Acquisition of CD80 (-1) by T Cells

Helen Sabzevari,* Judy Kantor,* Adnan Jaigirdar,* Yutaka Tagaya,† Mayumi Naramura,‡ James W. Hodge,* John Bernon,* and Jeffrey Schlom1*

Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4؉ T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different populations. The Journal of Immunology, 2001, 166: 2505–2513.

cell activation usually requires at least two different sig- reported increased levels of CD80 expression on both CD4ϩ and nals: 1) an Ag-specific signal (signal 1); and 2) a second, CD8ϩ cells in HIV-infected patients and revealed that these T cells T or costimulatory, signal (signal 2) that is provided by re- with accessory cell properties can costimulate proliferation in pu- ceptor ligand interaction between T cells and APCs, such as den- rified responder T cells from healthy control donors. Further stud- dritic cells (DCs).2 Although a variety of molecules reportedly ies of patients have demonstrated that these individuals have have costimulation function, the majority of studies to date have increased amounts of CD80 on PBLs, including T cells (14). How- been focused on costimulation delivered via CD28 and CD80 in- ever, although all of these studies reported increased levels of teractions (1, 2). CD80 is a member of the Ig gene superfamily and CD80 molecules on T cells, none of the studies have actually dem- is expressed mainly on professional APCs; its receptor, CD28, is onstrated an endogenous up-regulation of CD80 molecules on T constitutively expressed on thymocytes and on most resting and cells (6–14). activated T cells. CD80 has also been shown to react with CTLA-4 In parallel murine studies, the amount of CD80 on T cells was on the surface of activated T cells; however, CTLA-4 has been reported to vary with the state of activation. Resting T cells dis- shown not to be expressed on resting T cells (3, 4). Interaction played little or no CD80; however, upon activation, CD80 levels of CD28 and CD80 prevents the induction of and on the cell surface increased concomitantly, peaking at 72 h (15). apoptosis of T cells (5). In a recent study, Weintraub et al. (16, 17) demonstrated that al- Various studies have reported that CD80 can also be detected on though splenic T cells from normal B6 mice were negative for activated T cell clones, T cells activated repeatedly in vitro, CD80, freshly isolated T lymphocytes from the spleens of auto- and T cells that have infiltrated skin lesions and are in rheumatoid immune B6/gld and B6/lpr mice showed higher levels of CD80. synovial fluid (6–11). Studies by Kochli et al. that analyzed the This phenomenon was age dependent and may have contributed to expression of CD80 and MHC complex class II molecules on cir- lymphoproliferation and autoimmunity. Although these studies culating T cells of HIV-infected individuals have suggested that mentioned “increases in CD80 expression in T cells,” no data were these T cells develop an Ag-presenting phenotype by up-regulating actually presented in these studies as to whether these increases expression of HLA and CD80 (12). Moreover, Wolthers et al. (13) were due to up-regulation of CD80 in T cells or acquisition of CD80. *Laboratory of Tumor Immunology and Biology, Division of Basic Sciences, Na- Previous studies have demonstrated that T cells and thymocytes tional Cancer Institute; †Metabolism Branch, Division of Clinical Sciences, National can acquire MHC class I and class II determinants from other cells Cancer Institute; and ‡Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 present in the local environment (18, 19). Furthermore, it has been demonstrated that double-negative thymocytes can passively ac- Received for publication August 24, 2000. Accepted for publication December ϩ ϩ 4, 2000. quire a CD4 /CD8 determinant from other cells in the thymus ϩ ϩ The costs of publication of this article were defrayed in part by the payment of page that are actively expressing their CD4 /CD8 genes (20). A re- charges. This article must therefore be hereby marked advertisement in accordance cent study by Huang et al. (19) demonstrated that when peptide- with 18 U.S.C. Section 1734 solely to indicate this fact. specific T cells interacted with APCs, MHC molecules on APCs 1 Address correspondence and reprint requests to Dr. Jeffrey Schlom, Laboratory of formed clusters at the site of T cell contact. Afterward, these clus- Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 8B09, Bethesda, MD 20892-1402. E- ters were acquired by T cells and internalized through TCR endo- mail address: [email protected] cytosis. These studies also showed that the presence of APC-de- 2 Abbreviations used in this paper: DC, ; KO, knockout; B7dKO, CD80/ rived peptide-MHC molecules on the T cell surface could make CD86 double-knockout; PCC/TCR-Tg, TCR transgenic for pigeon cytochrome c88–104; rF-CD80, recombinant fowlpox virus expressing CD80; FP-WT, wild-type fowlpox; these T cells susceptible to lysis by neighboring T cells. Moreover, GFP, green fluorescence ; CHX, cyclohexamide. it has been reported recently that the activation of T cells with

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2506 ACQUISITION OF CD80 BY T CELLS

Drosophila cells as APCs may result in the rapid transfer of MHC cells) and cultured with 1 ϫ 106 effector/memory T cells. The APCs were molecules and other costimulatory molecules, such as CD80, from separated from T cells 24 h later by a magnet. This procedure yielded a APCs to T cells (21). preparation of T cells devoid of any APCs. Although the concept of molecule acquisition by T cells origi- nated 20 years ago, the actual interactions of receptor ligands and Recombinant fowlpox viruses and of DCs the factors affecting ligand acquisition still remain unclear. In this Recombinant fowlpox virus expressing CD80 (rF-CD80) and wild-type regard, we analyzed CD80 levels on murine T cells upon stimu- fowlpox (FP-WT) have been described previously (24). DCs were har- lation with various APCs expressing different levels of CD80 on vested on day 6 of culture and washed with OptiMem (Life Technologies, Gaithersburg, MD). The cells (1.2 ϫ 106/ml) were infected with FP-WT or their surface. Although CD80 was previously thought to be endo- rF-CD80 at 50 MOI (multiplicity of infection; PFU/cells) for2hat37°C. genously up-regulated, results of studies of CD80/CD86 double- Cells were first washed and then incubated in condition medium at 37°C knockout (B7dKO) mice reported here demonstrate that CD80 is overnight. After 18 h, DCs were harvested for in vitro assays. physically acquired by T cells from APCs shortly after T cell ac- tivation. We also demonstrate here that CD80 acquisition by T Peptide cells is mediated by the TCR and its CD28 ligand (confirming the The I-Ek-restricted PCC peptide was synthesized and HPLC-purified by recent data by Hwang et al. (21)) and that the level of CD80 ac- American Peptide (Sunnyvale, CA). quisition by T cells is related to both the level of CD80 expression Cell preparation and culture on APCs and the strength of signal 1. Moreover, our data for the first time suggest that acquisition of CD80 by T cells might be of CD4ϩ cells were purified using microbeads conjugated to anti-CD4 mAb, importance for the Ag-presenting capacity of T cells. Thus, these according to the manufacturer’s instructions (MiniMACS, Miltenyi Bio- tec), on MACS columns. Isolated CD4ϩ cells (5 ϫ 105 cells/ml) were findings might have important implications for a further under- cultured with APCs (1 ϫ 105 cells/ml) with or without signal 1, with either standing of immune regulation and the pathogenesis of immuno- anti-CD3 Ab or PCC peptide (at concentrations indicated in each figure pathological diseases. legend), in RPMI 1640 supplemented with the following: 10% heat-inac- Ϫ5 tivated FBS, 5 ϫ 10 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, Materials and Methods 100 ␮M/ml streptomycin, and 10 mM HEPES (all obtained from Life Technologies, Gaithersburg, MD), and 15 ␮g/ml gentamicin (BioWhit- Animals taker, Walkersville, MD). BALB/c, C57BL/6, B10.A, and CD28KO mice were obtained from The Effector/memory CD4ϩ T cells Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen- free conditions. B7dKO mice were provided by R. Hodes (National Insti- Effector/memory CD4ϩ T cells were generated by activating naive splenic tute on Aging, National Institutes of Health, Bethesda, MD) and were CD4ϩ T cells from PCC/TCR-Tg mice in culture with 10 ␮g/ml PCC originally obtained from A. Sharpe (Harvard University, Boston, MA). peptide for 3 days. Following centrifugation with Ficoll gradient, the live Pigeon cytochrome c88–104-transgenic (PCC/TCR-Tg) (5CC7 TCR-Tg) cells were washed and then rested for 4–6 days in six-well plates with 5 mice, which are TCR transgenic for PCC, were obtained from Taconic IU/ml of murine IL-2 medium. The cells were washed and given IL-2 every Farms (Germantown, NY) and bred in the National Cancer Institute animal 3 days following the 3-day PCC activation. The effector/memory CD4ϩ facility under pathogen-free conditions. PCC/TCR-Tg IL-2 green fluores- cells were analyzed for markers as follows: FITC-conjugated CD62 ligand, cence protein (GFP)ki (PCC/I-Ek-specific) mice were provided by M. Nara- PE-conjugated CD44, FITC-conjugated CD45RB, and FITC-conjugated mura (National Institute of Allergy and Infectious Diseases, National In- CD69. Appropriate isotype control was used for all of the markers. stitutes of Health). Apoptosis assay mAbs and flow cytometry Effector/memory CD4ϩ cells were cultured for 24 h in the presence of To evaluate CD80 expression on T cells, cell suspensions were stained with various concentrations of peptide as described in Results. Apoptosis was directly conjugated mAbs (anti-CD4PE; anti-CD80 FITC). The proportion assessed using the TUNEL (PharMingen) according to manufacturer’s ϩ ϩ of CD4 cells expressing CD80 was determined by gating on CD4 T instructions. cells, excluding the dead cells. Fluorochrome mAb Abs to CD4, CD80, MHC class II I-Ek, CD28, and purified anti-CD3, anti-CD80, hamster IgG, RNA extraction and PCR and anti-CD28 Abs were purchased from PharMingen (San Diego, CA), RNA was prepared using an RNA STAT-60 kit (Tel-Test, Friendswood, and samples were analyzed with a FACSCaliber (Becton Dickinson, TX), and 2 ␮g of total RNA was used to synthesize cDNA with the Su- Mountain View, CA) using CellQuest software for the Macintosh. perScript Preamplification System (Life Technologies, Gaithersburg, MD), APCs using oligo(dT) according to the manufacturer’s instructions. Oligonucle- otides specific for CD80 and ␤-actin were prepared by Cruachem (Dulles, DCs were prepared as described previously (22). Briefly, bone marrow VA) and Invitrogen (Carlsbad, CA). Primers for CD80 (517-bp fragment) cells from 6- to 8-wk-old mice were depleted of lymphocytes using a were 5Ј-ATCCAGGATACACCACTCC-3Ј (sense) and 5Ј-TCCAACCAA mixture of magnetic beads specific for CD4, CD8, MHC class I, and MHC GAGAAGCGAGG-3Ј (antisense). Primers for ␤-actin (1-kb fragment) class II (MiniMACS, Miltenyi Biotec, Auburn, CA). Cells were incubated were 5Ј-GCTCACCATGGATGATGATATCGC-3Ј (sense) and 5Ј-GGA in six-well plates (5 ϫ 106 cells/well) with medium supplemented with 10 GGAGCAATGATCTTGATCTTC-3Ј (antisense). cDNA samples were ng/ml GM-CSF and 10 ng/ml IL-4 (R&D Systems, Minneapolis, MN). subjected to amplification using PCR (model 9600; Perkin-Elmer Cetus, Cells were replated in fresh -supplemented medium on days 2 and Norwalk, CT). Samples were initially denatured for 2 min at 94°C. CD80 4. On day 6, cells were harvested for infection and analysis. The following and ␤-actin-specific amplification was conducted for 25 cycles, where each cell lines were provided by R. Germain (National Institute of Allergy and cycle consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, Infectious Diseases, National Institutes of Health): fibroblast cell line and polymerization at 72° for 30 s, followed by a finishing step at 72° for DCEK, which expressed a low level of CD80; MHC class II I-Ek and 13.9 10 min. Samples (20 ␮l) were resolved on 1% agarose gels in Tris-borate- fibroblast cell line, which expressed a high level of CD80; and COS (mon- EDTA (TBE), electrophoresed at 100 V, and stained with ethidium bro- key-kidney) cells, which expressed MHC class II I-Ek. Murine colonic mide; the cDNA fragments were visualized directly by UV . adenocarcinoma cells (MC38) and the retrovirally transduced MC38/CD80 cell line have been described previously (23). A20 cells are murine GFP-CD80 fusion protein lymphoma cells expressing 37% CD80 on their surface. A cDNA clone of murine CD80 was cloned and sequenced as described Separation of APCs by beads previously (25). Restriction endonuclease digestion of this clone with Kpni and Apai released a 1-Kb fragment that was gel purified and ligated into the Fibroblast cells (20 ϫ 106) were cultured with 250 ␮l of goat anti-mouse Kpni/Apai sites of pEGFP-C1, the C-terminal protein-fusion expression Dynabeads (Dynal, Lake Success, NY) in the presence of various concen- vector (Clontech Laboratories, Palo Alto, CA). DH5␣ Escherichia coli trations of peptide. After 2 days, the fibroblasts that had absorbed the beads cells were transformed with this construct (Life Technologies, Rockville, were separated by a magnet. These cells were then used as APCs (2 ϫ 105 MD), and kanemycin-resistant colonies were selected and sequenced for The Journal of Immunology 2507

CD80 expression. pEGFP-CD80 DNA was CsCl-banded and subsequently signal 1, incubation of T cells from B7dKO mice with both regular used to transfect COS cells for confocal studies. DCs and those infected with WT-FP led to the acquisition of rel- atively low levels of CD80 (7–15%) (Table I). However, in the Results presence of signal 1 (10 ␮g/ml anti-CD3), 55–57% of CD4ϩ cells CD80 acquisition by T cells in B7dKO mice acquired CD80 on their surface (Table I). When B7dKO CD4ϩ ϩ To establish the innate expression of CD80 on naive T cells, CD4ϩ cells were incubated with rF-CD80-infected DCs, the CD4 cells cells from four strains of mice were analyzed by two-color FACS acquired more CD80 in both the absence (53%) and presence analysis. T cell expression of CD80 is a relatively rare occurrence (87%) of signal 1 (Table I). These results using T cells from in these strains, as determined by immunofluorescence and flow B7dKO mice further demonstrate that CD80 levels on T cells after cytometry. CD80 expression levels on T cells varied among the a short period of activation are due to the acquisition of CD80 from different strains of mice, ranging from 2 to 9% in C57BL/6 mice, APCs, and not the constitutive expression and/or up-regulation of to very small (1–2%) or undetectable levels in B7dKO, CD28KO, endogenous CD80. Moreover, these results demonstrate that both PCC/TCR-Tg mice. the presence of signal 1 and the level of CD80 expression on APCs Previous studies have reported that activated T cells express influence this phenomenon. CD80 molecules on their surface 3–7 days after activation (15). However, none of these studies used B7dKO mice as the source of CD80 is acquired by T cells through CD28 interaction T cells. CD4ϩ naive T cells from B7dKO mice were used to in- CD80 acquisition by T cells raises the question of the role of CD28 vestigate whether CD80 expression on T cells shortly after acti- in this process. To examine the role of the CD28 molecule in vation is induced endogenously or acquired from APCs. Spleno- acquiring CD80, CD4ϩ cells from CD28KO (CD28Ϫ/Ϫ) mice and cytes from B7dKO mice lack functional genes for both CD80 and normal C57BL/6 mice were incubated for 24 h with MC38/CD80 CD86; upon activation, these cells are unable to express CD80 or cells; MC38/CD80 are retrovirally transduced murine carcinoma CD86 on their surface (26). CD4ϩ cells from B7dKO mice were cells expressing CD80. These experiments were conducted either incubated with 10 ␮g/ml of anti-CD3 as signal 1 and A20 cells (a in the presence or absence of anti-CD3 (10 ␮g/ml) as signal 1. As B cell line expressing 37% CD80) as APCs. The T cells were shown in Table II, T cells from CD28KO mice did not acquire monitored for 24 h. As shown in Fig. 1A, CD4ϩ cells from B7dKO CD80 upon incubation with MC38/CD80 in the absence or pres- mice did not express CD80; levels were similar to isotype control. ence of signal 1 (1–2% CD80 acquisition). However, 19% of Activation of B7dKO CD4ϩ cells with 10 ␮g of anti-CD3, in the CD4ϩ cells from C57BL/6 mice (expressing constitutively high absence of APCs (A20 cells), did not change CD80 expression levels of CD28) did acquire CD80 upon stimulation with MC38/ (Fig. 1B). As a result of incubating B7dKO CD4ϩ cells with A20 CD80 in the presence of signal 1. These studies demonstrate that cells in the absence of signal 1, 4% of CD4ϩ cells acquired CD80 the acquisition of CD80 by T cells is mediated through the CD28 (Fig. 1C). However, when CD4ϩ cells were incubated with A20 ligand and not by way of a random process. Experiments were also cells as APCs in the presence of anti-CD3 (10 ␮g/ml) as signal 1, conducted to show that the ligand on resting T cells for CD80 30% of the CD4ϩ cells from the B7dKO mice acquired CD80 (Fig. acquisition is not CTLA-4. T cells from PCC/TCR-Tg mice were 1D). incubated with fibroblasts expressing CD80 in the presence and To further address the hypothesis that CD80 is acquired by T absence of anti-CTLA-4 Ab. In both cases, there was no reduction cells upon activation, CD4ϩ cells from B7dKO mice were then in the acquisition of CD80 by T cells (data not shown). incubated for 24 h with either normal DCs or DCs that were in- fected with WT-FP virus or rF-CD80. Normal DCs expressed high Physical nature of CD80 transfer to T cells levels of CD80 (70%), and infection with WT virus did not affect To investigate the physical nature of the CD80 binding to the sur- CD80 expression. However, infection of DCs with rF-CD80 in- face of T cells, COS (monkey-kidney) cells expressing I-Ek MHC creased expression of CD80 on 90% of cells. In the absence of class II were transfected with a construct containing GFP-CD80. As determined by FACS analysis, 30% of COS cells expressed membrane-bound GFP-CD80 fusion protein (data not shown). Confocal microscopy studies of COS cells demonstrated that when these cells were transfected with GFP-CD80 construct, CD80 was

Table I. CD80 acquisition by CD4ϩ T cells from double knockout micea

Signal 1 APC (anti-CD3 ␮g/ml) % CD80

None 0 1.0 None 10 1.1 DC 0 7.5 10 55 FIGURE 1. Acquisition of CD80 from the A20 cell line by B7dKO T DC (FP-WT) 0 15 cells. Two-color FACS analysis was performed on T cells for CD4 and 10 57 CD80. A, Expression of CD80 on CD4ϩ T cells from B7dKO mice; B, DC (rF-CD80) 0 53 ϩ 10 87 CD80 expression on CD4 cells upon stimulation with 10 ␮g/ml of anti- ϩ CD3 Ab for 24 h; C, acquisition of CD80 by B7dKO CD4 cells in the a CD4ϩ T cells from B7dKO mice were purified using magnetic beads. CD4ϩ presence of the A20 cell line as APCs, but in the absence of signal 1; or D, cells (5 ϫ 105/ml) were cultured with DCs (1 ϫ 105/ml) or DCs infected with either ϩ FP-WT or rF-CD80 (as described in Materials and Methods) in the absence or pres- acquisition of CD80 by B7dKO CD4 cells in the presence of A20 cells ϩ ence of anti-CD3 as signal 1. Two-color FACS analysis for CD80 and CD4 cells and anti-CD3 as signal 1. The data are representative of two independent was performed 24 h later. The data are representative of two independent experiments. All gates were set based on negative controls. experiments. 2508 ACQUISITION OF CD80 BY T CELLS

ϩ Table II. Analysis of CD80 acquisition by T cells from CD28ϩ vs B–D). When PCC/TCR-Tg CD4 cells were incubated with trans- CD28 knockout micea fected COS cells in the presence of PCC peptide (10 ␮g/ml) as signal 1, T cells were shown to acquire CD80 molecules (Fig. 2, Source of CD4ϩ T Cells (%) E–H). These results further demonstrate the physical acquisition of Signal 1 APC (anti-CD3 ␮g/ml) C57BL/6 CD28KO CD80 by T cells.

None 0.0 8.0 1.8 Effect of cyclohexamide (CHX) on CD80 acquisition MC38/CD80 0.0 8.0 1.4 10.0 19.0 0.8 Recent studies have demonstrated the existence of a new homolog of the B7 family of molecules (27). This observation raises the a CD4ϩ T cells were prepared from C57BL/6 mice or CD28KO mice using mag- ϩ possibility that there might be a CD80 homolog that can be endo- netic beads. CD4 cells (5 ϫ 105/ml) were cultured with 1 ϫ 105/ml MC38 carci- noma cells that had been transduced with CD80 (MC38/CD80) in the absence or genously up-regulated rapidly on T cells upon stimulation, and that presence of anti-CD3 for 24 h and analyzed with two-color FACS analysis for CD4 the molecule might interact with the anti-CD80 Ab. To exclude and CD80 expression. The data are representative of two independent experiments. this scenario and further rule out the de novo generation of CD80 in short-term activated T cells, CD4ϩ cells from PCC/TCR-Tg expressed on cell surfaces (Fig. 2A). To further demonstrate cell mice were treated with the protein synthesis inhibitor CHX (20 ␮ ϩ surface expression of CD80, these cells were double-stained with g/ml) for 2 h. After CHX treatment, CD4 cells were incubated PE-anti-CD80. Superimposition of red PE on GFP led to the ap- with irradiated fibroblast cells expressing high levels of CD80 and k pearance of a yellow/orange color on cell membranes (Fig. 2, I-E MHC class II molecules for 4 h and for 24 h. There were no significant differences in CD80 acquisition between the CHX- treated cells and the untreated CD4ϩ cells at either 4 h (22% for CHX-treated and 30% for untreated cells) or 24 h (99% acquisition of CD80 for both treated and untreated cells). These results further indicate that CD80 expression on T cells upon activation is indeed due to acquisition, rather than up-regulation, of CD80 or any other homolog of the B7 family.

Analysis of naive and activated CD4ϩ T cells for CD80 mRNA by PCR To further demonstrate that CD80 is acquired by T cells upon activation, and not by the endogenous up-regulation of CD80 mRNA, PCR studies were performed (Fig. 3). Purified CD4ϩ cells of PCC/TCR-Tg mice were incubated with fibroblast cells express- ing high levels of CD80 (Fig. 4C) and 10 ␮g/ml of PCC peptide as signal 1. After 24 h of incubation, CD4ϩ cells were carefully re- moved from supernatant fluid and cultured two additional times (each time recovering cells in the supernatant) to ensure that no fibroblast (an APC that expresses CD80) was contaminating the T cell population. Purity of CD4ϩ cells, as well as the expression of CD80, was checked by FACS analysis. The cells were 100% CD4ϩ, and 79–80% of these cells had acquired CD80 on their surface (Fig. 4D). Simultaneously, as controls, naive PCC/TCR-Tg

FIGURE 2. Physical transfer of CD80 from COS cells (transfected with GFP-CD80 fusion protein) to T cells. Binding and transfer of CD80 from FIGURE 3. Analysis of CD4ϩ cells for CD80 mRNA. A 517-bp frag- COS cells to CD4ϩ cells were visualized by fluorescence and confocal ment of CD80 cDNA was amplified by RT-PCR, as described in Materials microscopy. Transfection of COS cells with GFP-CD80 vector led to cell and Methods. Lane A, DNA-molecular size standard; lane B, fibroblast surface expression (green) of the CD80 molecule (A) and double-staining transfected with plasmid expressing CD80 (positive control); lanes C and for CD80 (using PE CD80 Ab) (B–D), demonstrating that CD80 is mem- D, two different preparations from naive (unstimulated) PCC/TCR-Tg brane-bound on the COS cell surface. Incubation of GFP-CD80 COS cells CD4ϩ cells; lane E, PCC/TCR-Tg CD4ϩ cells stimulated for 24 h in the with PCC/TCR-Tg CD4ϩ cells in the presence of PCC peptide (10 ␮g/ml) presence of APCs (fibroblasts expressing CD80) and 10 ␮g/ml of PCC led to physical acquisition of CD80 from COS cells (E and F). G and H, peptide that had acquired CD80 (see Fig. 4D). These cells were depleted of Higher magnifications of E and F, respectively. APCs as described in Materials and Methods. The Journal of Immunology 2509

Acquisition of CD80 by T cells is proportional to CD80 expression on APCs Studies were undertaken to investigate whether increased density of CD80 on APCs might further stabilize the interaction and, there- fore, lead to more acquisition of CD80 by CD28 on T cells. To address this question, purified CD4ϩ cells of PCC/TCR-Tg mice were incubated for 24 h with fibroblast cells expressing either rel- atively low levels of CD80 (Fig. 4A) or high levels of CD80 (Fig. 4C) and PCC peptide (10 ␮g/ml) as signal 1. CD80 acquisition on T cells was proportional to CD80 expression by APCs. Although CD4ϩ cells stimulated with fibroblast cells expressing a low level of CD80 acquired much less CD80 on their surface (26%, Fig. 4B), 79% of CD4ϩ cells that were incubated with fibroblast cells ex- pressing a high level of CD80 acquired CD80 (Fig. 4D). To further determine whether this level of CD80 on APCs in- fluenced CD80 acquisition by T cells, purified CD4ϩ cells from PCC/TCR-Tg mice were incubated in the presence of PCC peptide (10 ␮g/ml) with either uninfected or rF-CD80-infected DCs. Nor- FIGURE 4. CD80 acquisition by T cells is related to the level of CD80 mal DCs express very high levels of CD80 (75%); DCs infected expression on APCs. When PCC/TCR-Tg CD4ϩ cells (5 ϫ 105 cells/ml) with rF-CD80 showed 90% CD80 expression. Incubation of CD4ϩ were incubated with a fibroblast cell line with lower expression levels of ϩ ϫ 5 ␮ cells with uninfected DCs led to 35% of CD4 cells acquiring CD80 (1 10 /ml) in the presence of 10 g/ml of PCC peptide for 24 h ϩ (A), T cells acquired relatively lower levels of CD80 (B). Incubation of CD80 on their surface. Moreover, CD4 cells incubated with rF- these cells with a fibroblast cell line expressing higher levels of CD80 (C) CD80 DCs obtained markedly higher levels of CD80 (81%) on in the presence of 10 ␮g/ml PCC peptide led to higher acquisition of CD80 their surface. by T cells (D). CD4ϩ cells were double-stained with PE anti-CD4 Ab and Studies were then conducted to investigate T cell selectivity in FITC anti-CD80 Ab. These experiments were repeated three times. Gates the acquisition of CD80 molecules; toward this goal, the fate of were set based on negative controls. E and F, Selective acquisition of ϩ another surface marker, CD40, on APCs was investigated in terms CD80 by T cells. Naive PCC/TCR-Tg CD4 (5 ϫ 105) cells and non- ϩ ϩ k of acquisition by T cells. CD4 cells were incubated with either transgenic, non-PCC-restricted B10.A CD8 (MHC I-E -restricted) cells normal DCs or DCs infected with FP-WT or rF-CD80 in the pres- (5 ϫ 105/ml) were incubated with an irradiated 13.9 fibroblast cell line ence or absence of various concentrations of PCC peptide as signal expressing high levels of CD80 (1 ϫ 105 cells/ml) and 10 ␮gofPCC peptide in 48-well plates for 24 h. E, CD80 acquisition by B10.A CD8ϩ 1. FACS analysis was performed on gated T cells, based on size cells after 24 h. F, CD80 acquisition by PCC/TCR-Tg CD4ϩ cells after and cell surface markers for T cells. An average of 30–40% of 24 h. Acquisition of CD80 by T cells was monitored by three-color FACS DCs express CD40 on their surface, whereas only 3–4% of T cells ϩ analysis. The data are representative of two different experiments. All gates express CD40. Incubation of CD4 cells with APCs in the pres- were set based on negative controls. ence of different concentrations of signal 1 PCC peptide led to little or no increase in the acquisition of CD40 by CD4ϩ cells, which remained 3–4% positive for CD40. These results further demon- strate that CD80 expression on T cells is not due to contaminating ϩ CD4ϩ cells were also purified from PCC/TCR-Tg animals and APCs, and that CD4 cells show selectivity in the acquisition of were checked for CD80 expression by FACS. These cells had very molecules from APCs. little or no CD80 on their surface. Using RT-PCR, we then ana- To further investigate the selectivity in CD80 acquisition, naive ϩ lyzed the expression of CD80 mRNA on unstimulated PCC/ CD4 cells from PCC/TCR-Tg mice were incubated with a fibro- TCR-Tg CD4ϩ cells or on CD4ϩ cells that were stimulated for blast cell line expressing high levels of CD80 and PCC peptide (10 ␮ 24 h. As shown in Fig. 3, a 517-bp CD80 fragment could readily g/ml) as signal 1 for 24 h (as described previously) in the pres- ence of CD8ϩ cells from non-Tg mice (B10.A mice). As shown in be amplified from the APC fibroblast cell line that expresses CD80 ϩ (positive control, lane B). However, CD80 could not be amplified Fig. 4E, non-Tg CD8 cells acquired relatively little CD80 (18%) ϩ above their 6–7% normal level of expression, whereas PCC/ from two different preparations of unstimulated CD4 cells (Fig. ϩ 3, lanes C and D)orCD4ϩ cells that were stimulated for 24 h (Fig. TCR-Tg CD4 cells acquired high levels of CD80 (77%, Fig. 4F). 3, lane E). ␤-actin expression demonstrated that equal amounts of These results further demonstrate that CD80 acquisition by T cells RNA were added in each PCR. To establish the sensitivity of de- is specific and not due to random acquisition or incorporation. tection of CD80 in PCR, a template dilution experiment was per- formed on a CD80-transfected fibroblast cell line. cDNA encoding CD80 acquisition by T cells is directly related to the strength of CD80 was synthesized using 2 ␮g of RNA from transfected fi- signal 1 broblasts; serial dilutions of cDNA (1:10- to 1:320-fold dilutions) Naive CD4ϩ cells from PCC/TCR-Tg mice were incubated with were used as a template for PCR amplification. Analysis of the fibroblast cells expressing low and high levels of CD80 in the product on an agarose gel revealed that the DNA bands could not presence of various concentrations of PCC peptide to investigate be detected when the template was diluted Ͼ1:160-fold, which is the effect of the concentration of signal 1 on T cell acquisition of Յ1 ng of RNA. Therefore, we can conclude that the T cells that CD80. Acquisition of CD80 by CD4ϩ cells upon stimulation with have been activated and acquired CD80 express Յ1 ng of CD80 fibroblast cells expressing a low level of CD80 was shown at the ϩ RNA. These results illustrate that upon activation of CD4 cells, high level of signal 1 (Fig. 5A). When PCC/TCR-Tg CD4ϩ cells there is no up-regulation of detectable levels of mRNA for CD80 were stimulated with fibroblast cells expressing high levels of at the 24-h time point; a 24-h time point was used in the above CD80, a direct relationship between the strength of signal 1 and the studies to detect the acquisition of CD80. acquisition of CD80 by T cells could be more clearly defined (Fig. 2510 ACQUISITION OF CD80 BY T CELLS

cultured with the same APCs expressing high levels of CD80 (Fig. 6). Based on our observations, the probable reason for this differ- ence between effector/memory and naive T cell acquisition is that effector/memory T cells express higher levels of CD28, the CD80 ligand, than naive T cells. Indeed, the effector/memory CD4ϩ cells from PCC/TCR-Tg mice used here expressed much higher levels of CD28 than naive T cells from these mice (70% for memory vs 32% for naive). When both naive and effector/memory CD4ϩ cells were cultured with APCs (expressing high amounts of CD80) and various concentrations of peptide, the effector/memory cells al- ways acquired more CD80 at a given peptide concentration (Fig. 6). At a peptide concentration of 0.1 ␮g/ml, ϳ90% of naive T cells acquired CD80; no cell death was observed in these T cells.

Acquisition of CD80 by memory cells results in apoptosis To determine whether the cell death in effector/memory T cell populations (as shown in Fig. 6) was due to apoptosis, effector/ memory CD4ϩ T cells were generated by activating naive cells with PCC peptide for 3 days and then resting them with IL-2 for 4 days. These effector/memory cells were then divided into two groups. Cells in the first group were cultured alone with various concentrations of PCC peptide for 24 h. As shown in Fig. 7A–C, these cells demonstrated minimal apoptosis when cultured with or without peptide. The second group consisted of effector/memory T cells that were cultured with APCs (fibroblasts expressing high amounts of CD80) in the presence of 0.001 ␮g/ml of PCC peptide for 24 h. Approximately 80% of these T cells acquired CD80 (as shown in Fig. 6), and the T cells were then separated from the fibroblast APCs by magnetic beads and cultured further in the presence of various concentrations of PCC peptide (i.e., 0, 0.001, or 0.1 ␮g/ml) for 24 h. Upon acquisition of CD80, 34% of these effector/memory T cells underwent spontaneous apoptosis (Fig. 7D) in the absence of exogenous peptide. Although the addition of

FIGURE 5. Influence of signal 1 on acquisition of CD80 by T cells. Purified PCC/TCR-Tg CD4ϩ cells (5 ϫ 105/ml) were incubated with 1 ϫ 105/ml irradiated fibroblast APCs expressing low CD80 (A) or high levels of CD80 in the presence of different amounts of PCC peptide (B)for1h (open column), 4 h (hatched column), and 24 h (filled column). The data are representative of three independent experiments.

5B). These results further demonstrate that the acquisition of CD80 by T cells can be influenced by the level of signal 1.

Differential acquisition of CD80 by naive and effector/memory CD4ϩ T cells To investigate the biological consequence of CD80 acquisition by effector/memory T cells, effector/memory CD4ϩ cells were gen- FIGURE 6. Differential acquisition of CD80 by naive and effector/ ϩ ϩ erated from naive CD4ϩ cells, as described above, and then cul- memory CD4 T cells. Either naive (f) or effector/memory CD4 T cells 5 5 tured with fibroblasts expressing high levels of CD80 as APCs in (छ)(5ϫ 10 ) were cultured with 1 ϫ 10 fibroblasts expressing high the presence of various concentrations of PCC peptide for 24 h. levels of CD80 as APCs with varying amounts of PCC peptide (0–0.1 ␮g/ml) as signal 1 in 48-well plates for 24 h. CD4ϩ cells were carefully Before incubation with APCs, 8–9% of effector T cells were pos- removed without disturbing the adherent fibroblast layer, and were ana- itive for CD80 expression, as compared with 1–2% of naive T cells ϩ lyzed for the presence of surface CD4 and CD80 by two-color FACS anal- Denotes that a high number of effector/memory T cells were dead ,ء .data not shown). When effector/memory CD4 cells were cul- ysis) tured with fibroblasts expressing high levels of CD80 as APCs, when analyzed by FACS analysis and trypan blue assay (as shown in Fig. ϳ40% of the cells acquired CD80. This is in contrast to the rela- 7). No cell death was seen in naive CD4ϩ T cells. The data represent four ϩ tively low percent CD80 acquisition by naive CD4 cells when different experiments. The Journal of Immunology 2511

CD4ϩ T cells treated with anti-CD80 Ab demonstrated a mean- ingful decrease in their apoptosis (46%, Fig. 8C). Based on these results, we propose that CD80 acquisition can in part lead to apoptosis of effector/memory cells and the level of apoptosis is enhanced with increased levels of signal 1.

CD80 acquisition can lead to Ag presentation by T cells Studies were then conducted to determine the biologic conse- quence that results when naive T cells acquire CD80. We asked specifically whether T cells that have acquired CD80 can now function as APCs to naive CD4ϩ T cells. To this end, IL-2 pro- duction was measured as an indicator of T cell activation. How- ever, one cannot simply measure IL-2 levels in the supernatant, because IL-2 can be produced not only from T cells functioning as APCs but also from T cells receiving the costimulatory signal, if any. We took advantage of an IL-2-GFPki mouse (28) in which the IL-2 exon from one of the IL-2 alleles was replaced by GFP. In FIGURE 7. Analysis of apoptosis in effector/memory CD4ϩ T cells ϩ these mice, IL-2 production can be monitored by cell-associated before and after acquisition of CD80. Naive CD4 cells from PCC/ (of the producer cell) fluorescence from GFP expression driven by TCR-Tg mice were cultured with 1 ␮g/ml of PCC peptide for 3 days and ␮ the endogenous IL-2 promoter. then rested with IL-2 (5 g/ml) for 4 days to generate the effector/memory ϩ T cells. These cells were divided into two groups. Cells in the first group PCC/TCR-Tg CD4 cells that have acquired CD80 were pre- were cultured overnight with various concentrations of peptide (i.e., 0, pared in the manner described above. Fibroblasts were used as 0.001, or 0.1 ␮g/ml) (A–C, respectively). Cells in the second group were APCs of choice to minimize the possibility of contamination of ϩ first cultured with fibroblasts (which express high levels of CD80) for 24 h CD4 cells that had acquired CD80 from professional APCs be- to acquire CD80 and were then separated from APCs as described in Ma- cause they can easily be separated from T cells by magnetic bead terials and Methods. The T cells were then cultured alone in the presence separation. Isolated CD4ϩ cells (2–3% expressing CD80 sponta- of various concentrations of peptide for 24 h (0, 0.001, or 0.1 ␮g/ml) (D–F, neously) were irradiated and used as control APCs. In the absence respectively). All of the above cells (in groups I and II) were analyzed for of PCC peptide, these control APCs were unable to activate the apoptosis using the TUNEL assay. Percent apoptosis is given in each panel. responding T cells (Fig. 9A); in the presence of PCC peptide (10 The data represent three different experiments. ␮g/ml), they could activate 16% of PCC/TCR-Tg/IL-2-GFPki

0.001 ␮g/ml PCC peptide had minimal effect (Fig. 7E), the addi- tion of 0.1 ␮g/ml of PCC clearly augmented the occurrence of apoptosis of CD4ϩ T cells (Fig. 7F). To further ascertain that the acquisition of CD80 caused the effector/memory cells apoptotic death, CD80 Ab was included in the assay, with an even higher concentration (1 ␮g/ml) of peptide as signal 1. As seen in Fig. 8A and B, isotype control Ab showed no inhibitory effects on the apoptotic death of these CD4ϩ T cells, whereas effector/memory

FIGURE 9. T cells that have acquired CD80 can themselves become APCs. Naive CD4ϩ cells from PCC/TCR-Tg mice were purified, irradiated (as APCs), and cultured with CD4ϩ cells from PCC/TCR-Tg/IL-2-GFPki mice as effector cells for 24 h in the absence of PCC peptide (A) or pres- ence of 10 ␮g/ml of PCC peptide (B). Furthermore, PCC/TCR-Tg CD4ϩ cells that had acquired CD80 from a fibroblast cell line expressing high levels of CD80 were purified, irradiated, and used as APCs in culture with PCC/TCR-Tg/IL-2-GFPki CD4ϩ cells as effectors. C and D, Activation of the effector cells (PCC/TCR-Tg/IL-2-GFPki) in the absence of peptide (C) FIGURE 8. CD80 acquisition can lead to apoptosis of effector/memory and in the presence of 10 ␮g/ml of PCC peptide (D). Moreover, PCC/ CD4ϩ T cells. Cells were cultured with fibroblasts for 24 h to acquire TCR-Tg CD4ϩ cells that acquired CD80 were treated with either 10 ␮g/ml CD80 and were then separated from APCs as described in Materials and of isotype-control Ab (E) or purified anti-CD80 Ab (F) for 30 min on ice, Methods. The CD4ϩ cells were then treated with either no Ab (A), isotype irradiated, and then incubated with PCC/TCR-Tg/IL-2-GFPki CD4ϩ cells control Ab (hamster IgG) (B), or anti-CD80 Ab (10 ␮g/ml) (C) for 30 min in the presence of peptide (10 ␮g/ml) for 24 h. Two-color FACS analysis and replated with 1 ␮g/ml of PCC peptide for 24 h. Cells were analyzed for was performed to study the presence of activated PCC/TCR-Tg/IL-2-GFPki apoptosis using the TUNEL assay. Percent apoptosis is given in each panel. CD4ϩ cells. The data represent two different experiments. 2512 ACQUISITION OF CD80 BY T CELLS

CD4ϩ cells, possibly due to the spontaneous expression of CD80 action and, therefore, acquisition of higher levels of costimulatory (Fig. 9B). In contrast, CD4ϩ cells that had acquired CD80 from molecules in the presence of less signal 1. fibroblasts were much more potent as stimulators of PCC/TCR- Several groups have previously reported the expression of CD80 Tg/IL-2-GFPki cells to produce IL-2 at 24 h in the absence (Fig. on T cell clones as well as on T cells from patients with autoim- 9C) or presence (Fig. 9D) of peptide. The possible reason for this mune diseases or HIV infection (6–13). Interestingly, there has will be discussed below. To further demonstrate that CD80 acqui- been no study to address the up-regulation of CD80 message in T sition allows naive CD4ϩ cells to become APCs, anti-CD80 Ab cells. T cells in these studies have always been in the presence of was included in the assay. As seen in Fig. 9F, APCs treated with APCs either in vitro or in vivo; therefore, it is reasonable to infer anti-CD80 Ab led to a marked decrease of GFP-IL-2 CD4ϩ cells that reported CD80 expression on T cells in these studies might becoming activated (Fig. 9F), when compared with APCs treated have actually been due to acquisition, rather than up-regulation of with control Ab (Fig. 9E). expression, of CD80. The studies reported here demonstrate that at the time of acquisition of CD80 by CD4ϩ cells (24 h), no CD80 Discussion mRNA could be detected by PCR (Fig. 3). In this report, we demonstrate for the first time the acquisition of Although two different biological consequences of CD80 acqui- the CD80 costimulatory molecule by naive CD4ϩ T cells using sition by T cells are demonstrated here, this phenomenon will most syngeneic APCs. Several studies were conducted to rule out the likely be the subject of numerous investigations in the future. Dur- endogenous expression of CD80 by T cells. When T cells from ing an initial encounter with Ag, naive Ag-specific lymphocytes B7dKO mice were incubated with either a B cell line or DCs in the proliferate and differentiate to become activated effector/memory presence of signal 1, they acquired CD80 on their cell surface (Fig. T cells. The current dogma states that most of these activated ef- 1; Table I). Because the CD80 and CD86 genes are deleted in fector T cells die after a brief life span (35–37). Mechanisms un- B7dKO mice, neither one of these molecules can be expressed derlying the regulated expansion of effector/memory T cells are (26). In these studies, we also used normal DCs and a B cell line not well understood. However, two of the findings reported here as APCs to eliminate the possibility that CD80 acquisition might may indicate negative regulatory consequences upon acquisition of be due to some abnormal phenomenon on vector-transfected APC CD80 by memory cells: 1) effector/memory T cells can acquire membranes. The PCR studies (Fig. 3) and the studies with CHX- significantly higher levels of CD80 in the presence of low levels of treated T cells further demonstrated that the expression of CD80 signal 1; and 2) these cells undergo apoptosis upon acquisition of on T cells is not de novo. In view of the recent discovery of B7h CD80 and increased levels of signal 1. The apoptosis observed in (a new homolog of CD80 and CD86), these results also ruled out the results of Figs. 7 and 8 is most likely not due to a consequence the possibility of interaction between anti-CD80 Ab and this hom- of the way cells were handled because the apoptosis was depen- olog. The confocal microscopy studies using GFP/CD80 fusion dent on the amount of peptide present (including no apoptosis with protein further strengthened the notion that CD80 molecules are no peptide), and all cells were handled in the same way. Also, it actually acquired upon T cell/APC interaction (Fig. 2). Moreover, should be mentioned that our data do not exclude the possibility we demonstrated here that the level of acquisition by T cells is that effector/memory CD4ϩ cells could also acquire the MHC/ related to the strength of signal 1 (Fig. 5). peptide complex; therefore, these MHC complexes might play a It has been demonstrated that shortly after T cell/APC interac- role in the apoptosis of these cells. It has been demonstrated that tion, immunological synapses or supramolecular activation clus- activated T cells can process and present Ags to other T cells in ters form at the contact site (29). Formation of these immunolog- vitro (38). The studies reported here using CD4ϩ cells from PCC/ ical synapses is associated not only with TCRs and MHC TCR-Tg mice that have acquired CD80 show not only that these molecules, but also with various costimulatory molecules such as cells expressed MHC class II molecules, but also that they were LFA/ICAM, CD2/CD48, and CD28/B7 receptor ligands (30–32). able to present soluble PCC peptide to T cells from PCC/TCR- Wade et al. (33) have demonstrated that truncation of the intracy- Tg/IL-2-GFPki mice more efficiently than CD4ϩ cells from PCC/ toplasmic tail of MHC molecules enhances lateral movement of TCR-Tg mice that had not acquired CD80; this led to activation these complexes but decreases immunogenicity. Presentation of and production of IL-2. Interestingly, the CD4ϩ cells that had ac- MHC/peptide complexes in a cross-linked form can lead to overt quired CD80 were able to activate the PCC/TCR-Tg/IL-2-GFPki T cell activation (33). It has been suggested that the accumulation CD4ϩ cells without adding additional peptide. One possible ex- and interaction of costimulatory molecules with their receptors planation of this is that, because these CD4ϩ cells had been incu- and, perhaps, cytoskeletal may promote the stabilization bated with fibroblast cells in the presence of peptide and had up- of TCR/MHC interactions. In addition, costimulatory molecules at regulated their MHC class II molecules (data not shown), these T the early stages of T cell activation may function by enhancing cells actually also acquired peptide-MHC and were able to activate TCR cross-linking, therefore intensifying signal 1. In light of the PCC/TCR-Tg/IL-2-GFPki CD4ϩ cells in the absence of the addi- above-mentioned studies, it is possible that increased CD80 ex- tion of exogenous PCC peptide. These results suggest that acti- pression on APCs, along with increased signal 1 in our models, vated T cells that have acquired CD80 molecules have all the char- may stabilize TCR/MHC interactions, contribute to the overall acteristics of professional APCs. However, these functions are not avidity of T cell/APC interactions, and thus lead to the increased expressed constitutively; instead, they occur only after T cell ac- transfer of CD80 to T cells. The data reported here on CD80 ac- tivation, when class II synthesis is up-regulated and T cells have quisition by CD28Ϫ/Ϫ cells support the hypothesis by Hwang et al. acquired the CD80 ligand. Studies are now in progress investigat- (21) that a high enough affinity for adhesion between CD28 and ing whether the APC function of CD4ϩ cells that had acquired CD80 may actually result in the transfer of CD80 molecules from CD80 as reported here is dependent on other acquired molecules APCs to T cells. such as MHC/peptide complexes or other costimulatory molecules. Previous studies have shown that varying the strength of co- However, the data presented here provide preliminary evidence stimulation dramatically reduces the Ag dose required for T cell that upon acquiring CD80, T cells can more efficiently activate activation (34). Our observations indicate that increasing the den- bystander T cells. sity of CD80 on APCs may facilitate a strong interaction with There are at least two possible reasons why T cells become CD28, which would lead to stabilization of the T cell/APC inter- APCs in vivo. One potential advantage is that activated T cells The Journal of Immunology 2513 would deliver both signals 1 and 2 to other T cells, thus amplifying 13. Wolthers, K. C., S. A. Otto, S. M. A. Lens, D. N. Kohlbach, R. A. W. van Lier, an immune response; the other is that T cells could deliver only F. Miedema, and L. Meyaard. 1996. Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regu- signal 2 to T cells that have received signal 1 from nonprofessional lation by CD4ϩ T cells. Eur. J. Immunol. 26:1700. APCs (38). Consequently, although the initiation of the T cell re- 14. Takasaki, Y., K. Abe, Y. Tokano, and H. Hashimoto. 1999. The expression of LFA-1, ICAM-1, CD80 and CD86 molecules in lupus patients: implications for sponse is dependent on professional APCs, it is possible that T . Intern. Med. 38:175. cells may costimulate each other by acquiring CD80 under certain 15. Prabhu Das, M. R., S. S. Zamvil, F. Borriello, H. L. 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