Immunoregulatory activity of a T-cell receptor a chain demonstrated by in vitro transcription and translation

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor a (TCRa) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCRa chain (A1.1 TCRa) suspected of having such activity. We found that TCRa, but not TCR,6, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCRa cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the a chain (VJa), at least the first 25 amino acids of the a chain of the constant (C) region (Ca) were present. Addition of an irrelevant protein sequence to the VJa failed to impart activity to the molecule, suggesting that the Ca requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCRa molecules, and the possible physiological relevance of these observations is considered. By the late 1960s it was clear to most cellular immunologists that T cells are crucial in the immune response as effector cells in cell-mediated responses and as helper cells in both humoral and cell-mediated responses. It was at about this time that an additional immunologic role for T cells was proposed: the suppression of immune responses. Since the original descriptions by Gershon and Kondo (reviewed in ref. 1), T cells capable of suppressing immune responses have been described in a wide range of systems. One problem that arose in this regard was that the effects were all attributed to "suppressor T cells," implying that there was only one mechanism (and cell type) that could produce such effects. We now know that there are a number of ways in which T cells can inhibit immune responses (e.g., see refs. 1 and 2). Most T lymphocytes bear T-cell antigen receptors (TCRs) composed of an a and a 13 chain (TCRa and TCRj3), and it is this receptor that is responsible for recognition of a complex ligand composed of an antigen peptide held in a major histocompatibility molecule on antigen-presenting cells (3). Upon recognition of this specific ligand, CD4+ T cells respond by the production of cytokines that are often capable of inhibiting some types of immune responses (1, 4). This mechanism of antigen-specific T cell-mediated suppression is well established. A far more controversial mechanism of T cell-mediated immunosuppression involves antigen-specific T cell factors (reviewed in refs. 5-9) that bind antigen and produce an immunoregulatory effect. T-cell hybridomas producing such antigen-specific regulatory molecules were generated and analyzed, and attempts to characterize the antigen-specific factors were made. However, a failure to consistently detect rearrangement of the TCR,B chain genes in such hybridomas (10, 11) contributed to skepticism regarding the existence of such T cells and factors (12). The issue was further confused by the observation that suppressor T-cell hybridomas and transformed T-cell lines rearrange and express TCRa genes (13, 14). Therefore, proposals to the effect that suppressor T cells use a novel T-cell receptor (15) were not substantiated. Progress has nevertheless been made towards resolving the paradox posed by T cell-derived, antigen-specific immunoregulatory factors. Several laboratories have found that such factors bear determinants recognized by anti-TCRa antibody (16-19), and the molecule resolves as having a molecular mass of 46 kDa, as does TCRa (19). TCRa cDNA from cells capable of producing antigen-specific factors transfers the ability to make such factors when expressed in other T cells (20, 21). Thus, some TCRa molecules appear to have antigen-specific immunoregulatory activity and can apparently exist in a cellfree form, probably in a complex with other molecules. Charged residues within the TCRa transmembrane region permit the molecule to exist in either a membrane form (stabilized by other-molecules) or a soluble form (22) (again, probably stabilized by other molecules). Previously, we reported that a helper T-cell hybridoma, A1.1, specific for a synthetic polypeptide antigen called "poly 18"-poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)5]-plus I-Ad constitutively releases a poly 18-specific immunoregulatory activity detected in an in vitro assay (23). An examination of the antigenic fine specificity of this cell-free activity revealed a correspondence to the specificity of the A1.1 TCR (19, 23), and the activity was bound and eluted from an anti-TCRa antibody (19). A relationship with the TCR was further established by the use of antisense oligodeoxynucleotides corresponding to the TCR variable region a chain (Va) of A1.1 (24). Finally, we found that expression of A1.1 TCRa cDNA in other T-cell hybridomas conferred the ability to produce the soluble, antigen-specific factor (20). These data strongly implicate the AM. TCRa molecule as a central component of the soluble antigen-specific immunoregulatory activity from Al.l. Here we examine the immunoregulatory activities of A1.1 TCRa molecules produced by in vitro transcription and translation (IVTT). MATERIALS AND METHODS Peptides and Reagents. Sheep red blood cells (SRBC) were purchased from Colorado Serum (Denver). Peptides based on Abbreviations: TCR, T-cell antigen receptor; IVTT, in vitro transcription and translation; poly 18, poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)5]; SRBC, sheep red blood cells; cpVIII, phage coat protein VIII; C, constant, V, variable; J, joining. §To whom reprint requests should be addressed. 3004 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. the nonrandom synthetic polypeptide poly 18 were kindly provided by Bhagirath Singh (University of Western Ontario) or by Kirin Pharmaceuticals (Maeabashi, Japan). Peptides were coupled to SRBC by using chromic chloride as described (19, 23). TCR Expression. TCRa and TCR,B cDNA derived from A1.1 were cloned into Bluescript KS (Strategene). Truncations in TCRa cDNA were generated by digestion with Xba I plus Bst E II, Nco I, or PflMI; alternatively, truncated forms of TCRa cDNA were generated by polymerase chain reaction with a 5' primer corresponding to the translation start (in boldface letters): 5'-GAAGAGGGATCC ATG AAA TCC TTG AGT-3'. The following 3' primers were all 5' to 3': J (ending at the J/C junction, in boldface letters), ACG TGC ATT CCAAAC TAAGAAATTCAACCCA; Clo (ending after 10 codons of Ca), GAA CCT GCT GTG TAC TAAGAATTCGATCCT; C20, CGG TCT CAG GAC AGC TAAGAATTCCTGTTC; C25, ACC CTC TGC CTG TTC TAAGAATTCGACTCC; C30, ACC GAC TTT GAC TCC TAAGAATTCGTGCCG; and C40, AAA ACC ATG GAA TCT TAAGAATTCATCACT. cDNA (0.05 jig) was used as template in each PCR amplification on a TwinBlock thermal cycler (Ericomp, San Diego). The mixture consisted of 60 pmol of each primer, dNTPs (200 ,uM each), and 100 ,l of Taq polymerase buffer (Promega) containing 1.5 mM MgCl2 and 5 units of Taq polymerase. After 25 cycles (1 min at 94°C; 1 min at 30°C; and 1 min at 72°C), PCR products were hydrolyzed with BamHI and EcoRI and cloned into Bluescript KS. Generation of pComp8-TCRa constructs will be described in detail elsewhere (T.O., D. Laface, G.B., T.B., N. Honma, T. Mikayama, A. Altman, and D.R.G., unpublished data). Briefly, A1.1 VJa or VJCc (where J and C = joining and constant regions) was amplified by PCR and cloned into the pComb8 vector (25) (provided by R. Lerner of Scripps) to produce a fusion protein composed of the TCRa region and coat protein VIII (26). The inserts were cloned into Bluescript SK (Stratagene) for in vitro expression. In all cases, RNA was expressed from the phage T7 promoter of Bluescript, and protein was translated using TNT T7-coupled reticulocyte lysate system (Promega). [35S]Methionine (DuPont) was added tothe reactions, and protein was resolved by SDS/PAGE. In Vitro Assay for Antigen-Specific Immunoregulatory Activity. "Accessory supernatants" required for A1.1 suppressor activity analysis were prepared as described (19, 23). Briefly, C57BL/6 mice were immunized twice with 0.2 ml of 20% SRBC, and immunoglobulin-free splenocytes (1 X 107 cells per ml) were then cultured in RPMI 1640 medium with 10% (vol/vol) FCS. After 48 hr the supernatants were collected, and the material was then extensively absorbed with SRBC at 4°C. To assay for antigen-specific suppressor activity ofTCRa or supernatants of A1.1, a simple antigen-specific system was employed as previously described (19, 23). Spleen cells (1 x 107) from C57BL/6 mice were placed into 1-ml cultures in RPMI 1640 medium supplemented with 10% FCS and 50 ,M 2-mercaptoethanol. Each culture received 50 ,ll of 1% peptidecoupled SRBC. TCRa (diluted 1:1000) with or without accessory supernatant was added to the cultures and incubated at 37°C in humidified 92% air/8% CO2. Anti-SRBC plaqueforming cells (without peptides) were assessed 5 days later. In no case was any suppressive effect observed in the absence of accessory supernatant (not shown). All values (controls and experimental) reported in this paper are from cultures containing accessory supernatant. Objectivity of Results. Assessment of plaque-forming cells is visual and thus necessarily subjective; therefore, the majority of the experiments described in this paper were performed under masked conditions. For antigen-specificity controls, peptides or saline were coded prior to coupling to SRBC. For experiments with truncated or chimeric TCRa molecules, the 3005 products of the IVTT (including negative controls) were coded prior to addition to the assay culture. Binding of TCRa to Peptide-Coupled SRBC. Fifty microliters of 10% peptide-coupled SRBC (see above) was mixed with 8 ,ul of [35S]methionine-labeled, full-length A1.1 TCRa and incubated for 2 hr at room temperature with shaking. Cells were then washed, and cell pellets were directly lysed in reducing SDS sample buffer. Samples were incubated for 5 min at 95°C prior to separation on a SDS/PAGE 10-20% gradient gel (Bio-Rad).