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Ly-6A.2 Expression Regulates -Specific CD4؉ Proliferation and Cytokine Production1

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

Ly-6 appear to serve cell adhesion and function, but the precise role of Ly-6A.2 in CD4؉ T 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 -Ag. Moreover, the Ly-6A.2-overexpressing CD4؉ cells generated elevated levels of IL-4, a key factor that propels the differen tiation 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 . 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 events. In contrast to the observed Ag-specific hypore- sponsiveness, 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. The Journal of , 2002, 168: 118Ð126.

rowth and differentiation of T cells following an encoun- these cytokines (e.g., IL-2) play a central role in clonal expansion, ter with an Ag are critical for proper immune function. whereas other cytokines produced influence differentiation of na- G The specificity of the clonal expansion and differentia- ive CD4ϩ T cells into either Th1 (IFN-␥) or Th2 (IL-4) subset tion of lymphocytes are determined by the interaction of TCR with (reviewed in Refs. 12 and 13). Therefore, understanding the influ- its ligand, a peptide-MHC complex expressed on the APCs. Clonal ence of accessory proteins on naive or activated CD4ϩ T cells in expansion of CD4ϩ T cells is tightly regulated, as unchecked generation of cytokines (type and quantity) that direct T cell dif- growth can cause toxic effector functions and unbalanced immune ferentiation is critical, especially when cytokine-induced differen- responses to foreign Ags. Although the mechanism of regulation of tiation can determine the kind of generated T cell growth remains largely unknown, previous studies suggest against a pathogen or (reviewed in Refs. 14Ð17). The role that surface expression of CTLA-4, Fas/APO-1, IL-2R proteins of accessory molecules expressed on naive or activated T cells in may regulate CD4ϩ T cell growth (1Ð3). Mice lacking expression generation of cytokines that in turn influence T cell differentiation of CTLA-4, Fas, or IL-2R show lymphoproliferation and abnormal remains unresolved. accumulation of activated T cells, suggesting their importance in The mouse Ly-6 locus encodes a family of GPI-anchored, de- regulating homeostasis of either naive and/or cycling T cells (4Ð7). velopmentally regulated cell surface proteins (reviewed in Refs. 18 Moreover, single gene mutations of Cbl, SLP, HPK1, and Csk in and 19). Members of Ly-6 gene family are excellent markers of mice are known to cause hyperproliferation of T cells and there- different lineages of hemopoietic origin, including lymphocytes fore suggest a role of these kinases in inhibiting T cell stimulation (20Ð25), (26, 27), marrow cells (20, 27, 28), and (8Ð11). The cell surface proteins that regulate these intracellular (29). There are shared motifs among the mouse Ly-6 kinases have not been identified. Many of these regulatory mole- proteins, including 8Ð10 conserved residues which are cules or growth inhibitory pathways may function in different T also found in human CD59, epidermal , urokinase cell subsets or at different stages of clonal expansion or even work plasminogen activator , squid Sgp-2, SP-10 (sperm Ag) synergistically. Mechanisms underlying these intricate regulations (reviewed in Ref. 30), snake neurotoxins/cytotoxins (29), and Cae- of clonal expansion following an encounter with foreign Ag re- norhabditis elegans odr-2 (31). All these proteins from different mains unclear. species have been grouped together into Ly-6 supergene family ϩ Stimulation of CD4 T cells with Ag initiates production of a based on their limited similarity and the presence of variety of cytokines including IL-2, IFN-␥, and IL-4. Some of conserved cysteine residues. Published reports have suggested role of Ly-6 in T cell signaling (32, 33) and cell adhesion (34Ð 36). Surface expression of Ly-6A.2 is important for immunore- Department of Cellular Biology, University of Georgia, Athens, GA 30602 sponsiveness of both T-T hybridomas (37) and normal T cells (38). Received for publication June 21, 2001. Accepted for publication October 25, 2001. Interestingly, surface expression of TCR/CD3 expression on T-T The costs of publication of this article were defrayed in part by the payment of page hybridomas is important for stimulation through the Ly-6 protein charges. This article must therefore be hereby marked advertisement in accordance (39, 40). Moreover, ectopic expression of Ly-6A.2 transgene on with 18 U.S.C. Section 1734 solely to indicate this fact. CD4ϩCD8ϩ promotes maturation of CD4ϩ (not 1 This work was supported by Research Project Grant RPG-97-089-01-CIM from the CD8ϩ) T cells in the in the absence of TCR-MHC inter- American Society (to A.B.). action (41). These data suggest that Ly-6A.2 expression influences 2 Address correspondence and reprint requests to Dr. Anil Bamezai, Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, cell growth and differentiation that are dependent or independent GA 30602. E-mail address: [email protected] of signaling through the Ag receptor. Contrary to some published

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 119

reports, the CD4ϩ T cells from Ly-6A mutant mice show a mod- Cell preparation estly higher proliferation in response to anti-CD3 Ab than their CD4ϩ T cells from Ly-6A.2 Tg or non-Tg mice were prepared from the controls (42). Moreover, Abs against Ly-6A.2 inhibit anti-CD3- lymph nodes. Lymph node cells were incubated with 100 ␮l of anti-CD8 induced IL-2 production by T-T hybridoma (43). The role of Ly- (3.155) and anti-MHC class II (M5/114) Abs for 30 min at 4¡C. Samples 6A.2 expression in Ag-specific response of primary CD4ϩ T cells were washed three times with the PBS supplemented with 0.1% BSA. remains untested and the mechanism by which Ly-6A.2 expression Following the washing step, cells were incubated with Dynal beads M-450 coupled with sheep anti-mouse IgG Ab, as per the manufacturer’s instruc- may augment or inhibit the TCR-induced activation is unknown. tions (Dynal Biotech, Oslo, Norway) for 45 min at 4¡C. Depletion of con- Ly-6A.2 and other members of the Ly-6 gene family, including taminating cells was achieved by magnetic separation, and purity of CD4ϩ E48 protein and Ly-6C, participate in cell-cell adhesion (35, 36). cells ranged from 85 to 95%. We recently reported biochemical characterization of a candidate ligand that binds Ly-6A.2 (44). The Ly-6A.2 candidate ligand is ϫ 6 ϩ expressed on the majority of B cells and (Refs. 34 A total of 1 10 lymph node or purified CD4 cells were incubated with anti-CD4-PE, anti-CD8-FITC, anti-Ly-6A/E (D7) (BD PharMingen, San and 44 and our unpublished data). Moreover, a ligand for Ly-6 day Diego, CA), and anti-DO11 TCR (KJ1-26) (49) Abs followed by appro- (Ly-6dL), another member of the Ly-6 gene family, was recently priate fluorochrome-conjugated second step reagents. Cells were analyzed identified that shows expression in almost all mouse tissues ana- on an EPICS Elite Analyzer flow cytometer (Beckman Coulter, lyzed (45). Ly-6dL shows similarity to Fullerton, CA). domain of mouse notch (motch-1) protein, known for its role in ELISA for detection of anti-DNA Abs and cytokines directing cell fate decisions in a variety of cell types during de- For detection of anti-DNA Abs, the microtiter wells were coated with velopment (reviewed in Ref. 46). These observations raise the pos- poly-L-lysine (25 ␮g/ml) for 24 h at 4¡C. Excess of poly-L-lysine was sibility that Ly-6 proteins may mediate cell function by binding to removed by washing with 0.1 M TBS containing 0.1% Tween 20 before a ligand, but the consequences of these Ly-6-ligand interactions coating of dsDNA at 5 ␮g/ml for2hatroom temperature (RT). Sera from are unknown. 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 To examine the role of Ly-6A.2 expression on the function of ϩ with protein G-alkaline phosphatase at 1/4000 dilution for1hatRTand CD4 T cells, we bred the Ly-6A.2 transgenic (Tg)3 mice with the was developed in the presence of the substrate, p-nitrophenyl DO11 TCR-Tg mice. In this study we report that the overexpres- phosphate. sion of Ly-6A.2 on CD4ϩ T cells inhibits responses initiated by For cytokine ELISA, microtiter wells (Costar, Cambridge MA) were coated with appropriate capture Ab in 0.1 M Na HPO binding buffer (pH the TCR in the presence of peptide Ag presented on APCs. Sur- 2 4 ϩ 9.0) overnight at 4¡C. Plates were washed five times with PBS plus 0.05% prisingly, the same Tg CD4 T cells are hyperresponsive to a Tween 20 and blocked with 100 ml of 1% BSA in PBS for 30 min at RT. combination of anti-TCR/CD3 and CD28 Abs, in the absence of After five washes, cytokine standards and samples diluted in blocking buff- APC. Our Ab against a candidate Ly-6A.2 ligand reversed the er/Tween 20 were added to wells for overnight incubation at 4¡C. Plates Ag-specific hyporesponsiveness. These observations suggest that 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 Ly-6A.2 expression exerts both inhibitory and activating roles de- ␮l of streptavidin-HRP conjugate (Vector Laboratories, Burlingame, CA) pending on how T cells are stimulated. Interaction of Ly-6A.2 with was added at a 1/2000 concentration. Plates were incubated for 30 min at a candidate Ly-6A.2 ligand negatively regulates T cell prolifera- RT and washed eight times before addition of the substrate 2,2Ј-azino-bis- tion. Moreover, Ly-6A.2 Tg DO11 CD4ϩ T cell primary cultures (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO). The assay was read at 405 nm by an ELISA reader. produce large amounts of IL-4 in response to the OVA323Ð339 pep- tide, suggesting that Ly-6A.2 expression may participate in differ- Ab immobilization ϩ entiation of CD4 T cells into Th2 subset. Microculture wells (Costar) were coated with either purified anti-CD3 (145-2C11), anti-CD28 (37N) (50), or anti-DO11 TCR (KJ1-26) for2hat Materials and Methods 37¡C in carbonate-bicarbonate buffer, pH 9.6. Unbound Abs were removed Mice by washing with 0.1 M PBS, pH 7.4, five times before the addition of the purified CD4ϩ T cells. Ly-6A.2 Tghigh and Tglow mice (4Ð6 wk of age) used in this study have been previously reported (47). The Ly-6A.2 Tg mice were bred to the Ag stimulation and cell culture DO11 TCR-Tg mice (48) (generous gift from Dr. D. Loh) and MRL-lpr/lpr A total of 1 ϫ 105 purified CD4ϩ T cells were cocultured with 5 ϫ 105 (The Jackson Laboratory, Bar Harbor, ME) for testing the role of Ly-6A.2 irradiated APCs in RPMI 1640-based culture medium (Irvine Scientific, expression in Ag-specific responses. Heterozygous Ly-6A.2 Tghigh and low Santa Ana, CA). The cultures were conducted in the presence of either Tg mice were bred with homozygous DO11 TCR-Tg mice and screened stimulating chicken OVA (cOVA) (SQAVHAAHAEINEAGRE) or for the presence of Ly-6A.2 Tg sequence as described before (47). The 323Ð339 ϩ nonstimulating cOVA324Ð334 (QAVHAAHAEIN) (synthesized at heterozygous Ly-6A.2 Tg DO11 TCR double Tg mice were used for Molecular Genetics Instrumentation Facility, University of Georgia, Ath- experiments, and littermates from the same breeding that expressed only ens, GA). The precise culture condition is listed in the appropriate figures. DO11 TCR transgene served as controls. Expression of DO11 TCR trans- Cultures were pulsed after 72 h with 1 ␮Ci of [3H]thymidine for the last gene was confirmed by staining with KJ1-26, anti-DO11 TCR Ab (49) 20 h of culture. (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 Calcium responses eight generations were used in the experiments. Following each backcross ϩ the mice expressing the mutated Fas gene were screened using forward CD4 T cells purified from the lymph nodes of BALB/c mice were loaded ␮ (5Ј-GTAAATAATTGTGCTTCGTCAG-3Ј) and reverse (5Ј-TAGAAAG with Indo-1 AM (Molecular Probes, Eugene, OR) at 2 g/ml final con- Ј centration. The cell loading was conducted at a cell concentration of 2 ϫ CTGCACGGGTGTG-3 ) primers yielding mutant fas (lpr/lpr) of 212 bp 6 and a wild-type (ϩ/ϩ) 184-bp PCR fragment. Seven- or eight-generation 10 cells/ml in loading medium consisting of HBSS with 1% BSA for 30 backcrossed mice on MRL-lpr/lpr background that only expressed Ly- min at 37¡C. Following incubation, cells were centrifuged for 8 min at 6A.2 transgene and possessed mutations of fas gene on both chromosomes 1000 rpm, and pellet was resuspended in cell loading medium to a final ϫ 6 (yielding only a 212-bp PCR fragment) were used for our experiments. concentration of 2 10 cells/ml. Cells were then stored at 22¡C and Mice with Fas mutations on both chromosomes (FasϪ/Ϫ) or one of two protected from light until analysis. Indo-1 AM-loaded cells were warmed chromosomes (Fasϩ/Ϫ) that did not express the Ly-6A.2 transgene and to 37¡C for 5Ð10 min before analysis. Agonists were added at varying time were generated in the same breeding served as controls. 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 3 Abbreviations used in this paper: Tg, transgenic; RT, room temperature; cOVA, of 10 ␮g/ml. Rabbit anti-mouse IgG and ionomycin were used at final chicken OVA. concentrations of 25 and 2 ␮g/ml, respectively. 120 REGULATORY ROLE OF Ly-6A.2 IN CD4ϩ T CELL CLONAL EXPANSION

Results CD3 mAb (Fig. 1B). Similar levels of hyporesponsiveness of Ly- ϩ Ly-6A.2-overexpressing CD4ϩ T cells are hyporesponsive to 6A.2 TghighCD4 T cells were observed when mitomycin-treated

cOVA323–339 peptide presented by splenic APC splenic cells were used to present the cOVA peptide (data not shown). Previous studies have suggested both activating and inhibitory ef- ϩ ϩ To examine whether the reduced proliferation in Ly-6A.2 Tg fects of Ly-6A.2 expression on CD4 T cell responses. Moreover, ϩ ϩ the role of Ly-6A.2 expression on Ag-specific T cell activation of DO11 Tg CD4 T cells was not due to a general unresponsive normal T cells is unclear. To clarify these issues and to fully un- (refractory) status of these cells, we tested the ability of the Ly- high ϩ derstand the role of Ly-6A.2 expression in Ag-specific responses 6A.2 Tg DO11 Tg CD4 T cells to proliferate in the presence we bred the Ly-6A.2high Tg mice to DO11 TCR-Tg mice. CD4ϩ of PMA and calcium ionophore, which induce receptor-inde- ϩ high T cells were isolated from the lymph nodes of Tg mice and tested pendent signaling in T cells. CD4 T cells from Ly-6A.2 Tg mice were fully responsive to activation by PMA and calcium for their responsiveness to cOVA323Ð339 peptide in the presence of irradiated syngeneic APC. Ly-6A.2 Tghigh DO11 TCRϩCD4ϩ T ionophore (Fig. 1B). Moreover, cross-linking of Ly-6A.2 with ϩ cells proliferated 7- to 8-fold lower than CD4ϩ T cells from Ly- an anti-Ly-6A.2 Ab induced higher proliferation in the CD4 ϩ 6A.2 TgϪ DO11 TCRϩ control littermates (Fig. 1A). Hyporespon- Ly-6A.2 Tg T cells than the CD4 T cells from non-Tg mice siveness of Ly-6A.2 TghighCD4ϩ T cells was observed at 1 ␮M (Fig. 1C). Higher proliferation was observed in cultures with ␮ ϫ 5 ϫ 5 (Fig. 1A) and lower (0.5 and 0.25 M) cOVA323Ð339 peptide con- 1.25 10 cells as compared with cultures with 0.6 10 cells centration (data not shown). DO11 TCR-Tg CD4ϩ T cells did not as expected. These results indicate that the hyporesponsiveness ϩ ϩ respond to cOVA324Ð334 control peptide as expected (Fig. 1A). observed in CD4 Ly-6 Tg T cells is OVA323Ð339 peptide-APC Ly-6A.2 TgϩCD4ϩ T cells also showed reduced responses to anti- specific.

FIGURE 1. Ag-specific responsiveness of the purified Ly-6A.2 Tg CD4ϩ T cells. Variable numbers of purified CD4ϩ T cells from Ly-6A.2 TgϪ (Ⅺ) or Ly-6A.2 Tghigh (f and Œ) mice were cocultured with 5 ϫ 105 irradiated syngeneic spleen cells. A, The cultures were conducted in the presence of either Œ Ⅺ f ␮ cOVA324Ð334 (control, ) or cOVA323Ð339 (specific peptide, and )ata1- M concentration. B, Cultures were also set up with anti-CD3 (145-2C11; ␮ ␮ 1/100 dilution of culture supernatant), cOVA323Ð339, or cOVA324Ð334 (1 M), or a combination of PMA (20 ng/ml) and ionomycin (0.125 g/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 TgϪ or Tghigh Ͻ mice did not proliferate when cultured with OVA323Ð329 in the absence of the irradiated syngeneic APC ( 350 cpm). A representative experiment of at least five independent experiments is shown. The Journal of Immunology 121

High expression of the Ly-6A.2 transgene ameliorates the mice do not proliferate in the presence of syngeneic APC (Fig. lymphoproliferative disorder in lpr/lpr mice 2C). These results indicate that the overexpression of Ly-6A.2 sup- Lymphoadenopathy and in the MRL-lpr/lpr mice presses lymphoproliferation and autoimmune phenotype in are consequences of spontaneous mutation in the Fas gene (5). T MRL-lpr/lpr mice. lymphocytes lacking the expression of Fas protein undergo acti- vation-mediated cell death after recognizing an unidentified au- ϩ Reduced proliferation of Ly-6A.2 Tg CD4 T cells in response toantigen bound to an appropriate MHC class II molecule (51). to peptide Ag requires interaction with APCs Interaction of the TCR with the self-MHC proteins is critical for initiation of this autoimmune phenotype (52, 53). We sought to The finding that Ly-6A.2 overexpression decreased peptide-stim- examine whether the overexpression of the Ly-6A.2 on T cells ulated but not the PMA plus calcium ionophore responses might from the lpr mice might inhibit autoreactivity through the TCR and suggest that the Ly-6A.2 affects signaling through the TCR or that reverse lymphoproliferative disorder in these mice. For this exper- a Ly-6A.2 ligand on the APC mediates this inhibition. To distin- ϩ iment the Ly-6A.2 Tghigh mice were backcrossed to the MRL-lpr/ guish between these two possibilities, purified CD4 T cells were lpr mice, and Ly-6A.2 Tghigh lpr/lpr and Ly-6A.2 TgϪ lpr/lpr cultured with anti-TCR plus anti-CD28 Abs bound to the micro- mice were analyzed. The TgϪ MRL-lpr/lpr mice develop large titer wells, in the absence of APCs. Previous experiments have lymph nodes at ϳ16Ð18 wk of age as expected. Interestingly, the demonstrated that cross-linking of TCR and CD28 with the Abs expression of high levels of Ly-6A.2 in the MRL-lpr/lpr mice directed against them was necessary and sufficient to activate naive ϩ results in lymph nodes of normal cellularity (Fig. 2A). Consistent T cells (50). Fig. 3B shows that CD4 T cells from DO11 TCR ϫ with the previous observation (54), the MRL-lpr/lpr mice have an Ly-6A.2 double Tg mice proliferate in response to the stimulation abnormal subset of CD4ϪCD8ϪThy-1ϩ T cells in the lymph node through the TCR and costimulatory molecule. Indeed, much higher (Fig. 2B). The abnormal subset is not observed in the Ly-6A.2 proliferation was observed with DO11 ϫ Ly-6A.2 double Tg T ϩ Tghigh MRL-lpr/lpr and normal mice (Fig. 2B). Moreover, relative cells in comparison to the responses of CD4 T cells from DO11 amounts of anti-DNA Ab (Ab titers), which is a signature of the TCR single Tg mice. Similar results were obtained with a combi- autoimmune phenotype, were significantly decreased in the lpr/lpr nation of plate-bound anti-DO11 TCR (KJ1-26) and anti-CD28 mice overexpressing Ly-6A.2 (Fig. 2D). These results suggest that (Fig. 3B) or anti-TCR (H57) and anti-CD28 (data not shown). ϩ the overexpression of Ly-6A.2 in the MRL-lpr/lpr mice suppresses Taken together these data suggest that CD4 T cells overexpress- the proliferation of T cells in the lymph node and reverses the ing Ly-6A.2 are capable of proliferating well (even more than the ϩ autoimmune phenotype. Consistent with the above observation is non-Tg CD4 T cells) in response to anti-TCR stimulation in the the finding that CD4ϩ T cells from Ly-6A.2 Tghigh MRL-lpr/lpr absence of APC. The Ag-specific hyporesponsiveness of Ly-6A.2 Tg CD4ϩ T cells occurs by a non-cell-autonomous mechanism that requires interactions with the APC.

FIGURE 2. Ly-6A.2 transgene expression ameliorates lymphoadenopa- thy in MRL-lpr/lpr mice. A, Cellularity of lymph nodes from normal, Ly- 6A.2 TgϪ/ϩ lpr, Ly-6A.2 TgϪ 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. FIGURE 3. Responsiveness of CD4ϩ T cells from DO11 TCR Ly-6A.2 CD4ϩCD8Ϫ, CD4ϪCD8ϩ, and CD4ϪCD8Ϫ subsets were gated, and the double Tg mice through Ag receptor and costimulatory molecule in the number of cells present in the gates (n ϭ 3 experiments) are shown. C, absence of APCs. Purified CD4ϩ T cells (1 ϫ 105) from the Ly-6A.2 TgϪ Lymph node cells (5 ϫ 105) were cultured with 5 ϫ 105 irradiated syn- (open symbols) or the Ly-6A.2 Tghigh (closed symbols) mice were cultured geneic splenic APC for 6 or 8 days followed by pulsing with [3H]thymidine in wells coated with different concentrations of anti-CD3 (145-2C11) (A) prior to the last 6Ð8 h of the culture. The data are expressed as the arith- alone (circles) or in combination with anti-CD28 (37N) at 10 ␮g/ml con- metic mean counts per minute of [3H]thymidine incorporated Ϯ SD (n ϭ centration (diamonds); or anti-TCR (KJ1-26) (B) alone (circles) or in com- 3 experiments). D, Anti-dsDNA Ab titers (1/10 to 1/103) were analyzed bination with 10 ␮g/ml of anti-CD28 (37N) (diamonds). Cells were pulsed from the sera of Ly-6A.2 TgϪ lpr/lpr (Ⅺ) or Ly-6A.2 Tgϩ lpr/lpr (f) mice and harvested after 48 h of culture. The data are expressed as arithmetic by ELISA as described in Materials and Methods. The pooled normal mean counts per minute of [3H]thymidine incorporated Ϯ SD. A repre- mouse serum was used as control (E). sentative experiment of five experiments is shown. 122 REGULATORY ROLE OF Ly-6A.2 IN CD4ϩ T CELL CLONAL EXPANSION

Reduced Ca2ϩ responses in CD4ϩ 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 Ca2ϩ 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 fluorocy- tometer and data are displayed as a ratio of emission fluorescence at 395 and 510 nm as function of time. Lower Ca2ϩ responses were observed in Ly-6A.2 Tg CD4ϩ T cells than in non-Tg CD4ϩ T cell controls (Fig. 4B). Both the mean intensity of Ca2ϩ flux (Fig. 4B) as well as the total number of Ly-6A.2 TgϩCD4ϩ T cells responding (data not shown) to TCR/CD3 stimulation were sig- nificantly reduced. The lack of any Ca2ϩ response with the control Ab indicates the specificity of the responses observed (Fig. 4A). These results suggest that Ly-6A.2 expression affects early signal- ing events.

Cytokine production by Ly-6A.2 Tg CD4ϩ T cells Impaired anti-TCR-mediated Ca2ϩ responses and proliferation of Ly-6A.2 Tg CD4ϩ 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. 5A), IFN-␥ (Fig. 5B), and IL-4 (Fig. 5C). 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 CD4ϩ 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. 5A). In contrast, the FIGURE 5. Ly-6A.2 Tg CD4ϩ T cells produce elevated levels of IL-4 ϩ ϩ Ly-6A.2 Tg CD4 T cells produced comparable or even more in response to OVA323Ð339 presented by splenic APC. Purified CD4 T IFN-␥ than their non-Tg controls (Fig. 5B). To our surprise the cells (1 ϫ 105) from either non-Tg (᭛) or Ly-6A.2 Tghigh (᭜) were stim- ϩ CD4 T cells from Ly-6A.2 Tg mice generated more IL-4 in re- ulated with cOVA323Ð339 peptide as described in Fig. 1 and Materials and ␥ sponse to the OVA peptide than their non-Tg controls (Fig. Methods. IL-2 (A), IFN- (B), and IL-4 (C) were quantitated in superna- 323Ð339 tants from day-1 to -3 cultures by ELISA as described in Materials and 5C). High amounts of IL-4 were detected in day-2 and -3 cultures, Methods. Each data point is expressed as arithmetic mean value Ϯ SD (n ϭ suggesting that Ly-6A.2 transgene expression promotes generation 2). A representative experiment of three experiments is shown. of IL-4 that is undetectable in the primary cultures in which CD4ϩ T cells were stimulated through the Ag receptor (our detection sensitivity, 62.5 pg/ml). Taken together these results suggest that ϩ differentiation of naive T cells by producing elevated levels Ly-6A.2 expression influences CD4 T cell growth by inhibiting of IL-4. full production of IL-2. Ly-6A.2 expression may also influence the The elevated production of IL-4 by Ly-6A.2 Tg CD4ϩ 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 CD4ϩ T cell with APCs or reflected intrinsic ability of these cells to produce IL-4 in response to TCR stimula- tion we stimulated CD4ϩ cells with anti-CD3 and anti-CD28 in the absence of APC. Plate-bound anti-CD3 Ab alone or in combina- tion with plate-bound anti-CD28 induce large amounts of IL-4 in DO11 ϫ Ly-6A.2 double Tg CD4ϩ T cells and not in the DO11 TCR single Tg controls (Fig. 6A). Similar observations were made when anti-DO11 TCR Ab was used either alone or in combination with anti-CD28 (Fig. 6B). These observations strongly suggest that the ability of IL-4 production by Ly-6A.2 Tg T cells is induced by ϩ FIGURE 4. Overexpression of Ly-6A.2 on CD4 T cells inhibits cal- a cell-autonomous mechanism that does not require interaction ϩ cium responses mediated through the TCR complex. Purified CD4 T cells with APC. from either Ly-6TgϪ (dashed line) or Ly-6Tgϩ (solid line) were loaded with Indo-1 and exposed to control (anti-H-2Kk)(A)at10␮g/ml, Role of Ly-6A.2-ligand interactions in Ag-specific inhibition of or anti-CD3 (B) followed by rabbit anti-mouse IgG (cross-reactive with proliferation and IL-4 production by Ly-6A.2 Tg CD4ϩ T cells hamster IgG). Mean calcium response (ratio of 395:510 nm) by the stim- ulated cells is shown and total calcium present in the cells is evaluated by We have generated mAb against the Ly-6A.2 ligand. This Ab rec- addition of ionomycin at a 2 ␮g/ml concentration. One representative ex- ognizes a 66-kDa protein expressed in the majority of professional periment of three experiments is shown. APCs in the spleen and blocks the binding of a candidate ligand The Journal of Immunology 123

FIGURE 6. Ly-6A.2 Tg CD4ϩ T cells produce elevated levels of IL-4 by a cell-autonomous mechanism in the absence of APC. IL-4 was quan- titated in supernatants from day-3 cultures of either Ly-6TgϪ (open sym- bols) or Ly-6A.2 Tg (closed symbols) mice stimulated with either anti-CD3 FIGURE 7. Role of the candidate Ly-6A.2 ligand in OVA pep- alone (diamonds) or a combination of anti-CD3 and anti-CD28 (circles) in 323Ð339 tide-specific CD4ϩ T cell proliferation and IL-4 production. A, Purified the absence of APC by ELISA as described in Materials and Methods. CD4ϩ T cells from the Ly-6A.2 TgϪ (open bars) or Ly-6A.2 Tghigh Each data point is expressed as arithmetic mean value Ϯ SD (n ϭ 2). A (hatched bars) mice were cocultured with 5 ϫ 105 irradiated syngeneic representative experiment of three experiments is shown. spleen cells. These cultures were conducted in the presence of either ␮ cOVA324Ð334 (control) or cOVA323Ð339 (specific-peptide) at a 1 M con- expressing cells to Ly-6A.2-overexpressing Chinese hamster ovary centration either in the absence or in the presence of anti-ligand (9AB2, 1/4 cells (44). We sought to test whether the anti-Ly-6A.2 ligand Ab dilution of supernatant) or control hamster (9E3 at 1/4 dilution of super- natant) Ab. Cells were harvested after 72 h of culture. The data are ex- reversed the peptide-specific hyporesponsiveness that was ob- 3 served with Ly-6A.2 Tg CD4ϩ T cells. Fig. 7A shows that an Ab pressed as the arithmetic mean counts per minute of [ H]thymidine incor- porated Ϯ SD (n ϭ 2). A representative experiment of four independent against the candidate Ly-6A.2 ligand (9AB2), but not the hamster experiments is shown. B, IL-4 production was quantitated in the superna- control Ab (9E3), reverses this inhibition. These results strongly ϩ tants of these day-3 cultures with the OVA323Ð339 peptide as described in suggest that inhibition of CD4 T cells to peptide Ag may be Materials and Methods. A representative experiment of three experiments mediated through the interaction of the overexpressed Ly-6A.2 is shown. with a candidate Ly-6A.2 ligand expressed on the APC. In con- trast, the presence of anti-Ly-6A.2 ligand Ab did not alter the

production of IL-4 in response to OVA323Ð339 peptide (Fig. 7B). These later results are consistent with our observation that IL-4 Our previous reports suggest the presence of Ly-6A.2 ligand on production is independent of APC (Fig. 6). Taken together these the majority of B cells and macrophages (34, 44). APC-dependent data suggest that hyporesponsiveness and elevated production of inhibition of CD4ϩ Tg T cell responses to peptide Ag is consistent IL-4 show differential dependence on ligand interaction. with these published data, because splenic APC is comprised of B cells and macrophages. Suppression of lymphoproliferative disor- Discussion der and autoimmune symptoms in the Ly-6A.2 Tgϩ lpr/lpr mice Previous reports suggest that mouse Ly-6 proteins have both cell that are normally observed in MRL-lpr/lpr mice in the absence of adhesion (34) and cell signaling (32, 33) function. Although the Ly-6A.2 transgene supports these in vitro observations. The inhib- ligands that might bind Ly-6 proteins have been proposed (44, 45), itory responses are proportional to the level of Ly-6A.2 transgene the functional consequence of their interaction with Ly-6 proteins expression, inasmuch as 10-fold lower transgene expression on remains unclear. We demonstrate that Ly-6A.2 expression nega- peripheral CD4ϩ T cells (from Ly-6A.2 Tglow mice) showed re- ϩ tively regulates Ag-specific CD4 T cell responses by interacting duced proliferation in response to OVA323Ð339 peptide (ranging with a candidate ligand. Ly-6A.2 Tg CD4ϩ T cells show lower from 0 to 40% inhibition compared with the wild-type controls) Ca2ϩ fluxes and IL-2 production than CD4ϩ T cells from non-Tg (our unpublished observations). Consistent with this finding is the controls in response to TCR stimulation. To our surprise the pri- observation that Ly-6A.2low lpr/lpr mice did not show reduced mary cultures of Ly-6A.2 Tg CD4ϩ T cells generated IL-4 in re- lymphoproliferative disorder (our unpublished observation). These sponse to peptide stimulation that was independent of interaction results corroborate a previously published report that shows a with the candidate Ly-6A.2 ligand. These data suggest that Ly- modest hyperresponsiveness of T cells lacking expression of Ly- 6A.2 expression regulates T cell proliferation and possibly differ- 6A.2 knockout mice in response to anti-CD3 stimulation (35). entiation of CD4ϩ T cells after encountering an Ag. These data Taken together these observations demonstrate that Ly-6A.2 over- also provide functional evidence of a candidate ligand expressed expression inhibits Ag-specific responses of CD4ϩ T cells that are on APC that is recognized by our recently reported hamster primarily mediated by interaction of Ly-6A.2 with its candidate anti-ligand Ab. ligand. 124 REGULATORY ROLE OF Ly-6A.2 IN CD4ϩ T CELL CLONAL EXPANSION

Marked peptide-specific hyporesponsiveness and amelioration CTLA-4, Fas, and IL-2R on T cells exerts their role in regulation of the lymphoproliferative disorder in lpr/lpr mice was only ob- of homeostasis of naive or Ag-stimulated T cells. A number of served with high levels of Ly-6A.2 transgene expression. The ex- published studies have suggested a growth inhibitory role of GPI- pression of Ly-6A.2 on naive CD4ϩ T cells from Tghigh mice is anchored proteins (including Ly-6) (reviewed in Ref. 59). Our re- 100- to 200-fold higher than the expression of the endogenous sults suggest that interaction of Ly-6A.2 with the candidate ligand levels that could be considered as nonphysiological. However, en- inhibits T cell proliferation and therefore regulates clonal expan- dogenous Ly-6A.2 on non-Tg T cells is inducible and achieves sion of T cells following their encounter with a foreign Ag. These similar levels of expression as on Tg T cells when activated later observations are consistent with the expression pattern of Ly- through the Ag receptor or stimulated with IFN-␥ alone (Refs. 56 6A.2. Naive CD4ϩ T cells express low levels of Ly-6A.2 protein and 57 and our unpublished data). Therefore, we favor the phys- that is profoundly increased (100- to 200-fold) upon T cell acti- iologic relevance of our Tg model. We postulate that these high vation and by treatment with type I and type II IFNs (Refs. 46 and levels are achieved physiologically during the immune response 47 and our unpublished observations). We propose that Ly-6A.2- against a foreign Ag and that up-regulated Ly-6A.2-ligand inter- ligand interactions do not affect the initiation of T cell proliferation actions exert their inhibitory affect after sufficient clonal expansion but instead down-regulate their proliferation when high-level ex- and effector functions have been generated by the CD4ϩ Th cells. pression of endogenous Ly-6A.2 is achieved following T cell ac- Future in vitro and in vivo experiments will directly address this tivation. These results may suggest that the regulation of T cell issue. proliferation may occur once the proliferating T cells have per- Analyses of cell lines and normal T cells suggest either an ac- formed their effector function. Further studies are needed to de- tivating (38, 39, 58) or an inhibitory (42, 43) role of the Ly-6A termine the precise stage at which Ly-6A.2 expression contributes protein. It is unclear why the expression of Ly-6A.2 has these to T cell proliferation. opposing affects. Our data may provide some insights into these To begin to understand the mechanism of Ag-specific inhibitory apparently contradictory observations. Our results suggest that one responses of CD4ϩ T cells overexpressing Ly-6A.2 protein, we candidate Ly-6A.2 ligand expressed on splenic APC can inhibit focused on early signaling events. We report that Ca2ϩ responses TCR-mediated responses in CD4ϩ T cells by interacting with Ly- are significantly affected. Additional experiments are needed to 6A.2 (Figs. 1 and 2), whereas the same Ly-6A.2 Tg CD4ϩ T cell precisely determine the mechanism of reduced Ca2ϩ fluxes in Ly- shows hyperresponses to TCR signaling (an opposite outcome) in 6A.2 Tg cells. We suspected that reduced initial signaling in T the absence of Ly-6A.2-ligand interactions (Fig. 3). These data do cells would affect IL-2 production. To our surprise, the production not rule out the possibility that hyperresponsiveness may also be of IL-2 on days 1 and 2 of T cell response was unaltered. Signif- induced by interaction with another Ly-6 ligand. Interaction of icant effects were observed in the production of this growth factor Ly-6A.2 with a recently reported ligand for Ly-6d (45), another on day 3 of the culture. It is possible that production of IL-2 is member of Ly-6 gene family, has not been examined. Taken to- reduced on days 1 and 2 of the culture, but our assays were unable gether our results suggest that hypo- or hyperproliferation of Ly- to detect these differences. These results suggest that overall IL-2 6A.2 in Ly-6A.2 Tg CD4ϩ T cells may depend on how these cells production is significantly diminished, therefore reducing the are stimulated. clonal expansion of T cells. Reduced production of IL-2 was ob- We have used anti-candidate ligand Ab to block Ly-6A.2-ligand served on day 3 but not days 1 and 2 of the culture, suggesting that interactions, and it remains a possibility that these experimental regulation of T cell proliferation by Ly-6A.2 may occur at a later systems are nonphysiologic, even though the Abs have provided stage where normal expression is highly up-regulated (typically insights into the functions of a number of other reported receptor- 48 h after the initial culture with Ag). Alternatively, the Ly-6A.2- ligand interactions. Therefore, the use of soluble Ly-6A.2 protein ligand interactions alter signaling through the IL-2R and therefore to block interactions of Ly-6A.2 with its ligand should provide inhibit growth of T cells regardless of IL-2 production, as has been insights into the physiologic role of Ly-6A.2. Unfortunately, the previously observed with Abs against GPI-anchored proteins (re- Ly-6A.2-IgG (dimer) is unable to recognize the candidate ligand viewed in Ref. 59). Further experimentation is needed to com- on splenic APCs and does not inhibit Ly-6A.2-dependent cell-cell pletely and precisely address this question. adhesion, rendering this reagent unusable to address this issue. How cell proteins expressed on naive or activated T cells par- Whether the absence of Ly-6A.2-IgG1 binding to the candidate ticipate in T cell differentiation is not entirely clear. A number of binding is due to its low affinity is not known. Future studies with factors influence development of Th1 and Th2 effector T cells, a highly multimerized form of Ly-6A.2 may address this issue. In including relative concentration of cytokines present. IL-4 is a key results obtained with two independent experimental systems, our regulator of differentiation of naive CD4ϩ T cells into Th2 subset, overexpression (Tg) and Ly-6A knockout mice (42) are consistent whereas IL-12 and IFN-␥ promote differentiation into Th1 effector with each other, and Ab directed against the candidate ligand re- cell. The Th1 and Th2 differentiation factors are derived from var- versed the Ag-specific hyporesponsiveness (Fig. 7); therefore, we ied sources, including macrophages and dendritic cells (IL-12), favor the interpretation that interaction of Ly-6A.2 with the can- NK cells, and T cells (IFN-␥). T cells are known to generate initial didate ligand mediates inhibition of CD4ϩ T cell clonal expansion. bursts of IL-4, perhaps not in large enough amounts to be detect- The Ag-stimulated T cells double every 4.5 h and therefore have able in the primary cultures (60). In addition, the nature, dose, and a potential to generate 1 ϫ 1012 cells in 1 wk. This profound route of administration of the Ag, as well as the nature of inter- proliferation compounded with limited space available in the lym- acting APC, influence the generation of differentiation factors (re- phoid compartment may potentiate the toxic effects and autoim- viewed in Ref. 61). Some recent reports suggest that interaction of mune consequences; therefore, these processes are under tight reg- CD28 with B7-2 (62Ð65), CD4 with MHC class II (66, 67), and ulation. Up-regulation of CTLA-4 and Fas on activated CD4ϩ T OX-40 with OX-40 ligand (68, 69) promote differentiation to Th2 cells is known to negatively regulate T cell proliferation (1, 2). but not to Th1 cells. In contrast, interaction of LFA-1 with IL-2 has been recognized as a T cell growth factor, but recent ICAM-1 or -2 (70, 71), CD28 with B7-1 (62, 72), and CD40 with observations also suggest its importance in propriocidal regulation CD40L (73) promote Th1 differentiation. Moreover, naive T cells of T cell growth by inducing in cycling T cells (reviewed lacking expression of CTLA-4 differentiate into Th2 subset, sug- in Ref. 3). These results strongly suggest that the expression of gesting a role of CTLA-4 in the Th1 differentiation pathway (74). The Journal of Immunology 125

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