Characterization and Activity of Fas Ligand Producing CD5+ B Cells

  • Steven K. LundyEmail author
  • Matthew W. Klinker
Part of the Methods in Molecular Biology book series (MIMB, volume 1190)


B lymphocytes make several contributions to immune regulation including production of antibodies with regulatory properties, release of immune suppressive cytokines, and expression of death-inducing ligands. A role for Fas ligand (FasL)-expressing “killer” B cells in regulating T helper cell survival and chronic inflammation has been demonstrated in animal models of schistosome worm infection, asthma, and autoimmune arthritis. Interestingly, a population of CD5+ B cells found in the spleen and lungs of naïve mice constitutively expresses FasL and has potent killer function against T helper cells that is antigen-specific and FasL-dependent. Killer B cells therefore represent a novel target for immune modulation in many disease settings. Our laboratory has recently published methods of characterizing FasL+ B cells and inducing their proliferation in vitro. This chapter will describe detailed methods of identifying and expanding killer B cells from mice, detecting FasL expression in B cells, and performing functional killing assays against antigen-specific TH cells.

Key words

Killer B lymphocytes T cell apoptosis Antigen-specific tolerance Death ligands IL-10 Immune suppression IL-5 CD40 ligand Lethal exosomes 


  1. 1.
    Roths JB, Murphy ED, Eicher EM (1984) A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice. J Exp Med 159:1–20PubMedCrossRefGoogle Scholar
  2. 2.
    Sobel ES, Kakkanaiah VN, Cohen PL, Eisenberg RA (1993) Correction of gld autoimmunity by co-infusion of normal bone marrow suggests that gld is a mutation of the Fas ligand gene. Int Immunol 5:1275–1278PubMedCrossRefGoogle Scholar
  3. 3.
    Lynch DH, Watson ML, Alderson MR, Baum PR, Miller RE, Tough T, Gibson M, Davis-Smith T, Smith CA, Hunter K et al (1994) The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster. Immunity 1:131–136PubMedCrossRefGoogle Scholar
  4. 4.
    Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata S (1994) Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76:969–976PubMedCrossRefGoogle Scholar
  5. 5.
    Hahne M, Peitsch MC, Irmler M, Schroter M, Lowin B, Rousseau M, Bron C, Renno T, French L, Tschopp J (1995) Characterization of the non-functional Fas ligand of gld mice. Int Immunol 7:1381–1386PubMedCrossRefGoogle Scholar
  6. 6.
    Adachi M, Watanabe-Fukunaga R, Nagata S (1993) Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice. Proc Natl Acad Sci U S A 90:1756–1760PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Wu J, Zhou T, He J, Mountz JD (1993) Autoimmune disease in mice due to integration of an endogenous retrovirus in an apoptosis gene. J Exp Med 178:461–468PubMedCrossRefGoogle Scholar
  8. 8.
    Nagata S, Suda T (1995) Fas and Fas ligand: lpr and gld mutations. Immunol Today 16: 39–43PubMedCrossRefGoogle Scholar
  9. 9.
    Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, Puck JM (1995) Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935–946PubMedCrossRefGoogle Scholar
  10. 10.
    Rieux-Laucat F, Le Deist F, Hivroz C, Roberts IA, Debatin KM, Fischer A, de Villartay JP (1995) Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 268:1347–1349PubMedCrossRefGoogle Scholar
  11. 11.
    Bleesing JJ (2005) Sorting out the causes of ALPS. J Pediatr 147:571–574PubMedCrossRefGoogle Scholar
  12. 12.
    Infante AJ, Britton HA, DeNapoli T, Middelton LA, Lenardo MJ, Jackson CE, Wang J, Fleisher T, Straus SE, Puck JM (1998) The clinical spectrum in a large kindred with autoimmune lymphoproliferative syndrome caused by a Fas mutation that impairs lymphocyte apoptosis. J Pediatr 133:629–633PubMedCrossRefGoogle Scholar
  13. 13.
    Sneller MC, Straus SE, Jaffe ES, Jaffe JS, Fleisher TA, Stetler-Stevenson M, Strober W (1992) A novel lymphoproliferative/autoimmune syndrome resembling murine lpr/gld disease. J Clin Invest 90:334–341PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Magerus-Chatinet A, Stolzenberg MC, Loffredo MS, Neven B, Schaffner C, Ducrot N, Arkwright PD, Bader-Meunier B, Barbot J, Blanche S, Casanova JL, Debre M, Ferster A, Fieschi C, Florkin B, Galambrun C, Hermine O, Lambotte O, Solary E, Thomas C, Le Deist F, Picard C, Fischer A, Rieux-Laucat F (2009) FAS-L, IL-10, and double-negative CD4–CD8- TCR alpha/beta + T cells are reliable markers of autoimmune lymphoproliferative syndrome (ALPS) associated with FAS loss of function. Blood 113:3027–3030PubMedCrossRefGoogle Scholar
  15. 15.
    Stroncek DF, Carter LB, Procter JL, Dale JK, Straus SE (2001) RBC autoantibodies in autoimmune lymphoproliferative syndrome. Transfusion 41:18–23PubMedCrossRefGoogle Scholar
  16. 16.
    Wu J, Wilson J, He J, Xiang L, Schur PH, Mountz JD (1996) Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J Clin Invest 98: 1107–1113PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Kojima T, Horiuchi T, Nishizaka H, Sawabe T, Higuchi M, Harashima SI, Yoshizawa S, Tsukamoto H, Nagasawa K, Niho Y (2000) Analysis of fas ligand gene mutation in patients with systemic lupus erythematosus. Arthritis Rheum 43:135–139PubMedCrossRefGoogle Scholar
  18. 18.
    Atkinson EA, Bleackley RC (1995) Mechanisms of lysis by cytotoxic T cells. Crit Rev Immunol 15:359–384PubMedCrossRefGoogle Scholar
  19. 19.
    Oshimi Y, Oda S, Honda Y, Nagata S, Miyazaki S (1996) Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells. J Immunol 157: 2909–2915PubMedGoogle Scholar
  20. 20.
    Hahne M, Renno T, Schroeter M, Irmler M, French L, Bornard T, MacDonald HR, Tschopp J (1996) Activated B cells express functional Fas ligand. Eur J Immunol 26:721–724PubMedCrossRefGoogle Scholar
  21. 21.
    Lundy SK (2009) Killer B lymphocytes: the evidence and the potential. Inflamm Res 58: 345–357PubMedCrossRefGoogle Scholar
  22. 22.
    Lundy SK, Lerman SP, Boros DL (2001) Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection. Infect Immun 69:271–280PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Lundy SK, Boros DL (2002) Fas ligand-expressing B-1a lymphocytes mediate CD4(+)-T-cell apoptosis during schistosomal infection: induction by interleukin 4 (IL-4) and IL-10. Infect Immun 70:812–819PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Lundy SK, Berlin AA, Martens TF, Lukacs NW (2005) Deficiency of regulatory B cells increases allergic airway inflammation. Inflamm Res 54:514–521PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Lundy SK, Fox DA (2009) Reduced Fas ligand-expressing splenic CD5+ B lymphocytes in severe collagen-induced arthritis. Arthritis Res Ther 11:R128PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL (2001) Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol 167:1081–1089PubMedCrossRefGoogle Scholar
  27. 27.
    Montandon R, Korniotis S, Layseca-Espinosa E, Gras C, Megret J, Ezine S, Dy M, Zavala F (2013) Innate pro-B-cell progenitors protect against type 1 diabetes by regulating autoimmune effector T cells. Proc Natl Acad Sci U S A 110:E2199–E2208PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Bonardelle D, Benihoud K, Kiger N, Bobe P (2005) B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice. J Leukoc Biol 78:1052–1059PubMedCrossRefGoogle Scholar
  29. 29.
    Zuniga E, Motran CC, Montes CL, Yagita H, Gruppi A (2002) Trypanosoma cruzi infection selectively renders parasite-specific IgG+B lymphocytes susceptible to Fas/Fas ligand-mediated fratricide. J Immunol 168:3965–3973PubMedCrossRefGoogle Scholar
  30. 30.
    Minagawa R, Okano S, Tomita Y, Kishihara K, Yamada H, Nomoto K, Shimada M, Maehara Y, Sugimachi K, Yoshikai Y (2004) The critical role of Fas-Fas ligand interaction in donor-specific transfusion-induced tolerance to H-Y antigen. Transplantation 78:799–806PubMedCrossRefGoogle Scholar
  31. 31.
    Mincheff M, Loukinov D, Zoubak S, Hammett M, Meryman H (1998) Fas and Fas ligand expression on human peripheral blood leukocytes. Vox Sang 74:113–121PubMedCrossRefGoogle Scholar
  32. 32.
    Martinez-Lorenzo MJ, Anel A, Gamen S, Monle n I, Lasierra P, Larrad L, Pineiro A, Alava MA, Naval J (1999) Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. J Immunol 163:1274–1281PubMedGoogle Scholar
  33. 33.
    Jodo S, Xiao S, Hohlbaum A, Strehlow D, Marshak-Rothstein A, Ju ST (2001) Apoptosis-inducing membrane vesicles. A novel agent with unique properties. J Biol Chem 276: 39938–39944PubMedCrossRefGoogle Scholar
  34. 34.
    Sabapatha A, Gercel-Taylor C, Taylor DD (2006) Specific isolation of placenta-derived exosomes from the circulation of pregnant women and their immunoregulatory consequences. Am J Reprod Immunol 56:345–355PubMedCrossRefGoogle Scholar
  35. 35.
    McKechnie NM, King BC, Fletcher E, Braun G (2006) Fas-ligand is stored in secretory lysosomes of ocular barrier epithelia and released with microvesicles. Exp Eye Res 83:304–314PubMedCrossRefGoogle Scholar
  36. 36.
    Kang SM, Schneider DB, Lin Z, Hanahan D, Dichek DA, Stock PG, Baekkeskov S (1997) Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nat Med 3:738–743PubMedCrossRefGoogle Scholar
  37. 37.
    Takeuchi T, Ueki T, Nishimatsu H, Kajiwara T, Ishida T, Jishage K, Ueda O, Suzuki H, Li B, Moriyama N, Kitamura T (1999) Accelerated rejection of Fas ligand-expressing heart grafts. J Immunol 162:518–522PubMedGoogle Scholar
  38. 38.
    Gregory MS, Repp AC, Holhbaum AM, Saff RR, Marshak-Rothstein A, Ksander BR (2002) Membrane Fas ligand activates innate immunity and terminates ocular immune privilege. J Immunol 169:2727–2735PubMedCrossRefGoogle Scholar
  39. 39.
    Mariani SM, Matiba B, Baumler C, Krammer PH (1995) Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases. Eur J Immunol 25:2303–2307PubMedCrossRefGoogle Scholar
  40. 40.
    Powell WC, Fingleton B, Wilson CL, Boothby M, Matrisian LM (1999) The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr Biol 9:1441–1447PubMedCrossRefGoogle Scholar
  41. 41.
    Sano Y, Yamada J, Ishino Y, Adachi W, Kawasaki S, Suzuki T, Kinoshita S, Okuyama T, Azuma N (2002) Non-cleavable mutant Fas ligand transfection of donor cornea abrogates ocular immune privilege. Exp Eye Res 75:475–483PubMedCrossRefGoogle Scholar
  42. 42.
    Klinker MW, Reed TJ, Fox DA, Lundy SK (2013) Interleukin-5 supports the expansion of Fas ligand-expressing killer B cells that induce antigen-specific apoptosis of CD4(+) t cells and secrete interleukin-10. PLoS One 8:e70131PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Martin F, Kearney JF (2001) B1 cells: similarities and differences with other B cell subsets. Curr Opin Immunol 13:195–201PubMedCrossRefGoogle Scholar
  44. 44.
    Yang Y, Tung JW, Ghosn EE, Herzenberg LA, Herzenberg LA (2007) Division and differentiation of natural antibody-producing cells in mouse spleen. Proc Natl Acad Sci U S A 104:4542–4546PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Kantor AB, Stall AM, Adams S, Watanabe K, Herzenberg LA (1995) De novo development and self-replenishment of B cells. Int Immunol 7:55–68PubMedCrossRefGoogle Scholar
  46. 46.
    Yoshimoto M, Montecino-Rodriguez E, Ferkowicz MJ, Porayette P, Shelley WC, Conway SJ, Dorshkind K, Yoder MC (2011) Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential. Proc Natl Acad Sci U S A 108: 1468–1473PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Klinker MW, Lundy SK (2012) Multiple mechanisms of immune suppression by B lymphocytes. Mol Med 18:123–137PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Schultze JL, Michalak S, Seamon MJ, Dranoff G, Jung K, Daley J, Delgado JC, Gribben JG, Nadler LM (1997) CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. J Clin Invest 100:2757–2765PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Morita Y, Gupta R, Seidl KM, McDonagh KT, Fox DA (2005) Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis. J Gene Med 7:869–877Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Rheumatology, Department of Internal MedicineUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Immunology Training Program, Program in Biomedical Sciences, Rackham Graduate SchoolUniversity of MichiganAnn ArborUSA

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