HIV viraemic patients downregulate CD94/NKG2A inhibitory receptors on NK as well as CD8 T cells in comparison with aviraemic counterparts

Background: The CD94/NKG2 heterodimer is a C-type lectin receptor formed by the association of CD94 and one of the NKG2 molecules (namely NKG2A, -B, -C or –E). The interaction of CD94/NKG2A with non classical HLA-E molecules delivers inhibitory signals. CD94/NKG2A is normally expressed on most NK cells whereas less than 5% of peripheral resting CD8+ T cells are positive. Although several reports have clearly shown an upregulation of CD94 on CD8 T cells in HIV infection, the simultaneous expression of both subunits of the inhibitory receptor on NK and T cells and its relation with viral load is largely unknown. Methods: PBLs from 30 HIV-infected patients (16 viraemic and 14 aviraemic under HAART) and 18 healthy volunteers were analysed by flow cytometry after staining with the following monoclonal antibodies (Percp-conjugated anti-CD8, FITC-conjugated anti-CD3, APC-conjugated anti-CD94, PE-conjugated anti-NKG2A). Results: The proportion of CD8 T cells expressing the CD94/NKG2A inhibitory receptor was not significantly increased in HIV-infected patients (5.68 ± 3.72%) in comparison with non-infected controls (4.90±2.84%). Interestingly, patients with viral load < 50 copies/ml had a higher proportion of CD8 T cells expressing the inhibitory receptor (7.15 ± 3.63%) than patients with HIV viraemia (4.40 ± 3.40%), p= 0.041. The same pattern was observed for NK cells and was even more pronounced. In aviraemic individuals, 61.75 ± 20.39% of NK cells expressed the inhibitory receptor vs 42.88 ± 26.38% in viraemic patients. The proportion of CD94/NKG2A positive cells was correlated between NK and CD8 T cell subsets (p=0.0351) but there was no correlation with absolute or relative CD4 counts. Conclusions: Our results suggest that chronic stimulation with HIV antigens in viraemic patients could lead to decreased rather than increased expression of inhibitory receptors on NK and CD8 T cells. This could contribute to the abnormal activation of the immune system associated with advanced HIV disease

Downregulation of CD94/NKG2A inhibitory receptors on CD8+ T cells in HIV infection is more pronounced in subjects with detected viral load than in their aviraemic counterparts

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated CD8+ T lymphocytes. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In this study, CD94/NKG2A receptor expression on CD8+ T lymphocytes and NK cells was assessed in 46 HIV-1-infected patients (24 viraemic, 22 aviraemic) and 10 healthy volunteers. The percentage of CD8+ T lymphocytes expressing the CD94/NKG2A inhibitory heterodimer was very significantly decreased in HIV-1-infected patients in comparison with non-infected controls. Within the HIV infected patients, the proportion of CD8+ T lymphocytes and NK cells expressing CD94/NKG2A was higher in subjects with undetectable viral loads in comparison with their viraemic counterparts. No significant difference was detected in the proportion of CD8+ T lymphocytes expressing the activatory CD94/NKG2C heterodimer between the HIV-1 infected patients and the healthy donors, nor between the vireamic and avireamic HIV-1 infected patients. In conclusion, chronic stimulation with HIV antigens in viraemic patients leads to a decreased rather than increased CD94/NKG2A expression on CD8+ T lymphocytes and NK cells

Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR

BACKGROUND: The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. METHODS: In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. RESULTS: We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. CONCLUSION: These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy

Human bone marrow, umbilical cord or liver mesenchymal stromal cells fail to improve liver function in a model of CCl4-induced liver damage in NOD/SCID/IL-2Ry(null) mice

Background aims. Transplantation is the gold standard procedure for treating acute and chronic end-stage liver diseases. Given the shortage of organs, the development of cellular sources other than human liver is urgent. The main objective of this project was to examine the effect of mesenchymal stromal cell (MSC) (bone marrow, umbilical cord and liver MSCs) intravenous injection on liver regeneration in a model of hepatic damage in NOD/SCID/IL non-obese diabetic/severe combined immunodeficient/Interleukin-2Rg(null) (NSG) mice. Methods. Mice received 3 intraperitoneal injections of CCl4 Carbon tetrachloride per week for 4 weeks. Forty-eight hours after the last injection of CCl4, mice received 500,000 MSCs or phosphate-buffered saline by intravenous injection. We examined hepatic damage by means of quantitative image analysis and blood enzyme analysis 24 h, 1 week or 8 weeks after MSC or phosphate-buffered saline injection. We also examined MSC homing by means of real-time polymerase chain reaction of human albumin. Results. We adapted a model of liver injury in immunodeficient mice. In this model, accumulation of collagen in newly formed scar septa was apparent up to 8 weeks after CCl4 treatment. Human albumin DNA was found in all organs tested. However, intravenous MSC injection, even after CXCR4 C-X-C chemokine receptor type 4 transduction and whatever the origin of MSCs, failed to improve liver damage. Conclusions. In this liver injury model, MSCs were propagated in various tissues, particularly filtering organs. For the treatment of hepatic damage, intravenous administration of moderate doses of MSCs does not appear to be effective. Yet, this adapted liver injury model is appropriate for investigating engraftment of human cells

Prostanglandin E2 induces the expression of functional inhibitory CD94/NKG2A receptors in human CD8+ T lymphocytes by a cAMP-dependent protein kinase A type I pathway

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated T cells, predominantly of CD8+ phenotype. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In an attempt to identify the mechanisms leading to inhibitory NKR upregulation on T cells, we analyzed the expression of the CD94/NKG2A heterodimer on human CTLs activated with anti-CD3 mAb in the presence of PGE2 or with 8-CPT-cAMP, an analogue of cyclic AMP. As previously described, anti-CD3 mAb-mediated activation induced the expression of CD94/NKG2A on a small fraction of CD8+ T cells. Interestingly, when low concentrations of PGE2 or 8-CPT-cAMP were present during the culture, the proportion of CD8+ T cells expressing CD94/NKG2A was 2 to 5-fold higher. This upregulation was partially prevented by the PKA type I inhibitor, Rp-8-Br-cAMP. We also report that cAMP induces upregulation of NKG2A at the mRNA level. We further demonstrated that cross-linking of CD94 on CD8+ T cells expressing the CD94/NKG2A heterodimer inhibits their cytotoxic activity in a bispecific antibody redirected lysis assay. Our findings clearly demonstrate that the PGE2/cAMP/PKA type I axis is involved in the expression of CD94/NKG2A receptor on human CD8+ T lymphocytes

Glucocorticoid-induced leucine zipper (GILZ) is involved in glucocorticoid-induced and mineralocorticoid-induced leptin production by osteoarthritis synovial fibroblasts.

BACKGROUND: Glucocorticoid-induced leucine zipper (GILZ) is a mediator of the anti-inflammatory activities of glucocorticoids. However, GILZ deletion does not impair the anti-inflammatory activities of exogenous glucocorticoids in mice arthritis models and GILZ could also mediate some glucocorticoid-related adverse events. Osteoarthritis (OA) is a metabolic disorder that is partly attributed to adipokines such as leptin, and we previously observed that glucocorticoids induced leptin secretion in OA synovial fibroblasts. The purpose of this study was to position GILZ in OA through its involvement in the anti-inflammatory activities of glucocorticoids and/or in the metabolic pathway of leptin induction. The influences of mineralocorticoids on GILZ and leptin expression were also investigated. METHODS: Human synovial fibroblasts were isolated from OA patients during knee replacement surgery. Then, the cells were treated with a glucocorticoid (prednisolone), a mineralocorticoid (aldosterone), a glucocorticoid receptor (GR) antagonist (mifepristone), a selective glucocorticoid receptor agonist (Compound A), mineralocorticoid receptor (MR) antagonists (eplerenone and spironolactone), TNF-alpha or transforming growth factor (TGF)-beta. Cells were transfected with shRNA lentiviruses for the silencing of GILZ and GR. The leptin, IL-6, IL-8 and matrix metalloproteinase (MMP)-1 levels were measured by ELISA. Leptin, the leptin receptor (Ob-R), GR and GILZ expression levels were analyzed by western blotting and/or RT-qPCR. RESULTS: (1) The glucocorticoid prednisolone and the mineralocorticoid aldosterone induced GILZ expression dose-dependently in OA synovial fibroblasts, through GR but not MR. Similar effects on leptin and Ob-R were observed: leptin secretion and Ob-R expression were also induced by prednisolone and aldosterone through GR; (2) GILZ silencing experiments demonstrated that GILZ was involved in the glucocorticoid-induced and mineralocorticoid-induced leptin secretion and Ob-R expression in OA synovial fibroblasts; and (3) GILZ inhibition did not alter the production of pro-inflammatory cytokines by OA synovial fibroblast or the anti-inflammatory properties of glucocorticoids. CONCLUSIONS: The absence of GILZ prevents corticoid-induced leptin and Ob-R expression without affecting the anti-inflammatory properties of glucocorticoids in OA synovial fibroblasts. Mineralocorticoids also induce leptin and Ob-R expression through GILZ

In vivo administration of a PKA type I inhibitor (Rp-8-Br-cAMPS) restores T-cell responses in retrovirus-infected mice

peer reviewedMurine AIDS (MAIDS) is caused by infection with the murine leukemia retrovirus RadLV-Rs and is characterized by T-cell anergy and severe immunodeficiency with increased susceptibilty to several experimental opportunistic infections as observed in HIV infection. T cell anergy is associated with an increase of intracellular cAMP level, triggering a multistep pathway involving activation of PKA type I and resulting in inhibition of proximal TCR signaling. We have reviously demonstrated that blocking PKA type I using the selective inhibitor Rp-8-Br-cAMPS, restores T-cell function in vitro in MAIDS as well as in HIV infection. In the present report, we investigated the effect of parenteral administration of Rp-8-Br-cAMPS in mice with MAIDS. We show that the compound is not toxic and partially restores the ex vivo proliferative responses to anti-CD3 mAb, but that it has no effect on the lymphadenopathy and splenomegaly characterizing the MAIDS syndrome

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood.

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3+/CD19+/CD14+/CD38+-depleted MNC and CD133+- or LNGFR+-enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90+/CD73+/CD105+ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non-invasive and abundant source of MSC

Prostaglandin E2 induces the expression of functional inhibitory CD94/NKG2A receptors in human CD8+ T lymphocytes by a cAMP-dependent protein kinase A type I pathway.

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated T cells, predominantly of CD8+ phenotype. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In an attempt to identify the mechanisms leading to inhibitory NKR upregulation on T cells, we analyzed the expression of the CD94/NKG2A heterodimer on human CTLs activated with anti-CD3 mAb in the presence of PGE2 or with 8-CPT-cAMP, an analogue of cyclic AMP. As previously described, anti-CD3 mAb-mediated activation induced the expression of CD94/NKG2A on a small fraction of CD8+ T cells. Interestingly, when low concentrations of PGE2 or 8-CPT-cAMP were present during the culture, the proportion of CD8+ T cells expressing CD94/NKG2A was two- to five-fold higher. This upregulation was partially prevented by PKA inhibitors, such as KT5720 and Rp-8-Br-cAMP (type I selective). We also report that cAMP induces upregulation of NKG2A at the mRNA level. We further demonstrated that cross-linking of CD94 on CD8+ T cells expressing the CD94/NKG2A heterodimer inhibits their cytotoxic activity in a bispecific antibody redirected lysis assay. Our findings clearly demonstrate that the PGE2/cAMP/PKA type I axis is involved in the expression of CD94/NKG2A receptor on human CD8+ T lymphocytes