Antigen recognition in the adaptive immune system 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

Janeway’s Immunobiology Murphy • Travers • Walport Janeway’s Immunobiology Seventh Edition Copyright © Garland Science 2008

Features of antigen recognition by Igs and TCRs the Ig serves as both an antigen sensor and an effector molecule the TCR serves solely as an antigen sensor

A single peptide-MHC complex can trigger cytokine secretion by T cells 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 it’s 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 125I-labeled cell surface antigens C6XL extract immunoprecipitated using 124-40 Ab C6XL extracts protein migrating below the diagonal is disulfide-linked splenic T cells splenic 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

ab TCR locus (germline configuration)

V(D)J recombination of the a and b 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 TCRa and TCRb chains to generate a stable TCRab heterodimer TCRab-expressing thymocytes then undergo thymic education (positive selection, negative selection, lineage commitment) rearranged TCRb first pairs with invariant pre-Ta 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) 1992 - murine IgG against canine lymphoma cells 1998 murine IgG1 directed against the small molecule phenobarbital

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

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 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 Ricardo’s 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

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

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