RESUMEN
Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.
Asunto(s)
Galactosilceramidas/inmunología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Antígenos CD1d/inmunología , Línea Celular , Galactosilceramidas/farmacología , Glucuronatos/inmunología , Humanos , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.
Asunto(s)
Antígenos CD1d/inmunología , Galactosilceramidas/inmunología , Células T Asesinas Naturales/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Antígenos CD1d/química , Clonación Molecular , Cristalización , Galactosilceramidas/química , Humanos , Ratones , Mutagénesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Glucocorticoids (GCs) are powerful inhibitors of inflammation and immunity. Although glucocorticoid-induced cell death (GICD) is an important part of GCs actions, the cell types and molecular mechanisms involved are not well understood. Untranslated exon 1A3 of the human glucocorticoid receptor (GR) gene is a major determinant of GICD in GICD-sensitive human cancer cell lines, operating to dynamically upregulate GR levels in response to GCs. We measured the GICD sensitivity of freshly isolated peripheral blood mononuclear cells and thymocytes to dexamethasone in vitro, relating this to GR exon 1A3 expression. A clear GICD sensitivity hierarchy was detected: B cells>thymocytes/natural killer (NK) cells>peripheral T cells. Within thymocyte populations, GICD sensitivity decreased with maturation. Interestingly, NK cell subsets were differentially sensitive to GICD, with CD16(+)CD56(int) (cytotoxic) NK cells being highly resistant to GICD, whereas CD16(-)CD56(hi) (cytokine producing) NK cells were highly sensitive (similar to B cells). B-cell GICD was rescued by co-culture with interleukin-4. Strikingly, although no significant increases in GR protein were observed during 48 h of culture of GICD-sensitive and -resistant cells alike, GR 1A3 expression was increased over pre-culture levels in a manner directly proportional to the GICD sensitivity of each cell type. Accordingly, this is the first evidence that the GR exon 1A3 promoter is differentially regulated during thymic development and maturation of human T cells. Furthermore, human peripheral blood B cells are exquisitely GICD-sensitive in vitro, giving new insight into how GCs may downregulate immunity. Collectively, these data show that GR 1A3 expression is tied with GICD sensitivity in human lymphocytes, underscoring the potential for GR 1A3 expression to be used as a biomarker for sensitivity to GICD.
Asunto(s)
Apoptosis/fisiología , Glucocorticoides/toxicidad , Linfocitos/efectos de los fármacos , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/genética , Técnicas de Cocultivo , Regulación hacia Abajo , Expresión Génica , Humanos , Interleucina-4/inmunología , Linfocitos/inmunología , Regulación hacia ArribaRESUMEN
CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor γt positive, and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.
Asunto(s)
Antígenos CD1d/inmunología , Proteínas de Unión al ADN/inmunología , Interleucina-17/inmunología , Mutación Missense , Células T Asesinas Naturales/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Factores de Transcripción/inmunología , Animales , Antígenos CD1d/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Diferenciación Celular/inmunología , Proliferación Celular , Proteínas de Unión al ADN/genética , Expresión Génica/inmunología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Masculino , Ratones , Ratones Transgénicos , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/patología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Estructura Terciaria de Proteína , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Timo/inmunología , Timo/metabolismo , Timo/patología , Factores de Transcripción/genéticaRESUMEN
We showed previously that NKT cell-deficient TCR Jalpha18(-/-) mice are more susceptible to methylcholanthrene (MCA)-induced sarcomas, and that normal tumor surveillance can be restored by adoptive transfer of WT liver-derived NKT cells. Liver-derived NKT cells were used in these studies because of their relative abundance in this organ, and it was assumed that they were representative of NKT cells from other sites. We compared NKT cells from liver, thymus, and spleen for their ability to mediate rejection of the sarcoma cell line (MCA-1) in vivo, and found that this was a specialized function of liver-derived NKT cells. Furthermore, when CD4(+) and CD4(-) liver-derived NKT cells were administered separately, MCA-1 rejection was mediated primarily by the CD4(-) fraction. Very similar results were achieved using the B16F10 melanoma metastasis model, which requires NKT cell stimulation with alpha-galactosylceramide. The impaired ability of thymus-derived NKT cells was due, in part, to their production of IL-4, because tumor immunity was clearly enhanced after transfer of IL-4-deficient thymus-derived NKT cells. This is the first study to demonstrate the existence of functionally distinct NKT cell subsets in vivo and may shed light on the long-appreciated paradox that NKT cells function as immunosuppressive cells in some disease models, whereas they promote cell-mediated immunity in others.
Asunto(s)
Células Asesinas Naturales/inmunología , Sarcoma Experimental/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Antígenos CD4/metabolismo , Línea Celular Tumoral , Galactosilceramidas/inmunología , Inmunidad Celular , Interleucina-4/metabolismo , Células Asesinas Naturales/metabolismo , Hígado/citología , Hígado/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sarcoma Experimental/patología , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Timo/inmunologíaRESUMEN
The nuclear factor (NF)-κB signalling pathway is known to be critical for natural killer T (NKT) cell differentiation; however, the role of individual NF-κB transcription factors and the precise developmental stages that they control remain unclear. We have investigated the influence of the classical NF-κB transcription factors NF-κB1, c-Rel and RelA on NKT cell development and function, using gene-deleted mice. Individually, none of these factors were essential for the requirement of NF-κB signalling in early NKT cell development before NK1.1 expression, in contrast to earlier reports in which the classical NF-κB pathway was globally disrupted. Instead, we found that each factor played a non-redundant role in later stages of NKT cell maturation and function. Although NF-κB1 deficiency resulted in a moderate reduction in mature NK1.1+ NKT cells, this was found to be more subtle than previously reported. RelA deficiency had a more profound effect on the NK1.1+ stage of NKT cell development, whereas c-Rel-deficient mice had normal NKT cell numbers. All three factors (NF-κB1, RelA and c-Rel) were necessary for normal NKT cell cytokine production. Notably, IL-17, which is produced by a specific subset of NKT cells (NKT-17 cells), defined as NK1.1(-)CD4(-), was not impaired by a lack of these individual NF-κB transcription factors, nor was this subset depleted, suggesting that NKT-17 cells are regulated independently of the NF-κB pathway. Thus, individual NF-κB family members have a largely redundant role in early NKT cell development, but each of them has an important and distinct role in NKT cell maturation and/or function.
Asunto(s)
Diferenciación Celular/inmunología , FN-kappa B/metabolismo , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transducción de Señal/inmunología , Factor de Transcripción ReIA/metabolismo , Animales , Recuento de Células , Citocinas/biosíntesis , Ratones , Ratones Endogámicos C57BL , FN-kappa B/deficiencia , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/metabolismo , Timo/citologíaRESUMEN
NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4(-)NK1.1(-) NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2-3 h of activation. On intrathymic transfer these cells develop into mature CD4(-)NK1.1(+) but not into CD4(+)NK1.1(+) NKT cells, indicating that CD4(-)NK1.1(-) NKT cells include an IL-17-producing subpopulation, and also mark the elusive branch point for CD4(+) and CD4(-) NKT cell sublineages.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Interleucina-17/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Mediadores de Inflamación/inmunología , Células Asesinas Naturales/citología , Ratones , Especificidad de Órganos/inmunología , Subgrupos de Linfocitos T/citología , Factores de TiempoRESUMEN
NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4(+) (on average 67%) with smaller populations being CD8(+) (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4(+) NKT cells occurred in all six macaques infected with CXCR4-tropic SHIV(mn229) early after infection, with a concomitant rise in CD8(+) NKT cells in some animals. The depletion of CD4(+) NKT cells was tightly correlated with the depletion of total CD4(+) T cells. R5-tropic SIV(mac251) infection of macaques resulted in a slower and more variable decline in CD4(+) NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4(+) NKT cells. An inverse correlation between the depletion of total and CD4(+) NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4(+) NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.
Asunto(s)
Células T Asesinas Naturales/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Antígenos CD4/análisis , Recuento de Linfocito CD4 , Antígenos CD8/análisis , Macaca nemestrina , Células T Asesinas Naturales/química , Subgrupos de Linfocitos T/inmunología , Carga ViralRESUMEN
Type 1 NKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. Herein, we report the production and characterization of a NOD.Nkrp1(b).Nkt2b(b) congenic mouse strain, which has increased thymic and peripheral NKT cells, a decreased incidence of type 1 diabetes, and enhanced cytokine responses in vivo and increased proliferative responses in vitro following challenge with alpha-galactosylceramide. The 19 highly differentially expressed candidate genes within the congenic region identified by microarray expression analyses included Pxmp4. This gene encodes a peroxisome-associated integral membrane protein whose only known binding partner is Pex19, an intracellular chaperone and component of the peroxisomal membrane insertion machinery encoded by a candidate for the NKT cell control gene Nkt1. These findings raise the possibility that peroxisomes play a role in modulating glycolipid availability for CD1d presentation, thereby influencing NKT cell function.
Asunto(s)
Perfilación de la Expresión Génica , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/genética , Peroxisomas/inmunología , Animales , Antígenos CD1 , Antígenos CD1d , Citocinas/biosíntesis , Diabetes Mellitus Tipo 1 , Humanos , Células Asesinas Naturales/citología , Recuento de Linfocitos , Ratones , Ratones Congénicos , Ratones Endogámicos NODRESUMEN
Single nucleotide polymorphisms in the gene encoding the protein tyrosine phosphatase TCPTP (encoded by PTPN2) have been linked with the development of autoimmunity. Here we have used Cre/LoxP recombination to generate Ptpn2(ex2-/ex2-) mice with a global deficiency in TCPTP on a C57BL/6 background and compared the phenotype of these mice to Ptpn2(-/-) mice (BALB/c-129SJ) generated previously by homologous recombination and backcrossed onto the BALB/c background. Ptpn2(ex2-/ex2-) mice exhibited growth retardation and a median survival of 32 days, as compared to 21 days for Ptpn2(-/-) (BALB/c) mice, but the overt signs of morbidity (hunched posture, piloerection, decreased mobility and diarrhoea) evident in Ptpn2(-/-) (BALB/c) mice were not detected in Ptpn2(ex2-/ex2-) mice. At 14 days of age, bone development was delayed in Ptpn2(-/-) (BALB/c) mice. This was associated with increased trabecular bone mass and decreased bone remodeling, a phenotype that was not evident in Ptpn2(ex2-/ex2-) mice. Ptpn2(ex2-/ex2-) mice had defects in erythropoiesis and B cell development as evident in Ptpn2(-/-) (BALB/c) mice, but not splenomegaly and did not exhibit an accumulation of myeloid cells in the spleen as seen in Ptpn2(-/-) (BALB/c) mice. Moreover, thymic atrophy, another feature of Ptpn2(-/-) (BALB/c) mice, was delayed in Ptpn2(ex2-/ex2-) mice and preceded by an increase in thymocyte positive selection and a concomitant increase in lymph node T cells. Backcrossing Ptpn2(-/-) (BALB/c) mice onto the C57BL/6 background largely recapitulated the phenotype of Ptpn2(ex2-/ex2-) mice. Taken together these results reaffirm TCPTP's important role in lymphocyte development and indicate that the effects on morbidity, mortality, bone development and the myeloid compartment are strain-dependent.
Asunto(s)
Desarrollo Óseo , Remodelación Ósea , Eritropoyesis , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Cruzamientos Genéticos , Hiperplasia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Especificidad de la EspecieRESUMEN
Many autoimmune diseases exhibit familial aggregation, indicating that they have genetic determinants. Single nucleotide polymorphisms in PTPN2, which encodes T cell protein tyrosine phosphatase (TCPTP), have been linked with the development of several autoimmune diseases, including type 1 diabetes and Crohn's disease. In this study, we have identified TCPTP as a key negative regulator of TCR signaling, which might explain the association of PTPN2 SNPs with autoimmune disease. We found that TCPTP dephosphorylates and inactivates Src family kinases to regulate T cell responses. Using T cell-specific TCPTP-deficient mice, we established that TCPTP attenuates T cell activation and proliferation in vitro and blunts antigen-induced responses in vivo. TCPTP deficiency lowered the in vivo threshold for TCR-dependent CD8(+) T cell proliferation. Consistent with this, T cell-specific TCPTP-deficient mice developed widespread inflammation and autoimmunity that was transferable to wild-type recipient mice by CD8(+) T cells alone. This autoimmunity was associated with increased serum levels of proinflammatory cytokines and anti-nuclear antibodies, T cell infiltrates in non-lymphoid tissues, and liver disease. These data indicate that TCPTP is a critical negative regulator of TCR signaling that sets the threshold for TCR-induced naive T cell responses to prevent autoimmune and inflammatory disorders arising.
Asunto(s)
Enfermedades Autoinmunes/etiología , Tolerancia Inmunológica/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 2/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/enzimología , Animales , Anticuerpos Antinucleares/biosíntesis , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Inflamación/sangre , Inflamación/genética , Inflamación/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Procesamiento Proteico-Postraduccional , Proteína Tirosina Fosfatasa no Receptora Tipo 2/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Quimera por Radiación , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Timocitos/patología , Proteína Tirosina Quinasa ZAP-70/fisiología , Familia-src Quinasas/metabolismoRESUMEN
The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.
Asunto(s)
Comunicación Autocrina/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Animales , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/metabolismo , Supervivencia Celular/inmunología , Citocinas/inmunología , Galactosilceramidas/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Granzimas/biosíntesis , Granzimas/inmunología , Interleucinas/biosíntesis , Células Asesinas Naturales/metabolismo , Ratones , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismoRESUMEN
CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.
Asunto(s)
Inmunoterapia Adoptiva , Células Asesinas Naturales/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T Reguladores/trasplante , Animales , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/terapia , Comunicación Celular/inmunología , Línea Celular Tumoral , Interleucina-12/fisiología , Ligandos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Linfoma/inmunología , Linfoma/terapia , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK , Trasplante de Neoplasias/inmunología , Neoplasias Experimentales/terapia , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas Naturales , Bazo/citología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
NKT cells are a minor subset of T cells that have important roles in controlling immune responses in disease states including cancer, autoimmunity and pathogenic infections. In contrast to conventional T cells, NKT cells express an invariant TCR and respond to glycolipids presented by CD1d. In this study, we sought to investigate the role of NKT cells in regulating the response to infection with HSV-1, and the mechanism involved, in well-established mouse models. Previous studies of HSV-1 disease in mice have shown clear roles for CD4+ and CD8+ T cells. The role of NKT cells in the resolution of HSV-1 (KOS strain) infection was investigated through flank zosteriform or footpad infection in wild-type versus CD1d-deficient mice, by measurement of viral plaque-forming units at different sites after infection, lesion severity and HSV-1-specific T-cell responses. In contrast to a previous study using a more virulent strain of HSV-1 (SC16 strain), no differences were observed in disease magnitude or resolution, and furthermore, the T-cell response to HSV-1 (KOS strain) was unaltered in the absence of NKT cells. In conclusion, this study shows that NKT cells do not play a general role in controlling the resolution or severity of HSV-1 infection. Instead, the resolution or severity of the infection may depend on the HSV-1 strain under investigation.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1 , Células Asesinas Naturales/inmunología , Animales , Antígenos CD1/inmunología , Antígenos CD1d , Herpesvirus Humano 1/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Ensayo de Placa ViralRESUMEN
After being positively selected on CD1d-expressing thymocytes, NKT cells undergo a series of developmental changes that can take place inside or outside the thymus. We asked whether CD1d continues to play a role in late-stage NKT cell development and, in particular, during the functionally significant acquisition of NK1.1 that is indicative of NKT cell maturity. We report that CD1d is indeed crucial for this step, because immature NK1.1(-) NKT cells fail to fully mature when transferred to a CD1d-deficient environment. Surprisingly, however, the lack of CD1d did not greatly affect the long-term survival of NKT cells, and they continued to express CD69 and slowly proliferate. This directly contradicts the currently held view that these phenomena are caused by autoreactivity directed against CD1d/TCR-restricted self-Ags. Our findings demonstrate an ongoing role for TCR-mediated signaling throughout NKT cell development, but the characteristic semiactivated basal state of NKT cells is controlled by CD1d-independent factors or is intrinsic to the cells themselves.
Asunto(s)
Antígenos CD1/fisiología , Homeostasis , Células Asesinas Naturales/fisiología , Animales , Antígenos CD , Antígenos CD1/genética , Antígenos CD1d , Antígenos de Diferenciación de Linfocitos T , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Asesinas Naturales/citología , Lectinas Tipo C , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T , Transducción de Señal , Linfocitos TRESUMEN
Activation of NKT cells using the glycolipid alpha-galactosylceramide (alpha-GalCer) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how they respond to stimulation in vivo, which costimulatory pathways are important, and what factors (e.g., Ag availability and activation-induced cell death) limit their response. We have explored these questions in the context of an in vivo model of NKT cell dynamics spanning activation, population expansion, and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40L costimulatory pathway was necessary for cytokine production by activated NKT cells, either early (2 h) or late (3 days) after initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The proapoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting that the pool size is regulated by apoptotic death, similar to that of conventional T cells. Ag availability was not the limiting factor for NKT cell expansion in vivo, and a second alpha-GalCer injection induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to NKT cells and was not associated with inhibitory NK receptor signaling. Furthermore, NKT cells from mice pre-challenged with alpha-GalCer in vivo showed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to primary and secondary antigenic challenge in vivo.
Asunto(s)
Proteínas Portadoras/fisiología , Galactosilceramidas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Antígenos CD28/fisiología , Antígenos CD40/fisiología , Citocinas/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR alpha-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d-/- mice that are completely deficient for NKT cells. CD1d tetramer- CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer- CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.
Asunto(s)
Antígenos CD1/fisiología , Integrina alfa2/biosíntesis , Células Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD1/biosíntesis , Antígenos CD1d , Biomarcadores , Células Cultivadas , Citocinas/biosíntesis , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/citología , Bazo/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
The antimetastatic effect of the CD1d-binding glycolipid, alpha-galactosylceramide (alpha-GalCer), is mediated by NK1.1(+)T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-gamma (IFN-gamma) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-gamma, and NK cells has been shown in the antimetastatic activity of alpha-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of alpha-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-gamma induction and control of lung metastases by alpha-GalCer. IL-18 was unnecessary for alpha-GalCer-related suppression of liver metastases. Most importantly, after adoptive transfer of alpha-GalCer-reactive NKT cells or NK cells into NKT cell-deficient, IFN-gamma-deficient, or RAG-1-deficient mice, it was demonstrated that the sequential production of IFN-gamma by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of alpha-GalCer.
Asunto(s)
Galactosilceramidas/farmacología , Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Metástasis de la Neoplasia/prevención & control , Traslado Adoptivo/métodos , Animales , Citotoxicidad Inmunológica , Galactosilceramidas/administración & dosificación , Galactosilceramidas/inmunología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-12/genética , Interleucina-12/fisiología , Interleucina-18/genética , Interleucina-18/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMEN
Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B-1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B-1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant alphabetaTCR+CD24(int/low) thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death.