Overview B cell development Transcriptional cascades Amazing aspects of lineage plasticity Conventional (B2) B cell development What happens to an autoreactive B cell? B1 vs B2 cells
Key anatomical sites Embryo/fetus aorta-gonad-mesonephros (AGM) Yolk sac Fetal liver Adult Bone marrow - primary, B2 B Lymph node, Spleen - secondary, B2 B Fetal liver & adult peritoneal cavity - B1 B cells
Hematopoiesis myeloid lymphoid Required transcription factors PU.1 ikaros E2A EBF pax5
Key transcription factors in lineage fate commitment lymphoid vs myeloid PU.1 - graded levels lineage fate; controls IL7R and CD45R/B220 ikaros - stem cell renewal, TdT VpreB λ5 lymphoid - B lineage differentiation E2A - initiates a transcriptional hierarchy; ebf, rag expression, λ5, mb-1 EBF - pax5, VpreB, λ5 pax5 (BSAP) - CD19, V-DJ joining (accessibility), blnk
PU.1 lineage fate decisions graded expression mediates lineage fate decisions Science (2000) 288:1439
Transcriptional hierarchy int PU.1 lo/- eryth progenitor
pax5 suppresses alternative fates Nature (1999) 401:556 Pro B: D-J No V-DJ VpreB λ5 macrophage granulocyte osteoclasts
Lineage fate determination Binary Plastic (simplified) M-CSF fms myeloid NK Meg my HSC DC HSC B IL-7 rag B E Gr T Commitment is plastic NOT binary Transcription factors regulate suites of genes on/off So, when is a cell committed??
Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors Cobaleda et al. (2007) Nature 449:473
pax5 deletion leads to cancer
Key surface markers
Conditional deletion of pax5
Mature IgM + IgD + B cells give rise to T cells on loss of pax5
polyclonal T cells derive from oligoclonal B cells
B cells turned T cells have normal T cell function
Loss of the transcription factor Pax5 leads to dedifferentiation of committed mature B cells
Biological sig?: Reed-Sternberg cells in Hodgkin Lymphoma phenotype: rearranged BCR somatic hypermutation ~30% CD20 small proportion CD45 variable CD19 lack: CD79a/mb-1 CD79b/B29 associated signaling molecules can possess: CD15 (granulocytes) CD30 (T cells) perforin
Conventional B2 B cells: The first checkpoint Before antigen 1 2 3 4 5 6 7 8 9 10 ~50 x 10 6 produced/day After antigen 10 only 10% enter periphery Figure 11-1
Different stromal cells provide different microenvironments life in 3D Blood (1999) 93:140
The developmental microenvironment Two kinds of support 1. cell adhesion molecules ex. VCAM 2. cytokines ex. IL-7, soluble ex. SCF, membrane bound Changes during development 1. surface: CD19, CD20, CD45 2. inside: signal transducers ex. lyn, blk, fyn transcription factors ex. E2A, EBF ~Nurse Cells
Selection of productive rearrangements rag expression
Pre B cell receptor B CELL STAGE stem cell early pro-b late pro-b pre-b IgH gene config. germline D H to J H V H to DJ H VDJ H IgL gene config. germline germline germline germline Ig light chain gene has not yet rearranged So what s paired with heavy chain?
Surrogate light chains VpreB is a V-like region λ5 is a C-like region
STAGES of PRODUCTION (B2) 1. generation 2. elimination of self-reactive B cells 3. activation by antigen 4. differentiation
Elimination of self-reactive B cells H-2 d transgenics production of mature anti-k k B cells
Elimination of self-reactive B cells Clonal Deletion
Option 2: Receptor Editing
Receptor Editing rag rag can be re-upregulated light chain ONLY undergoes new VJ recombination
Option 3: Anergy µ + in anergy, IgM is retained inside cell
Elimination of self-reactive B cells in bone marrow 1. Clonal deletion (apoptosis) 2. Receptor editing 3. Anergy
What happens if Ag expressed only in periphery
clonal deletion (note: in this case, K b antigen is expressed only in liver)
Autoreactive B cell bone marrow: clonal deletion (apoptosis) receptor editing anergy periphery: clonal deletion (apoptosis) anergy
Cutting Edge of Immunology: B1 B cells mature and reside in distinct physiological locations relative to B2 secrete mostly IgM do not require IL-7 required for containment of Borrelia B2 cannot compensate for loss of B1
Relapsing Hemorrhagic Fever (Borrelia hermsii) American Museum of Natural History http://research.amnh.org/ http://www.radio.cbc.ca/programs/ideas/shows/bacteria/willy.html Pioneering work of Alugupalli et al. J. Immunol. (2003) 170:3819 & Immunity (2004) 21:379 (followed up by Haas et al. (2005) 23:7 with strep pneumoniae)
What cells clear the Borrelia infection? loss of function expt. wild-type mouse IL2Rγ (γc) knockout rag knockout
Role of T cells Bacteremia (mean ± SD) 37.9 ± 7.3 wild-type mouse 31.1 ± 15 (NS) TCRβ knockout 39.8 ± 8.6 (NS) TCRβxδ knockout
Role of B2 B cells wild-type IL-7 KO B2 B cells not required; it must be the B1 B cells (indirect proof)
It s the B1 B cells, stupid! gain of function expt. # bacteria/µl of blood (x10 3 ) rag knockout rag knockout + purified B1 B cells days post infection B1 B cells mediate clearance (gain of function study)
T-dependent vs T-independent responses Figure 11-6 isotype switching affinity maturation memory - - - + + +
B2 versus B1 B cells site of development B2 bone marrow B1 peritoneum IL-7 requirement Yes No antibody production reactivity role IgM to start, then active class switching complex proteins; multiple epitopes memory; antibodies of different functionality dominantly IgM (natural antibody) repeating polysaccharides rapid production of neutralizing antibodies
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Summary B1, B2, MZ cells distinct biological roles distinct developmental requirements different physiological locations possibly distinct precursors
Review lineage plasticity E2A, EBF, pax5 transcriptional cascade conventional (B2) B cell development surrogate light chains dealing with autoreactivity: clonal deletion, anergy, receptor editing B2 versus B1 B cells