Methods in Cellular Immunity

Paul ZhouInstitut Pasteur of Shanghai, CAS

November, 2005

Cells and molecules in cellular immune responsesT cells and denderitic cells (DCs)T cell receptors and TCR signalingCD28 and B7 family members

Major Histocompatibility Complex (MHC) and peptide complexUptake of antigens and pathogens - C-type lectin receptors (CLRs)Toll-like receptors (TLRs) Processing of antigens into MHC class I loading compartmentProcessing of antigens into MHC class II loading compartment

Experimental proceduresEx vivo generation of DCs and their characterization Ex vivo manipulation of DCs for vaccineMeasurements of T cell responses in vitroMeasurements of T cell responses in vivoAdaptive T cell-based immune therapies

T cells and denderitic cells

T cells• One of two major classes of lymphocytes• Derived from hematopoietic stem cells, undergoing

differentiation in thymus, and then seeded to the peripheral lymphoid tissues and to circulating throughout the body

• Two major subsets: α/β TCR and γ/δ TCR• Two sublineages in α/β TCR T cells: CD4 and CD8

T cells (differ in antigen recognition and regulatory and effector functions)

• Th1 and Th2 subtypes in CD4 T cells (differ in cytokine secretion and helper functions)

Dendritic Cells

• Initial discovery of Langerhans cells in skin by Paul Langerhans in 1868

• Dendritic cells (DCs) described by Steinman and Cohn in mouse spleen in 1973

• Special properties of DCs in initiating immunity (i.e. antigen and pathogen recognition, uptake, process and presentation as well as pathogen dissemination) were discovered after depletion of monocytes, macrophages and B cells

• Reside in most peripheral tissues, especially at sites interface with environment (skins and mucosa); but DCs migrate to draining lymphoid nodes to activate T cells

• Paradigm of immature (steady) and mature DCs in antigen capture and presentation, lymphocyte activation and tissue distribution

• Ex vivo generation of DCs• Heterogeneity of DCs

LC DDC-IDC moDC pDC_________________________________________________

CD1a + + + -CD1d - + + nrCD11b - + + -CD11c + + + -CD52 - - + +/-CD83 + + + +e-cadherin + - - -CD207, Langerin + - - -CD208, CD-LAMP + + + -CD123 + + + ++BDCA-2,4 nr nr -/+ +_________________________________________________LC, DDC-IDC and moDC are also termed myeloid DCs or conventional DCs. pDCs (plasmacytoid DCs) are also termed lymphoid DCs.

Immature DC Mature DC(steady state)

___________________________________

CD80 Low HighCD86 Low HighHLA-DR Low HighAg capture Efficient notAg processing Efficient notAg presentation not EfficientAllo-stimulation Weak Strong

The Dendritic Cells on cytospin

T cell receptor MHC and peptide complex

TCR, MHC and peptide tri-molecular complex

T Cell Receptors

• TCR consists of two subsets: α/β and γ/δ.• Through somatic gene arrangement, the TCR

displays extreme sequence diversity.• TCR expresses on the surface of T cells in a clonal

fashion, in which to a given T cells only one pair of α/β or γ/δ TCR is expressed.

• α/β or γ/δ TCR itself does not mediate signaling. Instead it is intimately associated with CD3 complex (γδ2ε2ζ). It is the latter that mediates TCR signaling.

• The signal mediated through TCR/CD3 complex is called the signal one.

Human TCR gene organization

TCR/CD3 complex

Human and mouse CD3 protein structure

ITAM motifs

Structure of PTKs

TCR signaling through PTKs

Downstream of TCR signaling

CD28 and CD80/CD86 family molecules

T cell activation and proliferation

Two step T cell activation model:Activating T cells - IL-2 expression and secretion - binding of IL-2 to its receptor on T cells - T cell proliferation

However, in some cases engaging TCR with a cognate peptide/MHC complex leads to no IL-2 expression and release and T cell anergy, which is refractory further activation, suggesting an additional signal (signal 2) is needed for T cell activation.

Co-stimulatory molecules between T cells and APCs

Effect of CD28 signaling on T cells

• Increase transcription and mRNA stability of IL-2• Increase expression of anti-apoptotic protein Bcl-XL• Decrease the threshold of T cell activation by promoting the

formation of an immunological synapse• Naïve T cells depend more on CD28 signaling than

activated and memory T cells• Regulate T cells towards Th2 differentiation (IL4, IL5, and

IL-10 producing cells)• Regulate T cell migration by Increasing CXCR4 expression

and MIP-1α and MIP-1β production; while decreasing CCR5 expression

• Maintain homeostasis of immuno-regulatory CD4+/CD25+ T cells

Effect of CTLA-4 (CD152) signaling on T cells

• Unlike CD28, CTLA-4 is expressed on surface activated T cells. It binds higher to CD80 and CD86 than CD28

• Terminate early events of TCR-mediated signaling through modulating TCRzeta phosphorylation and ERK activation

• Inhibit IL-2 production• Inhibit cell cycle progression• Affect helper T cells to differentiate into Th2 cells• Potential mechanisms of CTLA-4-induced immune

tolerance:1. Directly modulate TCR signaling2. Competitively block CD28/B7 interaction3. CD4+CD25+and CTLA-4+ regulatory T cells

IL-2 and its receptors

Cellular responses to TCR signaling

• Early changes1. Changes in pH

2. Changes in membrane potential (MTT assay)

3. Fluxes in cyclic nucleotides and calcium (Ca influx assay)

• Late changes1. Cyto-skeletal changes (morphologic change)

2. Activation of the cytolytic mechanism (CTL and tetramer staining)

3. Gene regulation: CD25, CD69, IL-2, IL-3, IFNγ, GM-CSF, CTLA-4, MHC class II, VLA-2, 4F2, transferrin receptors, and insulin receptors, etc. (Elisa, Elispots, intracellular cytokine staining, activation markers)

4. T cell proliferation (3H-thymidine and CFSE assays)

Major Histocompatibility Complex (MHC)

Genes encoding MHC class I and class II molecules are located in a MHC region. In each species this region covers several mega-bases of DNA (chromosome 6 in human, chromosome 17 in mouse).

Numerous genes in this region, including1) MHC class I genes (HLA-A, B, C, E, F, and G; H-2D, L

and K)2) MHC class II genes (HLA-DR, DP, and DQ; H-2A and E)3) genes involved in class I and II pathways 4) genes involved in other immunological functions 5) genes seems not to be directly involved in immunological functions

Genetic map of human and mouse MHC

Genetic Polymorphism of HLA-A locus

Serology Alleles Serology Alleles

A1 A*0101,0102 A32(19) A*3201A2 A*0201?217 A33(19)A*3301?303

A3 A*0301,0302 A34(10)A*3401,3402

A11 A*1101?103 A36 A*3601A23(9) A*2301 A43 A*4301A24(9) A*2402?410 A66 A*6601,6602A25(10)A*2501 A68(28) A*68011?803A26(10)A*2601?608 A69(28) A*6901A29(19)A*2901,2902 A74(19) A*7401A30(19)A*3001 ?004 A*8001A31(19)A*31012

Genomic organization of MHC class I and II genes

Structure of MHC class I

Top view of MHC class I or II peptide-binding cleft and bound peptide

HLA-DR I and Peptide HLA-A2 and Peptide

Uptake of antigens and pathogens by Dendritic Cells

Antigen and pathogen recognition and uptake

1. Receptor-mediated endocytosis FcRs: CD64, CD16, and CD32Complement receptors: CR3 and CR4Receptors for heat shock proteins: CD91Scavenger receptors: CD36C-type lectin receptors: MMR, DEC205,

Langerin, BDCA-2, and DC-SIGN

2. Phagocytosis: pathogens and apoptotic and necrotic bodies

3. Macropinocytosis – a constitutive, cytoskeleton-

dependent fluid-phase endocytosis which allows DCs rapidly and non-specifically to sample large amount of surrounding fluid.

Signals that induce DCs to mature

• Receptors for direct pathogen signals: TLRs

• Receptors for indirect sensing of infection through inflammatory cytokines, internal cellular compounds and on-going immune responses: cytokine receptors, TNF receptor family molecules, CD40, FcR, and sensors for cell death

TLRs recognize pathogen-associated molecular patterns

TRL1 (surface, mDC) Soluble bacterial factor (heterodimer with TRL-2)

TRL2 (surface, mDC) bacterial peptidoglycans, lipoproteins

TRL3 (intra, mDC) viral dsRNA, poly(I:C)TRL4 (surface, mDC) LPSTRL5 (surface, mDC) flagellinTRL6 (surface, mDC) diacyl lipopeptidesTRL7 (intra, pDC) viral ssRNATRL8 (intra, mDC) viral ssRNATRL9 (intra, pDC) unmethylated CpG motifsTRL10 unknownTRL11 unknown

Most of TLR-ligand interactions results in intracellular signaling through MyD88, which in turn up-regulates CD83, co-stimulatory molecules and CCR7 and leads to the secretion of IFNα by plasmacytoid DCs or IL-6, IL-10, TNFα and IL-12 by conventional DCs.

Signaling through TRL7/9 in pDCs

T cell-based adaptive immune therapy

PB

Tumor mass

(patients or identical twins)

T cell or TILs

With or without genetic manipulation

Propagation

a) w/cytokines

b) w/ anti-CD3 &anti-CD8 Abs

Large numbers (1011 cells) or more

infusion

In vitro assays to determine1) w/o RCV2) w/ immune functions3) viability

PB, Spleens,

Lymph nodes,

Others

(Immunized or non-immunized

Infected or non-infected)

Mononuclear cells

T cells or T cell subsets

Activated T cells

a) Cytokine production

b) Activation markers

c) Cell proliferation

d) Killing functions w/ target cells

Culture w/

a) APCs + Ags

b) Cytokines

c) Mitogens

d) Superantigens

e) Pharmacological agents

Measurements of T cell responses in vitro

Manipulation of DCs for Antigen processing & presentation

Immature DCs

1) Subunit Ag loading

2) Gene delivery through viral vectors

3) mRNA delivery

Mature DCs

Peptide Ag

loading

a) TLRs

b) Inflammatory cytokines

c) CD40/CD40L

d) Others

Induction through

a) TLRs

b) Inflammatory cytokines

c) CD40/CD40L

d) Others

Bone Marrow

Cord Blood

PB (cytokine immobilized)

CD34 Progenitor cells

Ex vivo generate DCs

GM-CSF

TNFα

Others Immature DCs

Induction through

Mature DCs

PB PBMCs CD14+ Monocytes

IL-4

GM-C

SF

Characterization:

a) Phenotypes

b) Functions

Utilities:

Antigen presentation to T cells in vivo for vaccine to induce T cell responses to cancer, infectious diseases