marmor, M. D., M. Julius. 2000. The function of GPI-anchored proteins in T cell development, activation and regulation of homeostasis. J. Biol. Regul. Homeostatic Agents 14:99.
  60. Noben-Trauth, N., J. Hu-Li, W. E. Paul. 2000. Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation. J. Immunol. 165:3620.[Abstract/Free Full Text]
  61. Constant, S. L., K. Bottomly. 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15:297.[Medline]
  62. Freeman, G. J., V. A. Boussiotis, A. Anumanthan, G. M. Bernstein, X. Y. Ke, P. D. Rennert, G. S. Gray, J. G. Gribben, L. M. Nadler. 1995. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 2:523.[Medline]
  63. Ranger, A. M., M. P. Das, V. K. Kuchroo, L. H. Glimcher. 1996. B7-2 (CD86) is essential for the development of IL-4-producing T cells. Int. Immunol. 8:1549.[Abstract/Free Full Text]
  64. Rulifson, I. C., A. I. Sperling, P. E. Fields, F. W. Fitch, J. A. Bluestone. 1997. CD28 costimulation promotes the production of Th2 cytokines. J. Immunol. 158:658.[Abstract]
  65. Corry, D. B., S. L. Reiner, P. S. Linsley, R. M. Locksley. 1994. Differential effects of blockade of CD28–B7 on the development of Th1 or Th2 effector cells in experimental leishmaniasis. J. Immunol. 153:4142.[Abstract]
  66. Brown, D. R., N. H. Moskowitz, N. Killeen, S. L. Reiner. 1997. A role for CD4 in peripheral T cell differentiation. J. Exp. Med. 186:101.[Abstract/Free Full Text]
  67. Fowell, D. J., J. Magram, C. W. Turck, N. Killeen, R. M. Locksley. 1997. Impaired Th2 subset development in the absence of CD4. Immunity 6:559.[Medline]
  68. Flynn, S., K. M. Toellner, C. Raykundalia, M. Goodall, P. Lane. 1998. CD4 T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1. J. Exp. Med. 188:297.[Abstract/Free Full Text]
  69. Ohshima, Y., L. P. Yang, T. Uchiyama, Y. Tanaka, P. Baum, M. Sergerie, P. Hermann, G. Delespesse. 1998. OX40 costimulation enhances interleukin-4 (IL-4) expression at priming and promotes the differentiation of naive human CD4+ T cells into high IL-4-producing effectors. Blood 92:3338.[Abstract/Free Full Text]
  70. Salomon, B., J. A. Bluestone. 1998. LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production. J. Immunol. 161:5138.[Abstract/Free Full Text]
  71. Luksch, C. R., O. Winqvist, M. E. Ozaki, L. Karlsson, M. R. Jackson, P. A. Peterson, S. R. Webb. 1999. Intercellular adhesion molecule-1 inhibits interleukin 4 production by naive T cells. Proc. Natl. Acad. Sci. USA 96:3023.[Abstract/Free Full Text]
  72. Kuchroo, V. K., M. P. Das, J. A. Brown, A. M. Ranger, S. S. Zamvil, R. A. Sobel, H. L. Weiner, N. Nabavi, L. H. Glimcher. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[Medline]
  73. Ruedl, C., M. F. Bachmann, M. Kopf. 2000. The antigen dose determines T helper subset development by regulation of CD40 ligand. Eur. J. Immunol. 30:2056.[Medline]
  74. Oosterwegel, M. A., D. A. Mandelbrot, S. D. Boyd, R. B. Lorsbach, D. Y. Jarrett, A. K. Abbas, A. H. Sharpe. 1999. The role of CTLA-4 in regulating Th2 differentiation. J. Immunol. 163:2634.