Cytotoxic T Lymphocytes (CTLs) and NK Cells. Effector T cells. After activation, naïve T cells differentiate into effector and memory T cells

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1 After activation, naïve T cells differentiate into effector and memory T cells Cytotoxic T Lymphocytes (CTLs) and NK Cells After activation, T cells remain in lymph nodes for 5-6 days Effector T cells Cell Function Released Effector Molecules Th1 Cytokines to activate CTLs and macrophages IL-2, IFN TNF, GM-CSF Membrane Effector Molecules TNF Th2 Activate B cells IL-4, IL-10, CD40L CTL Kill Target cells Perforin, Granzymes Fas Ligand

2 CTLs do not require costimulation to kill infected targets Chromium Release Assay Experiments demonstrating CTL killing of target cells are usually done with a chromium ( 51 Cr) release assay, which measures target cell lysis in 4-8 hours. Infect mouse with virus to generate CTLs specific for virus infected targets. Wait until mouse has generated immune response. Use Spleen as source of CTLs. Incubate with syngeneic targets infected with virus that are radioactively labeled with 51 Cr that will be released when cell is lysed. Chromium attaches to proteins in the cytoplasm of target cells Chromium Release CTL Assay Target cells mixed with effector cells at various ratios. Measure release of 51 Cr into media. Express as percentage lysis relative to non-specific effector cell.

3 How do CTL kill target cells? Perforin/ Granzyme B Pathway Directional release of granules (in red). CTL Killing Perforin Contained in CTL granules Pore forming protein. Pokes holes in target cell membrane. Homologous to C9. CTL Killing CTL Killing Granzymes A family of proteases which are involved in induction of apoptosis in target cells after entry through perforin pores. Granzyme = granule enzyme Perforin forms polymers that poke holes in membranes to allow enzymes inside cell. Granzyme B is most active granzyme.

4 CTL Killing Perforin/Granzyme pathway CTL Killing Selective (antigen specific) killing happens in minutes. CTLs can be serial killers (repeatedly killing). Antigen specific killing of infected target cells allows for reduced bystander killing of uninfected cells. Why don't CTLS kill themselves? Cathepsin B-- a protease which cleaves perforin!!! Perforin deficient mice can still kill virally-infected target cells. How? Virus Virus Wildtype Perforin KO Harvest splenocytes 51 Cr labeled targets Harvest splenocytes Anti-cathepsinB control Specific killing of virally infected targets Specific killing of virally infected targets

5 Fas/Fas Ligand Pathway Fas Induced Apoptosis Cascade of Caspases CTLs express Fas ligand interact with Fas expressed on the target cell surface. Proteases that cut at C-terminal side of an aspartate. Pro-enzyme form becomes active through cleavage into subunits. Proteolytic cascade must be activated for eventual DNA fragmentation. Fas Induced Apoptosis Bcl-2 Proteins Control Apoptosis Pro-apoptosis and anti-apoptosis proteins Bcl-2 proteins can inhibit or activate apoptosis. Proteolytic cascade must be activated and not inhibited by anti-apototic bcl-2 proteins. (Target cell has to want to commit suicide).

6 Summary Naïve CD8 T cells are activated in secondary lymphoid organs and differentiate into CTL effector cells. CTL Pathways of Cytotoxicity CTLs can kill targets independently of costimulation. Once a CTL encounters a target cell it releases cytotoxic granules containing perforin and granzymes. Perforin forms pores in the membrane of the target cell allowing granzymes to enter the cell. Granzymes induce apoptosis in the target cell by cleaving caspases. CTL also express FasL and can kill targets via Fas expressed on target cells. But CTLS are not enough.. Viruses are tricky! Class I MHC inhibition by viruses Virus Protein Effect on class I Adenovirus E3-k19 Retain in ER HSV-1,2 ICP47 Blocks TAP EBV EBNA1 Block peptides CMV US2 ER to cytosol CMV US3 Retain in ER CMV US6 Blocks TAP NK Cells Detect "Missing Self" Protection Against Viruses Immune evasion mechanism of viruses to decrease Class I MHC. NK cells preferentially kill cells that have lost expression of MHC class I. cig C M V infecte d a nti-cla ss I N on -in fecte d an ti-cla ss I CMV infection down-regulated MHC class I on human fibroblasts

7 NK Cells Detect "Missing Self" Immune surveillance for Tumors Tumor cells often have decreased expression of Class I MHC to escape T cell recognition. NK cells kill tumor cells. NK cells Distinct lineage of lymphocytes. Do not rearrange α,β,γ or δ TCR. CD3-,CD56+ in humans. CD3-,NKR-P1+ (NK1.1) in rodents Effector functions include cell-mediated cytotoxicity & cytokine secretion. NK and T cell Development TCR Rearrange ment NK Cells - Distribution Stem Cell T / NK Prog enitor Pre-T Pre -T Mature T Thymus CD34 ++ CD34 + CD3- CD4+, CD8+ Thymus not required for development. Normal NK cells in scid mice and mice without RAG1 or RAG2 Bone Marrow Stroma IL-2, IL-7, stem cell factor (SCF) CD3 + CD3 + Pre -NK CD3 + Mature NK CD3 + CD4 + or CD8 + CD16 +, CD56 + CD3-, CD4-, CD8- ~5-20% peripheral blood lymphocytes ~5% lymphocytes in spleen Rare in uninfected lymph nodes >90% of lymphocytes in placenta

8 NK Cells - Effector Functions Cell mediated-cytotoxicity Perforin granzyme pathway Secreted or membrane TNF-α Antibody-dependent cellular cytotoxicity (ADCC) Cytokine secretion Early γ-interferon production Secretion of TNF-α, LT-α, GM-CSF, IL-5, M- CSF, IL-3, IL-10, IL-13. Antibody-dependent cellular cytotoxicity (ADCC) Cells that perform ADCC must have FC receptors to bind Ig molecules and trigger killing of target cell. Cytokine secretion of NK cells Role for IFN-γ Natural Killer (NK) Cells Part of Early Immune Response Viruses Varicella zoster virus & CMV are lifethreatening in humans lacking NK cells. Bacteria NK cells protect against intracellular bacteria which tend to infect macrophages. (e.g. Listeria Toxoplasma, Leishmania)

9 NK are lymphocytes without traditional antigen receptors How do they get activated? NK cells express both activating and inhibitory receptors Inhibitory receptors recognize MHC class I (self) on target cells Activating receptors recognize ligands upregulated on infected cells or tumor cells NK cell Activating Receptors Have ITAMs Intracellular signal of lymphocyte receptors through ITAMs. + Immunoreceptor Tyrosine based Activating motifs (ITAMs). NK cell Activating Receptors Have ITAMs Ligand Class I MHC Specific NK Inhibitory Receptors Killer Inhibitory Receptors(KIR) genes Extensive allelic polymorphism KIR genes found in primates, but not rodents human Ly49 ~10 genes Extensive allelic polymorphism Deleted from the human genome. mouse STIMULATORY RECEPTOR (FcR, NKR-P1, NKG2D) DAP12 KIR CD94 NKG2A/C/E Ly49 Immunoreceptor tyrosine-based Activation motif (ITAM) YxxL x 2 Y Y X X X X L L P Protein Tyrosine Kinase e.g. SYK Phosphorylation of substrates KIR2DL Ig SF KIR3DL C-type lectinrelated family = Immunoreceptor tyrosine-based inhibitory motif (ITIM)

10 ITIMs on Lymphocyte Receptors ITIM Signaling through Inhibitory Receptors MHC Inhibitory receptors have ITIMs to prevent activation. (Act in opposition to ITAMs) Immunoreceptor tyrosine-based Inhibitory motif= ITIM V/IxYxxL/V Inhibitory receptor V X Y X X L P PROTE IN TYROSIINE PHOSPHATASE E.g. Shp-1 De-phosphorylation of signaling molecules NK cell activation is regulated by integrated positive and negative signals Ligands for NK cell activating receptors NK Cell + - Target Activate Inhibit Outcome No No No killing No Yes No killing Yes No Killing Yes Yes No killing/killing Very little known about NK activating receptors and their ligands NKG2D (activating receptor) Recognizes MHC-like ligands (β2m-independent) MIC-A, MIC-B (humans) Rae-1 family (mice) These ligands are induced during viral infection and cellular stress Ligands for many of the activating receptors have not been identified yet NK Cells kill cells expressing activating ligands but need to have inhibitory receptors to protect MHC expressing cells.

11 NK Cell- Opposing Signal Model Inducible Ligand Model Normal Cell NK cell MHC No killing + Infected Cell NK cell MHC Killing Ligand + Ligand induced by stress or infection In these situations, activating receptor can overcome inhibitory signal Summary NK cells activation is controlled by the balance between activating and inhibitory receptors. Inhibitory receptors bind MHC class I molecules and prevent inappropriate lysis of self cells. NK cells are activated by missing self, which can occur when viruses or tumor cells downregulate MHC class I to avoid recognition by CTL. Some ligands for activating receptors are constitutively expressed. Others are induced upon viral infection or stress.