B-Cell Mediated Immunity I (PDF)

11/20/2023 TH2 TFH TFH TH1...

11/20/2023 TH2 TFH TFH TH1 Adaptive Immunity: CD4 CD40L TCR B-Cell Mediated Immunity I CD4 IL-2R TCR MHC-II CD4 IL-2 T CELL DC MHC-II CD40 Y MCB 11338 – Immunology B CELL LS Meadows CR2 MHC-I Bm TCR CTC IL-2 IL-2R plasma CD8 cell m Activation of the Adaptive Immune Response 4. Naïve B Cells 1. Dendritic cells enter 2° lymphoid tissue enter 2° lymphoid tissue from blood/lymph present antigenic peptides with MHC-I and MHC-II use BCRs to “scan” unprocessed antigens on 2. Naïve CD4 T cells enter 2° lymphoid tissue “follicular” DC (or macs) activated by DCs w/MHC-II endocytose BCR:ag complexes 3. Naïve CD8 T cells enter 2° lymphoid tissue process & present peptides with MHC-II to TFH activated by DCs (or DC + TH1) w/MHC-I cells 4. Naïve B cells enter 2° lymphoid tissue activated B cells activated by TFH cells proliferate & differentiate into plasma cells and memory B cells TFH cells provide various cytokines (IL-21, IL-6, et al.) to influence this 4. Naïve B Cells (continued) Plasma Cells = Effector B Cells activation requires 3 signals: initially secrete low-affinity IgM antibodies BCRs cross-linked by intact antigens some undergo affinity maturation and class-switching {covered later} B cell coreceptor molecules (CR2, secrete high-affinity IgG (or other classes) antibodies CD19, CD81) bind C3d (on antigen) CD40 binds CD40L on TFH cells 1  11/20/2023 LECTURE OUTLINE B Cells: An Overview I. Overview generate unique B cells circulate B cells through body each with a unique BCR blood, lymph, 2° lymphoid tissues II. B Cell Receptor Diversity eliminate self-reactive B cells activate antigen-specific B cells III. B Cell Development negative selection proliferate into clone IV. B Cell Maturation produce mature B cells differentiate activated B cells positive selection plasma cells & memory B cells V. B Cell Abnormalities VI. B Cell Activation VII. Antibody Structure & Function VIII. Antibody Receptors BCR versus TCR BCR (Ig) Gene Segments B Cell Receptor (BCR) T Cell Receptor (TCR) germline configuration of B-cell receptor / immunoglobulin genes 4 polypeptide chains 2 polypeptide chains inherited from egg & sperm 2 identical “heavy” chains one alpha chain fragmented == several gene segments (L, V, D, J, C); multiple alleles μ, δ, γ, α, OR ε one beta chain 2 identical “light” chains expression requires segment rearrangement & assembly into one κ OR λ complete functional gene BCR (Ig) Gene Segments Constant (C) Region Genes λ light chain: 4 or 5 Cλ possible gene segments к light chain: only 1 Cк possible gene segments heavy chain: 9 CH possible gene segments 2  11/20/2023 Figure 4.17 The numbers of functional gene segments available to construct the variable and constant regions of human immunoglobulin heavy chains and light chains 40 Variable Region Genes Somatic Recombination λ light chain: ~30 Vλ options + 4 or 5 Jλ options [V+J] cutting & splicing of Ig gene segments during B-cell development к light chain: ~35 Vк options + 5 Jк options [V+J] light chain gene = 1 recombination event [VL + JL] heavy chain gene = 2 recombination events [DH + JH; then VH + DJ] heavy chain: ~40 VH options + ~23 DH options+ 6 JH options [V+D+J] creates diversity in antigen-binding sites in BCR & Ig Recombination Signal Sequences (RSSs) Recombination Signal Sequences (RSSs) short DNA sequences that “flank” V, J, and D gene segments 12/23 rule 3’ side of V segments recombination only occurs between two RSSs with different length spacers 5’ side of J segments both sides of D segments light chains V+J heptamer CACAGTG heavy chains spacer (23 or 12 nt) D+J nonamer V + DJ ACAAAAACC NOT V + J 3  11/20/2023 V(D)J Recombinase RAG Complex complex of enzymes & other proteins that Y-shaped complex formed from two RAG-1 performs somatic recombination in B cells and two RAG-2 proteins RAG-2 = cofactor for RAG-1 RAG-1 and RAG-2 == functional RAG complex precisely aligns the RSSs to be cut & spliced recombination-activating gene proteins each RAG-1 molecule has subunits that bind to only expressed in developing B cells and T cells heptamer & nonamer sequences on each RSS during antigen-receptor gene rearrangement Figure 4.19 Rearrangement of V gene segments is required to make immunoglobulin genes functional V(D)J Recombinase endonuclease cleaves RSS at end of each heptamer produces four broken ends DNA repair enzymes recombine four ends in new configuration form two new DNA joints coding joint = creates functional V region exon signal joint = circularizes excised DNA Figure 4.36 Gene rearrangement and the synthesis of cell-surface IgM in B cells How many unique antigen-binding sites, so far? lambda light chain = kappa light chain = heavy chain = (heavy)(lambda) = _______ antigen-binding sites (heavy)(kappa) = ________ antigen-binding sites TOTAL = _________ unique antigen-binding sites 4  11/20/2023 Junctional Diversity Junctional Diversity DNA sequence variations created in coding P (palindromic) nucleotides joints during the process of V(D)J generated by RAG complex https://youtu.be/QTOBSFJWogE recombination N (non-templated) nucleotides creates additional diversity in antigen-binding added by terminal deoxynucleotidyl transferase (TdT) sites (“hypervariable” regions) Figure 4.20 The generation of junctional diversity during gene rearrangement How many unique antigen-binding sites, now? B Cell Development ~ 1,800,000 unique antigen-binding sites begins in bone marrow from random combinations of V(D)J and light/heavy chain combinations B-cell precursor WITHOUT junctional diversity pro-B cell pre-B cell immature B cell junctional diversity == up to ___________________ more variations continues in 2° lymphoid tissues (1.8 million)(_______________) = _________________________ mature B cell activated B cell differentiated B cell ____________ theoretically possible unique antigen-binding sites plasma cell or memory B cell 5  11/20/2023 Bone Marrow Stromal Cells Bone Marrow B Cell Stages stroma = supportive, connective tissue cells (non-lymphoid cells) “early” pro-B cell == D & J heavy chain gene segments rearranging create specialized microenvironment contact developing B cells via adhesion molecules (VLA-4, VCAM-1, et al.) “late” pro-B cell == V & DJ heavy chain gene segments rearranging produce growth factors (SCF = stem cell factor, IL-7) “large” pre-B cell == μ heavy chain made & tested == pre-B cell receptor “small” pre-B cell == V & J light chain gene segments rearranging immature B cell == B cell receptor expressed on cell surface BCR = membrane bound IgM Bone Marrow B Cell Stages BCR Gene Rearrangement = Inefficient addition of N & P nts may shift reading frame (frameshift mutations) nonproductive rearrangement cannot be translated into functional protein productive rearrangement can be translated into functional protein pre-B-cell B-cell light chain genes only rearranged if productive receptor receptor heavy chain rearrangement occurs Heavy Chain Gene Rearrangement Heavy Chain Gene Rearrangement 2 chromosomes (maternal, paternal) productive rearrangement == production of μ heavy chain D + J = usually successful (all three ORF in D segment “work”) pro-B cell becomes large, dividing pre-B cell usually both chromosomes undergo D & J rearrangement simultaneously V + DJ = only 1/3 successful nonproductive rearrangement “positive” one chromosome randomly selected; other chromosome used if needed selection pro-B cell dies via apoptosis 40 6  11/20/2023 Pre-B-Cell Receptor Pre-B-Cell Receptor tests ability of μ heavy chain to bind to assembled in ER only IF μ heavy chain a “surrogate” light chain can bind to surrogate light chain components mainly located in intracellular vesicles μ heavy chain dimer low level cell surface expression 2 surrogate light chains does NOT bind antigen Igα & Igβ {covered later} no pre-BCR assembly == apoptosis surrogate light chain proteins VpreB λ5 Figure 6.7 The pre-B-cell receptor resembles the B-cell receptor Pre-B-Cell Receptor Expression cell signaling pathways activated H chain gene rearrangement halted RAG gene transcription stops RAG proteins degraded chromatin “hides” unused H chain locus (allelic exclusion) cell division stimulated large clone of small pre-B cells produced, all making μ heavy chains Bone Marrow B-Cell Stages Large Pre-B Cell → Clone of Small Pre-B Cells all produce same μ heavy chain no surrogate light chains made no pre-B-cell receptors expressed RAG genes reactivated for light chain gene rearrangement occurs independently in each small pre-B cell each small pre-B cell produces its own unique light chain pre-B-cell B-cell receptor receptor 7  11/20/2023 Figure 6.8 The organization of the light-chain loci allows nonproductive rearrangements to be followed by a productive rearrangement Light Chain Gene Rearrangement four light chain chromosomes available (2 kappa, 2 lambda) kappa chromosomes used first if nonproductive arrangements occur, then lambda chromosomes used multiple rearrangement attempts possible on each chromosome Light Chain Quality Check Light Chain Quality Check light chain produced from first successful good light chain == surface expression of productive rearrangement IgM == immature B cell gene rearrangement machinery shut down must be able to bind to heavy chain “positive” selection dimers in ER failure to bind == further light chain gene rearrangements if no good light chain after all four light chain chromosomes used == apoptosis Figure 6.10 Two checkpoints determine the fate of B cells during their development in the bone marrow Bone Marrow B-Cell Stages “positive” selection pre-B-cell B-cell receptor receptor 8  11/20/2023 Allelic and Isotypic Exclusion ensures that each B cell produces monospecific antibodies same antigen-binding site heavy chains made with same variable domain one type of light chain (kappa OR lambda) unrearranged heavy & light chain genes are “excluded” (not used) Self-Tolerant Immature B Cells Expression of Membrane-Bound IgM BCRs DO NOT bind to self-antigens in BM Following VDJ rearrangement long primary RNA transcript made leave BM to mature in 2° lymphoid tissues contains Cμ exons & Cδ exons alternatively splice heavy-chain mRNA RNA processing == mRNA made increase production of membrane-bound IgD poly-A tail added at pAμm site introns removed mature (naïve) B cell exons spliced together including M & C exons for express both IgM and IgD BCRs transmembrane region mRNA translated into μ heavy chain Figure 4.22 Coexpression of IgD and IgM is regulated by RNA processing Expression of Membrane-Bound IgD Following VDJ rearrangement long primary RNA transcript made contains Cμ exons & Cδ exons RNA processing == mRNA made poly-A tail added at pAδm site introns removed exons spliced together including M & C exons for transmembrane region mRNA translated into δ heavy chain 9  11/20/2023 Figure 6.23 Summary of the main stages in B-cell development Self-Reactive Immature B Cells: Two Types Bind to Multivalent Self-Antigens Bind to Monovalent Self-Antigens on cells found in BM on soluble proteins in BM retained in BM & undergo stimulated to become anergic receptor editing die within a few days “negative” selection Figure 6.16 Immature B cells with specificity for multivalent self antigens are retained in the bone marrow Receptor Editing main mechanism for inducing self-tolerance in immature B cell population immature B cells recognizing multivalent self-antigens decrease IgM production continue to express RAG complex proteins continue to rearrange light chain genes Receptor Editing Anergic Immature B Cells if new BCR is self-tolerant, immature B cell … immature B cells recognizing monovalent self-antigens leaves BM and enters blood make IgM travels to secondary lymphoid tissue to finish maturation most retained inside cell (does not form BCR) make IgD if new BCR is self-reactive, process continues until … forms BCR but does NOT activate B cell upon antigen binding a self-tolerant BCR is produced OR leave BM and enter blood cell runs out of light chain genes == apoptosis most die within 1 to 5 days clonal deletion selective elimination of self-reactive antigen receptor specificity from B cell repertoire 10  11/20/2023 Types of Self-Tolerance Autoimmune Diseases Central Tolerance Peripheral Tolerance can occur when mature B cells are exposed induced w/in 1° lymphoid induced outside 1° lymphoid to normally inaccessible self-antigens tissues tissues stress, disease, trauma to self-antigens accessible to to self-antigens accessible to immature B cells in BM circulating immature B cells systemic lupus erythematosus (SLE) autoantibodies made to proteins and nucleic mechanisms: mechanisms: acids in ribosomes & nucleosomes receptor editing apoptosis apoptosis anergy anergy (NO receptor editing – too late) B Cell Maturation & Survival Self-Tolerant Immature B Cells occurs in lymphoid follicles in 2° lymphoid tissues leave BM; recirculate through blood, 2° lymphoid tissues, lymph immature B cells express high level of IgM BCRs; low level of IgD must interact with follicular dendritic cells (FDC) to BCRs complete maturation complete maturation in 2° lymphoid tissue mature B cells lymph node must interact periodically with FDCs to survive spleen similar microanatomy = will die ~100 days if specific antigen not encountered Peyer’s patch facilitates maturation process tonsil etc. Figure 6.23 Summary of the main stages in B-cell development Immature B Cells in Lymph Node attracted to LN by chemokines CCL21 (from LN stromal cells) CCL19 (from LN dendritic cells) enter LN from blood via HEV high endothelial venules 11  11/20/2023 Immature B Cells in Lymph Node Immature B Cells in Lymph Node follicular dendritic cells (FDC) immature B cells & FDC interact specialized LN stromal cells stim. B cells to mature & survive attract B cells to 1° follicles (PF) by maintains integrity of FDC network secreting CXCL13 lymphotoxin (LT) immature & mature B cells surface protein & soluble factor compete to enter 1° follicles produced by B cells most immature B cells fail & die by apoptosis B cell activating factor in TNF family (BAFF) soluble factor sec. by many anergic B cells also enter LN and die by apoptosis different 2° lymphoid tissue cells Figure 6.20 Immature B cells must pass through a primary follicle in a secondary lymphoid tissue to become mature B cells Mature B Cells if specific antigen NOT encountered exit LN & circulate through blood, lymph, 2° lymphoid tissues must periodically interact with FDCs to survive will die ~100 days if specific antigen not encountered B Cell Abnormalities B Cell Deficiency Diseases B Cell Deficiencies B Cell Tumors X-linked agammaglobulinemia Common variable impaired antibody production uncontrolled B cell growth & X-linked immunodeficiency with immunodeficiency (CVID) proliferation hyper-IgM (XHM) Kappa/lambda light-chain Selective IgA deficiency deficiency causes: cutting, splicing, mutating Ig Immunodeficiency with intrinsic molecular B cell defects Selective IgM deficiency genes thymoma lack of interaction between B cells IgG subclass deficiency increases chance of malignant and T cells transformation in B cells IgE hypogammaglobulinemia Transient often results in chromosomal hypogammaglobulinemia of Hyperimmunoglobulin E (HIE) translocations & activation of infancy (THI) syndrome proto-oncogenes 12  11/20/2023 X-Linked Agammaglobulinemia immunodeficiency = no antibodies produced recurrent infections with H. influenzae, S. pneumoniae, S. aureus defective BTK (Bruton’s tyrosine kinase) essential for B cell development cell signally pathways B cells cannot develop & mature treated with intravenous immunoglobulin B Cell Tumors Proto-Oncogenes many associated with chromosomal can cause cancer when expressed at the wrong time and/or in the translocations and/or activation of wrong amounts proto-oncogenes become oncogenes provide opportunities to increase first oncogenes found in understanding of the regulation of RNA tumor viruses cell growth and cell division “evolved” from host proto-oncogenes Proto-oncogene to Oncogene Burkitt’s Lymphoma MYC gene (chromosome 8) cell cycle control gene; strictly regulated translocated of MYC to Ig gene chromosome (2, 14, or 22) == continual production of MYC protein B cells grow & divide uncontrollably may acquire additional mutations == cancer 13  11/20/2023 B Cell Lymphoma LECTURE OUTLINE BCL2 proto-oncogene I. Overview anti-apoptotic protects B cell lineage from premature II. B Cell Receptor Diversity apoptosis III. B Cell Development IV. B Cell Maturation if over-expressed, B cells live too long V. B Cell Abnormalities may acquire additional mutations == cancer VI. B Cell Activation VII. Antibody Structure & Function VIII. Antibody Receptors Figure 6.23 Summary of the main stages in B-cell development B Cells: An Overview generate unique B cells circulate B cells through body each with a unique BCR blood, lymph, 2° lymphoid tissues eliminate self-reactive B cells activate antigen-specific B cells negative selection proliferate into clone produce mature B cells differentiate activated B cells positive selection plasma cells & memory B cells 14