How Cells of the Immune System “See” and Respond to Antigen

Class I MHCImmunoglobulin

Membrane Receptors for Antigen

• B-cell surface Receptor (BCR) – anchored Immunoglobulin

• T-cell receptor (TCR)

• Major Histocompatiblility Complex (MHC)

– Class I molecules

– Class II molecules

• NK receptors

B and T Lymphocytes

• B-cells make Immunoglobulin – both secreted and on the surface

• T-cells are lymphocytes that play a key role in the humoral (Ig) immune response, and also carry out their own recognition functions (cytotoxic lymphocytes)

• T-cells have receptors that are similar to Ig molecules in several respects …

Similarity of Ig and TCRs :• The TCR has two polypeptide chains, and that

together form the antigen-combining site (just as Ig has the H and L chains)

• The TCR chains each have two domains, a C and V, with folded structure that is similar to the C and V domains in the Fab arms of Ig.

Ig and TCRs are similar:• The TCR has two polypeptide chains, and that

together form the antigen-combining site (just as Ig has the H and L chains)

• The TCR chains each have two domains, a C and V, with folded structure that is similar to the C and V domains in the Fab arms of Ig.

• The antigen-combining region of the TCR are made up of 6 CDRs, three from each chain.

Immunoglobulin Fab, H+L(one chain) T-cell receptor,

and chains

CDRs on both the Ig and TCR variable regions

Ig and TCRs are similar:• The TCR has two polypeptide chains, and that together

form the antigen-combining site (just as Ig has the H and L chains)

• The TCR chains each have two domains, a C and V, with folded structure that is similar to the C and V domains in the Fab arms of Ig.

• The antigen-combining region of the TCR are made up of 6 CDRs, three from each chain.

• The high degree of variability in the variable regions (and CDRs) is mainly due to recombination in the DNA of developing T-cells in which one of several V, J and (in the b subunit) D regions are joined to create millions of different possible combinations.

Gene rearrangements in different combinations of V, D and J segments provide for a huge number of

different TCRs

Although there are these interesting similarities between the B-cell receptor (Ig) and the TCR, there is one really key difference:

While the B-cell receptor can recognize antigen alone, T-cell receptors recognize and antigen only when it is in the context of a Class I or Class II MHC molecule.

What is a MHC molecule, you ask? Well….

The Major Histocompatiblity Complex, or MHC

• BCRs (surface Igs) and TCRs both recognize a variety of chemical structures, but B-cells (as with the Igs they secrete) can “see” antigens in isolation

• T cells recognize antigens when they are associated with normal cells -- they recognize “foreign” in the context of “self”

• The “self” they see are molecules of the Major Histocompatilbity Complex, or MHC

• They are called “histocompatiblity” antigens because they were first recognized as provoking graft rejection, because they seen as “not self” by T-cells, and an immune response is elicited

• If you can find an individual with a similar (or better yet, identical) set of MHC antigens, they will be seen as “self” and not rejected

• Class I molecules are expressed in nearly every cell type in the body

– The TCRs of Cytotoxic lymphocytes see viral and tumor antigens when they show up on normal cells: foreign Ags “ in the context of Class I molecules “

• Class II molecules are expressed only on cells of the immune system, mostly macrophages and B cells

– When these cells take in Ag, some of it is put back out on the cell surface with a Class II molecule – a way of focusing attention on this unusual bit, and encouraging an immune response.

There are two kinds of MHC Molecule: Class I and Class II

Class I Class II

Both types of molecules have two polypeptides, and a domain structure similar to that of Ig and TCR, especially

in those domains that are closest to the membrane

Class I MHC molecules• Expressed on nearly all cells of the animal, though are more

abundant on some cells than others

• In humans there are three main genes, located on Chromosome 6, called HLA-A, HLA-B and HLA-C (HLA stands for Human Leukocyte Antigen – they were originally called antigens because animals make antibodies against MHC molecules from different individual)

• These genes are polymorphic, meaning that are many different alleles (or versions) of each gene in the human population.

Class I MHC proteins

The TCRs of Cytotoxic lymphocytes see viral and tumor antigens when they show up on normal cells: foreign Ags “in the context of Class I molecule” (CD8 is also involved)

Class II MHC molecules

• Expressed on cells of the immune system, mainly macrophages and B-cells

• In humans there are three main genes, located on Chromosome 6, called HLA-D – there are many genes, each with several alleles

• These are also important to match in tissue transplantation, especially if lymphocytes are transplanted.

• MHC Class II molecules are important in achieving good Antibody responses to antigen

T-cell help in Antibody generation

• Foreign antigens are engulfed by macrophages, non-specifically

• The macrophage partially degrade the antigen, and pieces are bound to Class II molecules in the internal membrane system of the cell

• These are put out on the surface: Antigen is presented in the context of Class II MHC molecules

• The TCR on CD4+ T cells can now “see the foreign antigen – it is in the context of the Class II molecule (which CD4 helps to bind)

• The T-cell is activated….

Class II molecules are important in the humoral immune response because they present antigen to T cells that enhance the immune response

The interaction stimulates both the T-cell and the macrophage:

• T-cell – Stimulated to secrete IL-2

• stimulates the T-cell to proliferate -- expansion of clones that can see this antigen

• Macrophage – stimulated to release IL-1

• further stimulates the T-cell• recruits other immune cells (inflammatory response)

Antigen can also be presented by B-cells

• Antigen can be bound to the Ig (BCR) on the surface of ‘virgin” B cells

• Binding causes endocytosis, and “processing” (partial digestion) of the antigen.

• Fragments of antigen bound to Class II molecules are put out to the surface

• Antigen + Class II engages T-cells

• The T-cell clones that can see this antigen are expanded, and secrete cytokines (IL-4 and IL-5) that stimulate the B-cells to proliferate and differentiate into plasma cells.

Besides being just interesting, this explains why some molecules can be good antigens and yet not be immunogenic …

• To elicit a good Ab response, a molecule must be able to be presented to T-cells

• If it is not big enough to bind to the Class II molecule and the T-cell receptor at the same time, it may fail to recruit T-cell help

• The B-cells that will recognize it may be activated, but without the help, the response will be poor.

• Small molecules, including peptides, need to be attached to carrier molecules in order to elicit a good responses.

Practical Issues in Ab Production (some may make more sense

now that we have looked at T-cell help)

• Purity and Integrity of Antigen

• Form of antigen

• Animals

• Adjuvant

• Route of injection, dose etc

Antigen• How pure should it be? …

– For polyclonals, purified is best.

– If you can “clean it up” in the serum, you do not need to start with a pure Ag

– Antigen-affinity column– Adsorb “out” other antibodies

– For monoclonals it is less of an issue (more unwanted clones, but if you can screen them out….)

Antigen, other considerations

• Native or denatured antigen (SDS-PAGE gel slices) – but if denatured, you may not get as many “native” epitopes

• Peptides

– Need carrier protein (T-cell help).

– Abs raised to peptides may not recognize native molecule well Depends on how much information you have

• Particulate Ags tend to work well because they are easily phagocytized (for Ag presentation)

Animals• Species considerations:

– Size/cost

– Phylogenetic distance (not good to use mouse for Abs to a mouse Ab. Chickens are sometimes used for this reason.

– Useful Ig properties, such as IgY (birds) or Ig (llamas)

– Monoclonals are usually mouse, unless phylogenetic distance is needed. (Also mice are more limited in the varriety of Class II molecules they have b/c they are inbred, but outbred strains are available )

• Several animals are generally done to provide more heterogeneity in Class II options

• (empirically animals differ in their response, so do >1

Injections• Adjuvant

– An emulsion of mineral oil/water (Freund’s) that will hold the Ag in place long enough for macrophages to get there

– May contain an irritant to cause local inflammation (“Complete” adjuvant) to recruit cells of immune response

• Route of injection, does per injection and period of time between boosts

– depend on the animal, whether adjuvant is used, the nature of the antigen, and experience (empirical) … people base their practices on history