Overview. Transcriptional cascades. Amazing aspects of lineage plasticity. Conventional (B2) B cell development

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1 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

2 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

3 Hematopoiesis myeloid lymphoid Required transcription factors PU.1 ikaros E2A EBF pax5

4 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

5 PU.1 lineage fate decisions graded expression mediates lineage fate decisions Science (2000) 288:1439

6 Transcriptional hierarchy int PU.1 lo/- eryth progenitor

7 pax5 suppresses alternative fates Nature (1999) 401:556 Pro B: D-J No V-DJ VpreB λ5 macrophage granulocyte osteoclasts

8 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??

9 Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors Cobaleda et al. (2007) Nature 449:473

10 pax5 deletion leads to cancer

11 Key surface markers

12 Conditional deletion of pax5

13 Mature IgM + IgD + B cells give rise to T cells on loss of pax5

14 polyclonal T cells derive from oligoclonal B cells

15 B cells turned T cells have normal T cell function

16 Loss of the transcription factor Pax5 leads to dedifferentiation of committed mature B cells

17 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

18 Conventional B2 B cells: The first checkpoint Before antigen ~50 x 10 6 produced/day After antigen 10 only 10% enter periphery Figure 11-1

19 Different stromal cells provide different microenvironments life in 3D Blood (1999) 93:140

20 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

21 Selection of productive rearrangements rag expression

22 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?

23 Surrogate light chains VpreB is a V-like region λ5 is a C-like region

24 STAGES of PRODUCTION (B2) 1. generation 2. elimination of self-reactive B cells 3. activation by antigen 4. differentiation

25 Elimination of self-reactive B cells H-2 d transgenics production of mature anti-k k B cells

26 Elimination of self-reactive B cells Clonal Deletion

27 Option 2: Receptor Editing

28 Receptor Editing rag rag can be re-upregulated light chain ONLY undergoes new VJ recombination

29 Option 3: Anergy µ + in anergy, IgM is retained inside cell

30 Elimination of self-reactive B cells in bone marrow 1. Clonal deletion (apoptosis) 2. Receptor editing 3. Anergy

31 What happens if Ag expressed only in periphery

32 clonal deletion (note: in this case, K b antigen is expressed only in liver)

33 Autoreactive B cell bone marrow: clonal deletion (apoptosis) receptor editing anergy periphery: clonal deletion (apoptosis) anergy

34 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

35 Relapsing Hemorrhagic Fever (Borrelia hermsii) American Museum of Natural History 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)

36 What cells clear the Borrelia infection? loss of function expt. wild-type mouse IL2Rγ (γc) knockout rag knockout

37 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

38 Role of B2 B cells wild-type IL-7 KO B2 B cells not required; it must be the B1 B cells (indirect proof)

39 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)

40 T-dependent vs T-independent responses Figure 11-6 isotype switching affinity maturation memory

41 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

42 * *

43 Summary B1, B2, MZ cells distinct biological roles distinct developmental requirements different physiological locations possibly distinct precursors

44 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