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

1. How Cells of the Immune System “See” and Respond to Antigen Class I MHC Immunoglobulin

2. 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

3. 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 …

4. Similarity of Ig and TCRs : • The TCR has two polypeptide chains, a and b 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.

5. Ig and TCRs are similar: • The TCR has two polypeptide chains, a and b 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.

6. CDRs on both the Ig and TCR variable regions Immunoglobulin Fab, H+L(one chain) T-cell receptor, a and b chains

7. Ig and TCRs are similar: • The TCR has two polypeptide chains, a and b 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.

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

9. 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….

10. 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

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

12. 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

13. 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 molecules

14. Class I MHC proteins

15. 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)

16. 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

17. 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 T-cell help in Antibody generation

18. 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)

19. 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.

20. 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.

21. 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

22. 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….)

23. 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)

24. 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

25. 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