Immunology Test 3

• SCID: No T cells (genetic)
• Lupis: Classic butterfly rash pattern
• DiGeorge's syndrome: Patients are born without a thymus and have less T cell maturation
• Genetic immunodeficiency (and lack of T cells) can be caused by loss of any of the following: RAG1/2, Artemis, cytokine receptors, Jak3, or Btk
• Plaque psoriasis, rheumatoid arthritis, and autoimmune diseases of the skin also involve T cells

• Common lymphoid progenitor cells produced in bone marrow migrate to thymus
• As they migrate through the thymus, these immature T cells receive signaling from DCs and cortical epithelial cells to progress from the double negative stage to the double positive
• The double positive T cells become CD4 or CD8 positive
• The mature TCR that is expressed during the DP stage is tested by medullary epithelial cells which express self peptides in the context of MHC I and II (looking for intermediate reactivity)

• Medullary epithelial cells of the thymus express self antigens in the context of MHC I and II.
• No signaling: Death by neglect or continued alpha chain rearrangement
• Intermediate: Becomes CD8 or CD4 based upon which MHC the cell responds to
• Strong: Death by negative selection (aka clonal deletion because we are narrowing the entire T cell population down to those specific to a single Ag)

• Following testing self reactivity by interactions with medullary epithelial cells, the double positive T cells downregulate CD8
• CD4-positive T cells that then receive competent TCR stimulation remain CD4 T cells
• CD4-positive T cells that do not receive competent TCR stimulation downregulate CD4 and upregulate CD8, becoming cytotoxic T cells

• A cell surface receptor that signals a common lymphoid progenitor cell to differentiate into a T cell lineage and not a B cell one. It also signals for the rapid proliferation characteristic of T cells
• Notch activation occurs when progenitor cells migrate to the thymus and receive signaling from the neighboring stromal cells that constitutively express Notch1, the ligand for the Notch receptor
• Clinical: Loss of Notch results in B cells in thymus; gain of Notch results in T cells in bone marrow

• A transmembrane receptor made of alpha and beta subunits, both of which contain a variable and a constant region
• Recognizes MHC-antigen complex
• Made from 8 proteins from 6 genes
• The TCR zeta subunit is associated with the TCR for intracellular signaling and is made of CD3 gamma, delta, and epsilon
• Clinical: Deficiencies in any of the TCR or CD3 subunits results in decreased TCR surface expression

• Each mature T cell expresses a single rearranged alpha and a beta chain
• Gene rearrangement occurs similarly to BCR production, except the alpha chain has only V and J sequences and the beta chain has VDJ
• These chains are thought to be duplicates of the locus for immunoglobulins
• RAG-1/2 are critical for TCR gene rearrangement by binding to RSS sequences that flank the TCR locus and cleaving one strand of DNA which can form a hairpin loop and be joined together to form the gene
• TCR beta is rearranged first during the double-negative, pre-TCR stage (DNIII)
• The beta chain then associates with the pre-TCR alpha chain which signals intracellularly via a tail to proliferate, express both CD4 and CD8 receptors, and begin alpha chain rearrangement
• The alpha chain is rearranged during the double positive stage, and can be rearranged multiple times
• Clinical: If we did not stop pre-TCR alpha from signaling (it is the signal for proliferation), we could develop leukemia

• Each V, D, and J segment can independently recombine, resulting in 5.8x10^6 possible recombinations
• Junctional diversity is responsible for most of the diversity, because cleavage of the DNA leaves hairpin loops that are randomly cut and filled in
• All together, we get around 10^18 possible TCRs
• Also: TCRs can bind more than one antigen by different orientations of binding and each TCR has different contact points with antigen-MHC complex

• A gamma-delta TCR also exists, and is used as a backup if TCR beta chain rearrangement is not fruitful
• If gamma-delta TCRs are made, they stop production of alpha-beta (and vice versa)

• Afferent lymphatics dump lymph fluid and DCs into the outer sinuses of the node
• The lymph flows inward to the efferent lymphatics
• Lymphocytes (T cells) can also enter the lymph node through the high endothelial venules (HEV) and enter into the T cell zone by CCR7/CCL21 where they scan resident dendritic cells before leaving the node or becoming activated
• T cells circulate every 24-36 hours

• Rolling: Mediated by L-selectin (T cells) binding to Gly-CAMs (ECs)
• Activation: The T cell is activated through the TCR or by chemokines to cause a conformational change in integrins; HEVs produce CCL21 and stromal cells produce CCL19 constitutively, which are bound by CCR7 (naive lymphocytes)
• Adhesion: This conformational change allows the integrin LFA-1 (lymphocytes) to bind their ligand ICAM-1 (ECs) with a much higher affinity
• Extravasation: Lymphocytes then enter the lymph node by diapedesis

• Lymphocytes recognize sphingosine-1-P (S1P) via a G-protein coupled receptor
• S1P is found in high concentrations in the blood and lymph and low concentrations in the tissues (S1P lyase destroys S1P in peripheral tissues)
• Lymphocytes follow the concentration gradient out of tissues/lymph nodes and into the blood/lymph circulation
• Once in the presence of high levels of S1P, the lymphocytes downregulate the S1P receptor so that they can be influenced by other chemokines to leave the bloodstream again

• Periphery: DCs take up antigen at sites of infection and are activated by TLR ligands or cytokines. They then migrate to the nearest lymph node by upregulating CCR7 and following the CCL19/CCL21 gradient and present Ag to T cells to fully activate them
• Lymph Node: DC residents of lymph nodes take up soluble antigen and are activated by soluble TLR ligand. T cells form transient contacts with the DCs and are partially activated since the interaction is transient and not long lasting; there is not as much antigen presentation
• DCs have ruffled membranes to increase the surface area exposed to lymphocytes

• Priming is the activation of naive T cells following their initial interaction with antigen
• Primed T cells immediately increase the IL-2 receptor affinity by upregulating the alpha subunit; the primed T cell can then respond to the cytokine IL-2 and can proliferate extensively after expressing CD25
• CD69 is also expressed; this molecule causes degradation of S1P1, causing the activated T cells to be trapped in the lymph node where they can proliferate
• Primed T cells require as few as 3 TCR/MHC interactions for activation
• Naive T cells need 10-20 interactions

• Classical: Important for antigen presentation; include HLA A, B, and C (all Class I); are polygenic and very polymorphic
• Non-classical: Polymorphic; are used to present antigen to αβ T cells, γδ T cells, NK cells and NK T cells
• Class I: Binds short peptides (8-9 AA) in the cytosol of any nucleated cell; alpha subunit binds Ag; the MHC Class I-antigen complex is presented to cytotoxic CD8 T cells, which cause cell death (or become activated if a DC is the one presenting MHC Class I-antigen)
• Class II: Binds longer peptides (12-20 AA) that have been endocytosed by macrophages or B cells (APC); both alpha and beta subunits bind Ag; MHC Class II-antigen complex is presented to CD4 on T cells, which then activate the macrophage or B cell
• The locus for MHC contains at least 200 genes

• The alpha chain is made in the ER and is bound by chaperones
• Peptides cleaved by peptidases are transported into the ER and processed by ERAP1
• The alpha chain binds antigen with the help of β2microglobulin, calreticulin, ERp57 and tapasin, and the alpha chain and β2microglobulin are transported to the cell surface
• Overall importance: MHC Class I binds short peptides from endogenous cellular proteins and only the alpha subunit comes into contact with the peptide

• Alpha and beta chains dimerize in the ER and bind calnexin (a chaperone), until the invariant chain is present and binds
• The dimer goes through the endosomal/lysosomal pathway because of a sequence in the invariant chain
• In the lysosome, the invariant chain is cleaved to CLIP which is released when a nonclassical MHC (DM) binds our Class II MHC
• DM is released when antigen binds, and the MHC Class II-antigen complex is sent to the cell surface
• Overall importance: MHC Class II binds longer peptides from exogenous cellular proteins and both the alpha and beta subunits come into contact with the peptide; Class II is also upregulated in activated DC

• CD4 and CD8 are coreceptors that bind invariant regions of MHC class II and I, respectively
• These coreceptors increase the responsiveness of the TCR to the antigen-MHC complex by 10-100 fold
• The coreceptors are associated with Lck, a tyrosine kinase, and when they bind the Ag-MHC complex, Lck is brought into proximity to initiate TCR-mediated signaling via phosphylation

• Used to treat patients like Emma Whitehead, these engineered receptors contain an extracellular antigen-specific scFv fragment, a constant Fc fragment, a costimulatory signaling region, and an intracellular component of the TCR (the zeta chain)
• This results in the specificity of an Ig and the activation of T cells through the TCR

• Protein modifications: Includes phosphorylation (tyr, ser, or thr), methylation, acetylation, glycosylation, lipidation, isomerization, ubiquitination, SUMOylation
• Complex formation: Often uses adaptor proteins, which connect receptors to downstream pathways, localize enzymatic proteins to sites of active signaling, or activate enzymatic signaling molecules; these adaptor proteins use modular interaction domains and binding motifs in order to recognize modified peptides, longer peptides, nucleic acids, domains, or phospholipids

• 1: TCR binds to the Ag-MHC complex and is recruited to the lipid raft that the CD4 coreceptor is found in by CD4 binding to MHC
• 2: Lck, which is found on the intracellular side of CD4, phosphorylates the 12 ITAM sites on the zeta subunit of the TCR
• 3: This phosphorylation recruits ZAP-70 to the TCR where it is activated
• 4: Activated ZAP-70 then goes on to phosphorylate LAT at 4 sites
• 5: LAT facilitates the formation of large complexes in the surface of the T cell

• Actin polymerization occurs to change T cell morphology to maximize T cell APC contacts
• 1: Nck is an adaptor protein that recruits WASP to LAT
• 2: Costimulation by binding of CD80/86 TO CD28 triggers activation of Vav1 through Gads and SLP-76; Vav1 is a GEF for activation of the GTPases Rac and CDC42
• 3: WASP, Rac, and CDC42 are all required for activation of the Arp2/3 complex, which induces actin branching

• Phosphorylated LAT causes binding of SLP-76 which then recruits ITK, a tyrosine kinase
• PLCgamma1, a phospholipase (the PL stands for that), also binds LAT, and is subsequently phosphorylated and activated by ITK
• Phosphorylated PLCgamma1 cleaves phosphatidyl inositol 4,5 phosphate (PIP3) into diacylglycerol (DAG) and inositol 1,4,5 phosphate (IP3)
• DAG signals PKC activation and IP3 causes calcium influx
• However, PIP3 can have all sorts of fun. It can be dephosphorylated by PTEN to become PI and can be phosphorylated again by PI3K
• PIP3 can then activate Akt, which is a protein heavily involved in metabolism, survival, and transcription

• Activation of the TCR and costimulatory receptors results in different waves of complex formation
• Initially, expansion occurs for about two minutes. Contraction follows over the course of ten minutes. Finally, a mature IS complex is formed in the center of the cell surface with surrounding microclusters

• G protein activation results in induction of kinase cascades: Recruitment of the GEF (Vav1) to LAT results in the stimulation of G proteins (CDC42 and Rac); their activation induces a cascade of protein kinase activation and gene transcription (also causes activation of Arp2/3 complex, which induces actin branching). Another example is the Erk pathway: G-protein activation recruits Raf which activates Mek which activates Erk which phosporylates transcription factors. The point: Provide sites for amplification and regulation
• Ca influx leads to activation of transcription factor NFAT: Calcium influx is triggered by cleavage of PIP3 into IP3. The calcium influx then causes activation of calmodulin which activates calcineurin which dephosphorylates NFAT, allowing it to go in the nucleus
• Protein degradation controls the activation of NF-kB transcription factors: Activated PKC (which is activated by PIP3 cleavage into DAG) affects the Bcl-10-MALT complex which activates IKK. IKK ubiquitinates the inhibitory subunit of NF-kB (I-kB), causing degradation of the I-kB. NF-kB is released and its nuclear translocation signal is exposed

• Central tolerance: Removal of self-reactive T cells in the thymus
• Peripheral tolerance: Removal or suppression of self-reactive T cells released from the thymus
• Most rearranged TCRs recognize self peptides, so we want to eliminate these self-reactive T cells through T cell tolerance

• Occurs in the medulla of the thymus
• Remember, T cells enter through the medulla and go to the cortical region, rearranging the beta TCR during the DN3 stage and testing the function of the beta chain during the DN4 stage. At this point, the cell rearranges the alpha TCR chain.
• The mature TCR in the DP thymocytes is then tested in the medulla by interacting with medullary epithelial cells expressing self antigens in the context of MHC I and II and DCs presenting tissue-specific Ag from food and gut flora.
• Death by neglect (no signaling) or negative selection (strong signaling) selects for intermediate-signaling cells

• Aire controls promiscuous expression of proteins by increasing the transcription/translation of tissue-specific proteins by removing epigenetic signals
• Aire expression is upregulated during the maturation of these cells
• Clinical: Subtle changes in Aire function may lead to increased autoimmunity. Humans with mutations in Aire have autoimmune polyendocrine syndrome type I

• T cells require three signals for full activation
• 1: TCR activation by binding to Ag-MHC complex; provides specificity to antigen --> TCR binds MHC Class II and Ag; CD4 binds MHC Class II
• 2: Costimulation provides a mechanism to prevent reactivity to self --> CD28 binds B7.1 and B7.2 (aka CD80/86)
• 3: Cytokines direct T cell differentiation into a specific T cell subset
• If signal 1 is received without signal 2 present, the T cell enters a nonreactive state called anergy or undergoes apoptosis

• CD28 binds B7.1 and B7.2: The primary costimulatory signal for T cells; CD28 is a homodimer expressed on all CD4 and most CD8 cells
• CD40L (T cells) is bound by CD40 (APC): Activation of TCR and CD28 results in upregulation of CD40L, which activates CD40 on APCs
• Positive feedback: Activated CD40 upregulates B7.1 and B7.2 (in the APC), resulting in increased T cell activation; in addition, activation of TCR and CD28 results in upregulation of activating and inhibitory receptors such as the TNF superfamily
• TNF superfamily: The TNF receptors are upregulated on the T cell by activation of TCR and CD28; the CD40 stimulation on the APC causes it to upregulate the TNF superfamily ligand expression (ex: Ox40R found in T cell is activated during a parasitic infection)
• CD2 superfamily: This family of costimulatory receptors is important for adhesive and costimulatory signals; some are constitutively expressed and others are inducible; receptors may be on T cells or on APCs
• This all results in a second wave of T cell activation and alters the function of the T cell depending on the infection

• Receptors can inhibit the binding of costimulatory receptors to their ligands by binding with a higher affinity or by inhibiting intracellular signals
• Example: CTLA-4 is upregulated in T cells following stimulation through TCR and CD28; this receptor binds B7.1 and B7.2 with a higher affinity than CD28, inhibiting T cell activation
• Example: PD-1 is also upregulated in T cells following stimulation through TCR and CD28; PD-1 is activated by binding ligands on DCs; this activation causes the intracellular tyrosine phosphatase activity to counteract the intracellular signals activated by the TCR and costimulatory receptors

There are at least 18 different receptors, all of which have 7 transmembrane regions and are G-protein coupled receptors

• Rare genetic disorder in which the beta2 integrin subunit is mutated, causing lack of firm adhesion of leukocytes
• Recurrent infections and high numbers of WBCs in blood
• Treated by a bone marrow transplant

• IFNgamma: Th1
• IL-4: Th2
• IL-17a: Th17

• T-bet: Th1
• GATA3: Th2
• RORgammat: Th17
• FoxP3: Tregs
• Bcl-6: Tfh

• Th1: Activate macrophages and help B cells produce antibodies; good for extracellular bacteria and intravesicular pathogens in macrophages
• Th2: Help B cells produce antibodies (especially IgE) and protect from parasites
• Th17: Enhance neutrophil respose and promote skin integrity; good for fungi
• Tfh: Help B cells produce antibodies and switch isotypes; good for all pathogens
• Tregs: Inhibit T cell responses

• Ca influx causes calmodulin to activate calcineurin which dephosphorylates NFAT, allowing it to enter the nucleus
• NFAT transcribes T-bet
• T-bet upregulates IFNgamma and Hlx
• Hlx also helps upregulate IFNgamma and downregulates IL-4R (which would be for Th2)
• IFNgamma causes expression of IRF-1 and IRF-2 which repress expression of IL-4 (for Th2)
• T-bet inhibits GATA3 (transcription factor for Th2) by binding it
• Th1 cells mediate their activity by IFNgamma release: Increased cytotoxicity of NK cells, increased survival of CD8 cells, increased macrophage activity, and increased B-cell-mediated complement fixation
• Th1 differentiation can also be caused by IL-12 and IL-18, both of which are produced by activated macrophages in order to stabilize the Th1 cell

• Lots of stuff going on here, but basically GATA3 and IL-4 are upregulated following TCR stimulation and ICOS binding from DCs causing c-Maf activation, and T-bet and IFNgamma are downregulated through unknown mechanisms
• Th2 cells cause smooth muscle contraction, mucosal epithelial cell proliferation, and increased IgA and IgE production from B cells, which causes activation of eosinophils/basophils/mast cells by binding of Fce to FceR, causing degranulation
• Th2 cells also release IL-4 and IL-13 to inhibit macrophage activity in order to promote tissue repair
• Remember: Th2 cells are important for parasites, so it makes sense to downregulate bacteriocidal activity to spare the tissues

• Bcl-6 is important (somehow)
• DCs express IL-12 and OX40L to cause upregulation of CXCR5 to allow the cells to migrate into the follicle where they can interact with B cells presenting antigen

• IL-17 and IL-23 cause expression of the transcription factor RORgammat
• IL-23 is a survival factor released by macrophages
• Activation of Th17 cells causes neutrophil activation; neutrophils die after attacking
• Macrophages take up dead neutrophils and that causes inhibition of IL-23 release and downregulates the Th17 response

• They are potent inhibitors of the adaptive immune response
• Express CD25, which is the high affinity IL-2 receptor; this causes them to soak up the IL-2
• Remember: IL-2 is the main proliferative signal for T cells
• Tregs release granzymes and perforin, which can actually destroy CD8 T cells

• X-linked syndrome in which FoxP3 does not work, and therefore Tregs do not work well and autoimmunity develops
• Limited success in bone marrow transplants treating the syndrome

• Produce IFNgamma or TNFalpha (which is also involved in target cell killing and activating macrophages) when they come across infected cells
• Induce apoptosis in infected cells by granzymes and perforin
• Granzymes: Serine proteases that activate apoptosis once in the cytoplasm
• Perforin: Pokes holes in the plasma membrane to facilitate granule delivery
• Cathepsin B protects CD8 cells from the effects of perforin
• Also express the FasL that can activate the Fas receptor on host cells, causing apoptosis

• 1: Activation
• 2: Expansion
• 3: Contraction - Mediated by Bim
• 4: Memory

• IL-10
• IL-35