Roslaniec Notes

Structure and Function of MHC and Antigen Presentation
Wednesday, 12/6/06, 8:30:00-9:50 AM
Ed Rosloniec- immunologist
in VA medical center. Contact information in UT directory.
3 lectures in 24 hours on immune response and steps to link innate and adaptive. 3 subjects listed in the Topics of MHC Structure and Function 1 slide are all 3 lectures.
T cell ontogeny is development and maturation of T cells.
2:Big picture (abridged)
We will focus on left. Need to understand how pathogenic antigens enter immune system, how presence is communicated to adaptive immune system, and how T cells are generated. Particulate and souble antigens are handled differently. We will focus on soluble protein antigen. We will discuss APCs, capable of grabbing pieces of pathogens and preparation for recognition by adaptive immune system. Today about T cell expression and path they follow.
Today looking at ability of APC to collect piece of pathogen and get it in context of MHC. T cell can survey, identify, mount response.
3: MHC most polymorphic locus in mammals. It has large numbers of allelic variants. Not hypervariable- not result of recombination. Human MHCs are called HLA. Mouse is H-2. Large loci with 3 classes, I,II,III. Not discussing III today. I and II have similar function despite different organization. DP, DQ, DR are class II. Class I are A, B , C.
Mouse: see slide.
within MHC- lots of other genes. They are chaperones and other genes with different function. For the most part they are involved in antigen presentation in one way or another.
4: Class I and II are heterodimeric glycoproteins. I has a single transmembrane region and a single small cytoplasmic chain.
II two of each (two transmembrane domains and two small cytoplasmic chains).
Short domains: 28 amino acids. Role in signalling limited. Primary function is to bind antigen peptide and display on the surface of the cell as a surveillance mechanism. Complex is ligand for T cell receptor.
5 and 6: Where are molecules expressed?
Class I is expressed in almost every cell.
II is expressed on Tcells, B cells, macrophages and APCs. It is also expressed on the epithelium of the thymus.
No class I or II on red blood cells.
Malaria affects red blood cells, but they cannot tell the immune system they are infected with no class I MHCs to present antigens.
7: Function of MHC molecules:
MHC is the link between innate and adaptive immunity. Regulates ability to produce a specific immune response. Ligand for T cell receptor.
8-10: Class I structure:
3 extracellular domains, 4th is beta2 microglobulin with few variants. It stabilizes molecule.
Beta-2 molecule noncovalently associates with alpha-domains. alpha- helical regions form binding cleft for peptides. All class I and II proteins look like the structure shown.
11-14: Class II:
gene organization is different, but crystal structure is almost identical to class I. No function in either chain alone. Must be paired to get to surface. DR is most represented of loci.
Ie and Dr are similar.
Class II crystal structure shows covalent linkage.
How to generate effective immune response?
specificity is interesting. Biochemical interactions tend to be highly specific. Well defined. MHC has variety, but there are rules. Each MHC can bind a wide variety of peptides. If you take one cell- isolate MHCs and kick off peptides- find at least 1000 different peptides. A single cell has associated with the same MHC molecule a large number of peptides. But there is specificity.
15: Different Class I and Class II function:
1. length peptide bound: Class I 8-10 AAs.
2. Specificity and mechanism of peptide binding: II has a different binding motif or pocket
3. where they pick up peptides
4. presentation to and stimulation of T cells
Peptide binding:
CLass I: short peptide, 8-10 AAs II: longer, extended peptide longer than 12-13 AAs, 30-
100AAs in vitro
closed ends, peptide buried into corners open ends on binding cleft
bind between int at N and C terminus of peptide much tighter definition of what it binds. Most 1,4,6,9 binding pocket motif with some variation.
to bind largermolecules- kink out molecule in Not all binding
middle pockets are always used.
too big- disrupts binding. Binding motifs- can see 1,4,6,9.
19-21:Class II:There are contacts with backbone as well as side chains of peptides. p1 has a conserved deep pocket and serves as the primary anchor position.
Binding motifs: p4-positive AAs.
Green are conserved residues. Rest have little conservation.
Peptides go into cleft in alpha helical or extended helical form.
2 crystal structures of peptides: both Y and F in p1. T cell specificity comes from the surface of the molecule from side chains and orientation presented to T cell.
23-24: Biosynthesis-
Class I is synthesized as one chain with beta-2 microglobulin associated for stability. Synthesized in ER, exit through Golgi to surface. Stabilized by calnexin, then beta-2 microglobulin. Class I also requires peptide in cleft for stability. Peptide enters ER through TAP molecules. They pump peptides into ER. Not MHC specific. Peptides come from large subunit called proteosome. Cleaves peptides into small molecules, picked up by TAP and pumped into ER, then bind if compatible to MHC.
25: Schematic diagram of TAP:
pumping is an energy dependent process . TAP has an ABC binding domain.
27: Class II totally different. It gets antigens through the endocytic process. Particulates in vesicles are brought in, the vesicles are acidified, proteases degrade, fuse with late endosomal vesicles with class II MHCs.
28-32: Details:
Class II is synthesized in ER, but stable in absence of peptide. Can grow in lab and purify. Has built in second domain. Biosynthesis- no peptides are there until late stages of biosynthesis and movement to surface of cell. Green- invariant chain Occupies peptide binding site during biosynthesis. Clip peptide in red binds all class II molecules. Shown in trimeric association. Invariant is called Type II molecule topogenically. Antiparallel interaction gives ability to bind.
Class 2 goes through stages- clip peptide is left at end. Clip binds tightly. How do we get rid of it? It gets displaced by second class II molecule with filled binding cleft. HLA-DM in mouse and human. Acts as chaperone to stabilize the molecule so clip can be released. Then molecule acquires antigenic peptides that have entered cell.
32:
Mechanisms of acquisition.
Can work for ingested bacteria in cell.
Also works for bound antigen on antibody from B cell. Extremely good antigen presenters.
33-34: Peptide binding facts:
1st point important. MHCs do not discriminate between self and pathogenic peptides. Lots of self bound.
2. Peptide with the right amino acid sequence will bind to MHC regardless of source.
3. Single MHC allele presents thousands of peptides at the same time.
Problem autoimmunity. Why don’t we generate response to all the peptides on the MHCs? Later.
MHC locus most polymorphic- multiple class I and II genes.
DR, DP, DQ: polymorphisms are significant. They are highly polymorphic, with polymorphic meaning 8-9 amino acids difference from one allele to another.
36-39: Polymorphisms have effect. Almost all surround peptide binding cleft and tend to be clustered.
Class II: polymorphisms grouped more tightly. Frequently referred to as 1,2,3,4 regions.
If you are completely heterogeneous at all alleles, express 6 different alleles for I and 6 for II. Expression of 12 different alleles is rare. Which alleles you express tends to depend on your ancestry and ethnicity.
Developing vaccines for therapeutic purposes- if alleles differ, different people will not mount a response to the same determinant. Can find peptide with broad binding specificity.
T cell receptor Restriction-
MHC complex is ligand for T cells. T cell receptor interaction with complex is highly specific and based on ability to recognize antigen peptide as well as to recognize the MHC presenting the antigen.
T cell receptor is restricted to only recognize specific MHC molecules. Develops in thymus.
DR1 with peptide:
significant portion of peptide is buried. Points of contact with T cell receptor are limited. >50% buried- usually 60-70% buried in class II. Specificity is high and limited in terms of contacts.
T cell receptor is specific for peptide only in context of MHC.
T cell receptor has to have both interactions. Interacts at an angle with the peptide and MHC.
45: Alloreactivity- unintended recognition of MHC by T cell receptor. Historically MHCs were first discovered in experiments with skin grafts in mice. Rejection led to observations of MHC. Rejection is not an intended function of MHC. Immune system has ability for T cell receptor to recognize nonself MHC class II and generate a response. Tcell from someone else- get response based on T cell receptor interacting with wrong MHC- high affinity with peptide or strong reaction with MHC withoout regard to peptide. Nonself MHC class II.
T cell receptor genes from mouse A can interact with mouse B’s MHC molecules. Plastic set of gene segments allow these molecules to interact even though they have never "seen" each other.
47: Last thing: superantigens.
Molecules with ability to put MHC and T cell receptor together and act as molecular clamp. Stimulate T cells for huge release of cytokines and chemokines. They are from bacteria and viruses. Discovered because of their role in toxic shock. The superantigen acts as a molecular vise to crosslink MHC and T cell receptor.

T Cell Development (Wed PM)
specialized process. MHC plays a pivotal role. None of this is generation of immune response- all preparing to respond to antigen.
Mostly occurs in thymus.
2: Big Picture- ability of cells to migrate and develop into T cells.
3:Class II interacts with CD4+ T cells.
Class I interacts with CD8- extra stability to increase affinity. Deciding and signalling to cell to be helpers or cytotoxic.
4-5: CD4 interacts with a small area of the beta subunit.
CD8 interacts with the alpha chain.
6-7:Where cells come from: T cells from bone marrow. Lineage diverges-see slide 6. Cells out of bone marrow- not distinct. Enter thymus and maturation starts from top. They mature as they move down pathway. Screening in thymic selection goes on as well. Identifies cells with receptors capable of interacting with CD4 and CD8 and eliminates cells that interact with self. T cell receptor has no concept of MHC. Somehow through recombination the T cell receptors become able to respond to MHC.
First screen: interact with MHC?- yes, then stimulated to differentiate and multiply. No- try again or apoptose.
Second screen: React too well? Recognize self ?yes-apoptose.no-mature into fully mature T cell.
Small numbers are released compared to what you start with.
8: Micrograph of cells in thymic stroma.
9: Australian immunology:
Importance of thymus:
scid mouse has a poor immune system, but a functioning thymus.
nude mouse- no thymus and no peripheral T cells as a result- has pre Ts, but they do not mature.
Combine- put nude mouse bone marrow into scid mouse- gets normal T cells –defect in bone marrow, not thymus.
Reverse- thymus from scid in nude mouse- T cells released. Transplanting thymus- can put it anywhere.
Adult thymus involutes, which means it shrinks over time. You have your maximum number of T cells in your teens. We produce lots of memory T cells, so it works out.
Properties- see slide 10.
After puberty- can live without the thymus if it is removed.
11: T cell life cycle in the thymus:
proliferate 7 days, develop lots of new cells, 98% die. Reasons they die: Nonfunctional receptor, untranslatable transcript, protein that does not recognize MHC, self recognition. You Produce 2 million cells per day.
Cells can be eliminated by apoptosis.
13: Stages and receptors:
14-15:: Pre-Ts emerge from bone marrow CD3,4,8 negative.
Into thymus, gammadelta and alphabeta pre-receptors form. Selection drives the cells into one developmental pathway or the other.
Cells that are double positive are CD4 and CD8 positive. Selection then occurs for that direction. Then commitment to lineage occurs and export to periphery.
Other changes at cell surface: CD25 is IL2 receptor for proliferation. Expressed just before serious proliferative phase in thymus.
17-23: delta locus is between valpha and jalpha locus.
1st you get CD25 expression and DJ rearrangement. This is on slides in series.
What receptor you get is a matter of statistical probability.
24:Important markers: RAG genes, TdT, preTalpha, signal transducers.
Allelic exclusion- once beta chain rearranges on one chromosome, shuts down other locus.
If T cell receptor doesn’t work, can go through other chromosome.
T cell can get 2 alpha chains expressed, but one will be nonproductive.
27: Gammadelta cells- less variability in T cell receptor cells, and found in interesting places like mucosal surfaces and lining reproductive tract. Not same variability. Ligands are different as well. Seem to be first line defense.
28: Irradiation-bone marrow chimeras-
F1 mouse with axb genotype- irradiate to wipe out bone marrow. Thymic epithelium is radiation resistant and expresses MHC I and II.
Irradiate type A, and give bone marrow from axb, repopulate APCs- T cell responses- only A because thymic cells select for A and are radiation resistant.
What role do the the resident epithelial cells of thymus have? Series of experiments addressed this with donors and acceptors. Periphery and thymus must match. Is this + or – selection? Giving mouse APCs of a type allows for selection of that type. Positive selection is mediated by thymic epithelial cells.
T cell receptor transgenic mouse- all T cells recognize MHCI CD8+. Receptor downregulates CD4 +T cells.
Negative selection examines response to self peptides. If self peptide is recognized, generates apoptotic signal.
Negative selection avoids producing self-reactive cells.
36:Thymus slide- orange spots are apoptotic cells. Peptide injected into thymus resulted in increased apoptosis of cells. Transgenic mouse- normal development is driven by endogenous T cell receptors.Added peptide was ligand for transgenic receptor expressed by almost every cell. Peptide is not recognized as self because of compartmentalization of +/- selection. In medulla- cells present foreign and self peptide with the injection. Part of thymus teaches what is self. Peptide is available to bone marrow derived cells- presented as self peptide and that is why apoptosis happens. Thymus epithelial cells work at top.
Bone marrow APC needed for negative selection- see slide.
skin graft not rejected- negative selection removed b responsive T cells.
38:Why are cells released at all?
39:Avidity hypothesis- avidity means movement of receptors together to act as a unit. It is the net of affinity of the individual receptors.
see slide. Weak signal is in part of thymus where the cell has few receptors anyway.
Cells go through differentiation after + selection, resulting in production of lots of receptor, so negative selection involves a much stronger signal.
Qualitative hypothesis- signalling events are different. Evidence hard to come by.
Other possibilities- thymic epithelial cells may process antigens differently.
Are all proteins from body expressed in thymus-no.
41-42: Overview slide is at end. Zap-70 and Lck are important for signalling.
Lecture 3:
T-Cell Activation and Cellular Immunity
Thursday, 12/7/06, 8:30:00-9:50 AM
2:Effector T cells are generated during immune response. Cells that have passed through selection but not been exposed to presented antigens outside the thymus are considered naive. They are released from the thymus and reside in spleen, lymph nodes, Peyer’s patches in the intestines, tonsils, etc. There they wait for signs of infection or required immune response. These T cells recirculate in the lymphatic system in a constant mechanism to be available for response.
T cells are activated by APCs: dendritic cells, macrophages, B cells.
3: Example of cell-mediated immunity:
Different types of T cells response to different pathogens. Viruses are intrracellular, so their proteins get into class I MHCs. Antigens encountered through extracellular mechanisms, including extracellular bacteria or bacteria in vesicles, generate TH1 T cell response mediated by class II complex.
Humoral- CD4 and class II activate B cell to make antibody.
4-5:Dendritic cells: most powerful and effective APC. Sit in surveillance mode. Become activated to be APC. Immature dendritic cells can be stained (green (class II)and red (lysosomal proteins)make yellow). Lysosomal proteins and classII MHC are colocalized. Sit in periphery (not lymphoid organs) until they encounter the antigen and change– separate green fluorescent signal from class II and red from lysosomal proteins are observed when the dendritic cells are activated. Surface of the dendritic cell becomes ruffled to allow increased surface area for more cells to interact so as many as possible will be activated. Dendritic cells take up by phagocytosis as much as possible. Once activated through innate receptor, they stop phagocytosis and start migration to lymphoid organs, and express class II on the cell surface. They migrate to lymph nodes- reside there- T cells scan.
6:Dendritic cells become antigen presenting machines.
7: Within lymphoid structure, where cells are found is shown on slide. Dendritic cells are in the cortex of lymph node, macrophages throughout, B cells in follicles. Antigen presenting cells may have different functions based on location, but whether this is true and to what extent is unknown.
8:T cells sample surface dendritic cell in lymph node. If there is no specific stimulation, they recirculate.
Recognize antigen- congregate around dendritic cell. A large number of T cells is necessary for proper response, but they number will be small compared to the total cohort of T cells in the body. Successful CD4 expression may be 1% of total repertoire. Small % in long run. Expansion and proliferation are important.
9: Adhesion molecules in dendritic sampling by T cells: no T cell receptor or MHC yet. T cell needs slowing down. LFAs, ICAMs, VCAMs get system started. ICAM3 and CD209 are specific to dendritic cells.
10: Once slowed down, T cell receptor samples MHC, CD4 comes in to enhance contact, interface determines whether T cell activates.
12-13: T cell activation requires 2 signals. This is a 2-signal process. Requires 2nd signalling event . One through MHC-T cell receptor, the other is co-stimulation through CD28:CD80/CD86 (B7.1,B7.2). APCs express B7.1 and B7.2.
14:Crosslink of CD28 gives costimulatory signal for proliferation and induces expression of CTLA-4 (or CD152) eventually. It binds B7 and sends negative signal to T cell to limit proliferative response. B7 only expressed by upregulation by stimulation of APC. Another layer of control.
Do not want uncontrolled proliferation of T cells. Superantigens do this, and it makes people really sick.
This is why costimulation is necessary.
Cell becomes cytotoxic- they kill other cells. No costimulation for effector function. Costimulation is only necessary for primary function.
16: Why is costimulation important?
Lack of B7 keeps cells from being injured by naive T cells. Cell encounters ligand without costimulatory signal. The cell is stimulated through T cell receptor- without signal through CD28- cell goes into nonresponsive state,and will not respond even to APC.
Get costimulation alone- no effect on T cell.
B7 must be induced on dendritic cells. Phenotype changes.
19: Macrophages- operate differently. Still have costimulatory system. Stimulated by bacterial infection. Toll-like receptor recognizes parts of bacteria, presents antigen, activates T cells.
Potential situation:
macrophage can encounter bacteria and get stimulated, but have some self peptides. Need only 10 MHCs on a cell with same peptide to stimulate in vitro. Say 20% occupied by bacterial proteins- others by self. It is possible to encounter a T cell which recognizes self peptides. Despite this. the link between bacterial infection and autoimmune response is weak. Some bacterial proteins are like ours, too- molecular mimicry could also cause autoimmune response.
20:B cells can endocytose antigen, act as affinity columns to sequester antigens, and bring them into cells through the Ig receptor. Large % MHC contain peptides. Efficient APC. Costimulatory molecule also required for induction on these cells.
21: Summary and comparison slide
Why don’t we develop autoimmunity with every bacterial infection?Epithelial cells are important to make self-reactive T cells anergic all the time. No phagocytic capacity or B7. Macrophages are within epithelium, but they have separate activity.
Lyme disease is a rickettsial disease that is poorly diagnosed. About 10% of cases progress to autoimmune arthritis due to a lack of treatment. Chronic infection yields autoimmune response.
22:T cell not activated- low affinity IL-2 receptor. Stimulate, upregulate alpha subunit, get a high affinity IL-2 subunit.
Add IL-2- get proliferation.
Class one and Cytotoxic T cell is major pathway for CD8 function.
25: CD4- Class II pathway- produces TH1 and TH2 cells. Primary means of identifying TH1 and TH2 cells is cytokines they produce.
Th2- IL-4,10, other cytokines. We cannot differentiate these cells phenotypically. No decent markers. we use what they produce.
24: Categories usually used in textbooks for TH1 and TH2 are not quite true. This slide is pretty good. Th1 can produce gamma interferon. Promotes IgG2A production (antibody) Th2 promote more IgG-1. Real differences- types of infection they combat.
Th1 predominates in autoimmunity and bact infection with intracellular aspects.
Th2 in allergy and parasitic infection. Both have uses and bad sides.
26-27: There may be crosstalk between 2 systems. CD4 T cells may help CD8 get stimulated. If you get class I and II on same cell, upregulates costimulatory molecules to encourage participation of both responses.APCs express all class I and II. Macrophages respond to gamma interferon to become bacteriocidal
Th17- appears to be separate lineage from T0 cells. TH17 cells produce IL-17. Seem to come out of Th0 lineage and play role in autoimmunity.
29: CTL function: Immunological synapse: Cytotoxic T cell has toxic granules- released to cell to kill.
30: Green is microtubules and red is cytotoxic granules.
31: SMAC-supermolecular adhesion complex. Includes T cell receptor, CD4, CD28, MHC:peptide, other proteins on slide. CTLs kill targets one at a time.
35-37: How they kill- perforin punches hole, granzymes and granulysin induce apoptosis.
In movie, dark area between peripheral and central adhesion zones is secretory zone where cytotoxic granules are delivered.
Gamma interferon causes macrophage to upregulate bacteriocidal effect.