Characterization of migratory activity and cytokine profile of helper and cytotoxic CMV-specific T-cell lines expanded by a selective peptide library.

OBJECTIVE: Reconstitution of cellular immunity by infusion of cytomegalovirus (CMV)-specific T lymphocytes is an attractive alternative to drugs currently used to control CMV reactivation in immunocompromised patients. For this purpose, we established a method for generating both anti-CMV CD4 and CD8 T cells following Good Manufacturing Practice indications, and we extensively characterized their immune functions. MATERIALS AND METHODS: For generating CD4 and CD8 CMV-specific lymphocytes, T cells from 11 CMV-seropositive donors were stimulated three times with dendritic cells (DC) pulsed with a library of selected CMV peptides, recognized by >85% of the Caucasian population. At the end of the culture, T cells were analyzed for their specificity, cytotoxicity, chemotactic migration, proliferation, and cytokine production. RESULTS: T cells were successfully expanded and enriched in CMV-specific subsets with an effector memory or an effector memory CD45 RA(+) phenotype. CMV-specific T-cell lines showed specific cytotoxicity (average lysis: 47%) against CMV peptides-pulsed DCs, and were depleted of auto- and alloreactivity. Moreover, the ability to proliferate following antigenic stimulation and the presence of functional CD4 lymphocytes producing Th1 and Th2 cytokines can ensure long-term antiviral immunity after in vivo injection. CMV-specific T lymphocytes also proved to be fully equipped to reach CMV-infected tissues, because they expressed CD49d and CCR1, CXCR3, CXCR4, necessary to recruit effector cells to inflamed sites. In accordance with this profile, they significantly migrated towards inflammatory chemokines and towards the supernatant collected from inflamed lung fibroblasts, frequently involved in CMV pathology. CONCLUSION: This strategy allows expansion of effector T cells capable to exert CD8 and CD4-mediated immune functions and, thus, is suitable for clinical use.

Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory T-cells in patients with graft-versus-host disease.

BACKGROUND: Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation. METHODS: We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n=4) or chronic (n=6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IL)-10 mRNA. RESULTS: ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P<0.05), with a simultaneous increase of glucocorticoid induced tumor necrosis factor receptor expression on CD4+CD25+ cells (from 15% to 40.8%, P<0.05). This increase was sustained after 12 procedures. T-regs expressed high levels of CD62L, CD45RO, and Foxp3. Sorted CD4+CD25+ T-regs were potently inhibitory toward the CD4+CD25- fraction, when matched with an allogeneic target (IFN-gamma secretion was reduced by 79%). Trans-well experiments showed that cell-to-cell contact was necessary to exert inhibitory activity. RQ-PCR revealed a significant expression of Foxp3 in CD4+CD25+ T-regs, but there was virtually no detection of TGF-beta and IL-10. GvHD improved in all patients, allowing tapering or discontinuation of immunosuppressive drugs. CONCLUSION: Our study shows a time correlation between ECP and increasing percentages of circulating functional T-regs. Albeit suggestive, our results need to be confirmed on larger series to determine the actual role of T-reg in mediating the clinical effect of ECP.

Chronic reboxetine desensitizes terminal but not somatodendritic alpha2-adrenoceptors controlling noradrenaline release in the rat dorsal hippocampus.

The slow onset of antidepressant drugs' effects is thought to reflect the time required for the development of adaptive changes such as desensitization of presynaptic autoreceptors controlling the release of neurotransmitters. Using in vivo microdialysis in conscious rats, we studied the effect of a continuous infusion of the selective noradrenaline (NA) reuptake inhibitor reboxetine on extracellular concentrations of NA. Doses of 10 mg/kg/day reboxetine through subcutaneous osmotic pumps for 2 days increased extracellular NA by 272% in the dorsal hippocampus (DH) of rats. NA rose significantly more in rats given reboxetine for 7 (469%) and 14 (437%) days. Intraperitoneal injection of 30 microg/kg clonidine, an alpha2-adrenoceptor agonist, reduced the release of NA to 49% of basal levels in rats given vehicle or reboxetine for 2 days, but this effect was markedly less in rats given reboxetine for 7 and 14 days. Likewise, the effect of intrahippocampal infusion of clonidine (0.05 and 0.2 microM) on extracellular NA was significantly attenuated in rats given reboxetine for 7 and 14 days, whereas the injection of 0.6 nmol clonidine into the locus coeruleus caused similar reductions of extracellular NA in the DH and prefrontal cortex (PFC) of rats infused with vehicle (DH -64%; PFC -42%) and reboxetine (DH -45%; PFC -28%) for 14 days. The results indicate that chronic treatment markedly enhances the effect of reboxetine on extracellular NA in the DH and suggest that this effect may be due to the desensitization of hippocampal alpha2-adrenoceptors.

Blockade of tachykinin NK1 receptors attenuates stress-induced rise of extracellular noradrenaline and dopamine in the rat and gerbil medial prefrontal cortex.

Substance P receptor antagonists cause antidepressant- and anxiolytic-like effects in rodents that are thought to involve brain monoamines. In the present study, we examined the effects of the NK1 receptor antagonist GR-205,171 on basal and stress-induced rise of extracellular noradrenaline (NA) and dopamine (DA) in the medial prefrontal cortex (mPFC) of conscious rats and gerbils with the in vivo microdialysis technique. GR-205,171 given intraperitoneally to rats (10 and 30 mg/kg) and gerbils (0.3 and 1 mg/kg) did not affect extracellular NA in either species and increased extracellular DA in rats. Forty minutes of immobilization increased extracellular NA and DA by, respectively, 179% and 188% of baseline values in rats and 222% and 316% of baseline values in gerbils. At 10 mg/kg, GR-205,171 attenuated the stress-induced increase of extracellular NA in the rat. At 30 mg/kg, GR-205,171 suppressed the effect of stress on extracellular DA but had no effect on NA. A lower dose (1 mg/kg) attenuated the stress-induced rise of extracellular NA and DA in the mPFC of gerbils. The results show that blockade of NK1 receptors marginally increased basal extracellular DA in rats but had no effect in gerbils, whereas the stress-induced rise of extracellular NA and DA was markedly attenuated in both species. It is suggested that catecholamines may contribute to the functional effects of GR-205,171.

The 5-HT receptor antagonist M100,907 prevents extracellular glutamate rising in response to NMDA receptor blockade in the mPFC.

We recently found that intracortical injection of the selective and competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-(R)-2-carboxypiperazin-4-propyl-1-phosphonic acid (CPP) impaired attentional performance in rats and blockade of 5-hydroxytryptamine (5-HT)2A receptors antagonized this effect. Here, we used the microdialysis technique in conscious rats to study the effect of CPP on extracellular glutamate (GLU) in the medial prefrontal cortex (mPFC) and the regulation of this effect by 5-HT2A receptors. Intraperitoneal injection of 20 mg/kg CPP increased extracellular GLU in the mPFC (201% of basal levels) but had no effect on 5-HT. Intracortical infusion of 100 microm CPP increased extracellular GLU (230% of basal values) and 5-HT (150% of basal values) in the mPFC, whereas 30 microm had no significant effect. The effect of 100 microm CPP on extracellular GLU was abolished by tetrodotoxin, suggesting that neuronal activity is required. Subcutaneous injection of 40 microg/kg M100,907 completely antagonized the effect of 100 microm cpp on extracellular GLU, whereas 10 microg/kg caused only partial attenuation. Likewise, intracortical infusion of 0.1 microm M100,907 completely reversed the increase of extracellular GLU induced by CPP. These findings show that blockade of NMDA receptors in the mPFC is sufficient to increase extracellular GLU locally. The increase of cortical extracellular GLU may contribute to CPP-induced cognitive deficits and blockade of 5-HT2A receptors may provide a molecular mechanism for reversing these deficits caused by dysfunctional glutamatergic transmission in the mPFC.