Ido Lubin

Human monoclonal antibodies specific to hepatitis B virus generated in a human/mouse radiation chimera: the Trimera system.

An approach to develop fully human monoclonal antibodies in a human/mouse radiation chimera, the Trimera system, is described. In this system, functional human lymphocytes are engrafted in normal strains of mice which are rendered immuno‐incompetent by lethal total body irradiation followed by radioprotection with severe combined immunodeficient (SCID) mouse bone marrow. Following transplantation, human lymphocytes colonize murine lymphatic organs and secrete human immunoglobulins. We have established this system as a tool to develop fully human monoclonal antibodies, and applied it for the generation of monoclonal antibodies specific for hepatitis B virus surface antigen. A strong memory response to hepatitis B surface antigen was elicited in Trimera engrafted with lymphocytes from human donors positive for antibodies to hepatitis B surface antigen. The human specific antibody fraction in the Trimera was 102–103‐fold higher as compared with that found in the donors. Spleens were harvested from Trimera mice showing high specific‐antibody titres and cells were fused to a human–mouse heteromyeloma fusion partner. Several stable hybridoma clones were isolated and characterized. These hybridomas produce high‐affinity, IgG, anti‐hepatitis B surface antigen antibodies demonstrating the potential of the Trimera system for generating fully human monoclonal antibodies. The biological function and the neutralizing activity of these antibodies are currently being tested.

Engrafted human T and B lymphocytes form mixed follicles in lymphoid organs of human/mouse and human/rat radiation chimera

BACKGROUND We recently described a new approach that enables the generation of human/mouse chimera by adoptive transfer of human peripheral blood mononuclear cells into lethally irradiated normal strains of mice or rats, radioprotected with bone marrow from donors with severe combined immune deficiency. In such human/mouse chimera, a marked humoral response to recall antigens, as well as a significant primary response to keyhole limpet hemocyanin, has been generated. METHODS In the present study, the organ distribution of the engrafted human cells in the human/mouse and human/rat chimera was investigated by immunohistochemistry. RESULTS Our results show that the T cells seem to be distributed throughout the reticular endothelial system, almost behaving like particles without any homing specificity. The B cells, however, can barely be found in internal organs, such as the liver or the pancreas, and are concentrated in the secondary lymphoid system (e.g., spleen, lymph node, and nonencapsulated lymphoid tissue). The B cells, together with the engrafted human T cells, form mixed lymphoid follicles. CONCLUSIONS The different homing patterns exhibited by the T and B lymphocytes indicate that the homing receptors on human B cells might be cross-reactive with their mouse counterparts, in contrast to the human T cells, which seem to be unable to interact with the mouse homing receptors. The presence of human B and T lymphocytes in close proximity to each other in the lymphoid tissues is in accordance with the ability of human/BALB radiation chimera to mount significant primary human antibody responses.

Conversion of normal rats into scid-like animals by means of bone marrow transplantation from scid donors allows engraftment of human peripheral blood mononuclear cells

We have recently shown that lethally irradiated normal strains of mice, radioprotected with SCID bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). We now demonstrate that lethally irradiated Lewis rats can also be radioprotected with a transplant of SCID bone marrow cells, administered 1 day after total body irradiation. Split chimerism was found in PBMC, 30 days after transplantation, with predominance of SCID donor-type cells. The average percentages of CD4 and CD8 T cells, of mouse or rat origin, were < 1%. This chimerism status could be maintained for over 3 months. When human PBMC (300-1000 x 10(6) cells) were transplanted intraperitoneally 1 day after the administration of SCID bone marrow, prompt engraftment of human CD4 and human CD8 T cells, as well as human CD20 B cells, was found in the peritoneum and in internal organ (such as liver, lung, spleen, thymus, and lymph nodes). T cell activation was high: about 50% of the cells expressed HLA-DR and almost all expressed CD45RO. High titers of human Ig (> 1 mg/ml) were initially found after 2 weeks; these levels were similar to those found in the irradiated mouse model and in the SCID model. Likewise, marked human anti-tetanus response, predominantly of the IgG type, was recorded 2 weeks after the immunization, reaching maximal levels at 4 weeks. The triple-chimeric SCID-like rats, which accept as much as 1000 x 10(6) human PBMC, can potentially be used to elicit both antibody responses and T cell responses against specific antigens, with the advantages of a larger animal.

Human → mouse radiation chimera do not develop Epstein-Barr virus lymphoma

It has been shown that engraftment of human peripheral blood lymphocytes (PBL) from Epstein-Barr virus (EBV) seropositive donors in C.B-17/SCID mice is associated with a high incidence of human B cell tumors. More recently, we described a new approach enabling engraftment of human PBL in normal strains of mice or rats receiving lethal split-dose radiation and radioprotected with SCID bone marrow. We now demonstrate that, in contrast to SCID recipients of human PBL, Balb/c and C3H/HeJ recipients of 50-100 x 10(6) human PBL did not develop any EBV lymphoma during a 7-month follow-up period, but were successfully engrafted with human B and T cells. On the other hand, lymphoma developed in 90% of the C.B-17/SCID mice infused with 70 x 10(6) human PBL from the same donor. Likewise, 36% of beige/nude/xid (BNX) mice, exposed to 12 Gy TBI, radioprotected with SCID bone marrow and then transplanted with human PBL developed lymphoma. Similar results were obtained when different strains were infused with PBL of the same donor. Immunohistochemical analysis indicated that the tumor cells were of human B cell origin and expressed the EBV-encoded latent membrane protein-1 and nuclear antigen 2. While further studies are required to understand the mechanisms which suppressed outgrowth of EBV lymphoma in human --> mouse radiation chimera, compared to human --> C.B-17/SCID or human --> BNX chimera, this marked resistance offers new possibilities for transplantation of hematopoietic tissues or cells from EBV-positive donors.

Lethally irradiated normal strains of mice radioprotected with SCID bone marrow develop sensitivity to low doses of staphylococcal enterotoxin B.

Normal strains of mice are rendered sensitive to small amounts (3-10 micrograms) of staphylococcal enterotoxin B (SEB) by transplanting bone marrow cells of SCID donor mice to lethally irradiated recipients. Four to 12 weeks post-transplantation, SEB induces 56-100% lethality. Transplantation of normal mouse bone marrow cells, either alone or with the SCID mouse selected bone marrow cells, does not confer SEB sensitivity. These data imply that either irradiation ablates certain cell population(s), that confer resistance to SEB in normal mice (populations that are absent in the SCID donor mice) or that the donor cells selectively repopulate recipients with SEB-sensitive cells. This model will help elucidate the cells, cytokines and the SEB peptide fragments responsible for SEB toxicity and will be useful in identifying promising vaccine candidates and in developing preventive medicines to protect against this potent toxin.

Natural antibodies do not inhibit xenogeneic transplantation of human PBL in lethally irradiated mice

Abstract: Attempts to transfer human peripheral blood lymphocytes (hu‐PBL) into lethally irradiated mice resulted in limited engraftment in recipients lacking natural antibodies (nAb) and could not be achieved in immunologically normal mice. It has been proposed that nAb with antihuman specificity play a major role in the rejection of the hu‐PBL graft. In the present study we demonstrate that, following intensification of the conditioning protocol (thymectomy, supralethal dose of TBI, and radioprotection with bone marrow for donors with severe combined immune deficiency (SCID), transplants of 50 to 70 × 106 hu‐PBL were successfully engrafted in BALB/c, CBA/J and C3H/HeJ mice—regardless of the initial high levels of nAb. The percentage of human CD45+ cells in peritoneal lavage was not statistically different from that obtained in congenitally immune‐deficient corresponding strains (SCID and CBA/N) lacking natural antibodies. Significant differences in engraftment of hu‐PBL, between different human donors, were related neither to the nAb content (r = 0.29) nor to the ABO(H) blood group. The transfer of serum with high level of nAb into SCID and CBA/N mice or incubation of hu‐PBL in such a serum prior to implantation, did not impede the engraftment and did not decrease the production of human immunoglobulins. These data demonstrate that the presence of nAb in supralethally irradiated normal mice does not inhibit the engraftment of hu‐PBL, emphasizing the role of cellular mediated mechanisms in xenograft rejection.