Lymphocyte Effector Functions Killing Major Lymphocyte Subpopulations in

Lymphocyte Effector Functions: Killing

Major Lymphocyte Subpopulations in Peripheral Blood and Selected Effector Functions B cells B Ab production Ag presentation T cells “Other” CD 8 CD 4 Cytotoxicity Help to B cells Help to CD 8 T cells Cytokine secretion

Peptide Loading on MHC Class I in the ER

What Cells Do When the Blitz is On

Antigen Presentation Pathways; Two Old: MHC Class I presentation of peptides MHC Class II presentation of peptides and Two New: Cross-priming of exogenous peptides (MHC Class I) CD 1 -mediated presentation of glycolipids

Question: How do viruses that don’t infect “professional APCs” such as dendritic cells elicit a primary immune response? After all, virally-infected cells don’t normally traffic to 2˚ lymphoid organs

“Classic” view of CTL response against virus-infected cells PVR expressed on non-hematopoietic cells. Infection with Poliovirus Endocytosis of virus, nuclear entry, synthesis of viral proteins in cytosol. Presentation of viral peptides on MHC Class I to CD 8+ cytotoxic T-cells Proliferation of cytotoxic T-cells (CTLs) Perforin/granzymemediated cell death Cross-priming of exogenous antigens by dendritic cells • PVR expressed on non-hematopoietic cells. Infection with Poliovirus Cytopathic changes; recognition and phagocytosis by dendritic cell Phagosome-to-cytosol protein export; ubiquitin-mediated proteolysis of viral proteins; Presentation of peptide via MHC Class I Perforin/granzymemediated cell death of DC; proliferation of CD 8+ CTL; Killing of virus-infected epithelial cells by CTL

Cross-priming: A Dendritic Cell Engulfs a Viral-infected Macrophages Influenza-infected MF Dendritic Cell From: Albert et al. , J. Exp. Med. 188: 1359, 1998

On the Job Training for CTLs

CD 8 T Cells Need Help With Their Memory CD 4 Help 1º effectors No help Memory 2º effectors

Cooperation in Killing: Granzyme and Perforin Tubulin X Barry & Bleackely. Nature Rev. Immunol. 2: 401, 2002 X Lieberman. Nature Rev. Immunol. 3: 361, 2003

Structure of Perforin

Cellular Events at the Synapse Between CTL and Target Cells Transfer of membrane proteins at the synapse. (a–e) A series of still frames from a movie showing CTLs killing a target cell, the membrane of which has been labeled with a fluorescent protein marker, and electron micrographs showing roughly equivalent stages of synapse formation. Granules polarize towards the immunological synapse as the CTL engages its target (a–c); as the CTL disengages, the granules withdraw from the synapse (d), where membrane from the target has accumulated. As the CTL detaches, target membrane is ripped off the dying target (e), and the CTL can engage a new target (color panel of e). Trambas & Griffiths. Nature Immunol. 4: 399, 2003

Human Diseases Involving Defective Granule Killing Disease Gene Clinical Manifestations Chediak-Higashi Syndrome CHS 1 Lysosomal inclusions in all leukocytes Recurrent bacterial infections Decreased NK cell function Oculocutaneous albinism (melanosome defect) Bleeding (platelet storage granule defect) Griscelli Syndrome Rab 27 a Partial albinism Hepatosplenomegaly (lymphohistiocytic infiltration) Decreased NK cell function Hermansky-Pudlak Syndrome HPS 1 Oculocutaneous albinism (melanosome defect) Bleeding (Platelet storage granule defect) Pulmonary fibrosis (Type II cell surfactant body inclusions) Familial Hemophagocytic Lymphohistiocytosis Perforin (30% of cases) Hepatosplenomegaly (accumulation of activated T-cell and macrophages) Decreased NK cell function Pancytopenia

Life (and Death) in the Fas Lane Barry & Bleackely. Nature Rev. Immunol. 2; 401 -409 (2002)

Viral Evasion of Immunity Barry & Bleackely. Nature Rev. Immunol. 2; 401 -409 (2002)

Cross-priming: DCs Fight Back Infected DC Infected tissue cell Cross-presenting DC

Innate-like Lymphocytes B cells B 1 NK, NKT, gd T cells

Time Course of Innate and Adaptive Immunity Time Course of the Primary. Acquired Immune Response immunity Innate immunity

Distinctions Between Innate and Adaptive Immunity Innate immune system Adaptive immune system Receptors Germline-encoded Somatically engineered Distribution Non-clonal Clonal Rapid Slow (requires clonal expansion) Kinetics Specificity self” Recognizes non-self “pattern recognition” Effector Cells All Recognizes “altered Primary structure (TCR) Higher order structure (Immunoglobulin; BCR) Primarily lymphocytes, DCs, Mf

Innate-like B Lymphocytes NK cells not MHC associated

B-1 Cells: B Prepared

Thymus-independent Antigens are Presented to Specialized B-cells Marginal zone B cells, like B-1 cells, respond to carbohydrate antigen and secrete mainly Ig. M MZ MZ Central arteriole GC MZ Like students who cram, after the initial encounter of Ag, they demonstrate little memory

Innate-like T Lymphocytes: NK Cells NK cells not MHC associated

Immunology Course-General Principles Natural Killer Cell

How do NK Cells Recognize Their Targets?

Genes Structure the Class Human MHC Major in theof MHC I Region The human MHC covers ~4 Mbp of DNA on chromosome 6 p 21. 3 and contains over 220 identified loci. It has been divided into three regions: class II (centromeric), class III, and class I (telomeric) with extended class I and class II regions on either side. This is one of the most gene-dense regions of the human genome. It encodes the most polymorphic human proteins known to date. Of the expressed loci in the MHC, roughly 40% are associated with the immune system. They include the classical class I, HLA-A, -B, and -C, nonclassical HLA-E, -F, and -G, as well as ''postmodern'' MICA and MICB genes (MHC class I chain-related genes). The products of classical polymorphic class I genes, HLA-A, B, and C, interact with T cell receptor (TCR) molecules as well as with the products of the killer immunoglobulin-like receptor (KIR) genes expressed on natural killer cells and some T cells. Trowsdale. , Immunity. 15: 363, 2001

One Mechanism of Triggering NK Cell Cytotoxicity The NKG 2 D-DAP 10 receptor complex and its ligands. A representation of NKG 2 D-DAP 10 receptor expression and the interaction of NKG 2 DDAP 10 with its ligands. Cytotoxicity requires the expression of ligands of NKG 2 D (e. g. , MICA, MICB) in the target cells. Lanier, Nature Immunol. 2: 23, 2001

Why do NK Cells Fail to Recognize Healthy Cells?

The Balance of Activating and Inhibitory Signals Determines the Outcome of the NK Effector Response KIR 2 DS KIR 2 DL NKG 2 D DAP 12 DAP 10 Y YP X X Y M Syk Y YP x x Y L PI 3 -kinase + Syk: a protein tyrosine kinase PI 3 -kinase: a phosphoinositide kinase SHP-2: A protein tyrosine phosphatase + - Cytotoxicity Cytokine Secretion SHP-2

X-linked Lymphoproliferative Disease Rare (prevalence of 1/1, 000) X-linked Defect in cytotoxicity Uncontrolled T-cell activation from excessive cytokine secretion, especially in response to EBV infection Defect apparent in NK cells and CTL Patients are treated with bone marrow transplantations of hematopoietic stem cells

Molecular Defect of X-linked Lymphoproliferative Desease Infected B cell NK cell

Innate-like T Lymphocytes NK cells not MHC associated

Structure of the CD 1 b Molecule-Look Familiar?

The NKT Cell Recognizes Glycolipid Antigen Presented by CD 1 on the APC DCs that are infected with intracellular bacteria present foreign bacterial lipid antigens on the cell surfac bound to CD 1 molecules. CD 1 -restricted T cells that are specific for the foreign microbial lipids are stimulated to carry out effector functions, including the secretion of cytolytic granules containing perforin and granulysin, which lyse the infected cells and have direct antimicrobial effects, respectively, and the production of IFN-g and TNF-a, which activate the microbicidal functions of macrophages.

Where Do NKT Cells Fit In? NKT CD 1 d-restricted High IL-4 production Rapid cytokine Production Non-MHC restricted CD 3/ab. TCR+ Thymus-dependent TCR-dependent Cytotoxic IFN-g production MHC-restricted T Thymus-independent NK

Summary 1. For cytotoxic CD 8 T-cells, ligation of the TCR by MHC I/peptide + co-stimulation results in release of granzymes and perforin and/or Fas. L, leading to apoptosis of the target cells. 2. Viruses evade host defense, in part, by down-regulating MHC Class I. Uninfected dendritic cells circumvent this by “cross-priming”: phagocytosis of virus-infected cell and presentation of “exogenous” viral antigens on MHC Class I. 3. The innate immune system has a rapid onset and recognizes molecular patterns in a non-clonal fashion. 4. NK cells lack TCRs, but instead express both activating and inhibitory (e. g. , KIRs) receptors at their surfaces. The relative expression and ligation of these receptors determines the outcome (i. e. , killing or not) of the NK effector response. 5. Innate immune B-cells (e. g. , B-1 cells and marginal zone B cells) recognize carbohydrate antigens, secrete Ig. M, and are not long-lived. 6. Innate immune T-cells (gd T-cells, and NK T cells) recognize non-peptide antigens in non-classical MHC-like molecules. They mediate cytotoxicity & rapid cytokine secretion.