RESUMEN
Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T(-)B(-)NK(+) SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T(-)B(-)NK(+) SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.
Asunto(s)
Inmunidad Adaptativa/genética , Enzimas Reparadoras del ADN/genética , Proteínas Nucleares/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Inmunidad Adaptativa/inmunología , Animales , Linfocitos B/inmunología , Secuencia de Bases , Mapeo Cromosómico , Haplotipos/genética , Células Asesinas Naturales/inmunología , Fenotipo , Tolerancia a Radiación/genética , Análisis de Secuencia de ADN , Sus scrofa , Linfocitos T/inmunologíaRESUMEN
The replication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in a line of pigs possessing a severe combined immunodeficiency (SCID). Real-time RT-PCR revealed a unique course of infection for the SCID group. During the course of infection, viremia was initially significantly lower than normal littermates, but by 21 days was significantly elevated. Deep sequencing of the viral structural genes at days 11 and 21 identified seven amino acid substitutions in both normal and SCID pigs. The most significant change was a W99R substitution in GP2, which was present in the inoculum at a frequency of 35%, but eventually disappeared from all pigs regardless of immune status. Therefore, amino acid substitutions that appear during acute infection are likely the result of the adaptation of the virus to replication in pigs and not immune selection.
Asunto(s)
Variación Genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Inmunodeficiencia Combinada Grave , Replicación Viral , Inmunidad Adaptativa , Sustitución de Aminoácidos , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Factores de Tiempo , Carga Viral , ViremiaRESUMEN
The intracellular roles of Granzyme B (GrB) in immune-mediated cell killing have been extensively studied. Recent data also implicate GrB in extracellular pathways of inflammation, cytokine activation and autoimmunity. Targeting (GrB) provides a new pharmaceutical agent for various inflammatory disorders. Serpina3n is a mouse extracellular inhibitor of GrB. There is no apparent equivalent in humans. In this study, we used a novel applied genetics approach to engineer a new extracellular GrB serpin. A chimeric protein was generated in which the reactive center loop (RCL) of human extracellular antichymotrypsin (ACT) was replaced with that of serpina3n. This serpin contained 27 amino acid residues from the serpina3n RCL and the remaining 395 residues from human ACT. The insertion converted human ACT into a GrB-inhibitory serpin. Several critical residues were identified by scanning mutagenesis on the chimera and serpina3n. Targeted mutagenesis was conducted on wild-type human ACT by specifically substituting those critical residues, creating a novel inhibitor that contains 99.3% human ACT sequence with only three point mutations. Wild-type human ACT had a kass for GrB of 2.26 × 10(4) M(-1) s(-1), whereas the novel inhibitor binds GrB with a kass of 7.65 × 10(5) M(-1) s(-1). This new drug candidate can be developed in animal models and further tested in clinical trials to help us understand the role of GrB in numerous disorders.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Granzimas/antagonistas & inhibidores , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Serpinas/metabolismo , Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/genética , Animales , Apoptosis , Humanos , Células Jurkat , Ratones , Mutación , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Serpinas/química , Serpinas/genéticaRESUMEN
The expression of the coinhibitor PD-1 on T cells is important for the establishment of immune homeostasis. We previously found that PD-1 is particularly critical for the control of self-tolerance during lymphopenia-induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD-1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD-L1, and not PD-1 itself. Herein we directly tested whether T-cell PD-1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia-potentiated autoimmunity caused by PD-1 KO RTEs. Employing the murine FoxP3(EGFP) reporter system to simultaneously monitor conversion of WT and PD-1 KO T cells to pTreg cells in the same animal, we found that PD-1 deficiency did not inhibit pTreg cell generation or lead to Th17-cell-mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD-1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD-1 is to restrain T-cell activation/proliferation to self-Ags rather than promote generation of Treg cells.
Asunto(s)
Proliferación Celular/fisiología , Activación de Linfocitos/fisiología , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Autoantígenos/genética , Autoantígenos/inmunología , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Recuento de Linfocitos , Linfopenia/inmunología , Ratones , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Linfocitos T Reguladores/citología , Células Th17/citologíaRESUMEN
Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1(-/-) pigs followed the same course as in SIGLEC1(-/+) and SIGLEC1(+/+) littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.
Asunto(s)
Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/fisiología , Animales , Animales Modificados Genéticamente , Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/virología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Receptores de Superficie Celular/fisiología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/deficiencia , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Sus scrofa , Porcinos , Acoplamiento Viral , Internalización del VirusRESUMEN
Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Granzimas/farmacología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Daño del ADN , Activación Enzimática , Proteína Ligando Fas/metabolismo , Granzimas/metabolismo , Células HeLa , Humanos , Células Jurkat , Células K562 , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Proteínas Recombinantes/farmacología , Receptor fas/metabolismoRESUMEN
LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated and nonstimulated CD4(+)CD25(+)FoxP3(+) T cells (regulatory T cells; Tregs) through apoptosis, while having no cytotoxic effect on CD4(+)CD25(-) T cells at the same LL-37 concentrations. Of interest, Tregs were much more sensitive to LL-37 than many other cells, dying at 10-fold lower concentrations than other cell types tested. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and apoptotic body formation, all indicative of an apoptotic form of cell death. The importance of granzyme family members in the apoptosis of Tregs after LL-37 treatment was analyzed by using C57Bl/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, and both the granzyme A and B genes. Granzyme A and granzyme B were both shown to play a role in LL-37-induced apoptosis of Tregs. Further analysis showed that apoptosis occurred primarily through caspase-dependent apoptosis at high LL-37 concentrations. However, grA-dependent/caspase-independent cell death was also observed. This suggests that LL-37 induces apoptosis in Tregs through multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply that LL-37 administered at the site of a tumor could influence the adaptive antitumor immune response by killing Tregs and thus inhibiting their suppressor activity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Granzimas/deficiencia , Linfocitos T Reguladores/efectos de los fármacos , Inmunidad Adaptativa/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Apoptosis/genética , Apoptosis/inmunología , Caspasas/genética , Cromatina , Granzimas/genética , Homocigoto , Humanos , Activación de Linfocitos , Linfocitos Nulos/citología , Linfocitos Nulos/efectos de los fármacos , Linfocitos Nulos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , CatelicidinasRESUMEN
This study compared serum antibody titers and granzyme B (GrzB) levels in virus-stimulated peripheral blood mononuclear cells following influenza vaccination. Twelve of 239 older adults who subsequently developed laboratory-diagnosed influenza illness (LDI) had significantly lower GrzB levels compared to subjects without LDI (p=0.004). Eight subjects with LDI in the previous year showed an enhanced GrzB response to vaccination (p=0.02). Serum antibody titers following vaccination did not distinguish those older adults who developed LDI from those who did not. These results suggest that GrzB levels could be combined with antibody titers to more effectively predict vaccine efficacy in older adults.
Asunto(s)
Anticuerpos Antivirales/sangre , Granzimas/sangre , Vacunas contra la Influenza/inmunología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Linfocitos T Citotóxicos/inmunología , VacunaciónRESUMEN
Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1ng/mL of granzyme B, compared to 1.5-2.5 microg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.
Asunto(s)
Apoptosis/fisiología , Granzimas/metabolismo , Células Asesinas Naturales/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Línea Celular , Humanos , Células JurkatRESUMEN
Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.
Asunto(s)
Proteínas de Fase Aguda/aislamiento & purificación , Granzimas/antagonistas & inhibidores , Serpinas/aislamiento & purificación , Células de Sertoli/metabolismo , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Animales , Factores Biológicos/inmunología , Factores Biológicos/aislamiento & purificación , Factores Biológicos/metabolismo , Células Cultivadas , Clonación Molecular , Humanos , Sistema Inmunológico , Células Jurkat , Masculino , Ratones , Unión Proteica , Serpinas/inmunología , Serpinas/metabolismo , Células de Sertoli/citología , Células de Sertoli/inmunologíaRESUMEN
We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.
Asunto(s)
Autoinmunidad , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Regulación hacia Abajo/inmunología , Tolerancia Inmunológica , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Granzimas , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplanteRESUMEN
It is commonly held that increased risk of influenza in the elderly is due to a decline in the Ab response to influenza vaccination. This study prospectively evaluated the relationship between the development of influenza illness, and serum Ab titers and ex vivo cellular immune responses to influenza vaccination in community dwelling older adults including those with congestive heart failure (CHF). Adults age 60 years and older (90 subjects), and 10 healthy young adult controls received the 2003-04 trivalent inactivated influenza vaccine. Laboratory diagnosed influenza (LDI) was documented in 9 of 90 older adults. Pre- and postvaccination Ab titers did not distinguish between subjects who would subsequently develop influenza illness (LDI subjects) and those who would not (non-LDI subjects). In contrast, PBMC restimulated ex vivo with live influenza virus preparations showed statistically significant differences between LDI and non-LDI subjects. The mean IFN-gamma:IL-10 ratio in influenza A/H3N2-stimulated PBMC was 10-fold lower in LDI vs non-LDI subjects. Pre-and postvaccination granzyme B levels were significantly lower in CHF subjects with LDI compared with subjects without LDI. In non-CHF subjects with LDI, granzyme B levels increased to high levels at the time of influenza infection. In conclusion, measures of the ex vivo cellular immune response to influenza are correlated with protection against influenza while serum Ab responses may be limited as a sole measure of vaccine efficacy in older people. Ex vivo measures of the cell-mediated immune response should be incorporated into evaluation of new vaccines for older adults.
Asunto(s)
Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Células Cultivadas , Granzimas , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/diagnóstico , Gripe Humana/prevención & control , Interferón gamma/sangre , Interleucina-10/sangre , Persona de Mediana Edad , Estudios Prospectivos , Serina Endopeptidasas/sangre , Linfocitos T/metabolismoRESUMEN
We have established novel ELISA- and ELISPOT-based assays specific for the detection of a potent cytotoxic mediator, granzyme B (GrB), for the assessment of antigen-specific cytotoxic T lymphocyte responses in mice. The sensitivity and specificity of our assays was demonstrated by ELISA using purified mouse GrB and supernatants and cell lysates of cytotoxic lymphocytes derived from GrB-deficient mice. No reactivity was observed by the GrB ELISA in GrB-deficient cells. The mouse GrB ELISPOT was successfully employed to detect antigen-specific effector cell responses of two CTL clones, producing GrB ELISPOT results that correlated strongly with target cell lysis, as assessed by 51Cr-release. Furthermore, we were able to demonstrate direct correlations between GrB ELISPOT and killing by LCMV gp33-specific effector and memory T cells generated in vivo. Thus, the mouse GrB ELISPOT may be used to detect cytotoxic responses, at the single-cell level, for the functional assessment of antigen-specific CD8+ T cell responses in mouse models of infection.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Serina Endopeptidasas/análisis , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos Virales , Degranulación de la Célula , Pruebas Inmunológicas de Citotoxicidad/estadística & datos numéricos , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Femenino , Granzimas , Memoria Inmunológica , Técnicas In Vitro , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Sensibilidad y Especificidad , Serina Endopeptidasas/biosíntesisRESUMEN
Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.
Asunto(s)
Calreticulina/deficiencia , Citotoxicidad Inmunológica , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación/metabolismo , Complejo CD3/metabolismo , Calreticulina/genética , Degranulación de la Célula , Línea Celular , Supervivencia Celular , Endopeptidasas/metabolismo , Granzimas , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/citologíaRESUMEN
Granzyme B, a neutral serine protease, has been demonstrated to be a pivotal molecule for protective immunity against viral infection and cellular malignant transformation. To facilitate monitoring of granzyme B levels, we have recently applied phage display technology to produce single-chain Fv antibodies specific for granzyme B, as versatile alternatives and complementary reagents to currently available monoclonal antibodies. Through four rounds of panning on purified human granzyme B-coated on solid phase, three unique clones were isolated. Expressed soluble scFv antibodies demonstrated specific immunological applications including ELISA, Western blotting, immunoprecipitation and intracellular staining. Based on sequence analyses and structural modeling, one scFv, Fv17, may have overlapping antigen binding specificity with monoclonal antibodies 2C5/F5 and GB11. Owing to the availability of its DNA sequence and large scale production capability, Fv17 should be a superior reagent for monitoring granzyme B expression in natural killer cells and antigen specific CD8+ T cell immunity.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Anticuerpos/química , Secuencia de Bases , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Granzimas , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have utilized the unique enzymatic properties of a key cytotoxic mediator in target cell destruction, Granzyme B (GrB), to establish an attractive alternative to 51Cr-release assays for the assessment of antigen-specific CTL responses. A number of potential colorimetric peptide substrates were compared to evaluate levels of GrB activity in cytolytic cells. The most specific and sensitive substrate for GrB was Ac-IEPD-pNA, as shown by the minimal enzymatic hydrolysis in apoptotic Jurkat cells and strong hydrolysis in human NK cells. When human peripheral blood lymphocytes were stimulated in vitro, elevated GrB levels were detected by both Ac-IEPD-pNA and a GrB ELISA. Analysis of allo-antigen-specific murine CTLs revealed that GrB exocytosis was only detectable upon challenge with appropriate allogeneic target cells and strongly correlated to 51Cr-release data. The validity of using Ac-IEPD-pNA in vaccine trials was demonstrated in mice immunized with allogeneic P815 cells, where GrB enzymatic activity was measurable in ex vivo splenocytes cell cultures only upon co-incubation with P815 targets. Additionally, influenza-infected mice were also assessed for GrB activity following in vitro peptide-stimulation of splenocytes and strongly reflected both peptide-specific tetramer staining and 51Cr-release results. The novel cytotoxic assay presented here should give investigators a sensitive, cross-species, nonradioactive alternative to 51Cr-release assays as a means to assess antigen-specific CTL responses in vaccine trials.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Serina Endopeptidasas/metabolismo , Linfocitos T Citotóxicos/inmunología , Anilidas/química , Animales , Antígenos/inmunología , Línea Celular , Colorimetría/métodos , Femenino , Granzimas , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Oligopéptidos/química , Orthomyxoviridae/inmunología , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Topical application of antigen induces antigen-specific humoral and cellular immune responses. In this study we examined whether expansion of dendritic cells (DC) by Flt3 ligand (Flt3L) treatment influences the induction of immune responses following transcutaneous immunization. Mice were treated intraperitoneally with Flt3L or phosphate-buffered saline (PBS) and immunized transcutaneously with hen egg lysozyme (HEL). Flt3L-treated mice developed lower HEL-specific cellular and humoral immune responses than PBS-treated mice. However, in the presence of cholera toxin (CT), a potent adjuvant for mucosal and transcutaneous immunization, Flt3L-treated mice developed significantly higher cellular and humoral immune responses to HEL when compared to PBS-treated mice. We assessed whether the immunomodulatory effects of CT were a result of activation of epidermal dendritic cells (Langerhans' cells; LC). Our results indicate that within 8-12 hr of topical application of CT, epidermal LC cells lose their dendritic morphology and become rounder in appearance. In addition, we observed enhanced expression of major histocompatibility complex (MHC) class II, and of adhesion molecules CD11c and intracellular adhesion molecule-1 (ICAM-1). Our observations support the concept that the state of activation of DC in the skin is central to the regulation of immune responses. This information is relevant to the design of effective transcutaneous vaccination strategies.