RESUMEN
Regulatory T cells (Tregs) are a subset of immune cells that suppress the immune response. Treg therapy for inflammatory diseases is being tested in the clinic, with moderate success. However, it is difficult to isolate and expand Tregs to sufficient numbers. Engineered Tregs (eTregs) can be generated in larger quantities by genetically manipulating conventional T cells to express FOXP3. These eTregs can suppress in vitro and in vivo but not as effectively as endogenous Tregs. We hypothesized that ectopic expression of the transcription factor Helios along with FOXP3 is required for optimal eTreg immunosuppression. To test this theory, we generated eTregs by retrovirally transducing total human T cells (CD4+ and CD8+) with FOXP3 alone or with each of the 2 predominant isoforms of Helios. Expression of both FOXP3 and the full-length isoform of Helios was required for eTreg-mediated disease delay in a xenogeneic graft-versus-host disease model. In vitro, this corresponded with superior suppressive function of FOXP3 and full-length Helios-expressing CD4+ and CD8+ eTregs. RNA sequencing showed that the addition of full-length Helios changed gene expression in cellular pathways and the Treg signature compared with FOXP3 alone or the other major Helios isoform. Together, these results show that functional human CD4+ and CD8+ eTregs can be generated from total human T cells by coexpressing FOXP3 and full-length Helios.
Asunto(s)
Factores de Transcripción Forkhead , Linfocitos T Reguladores , Factores de Transcripción Forkhead/genética , Humanos , Factor de Transcripción Ikaros/genética , Tolerancia Inmunológica , Isoformas de Proteínas/genéticaRESUMEN
PURPOSE: Poor preoperative nutritional status is associated with a higher complication rate after radical cystectomy in patients with bladder cancer. Given the short interval between diagnosis and radical cystectomy, we compared the effect of short-term specialized immunonutrition to that of a standard oral nutritional supplement on the acute inflammatory response and arginine status in patients treated with radical cystectomy. MATERIALS AND METHODS: In this prospective, randomized study in 29 men 14 received specialized immunonutrition and 15 received oral nutritional supplement. Each group drank 3 cartons per day for 5 days before and 5 days after radical cystectomy. The Th1-Th2 balance, plasma interleukin-6 and plasma amino acids were measured at baseline, intraoperatively and on postoperative days 2, 14 and 30. Body composition was measured by dual energy x-ray absorptiometry at baseline and on postoperative days 14 and 30. Differences in outcomes were assessed using the generalized linear mixed model. RESULTS: In the specialized immunonutrition group there was a 54.3% average increase in the Th1-Th2 balance according to the tumor necrosis factor-α-to-interleukin-13 ratio from baseline to intraoperative day, representing a shift toward a Th1 response. In the oral nutritional supplement group the Th1-Th2 balance decreased 4.8%. The change in the Th1-Th2 balance between the specialized immunonutrition and oral nutritional supplement groups significantly differed (p <0.027). Plasma interleukin-6 was 42.8% lower in the specialized immunonutrition group compared to the oral nutritional supplement group on postoperative day 2 (p = 0.020). In the specialized immunonutrition group plasma arginine was maintained from baseline to postoperative day 2 and yet the oral nutritional supplement group showed a 26.3% reduction from baseline to postoperative day 2 (p = 0.0003). The change in appendicular muscle loss between the groups was not statistically significant. CONCLUSIONS: Th1-to-Th2 ratios, peak interleukin-6 levels and plasma arginine suggest that consuming specialized immunonutrition counteracts the disrupted T-helper balance, lowers the inflammatory response and prevents arginine depletion due to radical cystectomy.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Cistectomía/efectos adversos , Suplementos Dietéticos , Complicaciones Posoperatorias/prevención & control , Neoplasias de la Vejiga Urinaria/terapia , Administración Oral , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Arginina/sangre , Cistectomía/métodos , Humanos , Recuento de Linfocitos , Masculino , Terapia Neoadyuvante/métodos , Estado Nutricional/efectos de los fármacos , Estado Nutricional/inmunología , Proyectos Piloto , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/inmunología , Cuidados Preoperatorios/métodos , Estudios Prospectivos , Células TH1/inmunología , Células Th2/inmunología , Resultado del Tratamiento , Vejiga Urinaria/cirugíaRESUMEN
Regulatory T (Treg) cells are a subset of immune cells that maintain homeostasis by promoting immune tolerance and suppressing the immune response via a variety of mechanisms such as secreting cytokines, killing reactive immune cells, and inducing anergy. Dysfunction of Treg cells has been implicated in inflammatory diseases such as autoimmunity and transplant rejection. Conversely, too many or hyperresponsive Treg cells has been observed in cancer and chronic infections. Treg cells have proven to be difficult to study as there are no definitive Treg surface markers. Additionally, Tregs can gain pro-inflammatory phenotype depending on stimuli. In this commentary, we discuss the expression and function of members of the Ikaros family of transcription factors during Treg cell development and activation.
RESUMEN
The Ikaros family of transcription factors includes five highly homologous members that can homodimerize or heterodimerize in any combination. Dimerization is essential for their ability to bind DNA and function as transcription factors. Previous studies showed that eliminating the function of the entire family blocks lymphocyte development while deletion of individual family members has relatively minor defects. These data indicate that multiple family members function during T cell development, so we examined the changes in expression of each family member as thymocytes progressed from the CD4-CD8- double negative (DN) to the CD4+CD8+ double positive (DP) developmental stage. Further, we compared the expression of each family member in murine and human thymocytes. In both species, Ikaros and Aiolos mRNA levels increased as thymocytes progressed through the DN to DP transition, but the corresponding increases in protein levels were only observed in mice. Further, Ikaros and Aiolos underwent extensive alternative splicing in mice, whereas only Ikaros was extensively spliced in humans. Helios mRNA and protein levels decreased during murine T cell development, but increased during human T cell development. These differences in the expression and splicing of Ikaros family members between human and murine thymocytes strongly suggest that the Ikaros family of transcription factors regulates murine and human T cell development differently, although the similarities across Ikaros family members may allow different proteins to fulfill similar functions.
Asunto(s)
Empalme Alternativo/genética , Expresión Génica/genética , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Empalme del ARN/genética , Timocitos/metabolismo , Adolescente , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Niño , Preescolar , Humanos , Lactante , Recién Nacido , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. METHODS: We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. RESULTS: Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient's cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients' cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. CONCLUSIONS: Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms.
RESUMEN
The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8+ thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8+ developmental stage, some thymocytes re-express CD4 and become CD3hi double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4+ thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4+ T-cell maturation. For thymocytes progressing toward the CD8+ T-cell lineage, TSP CD8+ thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8+ developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Factor de Transcripción Ikaros/metabolismo , Timocitos/inmunología , Timo/inmunología , Adolescente , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Niño , Preescolar , Selección Clonal Mediada por Antígenos , Humanos , Lactante , Recién NacidoRESUMEN
UNLABELLED: After radical cystectomy (RC), patients are at risk for complications including infections. The expansion of myeloid-derived suppressor cells (MDSCs) after surgery may contribute to the lower resistance to infection. Immune response and postoperative complications were compared in men consuming either specialized immunonutrition (SIM; n=14) or an oral nutrition supplement (ONS; n=15) before and after RC. MDSC count (Lin- CD11b+ CD33+) was significantly different between the groups over time (p=0.005) and significantly lower in SIM 2 d after RC (p<0.001). MDSC count expansion from surgery to 2 d after RC showed a weak association with an increase in infection rate 90 d after surgery (p=0.061). Neutrophil:lymphocyte ratio was significantly lower in SIM compared with ONS 3h after the first incision (p=0.039). Participants receiving SIM had a 33% reduction in postoperative complication rate (95% confidence interval [CI], 1-64; p=0.060) and a 39% reduction in infection rate (95% CI, 8-70; p=0.027) during late-phase recovery. The small sample size limits the study findings. PATIENT SUMMARY: Results show that the immune response to surgery and late infection rates differ between radical cystectomy patients receiving specialized immunonutrition versus oral nutrition supplement in the perioperative period. TRIAL REGISTRATION: ClinicalTrials.gov NCT01868087.
Asunto(s)
Cistectomía/efectos adversos , Nutrición Enteral/métodos , Huésped Inmunocomprometido , Células Mieloides/inmunología , Estado Nutricional , Infección de la Herida Quirúrgica/prevención & control , Neoplasias de la Vejiga Urinaria/cirugía , Anciano , Antígeno CD11b/análisis , Proliferación Celular , Nutrición Enteral/efectos adversos , Alimentos Formulados/efectos adversos , Humanos , Kansas , Masculino , Persona de Mediana Edad , Valor Nutritivo , Fenotipo , Proyectos Piloto , Factores de Riesgo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisis , Infección de la Herida Quirúrgica/inmunología , Infección de la Herida Quirúrgica/microbiología , Factores de Tiempo , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/fisiopatologíaRESUMEN
In human T cell development, the mechanisms that regulate cell fate decisions after TCRß expression remain unclear. We defined the stages of T cell development that flank TCRß expression and found distinct patterns of human T cell development. In half the subjects, T cell development progressed from the CD4(-)CD8(-) double-negative stage to the CD4(+)CD8(+) double-positive (DP) stage through an immature single-positive (ISP) CD4(+) intermediate. However, in some patients, CD4 and CD8 were expressed simultaneously and the ISP population was small. In each group of patients, CD3(-) ISP and DP thymocytes were subdivided into ISP1, ISP2, DP1, DP2, DP3, DP4, and DP5 developmental stages according to their expression of CD28, CD44, CD1a, CD7, CD45RO, and CD38. The ISP2, DP2, and DP3 thymocyte populations proliferated more robustly than ISP1 and DP1 and expressed markers consistent with TCRß expression. After the DP3 stage, proliferation returned to baseline levels. We then analyzed protein levels of Ikaros, Helios, and Aiolos, the three Ikaros family members most abundantly expressed in human thymocytes. Ikaros and Helios expression increased transiently at the ISP2, DP2, and DP3 populations. Aiolos expression also increased at the ISP2, DP2, and DP3 stages, but its expression remained elevated throughout the DP4 and DP5 stages. In summary, we propose a model of human T cell development that reflects the asynchronous nature of TCRß expression and we define the subpopulations of thymocytes that are highly proliferative and express Ikaros family members.
Asunto(s)
Selección Clonal Mediada por Antígenos , Factor de Transcripción Ikaros/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/metabolismo , Timocitos/metabolismo , Adolescente , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Niño , Preescolar , Selección Clonal Mediada por Antígenos/genética , Selección Clonal Mediada por Antígenos/inmunología , Expresión Génica , Humanos , Inmunofenotipificación , Lactante , Recién Nacido , Activación de Linfocitos/inmunología , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/inmunología , Timocitos/inmunologíaRESUMEN
GRB2 related adaptor protein downstream of Shc (GADS) is a member of the GRB2 family of adaptors and is critical for TCR-induced signaling. The current model is that GADS recruits SLP-76 to the LAT complex, which facilitates the phosphorylation of SLP-76, the activation of PLC-γ1, T cell adhesion and cytokine production. However, this model is largely based on studies of disruption of the GADS/SLP-76 interaction and murine T cell differentiation in GADS deficient mice. The role of GADS in mediating TCR-induced signals in human CD4+ T cells has not been thoroughly investigated. In this study, we have suppressed the expression of GADS in human CD4+ HuT78 T cells. GADS deficient HuT78 T cells displayed similar levels of TCR-induced SLP-76 and PLC-γ1 phosphorylation but exhibited substantial decrease in TCR-induced IL-2 and IFN-γ release. The defect in cytokine production occurred because of impaired calcium mobilization due to reduced recruitment of SLP-76 and PLC-γ1 to the LAT complex. Surprisingly, both GADS deficient HuT78 and GADS deficient primary murine CD8+ T cells had similar TCR-induced adhesion when compared to control T cells. Overall, our results show that GADS is required for calcium influx and cytokine production, but not cellular adhesion, in human CD4+ T cells, suggesting that the current model for T cell regulation by GADS is incomplete.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Calcio/metabolismo , Citocinas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos T CD4-Positivos/citología , Adhesión Celular , Línea Celular , Células Cultivadas , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Ratones , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/metabolismo , Transporte de Proteínas , Interferencia de ARNRESUMEN
BACKGROUND: Gastric disturbances such as dyspepsia are routinely encountered by multiple sclerosis (MS) patients, and these conditions are often treated with gastric acid suppressors such as proton pump inhibitors, histamine H2 receptor antagonists, or antacids. The proton pump inhibitor omeprazole can alter the gut flora and immune responses, both of which can influence the course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of the current study was to examine the effect of omeprazole treatment on the development of EAE. Bacterial microbiome analysis of mouse fecal pellets was determined in C57BL/6J EAE mice chronically treated with omeprazole, and spleen immune cell content, clinical scores, weight, rotarod latency, and histopathology were used as outcome measures in C57BL/6J and SJL/J mice with EAE. RESULTS: Omeprazole treatment resulted in decreases in Akkermansia muciniphila and Coprococcus sp. and an increase in unidentified bacteria in the family S24-7 (order Bacteroidales) in C57BL/6J mice with EAE. Omeprazole did not alter spleen immune cell content compared to vehicle in EAE mice, but differences independent of treatment were observed in subsets of T cells between early and advanced disease in C57BL/6J mice as well as between the two strains of mice at an advanced disease stage. Omeprazole caused no difference in clinical scores in either strain, but significantly lowered weight gain compared to vehicle in the C57BL/6J mice with EAE. Omeprazole also did not alter rotarod behavior or hindbrain inflammatory cell infiltration compared to vehicle in both strains of mice with EAE. Rotarod latency did reveal a negative correlation with clinical scores during active disease in both mouse strains, but not during clinical remission in SJL/J mice, suggesting that rotarod can detect disability not reflected in the clinical scores. CONCLUSIONS: Despite alterations in the gut microbiota and weight gain in the C57BL/6J EAE model, omeprazole had no effect on multiple measures of disease activity in C57BL/6J and SJL/J mice with EAE, supporting the notion that omeprazole does not substantially influence disease activity in MS patients.
Asunto(s)
Encefalomielitis Autoinmune Experimental/prevención & control , Omeprazol/farmacología , Inhibidores de la Bomba de Protones/farmacología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Linfocitos T/patologíaRESUMEN
The Gads adaptor protein is an essential component of the T cell signaling complex critical for T cell receptor-mediated calcium mobilization. After expression of TCRß in T cell precursors, Gads is required for optimal Bcl-2 expression and cell survival. Similarly, the IL-7 receptor chain CD127 is also necessary for optimal Bcl-2 expression and cell survival in TCRß-expressing thymocytes. Based on these observations, we tested whether Gads and CD127 might regulate convergent or linear signaling pathways by crossing Gads(-/-) mice with CD127(-/-) mice. Thymi from Gads(-/-)CD127(-/-) mice were barely detectable and many of the thymocytes were within the DN1 population. By contrast, B cell development in the Gads(-/-)CD127(-/-) mice was comparable to that of CD127(-/-) mice, indicating that the combined loss of Gads and CD127 did not lead to a global deficit in hematopoiesis. Analysis of Lin(-)Sca-1(+)c-kit(+) bone marrow cells and bone marrow chimera experiments indicated that Gads(-/-)CD127(-/-) T cell precursors either failed to migrate into the thymus or survive in the thymus. These data demonstrate that Gads functions at a stage of T cell development that had not been previously described.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Subunidad alfa del Receptor de Interleucina-7/genética , Ratones Noqueados , Timocitos/inmunologíaRESUMEN
Among the milestones that occur during T-cell development in the thymus is the expression of T-cell receptor-ß (TCR-ß) and the formation of the pre-TCR complex. Signals emanating from the pre-TCR trigger survival, proliferation and differentiation of T-cell precursors. Although the pre-TCR is essential for these cell outcomes, other receptors, such as Notch and CXCR4, also contribute. Whether interleukin-7 (IL-7) participates in promoting the survival or proliferation of pre-TCR-expressing cells is controversial. We used in vitro and in vivo models of T-cell development to examine the function of IL-7 in TCR-ß-expressing thymocytes. Culturing TCR-ß-expressing CD4(-) CD8(-) double-negative thymocytes in an in vitro model of T-cell development revealed that IL-7 reduced the frequency of CD4(+) CD8(+) double-positive thymocytes at the time of harvest. The mechanism for this change in the percentage of double-positive cells was that IL-7 promoted the survival of thymocytes that had not yet differentiated. By preserving the double-negative population, IL-7 reduced the frequency of double-positive thymocytes. Interleukin-7 was not required for proliferation in the in vitro system. To follow this observation, we examined mice lacking CD127 (IL-7Rα). In addition to the known effect of CD127 deficiency on T-cell development before TCR-ß expression, CD127 deficiency also impaired the development of TCR-ß-expressing double-negative thymocytes. Specifically, we found that Bcl-2 expression and cell cycle progression were reduced in TCR-ß-expressing double-negative thymocytes in mice lacking CD127. We conclude that IL-7 continues to function after TCR-ß is expressed by promoting the survival of TCR-ß-expressing double-negative thymocytes.
Asunto(s)
Interleucina-7/farmacología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Timocitos/efectos de los fármacos , Animales , Antígenos CD4/genética , Antígenos CD4/inmunología , Antígenos CD8/genética , Antígenos CD8/inmunología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/deficiencia , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/inmunología , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos T/efectos de los fármacos , Células Precursoras de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Timocitos/citología , Timocitos/inmunología , Timo/citología , Timo/efectos de los fármacos , Timo/inmunologíaRESUMEN
The Gads adaptor protein is critical for TCR-mediated Ca(2+) mobilization. We investigated the effect of Gads deficiency on the proliferation of CD8(+) T cells following peptide stimulation and in the context of infection with an intracellular pathogen. We stimulated CD8(+) T cells from Gads(+/+) OT-I and Gads(-/-) OT-I mice with cognate Ag (SIINFEKL) or altered peptide ligand. In vitro experiments revealed that Gads was required for optimal proliferation of CD8(+) T cells. This defect was most evident at the early time points of proliferation and when low doses of Ag were used as stimuli. Cell cycle analysis demonstrated that Gads(-/-) CD8(+) T cells had impaired TCR-mediated exit from the G(0) phase of the cell cycle. Furthermore, Gads(-/-) CD8(+) T cells had delayed expression of c-myc and CD69 upon the stimulation with SIINFEKL. We then investigated how Gads deficiency would impact CD8(+) T cell-mediated immunity in the context of infection with an intracellular pathogen. At early time points, Gads(+/+) and Gads(-/-) CD8(+) T cells proliferated to a similar extent, despite the fact that expression of CD69 and CD25 was reduced in the absence of Gads. After 5 d postinfection, Gads was required to sustain the expansion phase of the immune response; the peak response of Gads(-/-) cells was significantly lower than for Gads(+/+) cells. However, Gads was not required for the differentiation of naive CD8(+) T cells into memory cells. We conclude that the primary function of Gads is to regulate the sensitivity of the TCR to Ag ligation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Proliferación Celular , Inmunidad Celular/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ciclo Celular/inmunología , Femenino , Citometría de Flujo , Immunoblotting , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/genética , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Early in T-cell development, cells proceed through stages that are critically dependent on signaling through the Notch receptor. As cells mature, thymocytes transition from being Notch dependent to being Notch independent, but the stage of development during which this transition occurs is unknown. We used an in vitro differentiation system in which thymocytes can be cultured in the presence or absence of a Notch ligand to identify the stage of development in which thymocytes transition from being Notch responsive to Notch non-responsive. We identified the immature single-positive (ISP) CD8(+) stage of T-cell development as being this transition point. ISP thymocytes were responsive to Notch, but ISP cells responded to Notch ligation in a manner that was distinct from the response by double-negative (DN) thymocytes. Fewer ISP thymocytes proliferated and more ISP cells died in culture than DN thymocytes. Further, fewer double-positive (DP) thymocytes generated by culturing ISP thymocytes were in the S, G2 or M phase of the cell cycle as compared with DP thymocytes derived from DN thymocytes. These data indicate that the DP population created varied depending on the input population. In summary, the data presented here indicate that ISP thymocytes responded to Notch differently than DN thymocytes and ISP thymocytes represent the transition stage from Notch-dependent survival and proliferation to Notch-independent survival and proliferation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Receptores Notch/inmunología , Animales , Linfocitos T CD8-positivos/citología , Células Cultivadas , Técnicas de Cocultivo , Ligandos , Ratones , Ratones Endogámicos C57BLRESUMEN
Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5 kDa, 11 kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo-expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11 kDa is a more significant inducer of apoptosis than NS1, whereas 7.5 kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11 kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11 kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11 kDa expression using antisense oligos targeting specifically to the 11 kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11 kDa protein contributes to erythroid progenitor cell death during B19V infection.
Asunto(s)
Apoptosis , Células Precursoras Eritroides/metabolismo , Parvovirus B19 Humano/genética , Proteínas no Estructurales Virales/genética , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 10/metabolismo , Inhibidores de Caspasas , Línea Celular , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/virología , Citometría de Flujo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Células K562 , Peso Molecular , Parvovirus B19 Humano/metabolismo , Parvovirus B19 Humano/fisiología , Quinolinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/fisiologíaRESUMEN
Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4(+)CD8(+) double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4(+) thymocytes. Then, cells are selected to become either mature single positive CD4(+) or mature single positive CD8(+) thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads(+/+) mice, most TSP CD4(+) thymocytes are TCR(hi)Bcl-2(hi)CD69(+), suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads(-/-) mice could readily generate TSP CD4(+) thymocytes, many Gads(-/-) TSP CD4(+) cells were TCR(lo)Bcl-2(lo)CD69(-), suggesting that Gads(-/-) cells proceeded to the TSP CD4(+) stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4(+) thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4(+) stage are separable events and Gads is only required for positive selection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Animales , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Citometría de Flujo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Organismos Libres de Patógenos EspecíficosRESUMEN
The failure to reject the semi-allogeneic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. The T cell immunoinhibitory receptor, Programmed Death-1 (PD-1), and its ligand, B7-H1, maintain peripheral tolerance by inhibiting activation of self-reactive lymphocytes. Here, we investigated the role of the PD-1/B7-H1 pathway in maternal tolerance of the fetus. Antigen-specific maternal T cells both proliferate and upregulate PD-1 in vivo at mid-gestation in response to paternally inherited fetal antigen. In addition, when these cells carry a null deletion of PD-1, they accumulate excessively in the uterus-draining lymph nodes (P<0.001) without a concomitant increase in proliferation. In vitro assays showed that apoptosis of antigen-specific CD8(+) PD-1(-/-) cells was reduced following peptide stimulation, suggesting that the accumulation of these cells in maternal lymph nodes is due to decreased cell death. However, the absence of neither maternal PD-1 nor B7-H1 had detectable effects on gestation length, litter size, or pup weight at birth in either syngeneic or allogeneic pregnancies. These results suggest that PD-1 plays a previously unrecognized role in maternal-fetal tolerance by inducing apoptosis of paternal antigen-specific T cells during pregnancy, thereby controlling their abundance.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Linfocitos T CD8-positivos/metabolismo , Antígenos de Histocompatibilidad/inmunología , Histocompatibilidad/inmunología , Preñez/inmunología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Apoptosis/genética , Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Movimiento Celular/inmunología , Femenino , Desarrollo Fetal/genética , Desarrollo Fetal/inmunología , Histocompatibilidad/genética , Tamaño de la Camada/genética , Tamaño de la Camada/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Embarazo , Receptor de Muerte Celular Programada 1 , Transducción de Señal/inmunologíaRESUMEN
Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of DeltavpuSHIV(PPC), a live virus vaccine derived from SHIV(PPC). Macaques were administered two inoculations of DeltavpuSHIV(PPC), three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.
Asunto(s)
Vacunas contra el SIDAS/efectos adversos , Vacunas contra el SIDAS/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Inmunización Secundaria , Leucocitos Mononucleares/inmunología , Estudios Longitudinales , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Viremia/prevención & controlRESUMEN
TCRbeta expression in CD4(-)CD8(-) double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4(+)CD8(+) double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCRbeta(+) DN thymocytes and found that there are at least three functionally distinct subsets of TCRbeta(+) DN thymocytes: TCRbeta(+) DN3E, TCRbeta(+) DN3L, and TCRbeta(+) DN4. Survival and proliferation of TCRbeta(+) DN3E were independent of Gads, but survival and proliferation of TCRbeta(+) DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCRbeta(+) DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCRbeta(+) DN3L cells. Bcl-2 expression was not dependent on Gads in TCRbeta(+) DN4 cells, but proliferation of TCRbeta(+) DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads(-/-) DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCRbeta-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Linfocitos T/citología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Proliferación Celular , Supervivencia Celular/inmunología , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Mutantes , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Linfocitos T/inmunologíaRESUMEN
Nearly all hematopoietic receptors are dependent on adaptor proteins for the activation of downstream signaling pathways. The Gads adaptor protein is expressed in many hematopoietic tissues, including bone marrow, lymph node, and spleen. Using intracellular staining, we detected Gads protein in a number cells, including B cells, T cells, NK cells, monocytes, and plasmacytoid DC, but not in macrophages, neutrophils, or monocyte-derived DC. In the B cell compartment, Gads was first expressed after immature B cells leave the bone marrow and was down-regulated after B cell antigen receptor (BCR) ligation. Female Gads(-/-) mice had increased numbers of splenic B cells, as compared to female Gads(+/+) mice, suggesting a role for Gads in B cell homeostasis. Although B cell production and turnover of splenic B cell subsets appeared normal in Gads(-/-) mice, homeostatic proliferation was significantly impaired in Gads(-/-) B cells. Whereas BCR ligation can induce apoptosis in wild-type transitional stage 1 (T1) B cells, Gads(-/-) T1 B cells were resistant to BCR-induced apoptosis. Gads(-/-) B cells also showed increased BCR-mediated calcium mobilization. We conclude that Gads may have a negative regulatory role in signaling through survival pathways, and is necessary for normal homeostatic proliferation in B cells.