BAFF and Autoimmune BAFF and Autoimmune DiseaseDisease

Samia Ragheb, Ph.D.Samia Ragheb, Ph.D.

Wayne State UniversityWayne State University

Department of NeurologyDepartment of Neurology

Department of Immunology & Department of Immunology & Microbiology Microbiology

MG is a B-cell MG is a B-cell Mediated DiseaseMediated Disease

1. AChR-specific antibodies are present in 1. AChR-specific antibodies are present in the serum of 85% of all patients with the serum of 85% of all patients with generalized MG; these antibodies are generalized MG; these antibodies are responsible for the pathology of MG, responsible for the pathology of MG, leading to impaired neuromuscular leading to impaired neuromuscular transmission and subsequent muscle transmission and subsequent muscle weaknessweakness

2.2. In vitro, lymphocyte cultures from In vitro, lymphocyte cultures from MG patients produce AChR-specific MG patients produce AChR-specific antibodiesantibodies

3. EAMG can be induced in lab animals; 3. EAMG can be induced in lab animals; serum anti-AChR antibodies are present serum anti-AChR antibodies are present in these animalsin these animals

4. EAMG can be passively transferred, via 4. EAMG can be passively transferred, via antibodies, from immune animals to naïve antibodies, from immune animals to naïve animals and from man to mouse animals and from man to mouse

Overexpression of Overexpression of CXCL13, CD23, and Bcl-CXCL13, CD23, and Bcl-2 in the Myasthenic 2 in the Myasthenic

ThymusThymusCXCL13:CXCL13: promotes B-cell migration and promotes B-cell migration and

homing of B-cells to lymphoid homing of B-cells to lymphoid folliclesfollicles

CD23CD23: promotes the survival and : promotes the survival and differentiation of germinal center differentiation of germinal center B-cells through a mechanism that B-cells through a mechanism that involves upregulation of Bcl-2involves upregulation of Bcl-2

Bcl-2Bcl-2: pro-survival molecule that : pro-survival molecule that inhibits apoptosisinhibits apoptosis

BAFF = TNFSF13bBAFF = TNFSF13bMyeloid cells produce and secrete Baff (B-Myeloid cells produce and secrete Baff (B-cell activating factor of the tumor necrosis cell activating factor of the tumor necrosis factor superfamily) factor superfamily)

A membrane-bound form of Baff is also A membrane-bound form of Baff is also expressed on the surface of myeloid cells; expressed on the surface of myeloid cells; these include monocytes, macrophages, and these include monocytes, macrophages, and dendritic cells dendritic cells

Baff transgenic animals exhibit Baff transgenic animals exhibit hypergammaglobulinemia, lymphoproliferation, hypergammaglobulinemia, lymphoproliferation, and B-cell hyperplasia; and they develop and B-cell hyperplasia; and they develop autoimmune disease autoimmune disease

In Baff deficient animals, there are defects In Baff deficient animals, there are defects in peripheral B-cell maturation, and there in peripheral B-cell maturation, and there are decreased levels of circulating are decreased levels of circulating immunoglobulinsimmunoglobulins

Serum BAFF LevelsSerum BAFF Levels

Healthy MG MS ALS0

1

2

3

4

5

6

Effect of Clinical Extent Effect of Clinical Extent and Severity on BAFF Levelsand Severity on BAFF Levels

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Class I

Class II

Class III

Class IV

Baff (ng/ml), mean ± SD

Effect of Presence or Effect of Presence or Absence of Serum Anti-AChR Absence of Serum Anti-AChR Antibody on BAFF LevelsAntibody on BAFF Levels

Seronegative Seropositive0

1

2

3

456

Receptors for BAFFReceptors for BAFF• Baff-R deficient mice lack marginal Baff-R deficient mice lack marginal zone and follicular B-cellszone and follicular B-cells

• TACI deficient mice have an TACI deficient mice have an increased number of peripheral B-cells, increased number of peripheral B-cells, they develop autoimmune disease, and they develop autoimmune disease, and they exhibit fatal lymphoproliferationthey exhibit fatal lymphoproliferation

The pro survival The pro survival signals of Baff are signals of Baff are mediated by the Baff-R, mediated by the Baff-R, whereas the interaction whereas the interaction of Baff with TACI of Baff with TACI delivers inhibitory delivers inhibitory signalssignals

• CD3: mouse IgG1-FITCCD3: mouse IgG1-FITC• CD20: mouse IgG2a-FITCCD20: mouse IgG2a-FITC• Baff-R: goat IgG anti human Baff-R + rabbit Baff-R: goat IgG anti human Baff-R + rabbit F(ab)’F(ab)’22 anti goat IgG-Cy5 anti goat IgG-Cy5• TACI: biotinylated goat IgG anti human TACI + TACI: biotinylated goat IgG anti human TACI + streptavidin-Cy5streptavidin-Cy5

Immunofluorescent Staining of Immunofluorescent Staining of Peripheral Blood Mononuclear Peripheral Blood Mononuclear

CellsCells

Cell-Surface Expression of Cell-Surface Expression of BAFF-R and TACIBAFF-R and TACI

Thangarajh et al.:Thangarajh et al.:• MG thymus with hyperplasia: macrophages MG thymus with hyperplasia: macrophages express Baffexpress Baff• Germinal center: B-cells express Baff-RGerminal center: B-cells express Baff-R• Plasma levels of Baff were not differentPlasma levels of Baff were not different• No differences in the percentages of No differences in the percentages of BCMA, Baff-R, or TACI-positive B-cellsBCMA, Baff-R, or TACI-positive B-cells

Li et al.:Li et al.:• Increased percentage of CD19Increased percentage of CD19+ + Baff-RBaff-R++ B- B-cellscells

Kim et al.:Kim et al.:• Serum Baff levels are higher in MG Serum Baff levels are higher in MG patientspatients

Monocyte Dendritic cell

B-cell

Baff

Myeloid Cells Produce and Secrete BAFFMyeloid Cells Produce and Secrete BAFF

Immunofluorescent Staining Immunofluorescent Staining of Peripheral Blood of Peripheral Blood

MonocytesMonocytes

• CD14: mouse IgM-FITCCD14: mouse IgM-FITC• Baff: mouse IgG1 anti human Baff + goat F(ab)’Baff: mouse IgG1 anti human Baff + goat F(ab)’22 anti mouse IgG-PE anti mouse IgG-PE

BAFF Production by Adherent BAFF Production by Adherent MonocytesMonocytes

BAFF Production by BAFF Production by Adherent MonocytesAdherent Monocytes

BAFF Production by THP-1 CellsBAFF Production by THP-1 Cells

48 hrs 72 hrs 96 hrs0

100

200

300

LPS (1 g/ml)1 ng/ml 5 ng/ml 10 ng/ml

0

100

200

300

400

500 72 hrs

96 hrs

IFN-

THP-1:THP-1: human acute monocytic leukemiahuman acute monocytic leukemia1 year old male1 year old maleHLA: A2, A9, B5, DRw1, DRw2HLA: A2, A9, B5, DRw1, DRw2

BAFF Production by THP-1 BAFF Production by THP-1 CellsCells

PBMC are PBMC are treated with a treated with a mixture of mixture of mouse anti mouse anti CD2, CD7, CD2, CD7, CD16, CD19, CD16, CD19, CD56, and CD56, and CD235a CD235a followed by followed by anti mouse anti mouse IgG-coated IgG-coated magnetic beadsmagnetic beads

Enrichment of Enrichment of Monocytes by Monocytes by

Immunomagnetic Immunomagnetic SeparationSeparation

BAFF Production by BAFF Production by MonocytesMonocytes

Dendritic Cell LineagesDendritic Cell Lineages

Banchereau et al., 2000Banchereau et al., 2000

Differentiation of Monocytes Differentiation of Monocytes Into Dendritic CellsInto Dendritic Cells

Maturation of Dendritic Maturation of Dendritic Cells With sCD40L (CD154)Cells With sCD40L (CD154)

Cytokine Production by Cytokine Production by Dendritic Cells Dendritic Cells

sCD40LsCD40LImmature DCImmature DC Mature DCMature DC 254.0 pg/ml254.0 pg/ml IL-4 IL-4

192.0 pg/ml 192.0 pg/ml 5.05.0 IL-10 IL-10

5.4 5.4 00 IL-12 IL-12

1.3 1.3 00 IL-17 IL-17 0 0

BAFF Production by BAFF Production by Dendritic CellsDendritic Cells

Ueno et al., 2007Ueno et al., 2007

ContributorsContributorsBasic ScienceBasic Science ClinicalClinicalSamia Ragheb, Ph.D. Samia Ragheb, Ph.D. Robert Lisak, M.D.Robert Lisak, M.D.Felicitas Gonzales, B.S.Felicitas Gonzales, B.S. Richard Lewis, M.D.Richard Lewis, M.D.Kirk Simon, B.S.Kirk Simon, B.S. Gregory Van Stavern, M.D.Gregory Van Stavern, M.D.Yanfeng Li, M.S.Yanfeng Li, M.S.

Wayne State University School of MedicineWayne State University School of Medicine

Supported by the Muscular Dystrophy Association (MDA)Supported by the Muscular Dystrophy Association (MDA)