T Cell Activation/Function Ned Braunstein, MD ned_braunstein@merck.com nsb2@columbia.edu

The Major T Cell Subsets CD4+ T cells CD8+ T cells lck p56 lck p56 g z z z z d e g d e CD3 CD3 C a C b C a C b CD8 CD4 TCR V a V TCR b V a V b peptide peptide MHC II MHC I (1) Interacts with MHC class II expressing cells (B cells, macrophages) (2) Induce(help) B cells to synthesize antibody (3) Induce and activate macrophages (4) Secretes lymphokines (1) Interacts with MHC class I expressing cells (all nucleated cells) (2) Kill MHC class I expressing target cells (3) Suppress immune responses (4) Secretes lymphokines

Naïve T cells are activated by antigen in LNs where they mature into effector cells (1) Naive T cells home continuously from the blood to lymph nodes and other secondary lymphoid tissues. Homing to lymph nodes occurs in high endothelial venules (HEV), which express molecules for the constitutive recruitment of lymphocytes. (2) Lymph fluid percolates through the lymph nodes; the fluid is channeled to them from peripheral tissues, where dendritic cells collect antigenic material. In inflamed tissues, dendritic cells are mobilized to carry antigen to lymph nodes, where they stimulate antigen-specific T cells. On stimulation, T cells proliferate by clonal expansion and differentiate into effector cells, which express receptors that enable them to migrate to sites of inflammation. (3) Although most effector cells are short-lived, a few antigen-experienced cells survive for a long time. These memory cells are subdivided into two populations on the basis of their migratory ability: the effector memory cells migrate to peripheral tissues, whereas central memory cells express a repertoire of homing molecules similar to that of naive T cells and migrate preferentially to lymphoid organs.

The Biology of Dendritic Cells: Antigen capture and presentation to T cells Antigens are captured by DCs in peripheral tissues and processed to form MHC-peptide complexes. As a consequence of antigen deposition and inflammation, DCs begin to mature, expressing molecules that will lead to binding and stimulation of T cells in the T-cell areas of lymphoid tissues. If the antigen has also been bound by B cells, then both B and T cells can cluster with DCs. After activation, B blasts move to the lining of the intestine, the bone marrow, and other parts of the lymphoid tissue with some becoming antibody-secreting plasma cells. T blasts leave the blood at the original site of antigen deposition, recognizing changes in the inflamed blood vessels and responding vigorously to cells that are presenting antigen. This limits the T-cell response to the site of microbial infection. Banchereau, J. and Steinman, R. Nature 392, 245 - 252 (1998)

The T cell activation cycle Minutes Hours Days IL-2R P P Ca++ IP3 DAG c- fos IL-2 etc. c- jun Antigen recognition c- myc NF-KB NF-AT Cytokine production and autocrine stimulation Immediate events Effector functions: Help DTH Killing (CTL) regulation Proliferation

MHC + Ag CD4 CD3 Cytoplasm Nucleus ICAM-1 (CD54) CD80 LFA-1 (CD11a/CD18) CD3 CD28 IL2R lck CD45 fyn ZAP-70 PTK RAS Cytoplasm PLC PKC PIP2 DAG IP3 MAP-kinase Ca Nucleus NF-AT NF-KB OTF1 IL-2

MHC + Ag CD4 CD3 Cytoplasm Nucleus ICAM-1 (CD54) CD80 LFA-1 (CD11a/CD18) CD3 CD28 IL2R lck CD45 fyn ZAP-70 PTK RAS Cytoplasm PLC PKC PIP2 DAG IP3 MAP kinase Ca2+ P I-kB NF-kB Nucleus NF-AT NF-kB OTF1 IL-2

MHC + Ag CD4 CD3 Cytoplasm Nucleus CD80 ICAM-1 (CD54) LFA-1 (CD11a/CD18) CD3 CD28 IL2R lck CD45 fyn ZAP-70 PTK Cytoplasm PLC CNB Cal- modulin CNA PKC PIP2 DAG IP3 Ca2+ P NF-AT Nucleus Jun Fos P NF-AT NF-kB OTF1 IL-2

The T cell activation cycle Minutes Hours Days IL-2R P P Ca2+ IP3 DAG c- fos IL-2 etc. c- jun Antigen recognition c- myc NF-kB NF-AT Cytokine production and autocrine stimulation Immediate events Effector functions: Help DTH Killing (CTL) regulation Proliferation

Key molecular interactions between T cells and APCs MHC class II/ autopeptide CD3 TCR CD40 CD80 Activated T cell CD40 CD40L CD40 CD28 CD80 MHC class II (1) induction of cytokines/chemokines (IL-8, IL-12, TNF-a, MIP-1a) (2) stimulation of CD80 and CD86 expression and co-stimulatory function with activation of T cell growth (3) augmentation of antigen-presenting function

Naïve CD4+ T cells differentiate into Th1 and Th2 subsets Th1 Cells IL-2 IFN-g TNF IL-2 IFN-g, IL-12 Antigen + APC IFN-g (–) IL-4 IL-10 (–) Resting CD4+ cell “pTh” Activated CD4+ cell IL-4 IL-5 IL-6 IL-10 IL-4, IL-13 Th2 Cells

Functions of Th subsets Th1 Cells • Activate macrophages/dendritic cells augment antigen presentation • induce delayed type hypersensitivity (DTH) responses important in eradicating intracellular pathogens (TB, leprosy, listeria • mediate Th1 diseases (ie; rheumatoid arthritis, multiple sclerosis and type I diabetes IL-2 IFN-g TNF IL-4 IL-10 (–) IFN-g (–) Functions of Th2 subsets IL-4 IL-5 IL-6 IL-10 • Help B cells and induce humoral immunity • mediate allergic and immediate hypersensitivity responses • involved in antibody mediated immune diseases like SLE and ITP Th2 Cells

Major Functions of T Lymphocytes (1) Induction and Activation of B cells (Help)-required for most antibody responses (2) Delayed Type Hypersensitivity (DTH) - important in elimination of intracellular pathogens (virus, fungi and mycobacteria) (3) Cell mediated Cytotoxicity (Killer function)-important in the immune response to virus infected cells and cancer cells (4) Suppressor Cell Function- regulates the cell mediated and antibody responses

The Induction and Activation of B cells (Helper Function) Antigen binds specifically to SmIg, is internalized into vesicles and cleaved into peptides which displace CLIP and bind to MHC class II molecules in the endocytic compartment. The peptide/MHC complex is then transported to the surface membrane. The CD4+ T cell a,b TCR/CD4 complex binds to the MHC class II/peptide complex on the surface of B cells. The CD4+ Th2 cells are triggered to secrete IL-2, IL-4, IL-5, IL-6 and IL-10 and begin to express CD40L. These lymphokine and contact dependent signals (CD40L) induce B cells to proliferate, class switch and differentiate into antibody (IgM, IgG, IgA and IgE) secreting B cells and plasma cells.

Antigen Processing and Presentation by B cells MHC Class ll BCR (SmIg) B cell Peptide Internalization of antigen/Ig Antigenic peptides Bind to MHC class II molecules Antigen binds specifically to BCR (surface membrane Ig), is internalized into vesicles and cleaved into peptides which displace and bind to MHC class II molecules. The peptide/MHC complex is then transported to the surface membrane.

Antigen Presentation by B cells BCR (SmIg) hapten Antigen MHC class II carrier protein MHC class II/ carrier peptide complex B cell carrier peptides Antigen binds specifically to SmIg, is internalized into vesicles and cleaved into peptides which displace and bind to MHC class II molecules. The peptide/MHC complex is then transported to the surface membrane.

Resting EffectorT cell Expression of Membrane Proteins Following Antigen Specific Activation of T and B Cells BCR (SmIg) TCR Resting EffectorT cell Resting B cell MHC class II CD4 BCR (SmIg) CD23 IL-2R TCR Activated Effector T cell CD40 CD40L MHC class II CD4 CD80 CD86 Activated B cell MHC class II

CONSEQUENCES OF CD40L/CD40 INTERACTIONS DURING T-B CELL INTERACTIONS TCR Sm Ig CD4 CD40 Activated Effector T cell Activated B Cell Triggering of B cell proliferation Rescue from apoptosis Induction of Ig isotype class switching Up-regulation of CD80 and CD86 Germinal center formation Up-regulation of CD23 Downregulation of CD40L expression

Final Phases of B cell Differentiation are Mediated by Contact T cell signals (CD40L/CD40) and Lymphokines SmIg Activated B cell CD40 CD23 CD40L TCR CD4 Activated Effector T cell Lymphokines IL-2, IL-4, IL-5, IL-6, IFN-g, TGFb IgG IgA IgE Plasma Cell

The Hyper IgM Syndrome (HIM) The Hyper IgM Syndrome (HIM) is an X chromosome-linked Ig deficiency characterized by low serum levels of IgG, IgA and IgE with normal numbers of circulating IgM expressing mature B cells. Germinal centers and splenic follicles due not develop. Affected patients (usually males) are susceptible to pyogenic infections, autoimmune disease and lymphoproliferative disease. In addition, patients are also susceptible to Pneumocystis carini infections. The genetic defect in the majority of HIM patients is associated with mutations in the gene encoding CD40L and can be corrected functionally by soluble CD40 ligand, in vitro. A few HIM patients have normal CD40L but defects in CD40 signaling.

Major Functions of T Lymphocytes (1) Induction and Activation of B cells (Help)-required for most antibody responses (2) Delayed Type Hypersensitivity (DTH) - important in elimination of intracellular pathogens (virus, fungi and mycobacteria) (3) Cell mediated Cytotoxicity (Killer function)-important in the immune response to virus infected cells and cancer cells (4) Suppressor Cell Function- regulates the cell mediated and antibody responses

Delayed Type Hypersensitivity (DTH) a. DTH is initiated principally by CD4+ Th1 cells and is the primary defense mechanism against intracellular parasites including the mycobacteria (TB), fungi and intracellular bacteria (listeriae monocytogenes). b. The cognitive phase of DTH involves CD4+ T cell -macrophage/dendritic cell (MHC class II/peptide) interaction resulting in the local secretion of lymphokines. c. The effector phase of DTH is effected by lymphokines which activate macrophages to secrete lysozyme, TNF, IL-1 and IL-12 as well as chemotactic and migration inhibitory factors restricting granulocytes, macrophages and eosinophils to the site of inflammation. Delayed Type Hypersensitivity (DTH)

DTH Pathway of Contact Hypersensitivity

Th1 CD4+ T Cells Induce Inflammation and DTH Activated CD4+ Th1 cell APC CD4 TCR CD40L CD40 CD4+ Th1 cell CD40L dendritic cell CD40 Macrophage/ mesenchymal cell Inflammation inducing CD40L/CD40 interactions activated dendritic cell Activated macrophages, mesenchymal cells and endothelial cells Proinflammatory molecules Chemokines (IL-8, SDF-1) Lymphokines (IL-1, GM-CSF,TGFb IFNb, IL-12, IL-6 NO, proteolytic enzymes, PGE2

T Cell- Macrophage Interactions Fc receptor TCR a,b CD3 MHC class II CD4 Th1 Cell CD4 Macrophage CD28 B7 (CD80) IL-2 IL-1 IL-6 IL-12 TNF TGF-b IL-12 CD40L CD80 TCR a,b CD4 IL-2 Receptor IFN-g cytotoxic granules CD80 MHC II CD28 Activated Th1 Cell Activated Macrophage

Physiology of the DTH Response CD2 MHC II/peptide Antigen/IgG TCR a,b Phagocytosis killing CD4+ TH1 T Cell CD4 Fc Receptor Macrophage/ Dendritic cell IL-12 IL-2 TCR a,b IL-1, TNF, IL-6 IFN-g IL-2R CD4 endothelial cell fibroblasts hypothalamus granulocytes Mast Cell Eosinophil fever IL-3, IL-8 IL-3, IL-4, IL-5

Functions of Th subsets Th1 Cells • Activate macrophages/dendritic cells augment antigen presentation • induce delayed type hypersensitivity (DTH) responses important in eradicating intracellular pathogens (TB, leprosy, listeria • mediate Th1 diseases (ie; rheumatoid arthritis, multiple sclerosis and type I diabetes IL-2 IFN-g TNF IL-4 IL-10 (–) IFN-g (–) Functions of Th2 subsets IL-4 IL-5 IL-6 IL-10 • Help B cells and induce humoral immunity • mediate allergic and immediate hypersensitivity responses • involved in antibody mediated immune diseases like SLE and ITP Th2 Cells