Major Histocompatibility Complex Allopeptides in the Rat MOHAMED H
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Proc. Natd. Acad. Sci. USA Vol. 89, pp. 7762-7766, August 1992 Immunology Induction of immunity and oral tolerance with polymorphic class II major histocompatibility complex allopeptides in the rat MOHAMED H. SAYEGH*, SAMIA J. KHOURYt, WAYNE W. HANCOCKO, HOWARD L. WEINERt, AND CHARLES B. CARPENTER* *Laboratory of Immunogenetics and Transplantation, Renal Division, and tCenter for Neurologic Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; and tDepartment of Immunology and Pathology, Monash Medical School, Prahran, Victoria 3181, Australia Communicated by D. Bernard Amos, May 15, 1992 (receivedfor review, January 24, 1992) ABSTRACT We studied the immunogenicit and tolero- immunogenicity and tolerogenicity of orally administered genicity of class II major histocompatibility complex (MHC) synthetic MHC allopeptides in the rat. allopeptides in the rat. Inbred LEW (RT1') rats, used as responders, were immunized in the foot pad with a mixture of MATERIALS AND METHODS eight synthetic class H MHC allopeptides emulsified in com- plete Freund's adjuvant. These sequences represent the full- Animals. LEW, WF, and BN rats, 8-10 weeks old, were length second domain of RTl.Bu and RT1.Du (WF) (3 chains. obtained from Harlan-Sprague-Dawley or were bred in our In vitro, responder lymphocytes harvested from popliteal and own animal facility. inguinal lymph nodes of immunized animals exhibited signff- Allopeptides. We selected the RT1.B (3 and RT1.D (3 icant proliferation to the MHC allopeptide mixture. In addi- domains of RTlU (WF) and synthesized four overlapping tion, these responder lymphocytes had significantly increased peptides of 20-25 amino acids (residues 1-25, 20-44, 39-64, proliferation to allogeneic WF (RTl") stimulator cells, when and 68-92 for RT1.B and residues 1-25, 20-44, 39-64, and 60-84 for RT1.D) for each locus, by using published se- compared to naive controls in the standard one-way mixed quences of the class II ( chain (8). Fig. 1 shows these lymphocyte response. In vivo, peptide-immunized LEW ani- polymorphic sequences aligned with those of the (B chains of mals were challenged in the ear 2 weeks after immunization RT11 (LEW). Peptides that were used for in vitro proliferation with the allopeptide mixture, the individual allopeptide se- assays were purified by HPLC yielding >95% purity as quences, or allogeneic WF splenocytes. When compared to determined by amino acid analysis. controls, these animals had significant delayed-type hypersen- Proliferation Assays. Responder LEW rats were immu- sitivity responses to the allopeptide mixture, to the (-pleated nized subcutaneously in the foot pad with 100 ,ug of the sheet allopeptide sequences, and to allogeneic WF splenocytes mixture of the four RT1.Bu and four RT1.Du peptides (each but not to the a-helix allopeptide sequences, to syngeneic LEW at 12.5 ug) in complete Freund's adjuvant (CFA). Popliteal splenocytes, or to third party allogeneic BN splenocytes. Oral and inguinal lymph nodes were harvested 1 week after administration of the allopeptide mixture to LEW responder immunization and mashed through 60-gauge sterile stainless rats daily for 5 days before immunization effected significant steel sieves. The recovered cells were then washed twice and reduction ofdelayed-type hypersensitivity responses both to the resuspended into RPMI 1640 medium (Microbiological As- allopeptide mixture and to allogeneic splenocytes. This reduc- sociates), containing 10%o (vol/vol) fetal calfserum, penicillin tion was antigen-specific, since there was no reduction of (100 units/ml), streptomycin (100 ,gg/ml), 20 LM 2-mercap- delayed-type hypersensitivity responses to mycobacterium tu- toethanol, and 5 mM Hepes. T and B cells were separated by berculosis. These data demonstrate that lymphocytes from nylon adherence as described (9). Responder unseparated animals immunized with polymorphic class II MHC allopep- LEW lymphocytes (3 x 105 cells) were cultured in 96-well tides can recognize and proliferate to the same amino acid flat-bottom plates (Costar) with 10-50 ,g of the mixture of sequences on allogeneic cell surface MHC molecules. In addi- RT1.Bu, RT1.Du, or both, sets of allopeptides. In other tion, oral administration of these peptides down-regulates the experiments, 1 x 105 nylon-wool-adherant cells, used as systemic cell-mediated immune response in a specific fashion. antigen-presenting cells, were preincubated with 10-50 Pg of Synthetic MHC allopeptides should allow the study of alloim- the RT1.Bu, RT1.Du, or both, sets of allopeptides for 30 min munity in vivo, including induction of immune tolerance. at 370C. The cells were then washed twice to remove excess peptides before adding 2 x 105 nylon-wool-nonadherent Recent work with synthetic peptides representing portions of responder T cells. Negative control wells were set up with the polymorphic regions of mouse and human class I and II culture medium only. LEW x WF one-way mixed lympho- major histocompatibility complex (MHC) molecules indi- cyte responses (MLRs) were set up by using equal numbers cates that they can be bound to MHC molecules and elicit a of responder LEW and allogeneic WF stimulator lympho- T-cell response in vitro (1-5). There is no information on the cytes (prepared as described for LEW lymphocytes and induction irradiated with 3000 rads; 1 rad = 0.01 Gy) per well. The of immunity or tolerance by administration of plates were incubated at 370C with 5% C02/95% airfor 4 days synthetic MHC peptides in vivo. The oral route of adminis- before they were pulse-labeled for 6 h with [3H]thymidine (1 tration of antigens has been shown to induce immune hypo- ACi per well; 1 Ci = 37 GBq; NEN/DuPont) and harvested responsiveness (6). In alloimmunity, we have recently re- with a PHD cell harvester (Cambridge Technology, Cam- ported (7) that oral administration of allogeneic splenocytes bridge, MA). Proliferation was assayed by [3H]thymidine to inbred rats down-regulates the systemic cell-mediated incorporation measured by a Beckman liquid scintillation immune response in vitro and in vivo. We now report on the Abbreviations: MHC, major histocompatibility complex; MLR, The publication costs ofthis article were defrayed in part by page charge mixed lymphocyte response; DTH, delayed-type hypersensitivity; payment. This article must therefore be hereby marked "advertisement" CFA, complete Freund's adjuvant; EAE, experimental autoimmune in accordance with 18 U.S.C. §1734 solely to indicate this fact. encephalomyelitis. 7762 Downloaded by guest on September 26, 2021 Immunology: Sayegh et al. Proc. NatL. Acad. Sci. USA 89 (1992) 7763 10 20 30 40 50 A RT1 .Bpu QRLRRDFLVQFKPYCYFTNGTQRIRNVIRYIYNREEYLRYDSDVGEYRAV RT1.D ....................... ----------Q-0-------------Q --J-. 60 70 80 90 TELGRPSAEYFNKQ*Y*LERTRAELDTVCRHNYEKTEVPTSL RTL.B 10 20 30 40 50 Pep. Mix. RT1.D u RDPTPRFLGYLKPECHFYNGTNRVRLLARLIYNREEYARFDSDVGEYRAV 0 100,000 300,000 cpm RT1. ..............................Y-------T--------F--- Medium 60 70 80 90 TELGRPSAEYRNKQKEPMERRRATVDTYCRHNYEIFDRFL 0 20,000 40,000 60,000 80,000 cpm ----------Y-----Y--QI------A-K-D-....... B FIG. 1. Amino acid sequences ofthe synthetic MHC RT1.BU and RT1.D RT1.DU peptides aligned with those of RT11. Dots denote unknown sequences, dashes denote identical sequences, and asterisks denote absent sequences. RT1.B counter. Experiments were set up in quadruplicate, and results are expressed as cpm (mean SEM) or relative Pep. Mix. d1 response = (experimental cpm - background cpm)/(control cpm - background cpm). Delayed-Type Hypersensitivity (DTH) Response. LEW rats, Medium used as responders, were immunized subcutaneously in the 0I foot pad with 100 pg ofthe mixture offour RT1.BU (50 jug) and 0 20,000 40,000 60,000 80,000 100,000 four RT1.Du (50 pg) peptides (12.5 jug of each peptide) in cpm CFA. These animals were challenged subcutaneously 2 weeks later in one ear with 10 jug of the peptide mixture and FIG. 2. (A) Direct proliferation of lymphocytes harvested from in the other ear with 1 x 107 freshly prepared and irradiated LEW rats, naive (shaded bars) and immunized with the entire (3000 rads) splenocytes from WF (RTlu), syngeneic LEW allopeptide mixture (solid bars), that were incubated with the entire (RT11), or a third party, BN (RT1n). The DTH responses were peptide mixture (bars Pep.Mix) or RT1.B or RT1.D allopeptides. measured with micrometer caliper Field Tool Bars represent cpm (mean ± SEM) of a representative experiment (Mitutoyo, performed in quadruplicate (five experiments). (Inset) Concomitant Supply, Chicago) by a blinded observer as the change in ear LEW x WF MLR for the experiment shown. The calculated relative thickness before and 2 days after the challenge (inches x response for the MLR was 2.65 + 0.2 (n = 6). (B) Proliferation of 10-2; 1 inch = 2.54 cm). Experiments were performed using nylon-wool-non-adherant mononuclear cells harvested from immu- five animals in each study groups. P values were calculated nized (with the entire allopeptide mixture) LEW animals to the entire using the Student t test. peptide mixture (Pep.Mix) or RT1.B or RT1.D allopeptides pre- sented by syngeneic nylon-wool-adherant cells. Bars represent cpm (mean + SEM) of a representative experiment performed in qua- RESULTS druplicate (four experiments). Immunogenicity of Class II MHC Allopeptides Assessed in Vitro. To test the immunogenicity ofthe synthetic RT1.B and from animals immunized with these allopeptides proliferate RT1.D allopeptides, lymphocytes harvested from responder more vigorously to allogeneic cell surface MHC molecules. LEW animals immunized with the mixture of eight allopep- Immunogenicity of Class II MHC Allopeptides Assessed by tides 1 week earlier were compared to naive controls for their DTH in Vivo. LEW animals that were immunized with the ability to proliferate to the allopeptides in a standard 96-h entire allopeptide mixture and CFA had significant DTH proliferation assay. As shown in Fig. 2A, whereas naive responses both to the allopeptides and to freshly prepared lymphocytes had only minimal proliferation, immunized an- allogeneic WF splenocytes (Fig.