FR2777906A1 - Obtaining human dendritic cells from monocytes, useful for modifying immune response and for treating autoimmune and allergic diseases - Google Patents

Abstract

A process for obtaining human dendritic cells from monocytes (MODC) in the presence of interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF- alpha ) is new. A process for obtaining human dendritic cells from monocytes (MODC) in the presence of interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF- alpha ) is new and comprises several crucial technical parameters by cultivating the monocytes comprising: (a) the addition of 1-3 mg/ml bicarbonate to the culture medium; (b) maintaining the pH at 7.2-7.4; (c) culturing in Teflon pots; and (d) using 1-25% autologous/homologous human serum.

Description

DESCRIPTION

  Indication of the technical field The present invention relates to a process capable of generating cells

  human dendritics from monocytes.

  Dendritic cells play a critical role in the emergence of the immune response. In fact, only dendritic cells are capable of inducing a primary response from T lymphocytes. They are therefore the most important cells for the initiation of the immune response. Such a function is to be compared to many surface properties. Indeed, thanks to large expansions of their cytoplasmic membrane, the dendritic cells have a particularly large contact surface with the outside. In addition, they have on their surface a large number of class I and class II histocompatibility antigens. They are very rich in costimulation molecules, such as the molecules CD80, CD86, CD40 which activate the molecules CD28, CTLA-4 and CD40L respectively by initiating the immune response. They also have very many adhesion molecules, such as the CD54 molecule, the LFAI molecule, which facilitates cooperation between dendritic cells and T cells. Another particular characteristic of dendritic cells is to deploy different functions depending on of their stage of differentiation. Thus the capture of the antigen and its processing are the main functions of the dendritic cell while the capacities to stimulate the T cells increase as the dendritic cells migrate in

tissues and lymph nodes.

  Indication of the state of the art These dendritic cells are capable of presenting tumor antigens in experimental systems so as to induce a cytotoxic response against the cells

  tumor. These cells therefore have a very special interest in tumor immunotherapy.

  Until the description of our process, the isolation of dendritic cells from the blood

  peripheral was very difficult since less than I% of mononuclear cells belong to this category. An important advance was made when it was possible to generate dendritic cells from myeloid precursors and monocytes in the presence of different cytokines. The production of dendritic cells from monocytes can be done in the presence of GM-CSF and IL-4 to obtain a maturation after contact with TNFcx or with other

  agents such as CD40 ligand, LPS or media conditioned from macrophages.

  However, due to the differentiation of monocytes into macrophages in the presence of human serum, it has always been necessary to resort to bovine sera, which has restricted the use

  of dendritic cells in human therapy.

  Disclosure of the invention Our invention. by modifying the culture medium and conditions, aims to avoid this drawback and to be able to cultivate dendritic cells derived from monocytes, in the presence of human serum, in particular by controlling the pH and starting from purified monocytes devoid of T cells. more. our procedure prevents human serum from being an inhibitor of

  differentiation of dendritic cells.

  Similarly, our invention has established that the use of autologous plasma

  works as well, and sometimes even better, than the homologous serum.

  General procedures for producing dendritic cells Peripheral blood is taken from the subject concerned. This blood is then centrifuged for minutes at 200 g so as to reduce the contamination of peripheral blood cells by platelets. The upper part containing most of the platelets and the plasma is removed carefully before purifying the mononuclear cells by centrifuging them on a Ficoll separation gradient whose density is 1.077. The layer of mononuclear cells is recovered then washed twice in PBS buffer and deposited on a gradient containing 4 discontinuous densities of Percoll to isolate the monocytes. This gradient consists of dilutions of an isotonic percoll solution at a rate of 75% (6.5 ml), 50.5% (15 ml),% (3.5 ml) and 30% (3 ml) in a medium. from Dulbecco without magnesium or calcium and containing 5% human serum. 75 to 100 million cells are thus deposited on each tube and centrifuged at 1000 g for 25 minutes at 4 C. The low density cells, mainly the monocytes, are harvested at the 50.5% interface and washed twice in PBS. They are then resuspended in a culture medium, then deposited in culture plates containing 6 wells at a density of 5 × 10 6 per well in a final volume of 3 ml and left to adhere for 1 hour at 37 C. This can be substituted adhesion step by further purification of the monocytes using a negative purification system using a mixture of monoclonal antibodies combining an anti-CD3, CD7, CD19, CD45A, CD56 and anti-IGE using a Macs-type microbeads. This additional purification makes it possible to obtain concentrations of monocytes having a purity

greater than 90%.

  The cells are then cultured in culture wells at 37 ° C. under 5% CO 2. The medium which comprises RPMI contains 200 U / ml of recombinant human GM-CSF, 500 U / ml of recombinant human IL-4 in a final volume of 6 ml. On day 3 and on day 5, the cultures are renewed by eliminating 3 ml and adding 3 ml of fresh medium with the cytokines. On the 6th day, the cells are transferred into Teflon pots and cultured at a density of 5 × 10 5 cells per pot under 3 ml in the presence of recombinant human TNFcx at the rate of 200 U / ml for 2 days. The cells are harvested on the 8th day, washed and cultivated in

  absence of stimulation for 3 additional days.

  During this entire culture, the pH of the culture medium is closely measured and corrected with bicarbonate between 1 to 2%, ie an addition of between 1 to 3 mg / ml. It is advisable, in this technique, to closely monitor the pH which must be understood very exactly between 7.2 and 7.4 and especially never fall below 7.15 in the presence of 1 to 25% of human serum

  autologous or homologous (on average 10%).

  At the end of the culture, the quality of the maturation is evaluated by the expression of the molecules CD80, 83, 86, 14, la, HLA-DR on the surface of the cells. These markings reveal that more than 80% of viable monocytes have matured into dendritic cells characterized by the following phenotype: CD83 +, CD86 ++, CD80 ++, HLA-DR +++, CDla-, CD14-, CD401 +++,

  CD54 ++, which places them in the group of highly differentiated tissue dendritic cells.

  The use of this technique could be carried out in the following examples: Example 1: Production of dendritic cells to identify minor antigens

histocompatibility.

  This technique consists in recovering monocytes by making them differentiate into mature dendritic cells, then using them as stimulating cells in a mixed lymphocyte culture reaction between individuals of the same HLA compatible family. It turns out that, in this system, we manage to detect disparities concerning minor histocompatibility antigens and to be able to deepen the compatibility or incompatibilities between persons of the same family, which has definite advantages in the field of

  intra-family or even extra-family tissue and organ transplants.

  2nd example: Use of dendritic cells from MODC monocytes in

  the emergence of an antitumor response.

  To do this, we chose the system of a tumor antigen expressing itself strongly in medullary thyroid cancers. It is thyrocalcitonin which is relatively weakly immunogenic. In our system, we were able to produce T lymphocyte clones directed against thyrocalcitonin. These are cytotoxic clones capable of having anti-tumor activity in thyrocalcitonin cancers. A clinical study is underway for

validate this system.

  3rd example: Use of MODC in anti-infectious activities.

  By using tetanus toxoid as the antigen presented by the MODCs, it was possible to generate cytotoxic clones against tetanus toxoids, which clearly demonstrates that

  this system allows for primary immunization against infectious type antigens.

  4th example: Use of MODCs in inducing a tolerance response.

  Indeed, it is clear that dendritic cells, under certain culture conditions, can cause an anergy reaction. It turns out that by growing these cells in the presence of immunosuppressants such as cyclosporine or histamine, we cause a modification of the membrane antigens which will induce a tolerance response and not a cytotoxic response. In this system, it is therefore possible to educate dendritic cells to orient them towards a tolerance reaction. This finding could have important implications for organ transplants on the one hand, and treatments for autoimmune diseases on the other hand, such as rheumatoid arthritis, myasthenia gravis, diabetes.

  insulin dependent, multiple sclerosis, eczema, psoriasis, etc ... and allergic diseases.

  th example: Changes in the expression of surface antigens by the culture system In our culture system, the dendritic cells produced have a very specific assortment of surface antigens. Indeed, compared to a culture in the presence of fetal calf serum. in the presence of bicarbonate, there is a strong expression of the molecule CD54, CD80, especially CD83 and CD40. This is even clearer for molecules

CD83 and CD86.

  6th example: Use of MODCs to induce a mixed lymphocyte culture

allogeneic or even autologous.

  In the presence of MODC, allogenic lymphocytes. on the 6th and even on the 8th day, are endowed with an extremely significant proliferation, very much greater than that which is observed with the use of allogeneic B lymphocytes or allogenic monocytes. It

  the same is true for a mixed autologous culture.

Claims (5)

Claims   1) Process for obtaining human dendritic cells from monocytes (MODC) in the presence of IL-4 + GM-CSF + TNFc <comprising several crucial technical parameters characterized by the fact that the monocytes are cultured by combining: - the addition of bicarbonate to the culture medium at a rate of I to 3 mg / ml - the maintenance of the pH between 7.2 and 7.4 - the culture in Teflon pots   - the use of autologous or homologous human serum from 1 to 25%.   2) Method according to claim 1 characterized in that the addition of autologous or homologous human serum does not inhibit towards the phase of differentiation into dendritic cells tissue type.   3) Method according to claim 1 characterized in that the dendritic cells thus obtained can be used in techniques for detecting and characterizing antigens histocompatibility minors.   4) Method according to claim 1 characterized in that the MODC allow to obtain in vitro an immune response vis-à-vis tumor antigens used in antitumor immunotherapy. ) Method according to claim 1 characterized in that the MODCs are capable of presenting the antigens of microorganisms capable of being used in therapy anti-infectious.   6) Method according to claim 1 characterized in that the MODCs thus produced can modify the immune response in the sense of tolerance or treatment of diseases autoimmune and allergic.