MIMM 214 B CELLS

Humoral immunity

Antibodies promote pathogen neutralization, opsonization , complement activation

TFH cells

interact directly with B cells in response to all pathogen

TFH cytokines

• IL 21 every time

• IFN gamma for signal 1

• IL-4 for signal 2

• IL-17 forsignal 3

• signal will activate B cell to produce specific types of antibodies

IgM

Immunoglobulin M , an antibody class that serves as receptor on naive B cells.

B cells

• arise in bone marrow

• antigen specific

• clonotypic

• progenitors of plasmacells and plasmablasts

plasma cells

activated and differentiated B cells . The main Ab secreting cells

plasmablasts

• B cells that secrete antibody (igM)

• responsible for the early production

• found in primary focus

• most die after 5-10 days

• some can migrate to bone marrow and become plasma cells

BCR

• membrane bound receptor

• is secreted once the B cell is activated

B cells look for

an antigen, not p:MHC

Antigens in the lymph node

• ag arrive via afferent lymphatics

• can be covalently linked to complement components - opsonized

• can be retained in lymph nodes by SCS macrophages and follicular dendritic cells

subcapsular sinus macrophage (SCS)

• in lymph node

• express complement receptor on their surface

• can bind the complement on opsonized Ag and retain it

• low endocytic and degradative activity - hence the retaining

epitope

part of the Ag that B cells bind specifically

Signal 1

• Ag binds BCR

• B cell also expresses co-receptor , complement receptors CD19 and CD21

• Binds complement proteins - enhances signaling and activation

Igalpha and beta

• subunits associated with BCR

• have ITAMs motifs

singaling outcomes of BCR/ complement receptors

• transcription factors are activated

• survival signal

• cytoskeletal reorganization

internalization and presentation of Ag by B cell

• once signaling begins, BCR-Ag complexes are internalized

• internalized Ag are processed and presented on MHC

• this pMHC can then interact with TCR on a T cell

signal 2

interaction with T cell

• thymus-dependent antigens - TD

• thymus-independent antigens -TI

TD antigens

• signal 2 provided by an activated CD4+ TFH cell

• specific Ab and provide memory

Thymus-independent/ TI antigen

• signal 2 provided by TLR signaling

• such Ag are typically highly repetitive molecules, such as LPS

• only for some large B cells : B-1 and marginal zone b cells (less diversity, gives rise mostly to IgM antibodies)

TFH cells provide help to B cells

signal 2: signal from pMHC that has bound to TCR and co receptor on TFH cell

• signal from CD40 on B cells that has bound to CD40L on TFh cell

• results in signaling and activation of transcription factors

• leads to activation, proliferation, differentiation → antibody secretion

linked recognition

the rule that for a follicular helper T cell to be able to activate a B cell, the epitopes recognized by the B cell and the follicular helper T cell have to be derived from the same antigen

subcapsular sinus

where SCS macrophages are located and where they encounter the AG

T cell zone

where T cells get activated by interacting with DCs

B cell zone

where B cells encounter Ag and undergo later stages of proliferation and differentiation , including B cell follicles and germinal centers

T-B border

border between the B and T cell zones. This is where B cells first receive signal 2

follicle

development of B cells , where they get activated

germinal center

site of B cell proliferation and differentiation

activated B cell has choices

• form primary focus in sub capsular region

• migrate to follicle to form germinal center

germinal center reaction

give rise to plasma cells

primary focus gives rise to

plasmablasts- they can still proliferate and are differentiated B cells

primary focus main outcomes

• plasmablasts- early antibody production (mainly IgM)

• IgM+ Memory B cell : production of Igm

primary focus location

• near subcapsular zone

• interfollicular regions

• medullary cords

5 days post infection

when primary foci become apparent

two main outcomes of germinal center

• plasma cells- secrete large quantities of Ab

• memory B cells (important for memory response- maintain capacity to produce higher affinity antibodies)

germinal center aka

secondary lymphoid follicle

what happens in germinal center?

• B cells receive signal 1 &2 again

• undergo differentiation and processes to produce Ab that are more effective and have higher affinity

• size of germinal center peaks 7-12 days after Ag stimulation

germinal center processes

• class switching

• affinity maturation

• somatic hypermutation

early Ab production

• done by plasmablasts

• mostly in lymph node

• Antibodies have lower affinity - mostly IgM

plasma cells locations

• stay in lymph node (medulla)

• travel to bone marrow and reside there

immunoglobulin structure

• Y shaped glycoprotein

• 4 chains, 2 heavy and 2 light

• variable regions

• constant regions

Immunoglobulin

protein family to which antibodies and B cell receptors belong

• these are Ig like domains

variable regions function

• Ag binding

• Vl and Vh forms antigen binding sites

• binding can result in neutralization and other functions

constant regions function

• involved in complement activation (C1q → classical)

• constant region Fc binds to Fc receptors on phagocytes and other cell types

Fab fragment

• Two Fav fragments per antibody

• each has antigen-binding domain and part of the constant H and L chains

• Fab = fragment antigen binding

Fc fragments

• one Fc fragment

• Fc= fragment crystallizable

• constant region of the heavy chain

• receptors that bind Ab recognize the Fc region

CDR - complementarity determining region

• Antigen binding site

• 3 hypervariable loops

• not part of the beta strands

Ab binding

involves non covalent bonding between the Ig and the Ag epitope

• hydrogen bonds

• van der Waals

• hydrophobic

• ionic

five Ab classes

• IgM

• IgD

• IgG

• IgE

• IgA

different number of Ig-like domains - differences in the length of the constant region of the heavy chain - Fc fragments are different

IgM

• pentameric

• 5 antibodies linked together via disulphide bonds

• Heavy chain : one variable region and 4 constant regions

• mature naive B cells express TM IgM prior to activation

• part of first wave of secreted Ab

• most effective initiator of complement cascade

IgD

• heavy chain: one variable region and three constant regions

• IgD is part of first wave of secreted antibodies

IgG

• heavy chain: one variable region and three constant regions

• the most abundant in plasma

• 4 subclasses in humans - 1,2,3,4

• produced following differentiation in the germinal center

IgE

• heavy chain: one variable, 4 constant regions

• produced in response to helminth infections

• linked to TH2 response

IgA

• heavy chain: one variable and 3 constant

• monomer in plasma

• dimer in mucous secretions through the J chain

• important for mucosal immunity

• IgA2 and IgA1

experimental and clinical use of antibodies

• can be made to bind virtually any epitope- ex: TNF alpha for treatment of rheumatoid arthritis

• you can make an antibody that binds another antibody (fc region of IgG)

immunogenicity

it is possible for your immune system to mount an immune response agaisnt a therapeutic drug, including monoclonal antibodies

primary diversity

obtained through the process of somatic recombination, includes combinatorial diversity and junctional diversity events

Ag genetics

the heavy and light chain are encoded on different chromosomes

somatic recombination

process by which segments are arranged , tightly regulated machinery controls the recombination processes and many of the proteins are involved in DNA repair functions

light chain genes

• Variable V

• Joining J

• Constant C

heavy chain genes

• Variable V

• diversity D

• joining J

• constant C

CDR 1 and 2

encoded in the V segment of light and heavy chains

CDR3

the most variable CDR , is encoded in the joining of V-J segments of light chain, and V, D, J gene segments of heavy chain

variable region composed

in both heavy and light chains: segments V and J

in H chains, D segment as well

light chain genetics

two different loci on different chromosomes with different constant regions, only one will be expressed in the light chain of the antibody

• kappa chain

• delta chain

• each locus includes many different V and J regions, only one will be expressed and silence the other

heavy chain genetics

one locus, includes many different V, D and J regions

many different constant regions represent the different isotypes, correspond to IgM, IgD, IgE…

recombination signal sequences (RSSs)

flank each antibody gene segments

nonamer or heptamer sequence with 12 or 23 bp spacer. the sequences lie between heptamer and nonamer

spacing arrangement states that a 12 bp RSS must pair with a 23 bp RSS for recombination to occur

recombinase

enzyme, recognizes RSSs

RSS role

allow looping of DNA and binding by specific proteins. the loop part contain the segments not selected for recombination

rag

recombination activating gene

• Rag1 and 2 are necessary for recombination

• responsible for recognizing and cutting DNA at the immunoglobulin-encoding region and the RSS

• covalently closed DNA hairpin ends

junctional diversity

during recombination , nucleotides may be added or removed at the junctions between V, DJ and D& J or the V& J for the light chain

artemis

endonuclease, opens DNA hairpin at the coding ends of Ab genes

hairpin cleavage and junctional diversity

• the hairpin can be opened in three different ways by artemis

• addition of palindromic nucleotides at overhangs

• there is a template, allowing DNA repair enzymes to fill the complementary strand

• complementary strands of DNA read the same in both directions (5’ and 3’ end)

• 5’ to 3’ direction reads TCGA

• reading backwards on the bottom strand also yields TCGA

• happens mainly in light chain

exonuclease activity- junctional diversity

may remove nucleotides on each side of the coding joint

terminal deoxynucleotidyl transferase TdT

can add up to 20 N-nucleotides (non template coded) to the cleaved strands primarily in heavy chains- reason for why the cDRs vary in length

• repair enzymes will trim off nonmatching nucleotides, fill in remaining single stranded gaps and ligate the new DNA

junctional diversity main steps

• during recombination, nucleotides may be added or removed between V&D and D&J / V&J for light chain

• terminal deoxynucleotidyl transferase TdT adds nucleotides to the cleaved strands

• unpaired nucleotides are removed by an exonuclease

the mechanism of V (D) J recombination

mechanism generates BCR diversity in naive B cells

multiple gene segments - which gene segments are put together '

• heavy chain/light chain combinatorial diversity

• P nucleotide addition - templated nucleotides addition between joints, resulting from asymmetrical cleaving of hairpins by artemis

• exonuclease trimming - sometimes occurs at junctions- losing nucleotides

• non templated nucleotide additions - mediated by TdT , adding in random nucleotides between joints

TCR structure

• composed of alpha and beta chains

• each has a variable and constant region

alpha chain TCR

locus has multiple V and J segments

beta chain - TCR

locus has multiple V, D and J segments

TCR recombination

somatic recombination takes place in the thymus and is irreversible

VDJ recombination of TCR

• V,D and J segments are flanked by RSS

• Rag 1- recognize these sequences

• artemis cuts the DNA hairpin

• TdT adds non coded nucleotides in the joining regions

Ig vs TCR heavy chains

• ig heavy chain → D segment is surrounded by two RSS, both with 12-bp spacing

• TCR beta chain→ D segments have 5’ bp RSS and 3’ 23 bp RSS

TCR CDRs

• 3 CDRs per chain

• these are the sites with most diversity

• CDR3 is a particularity important source of diversity

• CDR 1 and 2 are encoded within the V segments of alpha and beta chains

• CDR3 is encoded in the D and J segments of the beta chain and between the V and J segments of the alpha chain

allelic exclusion

ensures that each B cell synthesizes only one allele for a heavy and one allele for a light chain

allelic exclusion mechanism

• once a BCR is expressed on surface of a developing cell → sends signal to silence the part of the gene that codes for other chromosome → other chromosome is methylated and inaccessible to transcription

• genomic silencing of the other chromosomes ensures each B cell will only express the same copy of BCRs have the same specificity

mature naive B cells

• express TM IgM and IgD

• initially, newly formed B cells express IgM as their primary B cell receptors

• later, some of these B cells switch to expressing IgD

• results in IgM and IgD being co expressed

B cell upon signals 1&2

some form primary focus to become plasmablasts = capable of secreting their Abs

processes to render Ab production more effective

in germinal center - lymphoid follicle

• somatic hypermutation

• affinity maturation

• class switching

secondary diversification B cell

occurs in germinal center after B cells have received signals 1&2 again

somatic hypermutation: higher affinity for it antigen, specificity remains the same

class switching : a process that replace one heavy chain constant region with one of a different isotype

these mechanism act on already rearranged ig genes- VD J recombination has occurred in the variable region, can’t go back

somatic hypermutation

• operates on activated B cells in peripheral lymphoid organs

• in the germinal center of lymph nodes

• high rates of point mutations in the V gene sequences that improves Ag binding

affinity maturation

selects for the survival of mutated B cells that have a high affinity for the antigen

• also occurs in secondary and tertiary responses → you get higher affinity antibodies

cytokines and Ab class

• cytokines secreted by TfH in germinal centers will inform class switching

• ex: type 2 response cytokine Il-4 induces IgE

class switching

• only occurs after B cell activation

• irreversible

• class switch recombination is guided by switch regions located upstream of each C gene

features of germinal center

• dark zone and light zone

• follicular helper T cells found in the light zone

• follicular dendritic cells FDCs retain Ag in the light zone

B cells that enter germinal center

• have already encountered signal 1 (Ag) and have been activated by a T cell at the B-T border (signal 2) and proliferated

• have the ability to produce IgM/IgD of a baseline affinity (TM)

light zone

thought to be primary site of plasma and memory cell differentiation

dark zone

thought to be site of hypermutation

role of FDCs/ TFh in germina; center

• FDCs serve as Ag concentration site for future selection and differentiation

• interaction of B cells with follicular helper T cells provides

cytokines secreted by TFH in germinal center

• inform class switching

• type 2 IL-4 will induce IgE

b cells in dark zone

undergo somatic hypermutation → same specificity but different affinity

• resulting B cells migrate to light zone where they compete to bind antigens trapped on FDCs

• higher affinity B cells will bind Ag → signal 1 = affinity msturation