Immunology

Paula Videira

Antibody and TCR

Function and Diversity

Immunoglobulins
T cell receptors
Genetic bases of diversity of antigen recognition

Learning outcomes:
- To name key structural features of the immunoglobulins and the
consequences of antigen and antibody interaction.
- describe subtypes of immunoglobulins and its main effector
functions.
- To depict the main characteristics of the organization and
expression of genes encoding immunoglobulins and T cell receptors.
- To explain the mechanisms of gene rearrangement leading to the
generation of receptor diversity.

ADAPTIVE IMMUNE RESPONSE

CELLS

RECEPTORS

ANTIBODY
IMMUNOGLOBULINS

TCR

Antibodies or immunoglobulins (Ig)

glycoproteins that bind antigen with high specificity and affinity. They can be
found in the B cell membrane receptors (BCR or the B cells) or soluble
(secreted by plasma cells).

Light chain
,
Variable
region
Heavy chain
, , , , .

Constant
region

The specificity is determined by

Complementarity determining
regions (CDRs) - present in the variable
region of the light chains and heavy
chains. The CDR regions show a higher
degree of variability (hypervariable
regions).

The Igs are bi-functional

molecules:
antigen-binding capacity
and biological effector
capacity.
The Fc region is the portion of the Ig responsible for functional characteristics

Fc receptors - determine the cell specificity of Ig response.

Cell
Immunoglobulin
Mononuclear phagocytic cells
Neutrophils
Mast cell , basophils
Eosinophils

Receptors
FcgRI, FcgRII, FcgRIII, FceRII
FcgRII, FcgRIII
FceRI
FceRII, FcgRII, FcgRIII

IgG1, IgG3, IgG2, IgG4, IgE

IgG1, IgG3, IgG2, IgG4
IgE
IgE, IgG1, IgG2, IgG3, IgG4

Fc receptors - determine the cell specificity of Ig response.

Cell
Mononuclear phagocytic cells
Neutrophils
Mast cell , basophils
Eosinophils

Receptors
FcgRI, FcgRII, FcgRIII, FceRII
FcgRII, FcgRIII
FceRI
FceRII, FcgRII, FcgRIII

Immunoglobulin
IgG1, IgG3, IgG2, IgG4, IgE
IgG1, IgG3, IgG2, IgG4
IgE
IgE, IgG1, IgG2, IgG3, IgG4

FcgRIII-NK cell

ADCC
FcgRI-phagocyte

Opsonization

Immunoglobulin
Heavy chain
[mg/dl]
Molecular weight
Half-life (days)
Carbohydrates (%)

IgG1 IgG2 IgG3

g1
g2
g3
9
3
1
146k 146k 170k
21
20
7
2-3
2-3
2-3

IgG4
g4
0.5
146k
21
2-3

IgM
m
1.5
970k
10
12

IgA1
a1
3.0
160k
6
7-11

IgA2 sIgA IgD

a2
a1/a2 d
0.5
0.05 0.03
160k 385k 184k
6
?
3
7-11 7-11 9-14

IgE
e
0.00005
188k
2
12

Structural differences:
-Size
-Amino acid composition
-Carbohydrate content

In contrast to other Ig classes,

IgA and IgM multimers formed
IgM
- Is a pentamer in plasma
IgA
- Is a dimer mucosal secretions
- Is a monomer in plasma

The J chain is an extra chain that promotes the polymerization of chains (IgA)
or (IgM)
The secretory component is a polypeptide that joins together
with J chain in the secreted form of IgA

 Isotype
Differences between constant regions of the heavy
chain
(using various C region genes)

Allotype
Differences due to different alleles of the same
gene C . (differences in 1-4 amino acids). May
occur in the light chain or the heavy chain.

Idiotype
Differences due to rearrangements of VH and VL.
All the antibodies produced by the same B cell
clone have the same idiotype.

Antigen (antibody Generator) - any substance that can be recognized by an antibody or T

cell receptor . Antibodies can recognize almost any type of biological molecule as Ag,
while only TCR peptides in the context of MHC.
Epitopes or Antigenic Determinants are immunologically active regions of an antigen,
discrete portions of a macromolecule that is recognized by antibodies or T cell receptors
Is the paratope of the Ab molecule that makes contact with the antigen or epitope
determinant.

Immunogenicity depends on:

Must be recognized as non-self

strange
> 10 kDa> immunogenicity.
size
chemical composition
complexity
dose
Susceptibility to antigen processing
and presentation
Route of administration and adjuvants

Immunogenicity depends on:

SIZE
> 100-kDa Strong immunogenic capacity
<5-10 kDa Low-immunogenic capacity

COMPLEXICITY

Proteins are generally more immunogenic

Heteropolymers> degree of immunogenicity than homopolymers (regardless of
size).

Ag/Ac Interaction

specific
reversible
Non-covalent

The ability of a specific-binding site of the

antibody to react with a single antigenic
determinant or the ability of a population
of antibodies reacting with only one
antigen.

Cross-Reactivity The antibodies may bind

to different antigens provided that their
epitopes are structurally similar, enabling
a connection with more or less force to
the hypervariable regions

- Monoclonal antibodies, derived from a single clone and are therefore

specific to single epitope.
- The population of antibodies produced in response to an antigen
(which contains multiple epitopes) is heterogeneous.
The set of monoclonal antibodies to the same antigen
give rise to a polyclonal antiserum

ANTI-IMMUNOGLOBULIN
Anti-isotype antibodies - antibodies against
the constant region,
Useful for: Quantification of Ig classes and
subclasses. Caracterization of various B cell
leukemias
and
diagnosis
of
immunodeficiency.
Anti-allotypic-antibodies - produced against
the same species allotypic variants. These
antibodies may be induced by injecting an Ig
in another person. In practice, one obtains
anti-allotypic sera after multiple pregnancies
in women or persons who have received
blood transfusions or from patients with
rheumatoid arthritis.
Useful in: forensic medicine and paternity
testing
-anti-idiotype antibodies - against specific
determinants for a particular antigen.
Useful in: Regulation of immune response;
vaccines; treatment of B-cell tumors .

Light chain
,
Variable
region
Heavy chain
, , , , .

Constant
region

To think?...

"It is estimated that each individual

is capable of producing about
10^15-10^18 different antibodies
..."

"Our genome contains about

30,000 genes ..."

Clonal selection

Tonegawa and Hozumi (1976) Theory of germline

recombination in the generation of antibody diversity - the
"birth of Molecular Immunology".

The same constant region can be associated with many

variable regions
Ex: IgG with different specificities
Same variable region may be associated with different
constant regions
Ex: IgG and IgM with the same idiotype

Dreyer and Bennet

each light or heavy chain is the product
formed by the combination of two
genetic sequences, several V region
genes can be combined with one C
region gene ...

Organization of the Ig genes in the germ line.

Three different loci encoding the heavy, light chains kappa and lambda.
Each locus consists of at least three different gene segments: V, C and J (joining). The heavy chain still has D
(diversity) segments. Within each locus has multiple copies of each of the non-coding regions separated by
segments.

Linha
germinal

Organization of the Ig genes in the germ line.

The V region, in a light chain is encoded by VJ segments. By a heavy chain VDJ segments.
Functional antigen receptors are expressed only after the rearrangement of germline genes that make the
contiguous segments V (D) J - recombination V (D) J

Germ line

The production of a functional

Ig gene rearrangement
involves germline sequence
and mRNA splicing.
The V-DJ recombination is one
of the critical control points.
Recombinations are sequential
and regulated by the
production of functional Ig
genes. Allelic exclusion
mechanisms prevent
recombination after the
generation of a functional Ig
gene.

Recombination V (D) J - involve recombinant

ion of DNA gene segments and nonhomologous regions mediated by multiple
enzymes.
Recombinase V (D) J - group of lymphocyte
specific enzymes and DNA repair enzymes.
Recognize recombination signal sequences
(RSS) present at 3 terminal of each V
segment; 5 'segment of each J; 5 'and 3' of
each segment D.
The RSS are composed of a conserved
heptamer and nonamer, 12 or 23 nucleotides
apart. The sequences of 12/23
corresponding to one or two turns of the DNA
helix.
The ONLY recombination occurs between
two segments in which 1 has a range of 12
and another of 23 nucleotides - Rule 12/23
or a turn / two turns.

The specific enzyme in lymphocyte

development recombinase complex V
(D) J recombination are-activating
gene 1 and 2 (RAG1/RAG2).
RAG1/RAG2 are specific lymphocyte
lineages and only in immature - mature
lymphocytes in no recombination of the
germline.
Other enzymes of the recombinase
complex are involved in the repair of
DNA - DNA-PK complex.

Ex. loci cadeia

RAG1 RAG2 complex binds to

the recombination signal
sequences (RSS) spaced 12:23
pb
The RAG complex bind to
each other, joining gene
segments

The DNA is cleaved hairpin

structures creating the ends of the
gene segments

Addition or subtraction of bases [TdT

and exonuclease]. Repair and ligation of
the coding region

Combinatorial diversity

Large numbers of genes, V, D, J

Recombination V (D) J
Association light + heavy chains

Junctional Diversity

Juxtaposition of VJ
regions (VD and DJ)
generated independently

If we consider ...
In the heavy chain:
VH DH 40 x 27 x 6,480 = 6JH combinations
D can be read in three frames: 6,480 x 3 = 19,440
combinations
In the light chains:
Vk 5 x Jk 29 = 145 combinations
Vl 4 x Jl 30 = 120 combinations
= 265 light chain
If the H and L chains anneal randomly as H2L2
19,440 x 265 = 5,151,600 possible

Addition of nucleotides between

the V-D-J segments
- During repair of recombination P-nucleotides
and N-nucleotide
Deletion of nucleotides with
exonucleases
Somatic hypermutation:
- Introduction of point
mutations in the V regions
(after being rearranged )
-occurs in secondary organs
in lymphocytes activated by
antigen
IDA (activation-induced
deaminase - transition
originates C -> T)
=> Affinity Maturation

The absence / RAG mutation leads to the

inability to produce mature B and T cells severe combined immunodeficiency
(Severe Combined Immunodeficiency SCID)

Since the Ig loci are sites of frequent recombination of DNA, "wrong" genes can be translocated to
these sites and be transcribed. In Lymphomas it is frequent finding chromosomal translocations
involving Ig genes and oncogenes.
eg t (8.14), t (8,22)

To think? ...

What happens to the other allele?

Why is only a heavy chain and a light
chain are expressed by the cell?
What happens if the cell fails this
genetic rearrangement, generating
"non-productive" genes?

REGULATION

Membrane and secretory

Class switching

TCR
T cell receptors

NO SOMATIC MUTATION ON TCR

Conclusions

Immunoglobulins and TCR are complex molecules generated by

multiple mechanisms of genomic recombination and transcription
control in order to generate maximum diversity (theory recombination of
germline).
The recognition of antigens is virtually unlimited and universal..