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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 24, 2005 as doi:10.1096/fj.04-1617fje. |
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Service de Recherches en Hémato-Immunologie, Commissariat à L’Energie Atomique-DRM-DSV, Institut Universitaire d’Hématologie, Hôpital Saint Louis, Paris, France
1Correspondence: Service de Recherches en Hémato-Immunologie, Commissariat à L’Energie Atomique-DRM-DSV, Institut Universitaire d’Hématologie, Hôpital Saint Louis, 1, Ave. Claude Vellefaux, Paris 75475 Cedex 10, France. E-mail: carosella{at}dsvidf.cea.fr
SPECIFIC AIMS
The nonclassical HLA class I molecule HLA-G is commonly viewed as a potent inhibitory molecule that protects the cells that express it from cytolysis. This function has been reported as being crucial for the protection of the fetal cytotrophoblast from destruction by the maternal immune system, for the protection of allografts against cytolysis by the recipient’s immune system, and for the protection of tumors against anti-tumor immunity. HLA-G exerts its inhibitory functions via three inhibitory receptors that are differentially expressible by immune cells (i.e., ILT2, ILT4, and KIR2DL4). Cells expressing HLA-G receptors and cells expressing HLA-G are often found in the vicinity of each other.
The purpose of this study was to investigate whether HLA-G had other functions than direct protection against cytolysis, and in particular, if a link existed between expression of HLA-G and expression of its receptors.
PRINCIPAL FINDINGS
1. HLA-G transfectants overexpress inhibitory receptors
To find out if exposure to HLA-G would induce up-regulation of inhibitory receptor expression, we compared ILT2 (LILRB1), ILT3 (LILRB4), ILT4 (LILRB2), and KIR2DL4 mRNA levels in antigen presenting cell lines (APC, i.e., lymphoblastoid LCL and promyelomonocytic KG1a lines) transfected with HLA-G1 (APC-HLA-G1, i.e. LCL-HLA-G1 and KG1a-HLA-G1) with those of controls (APC-RSV, i.e. LCL-RSV and KG1a-RSV). APC-HLA-G1 lines were used as models of APC that have had a long-term exposure to HLA-G1 since they express it themselves. Expression of ILT3 was investigated because even if not a receptor for HLA-G, it is a member if the ILT family and was shown to be up-regulated concomitantly with ILT4 in some circumstances. Results obtained for LCL-HLA-G1 vs. LCL-RSV cells showed up-regulation of ILT3 and ILT4 expression on all 3 LCL-HLA-G1 tested, and to a lesser extent, up-regulation of ILT2 and KIR2DL4 expression (Fig. 1
A). The results obtained for KG1a-HLA-G1 vs. KG1a-RSV were more striking and showed an induction of expression of the 4 inhibitory receptors tested in 3 independent KG1a-HLA-G1 lines (Fig. 1B
). For LCL-HLA-G1 and KG1a-HLA-G1 lines, up-regulation of ILT4 was the strongest and corresponded to a 6 to 60-fold increase in LCL-HLA-G1, and to a more than 106 fold increase in KG1a-HLA-G1. Up-regulation of ILT3 transcription by LCL-HLA-G1 cells and up-regulation of ILT2 transcription by KG1a-HLA-G1 cells correlated with up-regulation of cell surface expression.
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2. HLA-G directly up-regulates inhibitory receptor transcription in APC lines and monocytes
To determine whether HLA-G was involved in inhibitory receptor up-regulation in HLA-G1-transfected APC lines, we performed coculture experiments between HLA-G1-negative KG1a-RSV, KG1, and monocyte "responders" and HLA-G1-positive KG1a-HLA-G1 or HLA-G1- or HLA-G5-transfected melanoma "stimulator" cells. After 7 days of coculture, the responder cells were purified and transcription levels of inhibitory receptors were measured. Results show that ILT2 and ILT4 transcription was induced/up-regulated in responder cells cocultured with HLA-G-expressing cells but not in responder cells cocultured with HLA-G-negative controls.
To prove that HLA-G was directly responsible for inhibitory receptor up-regulation, the same coculture experiments as above were set up, in which HLA-G1 was masked by a blocking anti-HLA Class I mAb. Addition of the blocking antibody in the cocultures inhibited up-regulation of ILT4 in KG1a-RSV responder cells by HLA-G1-expressing stimulator cells by more than 80%. This showed that HLA-G1 was involved in the induction of inhibitory receptor up-regulation.
3. HLA-G1 and HLA-G5 up-regulate inhibitory receptor transcription and cell surface expression in APC, NK cells, and CD4+, but not CD8+ T cells
HLA-G receptors can be expressed by APC (ILT2 and ILT4), NK cells (ILT2 and KIR2DL4), and by T cells (ILT2 and KIR2DL4). These receptors have been described as HLA-G1 receptors. However, they are likely to bind the soluble counterpart of HLA-G1, HLA-G5. We therefore investigated the capability of HLA-G1 and HLA-G5 to induce up-regulation of inhibitory receptor transcription and cell surface expression in APC, NK, and T cells.
1)Stimulation of 3 different APC lines by HLA-G5-secreting cells induced cell surface up-regulation of ILT2 and ILT3 whereas stimulation of these APC lines by HLA-G-negative control cells had no effect. This showed that HLA-G5 was a more potent inducer of inhibitory receptor up-regulation than membrane bound HLA-G1 since stimulation of APC lines by HLA-G1 induced mainly transcriptional changes.
2) Stimulation of 2 NK lines that did not express ILT2 and KIR2DL4 by HLA-G1 expressing cells (M8-HLA-G1) induced cell surface up-regulation of KIR2DL4 in one line (YT2C2PR-IR-) and stimulation by HLA-G5-expressing cells (M8-HLA-G5) induced up-regulation of ILT2 and KIR2DL4 in the other (NKL-IR&-;) (Fig. 2
).
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3) Allo-stimulation of healthy donor peripheral blood mononuclear cells (PBMC) with HLA-G1-positive APC induced transcriptional up-regulation of ILT2 and KIR2DL4 in CD4+ T cells, but not in CD8+ T cells, when compared with PBMC allo-stimulated with HLA-G1-negative APC.
CONCLUSIONS AND SIGNIFICANCE
The nonclassical HLA Class I molecule HLA-G is generally viewed as a molecule involved in direct or HLA-E-mediated indirect protection of target cells against cytolysis by NK and T cells. The data presented here provide the first evidence that HLA-G1 can also act as a molecule capable of signaling transcriptional and phenotypical changes in immune cells, by itself, and even in the absence of activation. Given the data presented here, one can hypothesize that: 1) HLA-G acts as a cell-to-cell signaling molecule with potential inhibitory consequences; and 2) HLA-G-induced up-regulation of inhibitory receptors expression is a broad and early inhibitory function of HLA-G since it targets NK cells, T cells, and APC indiscriminately and might precede an immune reaction.
HLA-G is known to bind three inhibitory receptors with high affinity (i.e., ILT2, ILT4, and KIR2DL4). It is not clear whether any of these molecules were involved in HLA-G-induced up-regulation of inhibitory receptors. The effect was observed in cells that did not even transcribe ILT2, ILT4, or KIR2DL4. This seems to indicate that HLA-G might have other receptors than these three.
HLA-G-induced up-regulation of inhibitory receptors expression may very well have functional significance. For instance, HLA-G1 and HLA-G5 expressed by tissues such as fetal cytotrophoblast, tumors, or transplants might induce inhibitory receptors expression up-regulation in circulating/infiltrating immune cells even in the absence of an ongoing immune response. The potential consequences of such an up-regulation/induction might be multiple. 1) HLA-G-dependent up-regulation of HLA-G receptors might increase or induce the sensitivity of the responder cells to direct inhibition by tissue-expressed HLA-G. According to this hypothesis, the signaling functions of HLA-G would strengthen its well described inhibitory capabilities, and possibly be a prerequisite to HLA-G-mediated inhibition for cells that did not express HLA-G receptors originally. Up-regulation of inhibitory receptors expression might also 2) increase the sensitivity of the responder cells to inhibition by classical HLA class I molecules, since ILT2, ILT3, and ILT4 have other ligands than HLA-G, and 3) increase the activation threshold of the responder cells, as was reported for ILT4-overexpressing APC.
Physiologically, HLA-G-induced up-regulation/induction of inhibitory receptors should present advantages for HLA-G-expressing tissues that are potential targets for NK and cytotoxic T lymphocytes, and be part of immune escape mechanisms. Up-regulation of HLA-G receptors might strengthen the direct protection provided by HLA-G to classical HLA class I-negative tissues such as tumors, and strengthen the direct protection provided by HLA-G as well as classical HLA Class I molecules to cells expressing low levels of classical HLA molecules (e.g., tumors), and expressing self-HLA: "foreign" peptide complexes (e.g., tumors, transplants). Last, coupling the direct inhibitory effect of HLA-G and its effect on up-regulation of non-HLA-G-restricted inhibitory receptors might be of particular interest for allogeneic transplants, since these express HLA Class I molecules at a normal level and inhibitory receptors are capable of recognizing various alleles of different HLA Class I molecules. Thus, an HLA-G-expressing allogeneic transplant might use HLA-G as a direct inhibitory molecule, as well as a molecule capable of rendering immune effector cells sensitive or more sensitive to inhibition by its own HLA-Class I molecules.
Besides its direct inhibitory function, HLA-G seems to possess signaling functions aimed at actively strengthening the latter and broadening the scope of inhibitory mediators. The data presented here may explain why cells expressing HLA-G and cells expressing its receptors are often found in the vicinity of each other, and put an emphasis on the role of HLA-G in tumoral immune escape and tolerance.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1617fje;
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