Lecture 5 immunology

Humoral mediated immunity

• Humoral immunity refers to antibody production, and all the accessory processes that accompany it: – Th2 activation – cytokine production, – isotype switching, – affinity maturation – memory cell generation.

It also refers to the effector functions of antibody, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

Characteristics of the humoral immune response

• Antibodies secreted by terminally differentiated B cells following contact with an exogenous antigen constitute the humoral immune response.

• The response is characterized by distinct phases following exposure to antigen. – A lag phase immediately follows antigen exposure.

During this phase, B cells undergoclonal selection and differentiate into plasma cells.

– A logarithmic increase in levels of antigen-specific antibodies in the serum, which peak, plateau and

– Decline .

B cell activation• B cells need two signals to initiate activation • Most antigens are T-dependent, meaning T cell help is

required for maximal antibody production. • With a T-dependent antigen, the first signal comes from

antigen cross linking BCR and the second from the Th2 cell.

• T dependent antigens contain protein so that peptides can be presented on B cell Class II MHC to Th2 cells, which then provide co-stimulation to trigger B cell proliferation and differentiation into plasma cells.

• Isotype switching to IgG, IgA, and IgE and memory cell generation occur in response to T-dependent antigens.

• B cell activation is initiated by the cross-linking of BCR.

• Antigen must have at least two identical epitopes situated so that they can cross-link B cell surface Ig to activate B cells.

• Once antigen is bound, it is internalized with its BCR; antigen can then be degraded (processed), combined with Class II MHC, and presented to effector Th2 cells, which must bind specific antigen in order to provide help.

• Effector Th2 cells bind B cells via Cell Adhession Molecules (CAMs) and then specific peptide on Class II MHC with TCR.

• T cell CD40 ligand (CD40L) binds B cell CD40 and sends the co-stimulatory signal for B cell activation.

• The Th2 cell reorganizes its membrane molecules and golgi to concentrate CD40L binding and cytokine secretion towards the specific B cell to maintain the antigen-specificity of the response.

• Cytokines, predominantly IL-4, IL-5, and IL-6, signal the B cell to divide and differentiate into antibody-secreting plasma cells.

• In a primary immune response, IgM is secreted before isotype switching occurs and is the predominant isotype produced.

• Th cytokines direct isotype switching in activated B cells. Th1 cells induce isotype switching to certain isotypes.

• Th1 cells secrete mainly IL-2 and IFNg, while Th2 produce mainly IL-4, IL-5, and IL-6.

• IL-2 stimulates production of J chain. • IFNg stimulates switching to IgG2a. • IL-4 stimulates switching to IgE or IgG1,• IL-5 stimulates switching to IgA. • IL-6 promotes antibody secretion.• Each antibody isotype has specific effector

functions in humoral immunity.

Isotype switch• Isotype switching requires DNA recombination. It increases the

functional diversity of Ig molecules but does not affect their antigen-binding specificities.

• Human gene segments for CH are arranged linearly in the order Cm, Cd, Cg3, Cg1, yCe, Ca1, Cg2, Cg4, Ce, and Ca2. Each C gene segment, except Cd, is preceded by an intron containing a switch region sequence. This sequence is different from the recombination signal sequences found flanking the V region segments, and the recombinases mediating isotype switching are not encoded by RAG-1 and RAG-2.

• Rearranged VDJH is always expressed first with membrane Cm in the developing B cell, with both membrane Cm and Cd in the mature B cell, and with secreted Cm as the B cell begins responding to antigen. When the B cell receives the proper signals from antigen and cytokines to switch to IgG1 production, for example, recombination occurs between the switch regions Sm and Sg1, looping out the intervening DNA, including the coding sequences for m, d and g3 heavy chains.

• Isotype switching, affinity maturation, and generation of memory B cells occur late in a primary immune response. Antigen-binding specificity can be altered by somatic hypermutation, which occurs in rapidly dividing B cells in the germinal centers. Mutation occurs preferentially in the CDR (hypervariable regions) of H and L chains at a rate high enough that about half of B cells undergo mutation of their Ig antigen-binding regions.

• Affinity maturation occurs by clonal selection as antigen levels drop due to antigen clearance, resulting in survival of B cells with the highest antigen-binding affinity.

• Further Th2 cell interaction with these B cells signals them to become plasma cells or memory cells.

Plasma cells

• Plasma cells are antibody-producing cells that no longer divide or respond to antigen.

• They are larger than B cells and have more ribosomes, endoplasmic reticulum, and golgi, but no membrane Ig.

• Some survive only a few weeks, while others continue to produce antibody for longer periods, providing rapid protection against re-infection.

• Other B cells become memory cells, which can be rapidly re-stimulated by antigen and are present in higher frequency than the naïve resting B cells (more of the latter continue to be produced by the bone marrow).

• Memory B cells are functionally and physically distinguishable from naïve B cells. They often have membrane IgG, IgA, or IgE and higher levels of ICAM-1 and CR then naïve B cells, and are thought to live longer..

Isotype Distribution and Function

• IgM is secreted first in a primary response. No somatic hypermutation has yet occurred, so it is low affinity antibody.

• IgM avidity is high, however, because it is a pentamer, and IgM fixes complement very efficiently to promote inflammation and pathogen lysis.

• Because IgM is so large, it cannot enter the tissues very efficiently; but it is effective in controlling pathogens in the circulation

• Once isotype switching occurs, IgG predominates in serum and in tissues.

• IgG both neutralizes pathogens and their toxins and opsonizes them for phagocytosis by neutrophils and macrophages.

• IgG can also activate complement on the pathogen surface once concentrations are high enough for two IgG molecules to bind nearby epitopes.

• IgA is the predominant antibody that is secreted across epithelial cells of the respiratory, digestive and genital tracts to block pathogen entry into the body.

• IgE binds FceR on mast cells lining the blood vessels throughout the body. When pathogen binds to the mast cell IgE, the mast cells immediately release inflammatory mediators that trigger coughing, sneezing or vomiting to expel pathogens from the body.

• The selective transport of various Ig isotypes to particular regions of the body occurs because of isotype-specific Fc receptors on different tissues.

• Dimeric Ig A (and, to a lesser extent, pentameric IgM) bind to the poly Ig receptor on the body side of epitheilial cells in the intestines, respiratory tract, tear and salivary glands, and lactating mammary gland.

• The antibody-poly Ig receptor complex is endocytosed into the epithelial cell and travels in an endocytic vesicle across the cytoplasm (transcytosis) to be secreted on the outer surface of the epithelium (into the intestine or lung surface or tears, saliva, or milk).

• Maternal IgA in milk can neutralize pathogen in the infant's digestive tract until the infant's immune system is mature enough to take over that task.

• IgG which has crossed the placenta into the fetal circulation offers additional protection during the first few months of life.

FcR+ accessory cells

• In order to act as opsonins or to activate cells, antigen-bound antibodies bind to Fc receptors. Several different FcR have been identified on the membranes of granulocytes, dendritic cells, B cells, NK cells, and mast cells

• Some are there constitutively, while expression of others is induced during an immune response.

• Free antibody (unbound to antigen) binds to FcR with a very low affinity except in the case of IgE.

• Antibody aggregated by antigen, with several nearby Fc regions, binds much more tightly to FcR.

• Binding of ligand to FcR acts through signal transduction complexes to alter gene expression

• Bactericidal agents released in response to FcR binding include oxygen radicals and peroxides, nitric oxide, defensins, and lysozyme.

• Respiratory burst is the process by which phagocytes generate the toxic oxygen compounds that inactivate key microbial enzymes and structural proteins by oxidizing them.

Mechanisms of antibody action

Neutralization – covering up the toxic portions of bacteria viruses, or toxins

Agglutination – clumping of cells Precipitation – forming insoluble

substances (small molecules) Complement fixation – causes lysis

(rupture of cell membranes)

Agglutination• The clumping of cells such as bacteria or

red blood cells in the presence of an antibody.

• The antibody or other molecule binds multiple particles and joins them, creating a large complex.

Clonal selection theory– Clonal selection is basic operating principle of

adaptive immune response• Newly generated B cells have single type of

receptor with unique binding specificity generated pretty much at random

• Antigen binding triggers proliferation (clonal expansion) and further differentiation

• Cells produced by clonal expansion have same antigen specificity (except for somatic hypermutation, which generally increases affinity)

• Any self-reactive antibodies are eliminated