Lymphocyte Development & Generation of Lymphocyte Antigen Receptors Pin Ling ( 凌 斌 ), Ph.D. ext 5632; References: 1. Abbas, A, K. et.al, Cellular and Molecular Immunology (6th ed., 2007), Chapter 8

Questions What is the Bare Lymphocyte Syndrome? What is the advantage of MHC Polymorphism? Is that good if MHC is as diverse as Ig or TCR?  An immunodeficiency disease  Lacking Class-II MHC expression  Mutations in transcriptional factors regulating Class II expression, e.g. CIITA  MHC polymorphism may provide a broader protective immunity in a population during pathogen infection.

Outline Overview of Lymphocyte DevelopmentOverview of Lymphocyte Development Generation of Diversity in Lymphocyte Ag receptors B & T Lymphocyte Development Summary & Question

Key Concepts in lymphocyte development 1. Lymphocyte development-A process of differentiation of lymphoid progenitor cells into mature lymphocytes (T & B). 2. Rearrangement and expression of Ag receptor genes are associated with lymphocyte development. 3. Selection events are involved in preserving cells w/ correct Ag receptors and eliminating dangerous cells w/ self-recognition Ag receptor. 4. Proliferation in the early lymphocyte development is stimulated by IL-7.

Stages of Lymphocyte Development-I

Checkpoints in Lymphocyte Development

Selections in Lymphocyte Development

Pluripotent HSCs =>Lymphocyte Lineages

Outline Overview of Lymphocyte Development Generation of Diversity in Lymphocyte Ag receptors B & T Lymphocyte Development Summary & Question

Key Concepts in Diversity of lymphocyte Ag receptors 1. The germline organizations of Ig & TCR are similar. a. Multiple (V)ariable, (D)iversity, & (J)oining gene segments. b. These gene segments are spatially separated on the chromosomes 2. During lymphocyte development, gene rearrangement of Ag receptor genes occurs: a. Combinatorial diversity - Non-homologous DNA recombination (VDJ recombination) - Lymphoid-specific enzymes- RAG1 & RAG2 Other factors- DNA-PK, TdT, Ku, DNA ligase IV… b. Junctional diversity - Addition or removal of nucleotides among V (D)J joints - The largest contribution to diversity of Ag receptors

In 1970s, Scientists thought 10 5 genes in human body => Antibodies over ? => Limited gene segments  Hybrid-DNA nature (Recombination)  Diverse Ab products

Germline organization of Human Ig loci D segment in H chain

Germline organization of Human TCR loci D segment in  chain

Domains of Ig & TCR proteins HV3 or CDR3

Generation of Diversity in Ag receptor genes

1. Recombination Signal Sequence (RSS): Heptamer & Nonamer => separated by 12- or 23- spacers => Recognized by Recombinase 2. Deletion-VJ exons have the same orientation 3. Inversion – VJ have the different orientation V(D)J Recombination-I

V(D)J Recombination-II

V(D)J Recombination III – Junctional Diversity Junctional diversity=> the greatest variability at HV3 (CDR3)

Generation of Diversity in Ig or TCR

Overview of Ig gene rearrangement

Outline Overview of Lymphocyte Development Generation of Diversity in Lymphocyte Ag receptors B & T Lymphocyte DevelopmentB & T Lymphocyte Development Summary & Question

Pluripotent HSCs => lymphocyte lineages II 1. Regulated by transcriptional factors 2. Notch-1 & GATA3 => T EBF, E2A & Pax-5 => B

Features of B lymphocyte development 1. Maturation of B Lymphocytes development - Rearrangement & expression of Ig gene in a precise order - Selection & proliferation of pre-B cells via pre-Ag receptor 2. Selection of the mature B cell repertoire - Self Ag => Affect the strength of the BCR signal - Immature B cells => self Ag/high avidity => Receptor editing => Additional L chain recombination => Not Self-reactive Fail to receptor editing => Apoptosis 3. During this maturation, B cell lineage cells go through distinct stages => A specific Ig gene expression => Distinct surface markers 4. At Pre-B cell stage, H chain recombination occurs first and associates w/ Surrogate light chains (  & VpreB). - 5 & VpreB are similar to  & light chains but invariant - form pre-B cell receptor => Development

Stages of B lymphocyte development-I

Stages of B lymphocyte development-II

Pre-B cell & Pre-T cell receptors

Stages of B lymphocyte development-III

Receptor Editing – 2nd Chance

Features of T lymphocyte development 1. Maturation of T Lymphocytes development - Sequential Rearrangement & expression of TCR genes - Selection & proliferation of T cell repertoire 2. Selection of the mature T cell repertoire occurs in Thymus - Positive selection => Self MHC-restricted - Negative Selection => Self Ag-MHC/high avidity => Apoptosis => Central Tolerance 3. CD4 & CD8 are surface markers for differentiation of Thymocytes (immature T cells).

Stages of T lymphocyte development-I

Maturation of T lymphocytes in Thymus

CD4 & CD8 expression on Thymocytes

Selection of T lymphocytes in Thymus

Specificity & Avidity are different in T- cell Selection

TCR transgenic mouse model => T cell selection

Positive selection- Bone marrow chimera mice

Outline Overview of Lymphocyte Development Generation of Diversity in Lymphocyte Ag receptors B & T Lymphocyte Development Summary & QuestionSummary & Question

SUMMARY 1. Rearrangement and expression of Ag receptor genes regulate B & T lymphocyte development. 2. Selection events are involved in preserving cells w/ correct Ag receptors and eliminating dangerous cells w/ self-recognition Ag receptor 3. Gene rearrangement of Ag receptor genes occurs during lymphocyte development. - The basic mechanism is common to both B & T cells - Generate the Combinatorial diversity by randomly combining different V(D)J gene segments - Generate the Junctional diversity by filling the gap betweenjointed gene segments

Question What is happened to the immune system if RAG1 & RAG2 are mutated?