Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1209-1212, February 1988 Immunology Antigen presentation to HLA class II-restricted measles virus-specific T-cell clones can occur in the absence of the invariant chain (antigen processing/chloroquine/DNA-mediated gene transfer/HLA-DR restriction/major histocompatibility complex) RAFICK P. SEKALY*t, STEVEN JACOBSONt, JOHN R. RICHERT§, CECILE TONNELLE*¶, HENRY F. MCFARLANDt, AND ERIC 0. LONG* *Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; tNeuroimmunology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and §Department of Neurology, Georgetown University Medical Center, Washington, DC 20007 Communicated by William E. Paul, November 9, 1987 ABSTRACT A human fibroblast line expressing HLA- The requirements for cell-surface expression of class II a/3 DR1 antigen on its surface was generated by transfection with heterodimers have also been analyzed by DNA-mediated DRa and DRP cDNAs. Expression of the invariant chain gene gene transfer. It was recently shown that the invariant chain was not detectable in the transfected fibroblasts and was not that is transiently associated with a,8 heterodimers intracel- induced by infection with measles virus. Lysis of measles lularly is not required for expression of human MHC class II virus-infected cells occurred with DRM- but not with DR4- antigens at the cell surface (8, 9). These results suggested restricted measles virus-specific cytotoxic T-lymphocyte (CTL) that the invariant chain might be involved in the function of clones and was inhibited by a monoclonal antibody specific for MHC class II antigens, such as binding of processed antigen DR antigen. Therefore, the invariant chain is not required for fragments, rather than in their biosynthesis. In this report, DR-restricted presentation of measles virus antigens by this we have used clones of a transfected human fibroblast line fibroblast line. Transfected fibroblasts were lysed as efficiently that expressed DR antigens in the absence of the invariant as an autologous B-cell line even though they expressed much chain to test for their ability to present antigens. Presenta- less surface DR antigen. Lysis of both the transfected fibro- tion of measles virus antigens to specific CTLs occurred blasts and the B-cell line was insensitive to treatment with efficiently in these cells. chloroquine. These results demonstrate that expression of a DR a'p heterodimer at the surface of this fibroblast line is MATERIALS AND METHODS necessary and sufficient for presentation of measles virus antigens to specific CTL clones. Transfected Human Fibroblast Lines. The human simian virus 40-transformed fibroblast line 637B and two clones The interaction of a helper T cell with an antigen-presenting obtained after transfection with the DR1 a- and 8-chain cell that expresses surface class II antigens and a specific cDNAs have been described (9). Briefly, 637B cells were antigen results in lymphokine production by the T cell. transfected by the calcium phosphate-precipitation tech- Lymphokines in turn trigger effector cells of the immune nique with plasmids containing full-length DRa and DRP system (1). Human antigen-presenting cells (B cells, macro- cDNA clones together with the pSV2-neo plasmid for selec- phages) express class II antigens of the major histocompat- tion. Expression of cDNA inserts was controlled by the ibility complex (MHC)-namely, HLA-DR, -DQ, and -DP simian virus 40 early promoter. DR-positive 637B cells were (2). Recent advances in cloning the genes for the a and /3 isolated after selection in G418 by several cycles of cell subunits of class II antigens and their expression in cells that sorting and then were cloned (9). are normally class II negative have made it possible to study Viral Infections. Target cells were infected with the Ed- the structure-function relationships in class II antigens. monston strain of measles virus (6, 7) at a multiplicity of Murine fibroblasts transfected with the genes for murine or infection of 1 [for the B lymphoblastoid cell line (B-LCL)] or human MHC class II antigens can express the molecules on 3 (for 637B) for 90 min and incubated for 3 days in Eagle's the cell surface (3-5). Moreover, these class II-expressing minimal essential medium supplemented with 15% heat- transfectants are capable of stimulating several antigen- inactivated fetal calf serum and 2 mM glutamine. Target cells specific and allospecific T cells in a MHC-restricted manner were infected with influenza virus as described (7). (4, 5). RNA Blot Analysis. Poly(A) + RNA from the untransfected MHC class II antigens can also be involved in antigen 637B cell line, transfected cell lines, and control B-cell line presentation to cytotoxic T lymphocytes (CTLs). The CTL were denatured with glyoxal, size-fractionated in 1.5% aga- response to measles virus is predominantly MHC class II rose gels, and transferred to nylon membranes as described restricted and the majority of measles virus-specific CTL (9). Prehybridizations and hybridizations with DNA frag- cones are CD4+ (6, 7). To analyze the requirements for the ments from DRa, DR/3, or invariant chain cDNAs were recognition of measles virus-infected cells by CTLs, DNA- carried out as described (9). mediated gene transfer of human MHC class II genes was Effector Cells. The measles virus-specific CTL clones used to reconstitute a suitable target cell. In this report, we were generated and maintained in culture as described (6, 7). show that expression of HLA-DR at the surface of a human Clones TB30, TB34, TB38, and TB60 were shown to be fibroblast line infected with measles virus is sufficient for recognition and lysis by specific CTLs. Abbreviations: B-LCL, B lymphoblastoid cell line; CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex. tPresent address: Clinical Research Institute of Montreal, Montreal, The publication costs of this article were defrayed in part by page charge Quebec H2W1R7, Canada. payment. This article must therefore be hereby marked "advertisement" Present address: Centre d'Immunologie de Marseille-Luminy, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 13288 Marseille, France. Downloaded by guest on September 27, 2021 1209 1210 Immunology: Sekaly et al. Proc. Natl. Acad. Sci. USA 85 (1988) DR1-restricted by their ability to lyse a panel of DR1- transfected 637B lines was analyzed. The RNA transcripts positive B-LCL infected with measles virus and their inabil- encoded by the cDNA expression vector are =300 bases ity to lyse infected DR1-negative B-LCL. The DR4- longer than the natural transcripts because they contain restricted measles virus-specific CTLs were generated by additional vector sequences (9, 14). With this size difference, primary in vitro sensitization from a normal donor as de- it was possible to determine that measles virus infection did scribed (7). These CTLs cultured in bulk were shown to be not induce expression of the endogenous DRa and DRf3 predominantly CD4+ and HLA class II restricted (7, 10, 11). genes (Fig. 1). Longer transcripts from the transfected genes The DR4-restricted influenza virus-specific CTL line was were also detected; they most likely result from transcription generated by repeated stimulations with irradiated (6000 rad; through the simian virus 40 late gene transcription termina- 1 rad = 0.01 Gy) autologous influenza virus-infected periph- tion signal present on the vector and termination at down- eral blood lymphocytes as feeders and cultured in the stream flanking sequences. presence of 10% interleukin 2 and 5% human AB serum. The invariant chain gene is not expressed in 637B cells but After seven passages, this line expressed the CD4' pheno- it can be induced, together with other class II genes, by type and lysed specifically DR4-positive B-LCL targets treatment with y-interferon (9). It is also known that viral infected with influenza virus. particles can induce class II antigen expression on certain CTL Assays. An indium oxine ("1'In) release assay (12) cells (15). It was therefore important to test whether the was used with 637B cells because it offered a substantially 3-day infection with measles virus had induced expression of improved efficiency of labeling over 51Cr. Microtiter wells the invariant chain gene in 637B cells. No transcript from the containing confluent monolayers of transfected or untrans- invariant chain gene was detected in fected cells were washed twice with complete Hanks' bal- infected 637B-C9 cells anced salt solution and incubated with 200 jul of '11In at a (Fig. 1). Note that 5 gg of 637B-C9 poly(A)+ RNA was run final concentration of 2 gCi per well (1 Ci = 37 GBq), for 15 in parallel with only 0.5 ,ug of poly(A)+ RNA from a B-LCL. min at room temperature and then washed twice with Hanks' Antigen Presentation to Measles Virus-Speciflc DR1- balanced salt solution supplemented with bovine serum Restricted CTL Clones Occurs in the Absence of the Invariant albumin (40 mg/ml) fraction V. After several washes, the Chain. The ability of transfectants that lack invariant chain microwells were incubated in Hanks' balanced salt solu- expression to present antigens to measles virus-specific tion/bovine serum albumin for 90 min at 370C. Effector cells DR1-restricted CTL clones was tested. Results (Table 1) were added and incubated for 6 hr with the target cells. indicated that no specific lysis occurred when untransfected Supernatants were harvested and released 1"'in was mea- 637B cells were used as targets even when infected with sured by using a Micromedicine Plus model y-counter measles virus. Similar results were obtained with a G418- (Micromedic Systems, Horsham, PA). Percentage specific resistant DR-negative clone obtained from the same trans- release was calculated as follows: (cpm of experimental fection (data not shown).