RESUMEN

Cryopreservation of human T lymphocytes has become a key strategy for supporting cell-based immunotherapy. However, the effects of ice seeding on the cryopreservation of cells under relatively slow cooling have not been well researched. The cryopreservation strategy with a nontoxic, single-ingredient, and injectable cryoprotective solution remains to be developed. We conducted ice seeding for the cells in a solution of normal saline with 1% (v/v) dimethyl sulfoxide (Me2SO), 0.1 M trehalose, and 4% (w/v) human serum albumin (HSA) under different slow cooling rates. With the positive results, we further applied seeding in the solution of 0.2 M trehalose and 4% (w/v) HSA under the same cooling rates. The optimal concentration of trehalose in the Me2SO-free solutions was then investigated under the optimized cooling rate with seeding, with control groups without seeding, and in a freezing container. In vitro toxicity of the cryoprotective solutions to the cells was also tested. We found that the relative viability of cells (1% [v/v] Me2SO, 0.1 M trehalose and 4% [w/v] HSA) was improved significantly from 88.6% to 94.1% with ice seeding, compared with that without seeding (p < 0.05). The relative viability of cells (0.2 M trehalose and 4% [w/v] HSA) with seeding was significantly higher than that without seeding, 96.3% and 92.0%, respectively (p < 0.05). With no significant difference in relative viability between the solutions of 0.2 M trehalose or 0.3 M trehalose with 4% (w/v) HSA (92.4% and 94.6%, respectively, p > 0.05), the solution of 0.2 M trehalose and 4% (w/v) HSA was selected as the optimized Me2SO-free solution. This strategy could cryopreserve human T lymphocytes without any toxic cryoprotectant and boost the application of cell products in humans by intravenous injection, with the osmolality of the low-concentration cryoprotective solution close to that of human plasma.

Asunto(s)

Hielo , Trehalosa , Humanos , Trehalosa/farmacología , Linfocitos T , Crioprotectores/farmacología , Criopreservación/métodos , Dimetilsulfóxido/farmacología , Supervivencia Celular

RESUMEN

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.

Asunto(s)

Criopreservación , Hielo , Criopreservación/métodos , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Congelación , Humanos , Linfocitos T

RESUMEN

IFN-ß treatment is a commonly used therapy for relapsing-remitting multiple sclerosis (MS), while vitamin D deficiency correlates with an increased risk of MS and/or its activity. MS is a demyelinating chronic inflammatory disease of the central nervous system, in which activated T lymphocytes play a major role, and may represent direct targets of IFN-ß and vitamin D activities. However, the underlying mechanism of action of vitamin D and IFN-ß, alone or in combination, remains incompletely understood, especially when considering their direct effects on the ability of T lymphocytes to produce inflammatory cytokines. We profiled the expression of immune-related genes and microRNAs in primary human T lymphocytes in response to vitamin D and IFN-ß, and we dissected the impact of these treatments on cytokine production and T cell proliferation. We found that the treatments influenced primarily memory T cell plasticity, rather than polarization toward a stable phenotype. Moreover, our data revealed extensive reprogramming of the transcriptional output of primary T cells in response to vitamin D and IFN-ß and provide the bases for further mechanistic insights into these commonly used treatments.

Asunto(s)

Interferón beta/farmacología , Linfocitos T/efectos de los fármacos , Vitamina D/farmacología , Vitaminas/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/genética , Esclerosis Múltiple Recurrente-Remitente/inmunología , Linfocitos T/inmunología

RESUMEN

BAY 41-2272 increases guanosine 3', 5'-cyclic monophosphate (cGMP) levels by stimulating soluble guanylate cyclase (sGC). In this study, we evaluated the effect of BAY 41-2272 on human T lymphocyte functions. Pretreating T cells for 24 h with BAY 41-2272 at 3 µM and 30 µM, followed by activation with 90 nM phorbol myristate acetate (PMA), inhibited interferon-gamma (IFN-γ) production, with 3 µM and 30 µM BAY causing 16.5-fold and 12.1-fold inhibition, respectively, compared to PMA alone (p < 0.05, one-way ANOVA followed by Tukey's test). We also observed suppressive effects on the expression of CD69, with 30 µM BAY causing 3.55-fold lower expression than PMA/ionomycin (p < 0.001 one-way ANOVA followed by Tukey's test), and T-bet, with 30 µM BAY causing 1.47-fold lower expression than PMA/ionomycin (p < 0.05, one-way ANOVA test followed by Tukey's test). Additionally, T lymphocyte proliferation was reduced 2.13-fold and 4.3-fold, respectively, by 3 µM BAY and 30 µM BAY compared to PMA/ionomycin (p < 0.01, p < 0.001, one-way ANOVA followed by Tukey's test). BAY 41-2272 inhibits human T lymphocyte function and may be explored as an immunomodulatory drug in patients with autoimmune/inflammatory diseases and lymphoproliferative syndromes.

Asunto(s)

Pirazoles/farmacología , Piridinas/farmacología , Linfocitos T/efectos de los fármacos , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Proliferación Celular/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Humanos , Factores Inmunológicos/metabolismo , Interferón gamma/metabolismo , Ionomicina/farmacología , Lectinas Tipo C/metabolismo , Linfocitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate-poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of the bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (STR; 1-L scale). Two proprietary T lymphocyte culture media as well as a standard RPMI-based medium were used. As before, encapsulation coincided with the presence of only a low fraction of activated T lymphocytes (peripheral blood T cells) in the total population. Unexpectedly, growth rates were lower in well-mixed reactors than those in cultivations under static conditions, that is, in T-flasks. Switching the STR to low oxygen conditions (40% air saturation) improved the growth rates to the level of the static cultures and thus forms the potential basis for efficient scale-up of T lymphocyte expansion.

Asunto(s)

Reactores Biológicos , Técnicas de Cultivo de Célula , Proliferación Celular , Células Inmovilizadas/metabolismo , Linfocitos T/metabolismo , Células Inmovilizadas/citología , Medios de Cultivo/química , Humanos , Linfocitos T/citología

RESUMEN

CD8+ T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8+ T cells change throughout the human lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8+ T cells across anatomical sites and immunological phases of human life. We used ex vivo peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRß) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M158-66 (A2+M158). We dissected memory TCR repertoires directed toward A2+M158 CD8+ T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8+ T cell repertoires to A2+M158 CD8+ TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first ex vivo comparative analysis of paired antigen-specific TCR-α/ß clonotypes across different tissues and peripheral blood across different age groups. We show that human A2+M158 CD8+ T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2+M158 TCRαß repertoires reflect A2+M158 TCRαß clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2+M158 TCRαß repertoires displayed distinct features only in elderly adults, with large private TCRαß clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαß repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαß signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies.

RESUMEN

Indole-3-carbinol (I3C) and its dimer diindolylmethane (DIM) are bioactive metabolites of a glucosinolate, glucobrassicin, found in cruciferous vegetables. Both I3C and DIM have been reported to possess pro-apoptotic, anti-proliferative and anti-carcinogenic properties via modulation of immune pathways. However, results from these studies remain inconclusive since they lack thorough evaluation of these bioactives' physiological versus pharmacological effects. In the present study, we investigated I3C and DIM's dose-dependent effects on cytokines production in human T lymphocytes Jurkat cell line (Clone E6-1). The results showed that I3C and DIM pretreatment, at higher concentrations of 50 and 10 µM, respectively, significantly increased PMA/ionomycin-induced interleukin-2 (IL-2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) production, measured by real time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). As a plausible mechanism underlying such pronounced cytokine release, we found robust increase in downstream nuclear factor κB (NF-κB) and nuclear factor of activated T-cells 1 (NFAT1) signaling with I3C pretreatment, whereas DIM pretreatment only significantly induced NF-κB activation, but not NFAT1. We hypothesize that I3C/DIM pretreatment primes the T cells to become hyperresponsive upon PMA/ionomycin stimulation which in turn differentially induces two major downstream Ca2+-dependent inflammatory pathways, NF-κB and NFAT1. Our data show novel insights into the mechanisms underlying induction of pro-inflammatory cytokine release by pharmacological concentrations of I3C and DIM, an effect negligible under physiological conditions.

Asunto(s)

Antineoplásicos/farmacología , Indoles/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Humanos , Células Jurkat , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo

RESUMEN

The ex vivo expansion of primary human T cells is of considerable interest. Current protocols call for the addition of massive amounts of stimuli. This study presents as alternative the expansion of such cells in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (SCS/PDADMAC) polyelectrolyte microcapsules, which supports at least six cell divisions and results in >40 × 106 cells mLcapsule-1 within less than 10 d. Inside the microcapsules, the T cells are suspended in a viscous SCS-solution. The low molecular weight cut off (<10 000 Da) of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained, while nutrients and metabolites can pass. Expensive additives, such as interleukin-2 (IL-2), can be coencapsulated. Expansion then no longer requires specialized T-cell media. Moreover, these results suggest that an SCS with a low degree of sulfation has biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate.

Asunto(s)

Materiales Biomiméticos/química , Cápsulas/química , Proliferación Celular , Microambiente Celular , Linfocitos T/efectos de los fármacos , Materiales Biomiméticos/farmacología , Biomimética/métodos , Celulosa/análogos & derivados , Humanos , Polietilenos , Compuestos de Amonio Cuaternario , Linfocitos T/fisiología

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a]P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues.

Asunto(s)

Benzo(a)pireno/toxicidad , Daño del ADN/efectos de los fármacos , Mutagénesis/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Células Cultivadas , Daño del ADN/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Mutagénesis/fisiología , Pruebas de Mutagenicidad/métodos , Linfocitos T/metabolismo

RESUMEN

Although it is generally believed that nicotine accounts for the beneficial effect of smoking on ulcerative colitis, the underlying mechanisms remain not well understood. Our previous finding that nicotine inhibits inflammatory responses through inducing miR-124 prompted us to ask whether the miRNA is involved in the protective action of nicotine against UC. Our present study found that miR-124 expression is upregulated in colon tissues from UC patients and DSS colitis mice. Nicotine treatment further augmented miR-124 expression in lymphocytes isolated from human ulcerative colonic mucosa and ulcerative colon tissues from DSS mice, both in infiltrated lymphocytes and epithelial cells. Moreover, knockdown of miR-124 significantly diminished the beneficial effect of nicotine on murine colitis and IL-6-treated Caco-2 colon epithelial cells. Further analysis indicated that nicotine inhibited STAT3 activation in vivo and in IL-6 treated Caco-2 cells and Jurkat human T lymphocytes, in which miR-124 knockdown led to increased activation of STAT3. Blocking STAT3 activity alone is beneficial for DSS colitis and also abolished nicotine's protective effect in this model. These data indicate that nicotine exerts its protective action in UC through inducing miR-124 and inhibiting STAT3, and suggest that the miR-124/STAT3 system is a potential target for the therapeutic intervention of UC. KEY MESSAGE: Nicotine upregulates miR-124 expression in ulcerative colon tissues and cells. MiR-124 is required for the protective role of nicotine in DSS colitis mice and epithelial cells. The protective effect of nicotine in murine DSS colitis depends on blocking STAT3 activation. MiR-124 mediates the inhibitory role of nicotine on STAT3/p-STAT3. Targeting miR-124 and STAT3 represents a novel approach for treating ulcerative colitis.

Asunto(s)

Colitis Ulcerosa/tratamiento farmacológico , Colon/efectos de los fármacos , MicroARNs/metabolismo , Nicotina/farmacología , Factor de Transcripción STAT3/metabolismo , Animales , Células CACO-2 , Colitis Ulcerosa/metabolismo , Colon/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Nicotina/administración & dosificación , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Estadísticas no Paramétricas , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

OBJECTIVE Although it is generally believed that nicotine accounts for the beneficial effect of smoking on ulcerative colitis, the underlying mechanisms remain not well- understood. Our previous finding that nicotine inhibits inflammatory responses through inducing miRNA-124 prompted us to ask whether the miRNA is involved in the protective action of nicotine on UC. METHODS MiR-124 expression in colon tissues and cells was determined by q-PCR and in situ hybridization. The effect of miR-124 on protective role of nicotine in ulcerative colitis was evaluated in DSS-treated mice and IL-6-treated Caco-2 colon epithelial cells. Expression of p-STAT3/STAT3 was detected by immuno?histochemistry and Western blot analysis. RESULTS miR- 124 expression is upregulated in colon tissues from patients and DSS- induced colitis. Nicotine treatment further elevated miR- 124 level in colon tissues of the mice, in infiltrated lymphocytes and epithelial cells, and augmented miR- 124 expression in lymphocytes isolated from human ulcerative colon tissues. Administration of nicotine also reduced weight loss, improved DAI and decreased HE score in DSS-induced colitis. Moreover, knock?down of miR-124 in vivo significantly diminished the beneficial effect of nicotine, and in vitro on IL-6-treated Caco-2 colon epithelial cells. Further analysis indicated that nicotine inhibited STAT3 activation in vivo and in IL-6-treated Caco-2 colon epithelial cells and Jurkat human T lymphocytes, in which miR-124 knockdown led to increased activation of STAT3. CONCLUSION These data indicated that nicotine exerts its protective action in UC through inducing miR-124 and its effect on STAT3, suggesting that the miR-124/STAT3 system is a potential target for the therapeutic intervention of UC.

RESUMEN

Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1→3)(1→6)-ß-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H2DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H2O2. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion.

Asunto(s)

Anticarcinógenos/farmacología , Glucanos/farmacología , Linfocitos/efectos de los fármacos , Células Cultivadas , Humanos , Células Jurkat , Linfocitos/metabolismo , Metilmetanosulfonato/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética

RESUMEN

The differentiation of human CD4(+) T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel-based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC-MS/MS is required to provide a reference dataset for proteome-based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 (http://proteomecentral.proteomexchange.org/dataset/PXD001066).

Asunto(s)

Diferenciación Celular , Proteoma/metabolismo , Células TH1/metabolismo , Células Cultivadas , Cromatografía Liquida , Humanos , Proteoma/análisis , Proteómica , Espectrometría de Masas en Tándem , Células TH1/citología , Ubiquitinación

RESUMEN

The use of iron oxide nanoparticles (ION) for diagnostic and therapeutic purposes requires a clear favorable risk-benefit ratio. This work was performed with the aim of studying the ability of polyacrylic acid (PAA)-coated and non-coated ION to induce genotoxicity in human T lymphocytes. For that purpose, their influence on cell cycle progression and on the induction of chromosome aberrations was evaluated. Blood samples collected from healthy human donors were exposed to PAA-coated and non-coated ION, at different concentrations, for 48h. The obtained results showed that, for all culture conditions, the tested ION are not genotoxic and do not influence the cell cycle arrest. Their possible cumulative effect with the iron-dependent genotoxic agent BLM was also evaluated. Blood samples collected from healthy human donors were exposed to ION, at different concentrations, for 48h, in the presence of a pre-determined toxic concentration of BLM. The obtained results showed that, for all culture conditions, the tested ION do not potentiate the clastogenic effects of BLM.

Asunto(s)

Resinas Acrílicas/toxicidad , Compuestos Férricos/toxicidad , Compuestos Ferrosos/toxicidad , Nanopartículas del Metal , Linfocitos T/efectos de los fármacos , Bleomicina/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Cultivadas , Aberraciones Cromosómicas/inducido químicamente , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Mutagenicidad , Medición de Riesgo , Linfocitos T/patología , Factores de Tiempo

RESUMEN

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates immunosuppression caused by a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons or dioxins. Recent evidence suggests that AhR plays an important role in T-cell-mediated immune responses by affecting the polarization and differentiation of activated T cells. However, the regulation of AhR expression in activated T cells remains poorly characterized. In the present study, we used purified human T cells stimulated with anti-CD3 and anti-CD28 Abs to investigate the effect of T-cell activation on AhR mRNA and protein expression. The expression of AhR mRNA increased significantly and rapidly after T-cell activation, identifying AhR as an immediate-early activation gene. AhR upregulation occurred in all of the T-cell subtypes, and is associated with its nuclear translocation and induction of the cytochromes P-450 1A1 and 1B1 mRNA expression in the absence of exogenous signals. In addition, the use of an AhR antagonist or siRNA-mediated AhR knockdown significantly inhibited IL-22 expression, suggesting that expression and functional activation of AhR is necessary for the secretion of IL-22 by activated T cells. In conclusion, our data support the idea that AhR is a major player in T-cell physiology.

Asunto(s)

Núcleo Celular/inmunología , Activación de Linfocitos/fisiología , Receptores de Hidrocarburo de Aril/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunología , Transporte Activo de Núcleo Celular/fisiología , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/inmunología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/inmunología , Citocromo P-450 CYP1B1 , Técnicas de Silenciamiento del Gen , Humanos , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/inmunología , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/genética , Linfocitos T/citología , Linfocitos T/metabolismo , Regulación hacia Arriba/genética , Interleucina-22

RESUMEN

Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαß. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.

RESUMEN

Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses. HTLV-1 causes adult T cell leukemia and lymphoma, whereas HTLV-2 infection is not etiologically linked to human disease. The viral genomes of HTLV-1 and -2 encode highly homologous transforming proteins, Tax-1 and Tax-2, respectively. Tax-1 is thought to play a central role in transforming CD4+ T lymphocytes. Expression of Tax-1 is crucial for promoting survival and proliferation of virally infected human T lymphocytes and is necessary for initiating HTLV-1-mediated oncogenesis. In transgenic mice and humanized mouse model, Tax-1 has proven to be leukemogenic. Although Tax-1 is able to efficiently transform rodent fibroblasts and to induce lymphoma in mouse model, it rarely transforms primary human CD4+ T lymphocytes. In contrast, Tax-2 efficiently immortalizes human CD4+ T cells though it exhibits a lower transforming activity in rodent cells as compared to Tax-1. We here discuss our recent observation and views on the differential transforming activity of Tax-1 and Tax-2 in human T cells.