Down-Regulates IL-2 Receptor Association with Src Homology Protein-1 and and Proliferation in Human T Cells by Adhesion Molecule

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The Cell-Cell Adhesion Molecule Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Inhibits IL-2 Production and Proliferation in Human T Cells by This information is current as Association with Src Homology Protein-1 and of September 26, 2021. Down-Regulates IL-2 Receptor Charng-Jui Chen and John E. Shively

J Immunol 2004; 172:3544-3552; ; Downloaded from doi: 10.4049/jimmunol.172.6.3544 http://www.jimmunol.org/content/172/6/3544

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Cell-Cell Adhesion Molecule Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Inhibits IL-2 Production and Proliferation in Human T Cells by Association with Src Homology Protein-1 and Down-Regulates IL-2 Receptor1

Charng-Jui Chen and John E. Shively2

The cell adhesion molecule, carcinoembryonic Ag-related cellular adhesion molecule 1, shown by others to both activate and inhibit T cell proliferation, exhibits a reciprocal relationship to IL-2R expression over the time course of activation of PBMCs, and upon Ab ligation, inhibits both the production of IL-2 and cell proliferation. Carcinoembryonic Ag-related cellular adhesion Downloaded from molecule 1 associates with CD3 and is found in lipid rafts of PBMCs, is phosphorylated on the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of the -4L isoform, and associates with Src homology protein-1, providing an explanation for its inhibitory activity. When the ITIM-containing -4L and non-ITIM-containing -4S isoforms are transfected into Jurkat cells that produce, but do not depend on IL-2 for growth, both IL-2 production and cell proliferation are differentially inhibited, demonstrating that the two isoforms signal via different pathways. When the two isoforms are transfected into Kit-225 cells that depend on IL-2 for growth, IL-2R␤ and ␥, but not ␣ subunits are down-regulated, and the -4L, but not the -4S isoform inhibits cell http://www.jimmunol.org/ proliferation by 6-fold in an IL-2 dose-response study. The Journal of Immunology, 2004, 172: 3544–3552.

arcinoembryonic Ag-related cellular adhesion molecule by protein kinase C on Ser and Thr residues (11) and binds cal- 1 (CEACAM1)3 or CD66a is a type I membrane protein modulin (CaM) (11) and both actin and tropomyosin (12). Re- C and cell-cell adhesion molecule expressed in epithelial cently, we have shown that CEACAM1-4S reverts breast cancer and a variety of hemopoietic cells (1). CEACAM1 is expressed as epithelial cells to a normal morphology and mediates lumen for- four major isoforms, including three or four Ig-like ectodomains mation in a three-dimensional culture (13).

with short (e.g., CEACAM1-4S) or long (e.g., CEACAM1-4L) CEACAM1 functions as the cell surface receptor for mouse by guest on September 26, 2021 cytoplasmic domains (2). The distal N-terminal domain, an IgV- hepatitis virus in mouse (14) and a bacterial receptor for opacity- like domain, mediates its cell-cell adhesion properties (3). The associated (Opa)-positive neisseriae strains (15) and for Hae- 73-aa-long cytoplasmic domain contains two immunoreceptor ty- mophilus influenzae (16) in humans. CEACAM1 is up-regulated in rosine-based inhibitory motifs (ITIM) that have been shown to activated mouse and human T cells (17Ð19), suggesting a role in mediate inhibitory functions in epithelial cells (4). The long cyto- immunity. However, these studies have provided conflicting data plasmic domain has been shown to associate with actin and signal as to its role as either an activating or inhibitory molecule. The via the rho family of GTPases (5). Src family kinases have been binding of Neisseria to CEACAM1 on activated human T cells reported to phosphorylate the Tyr residues in the ITIM (6, 7). The was shown to arrest further activation and proliferation of T cells, inhibitory activity of the long cytoplasmic domain is most likely providing a case for inhibition (20). However, in intestinal T cells, mediated by the binding of Src homology protein-1/2 (SHP-1/2) a case has been made for both activation (21) and inhibition (22), via its Src homology 2 domains to the phosphorylated ITIM (4, 8). and decidual lymphocytes that express CEACAM1 are inhibited A critical Ser residue (S-503 in rat CEACAM1-4L) has also been by extravillous trophoblast cells that also express CEACAM1, per- identified in the long cytoplasmic domain that controls signaling haps leading to protection of embryonic tissue from immune attack via the insulin receptor in rat hepatocytes (9, 10). The 14-aa-short (23). These observations, plus the unexplained role of a ho- cytoplasmic domain lacks Tyr residues, but can be phosphorylated mophilic cell-cell adhesion in activated T cells, prompted us to further investigate the activity of CEACAM1 and its long and Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, short cytoplasmic domain isoforms in model systems. We demon- CA 91010 strate that the time course of expression of CEACAM1 vs IL-2R␣ Received for publication June 30, 2003. Accepted for publication January 15, 2004. in PBMCs follows reciprocal kinetics, that the -4L, but not the -4S The costs of publication of this article were defrayed in part by the payment of page isoform inhibits IL-2 production in transfected Jurkat cells, and charges. This article must therefore be hereby marked advertisement in accordance that the -4L, but not the -4S isoform inhibits proliferation in trans- with 18 U.S.C. Section 1734 solely to indicate this fact. fected Kit-225 cells. 1 This research was supported by National Institutes of Health Grant CA 84202. 2 Address correspondence and reprint requests to Dr. John E. Shively, Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: CEACAM1, carcinoembryonic Ag-related cellular Cells, cell culture, and transfections adhesion molecule 1; CaM, calmodulin; eGFP, enhanced green fluorescent protein; EVT, extravillous trophoblast; IP, immunoprecipitate; ITIM, immunoreceptor ty- Human PBMC were purified by Ficoll/Hypaque gradient centrifugation rosine-based inhibitory motif; mIg, mouse Ig; SHP, Src homology protein. and filtration through glass wool from whole blood or discard filters used

to collect platelets in the blood donor center. Puriﬁed PBMCs were stim- Immunoprecipitations ulated once with 5 ␮g/ml PHA plus 50 U of IL-2 (Roche, Basel, Switzer- land). Cells were split every 2Ð3 days and supplemented with 100 PBMCs grown in 100 U/ml IL-2 were treated for 10 min with 2 nM U/ml IL-2. calyculin A and 40 pM cypermetherin, or PMA (20 ng/ml), or pervanadate Jurkat cells were from American Type Culture Collection (Manassas, (1/100 dilution of 10 mM Na3VO4 plus1MH2O2), and then with T84.1 (2 ␮ VA), and Kit-225 cells were provided by J. Siegel (Center for Biologics g/ml, 2 min). Cells were lysed (50 mM Tris-HCl, pH 7.6, 150 mM NaCl2,2 ␮ ␮ Evaluation and Research, Food and Drug Administration). Stable cell lines mM EDTA, 1 mM PMSF, 1 g/ml leupeptin, 1 g/ml aprotinin, 1% Nonidet were obtained by transfection with human CEACAM1-4L or -4S (12, 13). P-40, 1 mM Na3VO4), precleared with protein A-agarose plus rabbit IgG, and Expression levels of the two isoforms were equivalent, as measured by treated with rabbit anti-CEACAM1 22-9. Immunoprecipitated proteins were FACS and Western blot analysis (data not shown). A CEACAM1-4L/en- resolved by SDS gel electrophoresis and Western blotted with appropriate hanced green ﬂuorscent protein (eGFP) fusion protein was constructed by Abs. Kit-225 cells were IL-2 depleted for 72 h, treated with IL-2 (500 U/ml, inserting eGFP after the C terminus of CEACAM1-4L with an intervening 30 min), lysed, precleared with protein G-agarose, and incubated with PY20- agarose. Immunoprecipitated proteins were resolved by SDS gel electrophore- Gly4 linker and stable tranfectants established. Expression levels were determined by FACS (see Fig. 5). sis and Western blotted with appropriate Abs.

Abs and confocal microscopy Results Kinetics of induction of IL-2R␣ and CEACAM1 on activated Anti-CD3⑀ was from BD Biosciences (San Diego, CA); rabbit anti-ZAP-70, anti-SHP-1, anti-SHP-2, anti-Jak 3, anti-IL-2R, and PY20 were from Santa PBMCs Cruz Biotechnology (Santa Cruz, CA); 4G10 was from Upstate Biotechnology Freshly puriﬁed PBMCs were activated with PHA and IL-2, and ␣ (Lake Placid, NY); anti-IL-2R was from BioSource International (Camarillo, the kinetics of induction of CEACAM1 and IL-2R␣ was deter- CA); and anti-IL-2R␤ and anti-IL-2R␥ were from BD PharMinigen (San Di-

ego, CA). Anti-CEACAM1 T84.1 (24) and rabbit Ab 22-9 to the long cyto- mined (Fig. 1A). Both receptors were barely detectable at the start Downloaded from plasmic domain (12) have been previously described. Anti-CEACAM1 4/3/17 of the treatment, with IL-2R␣ increasing more rapidly than was a gift from F. Grunert (VacGene, Freiburg, Germany) (25, 26). Confocal CEACAM1 through the ﬁrst 3 days of treatment. IL-2R␣ expres- microscopy was performed on a Zeiss (Oberkochen, Germany) model 310. sion peaked at 3 days, at which point CEACAM1 expression reached less than 20% maximal induction. As CEACAM1 expres- FACS analysis of PBMCs and transfected Kit-225 cells sion climbed from days 3 to 7Ð9 by 5-fold, IL-2R␣ expression CEACAM1 and IL-2R␣ on PBMCs were stained with 1 ␮g/ml anti- decreased by over 4-fold. Thus, the two receptors exhibit recipro- http://www.jimmunol.org/ CEACAM1 T84.1 or anti-IL-2R␣, followed by FITC-conjugated goat anti- cal kinetics, suggesting a possible causal relationship. Because ␣ mouse IgG (mIgG). Kit-225 cells were stained with FITC anti-IL-2R (2 IL-2R controls the response to IL-2, we investigated the IL-2 re- ␮g/ml) and PE anti-IL-2R␥ (2 ␮g/ml). CEACAM1 and IL-2␤ were stained with T84.1 (1 ␮g/ml) or anti-IL-2R␤ (2 ␮g/ml), followed by Alexa 488 sponse in more detail. goat anti-mIgG. Anti-CEACAM1 Abs inhibit IL-2 production and proliferation of Proliferation assays activated PBMCs PBMCs were grown in 96-well plates (2.5 ϫ 104cells/200 ␮L). Cells were PBMCs were stimulated with anti-CD3⑀ and/or anti-CD28 Abs in treated with anti-CD28 (2.5 ␮g/ml), anti-CEACAM1 (T84.1 or 4/3/17, 2.5 the presence or absence of anti-CEACAM1 Abs, and IL-2 produc-

␮g/ml) Ϯ anti-CD3⑀, plus mIgG to provide equivalent Ab concentrations. tion was measured (Fig. 1B). The results show that either anti- by guest on September 26, 2021 ␮ 3 After 3 days, 0.5 Ci of [ H]thymidine was added for 5 h and cells were CEACAM1 Ab T84.1 or 4/3/17 inhibit IL-2 production below max- harvested on glass fiber filters (Wallac (Gaithersburg, MD) Cell Harvester), and cpm incorporation was determined (Wallac 1450 Microbeta Trilux imal levels obtained with mIgG control-treated cells when stimulated Counter). Proliferation of transfected Jurkat cells was assayed similarly with either of two levels of anti-CD3⑀ Ab. As expected, combined with the addition of rabbit anti-mIgG (10 ␮g/ml) to induce cross-linking. treatment with anti-CD3⑀ and anti-CD28 gave maximal IL-2 produc- Kit-225 cells were IL-2 depleted for 72 h, plated in 96-well plates (2 ϫ 4 ␮ tion, while addition of anti-CEACAM1 Ab 4/3/17 inhibited stimula- 10 /150 L), incubated with increasing amounts of IL-2, and, after 24 h, ␮ ⑀ 0.5 ␮Ci of [3H]thymidine was added for 24 h. tion by 26%. In the case of stimulation with 0.50 g/ml CD3 , both T84.1 and 4/3/17 inhibited IL-2 production by 50%. The inhibition Cocapping and colocalization experiments results are remarkable in that only low levels of cell surface CEACAM1 are found on fresh PBMCs (see Fig. 1A). Activated PBMCs (2 ϫ 105 cells/200 ␮L) were incubated with biotinylated anti-CD3⑀ plus Texas Red-avidin (1 ␮g/ml, 4¡C, 1 h), then with Alexa 488 PBMCs were treated as above, and proliferation assays were T84.1 (40 min), and transferred to 37¡C for 30 min to induce capping. For performed (Fig. 1C). Combined treatment with anti-CD3⑀ and anti- ZAP-70, cells were permeabilized with PBS, 0.01% saponin, 0.25% gel- CD28 produced the highest level of proliferation, which was in- atin, and 0.1% Nonidet P-40 for 20 min at room temperature and stained hibited 33% by ligation of CEACAM1 with T84.1. However, the ␮ with rabbit anti-ZAP-70 (1 g/ml). Jurkat cells were incubated with Alexa effect of T84.1 treatment was minimal (ϳ10% inhibition) for cells 488 T84.1 and goat anti-mIgG (4¡C,1 h) followed by rhodamine-cholera ⑀ toxin B. Colocalization of CEACAM1-4L/eGFP with SHP-1 or SHP-2 was treated with anti-CD3 alone. Thus, CEACAM1 inhibition is most performed by permeabilizing as above and staining with mouse anti-SHP-1 pronounced in maximally activated T cells. or anti-SHP-2 and Texas Red goat anti-mIgG. Similarly, permeabilized cells were stained with 4G10 and Texas Red goat anti-mIgG. Kit-225 cells CEACAM1 is tyrosine phosphorylated in activated PBMCs were treated with anti-IL-2R␤ or anti-IL-2R␥ (1 ␮g/ml, 4¡C, 1 h), followed by staining with Alexa 488 T84.1 and Texas Red-avidin (1 ␮g/ml, 40 min). If CEACAM1 exerts its inhibitory activity on the TCR signal transduction pathway that depends on multiple ITAM phosphory- IL-2 production in PBMCs and CEACAM1-transfected Jurkat lations, then tyrosine phosphorylation of ITIMs in CEACAM1-L cells isoforms may oppose TCR signal transduction. To explore this possibility, a PBMC cell line was established and CEACAM1 li- PBMCs were stimulated with anti-CD3⑀ (0.1 ␮g/ml) plus anti-CD28 and/or anti-CEACAM1 4/3/17 or T84.1 (0.25 or 2.5 ␮g/ml), plus make-up gated with anti-CEACAM1 Ab T84.1, followed by immunopre- mIgG to provide equivalent total concentrations. After 3 days, aliquots cipitation and Western blotting (Fig. 2). The cell line had equiv- were assayed for IL-2 activity using the HT-2 indicator cell assay (detected alent expression levels of CEACAM1 as measured by both FACS with Alamar Blue; Trek, Medina, NY). Jurkat transfectants in 96-well and Western blotting (data not shown). When analyzed by PCR plates (1 ϫ 105cells/200 ␮L) were treated with anti-CD3⑀ (1 ␮g/ml), anti- CD28 (2.5 ␮g/ml), and/or anti-CEACAM1 (T84.1, CEA66, or 4/3/17; 2.5 using isoform-specific primers (27), the cell line expressed equiv- ␮g/ml), plus make-up mIgG, as before, and rabbit anti-mIgG (10 ␮g/ml) to alent levels of CEACAM1-4L and -3L, a minor amount of -4S, and induce cross-linking. After 2 days, aliquots were assayed for IL-2 release. no detectable -3S (data not shown). Controls included treatment 3546 CEACAM-1 IN HUMAN T CELLS

mediated its physiological effects (8, 10), we also included treatment with the pSer/pThr-phosphatase inhibitors cypermetherin and calyculin A. The results show that, except for pervanadate treatment, CEACAM1 is only tyrosine phosphorylated when CEACAM1 is ligated. Neither treatment with PMA nor the pSer/pThr phosphatase inhibitors alone was able to induce tyrosine phosphorylation on CEACAM1. The results with pervanadate treatment suggest that transient, reversible phosphorylation occurs in the absence of CEACAM1 ligation. Both PMA and pSer/pThr phosphatase inhibitor treatment affect the level of pTyr observed when CEACAM1 is ligated, raising the possibility that pSer plays a major role in controlling pTyr on CEACAM1. It should be noted that CEACAM1 occurs as two major ECD isoforms in these immunoprecipitates (IPs) (CEACAM1-4L and CEACAM1-3L), and that both isoforms contain ITIMs and are tyrosine phosphorylated. In addition, an 80-kDa phosphoprotein was coimmunoprecipitated with CEACAM1. Interest- ingly, a CEACAM1-associated phosphoprotein of the same size was previously observed to correlate with the growth-inhibitory activity of

rat CEACAM1 in epithelial cells (28), but its identity is unknown. Downloaded from Tyrosine phosphorylation of this band is strongly induced in the absence of CEACAM1 ligation plus pervanadate, but reduced when cells are treated with T84.1 plus pervanadate. Thus, CEACAM1 may interact with at least one other protein that is tyrosine phosphorylated. CEACAM1 associates with SHP-1 in activated PBMCs http://www.jimmunol.org/ Because the above results demonstrated that CEACAM1 is phosphorylated on its ITIMs in activated PBMCs, we then determined whether SHP-1 was coimmunoprecipitated with CEACAM1. Indeed, SHP-1 was found to coimmunoprecipitate with CEACAM1 on activated PBMCs both before and after CEACAM1 ligation; however, there was a 2- to 3-fold increase in the SHP-1 band after CEACAM1 ligation (Fig. 2). We also Western blotted for ZAP-70, a major constituent of the activated TCR complex, and CaM, which we (12) and Obrink and coworkers (29) have previously shown to associate by guest on September 26, 2021 with the cytoplasmic domain of CEACAM1. Low levels of ZAP-70 were found associated with CEACAM1 before and after ligation with T84.1 with a 2-fold increase after ligation. The association of CEACAM1 with both SHP-1 and ZAP-70 is of special significance FIGURE 1. Time course of IL-2R␣ and CEACAM1 induction on PHA/ given that SHP-1-deficient mice have hyperactive TCR responses (30, IL-2-activated PBMCs and their response to anti-CEACAM1 Abs. PBMCs 31) and ZAP-70 has been shown to be a target of SHP-1 (32, 33). The ␮ were stimulated once with 5 g/ml PHA plus 50 U/ml IL-2, and thereafter results for CaM were even more dramatic, with a 5-fold increase of split every 2 days with medium containing 100 U/ml IL-2. The results are binding after CEACAM1 ligation. The increased association with shown for a single donor, but similar results were obtained from other CaM after CEACAM1 ligation correlates with our previous finding donors. A, Cells were removed at each time point for Ab staining for CEACAM1 and IL-2R␣, and results are shown for an average of three that CaM inhibits the association of CEACAM1 with actin and determinations with SDs. B, PBMCs were stimulated with either 0.1 or tropomyosin (12). Because T cell activation involves Vav and cy- 0.5 ␮g/ml anti-CD3⑀ and 2.5 ␮g/ml additional Abs. IL-2 production was toskeletal reorganization (34), CEACAM1 may play a role in this measured after 3 days using HT-2 indicator cells (average of triplicates pathway too. Indeed, Vav is also coimmunoprecipitated with shown). Black bars, mIgG; dark gray bars, T84.1; light gray, 4/3/17; open CEACAM1 (data not shown). bars, CD28; open bars, with forward slash, CD28 ϩ T84.1; open bars, with backward slash, CD28 ϩ 4/3/17. Values of p were calculated for anti- IL-2 production and proliferation in CEACAM1-transfected p Ͻ Jurkat cells ,ءء ;p Ͻ 0.005 ,ء) CD3⑀ plus anti-CD28 Ϯ anti-CEACAM1 Abs Ͻ ءءء 0.0005; , p 0.00005). C, PBMCs were stimulated with increasing To probe the role of CEACAM1 cytoplasmic domain isoforms, amounts of anti-CD3⑀ and a fixed amount of additional Abs. Proliferation CEACAM1-transfected Jurkat cells were used as a model system. was measured after 3 days by [3H]thymidine incorporation and reported as the average of six determinations (ϮSD). Black bars, mIgG; dark gray Cell lines were selected by FACS for production of equivalent cell bars, T84.1; light gray bars, 4/3/17; open bars, CD28; open bars with for- surface levels of CEACAM1 isoforms and shown to have equiv- ward slash, anti-CD28 ϩ T84.1. Values of p were calculated for anti- alent amounts of CEACAM1 protein by Western blotting (data not p Ͻ shown). Jurkat cells produce, but do not respond to IL-2, and do ,ءء ;p Ͻ 0.005 ,ء) CD3-3 plus anti-CD28 Ϯ anti-CEACAM1 Abs p Ͻ 0.00005). not normally express CEACAM1 unless treated with PMA or ,ءءء ;0.0005 combined treatment with anti-CD3⑀/CD28 Abs for Ͼ2 days (data not shown). In vector-transfected controls, IL-2 production is min- with pervanadate, an inhibitor of tyrosine phosphatases, and PMA, imal for mIg-, anti-CD3⑀-, and anti-CD28-treated cells. However, known to stimulate critical protein kinase C in T cells, and immu- combined treatment with anti-CD3⑀ and anti-CD28 results in a noprecipitation with irrelevant rabbit IgG (data not shown). Be- substantial increase in IL-2 production (Fig. 3A). Addition of anti- cause previous studies on rat CEACAM1 had indicated that Ser503 CEACAM1 Ab T84.1 to the vector control cells results in a small, The Journal of Immunology 3547

FIGURE 2. CEACAM1 is tyrosine phosphorylated in activated PBMCs treated with anti-CEACAM1 Ab and coprecipitates with SHP-1, ZAP-70, and CaM. PBMCs were pretreated with pSer/pThr phosphatase inhibitors cypermetherin and calyculin A, or PMA, or sodium pervanadate for 10 min, and then Downloaded from incubated in the presence or absence of anti-CEACAM1 Ab T84.1 for 2 min. A, Cell lysates were precleared by rabbit IgG-protein A and immunoprecipitated by rabbit anti-CEACAM1-4/3L Ab 22-9, and separated proteins were probed ﬁrst with anti-phosphotyrosine Ab 4G10 and reprobed with T84.1 to determine the total amount of CEACAM1-4/3L in the IPs. The positions of CEACAM1-4L and -3L isoforms are marked with arrows. The band at 50 kDa is due to the presence of the H chain of T84.1 in those samples treated with T84.1. B, PBMCs were pretreated with either PMA or pervanadate for 10 min and incubated in the presence or absence of anti-CEACAM1 Ab T84.1, and lysates were precleared with rabbit IgG-protein A and immunoprecipitated with anti-CEACAM1 Ab 22-9. Western blot probes were: A, anti-CEACAM1 Ab T84.1; B, anti-SHP-1; C, anti-ZAP-70; D, anti-CaM. In lane

3, in which cells were treated with T84.1, the H chain of T84.1 is also detected. http://www.jimmunol.org/ but statistically insigniﬁcant reduction of IL-2. When Jurkat/ PBMC cell line was treated with biotinylated anti-CD3⑀ and avidin CEACAM1-4L cells were treated with anti-CD3⑀ and anti-CD28 to induce capping of CD3, and counterstained with anti- in the presence or absence of anti-CEACAM1 Ab, a 62% reduction CEACAM1 Ab T84.1, cocapping was demonstrated (Fig. 4, aÐf). in IL-2 production was observed (Fig. 3A). The results with Capping of CD3 in cholesterol-rich rafts is a hallmark of TCR CEACAM1-4S-transfected cells showed no or minimal inhibition, engagement and T cell activation (35). The fact that CEACAM1 demonstrating that the -4S isoform is not inhibitory in this assay. cocaps with CD3 suggests that it too is recruited to, or constitu- When control vector-transfected Jurkat cells were treated with tively resides, in the same lipid compartment (see later). by guest on September 26, 2021 anti-CD3⑀ or anti-CD28 Abs, cell proliferation as measured by ⑀ [3H]thymidine incorporation was in the range of 17Ð21,000 cpm Cocapping of CD3 and CEACAM1-4L/eGFP in Jurkat cells (Fig. 3B) with either no change or a slight increase observed for The expression pattern of a CEACAM1-4L/eGFP fusion protein, CEACAM1-4L-transfected cells when treated with three different like the parent CEACAM1-4L transfectants, is a beadlike pattern anti-CEACAM Abs. However, when CEACAM1-4S-transfected across the membrane, suggesting that the protein resides in discrete cells were treated with anti-CEACAM1 Abs plus either anti-CD3⑀ membrane domains (data not shown). CD3⑀ has an identical stain- or anti-CD28 Abs, a reproducible inhibition of cell proliferation ing pattern across the cell membrane (data not shown). When CD3 was observed (10Ð15%). When vector-transfected or CEACAM1- is capped with biotinylated anti-CD3⑀ plus avidin, a large portion transfected cells were treated with both anti-CD3⑀ and anti-CD28 in of the CEACAM1-4L/eGFP fusion protein is cocapped (Fig. 4, the absence of anti-CEACAM1 Abs, cell proliferation levels were gÐi). Because the cocapping results are similar to those obtained equivalent to those found in the groups above. However, when these with PBMCs, we conclude that CEACAM1-4L faithfully associ- groups (both anti-CD3⑀ and anti-CD28) were treated in the presence ates with the TCR signaling complex in the Jurkat model system. of anti-CEACAM1 Abs, the vector control production was uniformly We therefore, proceeded to determine whether CEACAM1-4L/ lowered compared with single treatments with anti-CD3⑀ or anti- eGFP also colocalized with SHP-1. CD28 alone. This reduction in baseline cell proliferation is most likely due to the aforementioned de novo production of CEACAM1. Thus, Colocalization of CEACAM1-4L/eGFP with SHP-1 in Jurkat we compared the added effects of CEACAM1 isoforms introduced by cells transfection vs the vector controls. All three anti-CEACAM1 Abs The CEACAM1-4L/eGFP-transfected Jurkat cells were treated reduced cell proliferation for both isoforms, 22Ð50% inhibition for with pervanadate, permeabilized, and stained for SHP-1, SHP-2, CEACAM1-4L, and 43Ð54% inhibition for CEACAM1-4S. These and pTyr (4G10). The results show good colocalization of results suggest that in this assay, CEACAM1-4S is a more potent CEACAM1-4L/eGFP with SHP-1, but not with SHP-2 (Fig. 4, jÐr). inhibitor than CEACAM1-4L. The pattern shows intense staining between the cells, as expected for a homophilic cell-cell adhesion molecule. A similar staining pattern is Cocapping of CEACAM1 with CD3 on activated PBMCs observed for pTyr (4G10), suggesting that proteins recruited to the CEACAM1 may mediate its inhibitory activity by associating with cell-cell membrane interface are highly phosphorylated on tyrosine. the CD3 signaling complex. This is an attractive possibility be- Based on the coimmunoprecipitation results obtained with PBMCs, it cause phosphorylation of its ITIMs would allow recruitment of the is likely that one of these proteins is CEACAM1. When the cells are inhibitory phosphatases SHP-1/2, which, in turn, could counteract not pretreated with pervanadate, little or no pTyr is detected with phosphorylation of the ITAMs on the CD3 complex or prevent 4G10 and there is no colocalization of CEACAM1-4L/eGFP with docking of downstream effectors such as ZAP-70. Indeed, when a SHP-1 or SHP-2 (data not shown). 3548 CEACAM-1 IN HUMAN T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 3. Inhibition of IL-2 production and cell proliferation in Jurkat cells transfected with CEACAM1-4L or -4S and treated with anti-CD3⑀ and anti-CD28 plus anti-CEACAM1 Abs. Jurkat cells transfected with CEACAM1-4L or -4S were treated with anti-CD3⑀, Ϯanti-CD28 and/or

anti-CEACAM1 (T84.1) in triplicate. Rabbit anti-mIgG was added to each FIGURE 4. Confocal analysis of CEACAM1 in PBMCs and transfected by guest on September 26, 2021 well to induce cross-linking. A, After 2 days of incubation, aliquots of cell Jurkat cells. aÐf, CEACAM1 cocaps with CD3⑀ on activated PBMCs. A culture medium were removed from each well and assayed for IL-2 activity cell line was established by treatment of PBMCs with PHA and IL-2 for 2 using the HT-2 cell proliferation assay (average of triplicates Ϯ SE). Black days and thereafter maintained in IL-2 only. The cells were treated with bars, mIgG; dark gray bars, anti-CD3⑀; light gray bars, anti-CD3⑀ ϩ biotin anti-CD3⑀, followed by Texas Red-avidin or Texas Red-avidin plus T84.1; open bars, anti-CD3⑀ ϩ anti-CD28; open bars with forward slash, Alexa 488 T84.1, all at 4¡C, and further incubated at 37¡C to induce cap- p Ͻ ping. Fixed and permeabilized cells were stained with rabbit anti-ZAP-70 ,ءء ;p Ͻ 0.005 ,ء :anti-CD3⑀ ϩ anti-CD28 ϩ T84.1. Values of p ,p Ͻ 0.00005. B, Cells were treated with Abs as indicated, and plus FITC anti-rabbit IgG. a, Anti-CD3⑀; b, anti-ZAP-70; c, merge; d ,ءءء ;0.0005 rabbit anti-mIgG was added to induce cross-linking. After 3 days, the cells anti-CD3⑀; e, anti-CEACAM1; f, merge. gÐi, Cocapping of CD3⑀ and were pulsed with [3H]thymidine for 5 h (average of triplicates Ϯ SEs, CEACAM1-4L/eGFP in Jurkat cells. Transfected cells were treated with cpm ϫ 10Ϫ3). Open bars, Jurkat/vector control; dark gray bars, Jurkat/ biotin anti-CD3⑀ Ab and anti-CEACAM1 Ab T84.1, followed by Texas p Ͻ Red-avidin, all at 4¡C, and transferred to 37¡C to induce capping. g, Texas ,ء :CEACAM1-4L; light gray bars, Jurkat/CEACAM1-4S. Values of p .p Ͻ 0.00005. Red channel (CD3⑀); h, eGFP channel (CEACAM1-4L/eGFP); i, merge ,ءءء ;p Ͻ 0.0005 ,ءء ;0.005 jÐr, Colocalization of CEACAM1-4L with SHP-1 in Jurkat cells transfected with CEACAM1-4L/eGFP. Transfected cells were either pretreated with sodium pervanadate or not, fixed, permeabilized, and stained with the CEACAM1-4L is found in GM1 lipid rafts indicated Abs. The results are shown for pervanadate-pretreated cells only. The beaded expression pattern of CEACAM1-4L in transfected jÐl, eGFP channel, Texas Red channel (anti-SHP-1), merge. mÐo, eGFP Jurkat cells suggested that it was localized to specific membrane channel, Texas Red channel (anti-SHP-2), merge. pÐr, eGFP channel, microdomains. Given its association with cross-linked CD3 (Fig. Texas Red channel (4G10), merge. sÐu, Cocapping of CEACAM1-4L and 4, gÐi), and the fact that cross-linked CD3 is recruited to lipid rafts GM1 in CEACAM1-4L-transfected Jurkat cells. Transfected cells were (35), we speculated that CEACAM1 is constitutively found in lipid incubated with Alexa 488 T84.1 at 4¡C, followed by goat anti-mIgG at 37¡C to induce capping, and stained with rhodamine-cholera toxin B. s, rafts. Using GM1 as a marker for cholesterol-rich lipid rafts (36), Alexa 488 T84.1 channel; t, rhodamine-cholera toxin B channel; u, merge. we performed cocapping studies on CEACAM1 and GM1 in Note the presence of cells with punctate staining observed during the cap- CEACAM1-4L/Jurkat cells. Fig. 4, sÐu, illustrates several cells ping process and several cells with defined caps. caught in early and late stages of capping, showing that CEACAM1-4L is associated with the GM1-positive microdomains at all stages of the capping process. Similar results were obtained for CEACAM1-4L/eGFP-transfected cells and CEACAM1-4S- IL-2R expression is reduced in CEACAM1-transfected Kit-225 transfected cells (data not shown). We conclude that CEACAM1 cells expression is localized to lipid rafts and is in position to interact To explore the mechanism responsible for our initial observation with CD3 and other signal transduction complexes that are re- regarding the reciprocal kinetics of IL-2R and CEACAM1 expres- cruited to lipid rafts. sion on activated PBMCs, we transfected Kit-225 cells, a human T The Journal of Immunology 3549 cell line that requires IL-2 for proliferation, with CEACAM1-4L sponse curve shows a 50% maximal response for the vector con- and -4S. Initially, we demonstrated that these cells have no cell trols and CEACAM1-4S transfectants at 5.0 U/ml IL-2, while that surface expression of CEACAM1 by FACS analysis, nor detect- of the CEAMCAM1-4L transfectants is 0.8 U/ml. This Ͼ6-fold able levels of CEACAM1 mRNA by PCR analysis (data not reduction in the 50% maximal response level for the shown). In contrast, these cells expressed high levels of all three CEACAM1-4L transfectants does not appear to depend on the ab- subunits of the IL-2R before transfection with CEACAM1 iso- solute levels on the IL-2R subunits, because the reduction in levels forms. After transfection, six cell lines were derived and analyzed was similar for the CEACAM1-4S transfectants (Fig. 6A). Thus, for the expression levels of CEACAM1 isoforms and IL-2R sub- we conclude that transfection with either isoform reduces the ab- units (Fig. 5). One line was a vector-transfected control, two lines solute levels of IL-2R␤ and ␥ subunits, but only in the case of the had high expression of the -4L isoform (lines 4L-1 and 4L-2), one CEACAM1-4L transfectants do we observe a shift in the 50% line had low expression of the -4L isoform (4L-3), and two lines maximal proliferative response to IL-2. had high expression of the -4S isoform (lines 4S-1 and 4S-2). For each line, the expression level of IL-2R␣ was unchanged vs the vector-transfected controls (Fig. 5B). However, the expression lev- Colocalization of CEACAM1-4L with IL-2R␤ and IL-2R␥ els of both IL-2R␤ and IL-2R␥ subunits were reduced by 50% in both CEACAM1-4L and -4S transfectants in which high levels of We next asked whether the inhibition of the proliferation response CEACAM1 were expressed (Fig. 5, C and D). In the case of the of the CEACAM1-4L transfectants was due to a direct association low expressing line for CEACAM1-4L, the IL-2R␤ and ␥ subunits of this isoform with IL-2R. As shown in Fig. 6B, CEACAM1-4L were reduced less. colocalizes with both the IL-2R␤ and ␥ subunits, and as shown for Downloaded from other cells, is found in discrete membrane domains and at cell-cell Both CEACAM1-4L and-4S inhibit proliferation of Kit-225 cells boundaries when two cells are found together. In one report, the To test the hypothesis that CEACAM1 isoforms affect the IL-2 localization of IL-2R␣, but not IL-2R␤ and ␥ to membrane rafts response in these cells, proliferation was measured for each of the has been reported (37), but in another report all three subunits were transfectants after prior IL-2 depletion (Fig. 6A). The dose-re- localized to lipid rafts (38). In addition, one report showed that http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Cell surface expression of CEACAM1-4L or -4S on transfected Kit-225 cells. Transfected Kit-225 cells were stained with: A, anti-CEACAM1 Ab T84.1; B, anti-IL-2R␣; C, anti-IL-2R␤; D, anti-IL-2R␥. Black, no primary Ab. Green, Kit-225/vector control. Magenta, Kit-225/CEACAM1-4L line 4L-3. Orange, Kit-225/CEACAM1-4L line 4L-1. Dark blue, Kit-225/CEACAM1-4L line 4L-2. Light blue, Kit-225/CEACAM1-4S line 4S-2. 3550 CEACAM-1 IN HUMAN T CELLS

FIGURE 6. CEACAM1-4L inhibits proliferation of Kit-225 cells and colocalizes with IL-2R␤ and ␥. A, Transfected Kit-225 cells were cultured in the absence of IL-2 for 72 h, and increasing amounts of IL-2 were added to each well. Cells were pulsed with [3H]thymidine for 24 h (average of quadruplicates Ϯ SE). Squares, vector- transfected cells. ‚, CEACAM1-4L line 4L-1. Œ, CEACAM1-4L line 4L-2. छ, CEACAM1-4S line 4S-1. ࡗ, CEACAM1-4S line 4S-2. B, Kit-225/CEACAM1-4L cells were treated with either biotinylated anti-IL-2R␤ or ␥ anti-IL-2R , followed by Alexa 488 T84.1 and Texas Downloaded from Red-avidin. aÐc, Cells treated with Alexa 488 T84.1 (a), biotinylated anti-IL-2R␤ and Texas Red-avidin (b), and merged images (c). dÐf, Cells treated with Alexa 488 T84.11 (d), biotinylated anti-IL-2R␥ and Texas Red-avidin (e), and merged images (f). http://www.jimmunol.org/ by guest on September 26, 2021 Abs to GM1, which is localized to lipid rafts, inhibited IL-2-me- 4L-transfected Jurkat cells. When phosphotyrosine-containing diated proliferation (37). Based on our study, we predict that IL-2R proteins were immunoprecipitated from cells either treated or not clustering will lead to recruitment of the ␤ and ␥ subunits into lipid with IL-2 and the separated proteins probed for the presence of raft domains, because CEACAM1 appears to reside within these Jak3, the level of phosphorylated Jak3 was reduced by 2.5- to microdomains, and the GM1 inhibition data are consistent with 3.0-fold in two lines of the CEACAM1-4L transfectants vs the this conclusion. vector control (Fig. 7, C and D). Thus, transfection with CEACAM1-4L can dramatically reduce the ability of IL-2R to ␤ Coimmunoprecipitation of CEACAM1-4L with IL-2R and signal via its most immediate associated kinase, Jak3. We con- ␥ IL-2R clude that even though the absolute levels of CEACAM1-4L im- To further demonstrate that CEACAM1 isoforms may associate munoprecipitated with IL-2R are low (Fig. 7, A and B), they are with IL-2R receptor subunits, we performed immunoprecipitation sufﬁcient to affect the levels of phosphorylated Jak3. studies (Fig. 7, A and B). When cell lysates were immunoprecipitated with anti-CEACAM1 Ab T84.1, both ␤ and ␥ subunits of IL-2R were detected. When cell lysates were immunoprecipitated Discussion with either anti-IL-2R␤ or ␥ Abs and IPs probed with T84.1, both Our data lend further strength to the accumulating evidence that isoforms of CEACAM1 were detected, depending on the transfec- the role of CEACAM1 in activated T cells is inhibitory, rather than tant analyzed. However, in both experiments, the relative amounts activating. Although this result is not surprising for CEACAM1- of coimmunoprecipitated proteins were low compared with total 4L, which contains two ITIMs, it had not been formally proved by protein detected in equivalent amounts of lysates. convincing biochemical data. We show in this study that activated PBMCs express the ITIM-containing isoforms of CEACAM1 that CEACAM1-4L down-regulates Jak3 tyrosine phosphorylation are both tyrosine phosphorylated and associate with their cognate Because only the CEACAM1-4L isoform was able to inhibit IL- inhibitory Src homology 2-containing phosphatase, SHP-1. How- 2-dependent proliferation in Kit-225-transfected cells, we decided ever, in order for this association to be inhibitory, the complex to further probe its effect on Jak3, the immediate downstream me- must, in turn, associate with the TCR complex, the signal trans- diator of signal transduction of IL-2R. Furthermore, a study by duction pathway that leads to both IL-2 production and prolifera- Leon et al. (39) had shown that SHP-1 expression had a profound tion. In this respect, we have shown by cocapping experiments that effect on Jak3 signaling linked to the IL-2R. Thus, we reasoned CEACAM1 does indeed associate with the TCR complex, and that that recruitment of SHP-1 to CEACAM1-4L phosphorylated on its small amounts of ZAP-70 are coimmunoprecipitated with ITIM could play a similar inhibitory role in the IL-2R, as was CEACAM1 from activated PBMCs. These results agree with stud- shown for TCR ligation in either activated PBMCs or CEACAM1- ies by Mustelin and coworkers (33), who showed that SHP-1, but The Journal of Immunology 3551

levels also contributes to CEACAM1 inhibition. Indeed, we show that CEACAM1 directly associates with IL-2R and is responsible for its down-regulation. Because CEACAM1 is a cell-cell adhesion molecule, we speculate that when activated T cells proliferate and express maximal amounts of CEACAM1 in a conﬁned space such as a lymph node, cell-cell contact increases, leading to inhibition of proliferation. Similarly, when activated T cells expressing CEACAM1 reach tissues like intestinal epithelium and EVT rich in CEACAM1, T cell inhibition is a likely outcome. In the case of the intestinal epithelium, this outcome sets an upper threshold to further activation in which foreign Ags abound. In the case of EVT, the inhibition protects embryonic tissue from maternal T cell attack. In the case of cancer cells, as shown in a recent study by Markel et al. (40), melanoma cells that express CECAM1 are protected from attack by activated T cells. However, in all of this work, no attempt was made to distinguish between the effects of the ITIM- vs the non-ITIM-containing isoforms of CEACAM1. In our model system in which CEACAM1-4L/eGFP was introduced

into Jurkat cells, the -4L isoform accumulated at cell-cell bound- Downloaded from aries, and when treated briefly with pervanadate to mimic activation, SHP-1 and phosphotyrosine-positive receptors (including FIGURE 7. Coimmunoprecipitation of CEACAM1-4L or -4S with IL-2R CEACAM1) were also recruited to the cell-cell boundaries. This is subunits and phosphorylation of Jak3. IL-2-depleted Kit-225/CEACAM1 further evidence that tyrosine phosphorylation and recruitment of transfectants were treated with IL-2 for 30 min, and lysates were immunopre- SHP-1 are involved in the inhibitory activity of CEACAM1-4L, cipitated and Western blotted, as shown. A, IP with T84.1 and Western blotted and suggests that the inhibitory activity is generated at the cell-cell http://www.jimmunol.org/ with anti-IL-2R␥ or anti-IL-2R␤ Abs. B, IP with anti-IL-2R␥ or anti-IL-2R␤ contact sites where CEACAM1-4L accumulates. Thus, it makes Abs and Western blotted with T84.1. V ϭ vector-transfected cells. L ϭ sense that CEACAM1-4L exerts its inhibitory effect late in T cell CEACAM1-4L-transfected cells. S ϭ CEACAM1-4S-transfected cells. IP ϭ activation when cell density is at a maximum. ϭ Ab used to immunoprecipitate. Probe Ab used for Western blotting. As a Our studies demonstrate that the two cytoplasmic domain iso- control, total lysates were Western blotted to determine the total amount of forms of CEACAM1 confer different phenotypes to T cells. In the probed protein in the cell. C and D, Proteins phosphorylated on tyrosine were model Jurkat system, CEACAM1-4L inhibited IL-2 production immunoprecipitated with anti-phosphotyrosine Ab PY20, separated by SDS gel electrophoresis, and Western blotted with either phosphotyrosine Ab 4G10 more effectively than CEACAM1-4S, while the opposite was true (C) or an anti-Jak 3 Ab (D). V ϭ vector control, L1 ϭ CEACAM1-4L line for cell proliferation. Thus, one may predict that the absolute levels 4L-1, L2 ϭ CEACAM1-4L line 4L-2. The ratio of phospho-Jak3 to total Jak3 of the two isoforms in activated T cells may fine tune the inhibitory by guest on September 26, 2021 for IL-2-treated vector transfectants was 0.45, 0.18 for line 4L-1, and 0.15 for response or allow different signaling pathways to operate under line 4L-2. different conditions. The role of the two isoforms in controlling the expression of IL-2R subunits in Kit-225 cells is equivalent in that the IL-2R␤ and ␥ subunits, but not the ␣ subunit, are strongly down- not SHP-2, can dephosphorylate ZAP-70 and inhibit T cell acti- regulated. However, the IL-2 dose-response curves for proliferation vation. In other studies, these investigators showed that SHP1 re- are very different for the two isoforms, with only the -4L isoform mains in the cytosol and requires activation for its inhibitory ac- conferring strong inhibition. Thus, the -4L isoform is directly tied to tivity (32). We show in this study that translocation of SHP-1, but both the TCR and IL-2R in terms of T cell proliferation, while the -4S not SHP-2, to the plasma membrane depends on the expression of isoform only inhibits IL-2-independent proliferation. CEACAM1-4L. Thus, activation of SHP-1 may involve translo- The fact that CEACAM1 expression is associated with the cation to an appropriate ITIM-containing molecule that is phos- down-regulation of IL-2R␣ in PBMCs and not with IL-2R␣, but phorylated and present in the plasma membrane. rather with the ␤ and ␥, subunits in Kit-225 cells, is intriguing. We At least three other studies also conclude that CEACAM1 ex- conclude that the functional IL-2R is differentially regulated in pression leads to an inhibitory T cell response. In the case of hu- Kit-225 cells vs PBMCs. Nonetheless, the end result is the same: man decidual lymphocytes that highly express CEACAM1, an in- less functional IL-2R. Despite the strong down-regulation of func- hibitory response was observed for both T and NKT cells (23). tional IL-2R, both PBMCs and Kit-225 cells still respond to IL-2, These authors speculated that the CEACAM1-CEACAM1 inter- but as we show in this study, the CEACAM1-4L isoform is further actions between the lymphocytes and the extravillous trophoblasts capable of inhibiting Jak3 kinase phosphorylation, the critical down- (EVT) were responsible for the inhibitory activity. In the case of stream mediator of the IL-2R response. Thus, CEACAM1-4L is murine splenic T cells, CEACAM1 expression required activation capable of both lowering the cell surface levels of functional IL-2R with anti-CD3⑀ and anti-CD28 Abs, and ligation of CEACAM1 and its ability to activate T cells. This outcome is likely to have resulted in inhibition of both an allogenic MLR and a delayed-type significant in vivo effects on the ability of T cells to respond to hypersensitivity response (22). In the case of Neisseria Opa inter- activating signals. The fact that CEACAM1-4S can lower cell surface actions with CEACAM1 on activated human T cells, the result is levels of functional IL-2R without affecting signal transduction, as also inhibitory (20). Thus, it seems likely that CEACAM1 inhibi- measured in the proliferation assay, suggests that this isoform can tion is tied to TCR signaling, and according to our data, especially modulate T cells in more subtle ways, for example making them when both the CD3 and CD28 pathways are engaged. The inhibi- dependent on higher levels of IL-2 for continued activation. tion results in both decreased production of IL-2 and proliferation. Immunoprecipitation data support the idea that CEACAM1-4L The inverse kinetics associated with CEACAM1 and IL-2R ex- can directly associate with the IL-2R and inhibit Jak3 phosphor- pression in activated PBMCs suggests that regulation of IL-2R ylation presumably via the SHP-1-CEACAM1-4L association. 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