Immunology:

Antigen recognition in the adaptive immune system

(TCR and Ig Rearrangement)

Peter A. Savage, Ph.D.

Assistant Professor
Department of Pathology
psavage@bsd.uchicago.edu

PATH 30100 01 Cell Pathology / Immunology (Spring 2016)

Monday, April 11, 2016
 Murphy Travers Walport

Janeways Immunobiology
Seventh Edition

Copyright Garland Science 2008

Features of antigen recognition by Igs and TCRs

the Ig serves as both the TCR serves

an antigen sensor and solely as an
an effector molecule antigen sensor
A single peptide-MHC complex can trigger cytokine secretion by T cells

single peptide-MHC
labeled with
quantum dot
on APC surface

antigen-specific
T cell

digital production
of cytokine triggered
by single pMHC

A single peptide-MHC ligand triggers digital cytokine secretion in CD4 + T cells

Huang et al., Immunity 39:846, 2013
Structure of the T cell antigen receptor (TCR)
hypervariable
CDR3 loops
Recognition of peptide-MHC complex by TCR

hypervariable CDR3 loops

sit directly over peptide
Identification and characterization of the TCR protein

Immunized Balb/c mice with T-cell lymphoma C6XL derived from C57BL/Ka strain
T-cell lymphoma is presumed to express a unique TCR on its surface

Generated hybridomas producing antibodies reactive to C6XL cells

One antibody, clone 124-40, reacted with C6XL cells but did not react with normal spleen cells

Identified disulfide-linked cell-surface heterodimeric protein (the TCR ?!?!?)

Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody

Allison et al., Journal of Immunology 129:2293, 1982
 Identification and characterization of the TCR protein
Two-dimensional SDS-PAGE analysis of cell extracts
125
I-labeled cell surface antigens
C6XL extract immunoprecipitated
using 124-40 Ab C6XL extracts

protein migrating
below the diagonal is
disulfide-linked
splenic splenic
T cells B cells
found to be a
heterodimer comprised
of 39 and 41 kDa chains

thymocytes bone marrow

cells

Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody

Allison et al., Journal of Immunology 129:2293, 1982
 TCR locus (germline configuration)
V(D)J recombination of the and TCR genes
 TCR gene segments are flanked by
recombination signal sequences (RSS)
The 12/23 rule governs recombination fidelity
of RSS elements during V(D)J recombination
 Induced asymmetry of the (RAG1-RAG2)2 complex
underlies the molecular mechanism of the 12/23 rule

Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures

Ru et al., Cell 163:1138, 2015
V(D)J recombination of antigen receptors
V(D)J recombination of antigen receptors
coding joints signal joints
 Steps in maturation and selection of T lymphocytes
T cells attempt to re-arrange functional in-frame TCR and TCR chains to generate a stable TCR heterodimer
TCR-expressing thymocytes then undergo thymic education (positive selection, negative selection, lineage commitment)

rearranged TCR first

pairs with invariant
pre-T chain
Structure of antibodies
Structure of Ig V domain

during B cell responses,

somatic hypermutation of the
CDR loops drives
affinity maturation
Features of the major isotypes (classes) of antibodies

J chain induces
multimerization
of IgA and IgM
 Binding of an antigen by an antibody
antigen-binding site can recognize soluble macromolecules in their native conformation
 Structures of full-length antibody molecules

Harris et al, 1992 &

1998

Saphire et al, 2001

(anti-HIV)
Immunoglobulin locus (germline configuration)
V(D)J recombination of the heavy and light chain Ig genes
 Ig gene segments are flanked by
recombination signal sequences (RSS)
The 12/23 rule governs recombination fidelity
of RSS elements during V(D)J recombination
Steps in maturation and selection of B lymphocytes
Steps in maturation of lymphocytes

self-reactive
B cells undergo:
1. receptor editing
2. deletion
3. anergy
Features of the major isotypes (classes) of antibodies

J chain induces
multimerization
of IgA and IgM
Generation of hybridomas and monoclonal antibodies
Nobel Prize in Physiology and Medicine, 1984
development of monoclonal antibody technology

immune theory production of monoclonal antibodies

Properties of antibodies and T cell antigen receptors (TCRs)
Potential diversity of Ig and TCR antigen receptors
Differences between Ig and TCR receptors
 Case 7: Omenn Syndrome
A defect in V(D)J recombination results in severe immunodeficiency

The case of Ricardo Reis: a bright red rash betrays an immunodeficiency

By day 10 after birth, had 10 loose bowel movements per day

By day 21, developed rash over entire body

Normal weight and size

Scaly rash and blisters on face, trunk, extremities
Spleen and liver not enlarged

Ricardos parents had had two other children, a boy and a girl, who had died
after the onset of a similar rash at 1 month of age
Parents were first cousins of Portugese extraction

White blood cells had high % of eosinophils and monocytes, but few lymphocytes
Low levels of serum IgG, IgA, IgM but high levels of IgE
Dermis infiltrated by large numbers of eosinophils, lymphocytes, macrophages,
with large numbers of cells around blood vessels

Condition worsened in hospital, developed bacterial and fungal infections

Blood was devoid of B cells and contained only activated T cells

Sequencing revealed hypomorphic R229Q (Arg to Gln) mutation in RAG2 gene

While studies were carried out, Ricardo developed pneumonia and died of
respiratory failure
 Case 7: Omenn Syndrome
A defect in V(D)J recombination results in severe immunodeficiency
 Case 7: Omenn Syndrome
A defect in V(D)J recombination results in severe immunodeficiency