Iwaki, Y

Murase, N

Starzl, TE

Takahashi, S

Yagihashi, A

PubMed

D-Scholarship@Pitt

A Monoclonal Antibody (L21-6) Recognizing an Invariant Chain Expressed on the Cell Surface in Rats With the Exception of the BN (RT1n): A Study of Tissue and Strain Distributions A. Yagihashi, S. Takahashi, N. Murase, T.E. Starzl, and Y. Iwaki I T IS an established fact that class II molecules play a crucial role in the initial recognition of foreign anti-gens.1.2 Exogenous antigen peptide and these class II molecule a-, /3-chain complexes are recognized by the T-cell receptor. On the other hand, in cytoplasm, the invariant chain, which is a nonpolymorphic peptide, binds the class II molecules and prevents the binding of endogenous pep-tides.3 In addition, it has been reported that an invariant chain is expressed on the cell surface in the human4 and mouse,s but .js not reported in the rat. The distribution of the invariant chain on the cell surface is very similar to that of class II molecules, but the invariant chain was observed without class II molecules in certain cells. The differential expression of class II and invariant chain with interferon-'Y treatment in the mouse was reported.f> Therefore, we developed a unique monoclonal antibody (MAb), L21-6, recognizing an invariant chain on the cell surface in the rats and studied the tissue and strain distributions. MATERIALS AND METHODS Animals All inbred strains of rats used in this study. LEW (RTl'), BN (RTI"). ACI (RTJD"'). F344 (RTlM ). WWFF(RTlU). BUF (RTl b ). and DA (RTID'·'). were purchased from Harlan Sprague-Dawley Inc (Indianapolis. Ind) as were the BALB/c mice. MAb BALB/c mice were immunized four times intraperitoneally (IP) with 1 x to' LEW spleen cells at IO-day intervals. Four days after the last immunization. approximately I x IQ'I mouse spleen cells were fused with 4 x to' P-3 mouse myeloma cells by PEG-4000. in accordance with the method as described previously.' The supernatants were screened by indirect immunofluorescence for antibody activity against LEW and BN spleen cells. The positive hybrids were cloned by limiting dilution. In further studies. we used one of these hybridomas. designated as L21-6, Detection of Cell Surface Antigen by FACScan The cells were stained with 1..21-6 by indirect immunofluorescence. Analysis was performed after the cell viability (more than 95%) was tested by trypan blue dye exclusion, Immunohistochemical Staining Cryostat sectIons of brain. lung. heart. thymus. liver. spleen. pancreas. kidney. skin. small intestine. lymph node. and aorta from LEW rats were obtamed. These sections were stained b\ the ABC method,' L21-h or MRC-OX6 was used al> first antibody, Inhibition of Mixed Lymphocyte Reaction Lymphocytes from mesenteric lymph nodes were used. Responder cells (I X 1E}~/wellF wer$: cocultured with 2 X IOS/well 20-Gy-irradiated stimulator cells in the presence of 1..21-6 in round-bottomed 96-well plates for 5 days. The cells were pulsed with 3H-thymidine. and harvested 18 hours later. Results were obtained from triplicate cultures. Molecular Determination by SDS-PAGE The procedures for immunoprecipitation and surface iodination used in this study have been described elsewhere,R The LEW spleen cells were labeled with 1OK~f by use of lactoperoxidase. SDS-PAGE was performed in 12.5% polyacrylamide gel under the reducing condition, RESULTS Reactivity of L21-6 to rat strains (LEW, ACI. DA, F344, WF, and BUF) was studied using spleen cells and positive staining patterns were obtained in all instances. The SDS-PAGE analysis provided a band at 32 kd (Fig 1). MAb L21-6 was capable of 95.2% inhibition of MLR of LEW against BN. The expression of MAb L21-6-defined antigen in normal LEW rat tissues was examined. Staining patterns are summarized in Table 1. DISCUSSION Our study demonstrated: (1) the MAb L21-6 recognized the determinant on the cell surface component of invariant chain because the band at 32 kd. which was different from an a- and l3-chain of class II molecules, was detected by SDS-PAGE using cell-surface iodinated material; and (2) tissue staining patterns of L21-6 were quite similar to those staine9 P.Y anti invariant chain antibodies reported earlier,b.9 but L21'-'6-defined determinant on the invariant chain was not expressed in BN rats. It was reported that the invariant chain, which was located between the a- and /3-chain in the slab gel, was coprecipitated with rat class II molecules recognized by HOK 7 (anti-class II) MAb using soluble glyco~ptein fraction from WKAH rat spleen cells. 1II How-From the Department of Surgery and Pathology, University of Pittsburgh. Pittsburgh. Pennsylvania; and the Department of Surgery. Sapporo Medical University. Sapporo. Japan. Address repnnt requests to Atsuhito Yagihashi. MD. PhD. Department of Surgery. Sapporo Medical University. S-1. W-16. Chuo-ku. Sapporo 060. Japan. <r. 1995 by Appleton & Lange 0041-1345/95/$3.001 + 0 Transplantation Proceedings, Vol 27. No 2 (April). 1995: pp 1519-1521 1519 -----_._._--------------1520 2 3 94 -67 -43 -30-20 -Fig 1. SDS-PAGE analysis of immunoprecipitates made with control (lane 1), MRC OX-6 (lane 2) or L21-6 MAb (lane 3) and LEW spleen cell lysate. Immune complexes were applied to 12.5% polyacrylamide gel under the reducing condition. ever, anti-rat invariant chain MAb was not reported. To our knowledge this is the first report of an anti-rat invariant chain MAb. It is unclear whether or not the invariant chain is expressed on the cell surface. Wraight et al noted that the possible explanation for the varying conclusion of the previous study on invariant chain cell surface expression was the previous absence of antibodies directed against C-terminal portions of the invariant chain. because the invariant chain is inserted into the membrane with its N-terminal on the cytoplasmic side:~ L21-6 is probably specific for the determinant on the C-terminal side (extra-cytoplasmic domain) of the invariant chain. Our result of SOS-PAGE also suggested that the cell surface inva.riant chain may be not associated with the class II a- and /3-chain as is the case in the cytoplasm. L21-6 was capable of 95.2% inhibition of MLR of LEW against BN suggesting the possibility that the invariant chain interacts with antigen presentation as an adhesion molecule. In addition, why the BN rat does not express the invariant chain must be studied. Two anti-class II antibodies were reported to express similar specificity on strain distribution. The alloantibody, RTl 8.9, which was raised by skin grafting of BN.IL VI (F344) onto BN, reacted with all haplolypes except RTr.lll The antibody HIS 19, which was produced by immunization YAGIHASHI. TAKAHASHI, MURASE ET AL Table 1. Tissue Distribution of Class II Antigen Recognized by L21-6 in Nonnal LEW Rat rlSSUe_ Tissue Central nervous system Brain GastrOintestinal system Ileum' Uver Hepatocyte Biliary epithelium Sinusoidal lining cell Artery, vein Interstitial dendritic cell Respiratory and cardiovascular system Lungt Bronchial epithelium Alveolar epithelium Heart Endocardium Epicardium Myocardium Aorta Interstitial dendritic cell Urogenital system Kidney Glomeruli Tubules Interstitial dendritic cell Miscellaneous Pancreas Exocrine portion Endocrine portion Skin _ Langerhans cell Interstitial dendritic cell Lymphoid tissue Lymph node Thymus: Medulla Cortex Spleen White pulp Red pulp Bone marrow! Peripheral blood lymphocyte' ( -) = no staining; (+) = posative staJntng. Reaction With [21-1, + + + + + + + + + + + + + "Lamina propna was stained dillusely. Payer's paten Showed the same staJning pattern as Iymp/1 nodeS. t AJveoIar macrophages w_ stained posrtovety. 'Corte)( of thymus was weakly staIning. fAppro)(lrnately. 10% of bone marrow cells w_ positive using Indirect immunofluorescence. "ApprolOmately. 20% of PBLs were posrtive USIng indirect immunofluores-cence. with lymphocytes isolated from AO (RTl") Peyer's patch, reacted with all haplotypes except RTl". II The polymorphism of the class II antigen might influence the expression of L21-6-defined antigen. Thus. L21-6 is useful to study the function of the invariant chain on the cell surface. L21-6 RECOGNITION OF AN INVARIANT CHAIN REFERENCES 1. Schwartz RH: Annu Rev Immunol 3:237, 1985 2. Townsend A. Bodmer H: ~nu Rev Immunol 7:601, 1989 3. Roche PA. Cresswell P: Nature 345:615, 1990 4. Wraight CJ, Enden PY, Moller P, et al: J Bioi Chem 265:5787. 1990 5. Koch N. Koch S, Hammerling GJ: Nature 299:664, 1982 6. Momburg F. Koch N, ·Moller P. et al: J Immunol 136:940. 1986 1521 7. Yagihashi A. Sato N, Torigoe T, et al: Cancer Res 48:2798, 1988 8. Epstein AL, Marder RJ, Winter IN, et al: J Immunol 133:1028, 1978 9. Ikeda H, Matsuno y, TsuchimolO S, et al: Transplant Proc 17:18202, 1985 10. Wettstein PJ: Immunogenetics 14:541, 1981 It. Stet RJM. Rozing J. Majoor GD. et al: Transplant Proc 17:1829. 1985 

A monoclonal antibody (L21-6) recognizing an invariant chain expressed on the cell surface in rats with the exception of the BN(RT1(n)): A study of tissue and strain distributions

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A monoclonal antibody (L21-6) recognizing an invariant chain expressed on the cell surface in rats with the exception of the BN(RT1(n)): A study of tissue and strain distributions

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