The Humoral Immune Response

Transcription

1 The Humoral Immune Response BBS755 March 19, 2015 Robert Woodland AS

2 Reading: Janeway Chapters 10 and 12

3 Starting point A naive B cell is recirculating resting lymphocyte with a selected receptor for antigen that secretes no immunoglobulin. This dormant state is the most efficient way to store vast immune potential at a reasonably low metabolic cost. This immune potential is assessed by B cell activation which is a complex process involving proliferation and differentiation facilitated by interactions with dendritic cells, cytokines and frequently T cells. How do we get from this resting cell to a fully active humoral immune response?

4 Lecture objectives A. The anatomy of secondary lymphoid organs B. Ag presentation and activation of B cells C. The germinal center reaction. D. Somatic hypermutation,class switching and memory E. Primary vs. secondary immune responses F. Antibody effector functions G. Fc Receptors and complement H. Mucosal immune system

5 Secondary lymphoid organs provide a scaffold for cell interactions and activation and a supportive environment for proliferation, selection and differentiation

6 Nature reviews IMMUNOLOGY 5: Spleen organization

7 Organization of lymphocytes around the central arteriole

8 Lymph node organization

9 Differences in lymphocyte recirculation patterns between spleen and lymph nodes

10 Antigen activation

11 B cell activation requires at least 2 signals 1. BCR + co-receptor (CD21/CR2-CD19-CD81) (signal 1) 2. Co-stimulation receptors: Best examples: CD40, Toll Like Receptors (TLRs) Dendritic cells and FDCs present antigen and express CD40 ligand (CD40L). They secrete cytokines, e.g. Type 1 IFN, that help B cells proliferate and differentiate. T H and T FH cells express CD40L and secrete cytokines (e.g. IL-4, IL-10, IL-2, IL-5, IL-6, IL-21) that help B cells proliferate and differentiate.

12 C3b split products (C3d and C3dg) bind to the BCR coreceptor CD21-CD19, greatly increasing B cell activation. CD21 is present on B cells and also on follicular dendritic cells (FDCs). Carroll and Isenman, 2012

13 Thymus independent antigens There are 2 classes of TI antigens: 1) TI-1 antigens such as LPS contain mitogenic moieties and activate toll-likereceptors. 2) TI-2 antigens are polyvalent, like bacterial capsular polysaccharides, and show no requirement for T cell help. They cross-link surface BCRs. TI responses: 1) are mediated mostly by marginal zone (MZ) and B1b B cells. 2) sometimes induce short-lived germinal centers. 3) are mostly IgM 4) show low levels of SHM. 5) can produce B cell memory 1. Antigen binding to BCR Nature of second signal not always known. Likely to be from TLR. 2. TLR ligand binding to TLR

14 Toll-like receptors (TLRs) are on B cells Can provide co-stimulation and can partially substitute for CD40, but they do not lead to formation of germinal centers. TLR and CD40 signals are both provided during infection. TLR provides a "danger signal". TLRs are rapidly induced on follicular B cell surfaces upon activation. MZ B cells have constitutively upregulated TLRs. TLR ligands are from pathogens. Other pattern receptors can also activate B cells.

15 Thymus dependent antigens Thymus-dependent (TD) antigens are proteins and B cells require help from T cells to mount a response. T cell help is by cell:cell contact and by the secretion of T cell derived cytokines. T cell help induces CSR, SHM, differentiation to antibody secreting cells and the induction of B cell memory.. IgM CD21-CD19-CD81 CD4+ T H 2 cell TCR MHC CD28 CD40L IL-4 B7 CD40 B cell

16 Stimulation from T helper cells is required for optimal antibody responses to protein antigens. IgM Protein Ag with C3d/C3dg fragments bound CD21-CD19-CD81 Antigen processing TCR MHC CD4 + T cell CD28 CD40L MHC B7 CD40 B cell NF-κB NF-AT B cell activation is by BCR-CD21- CD19-CD81 cross-linking. This increases B7 (CD80, CD86 expression). B cells bind Ag, endocytose it, present peptides to T cells on MHC class II. T cells with TCR specific for the peptide are then activated and up-regulate CD40L (CD154) expression. Induced on both TH1 and TH2 cells. (Dendritic cells and FDCs also express CD40L.) A special class of T helper cells T follicular helper cells is critically important for the stimulation of antibody secretion and memory B cell formation. CD40 - most important receptor for secondary (co-stimulatory) signals for B cells in T- dependent responses. Equivalent to CD28 for T cells. CD40 signaling induces NF-κB and NF-AT transcription activation factors (and others). CD40 signaling further induces B7 expression. Other ligand-receptor interactions also contribute.

17 CD40L or CD40 deficiency in humans and mice Hyper-IgM syndrome Little or no antibody class switching Little or no somatic hypermutation - no affinity maturation No germinal centers Patients require transfusions of IgG This demonstrates how important CD40 signaling is for B cell immune responses.

18 Cytokines are also important for B cell activation. IgM CD21/CD19 CD4+ T H 2 cell TCR MHC CD28 CD40L B7 CD40 B cell, IL-4 Most cytokines that help B cells are secreted by activated Th2 and Tfh cells: IL-4 increases viability, proliferation,. and stimulates switching to IgG1 and IgE in mouse B cells, and to most IgG isotypes and IgE in humans. IL-5 and IL-6 both promote differentiation to plasma cells, increase Ab secretion. IL-21 is required for plasma cell differentiation and IgG production. Th1 cytokines (IFNγ and IL-2) provide some help also. IFNγ stimulates proliferation and CSR to IgG2a in mouse. IL-2 helps B cells differentiate toward antibody secretion (similar to IL-5, IL-6).

19 Key Points Similar to T cells, B cells have an Ag receptor/co-receptor (CD21-CD19-CD81) that provides signal 1. Binds Ag-C3d and IgM-Ag-C3d complexes. Most important function of co-receptor is to help Ag internalization, processing and presentation to T H cells. Like T cells, B cells have co-stimulation receptors (CD40 and TLRs). Cytokines from T cells and Ag-presenting cells improve B cell responses, and regulate Ig isotype switching. Cognate interactions between B and T cells stimulate both interaction partners.

20 The germinal center reaction Basis for understanding humoral immunity Ag processing and presentation B and T cell activation Lymphocyte traffic and chemokines Somatic hypermutation and B cell affinity maturation Negative selection of autoreactive clones Generation of immune effectors Generation of precursor cell based immunological memory Generation of humoral memory Cellular regulation of immune responses

21 Histological analysis of germinal center formation

22 The need for a dynamic in vivo analysis

23 Multi Photon Microscopy

24 Comparison of one vs. two photon excitation Lower intensity photons lead to less photobleachi Cahalan etal (2002) Nat.Rev.Im

25 Visualization of cellular movement in living tissue intravital staining. Schwickert etal. (2007) Nature

26 Follicular B (B2) cells are initially activated by antigen presented by dendritic cells in lymph nodes. Naive B2 cells circulate from high endothelial venules and B cell follicles in lymph nodes, entering the T cell zone. If they encounter Ag that they can bind, presented on a dendritic cell, they stop moving, become activated (in the T cell zone), move to the T-B zone boundary, and proliferate. Initially, the frequency of Agspecific Th cells will be very low, so the B cells will interact with dendritic cells that present Ag (APCs). Schwartzberg et al. 2009

27 After B cells are activated by Ag in lymph nodes, they move to a B cell follicle. A few days after immunization or infection, activated Ag-specific Th cells will be present in the T cell zone. The B cells receive specific signals which further activate them to move to a nearby B cell follicle, to divide, and differentiate. There they interact with follicular dendritic cells (FDCs) and follicular helper T (Tfh) cells, and after ~ 7 days form a germinal center within the B cell follicle. Schwartzberg et al. 2009

28 Marginal zone (MZ) B cells are also activated by antigen from blood within the spleen marginal zone, and then they travel directly to B cell follicles. MZ macrophages, dendritic cells and neutrophils present non-degraded Ag from blood to MZ B cells.

29 Classic GC reaction Phase II

30 Anatomy of a germinal center Victora and Nussenzweig Annurev-Immunol. 30:2012

31 Two processes that modify the immunoglobulin gene of a naïve B cell after activation are : class switch recombination (CSR) which alters the Ig heavy chain expressed and somatic hypermutation (SHM) which alters the Ig heavy and light chain variable regions. These processes are initiated in the germinal center and both require activation induced cytidine deaminase (AID).

32 Antibody Class (or Isotype) Switching Ag Ag Ag Ag Variable regions Constant regions IgM or IgD IgG, IgE or IgA Naive B cells express IgM and IgD. After B cells are activated by immunization or infection, they switch from IgM and IgD to IgG, IgE or IgA expression. This switch improves the ability of the antibody to eliminate pathogens.

33 Isotypes and Allotypes Isotypes - related proteins encoded by different genes, i.e. different loci. Examples: µ H chain and α H chain Allotypes - proteins encoded by different alleles of the same locus. Examples: proteins encoded by the H2-D or Ig Cµ locus from the mother s and father s chromosomes (e.g. H2D d and H2D b or Cµ b and Cµ a ) S

34 Antibody Class Switch Recombination (CSR) Heavy chain genes in IgM/IgD expressing cell (mouse) VDJ Cµ Cδ Cγ3 Cγ1 Cγ2b Cγ2a Cε Cα S Switch recombination Heavy chain genes in IgEexpressing cell µ mrna S δ mrna Cδ VDJ VDJ S Cε Cε S AID (T. Honjo) Cγ2a Cα Cα S S S Germline transcript CSR allows a B cell that is activated Cγ1 by antigen binding (and co-stimulatory signals) to switch Cγ2b to expressing one of Cγ3 these downstream C H genes. Activation-induced cytidine deaminase (AID) is required for CSR and for SHM. B cells can switch to any Cµ of the downstream C H genes, depending on the antigen and co-stimulatory signals. Initiates 2-3 days after infection or immunization. AID KO mice created by T. Honjo s group.

35 Germline (GL) transcripts are required for CSR to make substrate for AID. Transcription directs CSR to a specific switch region and C H gene. Transcription is induced by cytokines that direct switch to that same C H gene. Examples: TH2 cytokine IL-4 induces GL γ1 and ε transcripts and CSR to IgG1 (mouse) and IgE. TH1 cytokine γ-interferon (γifn) induces GL γ2a transcripts and CSR to IgG2a (mouse). TGFβ induces GL γ2b and α transcripts and CSR to IgG2b and IgA.

36 CSR occurs by an intrachromosomal deletion between switch (S) regions located 5' (upstream) of each C H gene. Heavy chain genes in IgM/IgD expressing cell VDJ AID Cµ Cδ Cγ3 Cγ1 Cγ2b Cγ2a AID Cε Cα S µ mrna S δ mrna S Cγ1 S S S S Germline transcript Switch recombination Cγ3 Cδ Cµ Cγ2b Cγ2a AID (Activation-induced cytidine deaminase) is required for CSR and for SHM. VDJ Cε Cα AID KO mice were created by T. Honjo s group. Heavy chain genes in IgEexpressing cell VDJ Cε Cα ε mrna

37 AID initiates CSR by deamination of dc residues G C AID (activation-induced cytidine deaminase) G U (M.Neuberger et al)

38 Ung excises the du base during CSR AID G C Base Excision Repair (BER) Uracil-DNA Glycosylase (Ung2) G U G Ung -/- B cells: 95% reduced CSR (Rada, Petersen and Neuberger)

39 AID initiates CSR by deamination of dc residues AID (Activation-induced cytidine deaminase) Base Excision Repair (BER) Uracil-DNA Glycosylase (Ung) G C G U G Ung -/- B cells: 95% reduced CSR AP Endonuclease DNA pol β G G C Initiates class switch recombination if occurs on both strands.

40 Function of class switch recombination Changes the antibody H chain constant region, which changes the class (isotype) of the antibody. This changes the effector function of the antibody. The antibody class determines which Fc receptors it can bind to, its ability to bind and activate complement, its ability to pass through epithelial membranes, and the antibody stability. Certain antibody classes are better for resistance to extracellular bacteria; other classes for viruses, or parasites.

41 Antibody variable region (VDJ) genes undergo somatic hypermutation after activation by antigen. Somatic hypermutation (SHM) of the V genes initiates ~ 7 days after immunization or infection, and occurs in germinal centers found in both spleen and lymph nodes. SHM combined with selectionimproves the affinity of the antibody for antigen, by as much as 10 3 fold.

42 Somatic hypermutations are found in the first 1 kb of transcribed recombined V(D)J genes Site of transcription initiation VJ J3 J4 C This localization restricts mutations to V(D)J genes and surrounding introns, and results in unmutated C genes. Within this region, mutations are introduced into both CDRs and FRs, and can be deleterious or helpful.

43 AID initiates somatic hypermutation of V genes by deamination of dc residues G C G C A T replication Somatic hypermutation G U AID (activation-induced cytidine deaminase) Uracil-DNA Glycosylase (UNG) G C A T C G T A replication Somatic hypermutation G G AP Endonuclease Exonuclease and error-prone DNA synthesis mutates A:T bp.

44 Somatic hypermutation of antibody V regions 1 week after immunization or infection, nucleotide substitutions begin to be introduced into recombined V(D)J genes expressed in B cells that are activated by antigen binding. Mutations are introduced randomly and so they can destroy the antibody, or they can increase ability of BCR to bind antigen and these are selected. Example of sequences from mice immunized with phenyloxazolone Legend Replacement mutations Silent mutations

45 Timing of CSR and SHM Class switch recombination begins a few days after immunization or infection, first in T cell areas, and then in B cell follicles. This was discovered by immunizing mice expressing a transgenic BCR, so there were enough Ag-specific cells that could be detected with antibodies to IgG1. B cell follicles differentiate to form germinal centers about one week after immunization or infection, and this is when SHM initiates. After several weeks, germinal centers disappear because memory B cells and antibody secreting cells are dispersed.

46 Key Points: CSR and SHM CSR and SHM occur after activation of B cells in humans and mice by antigen and T cells. Both CSR and SHM require AID, which initiates DNA lesions that are not accurately repaired, leading to DSBs and mutations. Isotype to which B cells switch is determined by type of pathogen. The pathogen determines which cytokines are produced and which downstream C H genes are transcribed. Mutations are introduced into expressed V genes and surrounding intron sequences. B cells expressing BCR s with increased affinity due to mutations are selected. B cells with lower affinity die by apoptosis.

47 Affinity maturation requires selection of high affinity B cell clones.

48 Anatomy of a germinal center Victora and Nussenzweig Annurev-Immunol. 30:2012

49 Cellular dynamics within the germinal center

50 Positive selection of high affinity B cell clones

51 TfH cell based competition model for selection of high affinity clones Victora and Nussenzweig Annurev-Immunol. 30:2012

52 Generation of autoreactive responses Failure of T cell tolerance

53 Memory B cells and long-lived plasma cells are the end products of the germinal center reaction

54 Victora and Nussenzweig Annurev-Immunol. 30:2012

55 Memory B cells are generated in germinal centers. B memory cells are defined functionally. Functional definition: Cells that have divided in response to Ag and that can transfer memory to naïve recipients, and are present late after immunization/infection. B cells expressing switched isotypes on their surface, or that have undergone SHM and are present late after immunization are memory cells. However, there are also memory IgM+ cells. These are produced early in the response and do not require TfH. Like memory T cells, memory B cells are more easily activated than naïve B cells, and long-lived. Easier activation is partly due to changes in intracytoplasmic domains of the H chains. Memory B cells are found in spleen, lymph nodes, and they also traffic through the body. Human memory B cells express CD27 but other cells do too. This marker

56 Two types of plasma cells Shortly after B cell activation and prior to germinal center formation, shortlived IgM plasma cells can be generated. These plasma cells have not undergone SHM. Later, long lived plasma cells differentiate in germinal centers during a primary immune response. Long-lived plasma cells can also differentiate from memory B cells upon secondary immunization/infection. They usually have undergone class switching and SHM.

57 Plasma cells generated in germinal centers are very long-lived Long-lived plasma cells reside for years in the bone marrow, continuing to secrete antibodies. Bone marrow resident plasma cells mostly secrete IgG isotypes. Humoral memory lasts >20 yr in humans. Antibodies bathe tissues, forming part of first line of defense against re-infection. Evidence for longevity: 1. BrdU labeling in mice - measures new DNA synthesis in cells, find very slow rate of incorporation, no incorp. until 20 days after injection. 2.Transfer bone marrow cells from immunized mice to irradiated, naïve mice long after immunization (>14 weeks). Find Ag-specific IgG in serum. No Ag is required for Ab persistence. No response to Ag stimulation - so differ from memory B cells.

58 Key Points: B cell activation Naïve B and T cells travel around the body thru the blood, entering the secondary lymphoid organs to seek Ag from tissues (lymph nodes) or systemic infections (spleen). Initial stimulation of B2 cells is by Ag presented by dendritic cells in the T cell zones of lymph nodes and spleen. Subsequently, B2 cells interact with helper T cells that recognize the same immunogen/pathogen. Then B2 cells move to B cell follicles. Initial stimulation of MZ B cells is by particulate Ag complexes from blood and occurs in the MZ. Then MZ B cells move to the B cell follicles. In the follicles, B cells proliferate, differentiate and form germinal centers. Germinal centers are required for good humoral immune responses because SHM, affinity selection, and differentiation occur within them.

59 Effector functions of antibodies 1. Neutralization - bind to viruses/toxins - prevent their binding to cell surface receptors or entry into cytoplasm. 2. Opsonization - Abs bound to pathogen promote its phagocytosis. 3. Complement activation - facilitates uptake and destruction of pathogens by phagocytes.

60 Different heavy chain isotypes confer different functions to secreted antibodies IgM - large, has high avidity because of 10 combining sites, although low affinity. Neutralization, complement activation. Strongest complement activator (10 3 -fold relative to IgG). Helps to activate B cells by forming Ab-Ag-C3d complexes with the BCR/co-receptor. IgM has a short half life. Primarily found in primary responses. Mainly in blood > lymph. Too large to get into tissues. Natural IgM is constitutively secreted, by B1 cells mostly. It is highly cross-reactive, important for initiating B cell activation by activating complement, thereby forming ligands for CD21, and also by forming IgM-Ag complexes that can cross-link the BCR. Also very important for clearing apoptotic cells by complement activation, and for clearance by macrophage, or binding to erythrocytes. Natural sigm CD21-CD19-CD81 migm B cell

61 Class switched isotypes have different functions from IgM IgG - small, long half-life Principal class in blood and extracellular fluid bathing tissues. High affinity due to SHM and selection in germinal centers (GCs). Neutralization, opsonization by binding FcRs, some subclass activate complement. Primary protector against infection in extracellular spaces.

62 Class switched isotypes have different functions from IgM IgA - most abundant isotype in body, but very little in tissues or blood. At mucosal surfaces - it is transported thru epithelial cells. Protects mucosal membranes of all body orifices. Main function is neutralization to prevent infection, and neutralize toxins at mucosal surfaces. Poor opsonizer (No complement, few phagocytes at mucosal surfaces). High affinity due to SHM and selection in GCs in Peyer's patches. Dimerizes, which increases its avidity. IgE - mostly exists bound to mast cells and basophils via high affinity FcεRI. Mast cells reside just below body surfaces. Basophils in blood. Inhibits parasites, but its dysregulation causes 50% of allergy. Provides resistance to tick bites in mice.

63 Fc Receptors (FcRs) Many microorganisms cannot be neutralized by antibody binding, and thus the antibody binding must be linked to effector mechanisms. Binding to FcR facilitates phagocytosis of pathogens and/or release of stored mediators that kill the pathogens. FcRs bind the Ig C H region, which can be crystallized, thus the term Fc (crystallizable fragment).

64 Fc Receptors (FcRs) Activating FcRs have an α chain (binds Fc region) and one or more γ chains with 1 ITAM, or in one case the α chain has 3 ITAMS. Stimulate phagocytosis, activate bacteriocidal mechanisms of phagocytes and stimulate non-phagocytic cells (NK cells, eosinophils, basophils, mast cells) to secrete stored mediators. Present on macrophage, neutrophils, eosinophils, basophils, mast cells, NK cells. Inhibitory FcRs (FcγRII/CD32) have an ITIM on the α chain. The FcR ITIMs recruit SHIP (inositol phosphatase) resulting in inhibition of signaling. Only inhibits if cross-linked to an activating receptor, e.g. BCR on B cells, FcγRIII (CD16) on mast cells, macrophage, neutrophils. Present on B cells and macrophage, neutrophils and eosinophils.

65 Example of role of FcRs in immune response Opsonization of pathogens by binding of antibody-antigen complexes to FcRs on phagocytes. Opsonization increases binding of pathogen to phagocyte and also activates phagocytosis. Most important FcR-bearing cells in humoral immune response are the phagocytes - macrophage and neutrophils.

66 FcRs increase NK cell activity and specificity Binding of Abs crosslinked with Ag to FcγRIII (CD16) increases binding of targets to NK cells and activates secretion of stored granules. This is termed antibodydirected cellular cytotoxicity (ADCC).

67 IgE activity is mediated via the FcεRI Stable binding of Ab to FcRs requires Ag-Ab complexes, except for IgE, which binds FcεRI on the surfaces of mast cells and basophils prior to Ag binding (due to high affinity: M). Ag binding then cross-links Ab-FcεRI complexes, triggering release of inflammatory mediators stored in granules in the mast cells and basophils. Causes increase in local blood flow and vascular permeability, accumulation of Abs and blood borne cells in surrounding tissue. Role of IgE is to resist parasites, extracellular organisms which have repetitive surface epitopes that can crosslink Ab.

68 FcRs signal to activate or inhibit effector cell activities upon binding of Ag-Ab complexes. In lamina propria Endocytosis of Ab-coated bacteria Inhibitory receptors Activate SHIP - a phosphatase

69 FcR Key Points FcRs bind Ag-Ab complexes, although FcεRI can bind IgE that is not bound to Ag. However, FcεRI does not signal until it is cross-linked by Ag. Binding of Ag-Ab complexes to FcRs functions to provide an activating signal to the phagocyte, NK cell, granulocyte, mast cell or basophil to phagocytose and/or to kill the pathogen. This binding also helps NK cells recognize virus-infected cells. Exception: Inhibitory FcRs (FcγRII) when cross-linked to activating receptor, prevent activation. Roles are to inhibit further activation of B cells, mast cells, neutrophils or macrophage. Adjusts the threshold for activation.

70 The complement system Recognition of microbial surface Proteolytic signaling cascade All pathways lead to: 1) Covalent deposition of complement components on surface 2) Generation of pro-inflammatory peptides Membrane damage Target for destruction by phagocytes (opsonization) Inflammation

71 The complement system Protein components are named C1-C9 (in order of discovery, not order in the pathway). Each successive component is activated by proteolytic cleavage - analogous to any enzymatic signaling cascade. Each step leads to amplification of the response. Cleavage leads to the production of a and b fragments of each component. a s are small peptide fragments, some of which have proinflammatory activity: C5a>C3a>C4a. b s are either active proteases or become attached to pathogen surfaces: C3b is most important.

72 Three pathways of complement activation Figure 2-19 part 1 of 2 Cleavage of C3 and coating of microbe surface with C3b

73 Classical pathway is triggered by C1q binding to: 1) polyanions on bacterial surfaces (e.g. lipoteichoic acid) Figure ) C Reactive Protein (CRP) coated surfaces 3) antibody coated surfaces Conformational changes in C1q cause the activation of the proteolytic subunits C1r and C1s.

74 Both MBL and C1q-binding lead to the covalent attachment of C3b on cell surface Figure 2-22 Mechanism allows amplification - surface is coated with a small number of C4b and 1000 s of C3b molecules. C3 is most abundant complement protein in plasma.

75 Alternative pathway Figure 2-28 Initiated by spontaneous hydrolysis of C3 in plasma, and direct binding of C3b to nearby microbial surface. C3b non-specifically coats all microbial cell surfaces. Additional proteins bind, creating a C3 convertase. Thus, pathways converge at this point. Host cells make protective factors, e.g. DAF (decay accelerating factor) to protect themselves. Other host cell factors are also inhibitory. Any cell that cannot protect itself becomes coated with C3b. (Heat inactivate FCS.)

76 C3b coated surfaces recruit C5b to the surface leading to the formation of the membrane attack complex (MAC). Figure 2-35

77 Some effects of complement Direct lysis via MAC Inflammation and recruitment of immune cells Mediated by complement fragments (C5a>C3a>C4a) Targeting bacteria for phagocytosis: opsonization with C3b and C4b allows binding to complement receptors on phagocytes. B cell activation

78 C3b split products (C3d and C3dg) bind to the BCR coreceptor CD21-CD19, greatly increasing B cell activation. CD21 is present on B cells and also on follicular dendritic cells (FDCs). Carroll and Isenman, 2012

79 Complement deficiencies Classical MBL Alternative C3 MAC Opsonization Inflammation Losing single arms of complement isn t so bad, but losing C3 is REALLY bad: Results in recurrent bacterial infections. Most often Neisseria and Pneumococcus Most pathogens have acquired mechanisms to resist complement.

80 Complement Key Points Complement is widely distributed as precursor zymogens throughout body fluids and tissues. Most important function is to facilitate uptake (opsonization) of pathogens and their destruction by phagocytes. Due to complement receptors on phagoctyes. Complement cleavage generates two fragments, a larger fragment that binds covalently to the pathogen cell surface (C3b and C4b, being most important) and a smaller fragment that floats away and is often an inflammatory mediator and chemo-attractant. Most important and most abundant functional component is C3b. A great deal of C3b bound to pathogens is generated, and is very important for opsonization. Binds to CR1 (CD35) on macrophage and neutrophils. C3b split products (e.g. C3d and C3dg) that are bound to pathogens bind to the CD21-CD19-CD81 co-receptor on B cells, stimulating endocytosis of the Ag-C3d-BCR-CD21-CD19-CD81 complex. S

81 Difference between functions of complement receptors and Fc receptors FcR s are associated with ITAMs and ITIMs, whereas CR s on phagocytes are not. (However, CR2/CD21 is associated with CD19 on B cells). Therefore, binding of pathogen-c3b and -C4b complexes to CR1, CR3 or CR4 on phagocytes does not send signal to phagocyte. Complement components C5a, C3a and C4a signal by binding to G-coupled receptors. If these components bind to receptors on phagocytes along with binding of the larger components this stimulates phagocytosis.

82 Opsonization can occur by combination of binding to CR1 and FcRs.

83 Mucosal immune system Mucosal surfaces are particularly vulnuerable to infection -they are thin permeable barriers due to their functional requirements. Mucosal lymphoid organs are tonsils, adenoids, appendix, Peyer s patches of the small intestine, mesenteric lymph nodes, and solitary lymphoid follicles of the large intestine and rectum. Antibody secreting B cells reside in lamina propria of the intestine (microvilli in the gut wall). These are mostly IgA-secreting plasma cells. In addition, γδ T cells reside in the gut epithelium and provide a first line of defense. Mucosal immune system must be tolerant to food antigens. Gut is heavily colonized by commensal microorganisms, to which the mammal should not respond. DC priming in the gut does not induce inflammation. Intestinal epithelial cells produce retinoic acid which primes tolerogenic CD103 + DC which in turn suppress Th1 (γif) and Th17 (IL-17).

84 Mucosal lymphocytes recirculate separately from other lymphocytes Lymphocytes that were activated in mucosal lymphoid tissue recirculate separately from lymphocytes activated in peripheral lymph nodes. They recirculate throughout entire mucosal lymphoid system. This is due to expression of particular combinations of chemokine and homing receptors.

85 IgA production in the Peyer s patches

86 M cells actively transport antigen from the lumen of the small intestine into lymphoid follicles by endocytosis and phagocytosis. Although pathogens sometimes exploit M cells to gain entrance to the tissues (e.g. Helicobacter pylori, poliovirus, Salmonella typhi), the M cells deliver pathogens into the lymphoid system which can often clear the infection before it becomes systemic.

87 IgA neutralizes pathogens and toxins in the gut

88 Transport of IgA from basolateral surface of mucosal epithelium to the mucosal surface facing the lumen involves binding of IgA with the poly-ig receptor via a disulfide bond. At the apical surface the poly-ig receptor is cleaved, leaving the secretory component associated with IgA.

89 Key Points for Mucosal Immune System B cells specific for pathogens found at mucosal surfaces migrate specifically to mucosal lymphoid tissues due to their having specific homing and chemokine receptors. Peyer's patches are lymphoid tissues lining the small intestine, that function similarly to lymph nodes, i.e. Ag presentation/lymphoid cell activation/proliferation/differentiation. IgA is the predominant antibody at mucosal surfaces, due to its ability to bind the poly Ig receptor and to be transported across epithelial cells. The main function of IgA is to neutralize pathogens, i.e. prevent them from binding to and infecting epithelial cells at mucosal surfaces. Probably also important for maintaining the normal bacterial flora in the gut. IgA also binds to the FcRα and promotes phagocytosis by macrophage in lamina propria and germinal centers. IgA does not activate complement by the classical pathway.