RESUMEN
BACKGROUND: Quadrivalent human papillomavirus (HPV types 6/11/16/18) L1 VLP vaccine is highly effective in preventing HPV 6/11/16/18-related cervical and external genital disease. Herein, we evaluated the impact of the quadrivalent HPV 6/11/16/18 L1 VLP vaccine on prevention of HPV-associated cervico-genital lesions in a broad population of sexually active European women. METHODS: Female subjects (N = 9265) aged 16-24 with four or fewer lifetime sexual partners were enrolled and randomized to quadrivalent HPV vaccine or placebo. Subjects underwent cervicovaginal sampling for HPV DNA detection. Papanicolaou testing and anti-HPV 6/11/16/18 serology testing was also performed. RESULTS: Vaccine efficacy against lesions representing immediate cervical cancer precursors (cervical intraepithelial neoplasia grade 2/3 or adenocarcinoma in situ) related to HPV 6/11/16/18 in the per-protocol population was 100.0%[95% confidence interval (95% CI), 89.8-100.0]. Efficacy against external genital lesions (vulvar or vaginal intraepithelial neoplasia, condyloma, vulvar or vaginal cancer) related to vaccine HPV types in the per-protocol European population was 99.0% (95% CI, 94.4-100.0). CONCLUSION: These data demonstrate that quadrivalent HPV 6/11/16/18 vaccination programs in 16- to 24-year-old European women can be beneficial. NCT0009252, NCT00092534, NCT00092495.
Asunto(s)
Adenocarcinoma/prevención & control , Vacunas contra Papillomavirus/administración & dosificación , Displasia del Cuello del Útero/prevención & control , Neoplasias del Cuello Uterino/prevención & control , Adolescente , Adulto , Femenino , Vacuna Tetravalente Recombinante contra el Virus del Papiloma Humano Tipos 6, 11 , 16, 18 , Humanos , Placebos , Ensayos Clínicos Controlados Aleatorios como Asunto , Parejas Sexuales , Adulto JovenRESUMEN
Human papillomavirus (HPV) causes cervical, vulvar, and vaginal cancers, precancerous dysplasia, and genital warts. We report data for the longest efficacy evaluation to date of a prophylactic HPV vaccine. In total, 552 women (16-23 years) were enrolled in a randomised, placebo-controlled study of a quadrivalent HPV 6/11/16/18 L1 virus-like-particle vaccine with vaccination at months 0, 2, and 6. At regular intervals through 3 years, subjects underwent gynaecologic examination, cervicovaginal sampling for HPV DNA, serum anti-HPV testing, and Pap testing, with follow-up biopsy as indicated. A subset of 241 subjects underwent two further years of follow-up. At 5 years post enrollment, the combined incidence of HPV 6/11/16/18-related persistent infection or disease was reduced in vaccine-recipients by 96% (two cases vaccine versus 46 placebo). There were no cases of HPV 6/11/16/18-related precancerous cervical dysplasia or genital warts in vaccine recipients, and six cases in placebo recipients (efficacy = 100%; 95% CI:12-100%). Through 5 years, vaccine-induced anti-HPV geometric mean titres remained at or above those following natural infection. In conclusion, a prophylactic quadrivalent HPV vaccine was effective through 5 years for prevention of persistent infection and disease caused by HPV 6/11/16/18. This duration supports vaccination of adolescents and young adults, which is expected to greatly reduce the burden of cervical and genital cancers, precancerous dysplasia, and genital warts.
Asunto(s)
Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/inmunología , Vacunas contra Papillomavirus/uso terapéutico , Neoplasias del Cuello Uterino/prevención & control , Virión/inmunología , Adolescente , Adulto , Alphapapillomavirus/inmunología , Anticuerpos Antivirales/sangre , Condiloma Acuminado/prevención & control , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Infecciones por Papillomavirus/epidemiología , Displasia del Cuello del Útero/prevención & control , Frotis VaginalRESUMEN
Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4(+) T cells long before a quantitative decline in circulating CD4(+) T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4(+) T cells remains unclear. Both direct effects of cytopathic infection of CD4(+) cells and indirect effects in which uninfected "bystander" cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.
Asunto(s)
Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Herpesvirus Humano 1/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Glicoproteínas de Membrana/metabolismo , Virión/metabolismo , Antígeno B7-2 , Línea Celular , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 1RESUMEN
Increased levels of apoptosis are seen in human immunodeficiency virus (HIV) infection, and this has been proposed as an important mechanism contributing to HIV pathogenesis. However, interpretation of in vitro studies aimed at understanding HIV-related apoptosis has been complicated by the use of high concentrations of recombinant proteins or by direct cytopathic effects of replicating virus. We have developed an inactivation procedure that destroys retroviral infectivity while preserving the structural and functional integrity of the HIV surface proteins. These noninfectious virions interact authentically with target cells, providing a powerful tool to dissect mechanisms of HIV pathogenesis that do or do not require viral replication. Noninfectious CXCR4-tropic HIV-1 virions, but not microvesicles, partially activated freshly isolated CD4(+) and CD8(+) peripheral blood mononuclear cell T lymphocytes to express FasL and Fas, but not CD69 or CD25 (interleukin-2 receptor alpha) and eventually die via apoptosis starting 4 to 6 days postexposure. These effects required conformationally intact virions, as heat-denatured virions or equivalent amounts of recombinant gp120 did not induce apoptosis. The maximal apoptotic effect was dependent on major histocompatibility complex (MHC) class II proteins being present on the virion, but was not MHC restricted. The results suggest that the immunopathogenesis of HIV infection may not depend solely on direct cytopathic effects of HIV replication, but that effects due to noninfectious HIV-1 virions may also contribute importantly.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , VIH-1/patogenicidad , Activación de Linfocitos , Virión/patogenicidad , Proteína gp120 de Envoltorio del VIH/análisis , Antígenos de Histocompatibilidad Clase II/fisiología , Humanos , Receptores CXCR4/fisiología , Replicación ViralRESUMEN
Ag-specific CD8+ CTL clones require TCR stimulation to respond to IL-2 for growth. Because IL-2 may be produced in the vicinity of CD8+ CTLs when Ag is limiting at the end of an immune response, we have examined the effect of culturing viral-specific CTL clones in IL-2 in the absence of antigenic stimulation. Limiting dilution analysis revealed a high precursor frequency for CTL clones derived from IL-2 propagation (termed CTL-factor dependent (FD)) that are dependent upon exogenous IL-2 for growth and survival and no longer require TCR stimulation to proliferate. Culturing CTL-FDs with infected splenocytes presenting Ag and IL-2 did not revert the clones but did lead to a TCR-induced inhibition of proliferation. The derived CTL-FDs have lost the ability to kill via the perforin/granule exocytosis mechanism of killing, although they express similar levels of TCR, CD3epsilon, CD8alphabeta, CD45, and LFA-1 compared with the parental clones. The CTL-FDs retain Fas ligand/Fas-mediated cytotoxicity, and IFN-gamma production and regulate the expression of CD69 and IL-2Ralpha when triggered through the TCR. A parental CTL protected BALB/c mice from a lethal challenge of influenza virus, whereas a CTL-FD did not. These findings represent a novel regulatory function of IL-2 in vitro that, if functional in vivo, may serve to down-regulate cellular immune responses.
Asunto(s)
Antígenos Virales/fisiología , Citotoxicidad Inmunológica/inmunología , Interleucina-2/fisiología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/biosíntesis , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Administración Intranasal , Animales , Antígenos Virales/administración & dosificación , Técnicas de Cultivo de Célula/métodos , División Celular/inmunología , Células Clonales/citología , Células Clonales/inmunología , Células Clonales/metabolismo , Epítopos de Linfocito T/análisis , Virus de la Influenza A/inmunología , Interferón gamma/metabolismo , Dosificación Letal Mediana , Leucemia L1210 , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/prevención & control , Receptores de Superficie Celular/biosíntesis , Linfocitos T Citotóxicos/citologíaRESUMEN
Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Proteínas Bacterianas , Antígenos CD4/metabolismo , Proteínas Portadoras/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Conformación Proteica , Sitios de Unión , Epítopos de Linfocito B/metabolismo , Proteína gp120 de Envoltorio del VIH/química , VIH-1/crecimiento & desarrollo , Humanos , Fusión de Membrana , Pruebas de Neutralización , Solubilidad , Transcripción Genética , Células Tumorales Cultivadas , Virión/metabolismoRESUMEN
Although most viral vaccines used in humans have been composed of live attenuated viruses or whole killed viral particles, the latter approach has received little attention in research on experimental primate immunodeficiency virus vaccines. Inactivation procedures involving heat or formalin appear to adversely affect the viral envelope proteins. Recently we have inactivated human immunodeficiency virus type 1 (HIV-1) with the compound 2,2'-dithiodipyridine (Aldrithiol-2, Aldrich, Milwaukee, WI), which inactivates infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs. HIV-1 inactivated with Aldrithiol-2 retained the conformational and functional integrity of the viral and virion-associated cellular proteins on the viral membrane. We have extended our studies of zinc finger targeted inactivation to simian immunodeficiency virus (SIV) and evaluated the feasibility of applying the procedures to large scale (>30 l) production and purification of the primate immunodeficiency viruses. There was no detectable residual infectivity of SIV after treatment with 1 mM Aldrithiol-2 (>5 logs inactivation). Treatment with Aldrithiol-2 resulted in extensive reaction with the nucleocapsid protein of treated virus, as shown by immunoblot and high-performance liquid chromatography (HPLC) analysis. As expected, the virion gp120SU appeared to be completely unreactive with Aldrithiol-2. Sucrose gradient purification and concentration procedures resulted in little loss of viral infectivity or virion-associated gp120SU. When tested in a gp120-CD4 dependent cell binding assay, the inactivated virus bound to cells comparably to the untreated virus. Analysis of gp120-CD4 mediated postbinding fusion events showed that the inactivated virus could induce CD4-dependent fusion with efficiencies similar to the untreated virus. Inactivation and processing of primate immunodeficiency viruses by methods described here results in highly concentrated virus preparations that retain their envelope proteins in a native configuration. These inactivated virus preparations should be useful in whole killed-particle vaccine experiments as well as laboratory reagents to prepare antisera, including monoclonal antibodies, and to study noninfective virion-cell interactions.
Asunto(s)
VIH-1/patogenicidad , Proteínas de la Nucleocápside/metabolismo , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Virales , Virulencia/inmunología , Dedos de Zinc , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Antivirales/farmacología , Fusión Celular , Línea Celular , Cromatografía Líquida de Alta Presión , Disulfuros/farmacología , VIH-1/aislamiento & purificación , Humanos , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , TemperaturaRESUMEN
Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2'-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4(+) target cells and mediated virus-induced, CD4-dependent "fusion from without" comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.
Asunto(s)
VIH-1/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Virión/patogenicidad , Virulencia , VIH-1/genética , VIH-1/metabolismo , Humanos , Fusión de Membrana , Conformación Proteica , Transcripción Genética , Proteínas del Envoltorio Viral/química , Virión/genética , Virión/metabolismoRESUMEN
Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)-mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8(+) CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions.
Asunto(s)
Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfocitos T Citotóxicos/fisiología , Antígenos CD/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Células Clonales/metabolismo , Citotoxicidad Inmunológica/inmunología , Exocitosis/fisiología , Proteína Ligando Fas , Granzimas , Ionomicina/farmacología , Ionóforos/farmacología , Glicoproteínas de Membrana/fisiología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacología , Receptor fas/inmunologíaRESUMEN
IL-2 is a T cell growth factor that has pleiotropic functions in T cell differentiation, induction of lymphokine-activated killer cells, and regulation of immune responses. In studying TCR triggering of perforin or Fas ligand (FasL)/Fas (CD95 ligand/CD95) cytotoxicity in our influenza-specific T cell clones, we found that IL-2 can also induce FasL/Fas cytotoxicity. IL-2 induces FasL/Fas cytotoxicity in our CD8+ and CD4+ Th1 clones, but not in our CD4+ Th2 clones. IL-2 induction of cytolytic activity occurs when the CD8+ T cells are refractory to IL-2-induced proliferation. This killing is Ag independent, MHC unrestricted, and blocked by Fas.Fc fusion protein. IL-2 induces FasL/Fas cytotoxicity in a dose-dependent manner, but does not induce high levels of FasL expression as detected by flow cytometry. TCR triggered FasL/Fas cytotoxicity is detectable in CD8+ and Th1 clones by 3 h and peaks at 6 h; high levels of killing are maintained for at least 24 h. Similarly, IL-2 induces FasL/Fas killing in CD8+ and Th1 clones within 3 h of stimulation and maintains high levels for at least 24 h. TCR-triggered FasL/Fas killing is inhibited by emetine and cyclosporin A, whereas IL-2-induced FasL/Fas killing is inhibited by emetine, but not by cyclosporin A. These results demonstrate a second mechanism to induce FasL/Fas cytotoxicity in CD8+ and Th1 clones and may explain IL-2 induction of Ag-independent MHC-unrestricted lymphokine-activated killer cell activity.
Asunto(s)
Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interleucina-2/inmunología , Glicoproteínas de Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor fas/inmunología , Células Clonales , Ciclosporina/farmacología , Citotoxicidad Inmunológica , Emetina/farmacología , Proteína Ligando Fas , Humanos , Inmunosupresores/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
A diverse array of signals are generated in a cytotoxic T lymphocyte (CTL) after the T cell receptor (TCR) engages the class I major histocompatibility complex (MHC) peptide complex. These signals result in a multitude of CTL effector functions, including cellular cytotoxicity, cell surface receptor expression, and cytokine secretion. We have examined signaling through the TCR in a wild type CD8+, MHC-restricted, antigen-specific CTL clone, 14-7, and its interleukin 2-dependent variant clone 14-7FD. We report here that 14-7FD is unable to kill via the perforin mechanism of killing, yet is able to kill via the Fas ligand/Fas mechanism and secrete interferon-gamma in an antigen-specific manner. 14-7FD has cytolytic granules that contain perforin and serine esterases, which are secreted after phorbol ester and Ca2+ ionophore treatment. Lastly, to investigate which TCR signaling requirements were operational in 14-7FD, we examined TCR-triggered intracellular Ca2+ mobilization in the two clones. After TCR engagement, 14-7FD failed to mobilize intracellular Ca2+, which may be the cause for its inability to trigger the perforin/granule exocytosis mechanism of killing. These results indicate that the signal transduction events that trigger perforin killing and the signaling requirements to induce FasL expression are distinct. We hypothesize that these two distinct TCR signal transduction requirements allow for separate activation of these two mechanisms of killing relating to their role in eradication of infected cells or regulation of immune responses.
Asunto(s)
Citotoxicidad Inmunológica , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Animales , Linfocitos T CD8-positivos/inmunología , Calcio/metabolismo , Gránulos Citoplasmáticos , Exocitosis , Proteína Ligando Fas , Interferón gamma/metabolismo , Ionomicina/farmacología , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/inmunología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
T cells are normally activated when the peptide for which they are specific is presented to them in the context of the appropriate major histocompatibility complex (MHC) (class I and Class II for CD8+ and CD4+ T cells, respectively). An increasing body of evidence indicates that structural homologues of the immunogenic peptide can partially activate or antagonize CD4+ T cells. CD8+ T cells may also be partially antagonized by such peptides, and self-derived peptides of this type may play a role in CD8+ T cell selection in the thymus. Activated CD8+ T cells lyse their targets by perforin-dependent granule exocytosis and by inducing apoptosis mediated by CD95 (also known as Fas or APO1) with its ligand (CD95L). Here we show that a clone of Kd-restricted CD8+ T cells specific for influenza haemagglutinin, which can also be activated in a crossreactive manner by a peptide derived from a myeloma tumour immunoglobulin heavy-chain variable region (IgVH) to kill by both routes, kills only by the CD95-CD95L pathway when stimulated by the corresponding germline IgVH peptide. As this germline IgVH peptide differs from the tumour peptide only at a single position buried in the MHC-binding groove, this indicates that CD95-CD95L-mediated killing can be triggered independently of the perforin-mediated pathway, and can be selectively affected by changes in MHC conformation.