B Cell Activation and Antibody ProductionB Cell Activation and Antibody Production

Lecture 15Lecture 15

Overview of B CellDevelopment,

Activation,Antibody Production

B Cell AntigensB Cell Antigens

Proteins

Dependent Upon Helper T Cellsto Induce Antibody Production

Thymus Dependent Antigens

Polysaccharides, Lipids

Does Not Need Helper T Cellsto Induce Antibody Production

Thymus Independent Antigens

Antigens

Proteins

Dependent Upon Helper T Cellsto Induce Antibody Production

Thymus Dependent Antigens

Polysaccharides, Lipids

Does Not Need Helper T Cellsto Induce Antibody Production

Thymus Independent Antigens

Antigens

B Cell Responses to Thymus-Dependent Antigens (T Cell-

Dependent Antibody Responses)

Primary and Secondary Antibody Responses

Phases of the Humoral Immune Response

T Cell Epitope B Cell Epitope

Antigen

A T-Dependent Antigen Must Contain Both B and T Cell Epitopes

LINKED RECOGNITION

(B Cells)

(T Cells)

Follicles

Activation of B Cells by Antigen and Complement

1. Biochemical Signals2. Endocytosis of Antigen

Antigen Recognition Phase of T-Dependent Antibody Response

Interactions of B Cells with Helper T Cells

Initial Contact T-B Conjugate

TEM Picture

Note the broad area of membrane contact between B and T Cells.

B Cell B Cell

T Cell

Helper T Cell-Dependent Activation Of B Lymphocytes

B-Cell Activation by Thymus-Dependent Antigens

Cytokines

Linked Recognition

C’R

Activated B Cells(Following Interaction with TH Cells

Extra-follicular Site Follicle

Antibody SecretingCells

Germinal Center

Antibodies

Late Events in T Cell-Dependent Antibody Responses-Germinal Center Reaction

Late Events in T Cell-Dependent Antibody Responses-Germinal Center Reaction

• Affinity Maturation– Somatic Hypermutation

• Generation of Memory B Cells

• Affinity Maturation– Somatic Hypermutation

• Generation of Memory B Cells

Somatic Hypermutation and Affinity Maturation of Antibodies

Affinity maturation is the process that leads to increased affinity of antibodiesfor a particular antigen as a result ofsomatic mutation in the Ig genes followed by selective survival of B cells producingthe antibodies with the highest affinity

Affinity Maturation in Antibody Responses

Selection of High AffinityB Cells in Germinal Center

Phases of the Humoral Immune Response toT-Dependent Antigen

Anatomy of Humoral Immune Responses

Antibody Isotype Switching

Antibody Isotype Switching

Isotype Switching Under the Influence of HelperT Cell-Derived Cytokines

Mechanism of Ig Isotype Switching

CD4 T Cell-Dependent Effects in Antibody Responses

• Memory B Cell Development

• Isotype Switching

• Affinity Maturation

Thymus-Independent

Antigens

Thymus-Independent

Antigens

B-Cell Activation by Thymus-Independent and Dependent Antigens

Most TI antigens are polyvalent and induce maximal Crosslinking of membrane Ig on B cells, without a Need for T cell help.

Features of Antibody Responses toT-Dependent and T-Independent Antigens

Antibody Response to T-Dependent Antigens

Antibody Response to T-Dependent Antigens

• Role of Helper T Cells– Cytokines

– CD40/CD40L interactions

• Isotype Switching– Switch Recombination

– Cytokines and Isotypes

• Affinity Maturation– Somatic hypermutation

– Selection for B cells which produce High Affinity Antibodies

• Memory B Cells

• Role of Helper T Cells– Cytokines

– CD40/CD40L interactions

• Isotype Switching– Switch Recombination

– Cytokines and Isotypes

• Affinity Maturation– Somatic hypermutation

– Selection for B cells which produce High Affinity Antibodies

• Memory B Cells

Antibody Effector Functions

Antibody Effector Functions

Effector Functions of Antibodies

Neutralization of Microbes by Antibodies

Neutralization of Toxins by Antibodies

Opsonization of Microbes by Antibodies

Antibody-Dependent Cellular Cytotoxicity (ADCC)

Functions of Complement

Complement-Mediated Lysis of E. coli

Alive Killed

Cellular Interactions in Immune Responses

The Immune Response: A Summary

The Immune Response: A Summary

WHY can immune system recognize somany different epitopes??

Antibody heavy and light chains arecomposed of gene segments

Variable regions are uniqueA limited variety of constant region sequences

are used

They must be rearranged into functional genesbefore they can be transcribed

p. 106

Organization of Ig genes

Germline DNA- gene segments surrounded bynoncoding regions

These are rearranged to form functional genes

Light chains- V, J and C segments

Heavy chain- V, D, J, C

V regions rearrange first

A single V can rearrange to more than one C

Multigene families

or

In humans: 40 V, 5 J, 1 C

Similar number of genes in humans; this is rare in mice

Heavy-chain gene families are similar but morecomplex (D segment)

CH regions formed from exons

One of many possible combinations

p. 111

Heavy chain DNA

D-J and V-DJ rearrangements must occurseparately

On a mature B cell, both mIgM and mIgDare expressed on the cell surface

How does rearrangement occur?

Each V, D and J is flanked by RSS(Recombination signal sequences)

Mechanism is controlled by RAG-1 and RAG-2proteins and an enzyme TdT

If any of these proteins is defective no mature B cells can form; nor T cells

p. 112

“Junctional flexibility” contributes to diversity

But not all rearrangements are “productive”

p. 115

B cells are diploid and contain chromosomes from both parents

However, heavy chain genes are rearrangedfrom only one chromosome, as are light chaingenes.Therefore, any one B cell will contain

one VH and one VL (antigen specificity)

How? Allelic exclusion (Yancopoulos and Alt, 1986)

Model for allelic exclusion

p. 116

Generation of antibody diversity(why are there so many possible antigencombining sites?)

Multiple germline gene segments

In human germline:

51 VH, 27 D, 6 JH

40 V, 5 J

30 V , 4 J

Combinatorial V-J and V-D-J joining

57 V X 27 D X 6 J= 8262 possible combinationsfor VDJ joining

40 V X 5J = 200 possible V 120 possible V

8262 X (200+120) = 2.64 X 106 possible combinations

Without taking into account other sources of diversity

Junctional flexibility in V-J or V-D-J junction

Additional nucleotides added at junctions(P or N addition), if a single-strandedregion is created during the joiningprocess

Somatic hypermutationmutations occur AFTER rearrangementtends to occur in CDR regionsaffects antigen affinity (tends to increase):“affinity maturation”occurs in B but not T cells

Class switching

After antigen stimulation heavy-chain DNA canrearrange so VDJ joins to any isotype

Cytokines help determine the isotype

IgG2a or IgG3 (mice): IFN-IgM: IL-2, IL-4, IL-5IgE: IL-4

p. 122

Membrane-bound or secreted?

Alternative splicing, p. 124

Mature B cells express both mIgM and mIgD

No switch site between C and C

The VDJCC contains 4 polyadenylation sites

mIgM or mIgD can be generated dependingon which polyadenylation site is used

Regulatory elements of transcription

Promoters

Enhancers

Gene silencers

Gene rearrangement brings enhancers closeto the promoter they influence

Why aren’t Igs produced in B cells?

In T cells a protein may bind to the -enhancer and prevent V-J joining

Arrangement of immunoglobulin genes (andformation from exons) and greatlyfacilitated formation of geneticallyengineered antibodies