B Cells - Development and Functions PDF

Adaptive immunity (II) B cells – development and functions Prof. David Voehringer, Department of Infection Biology, UKER 1 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombinatio...

Adaptive immunity (II) B cells – development and functions Prof. David Voehringer, Department of Infection Biology, UKER 1 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 2 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 3 Discovery of B cells Max Cooper, Raymond Peterson and Robert Good found that there are two types of lymphocytes (Nature 1965) Removal of the Bursa fabricii in chicken leads to a lack of antibodies. Removal of the thymus leads to a lack of T cells B cells are produced in the B.f. in birds. But: no equivalent organ in mammals. B cells are produced in the bone marrow. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 4  General features of B cells B cells develop in mammals mainly from precursor cells in the bone marrow. Each developing B cell expresses one particular B cell receptor (BCR). Autoreactive B cells get deleted during development (clonal selection) to prevent autoimmunity. Stimulated B cells differentiate to plasma cells that produce antibodies with the same antigen- specificity as the BCR. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 5 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 6  B cell development in the bone marrow Pro-B cells do not yet express a BCR but they express IL-7R. 5 Pre-B cells express a pre-BCR and proliferate in IL-7-dependent manner (30-60-fold expansion) VLA-4: integrin α4β1 CAM: various cell adhesion molecules Kit: receptor tyrosine kinase, also expressed on hematopoietic stem cells and mast cells SCF: stem cell factor Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 7 Differences between pre-BCR and BCR The pre-BCR consists of two covalently linked VH heavy chains with 5 immunoglobulin domains CH1 (1 variable (V) and 4 constant (C)). Igα and Igβ are required for signal CH2 transduction. CH3 VpreB and λ5 constitute the surrogate light CH4 chain which is later replaced by the κ or λ light chain The BCR consists of two covalently-linked VH heavy chains and two covalently-linked light CH1 VL chains. CL CH2 Disulfide-bonds CH3 CH4 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 8 Germline configuration of immunoglobulin gene segments other C segments The genome contains 1 heavy-chain and 2 light-chain (κ, λ) loci V: variable gene segments D: diversity J: joining C: constant Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 9 Rearrangement of immunoglobulin gene segments D to the J and the V to the https://www.pixblick.de/wandtattoo-dj-musik-plattenspieler.html Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 10 Signaling through the BCR Igα and Igβ contain ITAMs Syk contains two SH2 domains that bind to phospho-ITAMs BLNK (SLP65) is an adaptor protein and corresponds to SLP76 in T cells Btk deficiency in humans causes XLA (x-linked agammaglobulinema) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 11 B cell co-receptor complex facilitates BCR signaling BCR signaling is greatly enhanced when the antigen is C3d tagged by complement fragments, engaging the B-cell co-receptor together with the B-cell antigen receptor antigen Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 12  Inhibition of BCR signaling Inhibitory receptors contain ITIMs that recruit SHIP (PIP3 phosphatase) or SHP- 1/-2 (Tyr phosphatases) which negatively regulate the BCR signaling pathway S/V/L/IxYxxL/V ITIM consensus sequence Nimmerjahn and Ravetch. Nat Rev Immunol; Immunology 2003 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 13 Regulation of B cell tolerance How does the immune system deal with self- reactive B cells? 14 Regulation of B cell tolerance to self antigen Receptor editing Secondary Light chain rearrangement Anergy: reversible state of unresponsiveness Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 15 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 16 B cell subsets Mature B cells can be categorized in two main groups: B-1 and B-2 B cells B-2 B cells constitute >85% of all B cells  In the spleen:  15% Marginal zone B cells (MZ B, CD23loCD21hiIgD-)  >70% Follicular B cells (Fo B, CD23hiCD21loIgD+) B-1 B cells originate mainly from fetal hematopoiesis and have a less diverse BCR repertoire  2% B-1a (CD5+)  1% B-1b (CD5-) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 17 Anatomical scheme of spleen architecture Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 18 B-2 cell maturation in the periphery  Immature B cells exit the bone marrow and migrate to the spleen (5-10% of the total B cell pool)  Immature B cells that do not reach the B cell follicle die within 2-3 days.  Transitional B cells (T1) enter the B cell follicle, express the BAFF receptor (TNFR superfamily member) but no CD21 or IgD. BAFF is critical for maturation and survival. BAFF APRIL TNF superfamily BAFF-R TACI BCMA TNFR superfamily B cell TACI is mainly expressed on plasma cells (antibody-producing cells) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 19   T2 B cells upregulate CD21 and either exit to the marginal zone (MZ B cells) or stay in the B cell follicle (FO B cells).  Mature FO B cells co-express IgM and IgD.  The chemokine receptor CXCR5 binds the ligand CXCL13 which is expressed mainly in the B cell follicle and retains mature B cells there. IgD IgM CD21 FOB T1 T2 MZB Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 20 Characteristics of major B cell subsets Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 21  What kind of antigens are recognized by B cells? Examples for B cell antigens  Various 3-dimensional structures  Proteins  Peptides  Carbohydrates  Lipids  Nucleic acids  Synthetic molecules What is an epitope?  Area of an antigen that is recognized by the antibody/BCR  Epitopes can be composed of aminoacids that are far apart in the primary sequence What is a hapten?  Small molecule that only when bound to a protein carrier elicits a strong humoral immune response Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 22 Alternative splicing leads to secreted antibodies BCR Antibody Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 23 Terms used to describe antibodies Isotype: different Fc regions of the heavy chain Idiotype: different V regions Allotype: Minor changes in the Fc region. Allotypic differences only occur between different individuals of a species while different isotypes and idiotypes occur also within one individual. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 24 Antibody fragments and their beneficial effect  Fab – Antigen-binding Fragment (Monomer)  F(ab`)2 – Dimer  Fc – crystallizing (constant) Fragment  What is the advantage of using Fab and F(ab´)2 fragments Pepsin-digest instead of complete antibodies for detection of antigens in tissue sections? F(ab`)2 fragments have a higher avidity as compared to Fab fragments Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 25 Engineered antibodies Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 26 B cell stimulation by antigens Thymus-dependent (TD) antigens  CD4 T cells see antigen on MHC-II on B cells and provide help via CD40L-CD40 and cytokines Thymus-independent (TI) antigens  TI-1: mitogens, activate B cells irrespective of their antigen-specificity. Examples: LPS, bacterial DNA  TI-2: Multivalent antigens (e.g. polysaccharides). X-linking of BCRs. Second signal often required to prevent activation by endogenous multivalent antigens (like DNA, collagen, actin). Second signal can be complement C3d or cytokines (from T cells or other cells). MZ B cells (express CR2 (CD21) which binds C3d) are important for TI-2 response (e.g. infection with Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis). Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 27 Comparison of three groups of B cell antigens Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 28 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 29 The germinal center reaction Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 30 Germinal center reaction Recirculating B cells enter the LN via high endothelial venules (HEV), migrate through the T cell zone and present peptide antigens on MHC-II. Cognate interaction leads to activation, migration into the B cell follicle and formation of a germinal center Activated B cells differentiate to plasma cells and memory B cells and leave the lymph node. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 31 The germinal center serves three major tasks Follicular T helper cell (TFH) CXCR3, PD-1, Bcl-6, IL-21 1. Immunoglobulin-class switch IL-4 (Th2) => IgE, IgG1 IFN-γ (Th1) => IgG2a, IgG3 TGF-β (Treg) => IgA 2. Affinity maturation of antibodies 3. Generation of memory B cells and plasma cells Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 32 The germinal center GC B cells cycle between light zone (LZ) and dark zone (DZ). In LZ they see their antigen on follicular dendritic cells (FDC) and present it to follicular T helper cells (TFH) to receive a positive signal. In DZ they proliferate and undergo somatic hypermutation to increase the affinity of the BCR. Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 33 Interaction between TFH cells and GC B cells TFH cells lose CCR7 and express CXCR5 to enter the B cell follicle. Multiple receptor-ligand interactions are required for GC B cell activation by TFH cells. Most importantly CD40-CD40L Short-lived long-lived when it finds suvival niche Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 34  Strong changes in metabolism Can divide every 4-6 hours Li et al. J Immunol Res. 2024 - Each subset of B cells has a unique metabolic profile/requirement. - Mitochondria, dynamically adjust to provide required energy but they are also involved in cell death. 35 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 36 Transcriptional changes during PC development Most plasma cells lose surface expression of the BCR Differentiation BLIMP-1 is a Zn-finger transcriptional repressor. Forced expression of BLIMP-1 induces PC PAX5 is a master differentiation. transcription factor for B XBP-1 is required to tolerate ER cell development stress response. (Also required for eosinophil development). Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 37 Features of plasma cells PCs secrete thousands of immunoglobulin molecules (antibodies) per second. PCs loose expression of CD19, CD20 and BCR but upregulate expression of CD138 (Syndecan-1), TACI, (CD27 in humans) and CXCR4 for migration into the bone marrow where they survive for years in a special niche. Columbia.edu 38  The PC survival niche in the bone marrow The survival niche for PCs in the BM is formed by stromal cells, megakaryocytes and eosinophils which produce IL- 6, APRIL and other survival factors to keep PCs alive for several months to years. TACI APRIL IL-6 Eosinophils Megakaryocytes 39  Longevity of humoral immunity by vaccines The half-life of specific antibodies after immunization with tetanus toxid or attenuated measles virus ranges from 11 to over 3000 years. N Engl J Med 2007; 357:1903-1915 40 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 41 Immunoglobulins – Generation of Diversity Class switch recombination (CSR) Gene conversion (e.g. in birds and rabbits) Somatic hypermutation (SHM) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 42 Immunoglobulins – Generation of Diversity Class switch recombination (CSR) Gene conversion Somatic hypermutation (SHM) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 43 Five different antibody isotypes Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 44 Mechanism of class switch recombination Switch regions (S) have to be transcriptionally active before CSR can occur. DSBR: Double strand break repair machinery AID (activation induced cytidin-deaminase) is required for CSR and somatic hypermutation Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 45 Multimerization of IgA and IgM by the J-chain J-chain: connects antibody monomers to form dimers (IgA) or pentamers (IgM) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 46 Epithelial transcytosis of IgA requires the J-chain Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 47 Characteristics of different antibody isotypes Anti-Inflammatory Activity of Human IgG4 Antibodies by Dynamic Fab Arm Exchange Van Der Neut Kolfschoten et al. Science 2007 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 48 Regulation of CSR by cytokines Th2 Th2 Th1 Treg Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 49 Immunoglobulins – Generation of Diversity Class switch recombination (CSR) Gene conversion (e.g. in birds and rabbits) Somatic hypermutation (SHM) 50  Somatic Hypermutation AID: activation induced Unprecise repair Cytidin-deaminase => Mutations 51 Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) Somatic Hypermutation FR: framework region HV: hypervariable region = CDR: complementarity determining region V D J V J Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 52 Memory response Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 53 B cells Discovery B cell development B cell receptor B cell subsets Germinal center reaction Plasma cells Antibody isotypes (Class switch recombination) Affinity maturation Antibody effector functions Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 54 Effector functions of antibodies Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 55 Fc receptors (affinities are from human data) Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 56 ADCC and CDC Antibody- dependent NK cell cellular Complement- cytotoxicity dependent (ADCC) cytotoxicity (CDC) MAC: membrane attack complex IMGT Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 57 Cytokine-producing B cells Some B cells produce pro-inflammatory cytokines like IL-6, IFN-γ or GM-CSF. „Regulatory“ B cells secrete IL-10 and/or IL-35 and prevent severe autoimmunity in a mouse model of multiple sclerosis. Some plasma cells can also produce cytokines Prof. D. Voehringer, University Hospital Erlangen and Friedrich-Alexander University Erlangen-Nuremberg (FAU) 58 Spectrum of B cell-regulated conditions 59 Summary B cells develop in the bone marrow via pro- and pre-B cells to mature B cells The BCR is generated by VDJ recombination and consists of two heavy and two light chains. Signaling occurs via ITAMs in Igα and Igβ. Two major B cell subsets: B-1 (from fetal development) and B-2. B-2 in the spleen mature to follicular or marginal zone B cells. B cells interact with FDCs and Tfh cells in germinal centers. The germinal center reaction is important for CSR, SHM and generation of memory B cells and plasma cells. PCs can be long-lived in the bone marrow where they reside in special niches. Five different antibody isotypes are generated and they have different effector functions by activation of the complement cascade or binding to Fc receptors. B cells and PCs can also produce cytokines that promote or inhibit immune responses. 60