Skip to main content
SpringerLink
Log in

Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus

  • Protocol
  • First Online:
Autoimmunity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 900))

Abstract

B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades, mainly in the context of autoantibody production. Both mouse and human studies reveal that different types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field of autoimmunity even further by establishing B cell signatures associated with disease severity, progression, and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for multiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together will likely be advantageous for improving the characterization, prognosis, and for possibly improving treatment regimens of chronic inflammatory diseases such as lupus.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pons-Estel GJ et al (2008) Understanding the epidemiology and progression of systemic lupus erythematosis. Semin Arthritis Rheum 39:257–268

    Article  Google Scholar 

  2. Borchers AT et al (2010) The geoepidemiology of systemic lupus erythematosus. Autoimmun Rev 9:A277–A287

    Article  PubMed  Google Scholar 

  3. Bertsias GK, Salmon JE, Boumpas DT (2010) Therapeutic opportunities in systemic lupus erythematosus: state of the art and prospects for the new decade. Ann Rheum Dis 69:1603–1611

    Article  PubMed  Google Scholar 

  4. Arbuckle MR et al (2003) Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med 349:1526–1533

    Article  PubMed  Google Scholar 

  5. Anolik JH et al (2009) Insights into the heterogeneity of human B cells: diverse functions, roles in autoimmunity, and use as therapeutic targets. Immunol Res (2009) 45:144-158

    Google Scholar 

  6. Wardemann H et al (2003) Predominant autoantibody production by early human B cell precursors. Science 301:1374–1377

    Article  PubMed  Google Scholar 

  7. Yurasov S et al (2005) Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med 201:703–711

    Article  PubMed  Google Scholar 

  8. Yurasov S et al (2006) Persistent expression of autoantibodies in SLE patients in remission. J Exp Med 203:2255–2261

    Article  PubMed  Google Scholar 

  9. Yurasov S et al (2005) B-cell tolerance checkpoints in healthy humans and patients with systemic lupus erythematosus. Ann N Y Acad Sci 1062:165–174

    Article  PubMed  Google Scholar 

  10. Yurasov S, Nussenzweig MC (2007) Regulation of autoreactive antibodies. Curr Opin Rheumatol 19:421–426

    Article  PubMed  Google Scholar 

  11. Tiller T et al (2007) Autoreactivity in human IgG+ memory B cells. Immunity 26:205–213

    Article  PubMed  Google Scholar 

  12. Sherer Y et al (2004) Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients. Semin Arthritis Rheum 34:501–537

    Article  PubMed  Google Scholar 

  13. Huang W et al (2002) The effect of anti-CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum 46:1554–1562

    Article  PubMed  Google Scholar 

  14. Erdei A et al (2009) Expression and role of CR1 and CR2 on B and T lymphocytes under physiological and autoimmune conditions. Mol Immunol 46:2767–2773

    Article  PubMed  Google Scholar 

  15. Casciola-Rosen LA, Anhalt G, Rosen A (1994) Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 179:1317–1330

    Article  PubMed  Google Scholar 

  16. Schutters K, Reutelingsperger C (2010) Phosphatidylserine targeting for diagnosis and treatment of human diseases. Apoptosis 15:1072–1082

    Article  PubMed  Google Scholar 

  17. Pugh-Bernard AE et al (2001) Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance. J Clin Invest 108:1061–1070

    PubMed  Google Scholar 

  18. Isenberg D et al (1993) Identification of the 9G4 idiotope in systemic lupus erythematosus. Br J Rheumatol 32:876–882

    Article  PubMed  Google Scholar 

  19. van Vollenhoven RF et al (1999) VH4-34 encoded antibodies in systemic lupus erythematosus: a specific diagnostic marker that ­correlates with clinical disease characteristics. J Rheumatol 26:1727–1733

    PubMed  Google Scholar 

  20. Bhat NM et al (2002) VH4-34 encoded antibody in systemic lupus erythematosus: effect of isotype. J Rheumatol 29:2114–2121

    PubMed  Google Scholar 

  21. Sanz I, Lee FE (2010) B cells as therapeutic targets in SLE. Nat Rev Rheumatol 6:326–337

    Article  PubMed  Google Scholar 

  22. Anolik JH et al (2004) Rituximab improves peripheral B cell abnormalities in human systemic lupus erythematosus. Arthritis Rheum 50:3580–3590

    Article  PubMed  Google Scholar 

  23. Calero I, Sanz I (2010) Targeting B cells for the treatment of SLE: the beginning of the end or the end of the beginning? Discov Med 10:416–424

    PubMed  Google Scholar 

  24. Shlomchik MJ et al (1994) The role of B cells in lpr/lpr-induced autoimmunity. J Exp Med 180:1295–1306

    Article  PubMed  Google Scholar 

  25. Chan OT et al (1999) A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J Exp Med 189:1639–1648

    Article  PubMed  Google Scholar 

  26. Ahuja A et al (2007) Depletion of B cells in murine lupus: efficacy and resistance. J Immunol 179:3351–3361

    PubMed  Google Scholar 

  27. Looney RJ et al (2004) B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab. Arthritis Rheum 50:2580–2589

    Article  PubMed  Google Scholar 

  28. Navarra SV et al (2001) Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebo-controlled, phase 3 trial. Lancet 377:721–731

    Article  Google Scholar 

  29. Chan O, Shlomchik MJ (1998) A new role for B cells in systemic autoimmunity: B cells promote spontaneous T cell activation in MRL-lpr/lpr mice. J Immunol 160:51–59

    PubMed  Google Scholar 

  30. Anolik JH et al (2007) Delayed memory B cell recovery in peripheral blood and lymphoid tissue in systemic lupus erythematosus after B cell depletion therapy. Arthritis Rheum 56:3044–3056

    Article  PubMed  Google Scholar 

  31. Sanz I et al (2008) Phenotypic and functional heterogeneity of human memory B cells. Semin Immunol 20:67–82

    Article  PubMed  Google Scholar 

  32. Wehr C et al (2004) A new CD21low B cell population in the peripheral blood of patients with SLE. Clin Immunol 113:161–171

    Article  PubMed  Google Scholar 

  33. Odendahl M et al (2000) Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus. J Immunol 165:5970–5979

    PubMed  Google Scholar 

  34. Wirths S, Lanzavecchia A (2005) ABCB1 transporter discriminates human resting naive B cells from cycling transitional and memory B cells. Eur J Immunol 35:3433–3441

    Article  PubMed  Google Scholar 

  35. Palanichamy A et al (2009) Novel human transitional B cell populations revealed by B cell depletion therapy. J Immunol 182:5982–5993

    Article  PubMed  Google Scholar 

  36. Pascual V et al (1994) Analysis of somatic mutation in five B cell subsets of human tonsil. J Exp Med 180:329–339

    Article  PubMed  Google Scholar 

  37. Wellmann U et al (2005) The evolution of human anti-double-stranded DNA autoantibodies. Proc Natl Acad Sci USA 102:9258–9263

    Article  PubMed  Google Scholar 

  38. Tsuiji M et al (2006) A checkpoint for autoreactivity in human IgM+ memory B cell development. J Exp Med 203:393–400

    Article  PubMed  Google Scholar 

  39. Cappione A 3rd et al (2005) Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus. J Clin Invest 115:3205–3216

    Article  PubMed  Google Scholar 

  40. Vinuesa CG, Sanz I, Cook MC (2009) Dysregulation of germinal centres in autoimmune disease. Nat Rev Immunol 9:845–857

    Article  PubMed  Google Scholar 

  41. Crotty S, Ahmed R (2004) Immunological memory in humans. Semin Immunol 16:197–203

    Article  PubMed  Google Scholar 

  42. Mamani-Matsuda M et al (2008) The human spleen is a major reservoir for long-lived vaccinia virus-specific memory B cells. Blood 111: 4653–4659

    Article  PubMed  Google Scholar 

  43. Anderson SM, Tomayko MM, Shlomchik MJ (2006) Intrinsic properties of human and murine memory B cells. Immunol Rev 211:280–294

    Article  PubMed  Google Scholar 

  44. Agematsu K et al (1997) B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production. Eur J Immunol 27:2073–2079

    Article  PubMed  Google Scholar 

  45. Tangye SG et al (2003) Intrinsic differences in the proliferation of naive and memory human B cells as a mechanism for enhanced secondary immune responses. J Immunol 170:686–694

    PubMed  Google Scholar 

  46. Good KL, Avery DT, Tangye SG (2009) Resting human memory B cells are intrinsically programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells. J Immunol 182:890–901

    PubMed  Google Scholar 

  47. Tangye SG, Avery DT, Hodgkin PD (2003) A division-linked mechanism for the rapid generation of Ig-secreting cells from human memory B cells. J Immunol 170:261–269

    PubMed  Google Scholar 

  48. Macallan DC et al (2005) B-cell kinetics in humans: rapid turnover of peripheral blood memory cells. Blood 105:3633–3640

    Article  PubMed  Google Scholar 

  49. van Gent R et al (2009) Refined characterization and reference values of the pediatric T- and B-cell compartments. Clin Immunol 133:95–107

    Article  PubMed  Google Scholar 

  50. Kruetzmann S et al (2003) Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J Exp Med 197:939–945

    Article  PubMed  Google Scholar 

  51. Griffin DO, Holodick NE, Rothstein TL (2011) Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+CD27+CD43+CD70−RDDDDD. J Exp Med 208:67–80

    Article  PubMed  Google Scholar 

  52. Wei C et al (2007) A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus. J Immunol 178:6624–6633

    PubMed  Google Scholar 

  53. Fecteau JF, Cote G, Neron S (2006) A new memory CD27-IgG+ B cell population in peripheral blood expressing VH genes with low frequency of somatic mutation. J Immunol 177:3728–3736

    PubMed  Google Scholar 

  54. Klein U, Rajewsky K, Kuppers R (1998) Human immunoglobulin (Ig)M+ IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med 188:1679–1689

    Article  PubMed  Google Scholar 

  55. Qian Y et al (2010) Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data. Cytometry B Clin Cytom 78(Suppl 1):S69–S82

    PubMed  Google Scholar 

  56. Dunn-Walters DK, Isaacson PG, Spencer J (1995) Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. J Exp Med 182:559–566

    Article  PubMed  Google Scholar 

  57. Weller S et al (2008) Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants. J Exp Med 205:1331–1342

    Article  PubMed  Google Scholar 

  58. Wu YC et al (2010) High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations. Blood 116:1070–1078

    Article  PubMed  Google Scholar 

  59. Seifert M, Kuppers R (2009) Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation. J Exp Med 206:2659–2669

    Article  PubMed  Google Scholar 

  60. Wasserstrom H et al (2008) Memory B cells and pneumococcal antibody after splenectomy. J Immunol 181:3684–3689

    PubMed  Google Scholar 

  61. Iwata Y et al (2011) Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood 117:530–541

    Article  PubMed  Google Scholar 

  62. Dogan I et al (2009) Multiple layers of B cell memory with different effector functions. Nat Immunol 10:1292–1299

    Article  PubMed  Google Scholar 

  63. Pape KA et al (2011) Different B cell populations mediate early and late memory during an endogenous immune response. Science 331:1203–1207

    Article  PubMed  Google Scholar 

  64. Weller S et al (2001) CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proc Natl Acad Sci USA 98:1166–1170

    Article  PubMed  Google Scholar 

  65. Mietzner B et al (2008) Autoreactive IgG memory antibodies in patients with systemic lupus erythematosus arise from nonreactive and polyreactive precursors. Proc Natl Acad Sci USA 105:9727–9732

    Article  PubMed  Google Scholar 

  66. Snapper CM, Mond JJ (1993) Towards a comprehensive view of immunoglobulin class switching. Immunol Today 14:15–17

    Article  PubMed  Google Scholar 

  67. Rodriguez-Bayona B et al (2010) Decreased frequency and activated phenotype of blood CD27 IgD IgM B lymphocytes is a permanent abnormality in systemic lupus erythematosus patients. Arthritis Res Ther 12:R108

    Article  PubMed  Google Scholar 

  68. Korganow AS et al (2010) Peripheral B cell abnormalities in patients with systemic lupus erythematosus in quiescent phase: decreased memory B cells and membrane CD19 expression. J Autoimmun 34:426–434

    Article  PubMed  Google Scholar 

  69. Houtkamp MA et al (2001) Adventitial infiltrates associated with advanced atherosclerotic plaques: structural organization suggests generation of local humoral immune responses. J Pathol 193:263–269

    Article  PubMed  Google Scholar 

  70. Kyaw T et al (2010) Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis. J Immunol 185:4410–4419

    Article  PubMed  Google Scholar 

  71. Sjoberg BG et al (2009) Low levels of IgM antibodies against phosphorylcholine-A potential risk marker for ischemic stroke in men. Atherosclerosis 203:528–532

    Article  PubMed  Google Scholar 

  72. Zhou X, Hansson GK (1999) Detection of B cells and proinflammatory cytokines in atherosclerotic plaques of hypercholesterolaemic apolipoprotein E knockout mice. Scand J Immunol 50:25–30

    Article  PubMed  Google Scholar 

  73. Ait-Oufella H et al (2010) B cell depletion reduces the development of atherosclerosis in mice. J Exp Med 207:1579–1587

    Article  PubMed  Google Scholar 

  74. Halliley JL et al (2010) Peak frequencies of circulating human influenza-specific antibody secreting cells correlate with serum antibody response after immunization. Vaccine 28:3582–3587

    Article  PubMed  Google Scholar 

  75. Gonzalez-Garcia I et al (2006) Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells. J Immunol 176:4042–4050

    PubMed  Google Scholar 

  76. Odendahl M et al (2005) Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response. Blood 105:1614–1621

    Article  PubMed  Google Scholar 

  77. Lee FE et al (2010) Circulating antibody-secreting cells during acute respiratory syncytial virus infection in adults. J Infect Dis 202:1659–1666

    Article  PubMed  Google Scholar 

  78. Fairfax KA et al (2008) Plasma cell development: from B-cell subsets to long-term survival niches. Semin Immunol 20:49–58

    Article  PubMed  Google Scholar 

  79. Oracki SA et al (2010) Plasma cell development and survival. Immunol Rev 237:140–159

    Article  PubMed  Google Scholar 

  80. Caraux A et al (2010) Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138− and CD138+ plasma cells. Haematologica 95:1016–1020

    Article  PubMed  Google Scholar 

  81. Bernasconi NL, Traggiai E, Lanzavecchia A (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298:2199–2202

    Article  PubMed  Google Scholar 

  82. Jacobi AM et al (2010) Effect of long-term belimumab treatment on B cells in systemic lupus erythematosus: extension of a phase II, double-blind, placebo-controlled, dose-ranging study. Arthritis Rheum 62:201–210

    Article  PubMed  Google Scholar 

  83. Moir S et al (2008) Normalization of B cell counts and subpopulations after antiretroviral therapy in chronic HIV disease. J Infect Dis 197:572–579

    Article  PubMed  Google Scholar 

  84. Jacobi AM et al (2008) Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95. Arthritis Rheum 58:1762–1773

    Article  PubMed  Google Scholar 

  85. Habib LK, Finn WG (2006) Unsupervised immunophenotypic profiling of chronic lymphocytic leukemia. Cytometry B Clin Cytom 70:124–135

    PubMed  Google Scholar 

  86. Diaz-Romero J et al (2010) Hierarchical clustering of flow cytometry data for the study of conventional central chondrosarcoma. J Cell Physiol 225:601–611

    Article  PubMed  Google Scholar 

  87. Cappione AJ et al (2004) Lupus IgG VH4.34 antibodies bind to a 220-kDa glycoform of CD45/B220 on the surface of human B lymphocytes. J Immunol 172:4298–4307

    PubMed  Google Scholar 

  88. Herzenberg LA et al (2006) Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol 7:681–685

    Article  PubMed  Google Scholar 

  89. Eisen MB et al (1998) Cluster analysis and ­display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:14863–14868

    Article  PubMed  Google Scholar 

  90. D’haeseleer (2005) How does gene expression clustering work? Nat Biotechnol 23:1499–1501

    Article  PubMed  Google Scholar 

  91. Quackenbush J (2001) Computational analysis of microarray data. Nat Rev Genet 2:418–427

    Article  PubMed  Google Scholar 

  92. Misra J et al (2002) Interactive exploration of microarray gene expression patterns in a reduced dimensional space. Genome Res 12:1112–1120

    Article  PubMed  Google Scholar 

  93. Lugli E et al (2007) Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data. Cytometry A 71:334–344

    PubMed  Google Scholar 

  94. Lee FE et al (2011) Circulating human antibody-secreting cells during vaccinations and respiratory viral infections are characterized by high specificity and lack of bystander effect. J Immunol 186:5514–5521

    PubMed  Google Scholar 

  95. Wei et al (2011) OMIP-003: Phenotypic analysis of human memory B cells. Cytometry A 79:894–896

    PubMed  Google Scholar 

Download references

Acknowledgments

We thank John Jung and Ravi Misra for reading the manuscript and members of the Sanz lab for help and advice. Supported by NIH R01 AI049660-01A1 and U19 Autoimmunity Center of Excellence AI56390 to I.S.

Author information

Authors and Affiliations

  1. Division of Allergy, Immunology, and Rheumatology, Department of Medicine, University of Rochester Medical Center, Rochester, NY, USA

    Denise A. Kaminski, Chungwen Wei, Alexander F. Rosenberg, F. Eun-Hung Lee & Ignacio Sanz

Authors
  1. Denise A. Kaminski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chungwen Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander F. Rosenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Eun-Hung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ignacio Sanz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ignacio Sanz .

Editor information

Editors and Affiliations

  1. College of Medicine, Division of Rheumatology, State University of New York, UMU, East Adams St. 750, Syracuse, 13210, New York, USA

    Andras Perl

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kaminski, D.A., Wei, C., Rosenberg, A.F., Lee, F.EH., Sanz, I. (2012). Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus. In: Perl, A. (eds) Autoimmunity. Methods in Molecular Biology, vol 900. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-720-4_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-720-4_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-719-8

  • Online ISBN: 978-1-60761-720-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation