Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 May 1;181(5):1715–1727. doi: 10.1084/jem.181.5.1715

Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing

PMCID: PMC2192002  PMID: 7722450

Abstract

We have developed a functional assay to identify processed antigen in subcellular fractions from antigen-presenting cells; stimulatory activity in this assay may be caused by either free peptide fragments or by complexes of peptide fragments and class II molecules present on organellar membrane sheets and vesicles. In addition, we have developed a functional assay to identify proteolytic activity in subcellular fractions capable of generating antigenic peptides from intact proteins. These techniques permit the direct identification of intracellular sites of antigen processing and class II association. Using a murine B cell line stably transfected with a phosphorylcholine (PC)-specific membrane-bound immunoglobulin (Ig), we show that PC- conjugated antigens are rapidly internalized and efficiently degraded to generate processed antigen within an early low density compartment. Proteolytic activity capable of generating antigenic peptide fragments from intact proteins is found within low density endosomes and a dense compartment consistent with lysosomes. However, neither processed peptide nor peptide-class II complexes are detected in lysosomes from antigen-pulsed cells. Furthermore, blocking the intracellular transport of internalized antigen from the low density endosome to lysosomes does not inhibit the generation of processed antigen. Therefore, antigens internalized in association with membrane Ig on B cells can be efficiently processed in low density endosomal compartments without the contribution of proteases present within denser organelles.

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. Amigorena S., Drake J. R., Webster P., Mellman I. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature. 1994 May 12;369(6476):113–120. doi: 10.1038/369113a0. [DOI] [PubMed] [Google Scholar]
  2. Beaufay H., Amar-Costesec A., Feytmans E., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J Cell Biol. 1974 Apr;61(1):188–200. doi: 10.1083/jcb.61.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blum J. S., Fiani M. L., Stahl P. D. Proteolytic cleavage of ricin A chain in endosomal vesicles. Evidence for the action of endosomal proteases at both neutral and acidic pH. J Biol Chem. 1991 Nov 25;266(33):22091–22095. [PubMed] [Google Scholar]
  4. Bomsel M., Parton R., Kuznetsov S. A., Schroer T. A., Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990 Aug 24;62(4):719–731. doi: 10.1016/0092-8674(90)90117-w. [DOI] [PubMed] [Google Scholar]
  5. Brodsky F. M., Guagliardi L. E. The cell biology of antigen processing and presentation. Annu Rev Immunol. 1991;9:707–744. doi: 10.1146/annurev.iy.09.040191.003423. [DOI] [PubMed] [Google Scholar]
  6. Buus S., Colon S., Smith C., Freed J. H., Miles C., Grey H. M. Interaction between a "processed" ovalbumin peptide and Ia molecules. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3968–3971. doi: 10.1073/pnas.83.11.3968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buus S., Sette A., Colon S. M., Jenis D. M., Grey H. M. Isolation and characterization of antigen-Ia complexes involved in T cell recognition. Cell. 1986 Dec 26;47(6):1071–1077. doi: 10.1016/0092-8674(86)90822-6. [DOI] [PubMed] [Google Scholar]
  8. Chain B. M., Kaye P. M., Shaw M. A. The biochemistry and cell biology of antigen processing. Immunol Rev. 1988 Dec;106:33–58. doi: 10.1111/j.1600-065x.1988.tb00772.x. [DOI] [PubMed] [Google Scholar]
  9. Chen J. W., Murphy T. L., Willingham M. C., Pastan I., August J. T. Identification of two lysosomal membrane glycoproteins. J Cell Biol. 1985 Jul;101(1):85–95. doi: 10.1083/jcb.101.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collins D. S., Unanue E. R., Harding C. V. Reduction of disulfide bonds within lysosomes is a key step in antigen processing. J Immunol. 1991 Dec 15;147(12):4054–4059. [PubMed] [Google Scholar]
  11. Davidson H. W., West M. A., Watts C. Endocytosis, intracellular trafficking, and processing of membrane IgG and monovalent antigen/membrane IgG complexes in B lymphocytes. J Immunol. 1990 Jun 1;144(11):4101–4109. [PubMed] [Google Scholar]
  12. Diment S. Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin. J Immunol. 1990 Jul 15;145(2):417–422. [PubMed] [Google Scholar]
  13. Diment S., Leech M. S., Stahl P. D. Cathepsin D is membrane-associated in macrophage endosomes. J Biol Chem. 1988 May 15;263(14):6901–6907. [PubMed] [Google Scholar]
  14. Diment S., Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem. 1985 Dec 5;260(28):15311–15317. [PubMed] [Google Scholar]
  15. Donermeyer D. L., Allen P. M. Binding to Ia protects an immunogenic peptide from proteolytic degradation. J Immunol. 1989 Feb 15;142(4):1063–1068. [PubMed] [Google Scholar]
  16. Drake J. R., Repasky E. A., Bankert R. B. Endocytosis of antigen, anti-idiotype, and anti-immunoglobulin antibodies and receptor re-expression by murine B cells. J Immunol. 1989 Sep 15;143(6):1768–1776. [PubMed] [Google Scholar]
  17. Germain R. N. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell. 1994 Jan 28;76(2):287–299. doi: 10.1016/0092-8674(94)90336-0. [DOI] [PubMed] [Google Scholar]
  18. Griffith I. J., Nabavi N., Ghogawala Z., Chase C. G., Rodriguez M., McKean D. J., Glimcher L. H. Structural mutation affecting intracellular transport and cell surface expression of murine class II molecules. J Exp Med. 1988 Feb 1;167(2):541–555. doi: 10.1084/jem.167.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Griffiths G., Hoflack B., Simons K., Mellman I., Kornfeld S. The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell. 1988 Feb 12;52(3):329–341. doi: 10.1016/s0092-8674(88)80026-6. [DOI] [PubMed] [Google Scholar]
  20. Griffiths G., Matteoni R., Back R., Hoflack B. Characterization of the cation-independent mannose 6-phosphate receptor-enriched prelysosomal compartment in NRK cells. J Cell Sci. 1990 Mar;95(Pt 3):441–461. doi: 10.1242/jcs.95.3.441. [DOI] [PubMed] [Google Scholar]
  21. Gruenberg J., Griffiths G., Howell K. E. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. doi: 10.1083/jcb.108.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
  23. Guagliardi L. E., Koppelman B., Blum J. S., Marks M. S., Cresswell P., Brodsky F. M. Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment. Nature. 1990 Jan 11;343(6254):133–139. doi: 10.1038/343133a0. [DOI] [PubMed] [Google Scholar]
  24. Harding C. V., Collins D. S., Kanagawa O., Unanue E. R. Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation. J Immunol. 1991 Nov 1;147(9):2860–2863. [PubMed] [Google Scholar]
  25. Harding C. V., Collins D. S., Slot J. W., Geuze H. J., Unanue E. R. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell. 1991 Jan 25;64(2):393–401. doi: 10.1016/0092-8674(91)90647-h. [DOI] [PubMed] [Google Scholar]
  26. Harding C. V., Geuze H. J. Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells. J Cell Biol. 1992 Nov;119(3):531–542. doi: 10.1083/jcb.119.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Harding C. V., Geuze H. J. Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment. J Immunol. 1993 Oct 15;151(8):3988–3998. [PubMed] [Google Scholar]
  28. Harding C. V., Unanue E. R. Cellular mechanisms of antigen processing and the function of class I and II major histocompatibility complex molecules. Cell Regul. 1990 Jun;1(7):499–509. doi: 10.1091/mbc.1.7.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Harding C. V., Unanue E. R. Low-temperature inhibition of antigen processing and iron uptake from transferrin: deficits in endosome functions at 18 degrees C. Eur J Immunol. 1990 Feb;20(2):323–329. doi: 10.1002/eji.1830200214. [DOI] [PubMed] [Google Scholar]
  30. Harding C. V., Unanue E. R., Slot J. W., Schwartz A. L., Geuze H. J. Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5553–5557. doi: 10.1073/pnas.87.14.5553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jensen P. E. Regulation of antigen presentation by acidic pH. J Exp Med. 1990 May 1;171(5):1779–1784. doi: 10.1084/jem.171.5.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jesaitis A. J., Naemura J. R., Painter R. G., Sklar L. A., Cochrane C. G. Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes. Biochim Biophys Acta. 1982 Dec 17;719(3):556–568. doi: 10.1016/0304-4165(82)90246-x. [DOI] [PubMed] [Google Scholar]
  33. Karasuyama H., Melchers F. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur J Immunol. 1988 Jan;18(1):97–104. doi: 10.1002/eji.1830180115. [DOI] [PubMed] [Google Scholar]
  34. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  35. Lanzavecchia A. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu Rev Immunol. 1990;8:773–793. doi: 10.1146/annurev.iy.08.040190.004013. [DOI] [PubMed] [Google Scholar]
  36. MacDonald R. G., Pfeffer S. R., Coussens L., Tepper M. A., Brocklebank C. M., Mole J. E., Anderson J. K., Chen E., Czech M. P., Ullrich A. A single receptor binds both insulin-like growth factor II and mannose-6-phosphate. Science. 1988 Mar 4;239(4844):1134–1137. doi: 10.1126/science.2964083. [DOI] [PubMed] [Google Scholar]
  37. Matovcik L. M., Goodhouse J., Farquhar M. G. The recycling itinerary of the 46 kDa mannose 6-phosphate receptor--Golgi to late endosomes--coincides with that of the 215 kDa M6PR. Eur J Cell Biol. 1990 Dec;53(2):203–211. [PubMed] [Google Scholar]
  38. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  39. Michalek M. T., Benacerraf B., Rock K. L. Weak base amines can inhibit class I MHC-restricted antigen presentation. J Immunol. 1991 Jan 15;146(2):449–456. [PubMed] [Google Scholar]
  40. Mitchell R. N., Barnes K. A., Grupp S. A., Sanchez M., Misulovin Z., Nussenzweig M. C., Abbas A. K. Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes. J Exp Med. 1995 May 1;181(5):1705–1714. doi: 10.1084/jem.181.5.1705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mitchell R. N., Shaw A. C., Weaver Y. K., Leder P., Abbas A. K. Cytoplasmic tail deletion converts membrane immunoglobulin to a phosphatidylinositol-linked form lacking signaling and efficient antigen internalization functions. J Biol Chem. 1991 May 15;266(14):8856–8860. [PubMed] [Google Scholar]
  42. Peters P. J., Neefjes J. J., Oorschot V., Ploegh H. L., Geuze H. J. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991 Feb 21;349(6311):669–676. doi: 10.1038/349669a0. [DOI] [PubMed] [Google Scholar]
  43. Qiu Y., Xu X., Wandinger-Ness A., Dalke D. P., Pierce S. K. Separation of subcellular compartments containing distinct functional forms of MHC class II. J Cell Biol. 1994 May;125(3):595–605. doi: 10.1083/jcb.125.3.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rabinowitz S., Horstmann H., Gordon S., Griffiths G. Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J Cell Biol. 1992 Jan;116(1):95–112. doi: 10.1083/jcb.116.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rock K. L., Benacerraf B., Abbas A. K. Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors. J Exp Med. 1984 Oct 1;160(4):1102–1113. doi: 10.1084/jem.160.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Roederer M., Bowser R., Murphy R. F. Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes. J Cell Physiol. 1987 May;131(2):200–209. doi: 10.1002/jcp.1041310209. [DOI] [PubMed] [Google Scholar]
  47. Ruud E., Kindberg G. M., Blomhoff H. K., Godal T., Berg T. Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle. J Immunol. 1988 Nov 1;141(9):2951–2958. [PubMed] [Google Scholar]
  48. Shaw A. C., Mitchell R. N., Weaver Y. K., Campos-Torres J., Abbas A. K., Leder P. Mutations of immunoglobulin transmembrane and cytoplasmic domains: effects on intracellular signaling and antigen presentation. Cell. 1990 Oct 19;63(2):381–392. doi: 10.1016/0092-8674(90)90171-a. [DOI] [PubMed] [Google Scholar]
  49. Stein P. E., Leslie A. G., Finch J. T., Carrell R. W. Crystal structure of uncleaved ovalbumin at 1.95 A resolution. J Mol Biol. 1991 Oct 5;221(3):941–959. doi: 10.1016/0022-2836(91)80185-w. [DOI] [PubMed] [Google Scholar]
  50. Stoorvogel W., Geuze H. J., Griffith J. M., Schwartz A. L., Strous G. J. Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol. 1989 Jun;108(6):2137–2148. doi: 10.1083/jcb.108.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tony H. P., Parker D. C. Major histocompatibility complex-restricted, polyclonal B cell responses resulting from helper T cell recognition of antiimmunoglobulin presented by small B lymphocytes. J Exp Med. 1985 Jan 1;161(1):223–241. doi: 10.1084/jem.161.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tulp A., Verwoerd D., Dobberstein B., Ploegh H. L., Pieters J. Isolation and characterization of the intracellular MHC class II compartment. Nature. 1994 May 12;369(6476):120–126. doi: 10.1038/369120a0. [DOI] [PubMed] [Google Scholar]
  53. Wall D. A., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. doi: 10.1083/jcb.101.6.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Watts C., Davidson H. W. Endocytosis and recycling of specific antigen by human B cell lines. EMBO J. 1988 Jul;7(7):1937–1945. doi: 10.1002/j.1460-2075.1988.tb03031.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weislow O. S., Kiser R., Fine D. L., Bader J., Shoemaker R. H., Boyd M. R. New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst. 1989 Apr 19;81(8):577–586. doi: 10.1093/jnci/81.8.577. [DOI] [PubMed] [Google Scholar]
  56. West M. A., Lucocq J. M., Watts C. Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells. Nature. 1994 May 12;369(6476):147–151. doi: 10.1038/369147a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES