Xenogreffe humain-souris scid et immunisation des lymphocytes humains en milieu murin
(1996)
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- The establishment of the hybridoma technology by Kohler and Milstein in 1975 gave the opportunity to use monoclonal antibodies (mAbs) in many areas oh human therapy and diagnosis. Presently, the mAbs available are of mouse or rat origin. Unfortunately the use of these rodent antibodies in human leads to xenogenic immunization limiting their efficacy. The production of human mAbs would solve this problem. It means that human B lymphocytes must be immunized and immortalized in order to produce human mAbs. But deliberate human in vivo immunization is restricted to vaccination for athical reasons. Thus, we decided to try to establish a human immunization system outside of human organism. <BR> Attempt to establish a human primary in vitro immunization system was unsuccessful in our experimental conditions. Thereupon, we devoted this work to the reconstitution of severe combined immunodeficient (scid) mice with human peripheral blood mononuclear cells (Hu-PBMC) in order to immunize human lymphocytes in vivo, in a murine environment. <BR> In this work; we coupled antigens [Canine albumin (Calb), or Dinitrophenyl (DNP)] with tetanus toxoid (TT), an antigenic protein against which our human donors had already memory T cells by vaccination. By this way, hu-scid immunised with couple DNP-tetanus toxoid (TT-DNP) or coupled Canine albumin-Tetanus toxoid (Calb-TT) mounted a specific human immune response anti-DNP or anti-Calb respectively. A secondary human immune response anti-tetanus toxoid was also detected in the sera f hu-scid immunised with products containing TT but not in the sera of those injected with phosphate buffer saline (PBS) alone. The scid mice grafted with Hu-PBMC from TT naive donor and challenged with Calb-TT or Calb alone failed to produce anti-Calb specific antibodies. These observations demonstrate that memory T cells can give a substantial help to naive B cells which are in contact with them for obvious B cell activation and differentiation to plasma cells. This model of immunization combined to a suitable system of immortalization might be useful for the other antigens of choice to obtain human monoclonal antibodies. Attempts to immunise human cells in scid mice against DNP couple to a rat monoclonal antibody anti-human IgM (LO-BM2) failed to induce a specific human response either anti rat immunoglobulins (Igs), or anti-DNP and led to a decrease of human Ig production in hu-scid. <BR> We also immunized hu-scid against ovalbumin alone but, only in some cases a low specific human immune response was observed and this system seems to be unreliable. <BR> In order to optimise the construction of hy-scid chimeras with human peripheral blood mononuclear cells (Hu-PBMC), the effect of two cytokines were studied. Hu-PBMC were incabuted in vitro with human recombinant interleukin-4 §HurIL-4) or human recombinant interleukin-2 (HurIl-2) and were intraperitoneally transferred into scid mice while HurIL-2 did not show any obvious effect. Thus, HurIL4 can be used as an adjuvant growth factor which improves successful engraftment of human peripheral blood lymphocytes in scid mice. <BR> The construction of human scid mouse has some limitations due to the variability in the graft take, the long experimental time, the cost of the system, and sometime the premature death of the mice, but also the development of human B lymphoproliferative syndrome (Hu-BLPS). The experiments reported in this work show how crucial is the presence of functional T lymphocytes for graft take and development f Hu-BLPS in Hu-PBMC-reconstituted scid mice since inhibition of T lymphocytes by a rat anti-human CD2 monoclonal antibody (LO-CD2a) during the first ten days of graft, prevent successful engraftment of human normal lymphocytes as well as Hu-BLPS in scid mice. The transfer of B cells alone or B cells plus monoclonal in scid mice does not permit either long-term engraftment or development of Hu-BLPS. We also demonstrate that L-Leucine methyl ester (LeuOME) treated Hu-PBMC are less susceptible to develop Hu-BLPS after engraftment in scid mice than untreated Hu-PBMC. The mechanism of action of LeuOME on these cells in discussed
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Bombil, F. (1996). Xenogreffe humain-souris scid et immunisation des lymphocytes humains en milieu murin. https://hdl.handle.net/2078.5/110981