Deletion of donor-reactive cells as a new conditioning regimen for allogeneic bone marrow transplantation.

Progress in the development of less toxic conditioning for bone marrow transplantation (BMT) came with the understanding that acceptance of mismatched BM does not require myeloablation of recipients. Lymphocyte deletion by a cocktail of immunosuppressive drugs is generally sufficient to ensure engraftment of compatible BM cells. However, reduced intensity conditioning (RIC) protocols available today do not provide robust tolerance to mismatched allogeneic BM. Herein we discuss 2 new experimental approaches to RIC protocols with the aim of facilitating allogeneic BM engraftment. Both conditioning regimens are based on selective deletion/inactivation of donor-reactive cells before BMT. Our data show that the first conditioning protocol, comprising priming of recipients by a donor-specific lymphocyte transfusion (DST) on day -2 and a single injection of cyclophosphamide, a drug that is predominantly toxic for proliferating cells, on day -1, consistently improves engraftment of allogeneic BM (day 0) in all experimental models tested. The second engraftment enhancing approach is based on the blockade by antagonistic reagents of the signaling pathways that govern the antigen-induced immune response. Combining the signaling blockade with the deletion of activated donor-reactive cells by cytoreductive agents provides additional benefits for transplantation across major histocompatibility barriers.

Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment.

Successful engraftment of hematopoietic stem cells requires a supportive hematopoietic stromal microenvironment (HSM). Defects in the HSM associated with aplastic anemia, myelofibrosis, or caused by intensive ionizing radiation and chemotherapy generally result in failure of bone marrow (BM) engraftment. Transplantation of donor BM within donor HSM may therefore provide optimal conditions for allogeneic BM transplantation. We have transplanted donor hematopoietic cells together with their own HSM to improve acceptance of allogeneic or xenogeneic BM. The non-myeloablative treatment used induced tolerance to murine allografts and provided conditions for the life-long acceptance of allogeneic HSM. Allogeneic BM transplanted within it's own HSM under the kidney capsule caused less graft-versus-host disease than BM transplanted i.v. Tolerance in mice to xenogeneic (rat) HSM was less complete. Ectopic ossicles were small and contained fewer hematopoietic cells. However, simultaneous transplantation of rat BM and HSM to preconditioned mice improved engraftment of rat BM compared with transplantation of BM alone. Donor hematopoietic cells survived longer on their own HSM than on HSM of recipients.

Nonmyeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice.

We recently described a new nonmyeloablative method to induce stable and specific transplantation tolerance to allogeneic tissues in adult mice. It included total lymphoid irradiation (TLI) of recipients with six fractions of 200 cGy each, inoculation with donor bone marrow (BM) cells and cyclophosphamide (Cy) for selective elimination or inactivation of residual donor-reactive cells of the host, and infusion with T-cell depleted donor BM cells after Cy. Here, we investigated the possibility to induce stable bilateral graft-vs-host and host-vs-graft transplantation tolerance using non-T-cell depleted allogeneic BM. Our results show that the dose of BM required for the induction of transplantation tolerance was inversely correlated with the intensity of the conditioning. Transfer of a low dose (3 x 10(6)) of total donor BM cells to recipients preconditioned with a less intensive regimen (two or three TLI fractions instead of six) diminished graft-vs-host disease (GVHD)-related mortality of recipients to 40% and converted 89% of the survivors into GVHD-free mixed hematopoietic chimeras that maintained donor skin allografts >180 days. A tenfold increase in the number of donor BM cells (3 x 10(7) instead of 3 x 10(6)) reduced the rate of GVHD-related mortality of recipients to 20% and resulted in bilateral transplantation tolerance in 100% of nonirradiated survivors.

Permanent and specific transplantation tolerance induced by a nonmyeloablative treatment to a wide variety of allogeneic tissues: I. Induction of tolerance by a short course of total lymphoid irradiation and selective elimination of the donor-specific host lymphocytes.

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.

Autoimmune response induction and regulation in rat erythrocyte-immunized mice.

The process of antibody formation to self-red blood cells (RBC) has been studied in rat RBC (rRBC)-immunized mice. A positive correlation was noted between antibody production to mouse RBC (mRBC) and rRBC in some mouse strains. The low responsiveness on both indices was overcome by s.c. injections of rRBC in low doses. rRBC-tolerant mice exhibited lower levels of antibody production to mRBC. Splenocytes from rRBC-immunized donors, when transferred to irradiated recipients, revealed enhanced and accelerated anti-mRBC and anti-rRBC antibody production in response to rRBC but not to autologous mRBC. Consequently, the autoimmune process is not accompanied by disordered immunologic tolerance to self-RBC and requires participation of Th responding to foreign epitopes of rRBC antigens. Splenocytes from rRBC-immunized donors, when transferred to non-irradiated recipients, inhibited antibody production to mRBC. The suppressive effect was not abrogated by pretreating donors or recipients with low doses of cyclophosphamide (CP) or by pretreating donors with antibodies to I-J. It was abrogated by the elimination of cells of donor origin within 8-9 days after transfer. Inoculation of antibodies to rRBC in immunized mice on a schedule imitating their splenocyte transfer dynamics inhibited antibody production to mRBC. Therefore, it can be assumed that the suppressive effect of cell transfer is accounted for, not by suppressors or their inducers, but by antibodies to rRBC on the basis of feedback regulation.

[Blood rheologic disorders in patients with polycythemia vera and their correction by therapeutic erythrocytapheresis]. / Gemoreologicheskie narusheniia u bol'nykh istinnoi politsitemii i ikh korrektsiia lechebnym éritrotsitaferezom.

The rheologic blood properties were studied in patients with polycythemia vera (PV) before and after erythrocytapheresis. The patients with PV showed a complex of hemorheologic disorders (high blood viscosity at different rates of deviation, intensified red blood cell aggregation, decreased deformability of these cells) found to be implicated in the disease pathogenesis. Erythrocytapheresis promoted the improvement of the rheologic characteristics such as dynamic blood viscosity and the red blood cell aggregation ratio.

[Antibody formation to autologous erythrocytes following the immunization with rat erythrocytes of normal animals and mice tolerant to the immunizing antigen]. / Formirovanie antitel k autologichnym éritrotsitam posle immunizatsii éritrotsitami krysy normal'nykh zhivotnykh i myshei, tolerantnykh k immuniziruiushchemu antigenu.

(CBA X C57B1/6)F1 mice immunized three times with rat erythrocytes produced antibodies both to this antigen and to autologous erythrocytes. Most of the antibodies to rat erythrocytes belonged to IgM isotype while antibodies to autologous red cells were of IgG isotype. Combined injection of thymectomized (CBA X C57B1/6)F1 mice with a massive dose of rat spleen cells and cyclophosphamide induced in animals stable tolerance to rat cells. Inducibility of antibodies to autologous red cells in tolerant mice injected 3-5 times with rat erythrocytes was drastically reduced. Nonspecific suppression (thymectomy and cyclophosphamide) did not prevent production of autoantibodies.

[Hemorheologic disorders in shock of diverse etiology]. / Gemoreologicheskie narusheniia pri shoke razlichnoi étiologii.

Comparative investigation of rats has been conducted to study rheologic blood properties during relative compensation and decompensation of the hemorrhagic, traumatic and burning shock. The most obvious hemorheologic disturbances have been revealed during a burning shock and feebly marked-during a hemorrhagic shock.

[Tolerance to a xenotransplant in an adoptive system]. / Issledovanie tolerantnosti k ksenotransplantatu v adoptivnoi sisteme.

Splenocytes of mice tolerant to rat neonatal heart graft were unable to respond to rat blood cells (RBC) when transferred adoptively to lethally irradiated syngeneic recipients 10 or 30 days after tolerogenic treatment. Early after induction of tolerance spleen cells of experimental mice were also unable to respond to sheep red blood cells. However, they responded vigorously to goose red blood cells. Later on (30 days after treatment) tolerance was found to be strictly RBC-specific. Cells suppressing anti-RBC response of intact cells were detected in the spleen of mice both 10 and 30 days after the induction of tolerance. Their suppressive activity was strictly RBC-specific. The results obtained show that early after tolerogenic treatment experimental mice are unable to respond due both to the deficiency of T-helpers involved in the response to mammalian blood cells and to activation of RBC-specific I-J+ T-suppressors. Thirty days after treatment tolerance is maintained solely by RBC-specific T-suppressor cells.

[Suppressors of the graft vs graft reaction in tolerance to alloantigens]. / Supressory reaktsii transplantat protiv transplantata pri tolerantnosti k alloantigenam.

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.

[Effectiveness of plasmapheresis on the PF-0.5 blood separator in patients with paraproteinemic hemoblastoses]. / Effektivnost' plazmafereza na fraktsionatore PF-0,5 u bol'nykh paraproteinemicheskimi gemoblastozami.

Plasmapheresis on a Soviet-made PF-0.5 blood cell separator was performed in 20 PPH patients (altogether 33 sessions of plasmapheresis). 1600 to 3300 ml of plasma were removed during one session. Rheopolyglucin was mainly used as plasma-substituting liquid. The procedure was well tolerated by all the patients. After plasmapheresis biochemical indices were within normal. In multiple myeloma paraprotein was decreased by 42%. Waldenström's macroglobulinemia by 47%. Plasma viscosity was decreased 1.2- and 2-fold, respectively. The investigations showed a high efficacy and reliability of plasmapheresis with the help of the Soviet-made PF-0.5.

Immunological status of mice with long-standing allogeneic or xenogeneic neonatal heart grafts.

Adult mice were thymectomized and given 1 X 10(8) allogeneic or xenogeneic spleen cells i.v. On the day following cell injection, the mice were treated with 200 mg/kg cyclophosphamide (Cy) i.p. Of the CBA mice treated according to this protocol, 87% accepted heterotopically transplanted C57BL/6 neonatal heart grafts and 61% accepted August rat heart grafts. Electrical activity of the grafts could be recorded in the majority of recipients for more than 5 months. Graft acceptance in most cases was specific. Some recipients with long standing rat heart grafts produced antirat hemagglutinins, but were negative for lymphocytotoxins. The absence of xenoantibodies correlated with specific unresponsiveness in the mixed lymphocyte culture (MLC) response.