Optimizing chimerism level through bone marrow transplantation and irradiation to induce long-term tolerance to composite tissue allotransplantation.

BACKGROUND: Mixed chimerism with long-term composite tissue allotransplant (CTA) acceptance can be achieved through allogeneic bone marrow transplantation (BMT). The present study investigated the optimal chimerism level by giving different irradiation dosages to recipients to induce tolerance to CTA. METHODS: Chimera were prepared using Brown-Norway and Lewis rats with strong major histocompatibility complex incompatibility. The Lewis rats received 5 mg antilymphocyte globulin (day -1 and 10) and 16 mg/kg cyclosporine (day 0-10) and were separated into groups 1, 2, 3, 4, and 5 according to the day -1 irradiation dosage: 0, 200, 400, 600, and 950 cGy, respectively. The Lewis rats were then reconstituted with 100 × 10(6) T-cell-depleted Brown-Norway bone marrow cells (day 0) and received vascularized Brown-Norway-CTA on day 28. Chimerism was assessed monthly by flow cytometry starting on day 28 after BMT. Graft-versus-host disease (GVHD) was assessed clinically and histologically. RESULTS: Chimerism, 4 weeks after BMT, averaged 0.2%, 9.2%, 30.7%, 58%, and 99.3% in groups 1 to 5, respectively. GVHD occurred as follows: groups 1 and 2, none; group 3, 1 case of GVHD; group 4, 7 cases of GVHD (of which 3 died); and group 5, 10 cases of GVHD (of which 6 died). The percentage of long-term CTA acceptance was 0%, 0%, 90%, 70%, and 40% in groups 1 to 5, respectively. The percentage of regulatory T cells was significantly lower in high-chimerism (≥ 20%, n = 15) than in low-chimerism (<20%, n = 5) rats that accepted CTA long-term . CONCLUSIONS: The chimerism level correlated positively with GVHD occurrence and long-term CTA acceptance but correlated negatively with regulatory T-cell levels. Optimal chimerism for CTA acceptance through pre-CTA BMT and irradiation occurs at 20-50% at day 28 after BMT in the rat model.

Radioresistant inhibition of lymphoma growth in congenitally athymic (nude) mice.

Several murine tumors were used to determine whether the phenomenon of tumor inhibition in athymic "nude" mice reported previously could be extended to other tumor systems in nude as well as conventional mice. The results with the L5MF-22 tumor line were confirmed, and similar data were obtained with the K36 leukemia of AKR mice and the LAF-17 leukemia of B10.A origin. This phenomenon of tumor inhibition has been called, tentatively, radioresistant inhibition of tumor and may be explained by one of several possibilities. The immunological origin of such tumor inhibition is supported by various observations. The data on tumor cell proliferation in spleens and liver of lethally irradiated mice were similar to previous findings on hemopoietic histocompatibility-incompatible lymphomas. Additionally, the nude mice were stronger responders against lymphoma cells than were conventional hosts. Another explanation is that the tumor inhibition is due to natural cytotoxicity.

Effects of X-irradiation on colony specificity in the compound ascidian, Botryllus primigenus Oka.

In a compound ascidian, Botryllus primigenus Oka, colony specificity or histoincompatibility has been known. Colony specificity is manifested by fusibility between colonies (fusion or nonfusion). It has been suggested that fusibility of a colony depends upon blood and that cellular and humoral factors may participate in the "nonfusion" reaction (NFR), which is induced in the contact area between two incompatible colonies. In the present study, effects of X-irradiation on colony specificity were studied. When the cut surfaces of two colony pieces previously irradiated were brought into contact, only weak NFR took place in the contact area, although fusion has never occurred. By the use of experimental alteration of fusibility, "fusibility alterable index" (FI) of the irradiated colonies were estimated. FI was inversely proportional to dose of X-irradiation. FI of a colony received 3,000 R corresponds to that of about one-fourth in size and FI of a colony received 5,000 R corresponds to that of about one-eighth in size. Therefore, a factor(s) controlling fusibility may be sensitive to X-irradiation.

[The effect of iron carrier proteins on the transplantation of H-2 locus-incompatible bone marrow in irradiated mice]. / Vliianie zhelezonesushchikh belkov na transplantatsiiu nesovmestimogo po H-2 lokusu kostnogo mozga obluchennym mysham.

Rabbit bone marrow supernatants were fractionated and purified by Ultrogel and Superose chromatography. A unique fraction promoted engraftment of allogenic bone marrow and enduring hemopoietic chimerism across the histocompatibility (H-2) barrier in lethally irradiated mice. This fraction analysed by reducing SDS-PAGE electrophoresis and transblotted on PVDF membrane or purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main pre-albumin band that was examined for primary structure by Edman degradation. It appeared to be rabbit transferrin. Iron saturated human transferrin, lactotransferrin and egg transferrin (conalbumin) were then tested in irradiated C57B1/6 mice transplanted with bone marrow from histoincompatible BALB/CJ donors. Most mice treated with iron-loaded transferrins survived and developed enduring allogeneic chimerism with no discernible signs of graft-versus-host disease at 10 months posttransplant. Observation of these animals is still carried on. Iron carrier proteins seem to provide a novel unexpected means for achieving a successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations and may open a new approach in the clinical area.

Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody.

Administration of a rat monoclonal antibody (M7/20) directed against the murine interleukin 2 (IL 2) receptor in combination with sublethal x-irradiation of the recipient significantly enhanced the survival of skin allografts, both when the grafts were MHC disparate from the hosts and when only minor histocompatibility differences were present compared with untreated controls. No prolongation in graft survival was seen with either treatment alone at the dose employed. M7/20 and x-ray-treated allograft recipients also displayed significantly decreased alloantigen-specific reactivity against donor-strain spleen cells in both delayed-type hypersensitivity and cytotoxicity assays. Thus, such combination treatment reduces expression of host immune reactivity against graft determinants by several criteria. This work provides additional evidence that monoclonal antibodies directed against the IL 2 receptor may be useful in clinical transplantation.

Prevention of graft-versus-host disease while preserving graft-versus-leukemia effect after selective depletion of host-reactive T cells by photodynamic cell purging process.

In this study, we investigated the possibility of selective depletion of donor alloantigen-specific T cells from C57BL/6 (H-2(b)) mice to prevent graft-versus-host disease (GVHD). These cells were first activated with irradiated BALB/c (H-2(d)) host spleen cells in a 5-day mixed lymphocyte culture. Following this activation, a photoactive rhodamine derivative called 4,5-dibromorhodamine 123 (TH9402), was added. This compound is selectively retained in the mitochondria of activated host-reactive cells but not tumor- or third-party-specific resting cells. The treated cells were subsequently exposed to visible light (514 nm) to deplete the TH9402-enriched activated host-reactive cells. Treatment with photodynamic cell purging process (PDP) inhibited antihost responses measured by cytotoxic T lymphocytes (CTL) by 93%, and interferon-gamma production by 66%. By contrast, anti-BCL1 (BALB/c-origin leukemia/lymphoma) and anti-third-party C3H/HeJ (H-2(k)) responses were preserved. PDP-treated primed C57BL/6 cells were further tested in vivo. All lethally irradiated BALB/c mice inoculated with BCL1 cells and T-cell-depleted bone marrow cells developed leukemia by day +30, with 50% mortality by 100 days. All mice died of GVHD after addition of 5 x 10(6) untreated primed C57BL/6 cells. However, addition of same numbers of PDP-treated cells allowed 90% of the recipients to survive more than 100 days without detectable BCL1 tumor cells and free of GVHD. Moreover, PDP-treated primed C57BL/6 cells retained the ability to induce GVHD in the third-party C3H/HeJ mice. These data suggest that PDP can selectively deplete host alloantigen-specific T cells for GVHD prevention and immune and antileukemia function preserve.