Acquisition of CD80 (B7-1) 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 apoptosis 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 T cell 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 lupus 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 clonal anergy 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 human 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, dendritic cell; 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 protein; 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 infection 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).