HE303 T cells PDF

The adaptive immune response T cells – receptors, activation, effector functions Adaptive immunity Remember: only lymphocytes (B cells and T cells) can mediate adaptive immune responses Adaptive immune responses Only found in animals with a jaw and backbone Enables a s...

The adaptive immune response T cells – receptors, activation, effector functions Adaptive immunity Remember: only lymphocytes (B cells and T cells) can mediate adaptive immune responses Adaptive immune responses Only found in animals with a jaw and backbone Enables a specific immune response against anything (diverse) A primary adaptive immune response results in immune memory Re-exposure to the same pathogen results in an escalating response (secondary immune response) This response activates faster than the primary immune response This response is stronger and more effective than the primary immune response How B cells and T cells recognize pathogens There are only two adaptive immune receptors: TCRs BCRs Both members of the immunoglobulin (Ig) superfamily Function: to recognize pathogens and initiate adaptive immune responses These receptors are randomly generated and highly diverse Self-reactive T cells are deleted in the thymus, self-reactive B cells are deleted in the bone marrow T cell receptors Most TCRs have alpha and beta chains (TCR⍺β) Some TCRs have gamma and delta chains (TCRɣ𝛿; joined together -> intervening DNA is removed TCR rearrangement TCR genes are made up of alpha and beta chains (most commonly) or gamma and delta chains TCR alpha and delta genes are found on chromosome 14 and contain multiple V, D, J exons TCR beta and gamma genes are found on chromosome 7 and contain multiple V and J exons (no D exons) Rearranged TCR express either ⍺β or ɣ𝛿 chains The Immune System, 4th ed.; Peter Parham ; ( 2014). Garland Science, Taylor & Francis Group, LLC, New York, NY. TCR receptor diversity Immature T cells (thymocytes) travel to the thymus to undergo receptor rearrangement there In the thymus to undergo a selection process but no further mutation Segment TCR alpha TCR beta TCR gamma TCR delta Variable (V) 70+ 52 14 8 Diversity (D) 0 2 0 3 Joining (J) 71 13 5 3 Constant (C) 1 2 2 1 http://omim.org/ Visualize beta chain somatic recombination Pause for a second: think about having 67 friends helping you out with this. You give each friend a ‘gene segment’ to hold and sort them into three groups. 52 friends will be holding a V gene segment, 2 friends will be holding a D gene segment and 13 will be holding a J gene segment. Then you are RAG-1/RAG-2 and you randomly pull out one friend from each group, one V, one D and one J. The rest of the genetic sequence is lost and the cell is now committed to make just that one receptor. Now imagine if you repeated the process as a different T cell, you could pick three different friends at random, you’d make a different TCR This is how lymphocyte diversity is generated! Thymic selection of mature T cells Immature T cells, called thymocytes, do not express a TCR Receptor rearrangement starts when the thymocytes reach the subcapsular region of the thymus Rearrangement is random TCRs could be self-reactive and need to pass the selection process T cell selection – two checkpoints Positive selection: Is the rearranged TCR useful? Lymphocytes expressing TCRs that can bind self MHC with low affinity receive a survival signal. TCRs that can’t bind to self MHC die Can the TCR recognize antigen in MHC? Negative selection: is the rearranged TCR dangerous? Lymphocytes expressing TCRs that bind to self MHC with high affinity receive a signal to die (apoptosis) The majority of thymocytes die during the selection process (~95%) This establishes central tolerance (eliminating self-reactive T cells during T cell development) What happens to the survivors? The remaining 5% of lymphocytes express TCR with intermediate affinity for self MHC This represents your T cell repertoire and is highly diverse (estimated to contain 1016-1018 unique TCRs) Activation of the adaptive immune system Now that our B cells and T cells have receptors that are capable of binding antigen but not ‘self’ they are ready to detect invading pathogens Hi, it's me, I'm the problem, it's me! Main cell types of the adaptive immune response Helper T cells: express the accessory molecule CD4 (which binds to an invariant portion of MHC class II) Cytotoxic (killer) T cells: express the accessory molecule CD8 (which can bind to the alpha 3 domain of MHC class I) TRegs: Regulatory T cells that suppress the activity of the above two populations and control immune responses Conventional B cells: have diverse BCRs (referred to as B2 cells) which rely on T cell help for activation Plasma cells: activated B cells that produce antibodies (the Ig molecule released has exactly the same specificity as the membrane bound BCR) Unconventional B cells: less diverse BCRs (referred to as B1 cells) which are less dependent on T cell help for their activation Lymphocytes that have never seen an antigen before are called ‘naïve’ lymphocytes Antigen presentation T cells are only activated in the presence of antigen-presenting cells (APCs) Dendritic cells (only APC for primary immune response) Macrophages, B cells APCs present pieces of the pathogen (8-24 amino acids) held in their MHC to the TCR Kambayashi, T., Laufer, T. Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell?. Nat Rev Immunol 14, 719–730 (2014). Antigen presentation Reminder: Dendritic cells exist in the tissue in an immature state Immature dendritic cells have a high ability to internalize antigens A steady rate of dendritic cells drain from tissue into lymph nodes This rate increases during inflammation Travelling to the lymph nodes the dendritic cells mature Mature dendritic cells are in the lymph nodes These cells cannot take up antigen but are very good at presenting antigen in their MHC class II receptors Present to T cells in lymph node What is MHC? Major histocompatibility complex ‘complex’ because genes for MHC all together in a single location on chromosome 6 Peptide receptors Peptide = a short chain of amino acids, cut from a pathogen- or host-derived protein There are two classes of MHC Class I MHC: comprised of an ⍺ chain non-covalently attached to β2 macroglobulin Class II MHC: comprised of an ⍺ and β chain  Similar tertiary structure, both MHC have a peptide binding groove Groove is 2 alpha helices above a beta pleated sheet MHC proteins don’t fold correctly/transit to cell membrane unless a peptide is bound within its groove MHC in humans are HLA Human leukocyte antigens Class I MHC are: HLA A, B and C Class II MHC are: HLA DR, DP and DQ MHC molecules are co-dominantly expressed Both maternal and paternal gene products are expressed at the same time in the same cells Each cell in your body expresses 2 copies of HLA-A, 2 copies of HLA-B and 2 copies of HLAC (6 different class I MHC) Specialized antigen-presenting cells express 2 copies of HLA-DR⍺, 2 copies of HLA-DR β, 2 copies of HLA-DP⍺, 2 copies of HLA-DPβ, 2 copies of HLA-DQ ⍺ and 2 copies of HLA-Daβ = 12 possible different class II MHC MHC genes are polymorphic Polymorphic means there are many different alleles (versions) of the gene found within the population The polymorphic region is the peptide binding groove MHC genes are actually the most polymorphic genes in the human population The chance you have the same MHC genotype as someone else is extremely small Different MHC will bind a different spectrum of peptides This depends on the specific peptide-binding properties of each allele The implications for this are HUGE  Ag presentation NK chaperone NK Peptide loading Ag presentation The Immune System, 4th ed.; Peter Parham ; ( 2014). Garland Science, Taylor & Francis Group, LLC, New York, NY. Polymorphic: many different alleles (for the same gene) found within a population Oligomorphic: a few different alleles found within a population Monomorphic: only one allele found within a population MHC haplotype Haplotype: the combination of HLA alleles found on an individual’s chromosome 6 Very little changes to the sequence during a single round of meiosis You inherit your haplotype from your parents Over time (10,000 generations) HLA alleles have been generated and recombined (made new combinations) Your haplotype are the HLA genes inherited from your parents, co- dominantly expressed on your cells 2 siblings have a 25% change of having an identical HLA haplotype, 50% chance of being HLA haploidentical (sharing one haplotype) and 25% chance of sharing no HLA haplotypes! https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628004/  Haplotype and antigen presentation Your MHC haplotype dictates what peptides your MHC bind best That means your cells are great at presenting some antigens to your T cells and will be bad and presenting other antigens to your T cells The Immune System, 4th ed.; Peter Parham ; ( 2014). Garland Science, Taylor & Francis Group, LLC, New York, NY. HLA-A*02:01 = HLA-A isotype, 02 (major allotype group), 01 (variant within group) If you express HLA-A*02:01 you will be better at fighting HIV If you express HLA-B*27:05 you will be better at fighting influenza Implications of this 1. Allelic advantage: Some alleles are better at presenting antigens then others 2. Heterozygotic advantage: Individuals that inherited diverse MHC haplotypes from their parents have an advantage More diversity = increased ability to bind different peptides to present to T cells 3. Individuals are attracted to others whose MHC haplotype is different from theirs  Heterozygotes (inherited diverse alleles from both parents) will be able to present a larger repertiore of Ag compared with homozygotes And if your alleles have very different binding abilities you will be able to present more Ags If haplotypes are closely related, you will bind The Immune System, 4th ed.; Peter more Ags than a Parham ; ( 2014). Garland monozygote, but not as Science, Taylor & Francis Group, many as if you had LLC, New York, NY. divergent haplotypes More diversity = more Ag presented = increased likelihood of an immune response Heterozygotic advantage: Red = heterozygote Blue = homozygote for 2-3 loci Yellow= homozygote for 1 loci More variability = better chance to fight HIV Allelic advantage: Some HLA alleles are associated with slow progression of HIV disease (HLA-B14, B27, B57, HLA-C8) Other alleles associated with rapid progression (HLA-A29, HLA-B22, HLA-C16, HLA-DR11) Almost all class I, as CD8 T cells are the principal control cell for virus infections The Immune System, 4th ed.; Peter Parham ; ( 2014). Garland Science, Taylor & Francis Group, LLC, New York, NY. MHC is not only responsible for antigen presentation It is also responsible for defining ‘self’ Remember: T cells ‘learn’ to recognize self-MHC in the thymus Positive selection: only T cells that recognize self-MHC with moderate affinity are allowed to survive MHC restriction: A T cell receptor will only recognize its matching antigen presented by ‘self’ HLA allotype It will not bind to the same Ag presented by a different allotype It will not bind a different Ag presented by the same allotype The combo has to be perfect  MHC restriction When T cells bind APCs they check: 1. Does my receptor bind the ligand? 2. Is the MHC a self MHC? Haplotype simplified: HLA-B8+ and HLA-B44+ Mismatch haplotypes mean T cell will not recognize antigen Mismatch antigen affinity means T cell will not recognize antigen MHC haplotype and antigen affinity must match MHC and Allorecognition T cells can detect ‘self’ vs ‘non-self’ by binding to MHC Recognizing non-self MHC will lead to T cell activation against the non-self MHC Principle behind transplantation rejection Front. Immunol., 05 November 2018 | https://doi.org/10.3389/fimmu.2018.02548 How are peptides presented by MHC? MHC class I and MHC class II have different pathways for loading peptides into their peptide-binding grooves MHC class I presentation Intracellular peptides ~9 amino acids long Remember: MHC class I presents to CD8+ T cells, so you want to present intracellular peptides to tell the killer T cell you’re infected Intracellular peptides Intracellular proteins are broken down into peptides in the cytoplasm by Proteasome Always present in the cell, breaks down damaged, mis-folded, unneeded proteins Immuno- proteasome Immunoproteasome APCs and some infected cells can start making a specialized proteasome Called the immunoproteasome Expression is induced by IFN-ɣ The immunoproteasome preferentially degrades proteins to produce MHC class I compatible peptides 8-10 aa in length, hydrophobic or basic C-terminus Cap changes to increase rate of peptide release Who makes IFN-ɣ?  TAP 9 amino acid peptides are actively transported into the endoplasmic reticulum by the Transporter associated with Antigen Processing (TAP) TAP favours peptides of 9 amino acids with hydrophobic or basic C terminal residues TAP is pre-optimized to transport MHC class I peptides MHC class I peptide loading  Calnexin is a chaperone protein that helps MHC alpha chain fold Peptide loading complex: Tapasin brings MHC class I to TAP and bends MHC so it is close to TAP for effective Ag binding Calreticulin Chaperone protein ERP57 Complex stabilizer ERAP (endoplasmic reticulum aminopeptidase) can trim peptides so they fit better into the MHC class I peptide binding groove A tight binding Ag changes the conformation of MHC and it breaks free from tapasin, leaves the peptide loading complex Leaves the RER → Golgi (glycosylated) → plasma membrane surface MHC class I antigen presentation Remember: expressed by almost all cells Normally self peptides are presented in class I MHC In the absence of an infection If a cell is infected, then the pathogen proteins are processed by the proteasome Peptide fragments are presented in class I MHC Different people will express different class I MHC, so they will present different pathogen peptides to their TCRs CD8+ T cells bind to MHC class I/peptide complexes MHC class II binding antigen Class II MHC binds extracellular peptides ~22-24 amino acids long  Newly synthesized MHC class II alpha and beta chains bind to protein called invariant chain while still in the ER The invariant chain presents peptides from binding Meanwhile extracellular pathogens/antigens are taken into the cell by phagocytosis or endocytosis As they travel away from the surface, the phagosome/endosome acidifies, merges with lysosomes, and the pathogen is degraded into peptides  The class II MHC+Ii complex goes through the Golgi and eventually will merge with a late endosome The invariant chain (Ii) is degraded by proteases inside the vesicle, leaving a small portion remaining in the class II peptide groove CLIP: class II associated invariant chain peptide HLA-DM catalyzes the release of CLIP from the peptide binding groove which is replaced by a pathogen-derived peptide HLA-DO acts as a negative regulator of this pathway, binding HLA-DM and blocking its function HLA-DM and HLA-DO non-classical MHC class II molecules  The class II MHC peptide complex is then transported to the plasma membrane Different class II MHC molecules will display a different spectrum of peptides based on the position of certain anchor residues which bind pockets in the MHC class II peptide binding groove Same concept as class I MHC CD4+ T cells bind to MHC class II/peptide complexes CD4+ T cells help with B cell activation, B cells make antibodies whose effector responses are against extracellular pathogens, so it makes sense that CD4+ T cells will be activated by extracellular pathogens But how to naïve CD8+ T cells get activated? Naïve CD4+ T cells get activated when they recognize extracellular pathogens derived peptides presented in the MHC class II by antigen presenting cells But what about naïve CD8+ T cells? The APC in the node isn’t infected How does a naïve CD8+ T cell in a lymph node get activated? Cross-presentation! 1st the APC activates a CD4+ T cell Remember: The dendritic cell in the infected tissue phagocytosed extracellular pathogens, broke them into pieces and put them into their class II MHC as they migrated from the tissue to the lymph node The DC activated a matching CD4+ T cell Via MHC class II presenting a pathogen peptide matching the TCR  The activated CD4+ T cell will release IL-2 IL-2 contributes to CD8+ T cell activation Also triggers DCs to cross-present Put extracellular antigens into their MHC class I A CD8+ T cell with a TCR that matches Ag in the MHC class I groove will become activated It will leave the node, travel to the infected tissue and bind infected cells by MHC class I  A nice summary image T cell activation Dendritic cell (green) interacting with a T cell (pink) T cell activation Activation of helper T cells in the first step to induce adaptive immune responses Three signals are required to activate a T helper (TH) cell These are provided by the APC The most potent APC that can activate naïve T cells is the dendritic cell This generally takes place in the secondary lymphoid organs Lymph nodes, spleen, Peyer’s patches T cells need more than MHC for activation 1. The TCR needs to match the peptide in MHC The MHC has to be self MHC 2. A co-stimulation signal is also needed CD28 on T cell binding CD80/86 on APC Together these trigger a signaling cascade that results in production of cytokines 1. Cytokines trigger T cell proliferation (IL-2) and differentiation (polarizing cytokines) When does an APC express a co-stimulatory molecule? T cells express CD28 It binds the co-stimulatory molecules CD80 or CD86 Only mature dendritic cells, activated macrophages or B cells can express CD80 or CD86 How is CD80/86 expression up-regulated? By APCs sensing PAMPs using their PRRs! https://www.researchgate.net/publication/267258375_Design_of_tumour- specific_immunotherapies_using_dendritic_cells_-_analyses_of_IL15-DC/figures?lo=1  APC presents pathogen-derived peptides in their MHC Also presents CD80/86 T cell with TCR that matches antigen will bind CD80/86 with their CD28 Cytokines are in the microenvironment that contribute to T cell activation, proliferation (IL-2) and polarization TH1: IL-12, IFNɣ TH2: IL-4 TCR signaling Signal 1 Specific binding of a TCR to an appropriate MHC/peptide complex Co-receptor (CD4 or CD8) bind to MHC class II or class I respectively  Signal 2 CD28:CD80/86 Leads to the activation of PI3 kinase which activates PLC-ɣ This allows for the production of second messengers Diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3) This leads to activation of transcription factors AP-1, NF-kB and NFAT  NFAT, AP-1 and NF-kB move to the nucleus and activate gene transcription IL-2 and the IL-2 receptor → T cell proliferation and differentiation Immunosuppressive drugs such as cyclosporin A (CsA) and tacrolimus (FK- 506) block calcineurin, stopping NFAT activation Used to stop solid organ transplants (ex. kidney) How it all ends… As the infection resolves, T cells start expressing CTLA-4 on their surface CTLA-4 is an inhibitory co-stimulatory molecule Has higher affinity for CD80/86 compared to CD28 CTLA-4: Inactivates the T cell Blocks antigen presentation Switches off the T cell response PD-1 also inactivates T cells Expressed on lymphocytes CTLA-4 and PD-1 blockers are used in cancer treatments, you will find out more in your discussion post this week! Anergy: still alive but not responsive to stimulants Signal 1 without signal 2? If a T cell get signal 1 without signal 2 the T cell becomes anergic The T cell is switched off and is much more difficult to activate NOTE: the T cell doesn’t die Let’s take a step back How is the adaptive immune response activated over space and time?  T cell activation Primary immune response Antigens are released by pathogens that have crossed our barriers The innate immune response will try to handle the infection first If the innate immune response cannot control the infection: Dendritic cells take the antigens up by phagocytosis, present in MHC (class II to begin with) and migrate to the T cell zone of the lymph node The DCs bind to the fibroblastic reticular cell (FRC) network and are scanned by naïve CD4 + and CD8+ T cells If the TCR on a CD4+ T cell matches the antigen in MHC class II on APC... A synapse forms between the naïve CD4+ T cell and the APC If the TCR matches the antigen (signal 1) and the T cell is recognizing the correct MHC AND there are co-stimulatory molecules (signal 2) THEN the T cell starts to activate Signal 3 = cytokines produced by the APC will dictate how the CD4+ T cell differentiates Signals 1 and 2 = proliferation Signals 1 and 2 will cause: An increase in survival signals Increased production of IL-2 and the high affinity IL-2 receptor This causes robust proliferation This produces a lot of clones of the original activated T cell, with the same TCR (who recognizes the antigen in the infection) Daughter cells can become memory cells (with self-renewal capacity) OR terminally differentiated effector cells Effector cells live for a few days-weeks and actively work to control the infection Memory cells live longer (months-years) and can self-renew Signal 3 – controls what kind of CD4+ T cell it will become PRRs on the surface of the APC and other cells will sense PAMPs from the pathogen This PRR/PAMP binding triggers cytokine production The cytokines produced depends on which PRRs are activated Cytokines are the 3rd signal They are sensed by the CD4+ T cell and will change how the T cell responds to the infection  Different TH subsets dependent on cytokine milieu during activation Polarizing cytokines Master regulators Different cytokines can activate different master regulator Transcription factors that are activated and control protein synthesis in the T cell IL-12 and interferon gamma will cause an increase in T-bet expression which will in turn increase production of IL-2, IFN gamma and TNF- alpha Promoting inflammation and macrophage and killer T cell activation IL-4 will cause an increase in GATA-3 expression which will in turn increase expression of IL-4, IL-5, IL-13 Promoting IgE production and anti-helminth responses Th1 and Th2 Th1 and Th2 CD4+ T cells are thought to be terminally differentiated Th1 CD4+ T cells produce IFN-gamma which suppresses Th2 development Th2 CD4+ T cells produce IL-10 which suppresses Th1 development Th17 are a subset of Th1 cells that are associated with inflammatory diseases Th9 are a subset of Th2 cells that are associated with allergic responses Th17 and T regs are not fixed in a particular phenotype and can change depending on the signals in the tissue High TGFbeta promotes Treg development, low TGFbeta and IL-6 promotes TH17 development TH1 Intracellular pathogens are sensed by PRRs that trigger production of cytokines (ex. IL-12, IFN-gamma, IL-18, as well as TGF-beta and IL-6) by APCs When the APC activates a CD4+ T cell, it also produced these TH1-polarizing cytokines The CD4+ T cell will not only proliferate, but also differentiate into a TH1 helper cell A Th1 CD4+ T cell will produce IFN gamma and TNF, encouraging inflammation, activation of more APCs and CD8+ T cells Everything you need to control an intracellular pathogen infection TH2 Extracellular pathogen PAMPs are sensed by PRRs on the APCs, triggering the production of IL-4 CD4+ T cells with matching TCRs will not only proliferation but differentiation into Th2 CD4+ T cells Th2 CD4+ T cells produce IL-4, IL-5 and IL-13 which encourages production of IgE (basophils and mast cells) all helpful to control worm infections Th2 also involved in allergic responses Th1 CD4+ T cells help activate CD8+ T cells Th1 CD4+ T cell produces cytokines that: 1. Encourage cross-presentation of antigens in MHC class I Allowing CD8+ T cells to recognize antigens 2. Can provide co-stimulatory molecules (CD40L) to bind CD40 on CD8+ T cells to help with CD8+ T cell activation 3. Produce IL-2 to cause CD8+ proliferation 4. IFN-gamma that aids in CD8+ T cell differentiation into cytotoxic T cells Activated T cells Activated killer T cells will leave the node, circulate through the body and enter the infected tissue Activated helper cells move to where they are needed to help modulate the immune response TFH to margin between T and B cell zones in the lymph node TH1 to CD8+ T cell to help with activation Memory T cells After activation of a T cell there is rapid, clonal expansion and production of effector cells that contribute to controlling the infection After the pathogen is cleared 90-95% of T cells die by apoptosis The leftover T cells are antigen-specific memory T cells Memory cells: React faster and with a stronger response than a naïve T cell to a re-infection with the same pathogen Represent 35% of circulating T cells in healthy young adults and 60% of T cells in people over 70 years old  Take home message: There are memory T cells throughout your body They are sentinels, waiting for a re- infection Cytotoxic T lymphocytes Remember: the only way to eliminate a virus infection is to kill all the virus infected cells CD8+ cytotoxic T lymphocytes (CTLs) specifically recognize and kill virus infected cells CTLs represent the third wave of antiviral responses within the body: 1. Type I IFNs 2. NK cells 3. CD8+ CTLs CTL mediated killing is a three-step process: 1. Activation 2. Recognition 3. Killing Timeline for antiviral immune responses Type I IFNs are produced within minutes to hours after infection Mount an antiviral state Recruit NK cells NK cells recognize virus infected cells and kill by apoptosis NOTE: review how NK cells recognize infected cells NK cells hold off the virus until CTLs are generated (~ 7 days pi) NOTE: virus infections can be controlled at either IFN or NK cell steps without CTLs too 1. Activation – two options CD4+ T cell binds APC first, ‘licenses’ the DC to cross-present Ab in MHC class I APC binds CD8+ T cell next, provides: Ag in MHC class I to bind TCR (signal 1), CD80/86 to bind CD28 (signal 2) and cytokines (ex. IL-2; signal 3) CD8+ T cell activates undergoes clonal expansion (rapid mitosis) and differentiates into a mature CTL (or memory cell) 1. Activation CD4+ T cell and CD8+ T cell bind to the APC at the same time CD4+ T cell not getting stimulated by APC but helping with CD8+ activation CD4+ T cell licenses the APC to put Ag in MHC class I as before, but IL-2 is now provided by the CD4+ T cell CD8+ T cell activates undergoes clonal expansion (rapid mitosis) and differentiates into a mature CTL (or memory cell) 2. Recognition The activated mature effector CTL leaves the secondary lymphoid organ and circulates throughout the body CTLs will enter inflamed tissue following chemokine gradients set up by resident cells CTLs will sample antigen presented in MHC class I by cells in the inflamed tissue If a CTL finds a cell with antigen in its MHC class I that matches its TCR it will create a synapse with that cell CTLs do not need 3 signals to activate to kill, 1 signal (MHC class I with Ag binding TCR) is sufficient A single signal is sufficient to kill 3. Cell death CTLs will induce target cells to die by apoptosis This is a multi-step process involving conjugate formation between the CTL and the target Two mechanisms of killing cells Just like NK cells 1. Granule exocytosis 2. Receptor mediated apoptosis https://www.youtube.com/watch?v=ntk8XsxV Di0 First the CTL needs to create a synapse with the target cell = kiss of death TCR binding causes conformational change in LFA-1 so it can bind tightly 10 minutes from initial contact to to ICAM-1 on surface of target cell movement of granules towards the target cell 1. Granule exocytosis Granules contain perforin and granzymes Granules localize to contact site between CTL and target cell Perforin is released as a monomer, and in the presence of calcium change shape and insert and polymerize in the target cell membrane Granzymes enter the target cell through the perforin pores and activate the caspase cascade to induce apoptosis 2. Receptor mediated apoptosis All cells express ‘death’ receptors (ex. Fas, TNFR-I) These death receptors have death domains (ex. FADD) which can activate the caspase cascade CTLs can express Fas ligand (FasL) which binds Fas or TNF-related apoptosis-inducing ligand (TRAIL) which binds TNFR-I Binding causes a conformational change in the cytoplasmic death domains on the target cell Recruiting procaspase-8 and cleaving it to caspase 8 → caspase cascade triggered Review CTLs recognize and kill one cell at a time They quickly attach, release kill signals and dissociated (