Proliferation by 2B4/CD48 Interactions T Cell

Cutting Edge: Regulation of CD8+ T Cell Proliferation by 2B4/CD48 Interactions Taku Kambayashi, Erika Assarsson, Benedict J. Chambers and Hans-Gustaf Ljunggren This information is current as of September 29, 2021. J Immunol 2001; 167:6706-6710; ; doi: 10.4049/jimmunol.167.12.6706 http://www.jimmunol.org/content/167/12/6706 Downloaded from References This article cites 23 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/167/12/6706.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Regulation of CD8؉ T Cell Proliferation by 2B4/CD48 Interactions1

Taku Kambayashi,2* Erika Assarsson,2* Benedict J. Chambers,* and Hans-Gustaf Ljunggren3*†

To date, the only known 2B4-binding molecule is CD48. Bind- The biological function of 2B4, a CD48-binding molecule ex- ing studies have shown that CD48 has a 5–10 times stronger af- pressed on T cells with an activation/memory phenotype, is not ﬁnity for 2B4 than for CD2 (7, 8). Anti-CD48 Abs have been clear. In this report, we demonstrate that proliferation of found to inhibit activation of CD4ϩ and CD8ϩ T cells (9, 10). ؉ Ϫ Ϫ CD8 T cells is regulated by 2B4. Proliferative responses of Whereas CD2 / mice are phenotypically similar to wild-type Downloaded from ؉ CD8 T cells were signiﬁcantly reduced by anti-2B4 Ab. The mice (11), T cells from CD48Ϫ/Ϫ mice show a severe reduction in effects were not potentiated by anti-CD48 Ab, suggesting that proliferation and IL-2 production in response to lectins, anti-CD3 the observed responses were driven by 2B4/CD48 interactions. Ab, and alloantigen (8, 12). The relatively normal phenotype of the Surprisingly, the 2B4/CD48-dependent proliferative responses CD2Ϫ/Ϫ mice suggests that CD48-binding ligands other than CD2 were also observed in the absence of APCs. This suggests that may compensate for the absence of CD2.

2B4/CD48 interactions can occur directly between T cells. Fur- In this report, we demonstrate that 2B4 is associated with an http://www.jimmunol.org/ ϩ thermore, when activated 2B4؉CD8؉ T cells were mixed with activation/memory phenotype of CD8 T cells. Furthermore, the 2B4؊CD8؉ TCR-transgenic T cells and speciﬁc peptide-loaded results suggest that 2B4 on activated/memory T cells serves as a APC, the proliferation of the latter T cells was inhibited by ligand for CD48, and by its ability to interact with CD48 provides anti-2B4 Ab. Taken together, this suggests that 2B4 on acti- costimulatory-like function for neighboring T cells. vated/memory T cells serves as a ligand for CD48, and by its ability to interact with CD48 provides costimulatory-like func- Materials and Methods tion for neighboring T cells. The Journal of Immunology, Mice Adult C57BL/6 (B6) and TCR-transgenic (Tg)4 (F ) mice speciﬁc for an

2001, 167: 6706Ð6710. 5 by guest on September 29, 2021 influenza nucleoprotein epitope, ASNENMDAM, presented on H-2Db (13) were bred at the Microbiology and Tumor Biology Center, Karolinska he 2B4 molecule (CD244) is expressed on NK cells, Institutet (Stockholm, Sweden). Animal care was in accordance with na- ϩ tional and institutional guidelines. monocytes, and basophils, and on subsets of TCR␥␦ T T cells and CD8ϩ T cells (1–4). Most of the information Reagents available on 2B4 comes from studies of NK cells. Cross-linking of All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless 2B4 on IL-2-activated NK cells leads to stimulation of lytic ac- otherwise specified. The influenza virus nucleoprotein-derived peptide (nu- ␥ tivity (1, 4, 5), IFN- secretion (1), and granule exocytosis (6). The cleoprotein (NP)366–374), ASNENMDAM, was purchased from Research biological function of 2B4 on CD8ϩ T cells remains largely un- Genetics (Huntsville, AL). All Ab used were purchased from BD PharM- clear. 2B4ϩCD8ϩ T cells have been associated with non-MHC- ingen (San Diego, CA) except for anti-CD2 (Southern Biotechnology As- sociates, Birmingham, AL). restricted cytotoxicity (6), but cross-linking of 2B4 on CD8ϩ T ϩ cells does not trigger redirected lysis of FcR-expressing targets, Preparation of CD8 T cells cytokine production, or proliferation (4). CD8␣ϩ cells were purified from spleens of mice using the MACS sepa- ration system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s guidelines and resuspended in complete medium (␣MEM, 10 mM HEPES, 2 ϫ 10Ϫ5 M 2-ME, 10% FCS, 100 U/ml pen- icillin, 100 U/ml streptomycin; Life Technologies, Paisley, U.K.) plus re- *Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden; † combinant human IL-2 (1000 U/ml; PeproTech, Rocky Hill, NJ) for 6 days. and Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, ϩ ϩ Huddinge University Hospital, Stockholm, Sweden FACS analysis of these cells demonstrated that only CD8 TCR cells were present in these cultures. Purification of CD8ϩ T cells from influenza- Received for publication June 1, 2001. Accepted for publication October 25, 2001. infected mice has been described previously (14). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance RT-PCR with 18 U.S.C. Section 1734 solely to indicate this fact. ϩ 1 2B4 transcripts were detected using RNA from naive or activated CD8 T This work was supported by the Swedish Foundation of Strategic Research, the cells by RT-PCR using the following primers: 2B4 short transcripts were Karolinska Institutet, the Swedish Medical Research Council, the Swedish Cancer Ј Ј Society, the Tobias Foundation, the Åke Wiberg Foundation, the Alex and Eva Wall- detected using 5 -ACTGTTTTTGTCCTGCTTGGTG and 3 -AGGAAAC stro¨m Foundation, and the Lars Hiertas Foundation. TGGTGGAGAAGAAAA (Cybergene, Stockholm, Sweden) encompassing nt 573-1093 of murine 2B4 (m2B4) short. m2B4 long transcripts were 2 T.K. and E.A. contributed equally to this study. 3 Address correspondence and reprint requests to Dr. Hans-Gustaf Ljunggren, Micro- biology and Tumor Biology Center, Karolinska Institutet, 171 77 Stockholm, Sweden. 4 Abbreviations used in this paper: Tg, transgenic; DC, dendritic cell; LAT cell, E-mail address: [email protected] lymphokine-activated T cell; NP, nucleoprotein; m2B4, murine 2B4.

● The Journal of Immunology 6707 detected using 5Ј-TGGAAGAAGACAGAACAGGG and 3Ј-TGGAAT their cytoplasmic tails while having identical extracellular domains CAGAAGGCTTGCAC encompassing nt 283-1136 of m2B4 long. cDNA (18, 19). As shown in Fig. 1B, both the short and long forms of ␤ quality was confirmed by amplification of a -actin gene fragment. 2B4 were equally up-regulated in CD8ϩ T cells after stimulation Flow cytometry with IL-2. Taken together, the data indicated that expression of 2B4 was increased in IL-2-activated CD8ϩ T cells both at the cell Following FcR-block, cells were stained with the specified Ab or its iso- b surface and at the transcriptional level. 2B4 expression was also type control or D -NP366-RED670 tetramer (14, 15). The fluorescence in- ϩ tensity was measured on a FACScan flow cytometer (BD Biosciences, observed on CD8 T cells on day 10 post-influenza infection (peak ϩ Mountain View, CA) and analyzed using CellQuest computer software of infection). Five to 10% of all CD8 T cells in the lungs of (BD Biosciences). infected mice expressed 2B4 compared with 1–2% in untreated CD8ϩ T cell proliferation assays mice (Fig. 1C). The expression of 2B4 on virus-specific T cells was confirmed using tetramers of MHC class I molecules refolded Bone marrow-derived dendritic cells (DC) were pulsed with ASNEN b 4 with an influenza nucleoprotein epitope (D -NP366) (Fig. 1C). Al- MDAM peptide overnight at 37°C and seeded at 2 ϫ 10 cells/well in Ϫ ϫ 4 ϩ though 2B4 was observed also on tetramer T cells, this could be U-bottom 96-well plates. A total of 3 10 TCR-Tg CD8 T cells (naive ϩ ϩ or IL-2-activated) or TCR-Tg lymphokine-activated T (LAT) cells (FACS related to the other CD8 2B4 T cells recognizing other flu sorted into NK1.1ϩ or NK1.1Ϫ) were added to each well with the indicated epitopes, or bystander activation, or both. azide-free Ab. When B6 LAT cells were mixed with naive TCR-Tg CD8ϩ T cells, 4 ϫ 104 B6 LAT cells (sorted into NK1.1ϩ or NK1.1Ϫ) were added 2B4/CD48 interactions are involved in Ag-specific and cytokine- with 3 ϫ 104 naive TCR-Tg CD8ϩ T cells to each well. In APC-free induced proliferation of preactivated CD8ϩ T cells b experiments, plates were coated overnight at 4°C with purified D -NP366 ϩ monomers (14, 15) at various concentrations with anti-CD28 Ab (10 ␮g/ The effect of anti-2B4 Ab on the proliferation of CD8 T cells was Downloaded from ϩ ml) in PBS. TCR-Tg LAT cells (3 ϫ 104) were added to each well. In examined using CD8 T cells from mice transgenic for a TCR cytokine-induced proliferation assays, 3 ϫ 104 B6 CD8ϩ T cells (naive or ϩ Ϫ specific for an influenza nucleoprotein epitope. The Ag-specific IL-2-activated) and TCR-Tg LAT cells (sorted into NK1.1 or NK1.1 ) response of purified naive TCR-Tg CD8ϩ T cells was compared were stimulated with IL-2 (1000 U/ml). All proliferation assays were for ϩ 72 h except the naive B6 CD8ϩ T cell cytokine-induced proliferation as- with that of TCR-Tg CD8 T cells which were first cultured in say, which was for 120 h. [3H]Thymidine (1 ␮Ci/ml) was added for the last IL-2 for 6 days (hereafter denoted as CD8 LAT cells). In response

18 h of all assays. The plates were harvested on glass fiber filters (Wallac, to peptide-pulsed DCs, anti-2B4 Ab had no effect on the prolifer- http://www.jimmunol.org/ Turku, Finland) and analyzed in a beta-scintillation counter (Wallac). ation of naive CD8ϩ T cells (Fig. 2A). However, using TCR-Tg LAT cells, the proliferative response to peptide-pulsed DCs was Results and Discussion ϩ significantly diminished by anti-2B4 Ab compared with isotype CD8 T cells acquire expression of 2B4 upon activation in vitro control Ab (Fig. 2B). and in vivo The effect of anti-2B4 Ab was also compared with that of anti- One to 2% of all splenic CD8ϩ T cells from normal uninfected CD48 and anti-CD2 Ab. Addition of anti-CD48 or anti-CD2 Ab mice express 2B4. In the present study, we show that activation by resulted in ϳ45 and ϳ20% reduction in the Ag-specific prolifer- IL-2, IL-4, and IL-15 induced expression of 2B4 on CD8ϩ T cells ation of naive TCR-Tg CD8ϩ T cells at peptide concentrations of ϩ ϩ (Fig. 1A and data not shown). CD8 2B4 T cells were predom- 0.01 ␮M and 0.1 ␮M, respectively (Fig. 2A). In contrast, the Ag- by guest on September 29, 2021 inantly CD44high and CD62Llow (data not shown), in line with specific proliferation of TCR-Tg CD8 LAT cells was suppressed recent findings showing that expression of NK cell receptors on by anti-CD48 but not by anti-CD2 Ab (Fig. 2B). However, both CD8ϩ T cells is associated with a memory phenotype (16, 17). anti-CD48 and anti-2B4 Ab caused a similar degree of inhibition Alternative splicing of the 2B4 mRNA has been shown to give rise (ϳ45%) in CD8 LAT cell proliferation at all peptide concentra- to two different polypeptides, 2B4 long and 2B4 short, differing in tions. Interestingly, the proliferation of CD8 LAT cells could not

FIGURE 1. Expression of 2B4 is induced on CD8ϩ T cells after activation in vitro and in vivo. A, Cell surface expression of 2B4 is increased on CD8␣ϩ T cells upon stimulation with IL-2 in vitro. CD8␣ϩ T cells were purified from spleens of B6 mice (left density plot), cultured in IL-2 (1000 U/ml) for 6 days (right density plot), and analyzed for surface expression of 2B4 by flow cytometry. The numbers represent the proportion of CD8␣ϩ cells expressing 2B4. B, RT-PCR analysis of 2B4 short (top panel) and 2B4 long (middle panel) was performed on RNA extracted from freshly purified splenic CD8␣ϩ cells from B6 mice pretreated with anti-NK1.1 Ab (left lane) and from the same CD8␣ϩ cells cultured in IL-2 (1000 U/ml) for 6 days (right lane). cDNA quality was confirmed by a ␤-actin control (bottom panel). C, Expression of 2B4 on virus-specific CD8ϩ T cells during influenza infection. Single-cell b ␣ suspensions were prepared from lungs of influenza-infected mice on day 10 postinfection and triple-stained with D -NP366 tetramer, anti-CD8 , and b ϩ b Ϫ anti-2B4 Abs. The cells were gated into D -NP366 tetramer and D -NP366 tetramer subsets and analyzed for expression of 2B4 (right panel) compared with isotype control (left panel)byflow cytometry. The results are representative of at least four independent experiments. 6708 CUTTING EDGE: REGULATION OF CD8ϩ T CELL PROLIFERATION BY 2B4/CD48

FIGURE 2. The effects of Ab against 2B4, CD2, and CD48 on CD8ϩ T cell proliferation. Naive TCR-Tg CD8ϩ T cells (A) or TCR-Tg CD8 LAT cells (B) were cultured with peptide-pulsed DCs in the presence of isotype control (E), anti-2B4 (F), anti-CD2 (‚), anti-CD48 (Œ), or anti-NK1.1 Ab ()at10␮g/ml as indicated. C, TCR-Tg CD8 LAT cells were cultured with peptide-pulsed DCs in the presence of isotype control (E), anti-2B4 (F), anti-CD48 (‚), or anti-2B4 Ab plus anti-CD48 Ab (Œ)at10␮g/ml as indicated. D, Naive B6 CD8ϩ T cells (ﬁrst set of columns), B6 CD8 LAT cells (second set of columns), B6 NK1.1ϩ CD8 LAT cells (third set of columns), or B6 NK1.1Ϫ CD8 LAT cells (fourth set of columns) were cultured in IL-2 (1000

U/ml) in the presence of isotype control, Downloaded from anti-2B4, anti-CD2, anti-CD48, or anti- NK1.1 Ab at 10 ␮g/ml as indicated. The proliferation of T cells in all assays was measured 3 days later by thymidine incorporation except for IL-2-induced proliferation of B6 naive CD8ϩ T cell, which was measured 5 days http://www.jimmunol.org/ later. Results are expressed as mean counts per minute Ϯ SD of triplicate p Ͻ 0.01 compared with ,ء .cultures isotype control Ab. The results are representative of at least three independent experiments.

be further inhibited by anti-2B4 Ab in the presence of anti-CD48 The 2B4/CD48 interaction takes place between CD8ϩ T cells by guest on September 29, 2021 Ab, suggesting that the effects of anti-2B4 Ab were mediated Initially, we believed that the 2B4/CD48 interaction took place through blocking of the 2B4/CD48 interaction (Fig. 2C). CD48 has between the APC and T cell, because others have reported that 2B4 been shown to have a 5–10 times stronger affinity for 2B4 than for ϩ cross-linking does not directly activate CD8 T cells and 2B4 was CD2 (7, 8). Thus, as 2B4 expression was increased on activated presumed to function as an adhesion molecule for T cells to APCs CD8ϩ T cells, it was likely that the CD2/CD48 interaction was (4, 21). However, as observed from the inhibition of IL-2-induced replaced by 2B4/CD48 interactions. This notion is supported by proliferation of CD8 LAT cells by anti-2B4 Ab, it was apparent the fact that anti-2B4 Ab blocks the Ag-specific proliferation of ϩ that the 2B4/CD48 interaction did not require APCs. Moreover, we CD8 LAT cells but not of naive CD8 T cells and that the opposite found that both anti-2B4 and anti-CD48 Ab inhibited the Ag-spe- is true for anti-CD2 Ab. cific proliferation of CD8 LAT cells in the absence of APCs where It has recently been suggested that the proliferation of memory proliferation was induced by cross-linking of the TCR using pu- T cells is MHC independent and probably regulated by cytokines (20). Because CD8ϩ2B4ϩ T cells in normal mice were primarily rified MHC class I-peptide complexes (Fig. 3A). These data sug- of memory phenotype, we examined whether 2B4 also played a gested that the 2B4/CD48 interaction does not necessarily take ϩ place at the T cell/APC interface, but can also occur between role in the cytokine-induced proliferation of CD8 T cells. In line ϩ with the above observations, a 40–50% reduction in proliferation neighboring CD8 T cells. ϩ These results led to the question of whether 2B4 was directly of naive B6 CD8 T cells was observed with anti-CD2 or anti- ϩ CD48 Ab, whereas no inhibition in proliferation was seen with triggering proliferation of 2B4 CD8 LAT cells or was affecting anti-2B4 Ab in response to IL-2 (Fig. 2D). However, a 20% de- the proliferation of T cells by virtue of its ability to function as a crease in the IL-2-stimulated proliferation of B6 CD8 LAT cells ligand for CD48. To answer this question, we first looked at which Ϫ ϩ was observed in the presence of anti-2B4 or anti-CD48 Ab but not population of cells, 2B4 or 2B4 , was proliferating in the CD8 anti-CD2 Ab (Fig. 2D). Furthermore, taking advantage of the fact LAT cell/DC cocultures. To do this, TCR-Tg CD8 LAT cells were ϩ Ϫ that NK1.1 is coexpressed with 2B4 (data not shown), B6 CD8 sorted into NK1.1 and NK1.1 subsets and cocultured with pep- Ϫ Ϫ LAT cells were sorted into NK1.1ϩ and NK1.1Ϫ subsets. The tide-pulsed DCs. Of the two subsets, only the NK1.1 (2B4 ) addition of anti-2B4 or anti-CD48 Abs to NK1.1ϩ CD8 LAT cells subset proliferated in response to specific Ag (Fig. 3B). In contrast, resulted in an ϳ40% attenuation of the IL-2-induced proliferation both subsets proliferated equally well to IL-2, indicating that the ϩ while no effect was observed by the addition of the same Ab to NK1.1 cells were not exhausted but were just unresponsive to NK1.1Ϫ CD8 LAT cells (Fig. 2D). Taken together, these results peptide (data no shown). This was also confirmed by FACS anal- suggested that non-MHC-restricted routes of T cell proliferation ysis of CFSE-labeled CD8 LAT cells, which showed that only the can also be regulated by 2B4/CD48 interactions and that such in- 2B4Ϫ cells proliferated after a coculture with peptide-pulsed DCs teractions may be involved in the propagation of memory T cells. (data not shown). Thus, from these results it was evident that the The Journal of Immunology 6709 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. 2B4/CD48 interactions occur between CD8ϩ T cells. A, Anti-2B4 and anti-CD48 Ab inhibit the proliferation of CD8 LAT cells in the absence of APCs. TCR-Tg CD8 LAT cells were cultured in plates precoated with various concentrations of MHC class I molecules refolded with b ␮ E F ‚ ASNENMDAM peptide (D -NP366) and anti-CD28 Ab (10 g/ml). Soluble isotype control ( ), anti-2B4 ( ), or anti-CD48 Ab ( ) were added to the p Ͻ 0.01 compared with isotype control Ab. B, TCR-Tg NK1.1ϩ (2B4ϩ) CD8 LAT cells do not proliferate in response to ,ء .cultures as indicated peptide-pulsed DCs. TCR-Tg CD8 LAT cells were sorted into NK1.1Ϫ (E) or NK1.1ϩ (F) subsets and cultured with peptide-pulsed DCs. C, Anti-2B4 Ab reduces the Ag-specific proliferation of naive TCR-Tg CD8ϩ T cells when mixed with B6 NK1.1ϩ CD8 LAT cells. B6 NK1.1ϩ LAT cells mixed with p Ͻ 0.01 compared ,ء .() naive TCR-Tg CD8ϩ T cells in the presence of isotype control (‚) or anti-2B4 Ab (Œ) and B6 NK1.1ϩ CD8 LAT cells alone with isotype control Ab of the respective T cell coculture. D, Anti-2B4 and anti-CD48 Ab reduce the Ag-specific proliferation of naive TCR-Tg CD8ϩ T cells when mixed with B6 NK1.1ϩ CD8 LAT cells. B6 NK1.1ϩ or NK1.1Ϫ LAT cells mixed with naive TCR-Tg CD8ϩ T cells in the presence of isotype control, anti-2B4, or anti-CD48 Ab (10 ␮g/ml). Anti-CD48 blocking in both cultures was related to the CD2 expression on the naive TCR-Tg CD8ϩ T cells. p Ͻ 0.01 compared with isotype control Ab of the respective T cell coculture. The proliferation of T cells in all assays was measured 3 days later by ,ء thymidine incorporation. Results are expressed as mean counts per minute Ϯ SD of triplicate cultures. The results are representative of three independent experiments. anti-2B4 Ab was not inhibiting the proliferation of 2B4ϩCD8ϩ T This suggested that the 2B4 expression on nonspecific CD8ϩ T cells, because these cells did not proliferate in response to Ag. cells (B6 CD8 LAT cells in this case) affects the proliferation of More compelling evidence came from experiments where B6 CD8 neighboring naive 2B4Ϫ Ag-specific CD8ϩ T cells (TCR-Tg LAT cells were sorted into NK1.1ϩ (2B4ϩ) and NK1.1Ϫ (2B4Ϫ) CD8ϩ T cells), because the B6 CD8 LAT cells did not proliferate subsets and mixed with naive TCR-Tg CD8ϩ T cells. The addition in response to specific Ag (Fig. 3, C and D). Furthermore, anti-2B4 of anti-2B4 Ab to B6 NK1.1ϩ CD8 LAT/TCR-Tg cocultures re- Ab had no effect on the proliferation of T cell/DC cultures when sulted in significant inhibition of Ag-specific proliferation, the B6 CD8 LAT cells were separated from the naive TCR-Tg whereas no effect was observed by the addition of the same Ab to CD8ϩ T cells by a cell-impermeable porous membrane, indicating NK1.1Ϫ CD8 LAT cells (Fig. 3, C and D). The addition of anti- that cell-cell contact was required (data not shown). Taken to- CD48 Ab to either B6 NK1.1ϩ CD8 LAT/TCR-Tg or NK1.1Ϫ gether, these results strongly suggest that 2B4 functions as a ligand CD8 LAT/TCR-Tg cocultures inhibited Ag-specific proliferation for an activating receptor on CD8ϩ T cells, most likely CD48, (Fig. 3D). This inhibition was related to the interaction of CD2 rather than being an activating receptor itself. Although CD48 is a expression on the naive TCR-Tg, as anti-CD2 Abs could also in- GPI-anchored receptor and lacks intracytoplasmic domains, it has hibit in both cocultures (data not shown). been shown to be physically associated to G proteins (22) or to the 6710 CUTTING EDGE: REGULATION OF CD8ϩ T CELL PROLIFERATION BY 2B4/CD48

SRC family member of tyrosine kinases in glycolipid-enriched mi- 7. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, and crodomains of the cell membrane (23). Thus, it is not surprising A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J. Exp. Med. 188:2083. that a signal is transduced by CD48 upon ligation with 2B4 and 8. Latchman, Y., P. F. McKay, and H. Reiser. 1998. Identification of the 2B4 mol- ϩ can affect CD8 T cell proliferation. However, exactly what sig- ecule as a counter-receptor for CD48. J. Immunol. 161:5809. nals lead to the enhancement of proliferation by 2B4/CD48 inter- 9. Reiser, H. 1990. sgp-60, a signal-transducing glycoprotein concerned with T cell activation through the TCR/CD3 complex. J. Immunol. 145:2077. actions is an issue that remains to be established. 10. Chavin, K. D., L. Qin, R. Yon, J. Lin, H. Yagita, and J. S. Bromberg. 1994. Anti-CD2 monoclonal antibodies suppress cytotoxic lymphocyte activity by the Concluding remarks generation of Th2 suppressor cells and receptor blockade. J. Immunol. 152:3729. In summary, we have shown that 2B4 plays a significant role in the 11. Killeen, N., S. G. Stuart, and D. R. Littman. 1992. Development and function of ϩ T cells in mice with a disrupted CD2 gene. EMBO J. 11:4329. Ag-specific and cytokine-induced proliferation of CD8 T cells. 12. Gonzalez-Cabrero, J., C. J. Wise, Y. Latchman, G. J. Freeman, A. H. Sharpe, and ϩ Although only a small proportion of CD8 T cells normally ex- H. Reiser. 1999. CD48-deficient mice have a pronounced defect in CD4ϩ T cell press 2B4 in the mouse, this molecule may play a significant role activation. Proc. Natl. Acad. Sci. USA 96:1019. 13. Mamalaki, C., T. Norton, Y. Tanaka, A. R. Townsend, P. Chandler, E. Simpson, in the propagation of T cells through interaction with CD48. We and D. Kioussis. 1992. Thymic depletion and peripheral activation of class I speculate that 2B4 could provide costimulatory signals necessary major histocompatibility complex-restricted T cells by soluble peptide in T-cell for the survival of memory T cells in response to low levels of Ag receptor transgenic mice. Proc. Natl. Acad. Sci. USA 89:11342. 14. Kambayashi, T., E. Assarsson, J. Michaelsson, P. Berglund, A. D. Diehl, or cytokines. B. J. Chambers, and H. G. Ljunggren. 2000. Emergence of CD8ϩ T cells expressing NK cell receptors in influenza A virus-infected mice. J. Immunol. 165: Acknowledgments 4964. 15. Assarsson, E., T. Kambayashi, J. K. Sandberg, S. Hong, M. Taniguchi,

b Downloaded from We thank Dr. T. N. M. Schumacher for providing us with the H-2D H L. Van Kaer, H. G. Ljunggren, and B. J. Chambers. 2000. CD8ϩ T cells rapidly ␤ chain construct, Dr. P. J. Travers for the murine 2-microglobulin construct acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in and Dr. D. Kioussis for the TCR-Tg (F5 transgenic) mice. We also thank vivo. J. Immunol. 165:3673. members of the H.-G. Ljunggren laboratory for fruitful discussions. 16. Coles, M. C., C. W. McMahon, H. Takizawa, and D. H. Raulet. 2000. Memory CD8 T lymphocytes express inhibitory MHC-speciﬁc Ly49 receptors. Eur. J. Im- munol. 30:236. References 17. Ugolini, S., and E. Vivier. 2000. Regulation of T cell function by NK cell re- 1. Valiante, N. M., and G. Trinchieri. 1993. Identiﬁcation of a novel signal trans- ceptors for classical MHC class I molecules. Curr. Opin. Immunol. 12:295. duction surface molecule on human cytotoxic lymphocytes. J. Exp. Med. 178: 18. Schatzle, J. D., S. Sheu, S. E. Stepp, P. A. Mathew, M. Bennett, and V. Kumar. http://www.jimmunol.org/ 1397. 1999. Characterization of inhibitory and stimulatory forms of the murine natural 2. Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, and killer cell receptor 2B4. Proc. Natl. Acad. Sci. USA 96:3870. A. Takashima. 1995. 2B4, a new member of the immunoglobulin gene super- 19. Stepp, S. E., J. D. Schatzle, M. Bennett, V. Kumar, and P. A. Mathew. 1999. family, is expressed on murine dendritic epidermal T cells and plays a functional Gene structure of the murine NK cell receptor 2B4: presence of two alternatively role in their killing of skin tumors. J. Invest. Dermatol. 105:592. spliced isoforms with distinct cytoplasmic domains. Eur. J. Immunol. 29:2392. 3. Colonna, M., H. Nakajima, and M. Cella. 1999. Inhibitory and activating recep- 20. Sprent, J., and C. D. Surh. 2001. Generation and maintenance of memory T cells. tors involved in immune surveillance by human NK and myeloid cells. J. Leu- Curr. Opin. Immunol. 13:248. kocyte Biol. 66:718. 4. Nakajima, H., M. Cella, H. Langen, A. Friedlein, and M. Colonna. 1999. Acti- 21. Tangye, S. G., J. H. Phillips, and L. L. Lanier. 2000. The CD2-subset of the Ig vating interactions in human NK cell recognition: the role of 2B4- CD48. Eur. superfamily of cell surface molecules: receptor-ligand pairs expressed by NK J. Immunol. 29:1676. cells and other immune cells. Semin. Immunol. 12:149. 5. Boles, K. S., H. Nakajima, M. Colonna, S. S. Chuang, S. E. Stepp, M. Bennett, 22. Solomon, K. R., C. E. Rudd, and R. W. Finberg. 1996. The association between by guest on September 29, 2021 V. Kumar, and P. A. Mathew. 1999. Molecular characterization of a novel human glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein ␣ natural killer cell receptor homologous to mouse 2B4. Tissue Antigens 54:27. subunits in lymphocytes. Proc. Natl. Acad. Sci. USA 93:6053. 6. Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, and V. Kumar. 23. Cebecauer, M., J. Cerny, and V. Horejsi. 1998. Incorporation of leucocyte GPI- 1993. A novel function-associated molecule related to non-MHC-restricted cy- anchored proteins and protein tyrosine kinases into lipid-rich membrane domains totoxicity mediated by activated NK cells and T cells. J. Immunol. 151:60. of COS-7 cells. Biochem. Biophys. Res. Commun. 243:706.