Basic research
Functions of the HLA-G molecule on iNKT cells
Our department described the functions of the HLA-G molecule on NK cells, Ta/b and Tg/d lymphocytes, B lymphocytes, antigen-presenting cells, and neutrophils [1].
iNKT cells are a sub-population of T lymphocytes characterised by the expression of TCR capable of recognising lipid antigens presented in the context of the CD1d molecule. iNKT constitute less than 1% of mononucleated cells in peripheral blood, but may play a major role in initiating immune responses, by their unique ability to secrete large quantities of pro- and/or anti-inflammatory cytokines. iNKT are actively studied for their ability to induce anti-tumoural immunity. We showed that ILT2 receptor surface expression at the surface of iNKT was greater than that of T lymphocytes and NK cells. It is therefore likely that the HLA-G molecule inhibits these cells and/or induces their differentiation into regulatory cells. This project aims to characterise the functions of the HLA-G molecule on iNKT cells, to determine their impact on the anti-tumoural capacities of these cells, and to provide proof of concept of a strategy making it possible to control the effect of the HLA-G molecule during anti-tumoural immunisation.iNKT cells are a sub-population of T lymphocytes characterised by the expression of TCR capable of recognising lipid antigens presented in the context of the CD1d molecule. iNKT constitute less than 1% of mononucleated cells in peripheral blood, but may play a major role in initiating immune responses, by their unique ability to secrete large quantities of pro- and/or anti-inflammatory cytokines. iNKT are actively studied for their ability to induce anti-tumoural immunity. We showed that ILT2 receptor surface expression at the surface of iNKT was greater than that of T lymphocytes and NK cells. It is therefore likely that the HLA-G molecule inhibits these cells and/or induces their differentiation into regulatory cells. This project aims to characterise the functions of the HLA-G molecule on iNKT cells, to determine their impact on the anti-tumoural capacities of these cells, and to provide proof of concept of a strategy making it possible to control the effect of the HLA-G molecule during anti-tumoural immunisation.
Characterisation of molecules interacting with HLA-G protein and their role in its functions.
The action of the HLA-G molecule through the ILT2, ILT4, and KIR2DL4 receptors has been clearly described. Nevertheless, we showed that this molecule was capable of inducing the expression of inhibitory receptors and blocking the multiplication of liquid tumours in tumour lines not expressing known receptors for HLA-G [2, 3]. In order to comprehend the tolerogenic functions of the HLA-G molecule, and also to use it in a clinical context, it is crucial to identify the molecules through which it acts, together with those modulating its activity. With this aim in mind, we will be pursuing our studies aiming to identify the molecules interacting with HLA-G and to characterise their role in its functions. We use the various recombinant proteins that we synthesised in order to identify still unknown HLA-G receptors at the cell surface, and plasma proteins that associate with HLA-G in normal blood donors, and transplanted and cancer patients. Cell lines transfected with the newly identified proteins will enable us to validate in vitro the association between HLA-G and each newly identified protein. We will then study the consequences of the association with HLA-G in terms of signalling, and, if possible, at a functional level.
Applied research
Use of HLA-G for bladder cancer diagnosis and therapy
Bladder cancer is the most common type affecting the urinary system, the 7th in males and the 17th in females. Its incidence is increased in smokers and patients having received prior radiotherapy for prostate cancer. Radiotherapy doses recently increased, rising from the conventional level of 65/70gy to 80gy at present, increasing the risk of radiation-induced tumour. Previous studies demonstrated the expression of the HLA-G molecule in this disease. As for other solid tumours, HLA-G could therefore constitute an immune escape mechanism. Blocking its action could therefore restore anti-tumoural immunity. This approach is particularly relevant since tumours not invading muscle are sensitive to immunotherapy. Intravesicular instillation of BCG is, in fact, recommended for high-grade tumours (pT1G3). This therapeutic approach makes it possible to delay but not to eradicate tumour regrowth. During this project, we propose to confirm the expression of the HLA-G molecule by bladder tumours, the expression of its receptors by invasive cells, and to determine whether this type of expression is associated with a poorer prognosis. Furthermore, in order to demonstrate formally that HLA-G expression by tumour cells constitutes an immune escape mechanism, we will attempt to restore the cytolytic capacity of NK and T effectors derived from the peripheral blood of the same patient with regard to tumour cells, by using blocking anti-HLA-G monoclonal antibodies (see part 3.3). In a therapeutic context, it is possible that the stimulation of immune effectors by BCG may be increased, or indeed replaced by blockade of the immuno-inhibitory functions of the HLA-G molecule, and that longer-term effects may be observed. As mentioned above, BCG immunotherapy is only able to delay relapse. Regular monitoring of patients having undergone surgery for bladder cancer is therefore of paramount importance; however, due to the lack of tumour markers, monitoring takes place by endoscopy. During this study, we will therefore implement an assay method for the HLA-G molecule in urine, and will evaluate the value of this assay in postoperative monitoring.
Therapeutic applications
HLA-G is a molecule with decisive functions in disorders associated with tolerance and immune escape. It is therefore endowed with high therapeutic potential. The most obvious applications on the one hand involve the use of its tolerogenic functions for transplantation or its immune reaction inhibition properties (autoimmunity, chronic inflammatory disease), and also blockade of its functions to avoid immune escape (solid tumours, viral infections). With a view of the use of the HLA-G molecule in human therapy, our development activity is organized according to three themes directly resulting from our fundamental research: (i) the use of cells expressing HLA-G in cell therapy, (ii) the production of exogenous tolerogenic proteins or proteins inhibiting cell multiplication for transplantation and oncology, respectively, and (iii) anti-HLA-G DNA immunisation in addition to anti-tumoural DNA immunisation protocols.
Cell therapy
One of the pitfalls of cell therapy is the rejection of transplanted cells. In view of the tolerogenic properties of the HLA-G molecule, the therapeutic challenges are either the use of its immuno-tolerogenic properties in order to promote the implantation of transplanted cells, or the generation of cells expressing HLA-G for the tissue graft.
Induced pluripotent stem (iPS) cells are endowed with similar properties to those of embryonic stem cells and are obtained by the transduction of reprogramming genes into somatic cells, which justifies the interest they have gained in regenerative medicine Nevertheless, their use is constantly faced with the problem of the immunogenicity of their differentiated progeny, and rejection by the recipient. Expression of the HLA-G molecule by cells and tissues differentiated from iPS could increase their tolerance after transplantation.
The objectives of this project are as follows: (i) to study the expression of the HLA-G molecule by iPS and the mechanisms which lead to its re-expression during generation from HLA-G negative somatic cells, (ii) to transduce HLA-G into iPS in order to ensure its expression after differentiation and to demonstrate that this expression promotes their tolerance after transplantation in an animal model, and (iii) to investigate the function of HLA-G on stem cells.
Exogenous HLA-G proteins
Our objective is to generate proteins derived from the HLA-G molecule, with tolerogenic properties able to be developed for transplantation, on the one hand, and cytostatic properties able to be used for the treatment of liquid tumours, on the other hand.
The studies conducted over the period 2008-2013 enabled us to select an initial protein of interest: dimeric synthetic protein (a1-a3)x2 for its tolerogenic function in vivo and its inhibitory function on cell multiplication with regard to liquid tumours in vitro [3]. During the period 2013-2018, we will study other truncated HLA-G proteins and will continue our efforts along the lines of characterising the structures of this molecule which are involved in each of its functions in order to generate exogenous proteins capable of targeting either the HLA-G tolerogen, or its inhibitory function on cell multiplication in vivo.
Anti-HLA-G DNA immunisation
Expression of the HLA-G molecule by solid tumours constitutes an immune escape mechanism, which is also the case for the expression of HLA-G by cells invading the tumour. In the context of anti-tumoural immunotherapy, expression of HLA-G may thus constitute a factor for failure. Our studies, conducted in partnership with INVECTYS-Institut Pasteur, aim to develop an anti-HLA-G DNA immunisation protocol in order to generate blocking antibodies, and anti-HLA-G cytotoxic T lymphocytes. The aim of this research is to block the inhibitor effect of the HLA-G molecule during anti-tumoural DNA immunisation, such as that developed jointly with INVECTYS-Institut Pasteur. Our previous research enabled us to characterise the immunogenic sequences of the HLA-G molecule and to develop an anti-HLA-G DNA immunisation protocol in order to generate blocking antibodies, and also to demonstrate the generation of specific CTLs for HLA-G. We will be continuing this partnership in order to obtain the data necessary for a pre-clinical study. In particular, we will demonstrate the blocking character of the anti-HLA-G antibodies generated, and the ability of anti-HLA-G CTLs specifically to destroy a tumour target expressing this molecule. In vivo, we will establish that anti-HLA-G DNA immunisation is able to eliminate human and murine tumours expressing HLA-G, in humanised, class I, HLA, transgenic, immunocompetent mice.