Chapter 3

1. Chapter 3 Antigen Capture and Presentation to Lymphocytes (What Lymphocytes See)

2. Nature of Antigens • B- and T-cells recognize different types of Ag • B-cells have a membrane bound Ab that recognize a wide variety of macromolecules (sugars, lipids, proteins, nucleic acid), small chemicals in soluble or cell membrane associated forms – humoral response • T-cells see only peptide fragments and only when displayed in special display molecules on the host cell • Chapter 4 = Receptors

3. Barriers to Adaptive Immunity • Very low frequency of naive lymphocytes with specificity for any 1 Ag, less than 1 in 100,000, must recognize and activate when Ag is present • Different microbes need different adaptive immune responses • virus in the blood is B cell response • virus in a cell is a T cell response

4. 2 Questions • How do naive lymphocytes find the microbes? • How does immune system produce the response best to eradicate microbe? • Highly specialized system of capture and display of Ag to lymphocytes • understand T-cell Ag presentation better but not a lot about B-cell Ag presentation

5. T-cell Ag Recognition • Majority of T-cells recognize peptides bound to and displayed by MHC on APC • MHC (major histocompatibility complex) is a genetic locus responsible for peptide display molecules of the immune system • T-cell receptor must recognize Ag and MHC molecule, • small subset may recognize lipid and non-protein Ag on uncommon class I MHC-like molecules (non-polymorphic) – poorly understood • Display of Ag is done by the APC that help naive T-cells form the immune response • professional APC will display Ag and can also provide extra signals to activate T-cells • Differentiated effector T cells must see Ag on APC

6. MHC and TRC Interaction • T-cell has dual specificity • recognizes residues of peptide Ag and residues of MHC molecule presenting the Ag • MHC restriction – different T cells can see peptides on MHC either class I or II

7. Capture of Ag • Ag is concentrated by APC in peripheral lymphoid organs where the immune response is initiated • T-cell rich areas • Microbes enter by contact (skin), ingestion (GI tract) and inhalation (respiratory); occasionally injected by insects into the blood stream • all are lined with epithelium – physical barrier that contains professional APC

8. Capture and Display ***

9. APC – Dendritic Cells • Present in the epithelial barrier and in lymphoid tissue • Langerhans cells in skin are immature and inefficient at stimulating T-cells • Capture by phagocytosis (particulate) or pinocytosis (soluble) using receptors for microbe Ag such as terminal mannose residues • May also initiate innate system by binding to the TLR resulting in production of TNF and IL-1 • cause changes in dendritic cells

10. Dendritic Cell Migration • Activated dendritic cells lose adhesiveness and make CCR7 surface receptor that will recognize chemokines that cause them to move into the lymph nodes • respond to TNF and IL-1 • similar to what happens to T-cells that re-circulate thru lymph node daily • Maturation into APC happens during igration • increased synthesis and stable expression of MHC on surface to display Ag to T-cells

11. Maturation

12. APC’s Functions • Dendritic cells are most potent APC • influence response of T-cell and nature oF response – subsets of dendritic cells can activate CD4+ T-cell into distinct populations (Chapter 5) • Macrophage are APC and prevalent in all tissues • in cell-mediated responses macrophage phagocytose microbes and display Ag to effector T-cell – activating macrophage to kill microbes (Chapter 6) • B-cells ingest protein Ag and display them to helper T-cell in lymphoid tissue • important for humoral immunity (Chapter 7)

13. CD8+ T-cells Activation • All nucleated cells can present Ag to CTLs • Dendritic cells initiate response of CD8+ T-cells to Ag of intracellular microbes • viruses can rapidly infect cells and only be eradicated by CTLs – CD8+ T-cells must be able to respond to Ag of intracellular microbes • may infect other cells that may not produce all the signals to activate T-cells – how do we clear infection??

14. Cross-Presentation or Priming • Mechanism unknown but likely dendritic cells ingest infected cells and display Ag for recognition by CD8+ cells • may also present Ag to CD4+ T-helper cells as CD8+ may require helper T-cell (Chapter 6) • Primed CTL can kill infected cells without APC present

15. MHC Molecules • Membrane proteins on APC that present peptide Ag to T-cells • major determinant of tissue graft acceptance or rejection – identical MHC locus can accept grafts (identical twins or inbred animals) but if locus isn’t same, will lead to rejection • reason for MHC restriction of T-cells • MHC is a genetic locus – a collection of genes found in all mammals which includes genes for MHC and other proteins • Human leukocyte antigens (HLA) found on WBC • 2 sets of highly polymorphic genes that create MCH class I and II to display Ag to T-cell • polymorphic – 2 or more alternative forms, stable frequency, 1 allele from each parent • also have non-polymorphic genes that function in Ag presentation and some with unknown function

16. MHC Locus

17. MHC I / MHC II • Contain peptide binding site at the amino terminus – different subunits but overall structure is similar

18. MHC I – CD8+T-cell •  chain is non-covalently linked to 2 microglobulin (gene outside the MHC locus) • 1 and 2 form the peptide binding cleft or groove to hold a peptide 8-11 AA long • sides and top of MHC contact the T-cell receptor and includes the peptide in the cleft • polymorphic genes encode different residues in cleft so can bind different peptides, additional changes made so MHC can recognize different T-cells • 3 region is invariant and has the binding site for the co-receptor – CD8 • T-cell activation – MHC associated peptide Ag by T-cell receptor and simultaneous recognition of MHC by the co-receptor (Chapter 5)

19. MHC II – CD4+ T-cell • 2 chains  and  • Amino terminus is made up of 1 and 1 domains • polymorphic and form a cleft that is large enough for 10-30 AA residues; also recognized by CD4+ cell • 2 domain binds co-receptor for CD4 • non-polymorphic

20. Must Remember • CD4+ T-cells recognizes Ag on MHC class II becoming a T helper cell • CD8+ T-cells recognizes Ag on MHC class I becoming a CTL cell • Also must have the appropriate co-receptors

21. MHC Properties and Functions

22. MHC Inheritance • Genes are co-dominant – inherited alleles from both parents and are expressed equally • 3 genes for MHC class I • HLA-A, HLA-B and HLA-C are one set of genes so cell can express 6 different MHC I molecules • 3 pairs of genes for MHC class II • HLA-DR, HLA-DQ and HLA-DP make up the  and  chain and both are polymorphic • MHC genes are highly polymorphic – many different alleles present among different individuals in the population • no 2 individuals are alike (except twins and inbreds) • existence of multiple alleles allows for any MHC molecule to present any peptide – helps prevent new or mutated microbes from causing problems • Not gene recombination as seen in B cell receptors (Chapter 4)

23. Location of MHC • MHC class I is on all nucleated cells • MHC class II on professional APC (dendritic cells, macrophages and B cells)

24. Peptide Binding Clefts • Side chains of the AA in the peptide fit in the pockets formed in the cleft and anchor the peptide by anchor residues • Some residues are pushed upward and are recognized by the T-cell receptor

25. Features of Peptide Binding Must know these things

26. Peptide Binding • 1 MHC molecule can display 1 peptide at a time but each MHC can display many different peptides • dependent on anchoring residues fitting cleft • “broad” specificity – bind many but not all peptides • few MHC to do many peptides • rarely binds anything but protein Ag • peptide to MHC is a low affinity interaction (stay bound in decrease concentration of Ag) • slow peptide off rate • display peptide a long time to increase chance of finding the right T-cell receptor

27. Acquiring Peptides • Acquire peptide during biosynthesis of MHC inside the cell – microbe Ag from inside the host cell and T-cell recognize cell associated microbes • MHC class I – cytosolic proteins • MHC class II – proteins in intracellular vesicles • Only loaded MHC molecules are stably expressed on surface – empty molecules are destroyed before the leaving the cell • display only useful MHC molecules

28. Self Recognition • MHC molecules can display foreign and self Ag so why don’t we develop self Ag activated immunity? • T-cell may need to see only ~0.1 to 1% of its Ag on the 105 MHC molecules on APC • T-cells that recognize self-Ag are either killed or inactivated (Chapter 9) • MHC can’t display whole Ag but only peptides – Ag processing

29. Processing Pathways • MHC II – extracellular proteins that come into the APC and are in vesicles • MHC I – proteins in cytosol of nucleated cells such as viral proteins, microbial proteins

30. Different Outcomes • Require different cellular organelles and proteins • Sample all extra- and intra-cellular proteins • Activate different classes of T-cell by different pathways due to separation of processing

31. Loading of MHC II • Endosomal peptides – proteins enter intracellular vesicles and may fuse with lysosome, proteins broken down by proteolytic enzymes to peptides • APC synthesize MHC II in ER and each MHC II has a sequence called class II invariant chain peptide (CLIP) attached to the invariant chain which binds tightly to the cleft • MHC II/CLIP complex begins way to cell surface in an exocytic vesicle which fuses with endosomal vesicle of peptides, also contains DM protein that removes CLIP so peptide can enter into cleft • if no peptide picked up, MHC II is degraded in endosome • MHC II/peptide complex is now stable and can move to the surface • Only the immunodominant epitopes of Ag enter MHC II – maybe 1-2 peptides

32. Loading of MHC I • Cytosolic peptides – proteins from cytoplasmic viruses and from phagocytosed microbes that may break thru the vesicle and escape to the cytoplasm • proteolysis – ubiquitin binds to protein and leads to proteosomes for digestion • some cleaved peptides are small enough to fit into MHC I • MHC I made in the ER while peptides are in cytoplasm – requires special transport proteins called TAP (transporters associated with Ag presentation) in the ER membrane • TAP pumps peptides into ER so can get on MHC I – loose association between TAP and MHC on inner surface of ER • right fit of peptide and MHC will stabilize the complex and move it to the cytoplasm • no peptide leads to MHC degradation

33. No Cross-Loading Peptides • Even though MHC I and MHC II are in ER, won’t cross load peptides because CLIP is in MHC II and not vacated until reaches the endosome

34. Viral Avoidance • Some viruses can avoid the Ag presentation by preventing MHC I pathway • remove MHC I from the ER • inhibit transcription of MHC I gene • block peptide transport function of TAP • Decrease CD8+ T-cell activation because no MHC I • Counterbalanced by NK cells to recognize virally infected cells – innate response, recognizes decrease in MHC I on surface (Chapter 6)

35. Physiological Significance • T-cell recognition of MHC-association peptide is that T-cells will see and respond only to cell-associated Ag • MHC can be loaded inside cells so can only recognize the Ag of phagocytosed and intracellular microbes  combated by T-cell mediated immunity • MHC I and MHC II pathways of Ag presentation so can handle Ag in best possible way • extracellular microbes are captured by APC including B-cells and macrophages – presented by MHC II – CD4+ T cells (help produce Ab by B cells and make macrophages better at microbe destruction) • cytosolic Ag displayed by MHC I on nucleated cells – associated peptides are recognized by CD8+ cells that differentiate into CTLs to kill infected cells and eradicate infection

37. Other APC Functions • Present 2nd signal for T-cell activation • insure adaptive response is to microbe and not self proteins (Chapters 5 and 7) • Different types of microbial products and innate immune response may activate APC to produce 2nd signal – response to LPS from bacteria • express co-stimulators recognized by T-cell receptors • APC will secrete cytokines recognized by T cell receptor • activates T-cell proliferation and differentiation

38. Ag Recognition by B-cells • B-cells use membrane bound antibodies to recognize many Ag, soluble or microbial surface – DOES NOT REQUIRE PROCESSING • B-cells differentiate in response to Ag and other signals to secrete Ab (Chapter 7) • secreted Ab bind to Ag and lead to neutralization and elimination • Not understood how Ag finds that appropriate B-cell • Lymphoid follicles of LN and spleen contain a population of cells called follicular dendritic cells (FDC) that use Fc receptor to bind Ag that are coated Ab and receptor for C3d complement protein to bind Ag with attached complement (Chapter 7)

39. Summary • MHC class I on all nucleated cells and recognize cytosolic proteins – CD8+ T cell - CTL • MHC class II expressed mainly on professional APCs (dendritic cells, macrophages and B-cells) and recognize vesicular proteins – CD4+ T cell – T helper cell