Immunoglobulin structure and function Presented by Kullapornpas Benyajirapach, MD. May17, 2019

1. Kullapornpas Benyajirapach, M.D.

2. Outline
• B-Cell Receptor Structure and Signaling
• Humoral Immune Response
• Immunoglobulin Protein Structure
• Generation of Immunoglobulin Diversity and Class Switch
• Immunoglobulin Function
• Immunoglobulin Fc Receptor
• Immunoglobulin and Human Disease
• Therapeutic Applications of Immunoglobulins

3. References
Diane F. Jelinek et al. Middleton’s 8th Edition
Abbas 9th Edition

4. Introduction
• Hallmark features of “Acquired Immune Response” = B and T lymphocytes and
their ability to recognize specific antigens
• T cells - T cell antigen receptor (TCR) as transmembrane molecule, recognizes only
antigens processed and presented in context of MHC by an antigen-presenting cell
• B cells - Immunoglobulin as transmembrane molecule, later as secreted molecule,
recognizes antigens in their native, properly folded form

6. Introduction
• Monomeric antibody molecule: 2 identical heavy chains (HCs) & 2 identical light
chains (LCs)
• Humoral immune response exhibits remarkable diversity up to billions different
and specific-to-target antibodies
• Antibody functions:
• Recognize and bind enormous variety of antigenic determinants
• Mediating variety of diverse, isotype-dependent biologic effects of the immunoglobulin,
eg. complement fixation or antibody-dependent cellular cytotoxicity (ADCC)

8. B Lymphocytes and
Humoral Immune Response
• Expression of a functional BCR is essential for B cell development, maturation, and
release from the bone marrow
• BCR - critical role during antigen-induced activation of mature B cells in secondary
lymphoid tissues
• After differentiation into antibody-secreting plasma cells, immunoglobulin
molecules are largely expressed as secreted molecules, and the rate of antibody
production significantly increases

9. B Lymphocytes and
Humoral Immune Response
• Transmembrane and secreted
forms of immunoglobulin from
same B or plasma cell exhibit
same antigenic specificity
• Different at carboxyl (C)
terminus due to alternative
messenger RNA (mRNA) splicing
 presence or absence of
hydrophobic transmembrane
region (tail)

10. B Cell Receptor Structure and Signaling

11. B Cell Receptor Structure and Signaling
• Transmembrane forms of all immunoglobulin HC
isotypes lack signaling motifs due to their short
length  association of one transmembrane
immunoglobulin molecule with one disulfide-linked
heterodimer consisting of 2 polypeptides, Igα
(CD79a) and Igβ (CD79b)
• Igα and Igβ are transmembrane glycoproteins -
extracellular and intracellular C-terminal cytoplasmic
domain that is obligatory for BCR signaling, contains
important immunoreceptor tyrosine-based
activation motif (ITAM) sequence  BCR transmit
downstream signals

12. B Cell Receptor Structure and Signaling
• Igα/Igβ heterodimer - essential for normal transport and B cell membrane
expression of all nine immunoglobulin (Ig) isotypes, has a role in early B cell
development
• After rearrangement of IgM (μ) HC gene  pre-B cells express μ HC on cell surface
+ surrogate LC + Igα-Igβ heterodimer = “Pre-BCR”  stimulates pre-B cell to
undergo proliferation and allelic exclusion  maturation to immature  then
mature B cell involves production of κ or λ LCs that complex with μ HCs and the
Igα/Igβ heterodimer  mature BCR

14. B Cell Receptor Structure and Signaling
• Engagement of the BCR by antigen  receptor aggregation at cell surface  recruitment
lipid rafts (specialized membrane microdomains that facilitate assembly and activation of
downstream signaling molecules)
• This step places the complex in proximity to LYN tyrosine kinase, which phosphorylates
tyrosine residues in the Igα/Igβ ITAM motifs & triggers recruitment of spleen tyrosine
kinase (SYK) and Bruton tyrosine kinase (BTK)
• Activated SYK phosphorylates & recruits the B cell linker (BLNK) protein, which provides
binding sites for phospholipase Cγ2 (PLCγ2), BTK, and VAV proteins (guanine nucleotide
exchange factors)
• PLCγ2 generates second messengers inositol triphosphate and diacylglycerol  calcium
release from intracellular stores and protein kinase C activation
• These signaling events lead to activation of transcription factors: nuclear factor of activated
T cells (NFAT), nuclear factor-κB (NF-κB), and activator protein 1 (AP-1)

15. Peripheral B Cell Maturation and
Homeostatic Regulation
• Immature, BCR-expressing B cells newly released from bone marrow = “Transitional
B cells”
• Regulating immature B cells transition: BCR signals, limited space in the peripheral
compartment due to homeostatic regulation, and specific cytokines
• Crucial cytokine is B cell activating factor belonging to the tumor necrosis factor
superfamily (BAFF, [TNFRSF13B])
• BAFF binding to BAFF receptor (BAFFR)  survival signals delivered to maturing B
cells.
• High BAFF levels - autoimmune and allergic diseases, infections, and B cell lineage
malignancies

17. Peripheral B Cell Maturation and
Homeostatic Regulation
• Transmembrane activator and calcium-modulating cyclophilin ligand interactor
(TACI) and B cell maturation antigen (BCMA) - two additional BAFF-binding
receptors (BBRs), and another is called a proliferation-inducing ligand (APRIL)
• TACI and BCMA bind BAFF with intermediate and low affinity, respectively
• APRIL binds only to TACI and BCM, effectively creates higher-order ligand by
binding heparan sulfate proteoglycans such as syndecans, CD138
• BAFFR - necessary for mature B cell survival
• BCMA and its high affinity ligand (APRIL) are required for survival of long-lived
plasma cells
• TACI is important for class switch recombination and for regulation of B cell
proliferation

18. T Cell-Independent VS T Cell-Dependent
B Cell Responses
3 types of antigens
• Type 1 TI (T cell-independent) antigens  deliver first signal through BCR &
second signal through Toll-like receptors
• Type 2 TI antigens - repeating epitopes  BCR to become extensively cross-linked
and to activate B cells, no second signal
• Both types of TI antigens  low-affinity IgM responses and generation of poor, no
memory B cells
• Thymus dependent (TD) - most prevalent, require T cell help for full B cell
activation

20. T Cell-Independent VS T Cell-Dependent
B Cell Responses

21. T Cell-Independent VS T Cell-Dependent
B Cell Responses
• TI and TD antigens deliver signal 1 through the BCR
• Second signal results from 3 B and T cell receptor-ligand interactions
• BCR internalizes antigen, processes, displays antigenic peptides on B cell MHC
class II molecules recognized by the TCR of antigen-specific T cells
• B7-1 (CD80) and B7-2 (CD86) expressed on B cells and CD28 molecule expressed
on T cells  induces expression of CD40 ligand (CD154) - activation of CD40
molecule on B cells.
• These 3 signals trigger T cell cytokine secretion  immunoglobulin class switch, B
cell proliferation and differentiation into memory B cells, short-lived plasma cells
that die within the lymph nodes, and long-lived plasma cells that migrate to the
bone marrow

22. TI Vs TD B Cell Responses
• IgM production - early primary immune response,
then class switch to IgG (or IgA or IgE)
• At the end of primary immune response, levels of
IgM and IgG decrease
• On subsequent challenge with same antigen 
secondary response: much faster and greater
production of antigen-specific IgG with higher
affinity due to activation of memory B cells

23. Immunoglobulin Structure

24. Immunoglobulin Protein Structure
• Disulfide bonds
• 2 major domains of HC and LC: constant
region (C) and variable region (V)
• Enzymatic proteolysis: Papain cleaves IgG
hinge region at amino (N)-terminal side of HC
disulfide bonds  2 Fab fragments (bind
antigen) + Fc fragment (bind complement or
Fc receptors)

25. Immunoglobulin Protein Structure
• Pepsin cleavage  F(ab′)2 fragment
• Identical antigen specificity of 2 Fab regions
permits antigen mediated cross-linking and
importance to BCR-mediated activation of B
cells and effector activity of secreted
antibodies

27. Immunoglobulin Protein Structure
• HCs have 1 variable domain, but have 3 – 4 constant-region domains (i.e., CH1,
CH2, CH3, and CH4) depending on the immunoglobulin HC isotype
• Sequence analysis of the VL and VH regions from multiple antibodies revealed
three regions that have hypervariable sequences called complementarity-
determining regions (CDRs)
• HC CDR3 region - prominent role determining antibody specificity
• Immunoglobulins = glycoproteins
• Glycosylation affects biologic function and structure of human immunoglobulins

28. Immunoglobulin Protein Structure

30. Human Light- and Heavy-Chain
Immunoglobulin Gene Loci
• Immunoglobulin HC locus - on chromosome 14
• κ LC locus - on chromosomes 2
• λ LC locus - on chromosomes 22

31. Human Light- and Heavy-Chain
Immunoglobulin Gene Loci
• Antibody diversity derives from multiple HC and LC variable (V) gene segments
• HC variable regions are encoded by one V gene, 1 of 23 diversity (D), and 1 of 6
joining (J) gene segments
• In contrast, LC variable regions are encoded by only two types of genes: V genes
and J genes
• Immunoglobulin HC locus encodes 9 constant-region genes
• 3 or 4 exons encode the bulk of constant region, and 2 additional exons are
variably processed at the mRNA level, resulting in transmembrane or secreted
antibodies

32. Generation of
Immunoglobulin Diversity
and Class Switch

33. V(D)J Light- and Heavy-Chain
Gene Rearrangement
• Transcription of immunoglobulins or TCRs  directed DNA breakage,
rearrangement, and repair
4 sources of immunoglobulin diversity sources:
1. Multiple V(D)J genes in the germline
2. Random assortment of HCs and LCs
3. Junctional nucleotide variability introduced during pre-B cell immunoglobulin
gene rearrangement
4. Somatic hypermutation of immunoglobulin variable regions after encounters
with TD antigens.

34. V(D)J Light- and Heavy-Chain
Gene Rearrangement
• To generate functional LC and HC genes, discontinuous V(D)J coding sequences
must first be rearranged at DNA level
• Process unique to B cells and T cells
• Immunoglobulin HC rearranges first during B cell development  random use of
one V, one D, and one J gene  encodes complete HC variable region
• If the rearranged V(D)J exon encodes “open reading frame”  pre-B cells make μ
HC protein  trigger LC gene rearrangement

35. V(D)J Light- and Heavy-Chain
Gene Rearrangement
• Rearrangement requires 2 recombination signal sequences (RSS-12 and RSS-23)
• RSS - critical for double-strand breaks mediated by the lymphoid-specific genes,
called recombination-activating gene 1 (RAG1) and RAG2
• Expression of RAG1 and RAG2 proteins - regulated during B cell and T cell
development
• Deficiencies in RAG genes  severe combined immunodeficiency (SCID) syndrome
(inability of B cells and T cells to generate antigen receptors)

36. V(D)J Light- and Heavy-Chain
Gene Rearrangement
• After μ HC signaling + surrogate LCs + Igα and Igβ  one of the κ LC alleles is
triggered to rearrange one V gene next to one J gene analogous to HC V regions
• Final coding sequence for full HC or LC requires RNA processing to yield functional
mRNA coding for mature HC or LC
• Significant additional diversity is introduced during the joining process and is
referred to as junctional diversity.

37. อิงอร กิมกง. ความหลากหลายของยีนอิมมโนโกลบลิน (Diversity of immunoglobulin genes)

38. Immunoglobulin Diversity
• Junctional diversity termed “Nucleotide (N) Diversity” = random, nontemplated
nucleotides may be added at V-D-J junctions by terminal deoxynucleotidyl
transferase
• Insertion of Palindromic (P) Nucleotides - short inverted-repeat sequences
identified at V(D)J junctions
• Each of these mechanisms specifically affects the CDR3 region, if the nucleotide
changes result in amino acid changes, they can have a dramatic effect on the
antigenic specificity of the final immunoglobulin molecule

39. Immunoglobulin Somatic Hypermutation
• All 3 CDRs  additional diversity during antigen-dependent activation in secondary
lymphoid organs through the process of immunoglobulin somatic hypermutation
• Affinity maturation  selective clonal expansion of B cells expressing high-affinity
BCRs through preferential introduction of mutations in all three immunoglobulin
HC and LC CDRs
• Somatic hypermutation and class switch recombination require expression of
activation-induced cytidine deaminase (AID), highly mutagenic enzyme

40. Immunoglobulin Somatic Hypermutation
• CD40 signals delivered by CD40L-expressing activated T cells  AID expression
• AID-mediated mutations are random
• Mutations that increase affinity of BCR for antigen afford activated B cell a
competitive advantage  preferential clonal expansion  higher-affinity
antibodies are prevalent in the secondary and later immune responses

42. Immunoglobulin
Class Switch Recombination
• 9 human constant-region genes: μ, δ, γ3, γ1, α1, γ2, γ4, ε, and α2
• Exons encoding the μ constant region are located closest to JH cluster - reason that
IgM is the first antibody isotype expressed
• Exons encoding the δ constant-region gene are located just downstream of the μ
constant-region genes, and most mature naïve B cells co-express surface IgM and
IgD
• Classic class switch recombination requires AID and involves switch from IgM
expression to any of the downstream HC constant-region genes other than δ
• Class switch recombination occurs at the DNA level and involves deletion of
intervening genomic DNA

43. Immunoglobulin
Class Switch Recombination
• B cell class switching is antigen dependent
• Occurs during activation in lymphoid tissue germinal centers
• Switch in immunoglobulin class depends on nucleotide sequences called switch
regions, located at the 5′ ends of each HC constant-region gene except δ
• As immunoglobulins switch classes  HC genes between recombined V(D)J
segments and current CH gene are permanently deleted and can never be used
again

44. Immunoglobulin
Class Switch Recombination
• Cytokines play a major role in class switch recombination
• Cytokine-specific response elements reside in promoter region of each constant-
region gene S region
• T helper cell type 2: cytokines IL-4 and IL-13  class switching to IgG4 and IgE,
whereas interferon-γ inhibit class switching to these two isotypes
• Transforming growth factor-β  class switch to IgA
• Isotype switching is highly T-cell dependent

46. Immunoglobulin Function

47. Specialized Effector Functions of
Immunoglobulin Molecules
65% 20% 10% 5% 90% 10%

48. Immunoglobulin M
• Transmembrane, monomeric IgM - earliest
immunoglobulin expressed by developing B cells
• Antigen binding  B cell activation & differentiation 
IgM secretion
• Increase in antigen-specific IgM levels indicates primary
immune response
• Secreted as pentamer of five IgM monomers linked
together by disulfide bonds with J chain
• Because of its size, pentameric IgM is largely found in
serum

49. Immunoglobulin M
• Extremely effective at fixing complement and neutralizing
antigens, and can bind to effector cells expressing the Fc
receptor, Fcα/μR
• Because pentameric IgM includes J chain, it can bind to the
poly-Ig receptor on the basolateral surface of mucosal
epithelial cells  facilitates immunoglobulin transport
through the epithelial barrier, cleaved and remains bound
to mucosal immunoglobulins as the secretory component

50. Immunoglobulin M

51. Immunoglobulin D
• Co-expressed with IgM on the surface of mature B cells
before antigenic stimulation
• Evidence that IgD signaling may protect B cells from
being tolerized
• Very low concentration in serum
• Small subset of B cells in humans expresses IgD in the
absence of IgM due to noncanonical form of class switch
recombination that occurs in upper respiratory mucosa
• Secreted IgD binds to basophils and other innate
immune cells through unknown receptors  cross-
linking promotes antimicrobial activities

52. Immunoglobulin G
• Secreted as monomer
• Major class of antibody in serum and non-mucosal tissues
• IgG1 - most prevalent, followed by IgG2, IgG3, and IgG4
• Although IgG subtypes are 90 - 95% homologous, they have
different functional properties
• IgG1, IgG2, and IgG3 (but not IgG4) can activate complement
cascade
• Providing passive immunity to infants - only immunoglobulin
isotype that can cross placenta by neonatal Fc receptor (FcRn)
• IgG2 - relatively inefficient at crossing the placenta, other three
subclasses constitute most of the passively transferred maternal
antibody

53. Immunoglobulin A
• Humans produce more IgA than any other class
• Mucosal immunity
• 2 subclasses: IgA1 and IgA2, can exist as monomers or dimers
tethered together by J chain
• Most monomeric serum IgA is IgA1
• Like IgM, polymeric IgA can bind to poly-Ig receptor and be
transported across mucosal epithelium, primary antibody found
in mucosal tissue secretions
• Shorter hinge region of IgA2 - resistant to bacterial proteases
than IgA1  advantage in mucosal environment

54. Immunoglobulin E
• Secreted as monomer
• Responses to parasitic infections
• Most IgE bound to high-affinity IgE Fc receptors (FcεRI)
on mast cells or basophils  type I hypersensitivity
• Elevated in atopic persons

55. Ontogeny of Serum Immunoglobulin
Harry W Schroeder

56. Antigenic Epitopes and
Antibody-Antigen Interactions
• Antibodies usually bind to complex macromolecules: proteins, polysaccharides,
phospholipids, and nucleic acids
• Immunoglobulins: their affinity and their avidity for particular antigen
• Affinity reflects antibody binding with single epitope or binding site = sum of the
noncovalent forces between the antigen-binding site of the immunoglobulin and
the antigen itself
• Avidity, semiquantitative describe overall binding of antibodies to antigen =
average measure of affinity of antibodies for a particular antigen
• Somatic hypermutation and subsequent selection of high-affinity B cells 
increases in avidity during immune response

57. Immunoglobulin-Mediated
Clearance of Antigens
3 major effector functions after binding antigen:
• Neutralization of the antigen or toxin by formation of soluble complexes prevents
further binding of that antigen to other cells
• Promote phagocytosis of antigens through complement cascade activation or
interactions with FcRs
• Cross-linking of antibodies bound to FcRs on effector immune cells, such as
natural killer (NK) cells or macrophages  ADCC

58. Immunoglobulin Fc Receptors

60. Fc Receptors
• 4 types of Fc receptors for IgG: FcγRI, FcγRII, FcγRIII, and FcRn, expressed in
neonates - responsible for binding maternal IgG for transport across the placenta
• FcRn also expressed on surface of several adult cell types, including endothelial
cells
• When IgG binds to FcRn  internalized into endosomes before being released
back into the circulation (longer serum half-lives of IgG antibodies, particularly
IgG1, IgG2, and IgG4)
• FcγRI (CD64) expressed on macrophages, monocytes, neutrophils, and dendritic
cells = major phagocytic FcγR and the only FcγR that binds monomeric IgG1 and
IgG3 with high affinity

62. Fc Receptors
• 3 forms of FcγRII (CD32): FcγRIIA, FcγRIIB2, and FcγRIIB1
• Each recognizes same ligands
• FcγRIIA - activating receptor, but FcγRIIB1 and FcγRIIB2 - inhibitory receptors
• All three forms of FcγRII bind monomeric IgG poorly but do bind immune
complexes containing IgG1 and IgG3 with higher affinity
• FcRγIII (CD16) binds IgG1, IgG3, and lectins and highly expressed on neutrophils
FcγRIII has low affinity for monomeric IgG
• FcγRIIIA - primarily expressed on macrophages and NK cells
• FcγRIIIB - expressed on neutrophils and eosinophils

63. Fc Receptors
• At least three distinct IgA receptors
• FcαRI (CD89) binds IgA1 and IgA2, expressed on macrophages, neutrophils, and
eosinophils
• FcαR expression on eosinophils is upregulated in atopic individuals and likely plays
key role in eosinophil activation
• Neutrophils account for most CD89+ cells
• IgA can also bind to Fcα/μR, an Fc receptor that also binds to IgM  has higher
affinity for IgM than IgA, may mediate phagocytosis of IgM- and IgA-coated
bacteria

64. Fc Receptors
• 2 IgE receptors: FcεRI - high-affinity IgE receptor, expressed by mast cells and
basophils and by dendritic cells and Langerhans cells in the skin
• In contrast to B cells, which express only single type of immunoglobulin, FcεRI-
expressing cells can bind wide variety of IgE antibodies, with different specificity
• Cross-linking of FcεRIs on exposure to allergen  mast cell and basophil mediator
release  allergic reactions
• Low-affinity IgE receptor FcεRII (CD23) - expressed on monocytes, B cells, and
dendritic cells
• CD23 acts as a buffer against accumulation of excessive levels of IgE, and its soluble
form can prevent binding of IgE to FcεRI

65. Immunoglobulin and
Complement Activation
• Activation of classic complement pathway: specific isotypes of antibody bound to
microbes
• In humans, IgM, IgG1, and IgG3 efficiently activate complement cascade
• Activation of C1q, which depends on binding to Fc region of at least 2
immunoglobulin molecules
• Fc regions possess a single C1q-binding site, complement pathway activated only
by antibodies in immune complexes and not by circulating free antibody
• Because of pentameric structure of IgM  effective at complement activation
• Complement cascade  deposition of additional complement components
recognized by complement receptors expressed by macrophages, neutrophils, NK
cells, and erythrocytes

66. Immunoglobulin and
Complement Activation
• Complement bound to immune complexes in circulation or antibodies bound to
certain antigenic determinants  cell activation and release of mediators 
damage tissue and recruit and activate additional inflammatory cells
• Cause type II (antibody-mediated) or type III (immune complex–mediated)
hypersensitivity

69. Immunoglobulin and Human Disease

71. Therapeutic Applications of
Immunoglobulins

72. Therapeutic Applications of
Immunoglobulins
Polyclonal Antibody
1. Immunization
2. Pooled polyclonal immunoglobulins (primarily IgG)
• Provide passive immunity, also used to treat antibody-mediated inflammatory and
hypersensitivity diseases
• Effectiveness may result from ability of IgG to bind to Fc receptors  inhibiting the
patient’s pathologic antibodies from binding
• IVIG may also compete with patient’s own antibodies for FcRn-mediated recycling
of IgG  increased rate of pathologic antibody degradation

73. Therapeutic Applications of
Immunoglobulins
Monoclonal Antibody
• Cloning individual B cells and expanding one particular antibody-secreting plasma
cell  all antibodies are identical
• Therapy with mouse monoclonal antibodies severely limited by rapid production of
anti-mouse antibodies  drastically minimized by using genetic engineering
techniques to humanize monoclonal antibodies