Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1988 Apr 1;167(4):1442–1458. doi: 10.1084/jem.167.4.1442

Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients

PMCID: PMC2188916  PMID: 3128633

Abstract

We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams T. E., Bodmer J. G., Bodmer W. F. Production and characterization of monoclonal antibodies recognizing the alpha-chain subunits of human ia alloantigens. Immunology. 1983 Dec;50(4):613–624. [PMC free article] [PubMed] [Google Scholar]
  2. Babbitt B. P., Allen P. M., Matsueda G., Haber E., Unanue E. R. Binding of immunogenic peptides to Ia histocompatibility molecules. 1985 Sep 26-Oct 2Nature. 317(6035):359–361. doi: 10.1038/317359a0. [DOI] [PubMed] [Google Scholar]
  3. Barnstable C. J., Bodmer W. F., Brown G., Galfre G., Milstein C., Williams A. F., Ziegler A. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell. 1978 May;14(1):9–20. doi: 10.1016/0092-8674(78)90296-9. [DOI] [PubMed] [Google Scholar]
  4. Bloom B. R., Godal T. Selective primary health care: strategies for control of disease in the developing world. V. Leprosy. Rev Infect Dis. 1983 Jul-Aug;5(4):765–780. doi: 10.1093/clinids/5.4.765. [DOI] [PubMed] [Google Scholar]
  5. Bontrop R., Ottenhoff T., Van Miltenburg R., Elferink D., De Vries R., Giphart M. Quantitative and qualitative differences in HLA-DR molecules correlated with antigen-presentation capacity. Eur J Immunol. 1986 Feb;16(2):133–138. doi: 10.1002/eji.1830160205. [DOI] [PubMed] [Google Scholar]
  6. Brodsky F. M., Parham P., Bodmer W. F. Monoclonal antibodies to HLA--DRw determinants. Tissue Antigens. 1980 Jul;16(1):30–48. doi: 10.1111/j.1399-0039.1980.tb00285.x. [DOI] [PubMed] [Google Scholar]
  7. Buus S., Sette A., Colon S. M., Miles C., Grey H. M. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science. 1987 Mar 13;235(4794):1353–1358. doi: 10.1126/science.2435001. [DOI] [PubMed] [Google Scholar]
  8. Charron D. J., McDevitt H. O. Analysis of HLA-D region-associated molecules with monoclonal antibody. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6567–6571. doi: 10.1073/pnas.76.12.6567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cowan E. P., Coligan J. E., Biddison W. E. Human cytotoxic T-lymphocyte recognition of an HLA-A3 gene product expressed on murine L cells: the only human gene product required on the target cells for lysis is the class I heavy chain. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4490–4494. doi: 10.1073/pnas.82.13.4490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elferink B. G., Ottenhoff T. H., de Vries R. R. Epstein-Barr virus-transformed B cell lines present M. leprae antigens to T cells. Scand J Immunol. 1985 Nov;22(5):585–589. doi: 10.1111/j.1365-3083.1985.tb01918.x. [DOI] [PubMed] [Google Scholar]
  11. Gay D., Maddon P., Sekaly R., Talle M. A., Godfrey M., Long E., Goldstein G., Chess L., Axel R., Kappler J. Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature. 1987 Aug 13;328(6131):626–629. doi: 10.1038/328626a0. [DOI] [PubMed] [Google Scholar]
  12. Germain R. N., Malissen B. Analysis of the expression and function of class-II major histocompatibility complex-encoded molecules by DNA-mediated gene transfer. Annu Rev Immunol. 1986;4:281–315. doi: 10.1146/annurev.iy.04.040186.001433. [DOI] [PubMed] [Google Scholar]
  13. Golde W. T., Kappler J. W., Greenstein J., Malissen B., Hood L., Marrack P. Major histocompatibility complex-restricted antigen receptor on T cells. VIII. Role of the LFA-1 molecule. J Exp Med. 1985 Mar 1;161(3):635–640. doi: 10.1084/jem.161.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gorski J., Tosi R., Strubin M., Rabourdin-Combe C., Mach B. Serological and immunochemical analysis of the products of a single HLA DR-alpha and DR-beta chain gene expressed in a mouse cell line after DNA-mediated cotransformation reveals that the beta chain carries a known supertypic specificity. J Exp Med. 1985 Jul 1;162(1):105–116. doi: 10.1084/jem.162.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gougeon M. L., Bismuth G., Theze J. Differential effects of monoclonal antibodies anti-L3T4 and anti-LFA1 on the antigen-induced proliferation of T-helper-cell clones: correlation between their susceptibility to inhibition and their affinity for antigen. Cell Immunol. 1985 Oct 1;95(1):75–83. doi: 10.1016/0008-8749(85)90296-5. [DOI] [PubMed] [Google Scholar]
  16. Goyert S. M., Silver J. Further characterization of HLA-DS molecules: implications for studies assessing the role of human Ia molecules in cell interactions and disease susceptibility. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5719–5723. doi: 10.1073/pnas.80.18.5719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grosveld F. G., Lund T., Murray E. J., Mellor A. L., Dahl H. H., Flavell R. A. The construction of cosmid libraries which can be used to transform eukaryotic cells. Nucleic Acids Res. 1982 Nov 11;10(21):6715–6732. doi: 10.1093/nar/10.21.6715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  19. Hildreth J. E., Gotch F. M., Hildreth P. D., McMichael A. J. A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur J Immunol. 1983 Mar;13(3):202–208. doi: 10.1002/eji.1830130305. [DOI] [PubMed] [Google Scholar]
  20. Hirayama K., Matsushita S., Kikuchi I., Iuchi M., Ohta N., Sasazuki T. HLA-DQ is epistatic to HLA-DR in controlling the immune response to schistosomal antigen in humans. Nature. 1987 Jun 4;327(6121):426–430. doi: 10.1038/327426a0. [DOI] [PubMed] [Google Scholar]
  21. Jacobson S., Nepom G. T., Richert J. R., Biddison W. E., McFarland H. F. Identification of a specific HLA DR2 Ia molecule as a restriction element for measles virus-specific HLA class II-restricted cytotoxic T cell clones. J Exp Med. 1985 Jan 1;161(1):263–268. doi: 10.1084/jem.161.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kasahara M., Ogasawara K., Ikeda H., Okuyama T., Ishikawa N., Takenouchi T., Wakisaka A., Kikuchi Y., Aizawa M. A monoclonal antibody that detects a polymorphic determinant common to HLA-DR1 and 2. Tissue Antigens. 1983 Feb;21(2):105–113. doi: 10.1111/j.1399-0039.1983.tb00378.x. [DOI] [PubMed] [Google Scholar]
  23. Langdon N., Welsh K. I., van Dam M., Vaughan R. W., Parkes D. Genetic markers in narcolepsy. Lancet. 1984 Nov 24;2(8413):1178–1180. doi: 10.1016/s0140-6736(84)92742-9. [DOI] [PubMed] [Google Scholar]
  24. Lechler R. I., Norcross M. A., Germain R. N. Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells. J Immunol. 1985 Nov;135(5):2914–2922. [PubMed] [Google Scholar]
  25. Lee B. S., Rust N. A., McMichael A. J., McDevitt H. O. HLA-DR2 subtypes form an additional supertypic family of DR beta alleles. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4591–4595. doi: 10.1073/pnas.84.13.4591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matis L. A., Glimcher L. H., Paul W. E., Schwartz R. H. Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and Ia molecules. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6019–6023. doi: 10.1073/pnas.80.19.6019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norcross M. A., Bentley D. M., Margulies D. H., Germain R. N. Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes. J Exp Med. 1984 Nov 1;160(5):1316–1337. doi: 10.1084/jem.160.5.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  29. Ottenhoff T. H., Neuteboom S., Elferink D. G., de Vries R. R. Molecular localization and polymorphism of HLA class II restriction determinants defined by Mycobacterium leprae-reactive helper T cell clones from leprosy patients. J Exp Med. 1986 Dec 1;164(6):1923–1939. doi: 10.1084/jem.164.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Qvigstad E., Moen T., Thorsby E. T-cell clones with similar antigen specificity may be restricted by DR, MT(DC), or SB class II HLA molecules. Immunogenetics. 1984;19(5):455–460. doi: 10.1007/BF00364648. [DOI] [PubMed] [Google Scholar]
  31. Rees A. J., Peters D. K., Compston D. A., Batchelor J. R. Strong association between HLA-DRW2 and antibody-mediated Goodpasture's syndrome. Lancet. 1978 May 6;1(8071):966–968. doi: 10.1016/s0140-6736(78)90252-0. [DOI] [PubMed] [Google Scholar]
  32. Rothlein R., Dustin M. L., Marlin S. D., Springer T. A. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol. 1986 Aug 15;137(4):1270–1274. [PubMed] [Google Scholar]
  33. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  34. Shastri N., Malissen B., Hood L. Ia-transfected L-cell fibroblasts present a lysozyme peptide but not the native protein to lysozyme-specific T cells. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5885–5889. doi: 10.1073/pnas.82.17.5885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shimonkevitz R., Kappler J., Marrack P., Grey H. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing. J Exp Med. 1983 Aug 1;158(2):303–316. doi: 10.1084/jem.158.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sone T., Tsukamoto K., Hirayama K., Nishimura Y., Takenouchi T., Aizawa M., Sasazuki T. Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules. J Immunol. 1985 Aug;135(2):1288–1298. [PubMed] [Google Scholar]
  37. Takenouchi T., Kasahara M., Ogasawara K., Ikeda H., Ishikawa N., Hawkin S., Wakisaka A., Aizawa M. Electrophoretic analysis of HLA-DR2 molecules isolated from HLA-Dw2 and HLA-Dw12 cell lines. J Immunogenet. 1985 Feb;12(1):65–69. doi: 10.1111/j.1744-313x.1985.tb00830.x. [DOI] [PubMed] [Google Scholar]
  38. Trowsdale J., Young J. A., Kelly A. P., Austin P. J., Carson S., Meunier H., So A., Erlich H. A., Spielman R. S., Bodmer J. Structure, sequence and polymorphism in the HLA-D region. Immunol Rev. 1985 Jul;85:5–43. doi: 10.1111/j.1600-065x.1985.tb01129.x. [DOI] [PubMed] [Google Scholar]
  39. Watson A. J., DeMars R., Trowbridge I. S., Bach F. H. Detection of a novel human class II HLA antigen. 1983 Jul 28-Aug 3Nature. 304(5924):358–361. doi: 10.1038/304358a0. [DOI] [PubMed] [Google Scholar]
  40. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  41. Wu S. K., Yabe T., Madden M., Saunders T. L., Bach F. H. cDNA cloning and sequencing reveals that the electrophoretically constant DR beta 2 molecules, as well as the variable DR beta 1 molecules, from HLA-DR2 subtypes have different amino acid sequences including a hypervariable region for a functionally important epitope. J Immunol. 1987 May 1;138(9):2953–2959. [PubMed] [Google Scholar]
  42. Young R. A., Mehra V., Sweetser D., Buchanan T., Clark-Curtiss J., Davis R. W., Bloom B. R. Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae. Nature. 1985 Aug 1;316(6027):450–452. doi: 10.1038/316450a0. [DOI] [PubMed] [Google Scholar]
  43. de Kretser T. A., Crumpton M. J., Bodmer J. G., Bodmer W. F. Demonstration of two distinct light chains in HLA-DR-associated antigens by two-dimensional gel electrophoresis. Eur J Immunol. 1982 Mar;12(3):214–221. doi: 10.1002/eji.1830120309. [DOI] [PubMed] [Google Scholar]
  44. van Eden W., de Vries R. R., D'Amaro J., Schreuder I., Leiker D. L., van Rood J. J. HLA-DR-associated genetic control of the type of leprosy in a population from surinam. Hum Immunol. 1982 Jul;4(4):343–350. doi: 10.1016/0198-8859(82)90007-6. [DOI] [PubMed] [Google Scholar]
  45. van Eden W., de Vries R. R., Mehra N. K., Vaidya M. C., D'Amaro J., van Rood J. J. HLA segregation of tuberculoid leprosy: confirmation of the DR2 marker. J Infect Dis. 1980 Jun;141(6):693–701. doi: 10.1093/infdis/141.6.693. [DOI] [PubMed] [Google Scholar]
  46. van Heyningen V., Guy K., Newman R., Steel C. M. Human MHC class II molecules as differentiation markers. Immunogenetics. 1982;16(5):459–469. doi: 10.1007/BF00372104. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES