T and B cell activation and signaling

Leslie Berg

S3-143B 6-8371

leslie.berg@umassmed.edu

Activation of naïve lymphocytes

• T cell development in the thymus and B cell development in the bone marrow produce populations of naïve circulating lymphocytes

• Naïve T and B cells are cells that have not yet encountered their antigen

• For any particular antigen (pathogen), the frequency of naïve T and B cells is very low

• Naïve T and B cells do not produce effector molecules

B cell activation, differentiation, and antibody production

1. Antigen recognition and activation 2. Proliferation and new gene expression (differentiation) 3. Effector response to clear infection (antibody production)

T cell activation, proliferation, and differentiation

1. Antigen recognition and activation 2. Proliferation and new gene expression (differentiation) 3. Effector response to clear infection

Two major subsets of effector T cells

• Cytolytic T cells: naïve CD8+ T cells differentiate into effector cytolytic T cells (CTLs) that can kill infected target cells

• Helper T cells: naïve CD4+ T cells differentiate into one of two types of effector cells

– TH1 cells that activate macrophages

– TH2 cells that help B cells produce antibodies

CD8+ CD4+

Summary of key points

• Naïve T and B cells are activated after encounter with antigen, then proliferate and differentiate into effector cells

• B cells differentiate into antibody-secreting cells

• CD8+ T cells differentiate into effector CTLs, CD4+ T cells into effector “helper” (TH2) or “inflammatory” (TH1) T cells

Biochemical analysis of antigen receptor complexes

• Lyse T or B cells in a mild detergent (e.g., digitonin)

– Retains protein-protein interactions more efficiently

• Two other polypeptides immunoprecipitate with the BCR (membrane-bound IgM)

– Igα and Igβ

• Four other polypeptides immuno-precipitate with the TCR αβ dimer

– CD3ε, δ, γ, and TCR ζ

TCR complex BCR complex

TCR or BCR aggregation lead to signaling

APC

T cell

TCR or BCR aggregation lead to signaling

T cell

Can mimic with Ab cross-linking Y

Anti-CD3 Ab

Secondary Ab (or Streptavidin)

Co-receptor complex for B cells

• Consists of three chains – CR2/CD21 is a complement receptor – CD19 is the signaling component – CD81 may help traffic complex to cell surface

• Co-receptor is engaged when antigen has complement proteins attached to it • Co-receptor is aggregated together with BCR and increases signaling

Co-receptor for T cells: CD4 or CD8

• CD4 is expressed on helper T cells

– CD4 binds to MHC class II

• CD8 is expressed on cytotoxic T cells

– CD8 binds to MHC class I

• CD4 and CD8 have two functions

– Adhesion (binding to MHC)

– Signaling

• Cytoplasmic tail associated with tyrosine kinase, p56lck

• CD4 >>> CD8

TCR - coreceptor - MHC interaction

T cell

APC

CD4

TCR

MHC Class II

B cell

Co- receptor BCR

BCR - coreceptor - Ag interaction

C’

Summary of key points

• Membrane-bound IgM is in a complex with invariant chains called Igα and Igβ, forming the BCR

• The TCR is in a complex with invariant transmembrane proteins called CD3ε, δ, γ, and TCRζ

• Igα, Igβ, CD3ε, CD3δ, CD3γ, and TCRζ each contain ITAM motifs

• B cells express a co-receptor composed of CD21, CD18, and CD81 that binds complement, increasing B cell signaling

• T cells also express a co-receptor, CD4 or CD8, that aids in MHC/peptide recognition and provides signaling function

Naïve B and T cells need two signals for activation

• B cell activation requires two independent signals – BCR binding to antigen – “co-stimulatory” signal provided by either T cell help or

by TLR signaling

• Activation of a naïve T cell requires two independent signals – TCR binding to specific MHC/peptide complex – “co-stimulatory” signal

• CD28 binding to B7 on the antigen-presenting-cell

• Ensures B and T cells only activated during an infection

Two signals for B cell activation

1. Antigen binding to BCR 2. TLR ligand binding to TLR

1. Antigen binding to BCR 2. T cell help via CD40 and cytokines

Only “professional” APCs express co-stimulatory signals for naive T cells

• Since naïve T cells need both TCR signals and co-stimulatory signals for activation, only APCs expressing B7 are able to activate a naïve T cell

• Three types of cells express B7 – Dendritic cells

– Activated macrophages

– Activated B cells

• All three cell types upregulate B7 after encounter with pathogens

Activation of naïve B cells

• Resting naïve B cells express surface IgM

• B cells also express receptors to interact with T cells and their products

– CD40

– Cytokine receptors

• Binding of antigen to IgM plus T cell help leads to B cell activation, including antibody secretion

Activation of naïve B cells

Activation of naïve T cells

• Resting naïve T cells express low affinity receptors for interleukin-2 (IL-2)

• Activation of naïve T cells through TCR induces transcription of cytokine and cytokine receptor genes

– IL-2

– High affinity chain of IL-2 receptor

• IL-2 is a T cell growth and differentiation factor that promotes T cell proliferation and the production of “effector” molecules needed to combat infection

Signal transduction pathways leading to B cell and T cell

activation

• TCR and BCR complexes use nearly identical signal transduction pathways

• Some proteins come in two flavors: a T cell version and a B cell version

• Details below focus on the TCR, but similar concepts and mechanisms apply to B cell activation

Naïve T cell activation

• Leads to IL-2 secretion and synthesis of high affinity IL-2 receptor – Drives T cell proliferation and differentiation

• Biochemical signaling pathways well worked out (using “sledgehammer” to activate TCR) – Aggregation of TCR using antibodies to

conserved CD3 proteins

T cell signaling

Stimulus

?

TCR/CD3 complex

TCRαβ

signaling proteins (CD3)

T cell signaling

Stimulus

TCR/CD3 complex

Tyrosine motifs (ITAMs)

phosphorylated

-S-S-

ζ−ζ δε γε

αβ TCR

-S-S-

(T Cells)

β

α

γ

FcεRI

-S-S-

(Mast Cells and Basophils)

Igα Igβ

mIgM

-S-S-

(B Cells)

-S-S-

-S-S- DAP12

KIR2DS (NK Cells)

Immune receptors that contain the ITAM: Immunoreceptor Tyrosine-based Activation Motif (ITAM; YxxL/Ix6-8YxxL/I)

(also FcαR, FcγRII/III associate with ζ-like subunits)

Big picture

• Goals of Ag receptor signaling on B and T cells – Entry into cell cycle – Increase metabolism – Establish differentiation pathways – Elicit effector functions

TCR

transcription

adhesion metabolism

survival actin polymerization

TCR stimulation leading to gene expression

• T cell is “activated” – T cell secretes cytokines (growth

factors) – T cell proliferates – T cell differentiates into an effector cell

• Mechanism: TCR signal turns on “latent” transcription factors leading to new gene expression

TCR aggregation leads to tyrosine phosphorylation of the TCR

Phosphorylation of adapter proteins leading to formation of signaling complex

CD4

Lck

TCR/CD3

ZAP-70

Tyrosine phosphorylation of receptor or adaptor proteins

recruits new substrates

TCR phosphorylation recruits Zap-70

Adapter complex assembled following TCR signaling

BCR phosphorylation recruits Syk

TCR and BCR signaling lead to PLC-γ activation, and the production of two

critical second messengers

TCR signaling activates Phospholipase C-γ (similar for

BCR)

Map-kinase

Activate pre-existing TFs

PKC + Ras-GRP

Can mimic with PMA + Ionomycin

Summary of key points

• TCR and BCR signaling activate latent transcription factors leading to new gene expression

• TCR and BCR signaling activate tyrosine kinases

• A key event downstream of the TCR and BCR is the activation of PLCγ

• PLCγ cleaves PIP2 into two important second messengers, DAG and IP3

TCR or BCR Signaling

• Activated Zap-70 or Syk phosphorylates downstream targets – Second messengers are generated, e.g., Ca+2, small

lipid co-factors – Additional protein kinases and phosphatases are

activated

• Outcome is the activation of latent transcription factors, leading to new gene transcription

• Examples of latent transcription factors activated by TCR signaling: Elk-1, c-Jun, NF-kB, NFAT

Mechanisms of transcription factor activation

• Phosphorylation activates transcription factor activity – Elk, Jun family members

• Phosphorylation leads to dimerization and nuclear transport – STAT proteins (activated by IL-2R signaling)

• Degradation of inhibitory subunit – NF-kB

• Dephosphorylation leads to nuclear transport – NFAT

The MAP-kinases Erk and Jnk phosphorylate transcription factors, thereby activating their

transcriptional activity

Cytokine receptor signaling activates Jak kinases that phosphorylate the STAT transcription factors,

thereby inducing dimerization and nuclear transport of the STAT dimers

TLR signaling induces degradation of IkB, thereby releasing NF-kB to enter the

nucleus and activate transcription

Activation of NFAT

• The NFAT transcription factors are phosphorylated and inactive in resting T and B cells

• T cell activation leads to increased activity of a protein phosphatase, Calcineurin

• Calcineurin dephosphorylates NFAT, leading to NFAT migration into the nucleus

Ca+2

TCR/CD3

CD4

CD28 NFAT Nucleus

IL-2 gene

Calcineurin

Zap-70 Lck

T cell

Summary of key points

• Latent transcription factors are activated by TCR or BCR signaling and mediate new gene expression

• Mechanisms for activating latent transcription factors include phosphorylation, dephosphorylation, and degradation of an inhbitor

• This strategy allows cells to have the necessary transcription factors present, but to keep them in an inactive state until TCR or BCR signals occur