Immunoglobulins

IMMUNOGLOBULIN

RETURN OF BASIC IMMUNOLOGY

นพ. สุ�รสุฤษดิ์ ขาวละออ ,19/08/52

IMMUNOGLOBULIN

CONTENTS

-Immunoglobulin structure-Immunoglobulin epitope-Immunoglobulin Fc receptors-Immunoglobulin gene diversity-Affinity maturation-heavy chain class switching-therapeutic application

IMMUNOGLOBULIN STRUCTURE

N Franklin. Middleton’s allergy 7th edition 2009

-Y or T-shaped molecule-composed of 4 polypeptides

-2 identical heavy chains-2 identical light chains

-each heavy & light chain has 2 major functional domains (C & V) each domain composes of 110 aa ม้�วนไปม้าเป�นกล��ม้ก�อน-this domains found in many molecules involved immune response (Ig-like domains)

Abul K. Abbas. Cellular and molecular immunology 6th edition 2007

-molecules with theses domains belong to Ig superfamily

-criterion for inclusion 1.presence of >=1 Ig domains (called Ig homology units)2.Ig homology unit = regions of 70-100 aa homologous to either Ig variable / Ig constant

domains

IMMUNOGLOBULIN SUPERFAMILY

-V = V like , C = C-like -H (C2) has length similar to C domains but sequences typical of both C & V domains-carbohydrate group attach to C-regions

-without impair complement binding Fc receptor binding ADCC activity

-Antigen-binding fragment(Fab)-recognize Ag-contain Ig idiotype -unique protein sequence that

identifies each Ab-Crystallizable fragment(Fc)

-define isotype of Ig-bind FcR for all functional attributes of Ab

-Fab link to Fc by hinge region

อรวดี� หาญวว�ฒน์�วงศ์� . อมม�โน์โกลบุ�ลน์ . อรวดี� หาญวว�ฒน์�วงศ์� , บุรรณาธิการ . วทยาภู�มคุ้��มก�น์พื้ !น์ฐาน์ และคุ้ลน์ก . กร�งเทพื้มหาน์คุ้ร 2551: -85101

-Variable-region domains -80-85% of aa ไม้�เปล��ยนแปลง -พบเพ�ยง 3 ตำ!าแหน�งที่��เปล��ยนแปลงค่�อนข�างม้ากค่%อ

บรเวณ aa ใกล�ก(บล!าดิ์(บที่�� 30,50,95 แตำ�ละตำ!าแหน�ง

ประกอบดิ์�วย aa ประม้าณ 10 ตำ(ว เร�ยกhypervariable regions(HVRs) เร�ยกอ�กชื่%�อว�าcomplementarity-determining regions (CDRs) : CDR1,CDR2,CDR3

-antigen binding site

-Constant-region domains

-IgG, IgA, IgD ม้� 3 domain-IgM, IgE ม้� 4 domain-CH1 & CL ที่!าให� VH & VL จั(บก(บ Agอย�างสุม้ดิ์�ล-CH2 ใน IgM, IgE ที่!าหน�าที่�� hinge

region-CH3 ใน IgM, IgE, CH2 ใน IgG,IgA จั(บ

ก(บ C1q

-Hinge region-composes of 10-60 aa-most proline ซึ่,�งจัะป-องก(นสุ�วนน�.ไม้�ให�เป�น globular domain และย(งที่!าให�สุ�วนน�.

ย%ดิ์หย��น สุาม้ารถโค่�งงอไดิ์�-เป�นตำ!าแหน�งเป1ดิ์ที่��เอ2นไซึ่ม้3ย�อยโปรตำ�นเข�า

ม้าย�อยไดิ์�-hinge region of IgD ยาวสุ�ดิ์จั,งที่!าให�ไม้� stable ถ4กย�อยไดิ์�ง�าย half life สุ(.น-IgE & IgM ไม้�ม้�สุ�วนน�.

Basic immunology overview

IMMUNOGLOBULIN ISOTYPES

-determined by Fc portion (constant region) of heavy chain : γ1, γ2, γ3, γ4, μ, α1, α2, δ, ε-immunoglobulin isotype (class/subclass) : IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, IgE

IMMUNOGLOBULIN A

-10-15% in serum-2 subclass

IgA1 & IgA2-mucosal immunity-found in both

monomeric and polymeric form-major monomeric IgA1, most in serum-major polymeric : dimer IgA2(secretory IgA)

Abul K. Abbas. Cellular and molecular immunology 6th edition 2007N Franklin. Middleton’s allergy 7th edition 2009

IMMUNOGLOBULIN A

-dimer เชื่%�อม้ตำ�อดิ์�วย j chain & secretory component(SC), can bind to poly-Ig receptor (like IgM) transported across mucosal epith.-Human produce more IgA than others (5-15 g/d)-selective IgA def. most common primary immunodef.-secretory IgA derived from locally produced IgA IV IgA replacement for IgA def. not restore IgA mucosal immunity

อรวดี� หาญวว�ฒน์�วงศ์� . อมม�โน์โกลบุ�ลน์ . อรวดี� หาญวว�ฒน์�วงศ์�, บุรรณาธิการ. วทยาภู�มคุ้��มก�น์พื้ !น์ฐาน์ และคุ้ลน์ก. กร�งเทพื้มหาน์คุ้ร 2551:85-101

IMMUNOGLOBULIN A

-sIgA : -neutralization-ป-องก(น colonization ของเชื่%.อโรค่

บน ผิวเซึ่ลล3เป-าหม้าย-ADCC by FcαR-activate complement by alternative pathway

อรวดี� หาญวว�ฒน์�วงศ์� . อมม�โน์โกลบุ�ลน์ . อรวดี� หาญวว�ฒน์�วงศ์�, บุรรณาธิการ. วทยาภู�มคุ้��มก�น์พื้ !น์ฐาน์ และคุ้ลน์ก. กร�งเทพื้มหาน์คุ้ร 2551:85-101

IMMUNOGLOBULIN E

-< 0.01% in serum-half life สุ(.นสุ�ดิ์ 2 ว(น-monomer-play role to parasitic infection

-bound to FcεRI on mast cells & basiophils type I hypersen.-ADCC by bound to FcεRI on eosinophil eosinophil degranule(ที่!าลายเชื่%.อปรสุตำ)

อรวดี� หาญวว�ฒน์�วงศ์� . อมม�โน์โกลบุ�ลน์ . อรวดี� หาญวว�ฒน์�วงศ์�, บุรรณาธิการ. วทยาภู�มคุ้��มก�น์พื้ !น์ฐาน์ และคุ้ลน์ก. กร�งเทพื้มหาน์คุ้ร 2551:85-101N Franklin. Middleton’s allergy 7th edition 2009

IMMUNOGLOBULIN M

-membrane-bound monomeric IgM earliest BCR on B cell in BM

-increase in Ag-specific IgMindicative of prim. immune response-pentameric IgM contain J chain, 15 kDa glycoprotein link 2 of 5, and 5 IgM-bind to poly-Ig receptor on basolat. surface of epith.

IMMUNOGLOBULIN M

-5-10 % in serum-activate complement by classical pathway เกดิ์ C3b, C4b จั(บก(บ CR1(CD35) บน phagocyte(opsonization)-ที่!าให�เกดิ์การรวม้กล��ม้ของ

เซึ่ลล3 หร%อเชื่%.อโรค่ไดิ์�ม้ากกว�า IgG 100-1000 เที่�า

-ม้(กพบใน Ab to natural

blood group, Ab to T-independent Ag, autoAb to self Ag

เชื่�น RF-hexamer พบน�อย, ย(ง

ไม้� ที่ราบหน�าที่��ชื่(ดิ์เจัน-find in colostrum

IMMUNOGLOBULIN G

-75-80 % in serum-4 subclasses

-IgG1(60-70%)-IgG2(14-20%)-IgG3(4-8%)-IgG4(2-6%)

-IgG1, IgG2, IgG3 considered Th-1 type antibodies-IgG4 ass. Th-2 type responses

-activate complement by classical pathway(IgG3 best, IgG4 no)

IMMUNOGLOBULIN G

-Bind to FcγR on Mononuclear phagocyte & neutrophil (opsonization)-ADCC : NK cell-pass placenta (IgG2 ineff. in crossing)-find in colostrum

-เป�นภู4ม้ค่��ม้ก(นเข�าสุ4�ล4กที่าง

FcRN ซึ่,�งเป�น receptor ในล!าไสุ�ของที่ารก-neutralize Ab : จั(บ ก(บจั�ลชื่�พ และผิลตำผิล

ของม้(น

IMMUNOGLOBULIN D

-< 0.5% in serum-monomer with long hinge region-most on mature B cell surface-role remains unknown

IMMUNOGLOBULIN EPITOPE

-Antigenic determinants(epitope) on Ab-glycoprotein immunogenic-Isotype : on constant region

class/subclass of heavy chain type/subtype of light chainspecies เดิ์�ยวก(น isotype เหม้%อนก(นใชื่�ในการตำรวจัสุอบปรม้าณ และชื่นดิ์

ของ Abใชื่�แยกชื่นดิ์ม้ะเร2งของ B cell

-Allotype : same isotypedifference of some aa onH & L chainในค่นพบไดิ์�ใน IgG เร�ยก Gm allotype,IgA ใน subclass

A2 เร�ยก A2m allotype,IgEเร�ยก Em allotype, บน κ lightchain เร�ยก κm allotype

-Idiotype : difference of each Abon variable region of H & L chain โดิ์ยเฉพาะสุ�วนhypervariable region(complementarity-determining region CDR})ม้าจัากเซึ่ลล3ตำ�นตำระก4ลเดิ์�ยวก(น

หร%อ monoclonal Ab ม้� idiotype เดิ์�ยวก(น

Allotypes

IMMUNOGLOBULIN Fc RECEPTORS

FcγR-4 Fc receptors for IgG : FcγRI, FcγRII, FcγRIII,

FcγRn-FcγRI(CD64) : -72 kDa glycoprotein

only FcγR that bind monomeric IgG high affinity (except IgG2) -on monocyte, MØ

-FcγRI(cont.) : -can be upregulated on neutrophils & eosinophils by IFNγ

-FcγRII(CD32) : -40 kDa glycoprotein -on placental trophoblasts, endothelial cell, all leukocytes(except NK) -bind monomeric IgG poor

-FcγRII(cont.) : -bind immune complexes containing IgG1 & IgG3 -binding to IgG2 is variable -upregulated on eosinophil by IFNγ & IL-3 -has 2 types : FcγRIIa

FcγRIIb

-FcγRIIa-considered an activating receptor-contain ITAM(immunoreceptor tyrosine-based activation motif)-expressed on human skin-derived mast cell

-FcγRIIb-contain ITIM ass.with down regulation

-human mast cells can respond not only to cross-linking of FcεR with allergen-specific IgE but also cross-linking mediated by allergen-specific IgG via FcγRIIa

-FcγRIII(CD16) : -50-80 kDa glycoprotein -bind IgG1, IgG3, lectin -expressed on neutrophils

FcγRn : -expressed in neonates -responsible for binding

maternal IgG for transport across placenta

FcαRI(CD89) : -55-75 kDa glycoprotein -bind both IgA1 & IgA2 express on monocyte, MØ, neutrophil, eosinophil -expression on eosinophil upregulated in atopic individuals & play role in eosinophil activation

-recently discovered Fcα/μR also bind IgMmediate phagocytosis of IgM-coated bacteria

FcεR : -2 IgE receptors -FcεRI(no CD)

-high-affinity IgER -expressed mast cells & basophils dendritic cells & langerhans cells -lead to mediator release

(in mild to severe allergic reaction)

-FcεRII(CD23) -low-affinity IgER -expressed on monocytes & B cells

IMMUNOGLOBULIN DIVERSITY

Factors cause diversity-multiple germ line segment

heavy chain(human) : 51VH, 27D, 6JH

κ light chain : 40VK, 5JK

λ light chain : 30Vλ, 4Jλ

-combinatorial association of H & L-V(D)J recombination

Factors cause diversity(cont.)-junctional diversity-somatic hypermutation & affinity maturation

V(D)J recombination -recombination signal sequences(RSSs): -located 3′ of each V gene segment, 5′ of each J segment, flanking each D segment both sides -consist of highly conserved of 7 nucleotides(heptamer) ; CACAGTG

V(D)J recombination(cont.) -followed by spacer of exactly 12 or 23 nonconserved nucleotides, followed by highly conserved AT-rich stretch of 9 nucleotides (nonamer) -mechanism

-deletion : most-inversion : 50% Ig κ locus

V(D)J recombination(cont.)-can be divided into 4 distinct events

-synapsis : 2 selected coding segments & their adjacent RSSs brought together bychromosomal looping

-clevage : double-strand breaks

V(D)J recombination(cont.)-coding end processing :

broken coding ends are modifiedby addition or remove of bases

-joining : signal ends brought together & ligated by repair process

V(D)J recombination(cont.)-recobination-activating gene-1 & gene-2

-Rag-1 protein recognizes DNA sequence at junction between hepatamer and coding segment cleaves it-Rag-1 enzymatically active only when complex with Rag-2 protein known as V(D)J recombinase

V(D)J recombination(cont.)-lack of Rag gene fail to produce Ig &

TCR protein lack of mature B &

T-Rag-1 & Rag-2 produced only in cell of B & T lymphocyte lineages and expressed only in immature lymphocyte

V(D)J recombination(cont.)-DNA-PK(DNA-dependent protein kinase) activate enzyme called Artemis (endonuclease) opens up hairpins at coding ends ; mutation of artemisabsence B, T-ligation of broken ends mediated by DNA ligase IV

V(D)J recombination(cont.)-consequences of V(D)J recombition bring promoters located in close proximity to downstream enhancers-enhancer maximize transcriptional activity of V gene promoter

Junctional diversity

-TDT(terminal deoxynucleotidyl transferase add nucleotides called N-nucleotides(non-template-encoded n.)-P-nucleotides(templated addition)

Somatic hypermutation & affinity maturation -mechanism for generation of high-affinity antibodies-involves changes in structure of V regions during T cell-dependent humoral immune reponses to protein Ag : process called somatic hypermutation

Somatic hypermutation & affinity maturation -antigen-stimulated B lymphocytes generates new V domain structures which bind Ag with greater affinity than original V domains-those C cells become dominant B cells with each subsequent exposure to Ag this process called affinity maturation

B CELL ACTIVATION

HEAVY CHAIN CLASS SWITCHING

-occur in peripheral lymphoid tissue-requirement of CD40 signaling to promote isotype switchinglack of CD40 & its ligand : Ab response to protein Ag is dominated by IgM-polysaccharide Ag do not elicit T cell help stimulate mainly IgM Ab

Mechanism of class switching-process called switch recombination-switch regions :

-located in J-C introns at 5′ ends of each CH locus, 1-10 kb long-contain numerous tandem repeats of GC-rich DNA sequences-found upstream of every heavy chain (except δ gene)

-upstream of each switch region is small exon called I exon-CD40 & cytokines trigger by inducing transcription through I exon, switch

region, CH exons-key enzyme to be required for switching & affinity maturation is AID (activation-induced deaminase)

-def. of AIDhyper-IgM syndrome similar to CD40L mutation-uracil N-glycosylase : enzyme which remove U residues, leaving abasic sites-Ape-1 endonuclease : enzyme which cleaves abasic sites, generating nick at each positioncontribute to double strand break

-existence of double-strand break in two switch regions results in deletion of DNA & joining together of two broken switch junctions-latter machinery used to repair double- strand DNA breaks during VDJ recombition

IMMUNOGLOBULIN

THERAPEUTIC APPLICATIONS

Polyclonal Ab-population of Ig molecules-all specific for particular Ag but do not have same idiotype(same isotype)-do not recognize same epitope-IVIG cold ethanol-fractionation of plasma multiple donor (10000-60000)

-supplied as 5% or 10% protein solution-95-99% IgG with trace amounts of IgM/IgA-play role in immune replacement

therapy & immunomodulationMonoclonal Ab

-genarated by cloning individual B cells and expanding one particular (clonal) Ab

Monoclonal Ab(cont.)-generic name ends in “-xmab” or “-zumab”-ending of “-omab” indicate mouse Ab “-umab” human origins

Nelson and Ballow J ALLERGY CLIN IMMUNOLFEBRUARY 2003

IMMUNOGLOBULIN

CONTENTS

-Immunoglobulin structure-Immunoglobulin epitope isotype, allotype, idiotype-Immunoglobulin Fc receptors-Immunoglobulin gene diversity V(D)J recombination, junctional diversity affinity maturation, heavy chain class switching-therapeutic application

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