the genetic basis of antibody structure · 2001-07-24 · dr. dov l. boros august 2, 2001 page 1...

Dr. Dov L. Boros August 2, 2001 (of 6)

Lecture 1 Benjamini et al., Chapter 6

THE GENETIC BASIS OF ANTIBODY STRUCTURE

Our immune response generates enormous diversity to antigens. The B and T

lymphocytes together may have specificities to 1015 – 1018 different antigens. Based on the one

gene-one peptide rule billions of genes would be required to code for such diversity. However,

humans have less than 200 genes to code for antibodies. Nature solved this problem ingeniously

by the generation of diversity in 5 different modes:

1. Gene rearrangement (VJ and VDJ combinatorial association)

2. Random assortment of H and L chains

3. Junctional and insertional diversity

4. Somatic hypermutation

5. Receptor editing

Organization and Rearrangement of L (light chain) genes

The κ and λ type L chains of an immunoglobulin consist of variable (VL) and constant (CL)

regions. The VL region is coded for by separate V (variable) and a smaller J (joining) gene

segments.

The entire L chain is formed by bringing together one V, one J gene to be joined to a C region

gene. This gene arrangement is termed: VJ recombination.

Rearrangement is mediated by recombinase enzymes. Recombination activating genes RAG-1

and RAG-2 are involved in coding for the cutting enzymes.

κ-chain synthesis. About 40 Vκ genes are present in the germ line. Genes are arranged linearly

separated by introns. Downstream at the 3′ end there are 5 Jκ gene segments. Figure 6.2

illustrates the genetic events.

Rearrangement – a randomly selected Vκ gene is joined to a Jκ segment. This is aided by

linking together the recognition sequences found at the end of the genes. A detailed illustration

of the rearrangement is shown in Figure 6.3. The strand of germline DNA loops out V2 and J4

are brought together and joined, the loop is cut and enzymatically degraded.


Lecture 1 Benjamin et al., Chapter 6

Fig. 6.2

Fig. 6.3



The rearranged DNA is now transcribed and the primary RNA transcript undergoes splicing (V1

and J5 are removed) and the mature messenger RNA is translated into the κ chain polypeptide in

the endoplasmic reticulum.

Gene rearrangement for the synthesis of the λ chain is essentially similar to that of the κ chain.

Organization and rearrangement of H (heavy chain) genes

The VH chain is constructed not from 2 but 3 gene segments: VH, DH (diversity) and JH.

The heavy chain locus is larger includes about 50 VH, 20 DH and 6 JH genes. Key feature: in the

germline configuration there are multiple genes coding for the C region of the immunoglobulin.

The C genes are flanked by introns. The C genes closest to the V region genes are the µ and δ

genes which are the first to be transcribed and translated into membrane immunoglobulins.

Fig. 6.4

Rearrangement of heavy chain germline DNA follows the previous L chain pattern. The first

rearrangement moves D to J segment, the second brings V segment next to the DJ unit. This unit

is transcribed with the Cµ and Cδ gene segments. The primary RNA transcript is alternatively

spliced producing either a µ or δ specific mature messenger RNA. This is translated into

VHCµCδ heavy chain polypeptide. Therefore an individual B cell will express both the µ and δ

chain polypeptides with identical specificity. (Figure 6.4)



Regulation of immunoglobulin gene expression. Potentially an immunoglobulin molecule could

be synthesized by genes on the paternal and maternal chromosomes. Indeed rearrangement of

the VDJ gene can occur on both chromosomes. Following successful rearrangement on the

paternal chromosome and the production of the H chain polypeptide, rearrangement of the H

chain gene on the maternal chromosome is stopped. This phenomenon is called the allelic

exclusion – which ensures a single antigen specificity for the B or T cell. If one parental gene

arrangement is unsuccessful, then the other is being rearranged. If both fail to rearrange the cell

that lacks Ig receptor undergoes apoptosis and dies.

Class or isotype switching

The antibody displayed on the early differentiating B cell is the product of gene rearrangement

made in the absence of antigen. The mature B cell that encountered antigen can switch its initial

IgM to different: IgG, IgA, IgE class antibodies with

preservation of the initial antigenic specificity. The switch is facilitated by factors (cytokines)

produced by T cells.

Mechanism: Each CH region gene has an S switch region at its 5’ end. This S region permits by a

recombinase enzyme any CH to be linked to an already arranged VDJ unit.

Thus a VDJCµCδ under the influence of cytokines is rearranged to be moved in front of the γ1

gene. The intervening C region DNA is spliced out then DNA is transcribed. Then the primary

RNA transcript is spliced and a final VDJγ1 mRNA is created for translation into a γ1 heavy

chain polypeptide.

Because the previous C region is deleted the cell is incapable of again making an IgM antibody.

(Figure 6.5)



Fig. 6.5

Generation of antibody diversity

1. Number of different genes in the V region

2. VJ, VDJ combinatorial association. 40 Vκ x 5Jκ genes form 200 κ chains etc.

3. Random association of H and L chains. Any L (κ or λ) chain can associate with any H chain.

Therefore all the H, κ and λ chains can provide about 2x106 different immunoglobulin

molecules.

4. Junctional and insertional diversity. Linkage of V to J or V, D, J genes has no set position,

may vary causing deletions. This results in changes of amino acids on the

hypervariable region of the polypeptides (junctional diversity). Moreover, some

nucleotides may be inserted at the V-D, D-J junctions (insertional diversity).

Insertion is mediated by terminal deoxynucleotidyl transferase (TdT) enzymes.

5. Somatic hypermutation. The antibodies of a primary response have low affinity. During the

secondary response mature B cell will have point mutations in the V(D)J



6. recombined unit. B cells with mutations have changed amino acid sequences in their H or L

chains. Sequences that are a better fit have higher affinity for the antigen. Such cells will be

selected for proliferation. This is the process of somatic hypermutation.

7. Receptor editing occurs when a B cell interacts with self antigen, to rescue itself from

apoptosis, there is a secondary rearrangement producing new V(D)J unit which will be

antigen reactive.

Study Questions

At the germline level B cell repertoire is generated in an antigen independent manner. Thismeans that:

A. Self reactive B cell receptors are not expressedB. B cells can be activated by antigen independent mechanismC. B cells are not reactive to antigenD. Generation of diversity is a random gene eventE. Antigen unexposed B cells show somatic hypermutation

Answer: D

After rearrangement of the heavy chain genes, the D and J segments code for:A. Kappa chainB. CD3C. CDR3D. µ chainE. J-S region

Answer: C

In the absence of RAG-induced enzymes activity:A. Isotype switch can still occurB. T, B cells do not developC. Only IgM is displayed on B cellsD. L chain synthesis is delayedE. H chains are not degraded

Answer: B

Consult Review Questions at the end of the relevant chapters in the book.

Dr. Dov L. Boros August 3, 2001 Page 1(of 6)

Lecture 2 Benjamin et al., Chapter 7

BIOLOGY OF THE B LYMPHOCYTE

The basic requirements for B and T lymphocytes are:

a. Specificity – response only to certain antigenic epitopes

b. Memory – recall of previous encounter with an antigen, rapid specific secondary response

c. Lack of response to self

Figure 7.1

Ontogeny of B lymphocytes (Figure 7.1)

B cell differentiation first occurs in the fetal liver, later the predominant site is within the bone-

marrow. In birds a special organ bursa of Fabricius is the site of B cell differentiation.

a. Stem cells: Are already committed to the B cell lineage by cytokine signals.

b. Pro-B cells: Have rearranged DH-JH genes

c. Pre-B cells: Have rearranged VH gene segments, form the VHDHJH unit which is moved next

to the Cµ gene. Now a µ chain is synthesized and displayed as a transmembrane molecule on

the cell surface. Two surrogate light chains λ5 and V pre B are also displayed. Close to the

Dr. Dov L. Boros August 3, 2001 Page 2 (of 6)


above chains are the Igα and Igβ transmembrane peptides. The µ chain, surrogate light

chains and two associated chains Igα and Igβ comprise the pre B cell receptor complex

(preBCR). Igα and Igβ act as signal transduction molecules both for the immature B and the

mature B cell after the antigen is bound by the variable regions of H and L chains of the

immunoglobulin. Failure to express preBCR causes cell apoptosis, while preBCR+ cells by

positive selection differentiate into immature B cells. (Figure 7.2)

Figure 7.2

d. Immature B cells

Light chains are now synthesized (κ or λ type), combine with µ chains and a monomeric IgM

is now inserted into the membrane. Such a receptor can recognize antigen – but the

interaction instead of activation results in inactivation of the B cell. Similarly such an

immature B cell can also interact with self molecules – these are inactivated and prevented

from responding to self. Mechanisms for acquisition of self tolerance:



1., deletion (apoptosis) 2., cell inactivation – anergy. 3., receptor editing – secondary

rearrangement of the H and L chain genes using unrearranged V, D, J genes. New specificity

arises for non-self antigen. (Figure 7.3)

Figure 7.3



e. Mature B cells

IgM and IgD immunoglobulins are now displayed on the B cell membrane. Differentiation

not only occurs in the bone marrow but also in the lymphoid organs. The IgD molecule is

generated by the alternate splicing of the RNA transcribed from VDJ and µ or δ genes

therefore IgM and IgD will have the same specificity. Mature B cells respond specifically to

antigen and within the germinal centers of the lymph node or spleen enlarge become blast

cells and then proliferate. The cells further differentiate into end stage plasma cells which

synthesize and secrete the antibody molecules. Plasma cells lack surface Ig and cannot

recognize or respond to antigen.

Isotype, or class switching occurs as the next step. During the early immune response

to an antigen only IgM is produced by the activated B cell. After a signal by T

lymphocytes in the form of cytokines and T-B cell interaction, B cells differentiate

into IgG, IgA or IgE producer cells. All subsequent isotypes have the same antigenic

specificity.

f. Memory B cells

Following antigenic activation some mature B cells become long lived, non-proliferating

memory cells capable of a rapid secondary response to the antigen. Such cells had

undergone class switching and somatic hypermutation within the germinal centers of the

spleen and lymph node. Within such centers dendritic cells with membrane bound antigen T

helper cells and activated B cells are present. This is

the site for the expansion, proliferation of B cells. Somatic hypermutation generates clones

with higher or lower affinity towards the antigen. Because all cells compete for the limited

amount of antigen, B cells with the highest affinity receptors will be selected for further

proliferation, while those with low affinity will be eliminated. This process is called affinity

maturation.

The B cell differentiation pathway provides a vast number of antigenically specific B cells –

this is the repertoire of an individual’s responses.



Distribution of B cell population

Mature B cells circulate in the blood stream and return to the lymphoid organs (spleen,

lymph nodes, Peyer’s patches in the intestines). Within the lymphoid organs it encounters

antigen presented by dendritic cells and properly activated it starts proliferating and

differentiating within the germinal center. Finally it differentiates into plasma cells which

are found in the medullary cords of the node.

CD markers

CD = cluster of differentiation antigens. These are cell surface proteins that are identified by

specific antibodies. CD cell surface markers appear at developmental stages on cells and

have important biological functions. (Figure 7.4)

Figure 7.4



B cell membrane proteins

1. The most important is the immunoglobulin molecule = Ig H+L – the B cell receptor, found

only on the mature B cell. The chains bind the antigen but due to their short intracytoplasmic

tail do not transmit signals.

2. Igα and Igβ which are non-covalently associated with the H+L chains transduce the

membrane signal into the interior, after undergoing protein kinase-mediated

phosphorylation of the tyrosine residues on their chains. These tyrosine residues are

within an amino acid sequence identified as: immuno-receptor tyrosine-based

activation motif (ITAM).

3. CD19, CD21 and CD81 are additional, BCR-associated signal transduction molecules.

CD21 is also the receptor for the C3d component of complement.

4. CD32 is the low affinity receptor for the Fc portion of IgG. It binds most efficiently

either aggregated IgG molecules or antigen-antibody complexes. May play a

regulatory role in B cell inactivation.

Mature B cells constitutively display MHC class II molecule and can present

antigenic peptides to CD4+ T cells. The co-stimulatory molecule CD40 is essential

for isotype switching interacts with CD154 on T cell. B7.1 and B7.2 are co-

stimulatory molecules interact with CD28 and CTLA-4 receptors on T cells.



THE ROLE OF THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)

IN THE IMMUNE RESPONSE

The MHC complex plays 2 major roles in the immune response: 1., binding and presentation of

antigen, 2., rejection of foreign transplants.

Variability of MHC genes and products

In humans the MHC region also known as Human Leukocyte Antigen (HLA) is located on

chromosome 6.

MHC class I and MHC class II genes are involved in the T cell response.

Fig. 8.1 shows a simplified map of the HLA regions.

Fig. 8.1



Class I genes: HLA-A, HLA-B and HLA-C.

Gene products expressed on cell surface as A, B, C peptides, all associated with β2-

microglobulin (not coded for by MHC genes).

Class II genes: DP, DQ and DR complex. Each contains A and B genes that code for α and β

polypeptide chains of the DP, DQ, DR proteins expressed on the cell surface.

Genetic polymorphism

Multiple forms-alleles of MHC class I and class II genes. Known as genetic polymorphism

causes tissue incompatibility between individuals. Probability that any two randomly selected

unrelated persons will share the same MHC genotype is 1 in 106.

Expression of the MHC molecules

Class I molecules are constitutively (permanent) expressed on every nucleated cell.

Class II molecules are constitutively expressed only on B lymphocytes, dendritic cells and

thymic epithelial cells. Macrophages, endothelial cells can be induced by cytokines to express

class II MHC.

Both class I and class II molecules are co-dominantly expressed (paternal and maternal gene

expression). Each individual has 6 MHC class I cell surface molecules (3 of each parent).

In the class II complex there is more than one DR-B gene and the α chain therefore can pair with

all the different β chains encoded by the B genes. Therefore 10-20 different class II molecules

are displayed on cell membranes.

STRUCTURE OF MHC MOLECULES

Function of MHC membrane molecules: 1., peptide binding 2., peptide presentation to the T cell

receptor (TCR).

Structure of MHC class I molecules

The class I molecule is a 43kDa single α chain which consists of α1, α2 and α3 extracellular

domains. It is non-covalently associated with the invariant 12kDa β2-microglobulin (β2m)

polypeptide. (Figure 8.2)



Figure 8.2



Sequence differences expressed by the allelic forms of genes are clustered in the α1, and α2

domains. The two domains create a deep groove with α helical walls and β pleated sheets on the

floor. Both act as binding sites for foreign peptides to be presented to the TCR. The groove can

I an 8-9 amino acids long peptide.

Shape and charge of the pockets at the bottom of the groove determine which amino acids of the

foreign peptide will be bound (anchor residues).

Structure of class II molecules

Class II molecules are built of 2 chains α = 35.000 and β = 28.000kDa size. Like class I

molecules, the class II molecules have an extracellular and a transmembrane portion with

cytoplasmic tail. The α1 and β2 portions of the chains together form the peptide-binding groove

(polymorphic region), which accommodates larger, 12-20 amino acid long peptides. The β2

chain represents the invariant portion-serves as binding site for the CD4 molecule on the T cell.

(Figure 8.3)



Figure 8.3

FUNCTION OF MHC MOLECULES

MHC peptides bind and display foreign peptides, trigger T cell responses. This is known as

antigen presentation. A complex foreign antigen has to be degraded within the cell (antigen

processing) and transported to the MHC molecules for display.

MHC class I molecules present protein antigen-derived peptides to CD8+ T cells



MHC class II molecules present protein antigen-derived peptides to CD4+ T cells

Therefore T cell responses are MHC restricted.

Antigen processing and presentation

a. Generation of MHC class II-peptide complexes

Figure 8.4



T cells respond only to 12-20 amino acid long linear peptides, therefore protein molecules first

are intracellularly degraded and then bound to the MHC molecule Exogenous antigens (bacteria,

proteins) are ingested by endocytosis or phagocytosis

Ingested antigen is stored in endosomal vesicle.

Endosomal vesicle fuses with lysosomal vesicles that contain degradative enzymes. (proteases,

lipases, nucleases)

In an acidic environment (pH ~ 4.0) within phagolysosome proteins are degraded to shorter

peptides and stored.

MHC class II α, β chains are synthesized in ribosomes within the rough endoplasmic reticulum

(ER). The two chains associate with the invariant chain (Ii, CD74) which blocks the groove and

prevents binding of endogenous self peptides. This complex is transported to the Golgi

apparatus and then to a late endosome. There most of the Ii peptide is degraded, the CLIP

fragment is left in the groove. The phagolysosomal and late endosomal vesicles intersect. In

acid vesicles CLIP is exchanged by the foreign peptide within the groove of the MHC. This

exchange is mediated by the HLA-DM molecule. The MHC class II-foreign peptide complex is

transported to cell surface and displayed. Binding of a foreign peptide is decided by the

sequence and charge of amino acids within the MHC groove. Some will bind to DR4 others to

DP2 of the HLA complex of an individual. Bound peptides are the immunodominant T cell

epitopes of an individual – trigger CD4+ T cell response. (Figure 8.4)



Figure 8.6

b. Generation of MHC class I-peptide complexes

Endogenous antigens of viral, parasitic origin are synthesized within the infected cells and

processed (degraded) in a cytosolic vesicle called proteasome. The 8-9 amino acid long

fragments are transported by TAP-1 and TAP-2 gene products into the ER.

Within the ER the MHC class I peptide and the β2 microglobulin chains are synthesized.

Chaperons arrange the correct folding of the synthesized chains.



The foreign peptide fragment binds to the groove of the MHC class I molecule.

This complex is transferred to the Golgi apparatus and finally is displayed on the cell surface.

MHC class I-peptide complex will interact with CD8+ T cells.

Peptides of self can bind to MHC molecules but do not activate T cells.

Reason: low concentration of peptides, T cells are tolerant of self. In certain individuals some

foreign peptides cannot bind to the MHC pockets. Such individuals will not respond to that

antigen.

Certain infectious agents can induce both MHC class I or class II responses. Ingestion of an

agent by a macrophage and processing within a phagolysosome will result in class II peptide

presentation. If the same agent is being intracellularly synthesized within an infected cell – the

class I presentation pathway will function. (Figure 8.6)

MHC AND RESISTANCE OR SUSCEPTIBILITY TO DISEASE

MHC polymorphism is beneficial against the vast array of infectious agents – enables individuals

to respond to numerous peptides.

Individuals who are MHC class I heterozygotes have slower progression to AIDS then

homozygotes.

The different polymorphic alleles of the MHC are decisive for exhibiting susceptibility or

resistance to bacterial (leprosy, tuberculosis) viral (hepatitis B, HIV) or parasitic (malaria)

diseases.

HLA alleles also influence the risk of developing autoimmune or chronic inflammatory diseases.

HLA-B27 is associated with ankylosing spondilytis

Hypotheses for the association of HLA and disease:

1. MHC molecules act as receptors for the attachment/entry of pathogens

2. Antigenic cross-reactivity between host MHC and pathogen molecules. (Molecular mimicry).

May lead to lack of response (pathogen’s molecule is considered self) or exaggerated

response to both self and pathogen peptide.



3. Different individuals with different MHC molecules will react differently because they bind

one but not another foreign peptide – cause for susceptibility due to different outcome of the

immune response.

4. Inability to recognize peptides of a certain pathogen “hole in the repertoire”

5. Alleles or single genes alone may not be decisive but linkage to other genetic loci will

generate susceptibility.

MHC “class III” genesSituated between class I and class II genes of MHC.

Code for C2, C4 and factor B components of serum complement and tumor necrosis

factors (TNF) α and β.

Study Questions

On a B cell membrane IgαA. Can bind antigenB. Is bound to VL chainC. Present on pro-B cellD. Absent on memory B cellE. Serves as signal transducer

Answer: E

Resting B cells are poor antigen presenting cells because:A. They bind antigen weaklyB. Cannot process ingested antigenC. Can easily shed bound antigenD. Have low levels of surface B7E. Recognize epitopes different from T cells

Answer: D

Congenital MHC class I deficiency is associated with deficient CD8+ T cell activity due to:A. Absent perforin production by CD8+ T cellB. Absence of MHC I on thymic epithelial cellC. Strong anti CD8 autoimmunityD. Defective CD4+ T cell helpE. Associated thymic aplasia

Answer: B




BIOLOGY OF THE T LYMPHOCYTE

T Cell receptor (TCR) complex

a., A two chain (α and β) heterodimer connected by a disulfide bond. Similar structurally

to the Ig molecule, arises from the immunoglobulin gene superfamily.

Extracellular portion of TCR has a variable (V) and constant (C) regions. The α and β

chain V regions together comprise three complementarity determining regions

(CDR1, 2, 3). This is the antigen-MHC binding site-TCR - unique to each T cell.

b., The α and β chains are associated with 3 other polypeptides γ, δ and ∈ - the CD3

molecule, which are also members of the Ig superfamily. They “chaperone”

intracellularly the newly synthesized TCR and guide it to the cell surface.

c., The TCR and CD3 chains are also associated with two ζ (zeta) chains.

The TCR + CD3 + zeta chains comprise the T cell receptor complex. The CD3 and

zeta chains do not bind antigen, but following antigen binding by the TCR serve as

signal transducers. The CD3 and zeta polypeptides contain tyrosine-containing

sequences called immunoreceptor tyrosine-based activation motif (ITAM) which bind

protein kinases after antigen-TCR interaction and mediate T cell activation. (Figure 9.1)

Fig. 9.1



CD4 and CD8 membrane molecules

A mature T cell displays either a CD4 or CD8 transmembrane molecule. The ratio of these

CD4+ to CD8+ T cell major subpopulations is usually > 2:1.

a., CD4 and CD8 peptides on the T cell serve as coreceptors bind to the invariant portion

of the MHC molecules on the antigen presenting cell and serve as adhesion molecules

which strengthen cell-cell interaction.

CD4+ binds only to the MHC class II and CD8+ only to class I molecules. This is the

basis of the MHC restriction of the T cell response.

b., CD4 and CD8 molecules also act as signal transducers. After antigen binding by the

TCR the protein kinases on the intracytoplasmic tail of the CD4 or CD8 chains get

activated and participate in T cell activation.

Interaction of the TCR with the MHC molecules

When a peptide antigen is bound in the groove of the MHC molecule its central portion protrudes

from the groove. This portion interacts with the CDR3 (the most variable portion of the V

region) of the TCR. The CDR1 region also interacts with the foreign peptide but the CDR2

region touches the walls of the MHC molecule and anchors the interaction between antigen

presenting and T cells. (Figure 9.3)

Figure 9.3



T cells

The γ, δ chains differ from α, β, but are also associated with CD3 and ζ. The γ, δ+ T cells lack

the CD4 membrane marker, some cells display the CD8 marker. The γ, δ+ T cells

are present in low numbers in the skin and pulmonary intestinal epithelia. Their protective

function is unclear, though during infections they multiply. They show a limited range of

responsiveness to protein antigens but lack a response to peptide – MHC

complexes. Production of cytokine and cytotoxic function to some antigens of mycobacteria

indicate role in first line of defense.

T cell receptor genes and receptor diversity

The α and γ chains gene complex comprises the V, J and C genes (similar to Ig L chain).

The β and δ chains gene complex comprises the V, D, J and C genes (similar to Ig H chain).

Genes are rearranged, transcribed and translated as for Ig genes.

Recombinase enzymes (activated by RAG-1 RAG-2 genes) and joining sequences are used to

create VJ or VDJ units. Allelic exclusion is also used.

Due to multiple germline V genes, random association of α, β or γ, δ chains and huge junctional,

insertional variability the TCR repertoire may exceed that of the Ig molecules: ∼ 1015 for α, β;

1018 for γ, δ TCR.

T CELL DIFFERENTIATION IN THE THYMUS

Site of differentiation of precursor cells is the thymus. Athymic children (Di George syndrome)

lacking mature T cells are severely immunodeficient. Intrathymic T cell differentiation

continues throughout life, but at puberty it is diminished concurrent with thymic involution.

Gene rearrangement for α β or γ δ type receptors occurs in the thymus. (Primary lymphoid

organ)

Steps in T cell differentiation

For differentiation intrathymic, non-lymphoid stromal cells are necessary for cell-cell interaction

and cytokine production. These cells are the cortical epithelial cells and interdigitating dendritic

cells. (Figure 9.5)



Figure 9.5



From the bone-marrow a stem cell programmed into the lymphoid pathway migrates to the

thymic cortex and starts to differentiate. TCR β and γ chains genes start to rearrange similar to

that of Ig genes. Successful γ chain-gene rearrangement initiates δ chain rearrangement;

followed by transcription and translation into polypeptides. This lymphocyte will be a γδ+ T cell

(separate lineage).

In cells with non-functional γ/δ rearrangement, β gene rearrangement continues and the β chain

polypeptide product is displayed on the cell membrane with an invariant molecule: pTα and

CD3. This is now a pre-T cell. Signaling through this precursor TCR arrests

further β gene rearrangement to ensure allelic exclusion of other β chains, and downregulates

pTα, and starts the rearrangement of the α chain gene.

Now αβ+ cells express CD4 and CD8 surface molecules to become the major population of

double positive (CD4+, CD8+, CD3+ αβ+ thymocytes within the cortex.

Such cells have to undergo double selections before leaving the thymus as mature T cells.



Figure 9.6



Thymic selection

A. Positive selection

The CD4+CD8+αβ+ thymocyte adheres to the thymic epithelial cell interacts with its MHC

molecules and further proliferates. This is called thymic education. Lack of interaction causes

cell death. The educated cell as mature T cell will respond only to an antigen if it is presented by

the same MHC encountered in the thymus. This is known as MHC restriction of the T cell

response.

B. Negative selection

To prevent the emergence of self-reactive T cells, the CD4+CD8+αβ+ T cell interacts with the

interdigitating dendritic cells at the cortico-medullary junction. Such cells carry self peptides

within their MHC class I or class II grooves. Thymocyte TCR that reacts with high affinity to

the MHC I or II + self peptide complexes is deleted by apoptosis to eliminate autoimmune T

cells. This process is the negative selection. Cells with lower affinity TCR constitute the

antigen-reactive population.

After successful negative selection the double (CD4+, CD8+) thymocyte downregulates one or

another of the CD molecules to become either a CD4+ or CD8+ mature T cell which leaves the

thymus and migrates to the lymphoid organs. (Figure 9.6)

Lymphocyte traffic

Lymphocytes circulate via the blood and lymphatics. They enter or leave the lymphoid organs

and can leave the circulation to migrate into the tissues (infection, inflammation). Naïve T

lymphocyte migrate (home) to peripheral and mucosal lymph nodes, activated T lymphocytes

migrate out of the lymph nodes to the tissues.

Adhesion molecules L-selectin on T cells and addressins on vascular endothelium mediate

homing of naïve T cells. Initial light binding between T cell and endothelial cell adhesion

molecules is followed by further adhesion molecule interaction and tight binding between LFA-1

on T cell and ICAM-1 on endothelium.

Within the lymph node antigen stimulated activated T cells downregulate CD62L and upregulate

another membrane adhesion molecule VLA-4 and CD44 expression.



These molecules facilitate homing to tissues of activated and memory cells.

Antigen presenting cells

Specialized antigen presenting cells are (APCs) are within the lymph nodes as well as in other

sites of the body. The APC takes up an antigen processes and presents it to the TCR of the

clonally selected T cell.

Dendritic cells are very efficient in antigen presentation, to CD4+ T cells because of the strong

display of MHC class II molecules. In the skin Langerhans cells (variety of dendritic cells) pick

up incoming antigen. Then migrate to the regional lymph node where they differentiate into

mature dendritic cells. Antigen presentation occurs in the T cell zone of the node.

In a primed (antigen-activated) system B lymphocytes can also function as APCs via their

receptors. Presentation to T cells occurs in the lymph node follicles (areas of B cells).



ACTIVATION AND FUNCTION OF T AND B CELLS

Antigen and T or B cells interaction results in cellular proliferation and functional differentiation.

Antigen – stimulated T cells make cytokines

Antigen – stimulated B cells make antibodies.

ACTIVATION OF CD4+ T CELLS

Figure 10.1 depicts the membrane molecular interactions between the APC and T cell.

Fig. 10.1



1. Initial necessary event: contact between peptide-MHC II and TCRα+β. This is the

first signal. This low affinity binding has to be stregthened by co-stimulatory and

adhesion molecules.

2. Coreceptor CD4 molecule on the T cell binds to the invariant non-polymorphic region of the

MHC class II molecule. This binding induces signal transduction.

3. Costimulatory molecules

Provide second signal. Pairs of molecules: CD40 and CD40L (ligand) and B7 and CD28 or

CTLA-4 ligands.

4. Adhesion pairs

CD2 on T cell binds to LFA-3(CD58) on APC

LFA-1 on T cell binds to ICAM-1 on APC

Adhesion molecules provide enhanced, prolonged binding. T cell can scan the antigen-MHC II

complex.

Intracellular events in CD4+ T cell activation

Figure 10.2



Membrane signal is sequentially conveyed into the interior of the cell and finally transduced

to the nucleus.

1. Engagement of the TCR transmits a signal to the CD3 and ζ (zeta) molecules which either

causes conformational change in the transmembrane region of the TCR or aggregates

multiple TCRs.

2. Tyrosine Kinases Fyn and Lck associated with the cytoplasmic portion of TCR and

CD4 molecules are activated.

The kinases phosphorylate the ITAMS (immunoreceptor tyrosine based activation

motifs) on the CD3 chains.

3. Phosphorylated ITAMs allow the binding of ZAP-70, a different tyrosine kinase.

4. Bound ZAP-70 is now activated by Lck and Fyn causing activation of phospholipase

C-gamma (PLC-γ) which breaks down membrane phospholipids such as

phosphatidylinositol biphosphate (PIP2) into diacyl glycerol (DAG) and inositol

triphosphate (IP3). Two major signaling pathways are created:

5.a. DAG activates protein kinase C which starts a cascade of kinases. Finally NF-KB

transcription factor is activated. b., IP3 elevates intracellular Ca++ level which activates

calcineurin that activates another major transcription factor: NF-AT. Both

transcription factors now enter the nucleus and bind to genes that code for cytokines

and cytokine receptors.

The first activated genes transcribe the α chain of IL-2 receptor (IL-2Rα, CD25) and

IL-2 cytokine. (Figure 10.2)

This is the first step in T cell activation. The cell secretes the translated gene product

IL-2, enlarges, synthesizes DNA and after 3 days starts dividing.

Changes in membrane adhesion molecules: L-selectin expression is down-regulated on activated

T cells which now do not home back to the lymph node, but move to sites of antigenic stimuli

(infection) in the body.



VLA-4 and CD44 expression is induced – these bind to endothelial cells and the activated T cell

now leaves the circulation.

Function of costimulatory signals

MHC II-peptide interaction with TCR upregulates CD40L(CD154) ligand on the T cell.

CD40L binds to CD40 on the APC. This binding is necessary for T cell activation.

Binding of B7 molecules on APC to CD28 on T cell, initiates phosphorylation and

activation of intracellular proteins such as phosphatidylinositol-3 kinase. Activated CD28

prolongs the half life of IL-2 mRNA-result: more sustained IL-2 production.

The CTLA-4 ligand on T cells is induced at a later stage of T cell activation. Interacts

with the B7 molecules on the APC. This transmits a negative signal, IL-2 production is shut off,

the T cell stops to proliferate, and differentiates into memory T cell.

CD4+ T cell subsets

All stimulated CD4+ T cells secrete cytokines; antigen non-specific soluble peptides

which affect the activity of CD4+, CD8+ T cells, B cells and other myeloid cells.

Based on the kinds of secreted cytokines Th(helper)0, Th1 and Th2 subsets are defined.

Th0 is the precursor cell for the differentiated Th1 and Th2 cells.

Fig. 10.3



Each subset has different functions.

Th1 cells secrete IL-2, Interferon (IFN)-γ and tumor necrosis factor (TNF)-β, which activate

cells in cell-mediated immunity: CD8+, NK cells, macrophages. Th2 cells secrete IL-4, IL-5, IL-

10, IL-13 activate B cells in Ig class switch to IgE and eosinophils. Th0 cells secrete a mix of

IL-2, IL-4 and IFNγ.

Antigens may induce Th0, Th1 and Th2 cell differentiation.

Viruses and bacteria preferentially induce Th1, allergens and parasites Th2 type cell response.

The mode of T cell stimulation and cytokine production is decisive for subset differentiation.

Antigen concentration and affinity for receptors as well as type of APC are contributory factors

to T subset development.

NK cells and macrophages are triggered during innate immunity, synthesize IL-12, IFNγ and IL-

18 induce Th1 subset development.

Mast cells or NK1.1 CD4+ T cells produce IL-4 and shunt differentiation into the Th2 pathway.

Cross-regulation exists between the subsets. Th1 cells that produce IFNγ inhibit Th2 cell

generation, Th2 cell-produced IL-4 and IL-10 inhibit Th1 cell generation. (Figure 10.3)

CD4+ T memory cell

Antigen activation during the immune response causes apoptotic death of many T cells. The

survivors develop into long lived memory T cells, that will respond efficiently during a recall,

secondary immune response. Changed membrane molecule expression: CD44 display is

enhanced, CD62L (L-selectin) expression is decreased. CD45RA phosphatase is changed to

CD45RO.

Function of CD8+ T cells

Major function is to kill cells (cytotoxicity) of self infected with viruses, bacteria, and foreign

transplanted cells = Cytotoxic T lymphocytes (CTL).

TCR of CD8+ cells recognizes antigenic peptides in association with MHC class I molecules.



Some CD8+ T cells also synthesize IFNγ, TNF-β cytokines (Th1-like phenotype) others make

IL-4 (Th2-like phenotype).

Preactivation of CTL – to several viruses is mediated by viral antigen-MHC-II complex that

activates CD4+ T cells, which secrete IL-2. IL-2 induces CD8+ T cell proliferation and

differentiation into mature CD8+ CTL.

If viral antigen is presented by dendritic cells in association with MHC class I molecules then

CD8+ CTL is directly activated.

CD8+, CTL activation (extracellular adhesion molecules, accessory signals, intracellular

phosphorylations) steps are similar to those of CD4+ T cells.

Two pathways of cytotoxic function: (Figure 10.4)

Fig. 10.4



a., After attachment of CTL to target cell, the killer cell moves its intracellular granules toward

the target, granular content is deposited onto the target membrane-exocytosis. One component

of the granules is monomeric perforin that polymerizes within the target membrane and creates

small ringlike channels, increasing cellular permeability and osmotic death. The others are the

granzymes-serine proteases that enter inside the target through the pores created by polymerized

perforin – cause cellular apoptosis.

b., Fas ligand (FasL) on the activated CTL interacts with Fas membrane molecule on the target

cell. A cascade of proteolytic enzymes called caspases inside the target cell cytoplasm is

generated that leads to apoptosis.

One CTL can kill repeatedly by attacking new target cells. After elimination of the target cells,

CTL themselves undergo apoptosis – (activation-induced cell death) while the survivors become

CD8+ memory cells.

CTL cells are also MHC restricted: the T cell of an HLA-A2 individual will kill virally infected

cells only if the viral peptide is presented within the MHC HLA-A2 context. Because MHC

class I antigens are displayed on almost all the cells of the body, any virally infected nucleated

cell will be killed by the CTLs.

T cell recognition of lipids, glycolipids

Bacterial cell wall lipids, glycolipids bind to the CD1 molecules on the APC. The CD1

molecules unlike the MHC are non-polymorphic, are associated with the β2-

microglobulin of dendritic and B cells. CD1 molecule has a large deep binding groove – the

hydrophobic tail of the lipid is bound there, and the polar region binds to the TCR of αβ+ or γδ+

T cells.

Non-antigen mediated alternative T cell activation

Unprimed T cells can be activated by superantigens and polyclonal activators (mitogens).

Superantigens activate T cells with a certain Vβ segment (Vβ3, Vβ11 etc) on their TCR. Up to

10% of the T cells may be activated.



Superantigens though presented by MHC class II on the APC are not bound in the peptide

groove, and are not presented to combined α β CDR3 region of the TCR. Staphylococcal toxin

acts as a superantigen.

Polyclonal activators

Can induce mitosis in practically all the T cell population. These are largely plant proteins:

Concanavalin A (Con A) and phytohemagglutinins (PHA) bind to the carbohydrate moiety on

the TCR. Antibodies specific for CD3 can also polyclonally activate T cells.

B CELL ACTIVATION AND FUNCTION

T-B Cooperation

Antigens with multiple repeating epitopes (carbohydrates) can directly activate B cells by cross-

linkage of their receptors. Most proteins have single epitopes that cannot crosslink the BCR,

therefore a CD4+ T cell signal is needed for B cell response. (T helper Th cells) TD (thymus

dependent antigens).

Antibody production to TD antigens requires cooperation between activated T and B cells, which

may recognize different epitopes on the same antigen. (linked recognition).

Antigen binding by T cells triggers cytokine production. Cytokines activate B cells to proliferate

and differentiate into antibody producing cells and also determine the immunoglobulin isotype

produced.

IFNγ induces switch from IgM to IgG, IL-4 from IgM to IgE antibody production

In the absence of T cell help B cells do not switch isotypes and produce only IgM (T

independent, TI antigens)

Interactions during the primary immune response: Antigens are processed by dendritic cells,

which present antigen to CD4+ T cell. CD4+Th cells activate B cells which had already

internalized and processed the antigen.

During the secondary immune response both T and B cells had already been previously antigen-

activated. Direct T-B cell cooperation occurs which does not need dendritic cells.



Fig. 10.6



B cells capture (by Ig), then internalize, degrade and present antigen to CD4+T cells with the

appropriate TCR.

T-B cell cooperation needs cell adhesion which facilitates paired molecular interactions on cell

membranes, and mutual activation.

Adhesion pairs:

CD2 – CD58, LFA-1 – ICAM-1, CD5 – CD72, CD45 – CD22 strengthen T-B cell interaction

and/or participate in signal transduction.

CD154 on activated T cell and – CD40 on activated B cell is a key molecular interaction:

a., upregulates B7 co-stimulatory molecule on B cell, enhances interaction with CD28 on T cell.

b., promotes B cell proliferation

c., required for B cell antibody class switch. In the absence of the paired interaction only IgM is

produced. (Figure 10.6)

The carrier effect

Linked recognition of an antigen is needed for both primary and secondary antibody responses.

B cells recognize a different portion of the antigen (polysaccharide) while T cells respond to the

peptidic portion. Primary immunization with a B cell epitope linked to a T cell epitope must be

followed by the immunization with the same B, T cell epitopes. The primed B cells display the

internalized processed antigenic peptide in context of the MHC-classII molecule. This

interaction triggers the T cell to secrete cytokines which induce B cell differentiation into plasma

cells. If the secondary immunization uses the same B, but a different T cell epitope then an

insufficient number

of primed T cell epitope-specific cells are produced, ineffective T-B cell cooperation is the

result.

Haptens are small univalent antigens which alone cannot induce an immune response. If coupled

with a carrier protein they become immunogenic, and induce a primary antibody response. A

secondary antibody response to the hapten is generated only if the hapten is linked to the same

carrier protein (carrier effect).



T independent responses

TI antigens are large polymeric molecules with multiple repeating epitopes (bacterial cell wall

lipopolysaccharides, capsular polysaccharides)

Some TI antigens are mitogenic at high concentrations act as polyclonal B cell activators. TI

antigens do not induce memory cell production.

Intracellular pathways of B cell activation

Following cross-linkage of BCR, the intracellular activation cascades are similar to those of T

cells. At the end transcription factors NF-AT and NF-kB enter the nucleus and promote the

transcription of immunoglobulin and cytokine receptor genes. B cells enlarge (blast cells)

proliferate and differentiate into the short-lived antibody-producing plasma cells.

Study Questions

Ingested antigens are degraded when the intracellular pH is acidic. This promotes:A. Degradation of peptides within endosomesB. Binding of peptides to MHC II moleculesC. Interaction between MHC II and the TCRD. Membrane expression of MHC IE. Transport of endogenous peptide to ER

Answer: A

Following the interaction of TCR with the MHC-peptide complex the major signal transductionpathway of the first signal is:

A. γδ heterodimer of the CD3 complexB. CD4 adhesion to T cellC. ξξ homodimer of the CD3 complexD. Cytoplasmic region of the TCR chainE. IL-2 receptor gene activation

Answer: C

For an allergic individual lymphocytes isolated from the blood upon allergen stimulation willsecrete:

A. IL-2, IFNγB. IL-2, IL-4C. IL-12, TNFαD. IL-4, TNFβE. IFNγ, TNFα

Answer: B


the genetic basis of antibody structure · 2001-07-24 · dr. dov l. boros august 2, 2001 page 1...

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