Lymphocyte Development & Generation of Lymphocyte Antigen Receptors• Pin Ling ( 凌 斌 ), Ph.D.

ext 5632; lingpin@mail.ncku.edu.tw

• References: 1. Abbas, A, K. et.al, Cellular and Molecular Immunology (6th ed., 2007), Chapter 8

QuestionsQuestions

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 .

OutlineOutline

• 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-IStages of Lymphocyte Development-I

Checkpoints in Lymphocyte DevelopmeCheckpoints in Lymphocyte Developmentnt

Selections in Selections in Lymphocyte DeLymphocyte Developmentvelopment

Pluripotent HSCs Pluripotent HSCs =>Lymphocyte Li=>Lymphocyte Lineagesneages

OutlineOutline

• 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

105 genes in human body => Antibodies over 1010 ? => Limited gene segments Hybrid-DNA nature (Recombination) Diverse Ab products

Germline organization of Human Ig lociGermline organization of Human Ig loci

D segment in H chain

Germline organization of Human TCR locGermline organization of Human TCR locii

D segment in chain

Domains of Ig & TCR proteinsDomains of Ig & TCR proteinsHV3 or CDR3

Generation of Diversity in Ag Generation of Diversity in Ag receptor genesreceptor genes

1. Recombination Signal Sequence (RSS): Heptamer & Nonamer => separated by 12- or 23- spacers => Recognized by Reco

mbinase

2. Deletion-VJ exons have the same orientation

3. Inversion – VJ have the different orientation

V(D)J RecombinV(D)J Recombination-Iation-I

V(D)J Recombination-IIV(D)J Recombination-II

V(D)J Recombination III –JuV(D)J Recombination III –Junctional Diversitynctional Diversity

Junctional diversity=> the greatest variability at HV3 (CDR3)

Generation of Diversity in Ig or TCRGeneration of Diversity in Ig or TCR

Generation of Diversity in Ig or TCRGeneration of Diversity in Ig or TCR

Overview of Ig gene rearrangementOverview of Ig gene rearrangement

OutlineOutline

• Overview of Lymphocyte Development

• Generation of Diversity in Lymphocyte Ag receptors

• B & T Lymphocyte DevelopmentB & T Lymphocyte Development

• Summary & Question

Pluripotent HSCs Pluripotent HSCs => lymphocyte lin=> lymphocyte lineages IIeages 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 => Addi

tional 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 TCR transgenic mouse model mouse model => T cell selecti=> T cell selectionon

Positive selectioPositive selection-n-Bone marrow chiBone marrow chimera micemera mice

OutlineOutline

• Overview of Lymphocyte Development

• Generation of Diversity in Lymphocyte Ag receptors

• B & T Lymphocyte Development

• Summary &Summary & Question Question

SUMMARY1. 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 betweenjo

inted gene segments

QuestionQuestion

What is happened to the immunesystem if RAG1 & RAG2 are mutated?