MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.

BACKGROUND: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. METHODS: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. RESULTS: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45-CD11b- non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C- cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C- cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells. CONCLUSION: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression.

TNFα suppresses IFNγ-induced MHC class II expression on retinal pigmented epithelial cells cultures.

PURPOSE: One major consequence of retinal pigment epithelium (RPE) cell activation during autoimmune uveitis is the induction of MHC II molecules expression at their surface. IFNγ is regarded as the main cytokine involved in this induction. As TNFα plays a central role in autoimmune uveitis, we investigated its effects on IFNγ-mediated MHC II induction on RPE cells. METHODS: Retinal pigment epithelium cells (ARPE-19) were stimulated with IFNγ, TNFα and the anti-TNFα antibody infliximab. The expression of MHCII and ICAM-1 was analysed by flow cytometry. The activation and expression of IRF-1 and STAT-1, two proteins involved in IFNγ-signalling pathway, were analysed by WB. Class II transactivator (CIITA) expression was monitored by qRT-PCR and immunoprecipitation. RESULTS: TNFα inhibits IFNγ-induced MHC II expression on ARPE cells in a dose-dependent manner. Infliximab completely reverses the inhibitory effect of TNFα. We did not observe an inhibitory effect of TNFα on the expression of ICAM-1 induced by IFNγ. Similarly, IFNγ-induced STAT1 phosphorylation and IRF1 expression were not affected by TNFα. On the contrary, we found that TNFα suppresses IFNγ-induced CIITA mRNA accumulation and protein expression. CONCLUSION: TNFα inhibits IFNγ-induced MHC II expression in RPE cells. This inhibitory effect was reversed by infliximab and was not because of a global inhibition of IFNγ -mediated RPE cell activation but rather to a specific down-regulation of CIITA expression. Those findings are consistent with the role of TNFα in the resolution of inflammation and might help to elucidate the complex development of autoimmune uveitis.

Development of a successful antitumor therapeutic model combining in vivo dendritic cell vaccination with tumor irradiation and intratumoral GM-CSF delivery.

Vaccination of dendritic cells (DC) combined with GM-CSF secreting tumor cells has shown good therapeutic efficacy in several tumor models. Nevertheless, the engineering of GM-CSF secreting tumor cell line could represent a tedious step limiting its application for treatment in patients. We therefore developed in rats, an "all in vivo" strategy of combined vaccination using an in vivo local irradiation of the tumor as a source of tumor antigens for DC vaccines and an exogenous source of GM-CSF. We report here that supplying recombinant mGM-CSF by local injections or surgical implantation of osmotic pumps did not allow reproducing the therapeutic efficacy observed with in vitro prepared combined vaccines. To bypass this limitation possibly due to the short half-life of recombinant GM-CSF, we have generated adeno-associated virus coding for mGM-CSF and tested their efficacy to transduce tumor cells in vitro and in vivo. The in vivo vaccines combining local irradiation and AAV2/1-mGM-CSF vectors showed high therapeutic efficacy allowing to cure 60% of the rats with pre-implanted tumors, as previously observed with in vitro prepared vaccines. Same efficacy has been observed with a second generation of vaccines combining DC, local tumor irradiation, and the controlled supply of recombinant mGM-CSF in poloxamer 407, a biocompatible thermoreversible hydrogel. By generating a successful "all in vivo" vaccination protocol combining tumor radiotherapy with DC vaccines and a straightforward supply of GM-CSF, we have developed a therapeutic strategy easily translatable to clinic that could become accessible to a much bigger number of cancer patients.

Synergy between dendritic cells and GM-CSF-secreting tumor cells for the treatment of a murine renal cell carcinoma.

Dendritic cell (DC) immunotherapy for cancer certainly holds promises but definitely needs improvements, especially for enhancing tumor-specific responses able to eradicate preexisting tumors. To this end, we investigated here, for the treatment of a preimplanted murine renal cell carcinoma Renca, a new vaccination approach combining injection of DC and granulocyte macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cells. When treatment by either DC or Renca-mGM-CSF cells alone had no therapeutic effect at all, combined vaccines induced therapeutic response in 50% of the tumor-bearing mice, in a GM-CSF dose-dependent manner. Importantly, all these cured mice were protected against a rechallenge with parental Renca cells, indicating the generation of memory immune response. The combined vaccines induced elevated cytotoxic responses in all the cured mice and half of the uncured ones and a stronger systemic CD4+ T-cell-mediated interferon-gamma production in the cured vaccinated mice as compared with uncured ones. In conclusion, vaccines associating DC and GM-CSF-secreting tumor cells induce high therapeutic effect in mice with preexisting renal cell carcinoma that are correlated to the induction of specific CD8 and CD4+ T-cell responses. This original vaccination approach should be further evaluated in a clinical trial for the treatment of metastatic human renal cell carcinoma.

Extracellular nucleotides and interleukin-8 production by ARPE cells: potential role of danger signals in blood-retinal barrier activation.

PURPOSE: RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATPgammaS, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors. METHODS: ARPE-19 cells were cultured with ATPgammaS, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay. RESULTS: Stimulation of ARPE-19 cells with ATPgammaS, UTP, and UDP induced IL-8 gene transcription and protein secretion. TNFalpha induction of IL-8 secretion was also increased by ATPgammaS, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium. CONCLUSIONS: ATPgammaS, UTP, and UDP stimulate both basal and TNFalpha-induced IL-8 secretion in RPE cells through an ERK 1/2-dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors.

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses, high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration.

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.

15-Deoxy-12,14-prostaglandin J2 inhibits interferon gamma induced MHC class II but not class I expression on ARPE cells through a PPAR gamma independent mechanism.

Retinal pigment epithelial (RPE) cells constitute the external part of the blood-retinal-barrier and play a pivotal role in the regulation of retinal immunity. In the present work, we investigated the effects of 15-deoxy-12,14-prostaglandin J2 (15 PGJ2), an endogenous ligand of PPARgamma, on the IFNgamma-induced expression of MHC class II on RPE cells. Indeed, pathological expression of MHC class II molecules at the surface of RPE cells is a common feature of many blinding conditions. We demonstrated that 15 PGJ2 inhibited the IFNgamma-mediated induction of MHC class II on RPE cells without affecting the level of MHC class I and CD54 expression. The other PPARgamma agonist rosiglitazone or troglitazone had no similar effects. Moreover, the inhibitory effect of 15 PGJ2 was not abrogated by co-incubation with PPARgamma antagonists and did not involve the modulation of STAT-1, AKT or ERK1/2 phosphorylation, nor CIITA, IRF1 or IRF2 transcription. In conclusion, 15 PGJ2 inhibits strongly and specifically the IFNgamma-induced MHC class II expression on RPE cells by a PPARgamma independent mechanism. Given the differential role of MHC classes I and II in the development of autoimmune uveitis and the potential toxicity of 15 PGJ2, our data's suggest that the development of novel small molecules targeting similar PPARgamma independent pathways would be useful for the future management of uveitis.

Generation of cell hybrids via a fusogenic cell line.

BACKGROUND: Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult. METHODS: We have established a stable cell line expressing a viral fusogenic membrane glycoprotein (FMG) that is not itself susceptible to fusion. This cell line has been used to generate hybrids and to evaluate the relevance of tools used for hybrid detection. RESULTS: This FMG-expressing cell line promotes fusion between autologous or allogeneic TC and DC in any combination, generating 'tri-parental hybrids'. At least 20% of TC are found to be integrated into hybrids. CONCLUSIONS: It is speculated that this tri-parental hybrid approach offers new possibilities to further modulate the anti-tumour effect of the DC/TC hybrids since it allows the expression of relevant immunostimulatory molecules by appropriate engineering of the fusogenic cell line.

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate.

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.

Immune characterization of clinical grade-dendritic cells generated from cancer patients and genetically modified by an ALVAC vector carrying MAGE minigenes.

Gene delivery into dendritic cells (DC) is most efficiently achieved by viral vectors. Recombinant canarypox viruses (ALVAC) were validated safe and efficient in humans. We aimed firstly to evaluate DC transduction by ALVAC vectors, then to investigate if such infection induced or not the maturation of the DC, and finally to assess the efficiency of ALVAC-MAGE-transduced DC to activate specific CTL clones. Clinical grade DC from melanoma patients were generated from blood monocytes and infected with a recombinant ALVAC virus encoding either a marker gene (EGFP) or the MAGE-1-MAGE-3 minigenes. According to the patient-donor, 22+/-16% of immature DC were successfully transduced. Flow cytometry analysis of surface markers expressed on DC after ALVAC infection did not reveal a mature phenotype. Moreover, ALVAC transduction did not interfere with the capacity of the DC to further mature under poly:IC stimulation. But most importantly, our results demonstrated that DC from HLA-A1 patient-donors infected with the recombinant ALVAC MAGE-1-MAGE-3 minigenes virus were capable of activating a MAGE 3/A1 CTL clone more efficiently than same DC loaded with MAGE 3/A1 peptide, as shown by increased IFN-gamma secretion. These results could be the basis for the development of a new clinical strategy in melanoma patient's immunotherapy.

Highly successful therapeutic vaccinations combining dendritic cells and tumor cells secreting granulocyte macrophage colony-stimulating factor.

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-gamma production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.

Assessment of in vivo chemotherapy-induced DNA damage in a p53-mutated rat tumor by micronuclei assay.

Production of DNA damage is the basis of cancer treatments such as chemo- and radiotherapy. Such treatments induce mitotic catastrophe, a form of cell death resulting from abnormal mitosis and leading to the formation of interphase cells with multiple micronuclei. In this study, we compared apoptosis induction and micronuclei formation to assess the DNA damage provoked in vivo by cytotoxic agents in established 9L rat gliosarcoma tumors expressing a mutated p53 gene. Results from TUNEL assays revealed the efficiency of local gamma-irradiation at the tumor site to induce apoptosis within 9L tumor mass. However, little or no apoptosis was detected after systemic (ip) injection of cisplatin (1 mg/kg). Interestingly, the micronuclei assays showed that not only gamma-irradiation but also cisplatin treatment led to an increase in the emergence of binucleated cells with micronuclei. Apoptosis induction and micronuclei emergence are thus not absolutely correlated. However, micronuclei assays, rarely performed on solid tumors, appear more sensitive than apoptosis assays in evaluating DNA damage linked to chemotherapy.

SHIP2 overexpression strongly reduces the proliferation rate of K562 erythroleukemia cell line.

SHIP2 belongs to the inositol 5-phosphatase family and is characterized by a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) 5-phosphatase activity. Evidence based on mice lacking the SHIP2 gene has demonstrated its predominant role in the control of insulin sensitivity. However, SHIP2 expression in both hematopoietic and non-hematopoietic cells suggests additional functions. SHIP2 was previously identified in chronic myelogenous progenitor cells, in which its constitutive tyrosine phosphorylation was reported by Wisniewski et al., [Blood 93 (1999) 2707-2720]. Here, we further investigated the function of SHIP2 in this hematopoietic and malignant context. A detailed analysis of the substrate specificity of SHIP2 indicated that this phosphatase is primarily directed towards PI(3,4,5)P(3) both in vitro and in K562 chronic myeloid leukemia cells. The SHIP2-mediated decrease in PI(3,4,5)P(3) levels and increase in phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) was accompanied by a reduction of cell proliferation, characterized by an accumulation of the cells in the G2/M phase of the cell cycle. Thus, in addition to its role in the control of insulin sensitivity, SHIP2 may also play a role in cell proliferation, at least in chronic myelogenous progenitor cells.

Extracellular adenine nucleotides inhibit the activation of human CD4+ T lymphocytes.

ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE(2), inhibit human CD4(+) T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4(+) T cells. The rank order of potency of the various nucleotides was: adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) approximately 2'- and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP >> dATP, 2-propylthio-beta,gamma-dichloromethylene-D-ATP, UDP, UTP. This effect did not involve the activation of A(2)Rs by adenosine or the synthesis of prostaglandins. ATPgammaS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPgammaS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-gamma; expression of CD25; and proliferation after activation of CD4(+) T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4(+) T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.