Catherine Bruyns

The P2Y11 Receptor Mediates the ATP-Induced Maturation of Human Monocyte-Derived Dendritic Cells

Recently, it has been shown that ATP and TNF-α synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-α and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5′-O-(3-thiotriphosphate) = 2′- and 3′-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

Critical role of interleukin 4 in the induction of neonatal transplantation tolerance

Neonatal injection of semiallogeneic cells is known to promote differentiation of donor-specific CD4+ T cells into TH2-like cells in the peripheral lymphoid organs. We reasoned that the propensity of neonatal T cells to synthesize high levels of IL-4 might be involved in this polarization of the alloreactive response and thereby in the development of neonatal transplantation tolerance. First, analysis of cytokine gene expression in lymph nodes after neonatal injection of 107 (A/J×BALB/c)Fl cells in BALB/c mice indicated that IL-4 but not IL-2 is rapidly produced by CD4+ cells after allogeneic challenge in vivo. To determine whether the early production of IL-4 was involved in the establishment of allotolerance, BALB/c mice neo-natally injected with (A/JxBALB/c)Fl spleen cells received on days 1 and 3 after birth 1 mg of anti-IL-4 mAb (11B11) or the same amount of control mAb. When grafted with A/J skin at 4 weeks, 88% of mice treated with control mAb retained their graft for more than 50 days, whereas rejection occurred within 30 days in 93% of mice treated with anti-IL-4 mAb. Analysis of T cell functions after in vitro restimulation with A/J spleen cells indicated that early IL-4 neutralization did not prevent donor-specific CTL unresponsiveness but allowed the emergence of alloreactive T cells secreting increased levels of IL-2 and IFN–y. We conclude that early production of IL-4 is critical for the establishment of neonatal transplantation tolerance in this strain combination, which has disparities across the entire H-2 region.

Transient expansion of peptide‐specific lymphocytes producing IFN‐γ after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage‐A1/A3‐positive tumors

Assessment of T‐cell activation is pivotal for evaluation of cancerimmunotherapy. We initiated a clinical trial in patients with MAGE‐A1and/or ‐A3 tumors using autologous DC pulsed with MAGE peptides aimedat analyzing T‐cell‐derived, IFN‐γ secretion by cytokine flowcytometry and ELISPOT. We also tested whether further KLH additioncould influence this response favorably. Monocyte‐derived DC weregenerated from leukapheresis products. They were pulsed with therelevant MAGE peptide(s) alone in group A (n=10 pts) andadditionally with KLH in group B (n=16 pts). A specific buttransient increase in the number of peripheral blood T lymphocytessecreting IFN‐γ in response to the vaccine peptide(s) was observed in6/8 patients of group A and in 6/16 patients of group B. We concludethat anti‐tumor vaccination using DC pulsed with MAGE peptides inducesa potent but transient anti‐MAGE, IFN‐γ secretion that is notinfluenced by the additional delivery of a nonspecific, T‐cellhelp.

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 PGE2, 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) (ATPγS) ≈ 2′- and 3′-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP ≫ dATP, 2-propylthio-β,γ-dichloromethylene-d-ATP, UDP, UTP. This effect did not involve the activation of A2Rs by adenosine or the synthesis of prostaglandins. ATPγS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPγS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-γ; 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.

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.

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

Abstract The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after longterm stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.

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.

Retinal pigment epithelial cells phagocytosis of T lymphocytes: possible implication in the immune privilege of the eye.

Aim: To investigate the capability of retinal pigment epithelium (RPE) cells to phagocytose T lymphocytes and to further analyse the immunobiological consequences of this phagocytosis. Methods: Human RPE cells pretreated or not by cytochalasin, a phagocytosis inhibitor, were co-cultured with T lymphocytes for different time points. Phagocytosis was investigated by optic microscopy, electron microscopy, and flow cytometry. T cell proliferation was measured by 3H thymidine incorporation. RPE interleukin 1β mRNA expression was quantified by real time PCR. Results: RPE cells phagocytose apoptotic and non-apoptotic T lymphocytes, in a time dependent manner. This is an active process mediated through actin polymerisation, blocked by cytochalasin E treatment. Inhibition of RPE cell phagocytosis capabilities within RPE-T cell co-cultures led to an increase of lectin induced T cell proliferation and an upregulation of interleukin 1β mRNA expression in RPE cells. Conclusions: It is postulated that T lymphocyte phagocytosis by RPE cells might, by decreasing the total number of T lymphocytes, removing apoptotic lymphocytes, and downregulating the expression of IL-1β, participate in vivo in the induction and maintenance of the immune privilege of the eye, preventing the development of intraocular inflammation.

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-γ 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.

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.