T-CELL DIFFERENTIATION IN THE PERIPHERY CD8 TCR APC CD8 TCR APC CD8 TCR APC CD4 TCR APC CD4 TCR APC CD4 TCR APC Ag Ag Ag Memory T-cell Activated T-cell Mature naive T-cell Naive or „resting” T cells… restless migration and recirculation, LN, lymph, blood…for several month, which is their lifespan

NO SIGNAL 1. for CD4+ Th activation PERIPHERAL TOLERANCE IMMUNE RESPONSES ARE NOT INITIATED IN THE PERIPHERY Normal tissue cells do not express MHC class II NO SIGNAL 1. for CD4+ Th activation Normal tissue cells do not express co-stimulatory molecules and do not produce T cell differentiating cytokines NO SIGNAL 2. for CD4+ Th activation Migration of naive T lymphocytes to normal tissues is limited Antigen presenting cells are not activated in normal tissues NO SIGNAL 3. for CD4+ Th activation PERIPHERAL TISSUES TOLERIZE THEMSELVES

T-CELL DIFFERENTIATION Environmental factors and interactions with APCs initiate distinct differentiation programs in naive T-cells

Arming of effector T cells Clonal selection and differentiation APC T IL-2 How can this cell give help to or kill cells that express low levels of B7 family costimulators? Activation of NAÏVE T cells by signal 1 and 2 is not sufficient to trigger effector function, but….. the T cell will be activated to proliferate and differentiate under the control of autocrine IL-2 to an effector T cell. These T cells are ARMED

Effector function or Anergy? Clonally selected, proliferating and differentiated T cell sees antigen on a B7 negative epithelial cell This contrasts the situation with naïve T cells, which are anergised without costimulation The effector programme of the T cell is activated without costimulation Armed Effector T cell CD28 Co-receptor TcR IL-2 Armed Effector T cell Naïve T cell Kill Epithelial cell Epithelial cell Epithelial cell

IL-12 FAVORS POLARIZATION TO TH1-TYPE EFFECTOR T-CELLS Virus, bacteria, protozoa, fungi DCMΦ CD8+ cytotoxic T cell IL-12 NK cell IL-12 IFNγ Th0 Th1 IL-12 Th2 IL-2 IFNγ TNF-β GM-CSF IL-3 IL-4 IL-5 IL-10 IL-13

IL-10 FAVORS POLARIZATION TO Th2 DC Self tissue, tumor cell IL-10 Macrophage Th2 IL-4 IL-5 IL-10 IL-13 Th0 IL-10 Th1 IL-2 IFNγ TNF-β TNF-α GM-CSF IL-3 TOLERANCE

POLARIZATION OF HELPER T LYMPHOCYTES IS DIRECTED BY DENDRITIC CELL-DERIVED AND AUTOCRINE CYTOKINES AND TRANSCRIPTION FACTORS

EFFECTOR FUNCTIONS OF TH1 CELLS Activation Killing Proliferation Feed back Entry to tissue Recruitment

Granulomas develop when intracellular pathogens resist elimination Long term persistance of infectious agent in a separated envitonment In some circumstances mycobacteria (red) resist the killing effects of macrophage activation (top panel). A characteristic localized inflammatory response called a granuloma develops (second panel). The granuloma consists of a central core of infected macrophages, which can include multinucleated giant cells formed by macrophage fusions, surrounded by large single macrophages often called epithelioid cells. Mycobacteria can persist in the cells of the granuloma. The central core is surrounded by T cells, many of which are CD4 T cells. The photomicrograph (bottom panel) shows a granuloma from the lung.

Responses to Mycobacterium leprae are sharply differentiated in tuberculoid and lepromatous leprosy. The photographs show sections of lesion biopsies stained with hematoxylin and eosin. Infection with M. leprae bacilli, which can be seen in the right-hand photograph as numerous small dark red dots inside macrophages, can lead to two very different forms of the disease. In tuberculoid leprosy (left), growth of the microorganism is well controlled by TH1-like cells that activate infected macrophages. The tuberculoid lesion contains granulomas and is inflamed, but the inflammation is localized and causes only local peripheral nerve damage. In lepromatous leprosy (right), infection is widely disseminated and the bacilli grow uncontrolled in macrophages. In the late stages there is severe damage to connective tissues and to the peripheral nervous system. There are several intermediate stages between these two polar forms.

EFFECTOR CD4+ HELPER T LYMPHOCYTES SECRETE DIFFERENT CYTOKINES IL-4, IL-5, IL-10 Th2 Th0 IFNγ, IL-2, TNF-β/LT Th1 IFNγ IL-4 Inflammatory cytokines CELLULAR IMMUNE RESPONSE Anti-inflammatory cytokines HUMORAL IMMUNE RESPONSE

SUBSETS OF HELPER T LYMPHOCYTES COLLABORATE WITH DIFFERENT PROFESSIONAL ANTIGEN PRESENTING CELLS B7 expression  antigen presentation Germinal center formation Affinity maturation Isotype switch Memory B cell generation Th2 B IL - 4 CD40L CD40 B7 expression  antigen presentation MHC-II expression  antigen presentation Mature dendritic cell Activated macrophage CD40L CD40 DCMΦ Th1 IFNγ

EPIGENETIC CHANGES, MEMORY CD4 TCR Th1 Intracellular pathogens Inflammation Tc activation IgG1 & IgG3 antibodies ADCC, opsonisation Complement activation EFFECTOR HELPER T-CELLS TCR CD4 Th2 Extracellular pathogens Multicellular parasites Secretory IgA IgE, allergy Naive CD4+ T cell Th0 blast TCR CD4 CD4 Th17 Extracellular pathogen Inflammation Autoimmune diseases Allergy TCR CD4 DC TCR TCR CD4 Treg Th1 inhibition Maintenance of immunological tolerance INTERACTION ACTIVATION INSTRUCTION CLONAL EXPANSION DIFFERENTIATION EPIGENETIC CHANGES, MEMORY

CHARACTERISTICS OF EFFECTOR HELPER T LYMPHOCYTES Th1 T-bet CCR1 CCR5 CXCR3 CD45RBlo IL-12Rα LAG3 Th17 RORγt IL-23R CCR6 TCR+ CD4+ CD28+ CD25+ CD127 IL-7Rα↓ CTLA4 B7 ligand GITR CD25 IL-2Rα Treg FoxP3 Th2 GATA3 CCR4 CCR3 CXCR4 CD45RBhigh IL-1R CD30

Th9 IL-9 HOWEVER, THIS IS NOT THE END OF THE STORY… AS OF YET… CHARACTERISTICS OF EFFECTOR HELPER T LYMPHOCYTES – Th9 CELLS TCR+ CD4+ CD28+ CD25+ Th9 PU.1 & IRF4 IL-4Rα IL-2Rγ Parasitic helminth infections Chronic allergic inflammation Asthma Autoimmune diseases Th9 Th0 TGF-β IL-4 Other cytokines that enhance Th9 polarization: Type I IFNs , IL-1β, IL-6 IL-9 IL-10 CCL17 CCL22

Annunziato and Romagnani (2009) Arthritis Research & Therapy 11:257

Neurath MF and Finotto S (2011) Cytokine Growth Factor Rev 22:83-89.

Th2 functions

Th2 cells stimulate the proliferation and differentiation of naive B cells

ISOTYPE SWITCH IN ACTIVATED B CELLS IS REGULATED BY HELPER T CELL - DERIVED CYTOKINES ISOTYPE SWITCH IS INFLUENCED BY site of antigen entry tissue microenvironment nature of professional antigen presenting cells polarization of helper T lymphocytes

REGULATION OF ISOTYPE SWITCH OF B CELLS IL-2 IL-4 IL-5 IgM Helper T cell IL-2 IL-4 IL-5 IL-2 IL-4 IL-6 IFNγ IgG B cell IL-5 TGFβ IgA IL-4 IgE B cell proliferation and differentiation – isotype switch

Human Ig classes and subclasses Complement activation Classical FcR binding IgM ++++ - IgG1 ++++ +++ IgG2 + + IgG3 +++ ++++ IgG4 +/- + IgA - +++ IgE - +++ IgD - -

Cytotoxic T-cells

PRIMING OF CD8+ CYTOTOXIC T CELLS

PRIMING OF CD8+ CYTOTOXIC T CELLS THROUGH COLLABORATION OF HELPER AND CYTOTOXIC T-CELLS ACTIVATION CD4+ Th CD40L Dendritic cells with high B7 expression activate CD8+ T cells directly Dendritic cells activate CD4+ T cells, which in turn enhance the co-stimulatory activity of dendritic cells Activated CD4+ T cells secrete cytokines (IL-2), which directly acts on activated CD8+ T cell B7 IL-2 CD28 CD8+ Tc

KILLING OF INFECTED CELLS BY CTL Recognition by CTLs induces secretion of cytotoxins to the target cell As shown in the panels on the left, initial adhesion to a target cell has no effect on the location of the lytic granules (LG) (top panel). Engagement of the T-cell receptor causes the T cell to become polarized: the cortical actin cytoskeleton at the site of contact reorganizes, enabling the microtubule-organizing center (MTOC), the Golgi apparatus (GA), and the lytic granules to align toward the target cell (center panel). Proteins stored in lytic granules are then directed onto the target cell (bottom panel). The photomicrograph in panel a shows an unbound, isolated cytotoxic T cell. The microtubules are stained green and the lytic granules red. Note how the lytic granules are dispersed throughout the T cell. Panel b depicts a cytotoxic T cell bound to a (larger) target cell. The lytic granules are now clustered at the site of cell-cell contact in the bound T cell. The electron micrograph in panel c shows the release of granules from a cytotoxic T cell.

PHYSIOLOGICAL AND PATHOLOGIC CELL DEATH Apoptotic signal Cell demage

APOPTOSIS AND NECROSIS Healthy cell Necrotic cell Apoptotic cell Late apoptotic cell HIGHLY REGULATED PROCESS Induction Excecution Mitochondrial function  * Activation of caspases Electron transport  * Serine protease,calpain, proteasome Oxidative phosphorylateion  * Redox potential ATP synthesis  * DNA degradation (endonuclease)

MECHANISM OF CELLULAR KILLING BY CD8+ CYTOTOXIC T LYMPHOCYTES Proteoglycans H2O, Ca++, ions Polymerized perforin Granzyme APOPTOSIS CYTOKINE RELEASE CD8+ T CELL TARGET CELL

TNF AND TNF RECEPTOR FAMILY MEMBERS AND THEIR LIGANDS Soluble TNF TNF-α TNF-β/LTα TNFRI TNFRII RECEPTOR TRIMERIZATION LTβ LTα LTβR Soluble FasL FasL FAS CD40L CD30L CD27L 4-1BBL Ox40L CD40 CD30 CD27 4-1BB Ox40 DEATH DOMAIN

MECHANISMS OF FAS RECEPTOR – MEDIATED PROGRAMED CELL DEATH

CONSEQUENCE OF T CELL-MEDIATED IMMUNE RESPONSES Cytotoxic T lymphocytes recognize virus-infected or tumor cells Activated cytotoxic T-lymphocytes kill virus-infected or tumor cells