Advertisement

Studying MHC class II Presentation of Immobilized Antigen by B Lymphocytes

  • M. I. Yuseff
  • A. M. Lennon-DumenilEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 960)

Abstract

The ability of B lymphocytes to capture external antigens (Ag) and present them as peptide fragments, loaded on Major Histocompatibility complex (MHC) class II molecules, to CD4+ T cells is a crucial part of the adaptive immune response. This allows T-B cooperation, a cellular communication that is required for B cells to develop into germinal centers (GC) and form mature high-affinity antibody producing cells and to further develop B cell memory. MHC class II antigen presentation by B lymphocytes is a multistep process involving (1) Recognition and capture of external Ag by B lymphocytes through their B cell receptor (BCR); (2) Ag processing, which comprises the degradation of Ag in internal compartments within the B cell and loading of the corresponding peptide fragments on MHC class II molecules and (3) Presentation of MHCII-peptide complexes to CD4+ T cells. Here, we describe how to study MHC class II antigen presentation by B lymphocytes at these three major levels.

Key words

B lymphocytes Immune synapse Antigen extraction Processing and presentation MTOC polarization Lysosome secretion 

Abbreviations

Ag

Antigen

BCR

B cell receptor

BSA

Bovine serum albumin

FBS

Fetal bovine serum

GC

Germinal center

Ig

Immunoglobulin

IS

Immune synapse

MHC

Major histocompatibility complex

MTOC

Microtubule organizing center

OVA

Ovalbumin

References

  1. 1.
    Cambier JC, Pleiman CM, Clark MR (1994) Signal transduction by the B cell antigen receptor and its coreceptors. Annu Rev Immunol 12:457–486. doi: 10.1146/annurev.iy.12.040194.002325 CrossRefPubMedGoogle Scholar
  2. 2.
    Reth M, Wienands J (1997) Initiation and processing of signals from the B cell antigen receptor. Annu Rev Immunol 15:453–479. doi: 10.1146/annurev.immunol.15.1.453 CrossRefPubMedGoogle Scholar
  3. 3.
    Mitchison NA (2004) T-cell-B-cell cooperation. Nat Rev Immunol 4(4):308–312. doi: 10.1038/nri1334 CrossRefPubMedGoogle Scholar
  4. 4.
    Lankar D, Vincent-Schneider H, Briken V, Yokozeki T, Raposo G, Bonnerot C (2002) Dynamics of major histocompatibility complex class II compartments during B cell receptor-mediated cell activation. J Exp Med 195(4):461–472CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Carrasco YR, Batista FD (2007) B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27(1):160–171. doi:S1074-7613(07)00334-2 (pii)CrossRefPubMedGoogle Scholar
  6. 6.
    Junt T, Moseman EA, Iannacone M, Massberg S, Lang PA, Boes M, Fink K, Henrickson SE, Shayakhmetov DM, Di Paolo NC, van Rooijen N, Mempel TR, Whelan SP, von Andrian UH (2007) Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450(7166):110–114. doi:nature06287 (pii)CrossRefPubMedGoogle Scholar
  7. 7.
    Suzuki K, Grigorova I, Phan TG, Kelly LM, Cyster JG (2009) Visualizing B cell capture of cognate antigen from follicular dendritic cells. J Exp Med 206(7):1485–1493. doi:jem.20090209 (pii)CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Harwood NE, Batista FD (2008) New insights into the early molecular events underlying B cell activation. Immunity 28(5):609–619. doi:S1074-7613(08)00197-0 (pii)CrossRefPubMedGoogle Scholar
  9. 9.
    Tolar P, Sohn HW, Pierce SK (2008) Viewing the antigen-induced initiation of B-cell activation in living cells. Immunol Rev 221:64–76. doi:IMR583 (pii)CrossRefPubMedGoogle Scholar
  10. 10.
    Fleire SJ, Goldman JP, Carrasco YR, Weber M, Bray D, Batista FD (2006) B cell ligand discrimination through a spreading and contraction response. Science 312(5774):738–741. doi:312/5774/738 (pii)CrossRefPubMedGoogle Scholar
  11. 11.
    Yuseff MI, Reversat A, Lankar D, Diaz J, Fanget I, Pierobon P, Randrian V, Larochette N, Vascotto F, Desdouets C, Jauffred B, Bellaiche Y, Gasman S, Darchen F, Desnos C, Lennon-Dumenil AM (2011) Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation. Immunity 35(3):361–374. doi:S1074-7613(11)00276-7 (pii)CrossRefPubMedGoogle Scholar
  12. 12.
    Vascotto F, Lankar D, Faure-Andre G, Vargas P, Diaz J, Le Roux D, Yuseff MI, Sibarita JB, Boes M, Raposo G, Mougneau E, Glaichenhaus N, Bonnerot C, Manoury B, Lennon-Dumenil AM (2007) The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation. J Cell Biol 176(7):1007–1019. doi:jcb.200611147 (pii)CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Le Roux D, Lankar D, Yuseff MI, Vascotto F, Yokozeki T, Faure-Andre G, Mougneau E, Glaichenhaus N, Manoury B, Bonnerot C, Lennon-Dumenil AM (2007) Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor. Mol Biol Cell 18(9):3451–3462. doi:E06-12-1114 (pii)CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Aluvihare VR, Khamlichi AA, Williams GT, Adorini L, Neuberger MS (1997) Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction. EMBO J 16(12):3553–3562. doi: 10.1093/emboj/16.12.3553 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lennon-Dumenil AM, Bakker AH, Wolf-Bryant P, Ploegh HL, Lagaudriere-Gesbert C (2002) A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr Opin Immunol 14(1):15–21. doi:S095279150100293X (pii)CrossRefPubMedGoogle Scholar
  16. 16.
    Wolf PR, Ploegh HL (1995) How MHC class II molecules acquire peptide cargo: biosynthesis and trafficking through the endocytic pathway. Annu Rev Cell Dev Biol 11:267–306. doi: 10.1146/annurev.cb.11.110195.001411 CrossRefPubMedGoogle Scholar
  17. 17.
    Villadangos JA, Bryant RA, Deussing J, Driessen C, Lennon-Dumenil AM, Riese RJ, Roth W, Saftig P, Shi GP, Chapman HA, Peters C, Ploegh HL (1999) Proteases involved in MHC class II antigen presentation. Immunol Rev 172:109–120CrossRefPubMedGoogle Scholar
  18. 18.
    Watts C (2001) Antigen processing in the endocytic compartment. Curr Opin Immunol 13(1):26–31. doi:S0952-7915(00)00177-1 (pii)CrossRefPubMedGoogle Scholar
  19. 19.
    Denzin LK, Cresswell P (1995) HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell 82(1):155–165. doi:0092-8674(95)90061-6 (pii)CrossRefPubMedGoogle Scholar
  20. 20.
    Muraille E, Gounon P, Cazareth J, Hoebeke J, Lippuner C, Davalos-Misslitz A, Aebischer T, Muller S, Glaichenhaus N, Mougneau E (2010) Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major. PLoS Pathog 6(10):e1001154. doi: 10.1371/journal.ppat.1001154 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lankar D, Briken V, Adler K, Weiser P, Cassard S, Blank U, Viguier M, Bonnerot C (1998) Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation. J Exp Med 188(5):819–831CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Boes M, Cerny J, Massol R, Op den Brouw M, Kirchhausen T, Chen J, Ploegh HL (2002) T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature 418(6901):983–988. doi: 10.1038/nature01004 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Institut Curie, Inserm U932ParisFrance

Personalised recommendations