B and T cells Notes

NOTES NOTES B & T CELLS ANTIBODY CLASSES osms.it/antibody-classes ▪ B cell receptor, major component of humoral immunity ▪ Heavy, light chain; fragment antigenbinding region; constant region (Fc) ▪ B cell develops into plasma cell → B cell receptor secreted as antibody ▪ Antibodies: monomers, polymers ▫ Valence: number of antigen-binding fragments FIVE TYPES ▪ Coded by heavy chain genes Immunoglobulin M (IgM) ▪ 1st antibody response ▪ Monomer as B cell receptor (valence: 2) ▪ Pentamer as antibody held together by joining (J) chain (valence: 10) ▪ Works against carbohydrate, lipid antigens ▪ Most effective at activating complement pathway ▪ Main immunoglobulin in mucosal sites; sometimes occurs as dimer (valence: 4) ▪ Two forms ▫ IgA1, IgA2 (differ in constant regions) Immunoglobulin E (IgE) ▪ Monomer (valence: 2) ▪ Production primarily induced by interleukin 4 (IL-4) ▪ Triggers granule release from mast cells, eosinophils, basophils ▪ Responds to nonpathogenic targets (e.g. peanuts) → allergies Immunoglobulin D (IgD) ▪ Monomer (valence: 2) ▪ Found alongside IgM antibodies, signals maturation of B cells Immunoglobulin G (IgG) ▪ Monomer (valence: 2) ▪ Four subclasses ▫ IgG1, IgG2, IgG3, IgG4 (differ in constant regions) ▪ Serves as opsonin ▪ Activates classical complement pathway Immunoglobulin A (IgA) ▪ Monomer (valence: 2) ▪ Serves as opsonin (eosinophils, neutrophils, some macrophages) Figure 45.1 B cell receptor components. OSMOSIS.ORG 377

Figure 45.2 Summary of the five classes of antibodies. IgM and IgD can act as B cell receptors. B CELL ACTIVATION & DIFFERENTIATION osms.it/b-cell-activation-and-differentiation ▪ Developing B cell receptor expresses μ heavy chain → B cell receptors IgM ▪ Alternative splicing → IgM, IgD expressed on surface → mature, naive B cell explores lymphatic system → B cells enter paracortical region of lymph nodes, migrate to cortical region → form primary follicle ACTIVATION ▪ On activation (antigen-binding), B cell forms germinal center → secondary lymphoid follicle ▪ Cross-linkage of two B cell receptors → Igalpha, Ig-beta, CD19 cluster ▫ Blk, Fyn, Lyn phosphorylate tyrosine residues on immunoreceptor tyrosine based activation motif (ITAM) units → transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), nuclear factor of activated T cells (NFAT) → gene expression of cytokines, upregulation of antiapoptotic cell surface markers 378 OSMOSIS.ORG DIFFERENTIATION ▪ B cells stimulated by cluster of differentiation 21 (CD21)/complement receptor Type II (CR2) (receptor for C3d complement fragment) ▪ Activated B cells differentiate into plasma cells, secrete antibodies ▫ Plasma cells initially secrete IgM, remain mainly in bone marrow, safeguard against future encounters with same antigen Activated CD4+ T cell → class switching ▪ B cells: antigen-presenting cells; present antigens on major histocompatibility complex (MHC) class II to helper T cells ▪ CD40 ligand on T cell binds to CD40 on B cell → cytokines instruct B cell on type of antibody to produce (by activation-induced cytidine deaminase) ▫ IL-4, IL-5 → IgE ▫ Interferon (IFN) gamma → IgG

Chapter 45 Immunology: B & T Cells ▪ Activation-induced deaminase removes constant regions during differentiation to leave desired antibody region Figure 45.3 Mature, naive B cells form a primary follicle in the cortical region of a lymph node. When the B cell binds an antigen, it activates and forms a germinal center. The follicle is now called a secondary lymphoid follicle. Figure 45.4 Series of events following antigen binding that lead to B cell activation. Ig-alpha, Igbeta, and CD19 are intracellular side chains of the B cell receptors that cluster when two B cell receptors are cross-linked by an antigen. OSMOSIS.ORG 379

Figure 45.5 Complement fragment C3d can bind an antigen and then be bound by molecule CD21/CR2 on a B cell. B cells can also be activated when they have a B cell receptor that is bound to an antigen, and a CD21 that’s bound to an antigen. Figure 45.6 B cell differentiation. 1: B cell presents an antigen to a CD4+ T cell. 2: If the T cell activates, it expresses CD40L on its surface, which binds to CD40 on the B cell. 3: CD40L and CD40 binding causes the B cell to express a cytokine receptor and the T cell to release cytokines. The type of cytokine determines what type of antibody the B cell will produce. 380 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells B CELL DEVELOPMENT osms.it/b-cell-development ▪ Lymphopoiesis: development of diverse set of lymphocytes with unique antigen receptors CREATION OF SUITABLE RECEPTOR ▪ B cell receptor contains two chains ▫ Heavy, light ▪ Antigen-binding site made of variable (V), diversity (D), joining (J) protein segments coded by genes of same name ▫ Heavy chain: all three segments ▫ Light chain: V, J segments Early pro-B cell ▪ Common lymphoid progenitor cell expresses recombination activating gene (RAG) 1, RAG2 → early pro-B cell Late pro-B cell ▪ Heavy chain D, J gene segments spliced together (allelic exclusion: 1st chromosome to complete splicing suppresses 2nd) → late pro-B cell Large pre-B cell ▪ Late pro-B-cell attaches D-J gene segment to V gene segment via V(D)J recombinase → binding site (heavy chain) recombined with mu gene → large pre-B cell ▫ Mu gene codes for IgM constant region protein Small pre-B cell ▪ Functionality of heavy chain tested by binding to surrogate light chain (VpreB, lambda 5) → if successful, cells proliferate → small pre-B cell Figure 45.7 Antigen binding site on heavy chain is composed of V, D, and J segments, while antigen binding site on light chain has only V and J segments. STAGES OF DEVELOPMENT ▪ Six stages: common lymphoid progenitor cell → early pro-B cell → late pro-B cell → large pre-B cell → small pre-B cell → immature B cell Immature B cell ▪ Light chain rearranged → functionality of light chain tested by autoimmune regulator (AIRE), identifies self-reactive cells by expressing bodily antigens in lymphoid organs → immature B cell ▪ Central tolerance/negative selection: elimination of self-reactive cells ▫ Strong binding to self-antigen → cell undergoes apoptosis ▫ Intermediate binding to self-antigen → light chain repeatedly rearranged with kappa gene on 1st, 2nd chromosomes, lambda gene 1st, 2nd chromosomes ▫ Failure to eliminate self-reactive cells → autoimmunity ▪ Immature B cells finally undergo alternative splicing on constant region → IgD constant region replaces IgM constant region → cells released into blood OSMOSIS.ORG 381

Figure 45.8 B cell development stages and the changes that move them to the next stage. 382 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells CELL MEDIATED IMMUNITY OF CD4 CELLS osms.it/cell-mediated-immunity-CD4-cells ▪ CD4 cells = T helper cells (support other immune cells) ▪ T cells initially naive ▪ In response to antigen, T cell primed → effector T cell ▫ Two signals: antigen (MHC molecule on antigen-presenting cell), costimulation (CD28 binds to B7 on antigenpresenting cells) ▪ Activated T helper cell → IL-2 → upregulates IL-2 alpha receptor ▪ T helper cell binds to IL-2 (autocrine stimulation) → clonal expansion FOUR TYPES OF T HELPER CELL ▪ Depends on cytokines in environment T helper Type I (Th1) ▪ Fights intracellular infections ▪ Macrophages → IL-12, natural killer (NK) cells → IFN-γ, infected cells → IFNα, IFNβ → transcription factors signal transducer and activator of transcription 1 (STAT1), STAT2 T helper Type II ▪ Fights parasites ▪ Eosinophils, basophils, mast cells → IL-4, IL-4, IL-10 → transcription factors STAT6, GATA-binding protein 3 (GATA3) T helper Type XVII ▪ Fights fungal, bacterial infections ▪ Fungi, bacteria → IL1, IL6, IL23, transforming growth factor (TGF)β → transcription factors ROR-γ, STAT3 T follicular helper (Tfh) ▪ Establishes memory B cells ▪ Antigen-presenting cells → IL6, IL21, IL27 → transcription factors B cell lymphoma protein 5 (BCL-5), cMaf Figure 45.9 T helper cells require two signals to be primed and become effector T cells: presentation of an antigen and binding of CD28 on T cell to B7 on antigen-presenting cell. OSMOSIS.ORG 383

CELL MEDIATED IMMUNITY OF NATURAL KILLER & CD8 CELLS osms.it/cell-mediated-immunity-NK-CD8-cells NATURAL KILLER (NK) CELLS ▪ Identify target cells; deliver perforin, granzymes ▪ Part of innate response → no need for specific antigen ▪ Activation receptors recognize surface molecules on infected cells; inhibitory receptors recognize molecules (e.g. native MHC class I molecules) ▪ Also activated by antibody-dependent cellmediated cytotoxicity ▫ IgG binds to virally-infected cell → CD16 on NK binds to antibody Figure 45.10 NK cells recognize cell surface molecules like MHC I to determine whether or not to kill a cell. They can also kill via ADCC. In this process, the NK cell is stimulated by binding to the constant chain of an IgG antibody attached to a virally infected cell. 384 OSMOSIS.ORG CD8 CELLS ▪ CD8 cells = cytotoxic T cells ▪ T cells initially naive ▪ In response to antigen, T cell primed → effector T cell ▫ Two signals: antigen (MHC molecule on antigen-presenting cell), costimulation (CD28 binds to B7 on antigenpresenting cells) ▪ Activated T helper cell → IL-2 → upregulates IL-2 alpha receptor ▪ T helper cell binds to IL-2 (autocrine stimulation) → clonal expansion ▪ Needs to see antigen in context of MHC I to kill cell (doesn’t need CD28) ▪ Binds nonspecifically to multiple cells with adhesion molecules → fails to bind to MHC I → disengages ▪ If antigen binds, cytoskeletal rearrangement → forms supramolecular activation cluster (SMAC) ▫ Includes central SMAC (cSMAC) for antigen recognition, peripheral SMAC (pSMAC) ▪ Cytotoxic cell releases granules with perforin, granzymes (caspases → apoptosis)

Chapter 45 Immunology: B & T Cells Figure 45.11 CD8 cells weakly bind a variety of cells with adhesion molecules. However, they only destroy cells with antigens on their MHC I molecules that allow the CD8 cells to bind tightly. CYTOKINES osms.it/cytokines ▪ Proteins secreted by all types of cells to communicate (bind to receptors, trigger response) FIVE TYPES Interleukins (ILs) ▪ Act as communication between leukocytes, nonleukocytes ▪ Promote development, differentiation of T, B cells ▪ Mostly synthesized by helper T cells Tumor necrosis factors (TNFs) ▪ Bind to cell receptors, cause cells to die (induce apoptosis) ▪ Heavily involved in inflammatory response (up-regulate expression of adhesion molecules, increase vascular permeability, induce fever) Interferons (IFNs) ▪ Type I ▫ Produced by virally infected cells → affect surrounding cells: degrade messenger RNA (mRNA), inhibit protein synthesis, express MHC ▪ Type II ▫ Interferon-gamma → promotes antiviral state, activates macrophages, CD4+ helper T-cells Colony stimulating factors (CSFs) ▪ Bind to surface receptors on hematopoietic stem cells → proliferation, differentiation Transforming growth factors (TGFs) ▪ Control proliferation, differentiation of cells MAIN FUNCTIONAL RESPONSES Pro-inflammatory ▪ Enhance innate, adaptive immune responses ▪ IL-1, IL-12, IL-18, TNF, IFN-γ Parasite/allergy ▪ Help immune system handle large parasites, induce allergic responses ▪ IL-4, IL-5, IL-10, IL13 OSMOSIS.ORG 385

Regulatory ▪ Immunosuppressive ▪ IL-10, TGF-β Growth and differentiation ▪ Replenish immune cells ▪ Granulocyte-macrophage colony- stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), IL-7 Chemotactic ▪ Help cells move towards site of inflammation ▪ IL-17, IL-8 MHC CLASS I & MHC CLASS II MOLECULES osms.it/MHC-class-I-MHC-class-II ▪ Major histocompatibility complex (MHC), AKA “human leukocyte antigen” ▫ Cell surface proteins, present antigens to T cells MHC CLASS I ▪ Found on all nucleated cells, presents antigens from inside ▪ Bound by CD8 molecules on cytotoxic T cells ▪ Includes HLA-A, HLA-B, HLA-C Structure ▪ Contains alpha, beta-2-microglobulin chains ▪ Alpha chain: peptide binding groove, transmembrane region ▫ Binding groove binds peptides 8–10 amino acids long; hydrophobic peptide residues ↔ hydrophilic groove amino acids ▪ Three extracellular domains: alpha-1, alpha-2, alpha-3 Figure 45.12 Structure of an MHC class I molecule. 386 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells Function ▪ Allows immune cells to sample cellular proteins (via endogenous pathway of antigen presentation) ▫ Marked protein sent to proteasome ▫ Proteasome degrades protein → short peptide chains ▫ Transporters of antigenic peptides (TAP) move peptide chains to endoplasmic reticulum ▫ TAP loads peptide onto MHC class I using tapasin ▫ MHC class I loaded into exocytic vesicle, sent to cell surface ▫ Cytotoxic T cells, NK cells interact with peptide (if necessary) Figure 45.13 Endogenous pathway of antigen presentation. MHC CLASS II ▪ Found on antigen-presenting cells, presents antigens from outside ▪ Bound by CD4 molecules on helper T cells ▪ Includes HLA-DP, HLA-DQ, HLA-DR Structure ▪ Contains alpha, beta chains ▫ Both penetrate cell membrane ▫ Binding groove binds peptides 14–20 amino acids long Function ▪ Engulfs, destroys pathogens; presents antigens to CD4+ T helper cells (via exogenous pathway of antigen presentation) ▫ Antigen-presenting cell ingests antigen → endosome ▫ Lysosome + endosome → phagolysosome; degrades protein → short peptide chains ▫ MHC class II binding groove filled temporarily with invariant chain (degrades during vesicular transportation) ▫ Vesicle fuses with phagolysosome ▫ MHC class II binds peptide, sent to cell surface ▫ CD4+ helper T cells interact with peptide (if necessary) OSMOSIS.ORG 387

Figure 45.14 MHC class II molecule structure. Figure 45.15 Exogenous pathway of antigen presentation. 388 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells SOMATIC HYPERMUTATION & AFFINITY MATURATION osms.it/somatic-hypermutation-affinity-maturation SOMATIC HYPERMUTATION ▪ Intentional mutation of antibody genes to create new antigen specificities → stronger, more specific response to antigen ▪ Occurs in activated B cells (germinal centers, spleen) ▪ CD40L on T cell binds to CD40 on B cell → cytokines instruct B cell to produce specific type of antibody ▪ Activation-induced cytidine deaminase (AID) turns cytidine into uridine (not usually found in DNA) → mismatch/base excision repair to remove uridine ▫ Mismatch repair proteins MSH2, MSH6 use nucleases to remove uridine; DNA polymerase replaces nucleotides → mutations ▫ Base excision: uracil-DNA glycosylase removes uracil from uridine → next round of replication, random nucleotide inserted → mutations ▪ Only some mutations increase affinity ▫ Low affinity B cells die naturally with time ▫ High affinity B cells live on (affinity maturation) AFFINITY MATURATION ▪ Process by which B cells increase affinity for antigen during an immune response ▪ Somatic hypermutation, clonal selection (only high affinity cells activated → only high affinity cells replicate) Figure 45.16 Somatic hypermutation only occurs in B cells which express enzyme AID. AID makes small mutations directly in antigen binding site of B cell receptor, which get expressed in daughter cells of a rapidly proliferating cell. These changes in the variable region change affinity (strength) that B cell receptor has for its antigen. As antigen becomes limited, B cells with lowest affinity will die off first, so only B cells with strongest affinity for their antigen remain. OSMOSIS.ORG 389

T CELL ACTIVATION osms.it/t-cell-activation ▪ Priming: T cell begins differentiation when exposed to antigen ▫ Two signals: antigen (MHC molecule on antigen-presenting cell), costimulation (CD28 binds to B7 on antigenpresenting cells) ▪ Signal sent to nucleus by CD3 peptide chains ▫ Lymphocyte-specific protein tyrosine kinase (LCK) phosphorylates tyrosine residues on immunoreceptor tyrosine based activation motif (ITAM) units ▫ Zeta-chain-associated protein kinase 70 (ZAP-70) phosphorylates LAT, SLP-76 → activation of transcription factors NF-kB, NFAT → gene expression of cytokines, upregulation of antiapoptotic cell surface markers ▪ Activated T cell → IL-2 → up-regulates IL-2 alpha receptor ▪ T helper cell binds to IL-2 (autocrine stimulation) → clonal expansion Figure 45.17 Summary of T cell activation. T cells need two signals to activate: first, presentation of its antigen by MHC class I (cytotoxic C cells) or class II (helper T cells), and costimulation, which is when CD28 and B7 bind. In helper T cells, this triggers a series of steps that lead to upregulation of the IL-2 alpha receptor and production of IL-2 for itself, causing clonal expansion, and CD8 T cells. 390 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells T CELL DEVELOPMENT osms.it/t-cell-development ▪ Lymphopoiesis: hematopoietic stem cell → common lymphoid progenitor cell → immature B cell (bone marrow) STAGES OF REARRANGEMENT CREATION OF SUITABLE RECEPTOR Double negative/DN stage ▪ Common lymphoid progenitor initially CD3-, CD4-, CD8- (double negative/DN stage; broken down into DN1, DN2, DN3, DN4) ▫ DN1 cell expresses RAG1, RAG2 → DN2 cell ▫ Beta chain D, J gene segments spliced together (allelic exclusion) → DN3 cell ▫ V gene segment combines with DJ gene segment by V(D)J recombinase → V-D-J gene segment bound to μ gene segment → DN4 cell ▫ Functionality of beta chain tested by binding to invariant pre-T alpha chain → if successful, cells proliferate ▪ T cell receptor contains two chains: alpha, beta ▫ Alpha: comparable to B cell’s light chain ▫ Beta: comparable to B cell’s heavy chain ▪ Antigen-binding site: V, D, J protein segments coded by genes of same name ▫ Beta chain: all three segments ▫ Alpha chain: V, J segments ▪ Tracked by CD3, CD4, CD8 cell surface markers Double positive/DP stage ▪ Daughter cells express CD3, CD4, CD8 (double positive/DP stage) Single positive/SP stage ▪ Central tolerance: eliminates potentially self-reactive cells by positive, negative selection ▫ Self-reactive cell elimination failure → autoimmunity ▪ Positive selection ▫ T cells recognize/bind to self-MHC molecules ▫ Binding failure → apoptosis ▪ Negative selection ▫ Autoimmune regulator gene (AIRE): allows primary lymphoid organs to express antigens normally found throughout body; aids in testing selfreactivity ▫ Excessively strong binding to selfantigens → apoptosis Figure 45.18 Structure of T cell receptor. Different combinations of V, D, and J segments provide T cell receptors with a wide variety of antigen specificities. OSMOSIS.ORG 391

Figure 45.19 T cell development summary. 392 OSMOSIS.ORG

Chapter 45 Immunology: B & T Cells ▪ DP cells recognize self-MHC but do not recognize self-antigen presented in MHC molecule → downregulate either CD4/CD8 receptor → further development into single positive (SP) cell ▫ Strong binding to MHC → CD4 downregulated → SP CD8+ T cell ▫ Weak binding to MHC → CD8 downregulated → SP CD4+ T cell VDJ REARRANGEMENT osms.it/VDJ-rearrangement ▪ Mechanism used to generate range of B, T cell receptors ▪ Antigen-binding sites: V, D, J protein segments coded by genes of same name ▫ Each cell inherits multiple V, D, J segments → randomly recombine → recombinational inaccuracy, random assortment of two chains (heavy/ beta chain rearranged first) → new specificities ▪ V(D)J rearrangement only affects V region (creates variability in hypervariable regions) HEAVY/BETA CHAIN REARRANGEMENT ▪ Recombination signal sequence ▫ Heptamer 5’-CACAGTC-3’, 12, 23 nucleotides, nonamer 5’-ACAAAAACC-3’ ▫ DNA loops to bring together two recombination signal sequences ▫ RAG1, RAG2 cut DNA at recombination signal sequence ▫ Recombinases (e.g. ku, artemis) reattach, recombine DNA ▪ Error-prone process ▫ Cut end placed onto terminal deoxynucleotide transferase (TdT) to add random nucleotides → alters antigen specificity ▪ Functionality of heavy chain tested → random assortment of chain LIGHT CHAIN REARRANGEMENT ▪ Rearranged into kappa/lambda light chain (kappa rearranged before lambda) Figure 45.20 Locations of hypervariable regions on BCRs and TCRs affected by V(D)J rearrangement. OSMOSIS.ORG 393

Figure 45.21 Summary of the process by which B and T cell receptors are made. 394 OSMOSIS.ORG