ImmunologyChapter 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

The Humoral Response

• Used for eliminating extracellular pathogens– produces many different antibody molecules– each specific for a certain epitope– may produce 1011 different antibodies

• In addition, the constant portion of the antibody may account for biological effector functions

• Humoral process requires participation of other cells– macrophages– B cells

– also important is the interaction between TH 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

Humoral effector functions

• Activate complement system

• Enhance phagocytosis via opsonins

• Neutralize bacterial toxins

• Neutralize viruses

• Prevent colonization at mucosal surfaces

• Involved in ADCC

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 107 B cells/day

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

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

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

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

• 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 37oC

• 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)

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

Associative (linked) recognition• This is a process where

TH 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

• The activated TH 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

• 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

Assignment

• Read Chapter 17, Hypersensitivity Reactions

• Review question 3 (pg 439)