RESUMEN
Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Antígeno B7-H1/efectos de los fármacos , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoterapia/métodos , Activación de Linfocitos/genética , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/efectos de los fármacos , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: The nature and extent of inflammation seen in multiple sclerosis (MS) varies throughout the course of the disease. Changes seen in CD4+ T-helper cells in relapsing-remitting (RR) MS and secondary progressive (SP) MS might differ qualitatively and/or quantitatively. OBJECTIVE: The objective of this paper is to study the frequencies of all major CD4+ T-helper subtypes - Th17, Th22 and Th1 lineage cells - in relapse, remission and secondary progression alongside CCR6 status, a chemokine receptor involved in migration of these cells into the central nervous system. METHODS: We compared 100 patients (50 RRMS and 50 SPMS) and 50 healthy volunteers and performed flow cytometric analysis of lymphocytes in blood samples. RESULTS: We demonstrated raised frequencies of various cell types along the Th17 axis; Th17, Th17.1 (IL-17+ interferon gamma+) and dual IL-17+ IL-22+ cells in RRMS. Th22 and CCR6+ Th1 cells (nonclassical Th1) were also increased in RRMS. All these cells were CCR6+. Only Th17 frequencies were elevated in SPMS. CONCLUSIONS: Increased frequencies of Th17 cells are implicated both in RRMS and SPMS. The CCR6 pathway includes Th17, Th22 and Th1 nonclassical cells, of which Th22 and Th1 cells represent the greatest subsets in MS.
RESUMEN
OBJECTIVE: To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts. METHODS: CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS (RRMS; n=50), primary progressive MS (PPMS; n=20) and other neurological disease controls, both inflammatory (ONID; n=23) and non-inflammatory (OND; n=114). CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available. RESULTS: There was an increased median CSF κ:λ free light chain (FLC) in all MS groups (CIS: 18.2, 95% CI 6.8 to 30.3; RRMS: 4.4, 95% CI 2.7 to 11.4; PPMS: 12.0, 95% CI 3.6 to 37.1) but not controls (OND: 1.61, 95% CI 1.4 to 1.9; ONID: 1.7, 95% CI 1.3 to 2.2; p<0.001). This ratio predicted Expanded Disability Status Scores (EDSS) progression at 5 years, with a lower median EDSS in the group with high (>10) CSF κ:λ FLC (0.0, 95% CI 0 to 2.5 vs 2.5, 95% CI 0 to 4, high vs low; p=0.049). CSF κ:λ FLC correlated with CSF IgG1 κ:λ (r=0.776; p<0.0001) and was intrinsic to CSF plasmablasts (r=0.65; p=0.026). CONCLUSIONS: These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification.
Asunto(s)
Cadenas Ligeras de Inmunoglobulina/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/líquido cefalorraquídeo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Adulto JovenRESUMEN
A number of members of the G protein-coupled receptor class of cell surface receptors are 'orphans' with no known endogenous ligand. One of these orphan receptors is GPR61; there are little data about its expression in human cells and tissues. In this study, we investigated the post-translational modification of GPR61 by N-glycosylation at an identified consensus N-glycosylation site (N12) and the impact of this modification upon the subcellular expression of the protein. The N-glycosylation inhibitor tunicamycin reduced the apparent molecular weight of immunoreactivity associated with myc-tagged GPR61 by 1-2 kDa, which was comparable to the evident molecular weight of the myc-tagged N12S GPR61 mutant with disrupted consensus N-glycosylation site. Analysis of GPR61 expression demonstrated that tunicamycin treatment reduced considerably heterologous expression of GPR61 in the cell membrane despite the N12S GPR61 mutant being readily expressed at the cell surface. These results demonstrate that GPR61 is subject to N-glycosylation but suggest this is not a prerequisite for cell surface expression, although N-glycosylation of other proteins may be important for cell membrane expression of GPR61. Expression of GPR61 protein was demonstrated at the cellular level in human hippocampus and human peripheral blood mononuclear cells. In the latter, there was a significantly higher expression of GPR61 in the Th17 cell subset in comparison with resting CD4+ cells, which may point toward a potential role for the GPR61 receptor in autoimmune diseases. This is the first report that GPR61 protein is subject to post-translational modification and is expressed in immune cell subsets and the hippocampus. These findings will help guide studies to investigate the function of GPR61.
RESUMEN
PURPOSE: Ocular mucous membrane pemphigoid (OcMMP) is a rare autoimmune disorder resulting in progressive conjunctival fibrosis and ocular surface failure leading to sight loss in up to 50%. This study was designed to optimize an ocular surface sampling technique for identification of novel biomarkers associated with disease activity and/or progressive fibrosis. METHODS: Fifty-seven patients with OcMMP underwent detailed examination of conjunctival inflammation and fibrosis using fornix depth measurement. Ocular surface impression cytology (OSIC) to sample superior bulbar conjunctiva combined with flow cytometry (OSIC-flow) profiled infiltrating leukocytes. Profiles were compared with healthy controls (HC) and disease controls (primary Sjögren's syndrome, pSS). Thirty-five OcMMP patients were followed every 3 months for 12 months. RESULTS: Overall neutrophils were elevated in OcMMP eyes when compared to pSS or HC (109 [18%] neutrophils/impression [NPI]; 2 [0.2%]; 6 [0.8%], respectively [P < 0.0001]) and in OcMMP patients with no visible inflammation when compared with HC (44.3 [7.9%]; 5.8 [0.8%]; P < 0.05). At 12 months follow-up, 53% of OcMMP eyes progressed, and this was associated with baseline conjunctival neutrophilia (P = 0.004). As a potential biomarker, a value of 44 NPI had sensitivity, specificity, and positive predictive values of 75%, 70%, and 73%, respectively. Notably, eyes with no visible inflammation and raised conjunctival neutrophils were more likely to progress and have a greater degree of conjunctival shrinkage compared to those without raised neutrophils. CONCLUSIONS: These data suggest that OSIC-flow cytometric analyses may facilitate repeated patient sampling. Neutrophils may act as a biomarker for monitoring disease activity, progressive fibrosis, and response to therapy in OcMMP even when the eye appears clinically uninflamed.
Asunto(s)
Cicatriz/patología , Conjuntiva/patología , Membrana Mucosa/patología , Neutrófilos/patología , Penfigoide Benigno de la Membrana Mucosa/diagnóstico , Anciano , Anciano de 80 o más Años , Cicatriz/etiología , Progresión de la Enfermedad , Femenino , Fibrosis/patología , Citometría de Flujo , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Penfigoide Benigno de la Membrana Mucosa/complicaciones , Factores de TiempoRESUMEN
Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors. 1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100nM) caused the death of approximately 40% DOHH2 cells after 24h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.
Asunto(s)
Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Calcitriol/farmacología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Vitamina D/análogos & derivados , Antígenos CD19/metabolismo , Apoptosis , Linfocitos B/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Células HL-60 , Voluntarios Sanos , Humanos , Leucocitos Mononucleares/citología , Factores de TiempoRESUMEN
BACKGROUND: Transforming growth factor ß (TGFß) is an important immunoregulatory cytokine in regulatory T cell (Treg) and Th17-mediated pathology, including uveitis due to Behçet's disease (BD). Of the three isoforms, TGFß2 is found at highest levels in the aqueous humour of uninflamed eyes. TGFß signals through a cell-surface receptor comprising three subunits (TGFBR1, 2 and 3). TGFBR3 is considered necessary for TGFß2 signal transduction, but not for other isoforms. A polymorphism in TGFBR3 (rs1805110) has previously been identified in Han Chinese patients with BD. We investigated the frequency of this polymorphism in a Caucasian population with BD and idiopathic intermediate uveitis (IIU). METHODS: The single-nucleotide polymorphism (SNP) rs1805110 in TGFBR3 was genotyped in 75 BD patients, 92 IIU disease controls and 85 disease-free controls. The association with both diseases was analysed using Fisher's exact test. RESULTS: No significant difference in rs1805110 allele or genotype frequency was observed. A low frequency of the T allele was observed (5.88% control, 9.33% BD, 10.33% IIU) with the TT genotype absent in patients with BD and IIU (1.18% control, 0% BD and 0% IIU). Stratification analysis according to clinical features of BD did not associate with the tested SNP. CONCLUSIONS: RS1805110 is not associated with BD or IIU in Caucasian patients. The T allele frequency is consistent with that presented for Caucasian populations in the HapMap database (p>0.05). Our results differ from the previous analysis in Han Chinese patients (p<0.0001), however, the possibility of having a much smaller effect due to the low minority frequency cannot be excluded.
Asunto(s)
Síndrome de Behçet/genética , Polimorfismo de Nucleótido Simple , Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Uveítis Intermedia/genética , Población Blanca/genética , Síndrome de Behçet/diagnóstico , Femenino , Frecuencia de los Genes , Genotipo , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Uveítis Intermedia/diagnósticoRESUMEN
Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (ß)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/inmunología , Células Madre/inmunología , Adulto , Amnios/citología , Células Cultivadas , Células Epiteliales/inmunología , Femenino , Humanos , Hipogravedad , Inmunomodulación , Trasplante de Islotes Pancreáticos/inmunología , Rotación , Trasplante Homólogo/métodosRESUMEN
PURPOSE: Conjunctival epithelial T cells are dominated by CD3(+)CD56-TCRαß(+)CD8αß(+) lymphocytes. In this study we explored the antigen experience status, mucosal homing phenotype, cytokine expression, and viral antigen recognition of conjunctival epithelial CD8(+) T cells from healthy individuals. METHODS: Following ocular surface impression cytology, conjunctival cells were recovered by gentle agitation and analyzed by flow cytometry for cell surface markers, cytokine production (stimulated by phorbol 12-myristate 13-acetate [PMA]/ionomycin), and Epstein-Barr virus (EBV)/cytomegalovirus (CMV) immunodominant epitope recognition using major histocompatibility complex (MHC) class I peptide tetramers. RESULTS: In contrast to peripheral blood, conjunctival epithelial CD8(+) T cells were dominantly CD45RA(-)CCR7(-) effector memory cells, and the vast majority expressed the mucosal homing integrin αEß7. Conjunctival memory CD8(+) T cells maintained effector functions with the ability to secrete IFN-γ and expression of Granzyme B, although they expressed significantly reduced amounts per cell compared to peripheral blood T cells. Interestingly, herpetic virus-specific CD8(+) T cells recognizing epitopes derived from EBV and CMV could be detected in the conjunctival cells of healthy virus carriers, although they were generally at lower frequencies than in the peripheral blood of the same donor. Virus-specific conjunctival CD8(+) T cells were dominated by CD45RA(-)CCR7(-) effector memory cells that expressed αEß7. CONCLUSIONS: These data demonstrate that the majority of conjunctival epithelial CD8(+) T cells are mucosal homing αEß7(+) effector memory T cells, which can recognize viral epitopes and are capable of secreting Granzyme B and IFN-γ.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Conjuntiva/inmunología , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Superficie/metabolismo , Antígenos Virales/inmunología , Estudios de Cohortes , Infecciones por Citomegalovirus/inmunología , Epitelio/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/inmunología , Adulto JovenRESUMEN
Although the inhibitory effects of therapeutic glucocorticoids (GCs) on dendritic cells (DCs) are well established, the roles of endogenous GCs in DC homeostasis are less clear. A critical element regulating endogenous GC concentrations involves local conversion of inactive substrates to active 11-hydroxyglucocorticoids, a reduction reaction catalyzed within the endoplasmic reticulum by an enzyme complex containing 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and hexose-6-phosphate dehydrogenase (H6PDH). In this study, we found that this GC amplification pathway operates both constitutively and maximally in steady state murine DC populations and is unaffected by additional inflammatory stimuli. Under physiologic conditions, 11ßHSD1-H6PDH increases the sensitivity of plasmacytoid DCs (pDCs) to GC-induced apoptosis and restricts the survival of this population through a cell-intrinsic mechanism. Upon CpG activation, the effects of enzyme activity are overridden, with pDCs becoming resistant to GCs and fully competent to release type I interferon. CD8α(+) DCs are also highly proficient in amplifying GC levels, leading to impaired maturation following toll-like receptor-mediated signaling. Indeed, pharmacologic inhibition of 11ßHSD1 synergized with CpG to enhance specific T-cell responses following vaccination targeted to CD8α(+) DCs. In conclusion, amplification of endogenous GCs is a critical cell-autonomous mechanism for regulating the survival and functions of DCs in vivo.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/inmunología , Deshidrogenasas de Carbohidratos/inmunología , Corticosterona/análogos & derivados , Células Dendríticas/inmunología , Receptores de Glucocorticoides/inmunología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Animales , Apoptosis/efectos de los fármacos , Trasplante de Médula Ósea , Antígenos CD8/genética , Antígenos CD8/inmunología , Deshidrogenasas de Carbohidratos/genética , Células Cultivadas , Corticosterona/metabolismo , Corticosterona/farmacología , Ciclopropanos/farmacología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Guanosina/análogos & derivados , Guanosina/farmacología , Interferón Tipo I/biosíntesis , Interferón Tipo I/inmunología , Ratones , Ratones Noqueados , Receptores de Glucocorticoides/genética , Transducción de Señal , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Irradiación Corporal TotalRESUMEN
PURPOSE: Ocular complications related to Stevens-Johnson Syndrome (SJS)-Toxic Epidermal Necrolysis (TEN) may persist and progress after resolution of systemic disease. This is thought to be related in part to persistent ocular innate-immune signaling. In this study, our aim was to characterize infiltrative conjunctival cellular profiles during acute (<12 months) and chronic (>12 months) disease. METHODS: Consecutive patients presenting with SJS-TEN over a 12-month period were followed for 1 year. Detailed clinical examination and conjunctival impression cell recovery was analyzed by flow cytometry for the presence of intraepithelial leukocytes and compared with healthy controls (n = 21). RESULTS: Ten patients were recruited of whom six had acute disease and five were classified as TEN (SCORTEN = 1, n = 4). Conjunctival inflammation was graded as absent/mild in a total of nine patients; but despite this, evidence of fornix shrinkage was observed in nine subjects. This inversely correlated with disease duration (P < 0.05). A reduction in percentage of CD8αß(+) T cells compared with controls (80% vs. 57%; P < 0.01) was associated with a corresponding increase in the number/percentage of CD45(INT)CD11b(+)CD16(+)CD14(-) neutrophils (186 vs. 3.4, P < 0.01, 31% vs. 0.8%, P < 0.001). Neutrophils inversely correlated with disease duration (r = -0.71, P = 0.03), yet there was no absolute change in the CD8αß(+) or neutrophil populations during the study period (P = 1.0). CONCLUSIONS: These data highlight that a neutrophilic infiltrate is present in mildly inflamed or clinically quiescent conjunctival mucosa in patients with ocular SJS-TEN, where neutrophil numbers inversely correlate with disease duration. Neutrophil persistence endorses the hypothesis of an unresolved innate-inflammatory process that might account for disease progression.
Asunto(s)
Antígenos CD/inmunología , Conjuntiva/patología , Enfermedades de la Conjuntiva/inmunología , Neutrófilos/inmunología , Síndrome de Stevens-Johnson/inmunología , Enfermedad Aguda , Adolescente , Adulto , Enfermedad Crónica , Estudios Transversales , Epitelio/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
PURPOSE: To identify clinical, demographic, immunologic, and health-related quality-of-life data from a cohort of vernal keratoconjunctivitis (VKC) patients with the onset of the disease after puberty (VKC-like disease). DESIGN: Retrospective, observational case series. METHODS: Forty-nine patients with late-onset VKC-like disease from among 600 consecutive VKC patients. History of disease, test results for allergen sensitivity, signs and symptoms, impact of disease on work productivity, health-related quality of life, and treatment satisfaction were assessed. In addition, multiplex bead analysis for Th1/Th2 cytokines were carried out in tear samples from 20 VKC patients (10 adults and 10 children) and from 10 normal subjects. RESULTS: A family history of allergy was positive in only 28% and positive prick test results were present in 55% of the 49 VKC-like adult patients. Based on typical signs and symptoms, 48% were affected by the limbal form, 33% were affected by the tarsal form, and 19% were affected by the mixed form. Corneal ulcer complicated the disease in only 2 adult patients. Although the disease was not considered a limiting factor for work, productivity was reduced by 26% and social activities were reduced by 31% during active flare-ups. No significant differences were found in tear cytokine pattern production between VKC in children and VKC in adults. CONCLUSIONS: A late onset VKC-like disease can appear in young adults with signs and symptoms similar to those in pediatric disease, but with less corneal involvement.
Asunto(s)
Conjuntivitis Alérgica/epidemiología , Córnea/inmunología , Citocinas/metabolismo , Inmunoglobulina E/análisis , Linfocitos T/inmunología , Lágrimas/química , Adulto , Conjuntivitis Alérgica/inmunología , Córnea/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Italia/epidemiología , Masculino , Pronóstico , Calidad de Vida , Estudios Retrospectivos , Lágrimas/inmunología , Adulto JovenRESUMEN
PURPOSE: Noninfectious uveitis is characterized by a dysregulated inflammatory or immune response in the eye. It is unclear whether this represents a failure of immune privilege or an overwhelming inflammatory drive that has exceeded the capacity of regulatory mechanisms that are still functioning. The authors investigated immune regulation in the human eye during intraocular inflammation (uveitis) and its impact on dendritic cell (DC) function and subsequent T-cell responses. METHODS: Myeloid DCs were isolated from the aqueous humor (AqH) and peripheral blood of patients with active uveitis and characterized by flow cytometry. The effect of uveitis AqH was interrogated in an in vitro model of peripheral blood monocyte-derived DCs from healthy controls. RESULTS: Myeloid DCs isolated from uveitic AqH were characterized by elevated major histocompatibility complex classes I and II (MHC I/II), but reduced CD86 compared with matched peripheral blood DCs. Exposure of peripheral blood monocyte-derived DCs from healthy controls to the inflammatory AqH supernatant recapitulated this phenotype. Despite interferon gamma (IFNγ)-dependent upregulation of MHC I, inflammatory AqH was overall suppressive to DC function, with reduced CD86 expression and diminished T-cell responses. This suppressive effect was equal to or greater than that induced by noninflammatory AqH, but was glucocorticoid independent (in contrast to noninflammatory AqH). CONCLUSIONS: These data indicate that the ocular microenvironment continues to regulate DC function during uveitis, despite IFNγ-driven upregulation of MHC expression, supporting the hypothesis that immune regulation within the eye is maintained during inflammation.
Asunto(s)
Humor Acuoso/fisiología , Células Dendríticas/fisiología , Inmunidad Celular , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Uveítis/inmunología , Humor Acuoso/citología , Células Cultivadas , Células Dendríticas/inmunología , Citometría de Flujo , Humanos , Uveítis/metabolismo , Uveítis/patologíaRESUMEN
The conjunctiva is a highly specialized ocular mucosal surface that, like other mucosa, houses a number of leukocyte populations. These leukocytes have been implicated in age-related inflammatory diseases such as dry-eye, but their phenotypic characteristics remain largely undetermined. Existing literature provides rudimentary data from predominantly immunohistochemical analyses of tissue sections, prohibiting detailed and longitudinal examination of these cells in health and disease. Using recovered cells from ocular surface impression cytology and flow cytometry, we examined the frequency of leukocyte subsets in human conjunctival epithelium and how this alters with age. Of the total CD45+ leukocyte population within the conjunctival epithelium, 87% [32-99] (median) [range] comprised lymphocytes, with 69% [47-90] identified as CD3 + CD56- T cells. In contrast to peripheral blood, the dominant conjunctival epithelial population was TCRαß + CD8αß + (80% [37-100]) with only 10% [0-56%] CD4+ cells. Whilst a significant increase in the CD4+ population was seen with age (r = 0.5; p < 0.01) the CD8+ population remained unchanged, resulting in an increase in the CD4:CD8 ratio (r = 0.5;p < 0.01). IFNγ expression was detectable in 18% [14-48] of conjunctival CD4+ T cells and this was significantly higher among older individuals (<35 years, 7[4-39] vs. >65 years, 43[20-145]; p < 0.05). The elevation of CD4+ cells highlights a potentially important age-related alteration in the conjunctival intra-epithelial leukocyte population, which may account for the vulnerability of the aging ocular surface to disease.
Asunto(s)
Envejecimiento/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/análisis , Linfocitos T CD8-positivos/inmunología , Conjuntiva/inmunología , Recuento de Linfocitos , Subgrupos Linfocitarios/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Relación CD4-CD8 , Enfermedades de la Conjuntiva/inmunología , Femenino , Humanos , Inmunocompetencia/inmunología , Interferón gamma/análisis , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Valores de Referencia , Adulto JovenRESUMEN
OBJECTIVE: Interleukin-6 (IL-6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL-6 receptor (IL-6R; CD126) or via trans-signaling, in which soluble IL-6R/IL-6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL-6 in the joints of patients with rheumatoid arthritis (RA). METHODS: Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients. RESULTS: Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans-signaling by soluble IL-6R/IL-6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down-regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL-6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up-regulated locally. Among a range of cytokines tested, only IL-10 induced CD130 expression in T cells. CONCLUSION: The inflamed microenvironment in the synovial tissue maintains responsiveness to IL-6 trans-signaling through the up-regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL-10.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-6/metabolismo , Líquido Sinovial/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Receptor gp130 de Citocinas/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-6/metabolismo , Articulaciones/inmunología , Masculino , Persona de Mediana Edad , Membrana Sinovial/inmunologíaRESUMEN
Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory process of the lung inducing persistent airflow limitation. Extensive systemic effects, such as skeletal muscle dysfunction, often characterize these patients and severely limit life expectancy. Despite considerable research efforts, the molecular basis of muscle degeneration in COPD is still a matter of intense debate. In this study, we have applied a network biology approach to model the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. Our model shows that failure to co-ordinately activate expression of several tissue remodelling and bioenergetics pathways is a specific landmark of COPD diseased muscles. Our findings also suggest that this phenomenon may be linked to an abnormal expression of a number of histone modifiers, which we discovered correlate with oxygen utilization. These observations raised the interesting possibility that cell hypoxia may be a key factor driving skeletal muscle degeneration in COPD patients.
Asunto(s)
Músculo Esquelético/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Biología de Sistemas/métodos , Anciano , Animales , Hipoxia de la Célula/fisiología , Citocinas/sangre , Metabolismo Energético , Femenino , Perfilación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-1beta/farmacología , Masculino , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Consumo de Oxígeno , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
CD248 (endosialin) is a transmembrane glycoprotein that is dynamically expressed on pericytes and fibroblasts during tissue development, tumour neovascularization and inflammation. Its role in tissue remodelling is associated with increased stromal cell proliferation and migration. We show that CD248 is also uniquely expressed by human, but not mouse (C57BL/6), CD8(+) naive T cells. CD248 is found only on CD8(+) CCR7(+) CD11a(low) naive T cells and on CD8 single-positive T cells in the thymus. Transfection of the CD248 negative T-cell line MOLT-4 with CD248 cDNA surprisingly reduced cell proliferation. Knock-down of CD248 on naive CD8 T cells increased cell proliferation. These data demonstrate opposing functions for CD248 on haematopoietic (CD8(+)) versus stromal cells and suggests that CD248 helps to maintain naive CD8(+) human T cells in a quiescent state.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/inmunología , Células del Estroma/inmunología , Animales , Western Blotting , Proliferación Celular , Citometría de Flujo , Humanos , RatonesRESUMEN
Aqueous humor (AqH) has been shown to have significant immunosuppressive effects on APCs in animal models. We wanted to establish whether, in humans, AqH can regulate dendritic cell (DC) function and to identify the dominant mechanism involved. Human AqH inhibited the capacity of human peripheral blood monocyte-derived DC to induce naive CD4(+) T cell proliferation and cytokine production in vitro, associated with a reduction in DC expression of the costimulatory molecule CD86. This was seen both for DC cultured under noninflammatory conditions (immature DC) and for DC stimulated by proinflammatory cytokines (mature DC). DC expression of MHC classes I/II and CD83 was reduced (mature DC only). Myeloid DC from peripheral blood were similarly sensitive to the effects of human AqH, but only under inflammatory conditions. The addition of α-melanocyte stimulating hormone and vasoactive intestinal peptide did not cause significant inhibition at physiological levels. However, the addition of exogenous cortisol at physiological levels recapitulated the AqH-induced reduction in CD86 and inhibition of DC-induced T cell proliferation, and blockade of cortisol in AqH partially reversed its suppressive effects. TGF-ß2 had an additional effect with cortisol, and although simultaneous blockade of cortisol and TGF-ß2 in AqH reduced its effectiveness, there was still a cortisol- and TGF-ß-independent component. In humans, AqH regulates DC maturation and function by the combined actions of cortisol and TGF-ß2, a pathway that is likely to contribute to the maintenance of immune privilege in the eye.
Asunto(s)
Humor Acuoso/inmunología , Células Dendríticas/inmunología , Ojo/inmunología , Hidrocortisona/fisiología , Tolerancia Inmunológica , Factor de Crecimiento Transformador beta2/fisiología , Presentación de Antígeno/inmunología , Humor Acuoso/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ojo/metabolismo , Antígenos HLA/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Hidrocortisona/antagonistas & inhibidores , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta2/antagonistas & inhibidoresRESUMEN
BACKGROUND/AIMS: Documentation of conjunctival forniceal foreshortening in cases of progressive cicatrising conjunctivitis (PCC) is important in ascertaining disease stage and progression. Lower fornix shortening is often documented subjectively or semi-objectively, whereas upper forniceal obliteration is seldom quantified. Although tools such as fornix depth measurers (FDMs) have been described, their designs limit upper fornix measurement. The purpose of this study was to custom-design a FDM to evaluate the upper fornix and to assess variability in gauging fornix depth. METHODS: A polymethylmethacrylate FDM was constructed using industry-standard jewellery computer software and machinery. Two observers undertook a prospective independent evaluation of central lower fornix depth in a heterogeneous cohort of patients with clinically normal and abnormal conjunctival fornices both subjectively and by using the FDM (in mm). Upper central fornix depth was also measured. Agreement was assessed using Bland-Altman plots. RESULTS: Fifty-one eyes were evaluated. There was 100% intraobserver agreement to within 1 mm for each observer for lower fornix measurement. The mean difference in fornix depth loss using the FDM between observer 1 and 2 was 1.19%, with 95% confidence of agreement (±2SD) of -15% to +20%. In total, 86% (44/51) of measurements taken by the two observers agreed to within 10% of total lower fornix depth (ie, ±1 mm) versus only 63% (32/51) of the subjective measurements. Mean upper fornix difference was 0.57 mm, with 95% confidence of agreement of between -2 and +3 mm. CONCLUSIONS: This custom-designed FDM is well tolerated by patients and shows low intraobserver and interobserver variability. This enables repeatable and reproducible measurement of upper and lower fornix depths, facilitating improved rates of detection and better monitoring of progression of conjunctival scarring.