Lecture 2 – Intro to Immunology
· Angiten: chemical substances that elicit a specific immune response
· Components of Innate Sys: phagocytes, NK cells, serum proteins (complement, CRP, MBP)
· Innate System recognizes well-conserved structures s/a LPS, PepG, dsRNA, etc
· Adaptive Sys made up of T cells and B cells. These cells express “clonally restricted, antigen-specific cell surface receptors”. (TcR and BcR). These receptors recognize antigen and activate their respective cells.
· While both B and T lymphocytes have Ag receptors, the mechanism by which they recognize Ag differs. BcR is similar in structure to antibody (Ig) and can recognize any chemical structure that is floating around. TcR requires Ag to be presented by MHC located on APCs.
· “Naïve” Lymphocyte: lymphocyte which has never seen Ag. (As opposed to memory lymphocyte)
· Cardinal features of adaptive response: specificity, diversity, memory. Memory = the 2nd time a T/B sees Ag, its response is both faster and stronger. Due to 1: clonal expansion during initial Ag exposure & 2)differences in functional responses between naïve lymphocytes and memory lymphocytes.
· Adaptive System is divided into humoral and cell-mediated immunity.
· Humoral: mediated by antibody (the secreted product of B cells). Largely directed against extracellular pathogens (eg bact, parasites)
· Cell-Mediated: mediated by T cells either directly or through action of secreted cytokines. Largely directed against intracellular pathogens (some bact, viruses). There are 2 types of T cells: helper T cells (TH, CD4+) and cytotoxic T cells (CTLs, CD8+). In response to antigen, CD4s secrete cytokines, while CD8s kill the cells directly.
· Primary Lymphoid Organs: where leukocytes develop and mature. For all leukocytes except T cells, this is the bone marrow. For T cells it is the thymus.
· FC: What are the secondary lymphoid organs? Ans: Lymph nodes, spleen, mucosal immune system, cutaneous immune system. It is to these secondary lymphoid organs that the naïve B and T lymphocytes home once they have matured. It is in these secondary lymphoid organs that the B and T initially encounter Ag and where the immune responses are generated.
· All secondary lymphoid organs are highly organized into specialized B and T areas. B areas are called follicles while T areas are known as interfollicular zones.
· T cells traffic through the secondary lymphoid organs using the blood and lymphatic system.
· Adjuvant: A vehicle used to enhance antigenicity. Generally speaking, adjuvants activate macrophages and dendritic cells in the tissues activating them to present Ag to T cells in the secondary lymphoid organs. Most microbes contains one or more natural adjuvants.
· Lymphocyte Recirculation: Lymphocytes, particularly naïve Ts, constantly recirculate through the secondary lymphoid organs. Naïve lymphocytes leave the blood through HEV and enter the T cell area. (B cells migrate to the follicle). If the lymphocytes don’t encounter Ag, they leave through the efferent lymphatics. They then either go to other lymph nodes via afferent lymphatics, or reenter circulation via the thoracic duct. Each T cell hits one lymph node each day (estimate). Ie: A given specific T cell does not remain in its one lymph node forever. It moves around to other lymph nodes too.
Lecture 3 – Antibody and TcR
· Assembly of Antibody: The variable region of IgH is made up of V,D,J gene segments. Once the VDJ region is formed, it is spliced together to the constant region to form a mature IgH. A similar process takes place with IgL, but there are only V and J (no D) gene segments.
· Somatic Gene Rearrangement: Antibody molecules are assembled from a large number of individual gene segments. This process is known as somatic gene rearrangement. It occurs during B development in the bone marrow. Gene segments are known as V, D, J.
· During the course of an immune response (c/o!!) antibody molecules undergo changes in their variable regions to increase affinity, and in their constant regions to change effector functions.
· TcR: Similar to antibody. a chain is analogous to IgL (a looks like L) while b chain is analogous to IgH (b looks like ‘H’). Like Ig molecules, the TcR is assembled from numerous gene segments by somatic gene rearrangement. b chain has V,D,J while a chain has only V and J.
· CDR1 and CDR2 are primarily responsible for interacting with MHC while CDR3 is responsible for interacting with peptide antigen.
· Unlike Ig, TcR does not undergo structural changes during the immune response.
· Defects in RAG Genes/Proteins: Somatic Gene Recombination is controlled by lymphocyte specific proteins known as RAG-1 (recombinase activating gene) and RAG-2. Defects in RAG genes prevent recombination which means that no mature B or T cells can be formed.
· D and J are joined first. Then V. The variable region is then spliced to the constant region (which is also variable—determines which Ig in B cells. Not as important in T cells).
· If for any reason rearrangement is unable to generate a functional BcR or TcR, the cell dies. (see previous bullet).
· Once a mature receptor has been generated, they still must go through some final checkpoints to eliminate those B and T that do things like recognize self-antigen (negative selection). TcR undergo additional selection to ensure that only those receptors that require MHCs can go on.
· Mecanisms of Generating Diversity: 1)Combinatorial association of VDJ regions. 2)Pairing of the two different receptor chains (a, b). 3)P-region addition 4)N-region diversification.
- P-region addition: imprecise cleavage/splicing of the VDJ regions with addition or deletion of nucleotides
- N-region diversification: addition of nucleotide sequences between gene segments by the enzyme terminal transferase.
- In all cases, the major location for diversity is at the junctions. It is the hypervariability of these junctional regions that gives rise to the CDR3 regions (principally responsible for Ag recognition).
· Allelic Exclusion: Receptors can only be expressed at the cell surface in dimer form. So the heavy chains are expressed along with pre-BcR or pre-TcR. Cell surface expression of a functional heavy chain with a pre-receptor chain means that successful rearrangement has occurred. A signal is sent down to the cell to inform it to cease further rearrangement. This ensures that only a single receptor is expressed.
· At this point the immature B and T cells (now expressing functional TcR/BcR) can undergo positive and negative selection.
· Negative Selection: those cells that recognize self-MHC with high affinity and therefore are potentially autoreactive are induced to die by apoptosis.
· Ig but not TcR undergo further molecular changes:
- Isotype switching: delete the intervening DNA between the VDJ and the new isotype C region.
- Somatic hypermutation: Random mutations in the V regions. Those that lead to lower affinity cells are induced to die.
- Switch to secreted Ab: Splice the Ig mRNA coding for the transmembrane exon protein.
6 – MHC and Antigen Presentation
· The principal function of MHC is presentation of protein antigen to TcR.
· MHC-I is present on nearly all cells (except RBCs). – antiviral immunity
· MHC-II is present only on pro-APCs (Bs, dendritic cells, Macrophages).- DTH/humoral immunity
· They are also responsible for self/non-self discrimination. (the principal cause of graft rejection)
· Awos: MHC alleles control immune responsiveness and graft rejection.
· MHC Restriction: T cells can only recognize antigen when presneted in combination with self-MHCs.
· Only MHC Ib (b2 microglobulin) chain is not polymorphic. Ia, IIa and IIb are all polymorphic. (Ie: The b2 chain on MHC-I is identical for all MHC-Is).
- Each variant MHC-I allele is encoded by a single a chain.
- Each variant MHC-II allele is encoded by both an a and a b chain.
- This means that you have 6 different MHC-I molecules (genes) in your halotype (eg: HLA-A13, HLA-A299, HLA-B27 (ankSpond), HLA-B5301 (resistance to malaria), HLA-C2, HLA-C348). Because MHC-II has two chains, you have 12 different possible molecules for that one.
· Certain polymorphic MHC alleles are linked to resistance for disease (eg HLA-B*5301 for malaria) as well as for autoimmunity (HLA-B27 – ankylosing spondylitis)
· In order for MHC-Ag to be presented, the Ag must first be “presented” to a T by an APC. ??
· Dendritic cells tend to capture antigen at initial site of infection. They then run off to a lymph node so they can present it to a naïve T cell.
· FC: What is the purpose (other than ID!) of CD4/8? The CD4/CD8 co-receptor interacts with the non-polymorphic chains during activation. Ie: CD4 interacts with a3 domain of MHCII and CD8 interacts with b2 chain of MHC-I. The Lck on the CD4/8 phosphorylates the ITAMs on CD3 copmlex allowing ZAP70 to come in and bind.
· MHCI presents to CD8+ and MHCII presents to CD4+.
· Each MHC molecule (I and II) are coded for by three different gene loci. MHC-I is coded for by HLA-A, HLA-B, HLA-C on chromosome 6. Each of those loci has many different possible alleles. Similarly, MHC-II is coded for by HLA-DP, HLA-DQ, HLA-DR also on chromosome 6.
· Each person has a specific HLA-A allele, HLA-B allele, HLA-C allele. If there were 5 different alleles of each, each halotype has a 1/125 chance of being identical. However, recall that there are 2 chromosomes… (MHC genes are expressed codominantly).
· Halotype: the entire set of MHC genes on each chromosome.
· Anchor Residues vs TcR Contact Residues: the part of the peptide that binds to the MHC is called the anchor residue. The part that binds to the TcR is called the contact residue.
· Only MHCs bound to peptide are stably expressed at the cell surface. (If inside the ER a peptide does not get bound, the MHC will be destroyed).
· MHCs are unable to discriminate between self and foreign peptides.
· IFN g (eg by NK, CD4) increases MHC expression and Ag presentation.(in addition to activating macrophages, etc)
· MHC Class I Pathway (cytosolic proteins):
1. cytosolic proteins are degraded by the proteosome
2. residues are transported into ER by TAP
3. protein is bound to MHC and sent off to the surface
- if a person is deficient in TAP, they are susceptible to viral infections
· MHC Class II Pathway (proteins in endosomes/phagosomes)
1. phago/endosome fuses with lysosome to form phagolysosome which degrades peptides and transported into the ER.
2. Meanwhile, inside the ER, class II MHCs are bound to invariant chain protein (occludes the peptide groove)
3. MHCII-Ii and peptide are transported into a vesicle called ‘MHC class II compartment’ (MIIC) where Ii is attacked by proteases leaving only a peptide called CLIP. Another protein called HLA-DM catalyzes removal of CLIP allowing the peptide to bind to the MHC.
- Question: what keeps these peptides from binding to MHC-I?
- “II = Ii”
7 – T Lymphocyte Activation
· TcR/CD3 Complex: The TcR dimer is expressed along with other molecules, most importantly, the CD3 complex. Because the TcR cytoplasmic tails are so short, the T cell needs the CD3 to delve into the cytoplasm to deliver the message.
· Signal Transduction in T activation: The CD3 complex has ITAM(s) attached to each chain. Phosphorylation of ITAM allows molecules containing SH-2 domains to bind. The most important of these molecules in T activation is ZAP70.
· When a T cell encounters Ag (with MHC, of course!), TcR and CD3 are recruited to the site of interaction. CD4/8 is recruited as well. On the cytoplasmic tail of CD4/8 is a tyrosine kinase called p561ck or Lck. It is Lck that phosphorylates the ITAMs on the CD3 which then allows ZAP70 to bind.
· Activated ZAP70 triggers a variety of events such as: Ca; activation of MAPK pathway; activation of calcineurin; Also transcription factors that promote cytokine gene expression. Eg: NFAT, AP-1, etc
· IOW: 1)T/Ag interact. 2)Recruit CD3, CD4/8. 3)Lck on CD4/8 phosphorylates ITAMs on CD3. 4)ZAP70 binds and is activated by ITAMs. 5)ZAP70 exerts effects by phosphorylating multiple substrates (eg Ras-MAPK).
· CD4/CD8 Coreceptors: see above…
· Adhesion Molecules: Provide adhesion of the TcR to the APC. (Requires > 4-6 hours for full activation). Eg The integrin LFA-1 (ICAM-1); CD2 (LFA-3). Adhesion molecules are not activated until the TcR is bound to an antigen.
· Costimulatory Molecules: Eg: CD28, CD40L. Also known as the second signal. Essential for proper activation of T cells. Without 2nd signal à anergy.
v CD28 is present on essentially all T cells and binds to B7 found on APCs.
v FC: Functions of CD40L (nreg of B7; Induce IL-12; isotype switching) is upregulated on activated Ts. Interacts with CD40 found on a variety of APC. CD40 activation on macrophages causes upregulation of costimulatory B7s, activates macorphages, induces IL-12, etc. CD40 activation on B cells upregulates B7 co-stimulation and induces B cells to undergo isotype switching.
v FC: Deficiency of CD40L? Note how CD40 signalling is important for both CMI and humoral immunity. Patients with deficiencies in CD40L (hyper-IgM) exhibit profound defects in both cell-mediated and humoral immunity.
v A “resting” APC is co-stimulator deficient. Initially, the APCs (eg macrophage) do NOT have B7, CD40, etc. However, if they are activated (eg by innate immune system), they will upregulate their co-stimulator molecules.
v FC: How is CD40 induced? Ans: By TcR signalling (following activation from an antigen).
· Role of IL-2: IL-2 causes clonal expansion of T cells When a T cell becomes fully activated it upregulates expression of IL-2 (“T cell growth factor”). It also upregulates expression of the high-affinity IL-2 receptor. (Ie: Autocrine action).
· ITAMs: found in a wide variety of cell-surface signalling molecules involved in signal transduction in the immune system.
· FC: Phases of the T cell immune response (from naïve T): Ans: 1)Ag is presented to naïve T by APC. 2)T (CD4 or CD8) is activated and secretes IL-2 and upregulates IL-2R. This causes proliferation 3) IL-2 also causes differentiation: CD4 into CD4 effectors and CD4 memory cells. Same thing with CD8. 4)CD4 effectors activate Bs/Ts; activate macrophages; inflammation. CD8 effectors lyse target cells and activate macrophages.
· Signal 1 vs Signal 2: The TcR provides signal 1. The costimulatory molecules are known as signal 2. Signal 2 is essential to achieve full activation. In fact, without signal 2, you get anergy.
· FC 3 Mechanisms for turning off T immune response: (no Ag, Ag, CTLA-4)