1 / 42

Host Responses to Viruses: Overview

Host Responses to Viruses: Overview. Background Slides: Levels of immune protection Cytokines, chemokines Complement Patterns of viral infection (Acute vs. Chronic) CD8 + CTL: mechanism of cytolytic action What are neutralizing antibodies? RNA viruses and error catastrophe

bryony
Download Presentation

Host Responses to Viruses: Overview

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Host Responses to Viruses: Overview • Background Slides: • Levels of immune protection • Cytokines, chemokines • Complement • Patterns of viral infection (Acute vs. Chronic) • CD8+ CTL: mechanism of cytolytic action • What are neutralizing antibodies? • RNA viruses and error catastrophe 2. Viral evasion mechanisms A. Viral mimicry of cytokines, chemokines B. Complement evasion • Inhibition of Natural killer cells • Antigenic variation • Escape from CD8+ CTLs • Escape from Neutralizing antibodies (Influenza, HIV) • Host susceptibility to virus infection • Persistence in immunologically privileged sites (e.g. neurons) • 3. Immunological techniques to explore host responses to viruses • References Pabio552 Lecture 17; NL Haigwood

  2. Learning Objectives: • What are the major mechanisms used by viruses to evade innate and adaptive immunity? • What is the association between types of viral infection (acute and chronic infection) and modulation of immune responses? • How does HIV trick the various arms of the immune system? • What are the major methods used to measure adaptive immunity? Pabio552 Lecture 17; NL Haigwood

  3. Complement NK Cells Levels of immune protection Source: http://mil.citrus.cc.ca.us/cat2courses/bio104/ChapterNotes/Chapter39notesLewis.htm Pabio552 Lecture 17; NL Haigwood

  4. Janeway, Immunobiology 5th Edition; Fig 1.12 Cytokine, Chemokines • Cytokines are small soluble proteins secreted by one cell that can alter the behavior or properties of the cell itself or of another cell. They are released by many cells in addition to those of the immune system. • Cytokines, such as interferons (IFNs) and tumor-necrosis factor (TNF), induce intracellular pathways that activate an anti-viral state or apoptosis, and thereby limit viral replication. • Chemokines are chemoattractant cytokines that regulate trafficking and effector functions of leukocytes, and so have an important role in inflammation and immune surveillance • Chemokines: divided into 4 classes, CC-, CXC-, C-, CX3C- • Expression of specific chemokines, together with differential expression of respective chemokine receptors by leukocytes, determines which immune cells migrate during inflammation See Flint Table 14.6 Blocking interferon action HIV utilizes chemokine receptors CCR5, CXCR4 for entry into target cells Pabio552 Lecture 17; NL Haigwood

  5. Viral Mimicry of cytokines, chemokines and their receptorsAlacami A. Nat Rev Immunol. 2003 Jan;3(1):36-50. • Properties of poxvirus-encoded soluble cytokine receptors or binding proteins. Pabio552 Lecture 17; NL Haigwood

  6. Factor H C3bH Factor I + + + + iC3b CD46 CR1 Factor I C4d + C4c (inactive) CD46 CR1 C3c (inactive) Factor S CD59 Favoreel HW., et al., J Gen Virol. 84: 1-15 (2003). Complement • Interacting set of enzymes (proteases) that upon activation give rise to cascade of reactions culminating in the destruction of pathogens and infected cells. • Three pathways: • Classical: implicated in controlling HIV, HTLV, CMV infected cells • Mannan binding lectin (MBL) : similar to classical; lyses HBV, HCV and infuenza infected cells • Alternative pathway: default pathway; spontaneous and indiscriminate deposition of complement factor C3b on host cell surface or foreign particles; complement activation will proceed unless downregulated by complement regulators CD59, DAF Pabio552 Lecture 17; NL Haigwood

  7. Favoreel HW., et al., J Gen Virol. 84: 1-15 (2003). Complement Evasion • Remove Ag:Ab complexes; express Fc receptors on the infected cells HSV gE-gI glycoprotein complex binds non-immune IgG and sterically hindered access of virus specific effector cells or neutralizing antibodies PRV gE cytoplasmic domain has two tyr residues that internalizes through clathrin mediated endocytosis, followed by degradation • Viral mimicry of complement regulators Poxvirus and -Herpesvirus infected cells secrete soluble viral encoded factors that are genetically similar to complement regulators; inhibit by inactivating C3b and C4b, C3 convertase -herpesvirus (HSV) gC glycoprotein acts as complement receptor for C3b, C5 and P factor • Incorporation or up-regulation of cellular complement control factors Enveloped viruses like HIV, Ebola, influenza incorporate complement control proteins CD46, CD55, CD59 (enriched in lipid rafts) during virus release HCMV upregulates expression of CD59 in infected cells leading to suppression of alternate pathway. Pabio552 Lecture 17; NL Haigwood

  8. NK cells perform immune surveillance Janeway, Immunobiology 5th Edition; Fig 8.19 ADCC of NK cells Janeway, Immunobiology 5th Edition; Fig 9.34 Natural Killer Cells • Lymphocytes that do not undergo genetic recombination events to increase their affinity for particular ligands (effectors of the innate immune system) • Capable of killing without previous stimulation • Characterized by the absence of conventional receptors for antigen (TCR); display CD3- CD16+ phenotype • CD16 (FcγRIII) is a low affinity receptor for IgG and is involved in Antibody Dependent Cell mediated Cytotoxicity (ADCC) • NK cell activating receptors: • Human natural cytotoxicity receptors NKp30, NKp44, NKp46 • LFA-1 and CD2 family • Inhibitory receptors: • receptors for MHC class I belonging to killer cell inhibitory receptor (KIR) family; KIR receptors recruit phosphatases (Src domain containing protein tyrosine phosphatase 1) to transduce the inhibitory signal • Immunoglobulin-like inhibitory receptors (ILT) • Lectin like heterodimer CD94-NKG2A • NK cell responses are also coordinated and regulated by cytokines such as IFN-, IFN-, IL-2, IL-12, IL-15 and IL-18 • Spontaneously active, unless they are inhibited by self-MHC molecules. Well-suited to perform immunosurveillance for nonself MHC-bearing targets (eg, transplanted cells, tumors, virally-modified cells) Pabio552 Lecture 17; NL Haigwood

  9. Orange JS. Et al., Nat Immunol. 3: 1006-12 (2002). Viral mechanisms for evading NK cells. • Viral homologs of MHC class I Binds to inhibitory NK cell class I receptors • Selective modulation of MHC class I allele expression Increase relative expression of HLA-C, HLA-E by down regulating HLA-A, HLA-B; NK cells inhibited through class I CD94-NKG2A and KIR HIV Nef causes endocytosis of Class I, except HLA-C, HLA-E HCMV UL40 enhances expression of HLA-E • Inhibition of activating receptor function • Viral cytokine binding proteins (ii) viral homologs (iii) induce host cell secretion of NK cell inhibitory cytokines HIV Tat blocks NK cell activation by specifically binding and blocking Ca2+ influx channel important for NK Cell cytotoxicity KSHV K5 protein ubiquitinates, and decreases surface expression of ICAM-1, B7-2  inhibits NK cell cytotoxicity • Secrete antagonists of NK cell receptor-infected cell ligand interactions KSHV vMIP-I, vMIP-II acts as a chemokine antagonist and intereferes with NK cell trafficking HPV E6, E7 binds IL-18 receptor and inhibits IFN- production by NK cells • Directly infects NK cells or ligates NK cell inhibitory receptors through envelope glycoproteins HIV in vitro infects NK cells through an unknown receptor HCV E2 glycoprotein binds CD81 on NK cells and sends inhibitory signal to NK cells Pabio552 Lecture 17; NL Haigwood

  10. CTL Ab titers Ab CTL General patterns of viral infection • Acute Infection • Rhinovirus • Rotavirus • Influenza virus Virus is cleared Memory responses established • Persistent Infection • Lymphocytic choriomeningitis virus • Latent, Reactivating infection • HSV • Slow virus infection • Lentivirus (HIV) Source: Flint, Principles of Virology 4th Ed Fig: 16.1 Pabio552 Lecture 17; NL Haigwood

  11. HIV: drives the host defenses into exhaustion, and ultimately failure • High rate of viral evolution • Early escape from CTL • Escape from neutralizing antibodies • Down regulation of MHC • CD4+ T helper cell destruction • Integration and reactivation Pabio552 Lecture 17; NL Haigwood

  12. Ferrantelli F., et al., Curr Opin Immunol. 14: 495 (2002). Immune responses to HIV • Control of primary HIV-1 infection coincides with the appearance of virus-specific Cytotoxic T lymphocytes (CTLs) • 2. Antibodies form early; neutralizing antibodies arise later in infection Strong immune responses to HIV infection control it for many years but ultimately host dies after erosion of T cells Pabio552 Lecture 17; NL Haigwood

  13. Domingo E. Virology 270, 251-253 (2000) RNA Viruses HIV and other RNA viruses thus exist as dynamic distributions of non-identical but related genomes – Quasispecies HIV: Antigenic Escape, Quasispecies, Error Threshold • E. coli: • Genome size 4.6 x 106 bp or ~107 bp • Error rate in DNA replication is 10-10 • 1 error for every 1000 genome replications • RNA Viruses: • Genome size ~104 nucleotides • Error rate of RNA replicases is 10-4 • 1 error with every replication Pabio552 Lecture 17; NL Haigwood

  14. DNA Distance 8 12 4 10 2 8 12 10 0 6 6 4 2 0 Years post seroconversion Shankarappa et al., J. Virol. 73:10489 (1999) HIV intrapatient viral evolution Pabio552 Lecture 17; NL Haigwood

  15. Viral Escape from CD8+ Cytotoxic T lymphocytes (CTLs)Flint Table 15.4 and Figure 15.7*RSV--less MHC class I transcription*HIV--Tat, Vpu interfer with MHC class I synthesis*Adenovirus--E3 and E1a reduce MHC in ER and transcription*CMV, HSV, EBV--Tap transporter inhibition, retention of peptides in ER Pabio552 Lecture 17; NL Haigwood

  16. Barry M., et al., Nat Rev Immunol. 2: 401-9 (2002). CD8+ CTLs: Mechanism of cytolytic action • Potent defenders against virus infection and intracellular pathogens • Mediates apoptotic death through Fas-FasL interactions • Upon interaction with an infected cell, an “immunological synapse” between the CTL and the target cell is formed • Targeted release of effector molecules • Perforin: forms pore in the target cell (release of intracellular contents from infected cell); translocation channel for granzymes • Granzyme B: belongs to family of serine proteases that mediate apoptotic cell death through the (i) direct cleavage of pro-caspase-3 or, (ii) indirectly, through caspase-8, (iii) cleavage of BID resulting in its translocation, with other members of the pro-apoptotic BCL2-family such as BAX, to the mitochondria, (iv) direct activation of DFF40/CAD (DNA fragmentation 40/caspase-activated deoxynuclease) — which damages DNA and leads to cell death — by granzyme-B-mediated proteolysis of the inhibitor ICAD. • CD8+ CTLs have non-cytolytic action too (today’s discussion paper) Pabio552 Lecture 17; NL Haigwood

  17. CD8+ CTL control of HIV infectionEvidence • Control of high viral loads associated with the acute phase of HIV infection is coincident with the appearance of anti-HIV CTLs • CTLs can be found at sites of HIV replication in vivo • The quantity of anti-HIV CTLs, as measured by MHC-I tetrameric complexes, correlates inversely with viral load (data conflicting) • When SIV infected macaques were immunodepleted for CD8+ T cells, a dramatic increase in SIV replication was observed • HIV vaccine strategies targeting CTL responses show decrease in viral load in SIV/ SHIV infected macaques • CTLs from Long-term nonprogressors secrete higher levels of perforin, granzyme B • BUT, vaccines that elicit only CTL cannot protect from infection, only control, and patients can be superinfected with another HIV-1 isolate, even if CTL are present Pabio552 Lecture 17; NL Haigwood

  18. Impairment of the cellular immune response • Interference with proteosomal degradation • Peptide transport associated with Ag presentation • Retention of MHC I molecules in subcellular compartments • Destruction of MHC class I heavy chains • Induction of functional paralysis of DCs • Downregulation of MHC Class II expression • Circumvent NK-cell mediated killing • Antigenic variation of T cell epitopes Pabio552 Lecture 17; NL Haigwood

  19. Viral load is critical to the control of disease Pabio552 Lecture 17; NL Haigwood

  20. Viral Escape from Neutralizing Antibodies Pabio552 Lecture 17; NL Haigwood

  21. What are Neutralizing antibodies? CD4 CCR5/ CXCR4 Target cell Pabio552 Lecture 17; NL Haigwood

  22. What are Neutralizing antibodies? CD4 binding induces a conformational change in envelope leading to exposure of the coreceptor binding site Target cell Pabio552 Lecture 17; NL Haigwood

  23. What are Neutralizing antibodies? Binding to CCR5 exposes the fusion domain leading to subsequent viral core entry Target cell Pabio552 Lecture 17; NL Haigwood

  24. What are Neutralizing antibodies? Most antibodies however, bind the virus but do not neutralize Neutralizing antibodies: Block virus infection in the target cells by directly binding to virion and prevent their interaction with receptor on the target cells Target cell Pabio552 Lecture 17; NL Haigwood

  25. Burton DR. Nat Rev Immunol. 2: 706 (2002). NAbs act at different levels Pabio552 Lecture 17; NL Haigwood

  26. Envelope modifications enable NAb escape • Influenza Virus No cross-protective immunity Escape from neutralizing antibodies Original antigenic sin: Abs made only to epitopes present on the initial viral variant when infected with a second variant Source: http://www.gak.co.jp/FCCA/glycoword/GD-A06/GD-A06_E.html Pabio552 Lecture 17; NL Haigwood

  27. HIV: Escape from Antibody-mediated NeutralizationReasons Schematic of envelope trimer • Transient exposure of neutralizing epitopes only at critical moments during the entry process • CCR5 binding site is highly protected and is exposed only after conformation changes has occurred upon binding CD4 • CD4 independent viruses are highly neutralization sensitive • Occlusion of conserved domains within the oligomer CD4 binding site CCR5 binding site • Wyatt R., et al., Nature 393: 705 - 711 (1998) Pabio552 Lecture 17; NL Haigwood

  28. Schematic of envelope trimer CD4 binding site CCR5 binding site • Wyatt R., et al., Nature 393: 705 - 711 (1998) Land A., et al., Biochimie. 83: 783-90 (2001). HIV: Escape from Antibody mediated NeutralizationReasons • Extrusion of variable domains from the exposed surface of the oligomeric complex • Serves as an antigenically variable shield and covers the more conserved envelope core • Removal of V2 loop opened up the CD4 binding site rendered the virus susceptible to neutralization from other clades, and also resulted in high titer neutralizing antibodies in a SHIV infected macaque and reduced viral burden (Stamatatos et al; J Virol. 1998. 72:7840) Pabio552 Lecture 17; NL Haigwood

  29. Land A., et al., Biochimie. 83: 783-90 (2001). HIV: Escape from Antibody mediated NeutralizationReasons • Extensive glycosylation of the envelope proteins • HIV/ SIV envelope contains 28-30 potential N-linked glycosylation sites that account for 50% of the mass of gp120 • Position of the glycans modulate antibody neutralization specificity • Removal of N-linked glycan in V3 loop increased HIV’s sensitivity to neutralizing antibodies • Longitudinal analyses indicate that the number of glycans remain fairly constant, while positions vary, with indels, and most commonly a shift by a couple of aa’s • Evolving Glycan shield Pabio552 Lecture 17; NL Haigwood

  30. HIV: Escape from Antibody mediated NeutralizationEvidence • Viral variants are resistant to plasma (NAbs) from the same time point • During chronic HIV infection, there are high levels of NAbs, however they are not directed towards autologous virus from the patient at the same time point • NAbs that recognize heterologous isolates arise late in infection (Broad) • The development of heterologous NAb hinders the development of autologous NAb responses that may control infection Richman DD., et al. PNAS. 100(7): 4144. (2003) Despite these changes, Env is by necessity relatively conserved in CD4, co-receptor binding regions Pabio552 Lecture 17; NL Haigwood

  31. Ferrantelli F., et al., Curr Opin Immunol. 2002 Aug;14(4):495-502. Broad NAbs do arise in some patients Pabio552 Lecture 17; NL Haigwood

  32. Passive immunization studies • Combination of broad human monoclonal NAbs act synergistically to confer protection against SHIV and SIV • Prevention of SHIV transmission to macaque monkeys when IgG1 b12 was mucosally applied • Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques • Potent cross-group neutralization (Clades A, C, D,E, and F) of primary HIV isolates with monoclonal antibodies - IgG1b12 (b12), F105,2G12, 2F5, 4E10, F424,and Clone 3 (CL3) Pabio552 Lecture 17; NL Haigwood

  33. Host susceptibility to viral infection • HLA haplotypes influences HIV disease progression • Associated with slower disease progression: A*11, B*27, B*57, B858, Cw*02, Cw*14 • Associated with rapid disease: B*35, B*53, Cw*04 • Maternally acquired protective alleles were no longer protective against disease progression in vertically infected infants • Mutant forms/ Polymorphisms of coreceptor modulates progression to AIDS • Individualshomozygous for a 32-bp deletion (32) in CCR5 are resistantto infection by HIV-1 • HIV-1-infected individualsheterozygous for CCR5 32 (-/+) show a slightly slowerdisease progression than CCR5+/+ homozygotes • Common polymorphism in the 3'-untranslated region of the stromalderived factor gene (SDF-1; natural ligand for CXCR4) associated with delayed diseaseprogression from some cohorts. In others,the same homozygous genotype has been associated with rapid progressionto AIDS, but with prolonged survival after diagnosis of AIDS • Polymorphism in 3’ UTR SDF-1 is associated with increased risk of vertical transmission Pabio552 Lecture 17; NL Haigwood

  34. Immunological techniquesNot an exhaustive list! • Humoral Immunity (B cells) • ELISA; Radioimmunoassay : checks for the development of virus-specific antibodies in plasma, other body fluids • Ab competitive inhibition assay • Antibody-dependent cellular cytotoxicity (ADCC) • How are monoclonal antibodies generated? • What is Biacore? What does it measure? • Neutralization assay – viral infectivity on target cells in the presence or absence of Ab • Cellular immunity (CD4, CD8 T cells) • How do you check for the presence of cytokine secreted by a T cell? • Antigen proliferation assay (also called lymphocyte proliferation assay, thymidine uptake assay) • MHC Tetramer Assay: Ag specific T cell staining reagent • Functional Assays • Cytokine production: ELIspot, intracellular cytokine staining • B- cell activation: What is an ELIspot? • Target Cell Killing: Chromium release assay: what does it measure? • Antibody dependent cell-mediated cytotoxicity (ADCC) • Chemotaxis assay Pabio552 Lecture 17; NL Haigwood

  35. References • Reviews • Alcami A. Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol. 3: 36-50 (2003). • Favoreel HW., et al. Virus Complement evasion strategies. J. Gen. Virol. 84: 1-15 (2003). • Orange JS., et al. Viral evasion of natural killer cells. Nat. Immunol. 3: 1006 (2002). • Vossen MTM., et al. Viral Immune evasion: am masterpiece of evolution. Immunogenetics. 54: 527 (2002). • Domingo E. Viruses at the edge of adaptation. Virology. 270: 251 (2000) • Barry M., et al. Cytotoxic T lymphocytes: all roads lead to death. Nat. Rev. Immunol. 2: 401 (2002). • Ferrantelli F, et al., Neutralizing antibodies against HIV -- back in the major leagues? Curr Opin Immunol. 14: 495 (2002). • Land A., et al., Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum. Biochimie. 83: 783 (2001). • Burton DR. Antibodies, viruses and vaccines. Nat Rev Immunol. 2: 706-13 (2002). • Wyatt R. et al., The antigenic structure of the HIV gp120 envelope glycoprotein. Nature. 393: 705-11 (1998). • Primary papers • Schlender et al. Inhibition of toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus. J Virol. 2005 May;79(9):5507-15. • Richman DD., et al. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. PNAS. 100: 4144 (2003). • Ferrantelli F., et al., Potent cross-group neutralization of primary human immunodeficiency virus isolates with monoclonal antibodies--implications for acquired immunodeficiency syndrome vaccine. J Infect Dis. 189: 71-4 (2004). • Mascola JR., et al., Protection of macaques against vaginal transmission of pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Medicine 6: 207-210 (2000). • Chackerian et al., Specific N-linked and O-linked glycosylation modifications in the envelope V1 domain of simian immunodeficiencyvirus variants that evolve in the host alter recognition by neutralizing antibodies. J Virol. 1997 Oct;71(10):7719-27. • Wei X et al. Antibody neutralization and escape by HIV-1. Nature. 2003 Mar 20;422(6929):307-12. Pabio552 Lecture 17; NL Haigwood

  36. Tat LTR - Gal Neutralization Assay • Target cells (Cell line/ PBMC) expressing viral receptors for entry (For ex, HIV-1 CD4, CCR5) and a reporter gene (-gal, luciferase) • Mechanism • Only infected cells produce  Gal  Easy visualization through X-gal staining • Neutralization assay: pretreatment of virus with Ab • Reduction in  Gal positive cells indicates virus neutralization • Calculate % neutralization Pabio552 Lecture 17; NL Haigwood

  37. Generation of monoclonal antibodies Janeway, Immunobiology 5th Edition; Fig A.15 Pabio552 Lecture 17; NL Haigwood

  38. 3. After a 5 day incubation period, pulse with H3thymidine 1. Incubate PBMCs with Antigen/ Stimulus 2. Ag specific Lymphocytes start proliferating Antigen proliferation assay 8-16 hours incubation 4. Harvest the cells onto filters using cell harvester Antigen: gp120, peptide pools (Gag, Env) AT-2 inactivated SHIV89.6 Contols: PHA SEB a-CD3 mAb irrelavant peptide Plain media 5. Read filters on a scintillation counter Calculate Stimulation index = Samplecpm Irr cpm Pabio552 Lecture 17; NL Haigwood

  39. ELIspot Source: www.bdbiosciences.com Pabio552 Lecture 17; NL Haigwood

  40. Tetramer staining • Stain antigen-specific T cells • MHC:peptide tetramers are formed from recombinant refolded MHC:peptide complexes containing a single defined peptide epitope • Recombinant MHC heavy chain is linked to a bacterial biotinylation sequence, a target for the E. coli enzyme BirA, which is used to add a single biotin group to the MHC molecule • Streptavidin is a tetramer, each subunit having a single binding site for biotin, thus the streptavidin/MHC:peptide complex is a tetramer of MHC:peptide complexes • While the affinity between the T-cell receptor and its MHC:peptide ligand is too low for a single complex to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple MHC:peptide complexes binding simultaneously, is able to bind to T cells whose receptors are specific for the particular MHC:peptide complex • Streptavidin is coupled to a fluorochrome, so that the binding to T cells can be monitored by flow cytometry. Janeway, Immunobiology 5th Edition; Fig A.32 Pabio552 Lecture 17; NL Haigwood

  41. Biacore • Based on Surface plasmon Resonance (SPR) technology • SPR arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index • SPR causes a reduction in the intensity of reflected light at a specific angle of reflection • Angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) • When molecules in the sample bind to the sensor surface, the concentration and therefore the refractive index at the surface changes and an SPR response is detected • Plotting the response against time (called sensorgram) during the course of an interaction provides a quantitative measure of the progress of the interaction Source: www.biacore.com Pabio552 Lecture 17; NL Haigwood

  42. Janeway, Immunobiology 5th Edition; Fig A.38 Chromium Release assay • Measures Cytotoxic T-cell activity • Target cells are labeled with radioactive chromium as Na251CrO4, washed to remove excess radioactivity and exposed to cytotoxic T cells • Cell destruction is measured by the release of radioactive chromium into the medium, detectable within 4 hours of mixing target cells with T cells. Pabio552 Lecture 17; NL Haigwood

More Related