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.

[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.

[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.

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.

Mechanisms of antigen-induced blockade of immune response and cyclophosphamide-promoted tolerance to Salmonella typhi Vi antigen.

Blockade of the immune response, caused by a high dose of Salmonella typhi Vi antigen (200 microgram i.v.) and cyclophosphamide (CY)-induced tolerance to Vi antigen, were analyzed. The results of the study show that blockade of the immune response cannot be attributed to masking of the response resulting from neutralization of antibodies by the excess of non-cell-bound antigen. A high dose of Vi-antigen induced triggering and proliferation of specific B precursors but reversibly suppressed synthesis or secretion of antibody by plaque-forming cells. A single injection of CY (200 mg/kg i.p.) 2 days after a high dose of Vi antigen markedly prolonged the antigen-induced state of unresponsiveness. CY-induced tolerance to Vi antigen is due to elimination or long-term inactivation of specific B precursors. Dissimilarities in the characteristics of immune response blockade and CY-induced tolerance are discussed as well as their possible implications for the mode of action of CY.

Tolerance to allogeneic and to xenogeneic heart grafts provided by thymectomy of adult mice combined with donor cell and cyclophosphamide inoculation.

A new method of induction of tolerance to allogeneic and to xenogeneic cells is presented. It includes thymectomy of adult mice followed 1 month later by the injection of 1 X 10(8) spleen cells i.v. and i.p. administration of 200 mg of cyclophosphamide per kg 1 day after cells. This method induced prolonged survival of heterotopically transplanted neonatal C57BL/6 murine heart grafts (more than 8 months) and of August rat heart grafts (more than 2 months) in CBA mice. Tolerance to allo- or xenoantigens was formed at the cell level. Experimental animals did not produce allo- or xenohemagglutinins after graft implantation. Spleen cells of mice with surviving C57BL/6 heart grafts did not respond to C57BL/6 cells in mixed lymphocyte culture (MLC) reaction. Lymphoid cell chimerism was not observed in animals tolerant to alloantigens.

[Immune response and tolerance to S. typhi Vi-antigen in T-lymphocyte depleted mice]. / Immunnyi otvet na Vi-antigen S. typhi i tolerantnost' k nemu u myshei, lishennykh T-limfotsitov.

Comparison of immune response to Vi-antigen in thymetomized letally irradiated and reconstituted with fetal liver cells mice and in control animals revealed no difference between the two groups. The absence of enchancement of antibody formation in T cell depleted mice favours thymic-independent regulation of immune response to optimal dose of Vi-antigen. The induction of cyclophosphamide tolerance to Vi-antigen did not depend on the presence of T cells: tolerogenic treatment was equally effective in T cell depleted mice and in control animals. Therefore cyclophosphamide tolerance was not due to the activation of T suppressors but to direct elimination of immunocompetent clones of B cells.

[Specific blockade of the immune response caused by an excessive dose of S. typhi Vi-antigen]. / Spetsificheskaia blokada immunnogo otveta, obuslovlennaia izbytochnoi dozoi Vi-antigena S. typhi.

Intravenous injection to adult mice of Vi-antigen (200 microgram) induces in them the state of a short-term (10--12 days) unresponsiveness. This is due to the block of immunocompetent cells, not to masking the antibodies production by the excess of free antigen. Double washing to spleen cells before the test of local passive hemolysis in gel failed to reverse the block of the immune response; besides, there was no free antigen in the spleen that could inhibit the antibodies produced by the cells of the immune animal. The immune response block can be reversed by the injection of heterologous antiserum to Vi-antigen 18 to 24 hours before the Jerne's test. The restoration of immune response by means of the antiserum is prevented by the administration of 6-thioguanine after Vi-antigen (200 microgram). Thus, administration of a massive dose of Vi-antigen failes to block the proliferation and differentiation of the antigen-recognizing cells, but depresses the synthesis or secretion of antibodies.

[Mechanism of tolerance to VI-antigen of Salmonella typhi induced by cyclophosphane]. / Mekhanizm tolerantnosti k VI-antigenu Salmonella typhi, indutsirovannoi s pomoshch'iu tsiklofosfana

High dose Vi-antigen treatment and injection of cyclophosphamide 46 to 48 hours later induced in mice a state of immunological unresponsiveness remaining stable in adoptive transfer. Only low amounts of the antigen were revealed in the blood and spleen of tolerant animals 2 to 3 weeks after the tolerogenic treatment. No T-suppressors were found in the spleen of tolerant mice--the cells of tolerant mice failed to suppress the immune response of normal lymphocytes when transferred together to the irradiated recipients, or to induce tolerance in normal mice. Normal spleen cells restored partially the immune responsiveness in tolerant animals. The results obtained suggest that cyclophosphamide tolerance was due to deletion or the long-term inactivation of the immunocompetent cells.

[The potential nuclease activity of the T antigen of SV-40 virus]. / O vozmozhnoi nukleaznoi aktivnosti T-antigena virusa SV-40

SV40 T-antigens were isolated from an extract of golden hamster tumours by precipitation with ammonium sulphate with subsequent fractionation on DEAE cellulose. The degree of purification of the preparation proved to be about 100-fold; it, however, contained an admixture of several cell proteins. Treatment of the DNA of the calf thymus with the T-antigen preparation in the presence of magnesium ions decreased the viscosity of the DNA solution during the first hour of incubation. T-antigen inactivated by heating, and also a fraction of normal hamster tissues analogous to it produced no such effect. In case of centrifugation in the saccharose gradient the constant of DNA sedimentation fell after the treatment with T-antigen from 285 to 165, this corresponding to about4--5-fold reduction of molecular weight of the DNA. The data obtained indicated that the partially purified T-antigen preparation possessed endonuclease activity.