RESUMEN
Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.
Asunto(s)
Apoptosis , Carcinogénesis , Neoplasias Colorrectales/metabolismo , Células Epiteliales/fisiología , Receptores de Lipopolisacáridos/fisiología , Receptor Toll-Like 4/fisiología , Animales , Células CACO-2 , Colon/metabolismo , Colon/fisiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células Epiteliales/metabolismo , Humanos , Ratones , Transducción de SeñalRESUMEN
In animal models of IgA nephropathy, the inevitable endogenous immune response to passively administered antigens alone or in complex with specific IgA mask the exact role each might play in pathogenesis. To delineate the role the immune response might play, we have developed a passive model with exclusive IgA-immune complex-mediated nephropathy in B-cell-deficient (BCD) mice. Glomerular IgA immune deposits were induced by administration of purified IgA antiphosphorylcholine and the specific pneumococcal C-polysaccharide (PnC) antigen daily for 2 weeks into BCD and wild-type (WT) mice. In BCD mice IgA+PnC deposits induced severe glomerular injury and renal dysfunction. In contrast, WT mice developed intense glomerular IgG and IgM and C3 co-deposits of the IgA+PnC with significantly less renal injury. Cytofluorometric analysis revealed that PnC induced in BCD, but not in WT, a rapid and dramatic increase in number of activated CD3(+)/CD69(+) T-cell population. The nuclear factor-kappa B (NF-kappaB) transcription factor was activated early and progressively increased in response to glomerular IgA+PnC deposits. These results suggest that nephritogenic IgA+PnC immune deposits induce glomerular and renal dysfunction through activation of the NF-kappaB. This inflammatory pathway is modulated by the endogenous cellular and antibody response to the antigen affecting the course of IgA nephropathy progression.
Asunto(s)
Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/patología , Sistema Inmunológico/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Inmunoglobulina A/inmunología , Interleucina-6/sangre , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/patología , FN-kappa B/metabolismo , Fosforilcolina/inmunología , Polisacáridos Bacterianos/inmunología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
The AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic alpha subunit and two regulatory subunits, beta and gamma. The gamma subunit is essential for enzyme activity by virtue of its binding to the C-terminus of the alpha subunit and appears to play some role in the determination of AMP sensitivity. We demonstrate that a gamma1R70Q mutation causes a marked increase in AMPK activity and renders it largely AMP-independent. This activation is associated with increased phosphorylation of the alpha subunit activation loop T172. These in vitro characteristics of AMPK are also reflected in increased intracellular phosphorylation of one of its major substrates, acetyl-CoA carboxylase. These data illustrate the importance of the gamma1 subunit in the regulation of AMPK and its modulation by AMP.
Asunto(s)
Proteínas Portadoras , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas Activadas por AMP , Sustitución de Aminoácidos , Animales , Células COS , Línea Celular , Activación Enzimática/genética , Expresión Génica , Humanos , Isoenzimas/genética , Fosforilación , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Relación Estructura-Actividad , Factores de Transcripción/genética , Transfección , LevadurasRESUMEN
The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Citotoxicidad Inmunológica , Neoplasias Experimentales/inmunología , Proteínas Oncogénicas Virales/inmunología , Neoplasias del Cuello Uterino/inmunología , Adenoviridae , Animales , Femenino , Vectores Genéticos , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales/genética , Vacunas SintéticasRESUMEN
An important goal of vaccination is to achieve long-term survival of functional memory T cells. Using a MHC-compatible adoptive transfer system, we show here that a short, 3-day IL-4 but not IL-2 or IL-12 exposure during in vitro T cell receptor stimulation of naive CD8(+) T cells induced long-lasting in vivo memory. Such long-term memory CD8(+) T cells expressed antigen-specific cytotoxicity and the potential for IFN-gamma and IL-4 production. Our results support the concept that functional T cell longevity can be regulated by cytokines during initial antigen encounter and provide a rational foundation for vaccine development. They also may have implications in formulating optimal therapeutic regimens of ex vivo expanded autologous cancer- and HIV-specific CD8(+) T cells. In addition, the availability of large numbers of memory CD8(+) T cells generated through our high-efficiency system should facilitate progress in the molecular dissection of CD8(+) T cell memory development.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Interleucina-4/farmacología , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Traslado Adoptivo , Animales , Secuencia de Bases , Linfocitos T CD8-positivos/inmunología , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242-2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Dependovirus , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas Oncogénicas Virales/inmunología , Papillomaviridae/inmunología , Vacunas contra Papillomavirus , Neoplasias del Cuello Uterino/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Fusión Artificial Génica , Northern Blotting , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , ADN Viral , Dependovirus/genética , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Vectores Genéticos/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Péptidos/genética , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The source of IL-4 required for priming naive T cells into IL-4-secreting effectors has not been clearly identified. Here we show that upon TCR stimulation, thymus NK1-CD4+8- T cells produced IL-4, the magnitude of which was inversely correlated with age. This IL-4 production response by Th2-prone BALB/c mice was approximately 9-fold that of Th1-prone C57BL/10 mice. More than 90% of activated NK1-CD4+8- thymocytes did not use the invariant V alpha 14-J alpha 281 chain characteristic of typical CD1-restricted NK1+CD4+ T cells. Stat6-null NK1-CD4+8- thymocytes produced bioactive IL-4, with induction of IL-4 mRNA expression within 1 h of stimulation. Our results support the possibility that TCR repertoire-diverse conventional NK1-CD4+ T cells are a potential IL-4 source for directing naive T cells toward Th2/type 2 CD8+ T cell (Tc2) effector development.
Asunto(s)
Envejecimiento/inmunología , Antígenos/biosíntesis , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Interleucina-4/biosíntesis , Glicoproteínas de Membrana , Biosíntesis de Proteínas , Proteínas , Subgrupos de Linfocitos T/metabolismo , Timo/fisiología , Transactivadores/fisiología , Envejecimiento/genética , Animales , Antígenos CD/biosíntesis , Antígenos de Superficie , Antígeno CD24 , Células Cultivadas , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/inmunología , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Subfamilia B de Receptores Similares a Lectina de Células NK , Factor de Transcripción STAT6 , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Células Th2/inmunología , Timo/citología , Transactivadores/deficiencia , Transactivadores/genéticaRESUMEN
Appropriately activated CD8+ T cells differentiate into cytotoxic effectors capable of eliminating cancer cells and virally infected cells. Successful generation and maintenance of effective CD8+ T-cell memory, through either natural infection or through vaccination, establishes long-term protection against various pathogenic agents and, therefore, contributes significantly to our health. This report is a review of recent advances in CD8+ T-cell memory research. The pool of memory CD8+ T cells is maintained through a mechanism of rapid turnover that is antigen-independent, class I major histocompatibility complex (MHC I) antigen-dependent, and, potentially, IL-15-dependent. Memory CD8+ T cells, in marked contrast to naive CD8+ T cells, constitutively express cytotoxic effector function in the absence of antigen stimulation. Furthermore, the vast majority of activated CD8+ T cells in mice infected with lymphocytic choriomeningitis virus are antigen-specific, prompting revision of the commonly held view that many antigen-nonspecific CD8+ T cells are activated in response to viral infection. These newly published results not only provide exciting insights into the inner workings of CD8+ memory maintenance, but they also establish a sound foundation for future investigation. As more detailed molecular and cellular mechanisms of the regulation of memory CD8+ T-cell survival emerge, the most exciting challenge will be to apply this understanding toward the rational design of vaccines and immunotherapies. These potentials are even more relevant in view of the critical nature of CD8+ T cells in combating viral infection and cancer, and the relative paucity of effective drugs against these diseases.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Animales , Diferenciación Celular , Supervivencia Celular , Citocinas/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Humanos , RatonesRESUMEN
Recent studies using IL-2R alpha knockout mice have generated conflicting results regarding the hypothesis that IL-2/IL-2R interaction is obligatory for the development of AICD, which plays a central and pivotal role in maintaining peripheral tolerance. A relevant consequence of AICD defect is the demonstrated development of autoimmune lymphoproliferative disease in IL-2, IL-2R alpha, and IL-2R beta knockout mice, but not in IL-4, IL-7, or IL-7R knockout mice. Whether IL-4, IL-7, or IL-15 can provide the required signal for AICD development is addressed here using IL-2 and IL-2R beta knockout mice. Lymph node T cells from knockout mice were stimulated with Con A plus rIL-1 for 3 days and then maintained in high concentrations of rIL-4, rIL-7, or rIL-15 for an additional 3 days before they were subjected to AICD analysis. Our study demonstrates that IL-4, IL-7, and IL-15 can transduce signals critical for AICD development in the absence of IL-2-mediated signals. The requirement for relatively high concentrations of these lymphokines suggests their limited role in maintaining peripheral T cell tolerance, thus explaining the differential expression of autoimmune lymphoproliferative disease in the targeted mutant strains described above.
Asunto(s)
Apoptosis/inmunología , Interleucina-2/genética , Activación de Linfocitos , Linfocinas/fisiología , Receptores de Interleucina-2/genética , Linfocitos T/inmunología , Animales , Apoptosis/efectos de los fármacos , Proteína Ligando Fas , Interleucina-15/genética , Interleucina-15/farmacología , Interleucina-2/deficiencia , Interleucina-4/genética , Interleucina-4/farmacología , Interleucina-7/genética , Interleucina-7/farmacología , Ligandos , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Ratones SCID , Receptores de Interleucina-2/deficiencia , Proteínas Recombinantes/farmacología , Linfocitos T/metabolismo , Receptor fas/fisiologíaRESUMEN
The retinoblastoma protein (Rb), an important ubiquitous cell cycle regulator, was initially identified as the retinoblastoma tumor suppressor. To further address the activities of Rb in proliferation and tumorigenesis in the hematopoietic lineage, we transplanted Rb-/- fetal liver cells into sibling mice and assessed the outcome of Rb-/- hematopoietic cells in both short-term and long-term studies. Rb-/- hematopoietic cells rescued lethally irradiated mice with an efficiency comparable to that of wild-type cells. In spleen colony-forming unit assays, proliferation rates of the Rb-/- cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Rb-/- embryonic livers. Recipients of Rb-/- cells lived for more than 15-18 months, and most blood cell lineages matured normally with the expected switch from fetal to adult hemoglobin. However, the continued presence of nucleated erythrocytes in the peripheral blood and extensive extramedullary erythropoiesis indicated that the Rb-/- erythrocytes were not completely normal. No erythroleukemia developed during the 15-18 month period following transplantation. These results demonstrate the mitogenic effect but not tumorigenic transformation in erythrocyte lineage in the absence of Rb, which is distinct from the effect of Rb deficiency in neuroectodermal cells. The study supports the prevalent model that loss of the ubiquitously expressed tumor suppressor gene predisposes to only a limited spectrum of tumors.
Asunto(s)
División Celular , Células Precursoras Eritroides/citología , Eritropoyesis , Genes de Retinoblastoma , Ratones Noqueados , Animales , Células de la Médula Ósea , Supervivencia Celular , Heterocigoto , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Mitosis , Fragilidad Osmótica , Distribución TisularRESUMEN
A comparative study of immune function and marker expression of CD4+ T cells from MHC class 1-restricted 2C TCR-transgenic (2C+) and control transgene-negative littermate (2C-) mice was performed. While 2C+CD4+ T cells resembled memory T cells on the basis of CD44highCD45RBlow expression, the majority of 2C-CD4+ T cells were of the CD44lowCD45RBhigh naive phenotype. Slightly lower levels of TCR-beta and CD3 were found on 2C+CD4+ T cell than 2C-CD4+ T cells. Vigorous proliferation by 2C-CD4+ T cells was observed upon stimulation with 1) anti-CD3 mAb presented through the FcR of macrophages; 2) immobilized (plate-bound) anti-CD3 + anti-CD28 mAbs; and 3) PMA + ionomycin. In marked contrast, all three mitogenic stimuli stimulated highly deficient proliferative responses by 2C+CD4+ T cells. However, significant IL-2 production was detected both in anti-CD3 and in PMA + ionomycin-stimulated cultures of 2C+CD4+ T cells. While intracellular calcium in 2C-CD4+ T cells rapidly increased following anti-CD3 addition, no such increase was observed for similarly stimulated 2C+CD4+ T cells. Anti-CD28, PMA, and coculture with 2C-CD4+ T cells each failed to significantly correct the deficient 2C+CD4+ T cells proliferation as induced by anti-CD3. In addition, IL-2, IL-4, and IL-7 supplements also failed to reverse the deficient proliferation of 2C+CD4+ T cells despite expression of IL-2R component alpha-, beta-chains and the gamma-chain common also to IL-4R and IL-7R. Thymus CD4+8- T cells from the 2C-transgenic mouse were similarly deficient in proliferation as spleen CD4+ T cells. A small subpopulation of CD4+ T cell from the 2C-transgenic mouse expressed the transgenic TCR alpha:beta heterodimer as detected by the 1B2 anti-2C clonotypic mAb; both 1B2+ and 1B2- subpopulations proliferated poorly in response to anti-CD3 and to PMA + ionomycin. These results raise the possibility that TCR engagement with MHC class 1 molecules during early intrathymic development can result in the emergence of CD4+ T cells characterized by unusual marker expression and function.
Asunto(s)
Linfocitos T CD4-Positivos/patología , Antígenos H-2/genética , Activación de Linfocitos , Linfopenia/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica/inmunología , Inmunoadsorbentes , Interleucina-2/biosíntesis , Interleucina-2/genética , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfopenia/patología , Linfopenia/terapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Although patients with refractory periodontitis have been widely reported, no clear biologic profile of these patients has been noted. The purpose of the present study was to evaluate host responsiveness of a well-defined group of refractory periodontitis patients by determining the effect of a lipopolysaccharide (LPS) challenge on monocyte surface receptor density and on the release of inflammatory mediators. Venous blood was obtained from 7 refractory periodontitis, 8 stable periodontal maintenance, and 8 gingivitis patients with no evidence of periodontitis. Mononuclear cells were cultured in either control media or media treated with Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), or Salmonella typhimurium (S. typh) LPS. At 0 and 24 hours supernatants were assayed for prostaglandin-E2 (PGE2) and interleukin-1 beta (Il-1 beta) release by ELISA. Using flow cytometry the density of specific monocyte surface receptors were assayed with Mo3e and LeuM3 monoclonal antibodies (mAb); T-cell CD4/CD8 ratios were assayed with OKT-3, OKT-4, and OKT-8 mAb. After 24 hours incubation with Pg or S. typh LPS, the upregulation of the Mo3e receptor was significantly decreased for refractory periodontitis patients (P < 0.05) when compared to gingivitis and to stable maintenance patients. In refractory periodontitis patients the T-cell CD4/CD8 ratio was decreased. Upon stimulation with Pg or S. typh LPS, monocytes from stable maintenance and refractory periodontitis patients released more Il-1 beta (P < 0.05) and PGE2 (P = 0.13 and 0.15) than monocytes from gingivitis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Periodontitis/inmunología , Adulto , Anciano , Análisis de Varianza , Relación CD4-CD8 , Distribución de Chi-Cuadrado , Enfermedad Crónica , Dinoprostona/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-1/metabolismo , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Receptores de Superficie Celular/inmunología , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Regulación hacia ArribaRESUMEN
Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.
Asunto(s)
Interleucina-2/biosíntesis , Linfocitos T Colaboradores-Inductores/metabolismo , Timo/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Linfocitos T CD4-Positivos/fisiología , Antígenos CD8 , Citometría de Flujo , Inmunoterapia Adoptiva , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta , Bazo/inmunología , Factores de TiempoRESUMEN
Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.
Asunto(s)
Interleucina-2/biosíntesis , Mitógenos/farmacología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/fisiología , Complejo CD3 , Calcio , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnicas In Vitro , Interleucina-2/fisiología , Ionomicina/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa C , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta , Bazo/inmunología , Acetato de Tetradecanoilforbol/farmacología , Timo/inmunologíaRESUMEN
Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.
Asunto(s)
Comunicación Celular/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos Ly/biosíntesis , Complejo CD3 , Linfocitos T CD4-Positivos/inmunología , División Celular/inmunología , Citotoxicidad Inmunológica , Interleucina-2/biosíntesis , Interleucina-2/fisiología , Isoantígenos/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/fisiología , Células Madre/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaAsunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Linfocitos T Citotóxicos/inmunología , Alelos , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/aislamiento & purificación , Antígenos Ly/fisiología , Antígenos CD8 , División Celular/inmunología , Línea Celular , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Ratones , MutaciónRESUMEN
A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T , Interleucina-2/biosíntesis , Activación de Linfocitos , Mitógenos , Linfocitos T/clasificación , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Complejo CD3 , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Fenotipo , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Interleucina-2/biosíntesis , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/anomalías , Timo/inmunologíaRESUMEN
A comparative study of the phenotype and immune functions of highly purified CD8+CD4- T cells obtained from the spleen and thymus of normal mice and from the spleen of athymic nude mice was conducted. Of seven individual normal and nude mice examined, the range of V beta 8+ cells among CD8+ T cells was a heterogeneous 4.3 to 30.5% for athymic nude mice and a much more uniform spread from 14.7 to 18.5% for normal mice. In six of the seven nude mice examined, the fraction of V beta 8+ cells was below the lower limit of the V beta 8 distribution in normal mice. However, one of the seven nude mice contained nearly twice the percentage of normal V beta 8+ cells. A reduction in the density of V beta 8 as well as CD3 Ag expression was also observed in athymic CD8+CD4- cells although an Ly-6-linked Ag, B4B2 displayed a highly increased expression. Considering the battery of Ag analyzed in entirety, athymic CD8+CD4- T cells were clearly distinct from their "counterpart" CD8+CD4- T cells isolated from either thymus or spleen of normal (euthymic) mice. Anti-CD3-mediated triggering of the TCR:CD3 complex caused extensive clonal proliferation in cultures to which single responding CD8+ T cells had been deposited. Under identical conditions, however, anti-CD3 caused little, if any clonal expansion in CD8+ cells from athymic nude mice. Highly purified athymic CD8+CD4- cells produced readily detectable IL-2R expression and IL-2 synthesis and secretion upon stimulation by anti-CD3 and by Con A. Production of IL-2 by purified athymic CD8+CD4- cells was due to CD8+CD4- cells and not due to a minor population of contaminating CD8- cells as anti-CD8 + C treatment completely abrogated the ability of athymic CD8+CD4- cells to produce IL-2. Despite IL-2 production and IL-2R expression by athymic nude CD8+CD4- T cells in response to anti-CD3 and to Con A, an impaired proliferative response followed.
Asunto(s)
Antígenos de Diferenciación , Células Clonales/inmunología , Ratones Desnudos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/fisiología , Antígenos de Diferenciación/inmunología , División Celular , Separación Celular , Células Clonales/clasificación , Concanavalina A/farmacología , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Fenotipo , Receptores Inmunológicos/biosíntesis , Receptores de Interleucina-2 , Bazo/citología , Linfocitos T/clasificación , Linfocitos T/metabolismo , Timo/citologíaAsunto(s)
Rechazo de Injerto , Linfocitos/inmunología , Cavidad Peritoneal/citología , Adenosina Desaminasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Citometría de Flujo , Riñón/patología , Trasplante de Riñón , Linfocitos/fisiopatología , Ratones , Bazo/inmunologíaRESUMEN
MRL-lpr mice and MRL-+/+ mice are identical except for the presence of an autosomal recessive lymphoproliferation gene (designated lpr) in the former. Mice bearing the lpr gene develop autoimmune and lymphoproliferative abnormalities. An antigenic marker designated 14D10, characteristically expressed on the surface of Lyt-2+ T cells and B cells of normal mice, is expressed in unusually high levels on Thy-1+ cells of lpr mice which are Lyt-2-. In younger lpr animals, 14D10+ cells are a minor subpopulation of Ia+ cells which, when expanded in diseased animals, continue to express Ia. 14D10+ cells from lpr mice are elevated in fetal spleen and adult bone marrow (BM) but are absent on pre-B cells in the BM. Medullary thymocytes of lpr mice are enriched in 14D10+ cells compared to congenic (+/+) controls. Although 14D10 appears to be present on the activated, proliferating T-cell population, coculture of lpr cells with IL-2 leads to minimal proliferation. 14D10+ Lyt-2- T cells can be isolated from normal spleens, indicating the lpr gene may be responsible for the disregulated proliferation of a minor cell subset. The functional significance of this molecular complex is still undetermined.