1. Immunology Chapter 16, Lecture 2 Richard L. Myers, Ph.D. Department of Biology Southwest Missouri State Temple Hall 227 Telephone: 417-836-5307 Email: rlm967f@mail.smsu.edu

2. The Humoral Response Used for eliminating extracellular pathogens –produces many different antibody molecules –each specific for a certain epitope –may produce 10 11 different antibodies In addition, the constant portion of the antibody may account for biological effector functions

3. Humoral process requires participation of other cells –macrophages –B cells –also important is the interaction between T H and antigen-class II MHC complex B cells are the principle cell in humoral immunity –they interact with antigen via a BCR –proceeds with receptor-mediated endocytosis unlike macrophage which phagocytizes anything –then antigen presented with a class II MHC on the membrane

4. Humoral effector functions Activate complement system Enhance phagocytosis via opsonins Neutralize bacterial toxins Neutralize viruses Prevent colonization at mucosal surfaces Involved in ADCC

5. Basic facts Immunocompetent B cells possess IgM and IgD membrane bound antibodies Clonal proliferation and differentiation occur after activation B cells have average cell cycle of 15 hr Unless activated by antigen, they will die in a few days (usually 90% will die) Marrow produces about 10 7 B cells/day

6. General response to antigen The response is characterized by the 1) production of antibody-secreting cells and 2) memory B cells –during the lag phase cells undergo clonal selection –then the logarithmic phase occurs increase in antibody; it eventually declines –for example, with SRBCs, lag phase lasts 4 days; peak plasma cell levels within 5 days; peak antibody within 7 days –IgM secreted initially, followed by IgG Referred to as the primary response

7. Primary response with formation of antibodies differs depending upon –nature of the antigen –route of antigen administration –presence of adjuvants –species or strain

8. Plasma cell Secondary response different from primary –response is more rapid –produces more antibody –lasts for a longer time maybe 1,000 times more antibody produced Secondary response occurs with second exposure to the antigen Depends upon the existence of memory B cells and memory T cells

10. Hemolytic plaque assay Assay to measure plasma cell numbers in mice primed with SRBCs –many modifications Assay can be used to quantitate plasma cells secreting antibodies specific for any antigen First, immunize mice with SRBCs

11. Prepare a spleen cell suspension from a primed mouse Mix in warm, melted agar to which SRBCs have been added Prepare a petri dish with a layer of hard agar Overlay with mixture above Allow to cool and solidify Incubate for 1 hr at 37 o C

13. During incubation, antibodies diffuse into agar and binds to the SRBC Guinea pig serum containing complement is added Complement reacts with the bound antibody –mediates lysis Lysis is indicated by a plasma cell surrounded by a clear plaque devoid of cells Plaques can be counted –referred to as direct plaque- forming cells (PFC)

14. Elispot assay Plasma cells quantitated without SRBCs Use antigen-primed splenocytes Plate in agar containing antigen Plasma cells secrete antibody which binds to the antigen Remove cells Visualize bound antibody with ELISA

15. Associative (linked) recognition This is a process where T H and B cells must see peptides on the same molecule for B cell activation to occur In the following example the epitope is a viral coat (spike) protein T cells recognize internal protein which allows B cells to make antibody to coat protein

18. The activated T H cell recognizes the processed peptide together with the class II MHC molecule Antibodies can then be produced to the peptide Binding of antibody to virus occurs There is also localized release of cytokines Cytokines allow B cell to proliferate/differentiate

19. There are other membrane receptors involved –LFA-1 and CD4 are involved in cellular adhesion Once in contact a signal generates the expression of CD40L on the T cell This interacts with CD40 on the B cell membrane This causes induction of cytokine receptors Results in fully activated B cells –these can proliferate

21. Assignment Read Chapter 17, Hypersensitivity Reactions Review question 3 (pg 439)