1. November 2, 2004

2. IMMUNITY ADAPTIVEINNATE CELL MEDIATEDHUMORAL ANTIBODIES EFFECTOR SYSTEMS Fc Receptors Complement RECEPTORS EFFECTORS Cells Molecules ANTIGENS TCR BCR Soluble Ab MHC B2 Cells B1 Cells

3. B1 (Ly1) B2 or conventional

4. Ly 1 (CD5) or B 1 B cells Are very long-lived and self-replenishing Develop early in ontogeny from fetal omentum and liver After birth the stems cells for B-1 are not present in the bone marrow In the adult are found predominately in the peritoneal and pleural cavities Rare in lymph node, peripheral blood and spleen Location

5. Ly 1 (CD5) or B 1 B cells Display a limited V region repertoire Lots of self-reactivity and recognition of bacterial antigens Reactivity is broad specificity, low affinity Produce large amounts of antibody; although <5% of B cells make >50% of all antibodies in serum

6. Ly 1 (CD5) or B 1 B cells Do not require T cell help Play a role in the innate immune response to infection; Abs mostly germline encoded Make antibody response to polysaccharide Ags found on bacteria (TI-2) Rapid response - make Abs within 48 hours of antigen exposure

7. IgM effective in activating complement thereby helping to remove bacteria Innate response

8. Ly 1 (CD5) or B 1 B cells Hypothesized to be primitive B cells Abundant in autoimmune disease - remember specificity is often self-reactivity In mice at birth most B cells are B-1 Most B cell leukemias are B-1 cells

9. Conventional B cell or B-2 B cells Are the principal B cells in the secondary lymphoid organs Stem cells continually generate Arise from fetal liver and bone marrow in adult Are the principal B cells in the adaptive immune response Have extensive variable region repertoire that is generated somatically Require T cell help and hence show memory

10. Ontogeny of Conventional B cells In the fetus, before there is a BM, hematopoiesis takes place in the liver Although the fetal liver shuts off around birth, it is often referred to as the source of stem cells In the adult, Ig gene rearrangement and the initial events in B cell differentiation occur in the bone marrow (BM)

11. Stem cells from the fetal liver persist for life around the periphery of the hollow bone space

12. Stem cells divide and migrate toward the large vein in the center of the bone

13. The spongy matrix of reticular cells macrophages and other supporting cells produce growth and differentiation factors that guide development

14. The adhesion molecule VLA-4 on the pro-B cells interacts with VCAM-1 on stromal cells

15. c-Kit on the pro-B cells then interacts with stem-cell factor (SCF) on the stromal cell activating c-Kit and causing the pro-B to divide and differentiate into a pre-B cell

16. Pre-B cells express the IL-7 receptor. IL-7 produced by the stromal cells drives the maturation process

18. Note there is an ordered sequence of events for H chains

19. B220 is a molecule present on B cells and marks all cells of the B lineage

20. Surrogate L chain is comprised of Vpre-B and 5 and is necessary to get expression of the pre-B cell receptor on the surface

21. The immature B cell expresses IgM on its surface

22. Mature B cells leave the bone marrow and populate the secondary lymphoid organs (spleen and lymph node) The mature B cell expresses both IgM and IgD on its surface

23. Up to this point events occur in the absence of antigen stimulation This development takes place in the bone marrow

24. Activation requires stimulation by antigen and occurs in the peripheral lymphoid tissue

25. The plasma cell is the terminally differentiated B cell and is committed to the production of large quantities of a specific antibody

28. Vector for making a knock-out mouse

29. Note that some mice will also be white if they are not from the ES cells

30. If you knock-out RAG1 or RAG2 the BCR and TCR remain unrearranged Both B cell and T cell development is arrested

31. Surface Ig is the receptor for the B cell Ig-  and Ig-  associate with membrane bound heavy chain Ig-  and Ig-  have immunoreceptor tyrosine-based activation motifs (ITAM) Activation through the BCR leads to phosphorylation of the ITAMs by Src family kinases and the recruitment and activation of Syk kinase with the signal transduced along several intracellular pathways

32. Cells lacking Ig-  or Ig-  do not have surface Ig and arrest in B cell development There is no affect on T cells

33. Before L chain rearrangement a surrogate L chain comprised of Vpre-B and 5 associates with the BCR Disruption of expression of the surrogate L chain blocks most B cell differentiation at the pre-B stage

34. Signaling through the BCR is important for establishing allelic exclusion and initiating L chain rearrangement Both require expression of the membrane form of IgM

35. Tra Transgenic mice have been used to show that expression of mIgM stops DNA rearrangement

36. Expression of a functional heavy chain on the membrane inhibits further heavy chain rearrangement Murine IgM

37. E Expression of a functional heavy chain on the surface of a B cell also initiates L chain rearrangement This was demonstrated by transfecting B cells and assaying for L chain rearrangement

41. When immature B cell encounter self-antigen within the bone marrow they die, presumably by apoptosis These mice are transgenic for an Ab specific for K k

42. Only a small fraction of the B cell produced in the bone marrow survive to exit and go to the peripheral lymphoid organs Cells which sustain non-productive Ig rearrangements and fail to express functional surface Ig are eliminated by apoptosis Immature bone marrow B cells expressing surface IgM that reacts with self-antigens are eliminated Mature B cells expressing IgM + IgD exit the bone marrow and populate the peripheral lymphoid organs Further development is antigen dependent

43. Production of cells which will grow forever and produce a single homogeneous (monoclonal) antibody These cells are called hybridomas

44. It is now possible to immortalize a B cell producing a monoclonal antibody The resulting cell lines are called hybridomas Spleen cells make antibody Myeloma cells grow forever

45. In the presence of aminopterin cells must use the salvage pathway for DNA synthesis For these experiments myeloma cells which can grow indefinitely but are deficient in either HGPRT or TK are used -if cells lack enzymes of salvage pathway they die