ANTIBODY DIVERSITY II

Macfarlane Burnet ( ) Macfarlane Burnet ( ) CLONAL SELECTION THEORY Antibodies are natural products that appear on the cell surface as receptors and selectively react with the antigen Lymphocyte receptors are variable and carry various antigen-recognizing receptors ‘Non-self’ antigens/pathogens encounter the existing lymphocyte pool (repertoire) Antigens select their matching receptors from the available lymphocyte pool, induce clonal proliferation of specific clones and these clones differentiate to antibody secreting plasma cells The clonally distributed antigen-recognizing receptors represent about ~10 7 – 10 9 distinct antigenic specificities

Cc. (minimum) 10 million various (10 7 ) B lymphocyte clones with different antigen-recognizing receptors Cc. (minimum) 10 – 1000 million various ( ) T lymphocyte clones with different antigen-recognizing receptors DIVERSITY OF LYMPHOCYTES Assumption 1 All lymphocytes have a different receptor Assumption 2 The receptor can be activated by many different antigens lymphocytes in our body ( B and T lymphocytes) How many SPECIFICITIES ?

How does somatic gene rearrangement (recombination) work? 1.How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2.How do V region find J regions and why don’t they join to C regions? rule 3.How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity Junctional diversity (P- and N- nucleotides, see above)

Hipervariable and framework regions exist within the variable domains of Igs HV3 in the light-chain is at the junction between rearranged V and J segments In the heavy chain HV3 is formed by the D segment and its the rearranged V and J segments.

Rearrangement of V, D, and J segments produces a functional heavy-chain gene. Separate gene segments encode the constant domains of the various Immunoglobulin isotypes

Gene rearrangement and the synthesis of cell-surface IgM in B cells SUMMARY

Coexpression of IgD and IgM is regulated by RNA processing.

Each B-cell produces immunoglobulin of a single specificity Both the heavy and the light chain coding sequences are present twice in the germline.. (maternal and paternal chromosome) Yet, the B-cell receptor (BCR) on each B-cell is mono-specific The B-cell produces monospecific antibodies This is important for the efficiency of clonal selection and to ensure specificity of the immune response. The process that ensures monospecificity is called: Allelic exclusion

A clever genetic model provides evidence for allelic exclusion Allotypes can be identified by staining B cell surface Ig with antibodies a/a b/b a/b Y B b Y B a Y B b Y Y B ab Y B a AND ALLOTYPE- a polymorphism in the Heavy chain C region of Ig

Allelic exclusion is needed for efficient clonal selection All daughter cells must express the same Ig specificity otherwise the efficiency of the response would be compromised Suppression of H chain gene rearrangement helps to prevent the emergence of new daughter specificities during proliferation after clonal selection S. typhi Antibody S. typhi

Y Y YY Suppression of H chain gene rearrangement ensures only one specificty of Ab expressed per cell. Allelic exclusion prevents unwanted responses B Self antigen S. aureus Y Y Y Y Y B Y Y Y Y Y Y Y Anti S. aureus Antibodies Y Y Y Y Y Y Anti self Abs One Ag receptor per cellIF there were two Ag receptors per cell Y Y Y Y Y Y Y Anti S. aureus Antibodies Prevents induction of unwanted responses by pathogens

Allelic exclusion is needed to prevent holes in the repertoire Exclusion of anti-brain B cells i.e. self tolerance Y Y B B One specificity of Ag receptor per cell S. aureus Anti-self Ig AND anti-S. Aureus Ig Y Y Y B B IF there were two specificities of Ag receptor per cell Anti-brain Ig B B Deletion Anergy OR anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” BUT Y Y Y B B

Allelic exclusion helps diagnose and monitor lymphoma: Due to clonal expansion of a single cell that contains a unique rearrangement the amount of cancer cells in blood or in bone marrow can be determined Can be used to monitor residual tumor cells upon treatment

Minimal residual disease (MRD) Childhood Acute lymphoblastic leukemia (ALL) is a heterogenous disease Prognosis varies Personalized treatment is required Response to therapy Early detection of relapse Bone marryw transplantation

Flow cytometry: -Detection of the aberrynt protein -Kvantitative method, but labour intensive, and less sensitive Real-time kvantitative PCR -Qquantitative, specificity and superior sensitivity, -Requires minimal amounts os marerial for the test Metods for detection of MRD - nyomonkövetésére használt módszerek

Diagnosis and monitoring of the treatment of childhood acute lymphoblastic leukemia (C-ALL) The sequence of the framework regions are well conserved. Step 1: design primers that amplify these regions together with the hypervariable regions Step 2: sequence the PCR fragments to obtain spec. Sequence info on the actual tumor (monoclonal, majority of amplification product is tumor-derived Step 3: based on the sequence info, design tumor-specific PCR primers Step 4: quantitate gene expression by Q-PCR. Follow success of therapy and detect minimal residual disease much earlier then by flow cytometry

I. Identification of monoclonal, i.e. blast-specific gene rearrangements (IGH, TCR , TCR  and IGK-KDE) II. Sequencing amplified DNA Result of IMGT junction analysis (http//imgt.cines.fr): IGH rearrangement of patient Q015 An algorythm is available for the identification of unique and conserved portions of the sequence ImMunoGeneTics information system (IMGT)

Consensus TaqMan probe and reverse primer Patient specific forward primer designed by us BIOMED2 Concerted Action: IgH, TCRg, TCRd, TCRb, IgK VN1DN2J Design patient-specific primers (red) and use them together with consensus primers