Allogeneic Chimerism Research Articles

Inhibition of resistance to hemopoietic allo-grafts in granulocyte colony-stimulating factor transgenic mice.

Transplanted allogeneic marrow cells often fail to engraft in a lethally irradiated host. This phenomenon, termed resistance to allogeneic marrow grafts or alloresistance, is well documented, although its mechanism is not yet understood. Transplantation of major histocompatibility complex disparate allogeneic marrow cells into mice transgenic for granulocyte colony-stimulating factor (G-CSF) showed donor-derived spleen colonies (CFU-S) and resulted in stable allogeneic chimerism with excellent survival (100% up to 40 days and 89% up to 120 days). Under the same experimental conditions, all the littermate controls failed to show CFU-S and died shortly after marrow transplantation. Thus, resistance to allogeneic marrow cells appeared to be severely impaired in this transgenic mouse. The observation that neutralizing antibody against G-CSF restored allo-resistance in G-CSF transgenic mice and that CFU-S was inducible upon administration of recombinant G-CSF using a mini-osmotic pump in nontransgenic recipients, suggests that an elevated level of this cytokine is important for the inhibition of allo-resistance. Thus, G-CSF was found to play a role in allogeneic resistance to marrow grafts and the G-CSF-transgenic mice provide a useful model to study the inhibition of the resistance. The inhibition of allo-resistance may be useful in preparing allogeneic bone marrow chimeras in both experimental and clinical settings.

A nonlethal conditioning approach to achieve durable multilineage mixed chimerism and tolerance across major, minor, and hematopoietic histocompatibility barriers.

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.

Mixed allogeneic chimerism achieved by lethal and nonlethal conditioning approaches induces donor-specific tolerance to simultaneous islet allografts.

We previously reported that donor-specific, but not third party, skin allografts were permanently accepted if mixed allogeneic (B10+BR-->B10) reconstitution and skin graft placement were performed sequentially or simultaneously in lethally conditioned (950 cGy) recipients. The purpose of the present study was to examine whether a similar outcome would occur if islets were placed coincident with the time of bone marrow infusion and to establish the minimum dose of cytoreduction sufficient to achieve chimerism and tolerance for simultaneous islet allografts. B10 (H-2b) mice were rendered diabetic using streptozotocin. After sustained hyperglycemia (> 300 mg/dl), diabetic B10 mice were irradiated (950 cGy) and reconstituted with 5 x 10(6) T cell-depleted (TCD) B10 + 15 x 10(6) TCD B10.BR bone marrow cells. Islet allografts genetically matched or disparate to the bone marrow donor were placed under the renal capsule within 24 hr following infusion of bone marrow cells. All donor-specific B10.BR mouse (H-2k) islet allografts were permanently accepted (n = 8; MST > or = 173 days), while 7 of 9 MHC-disparate third-party BALB/c mouse (H-2d) islet grafts were rejected. The other 2 allografts remained functional over 200 days posttransplantation. We recently established a nonlethal conditioning strategy to achieve multilineage mixed chimerism. We applied this model to examine whether simultaneous islet grafts matched to the donor would be permanently accepted if the donor was incompletely myeloablated. Diabetes was induced in B10 mouse recipients. Animals with hyperglycemia were conditioned with 500 cGy of TBI followed by an infusion of 15 x 10(6) untreated B10.BR bone marrow cells. A simultaneous islet allograft matched or MHC-disparate to the bone marrow donor was performed the same day. Two days following bone marrow transplantation, a single dose of cyclophosphamide (200 mg/kg) was injected via the intraperitoneal route. Islet allografts matched to the bone marrow donor were significantly prolonged (n = 9; MST > or 226 days) and showed no evidence for chronic rejection, while MHC-disparate grafts were rejected (n = 5; MST = 34 days). Animals that received islet grafts but no bone marrow also rejected their grafts with a similar time course. These data suggest that permanent donor-specific tolerance to islet allografts placed coincident with bone marrow transplantation can be achieved after lethal as well as incompletely myeloablative conditioning.

Allogeneic lymphocyte chimerism after clinical lung transplantation

Donor lungs contain large amounts of passenger leukocytes which are transferred to the recipient by organ transplantation. In this study we have analysed the fate of these cells and have studied the population of donor leucocytes detectable in the blood circulation of ten lung transplanted patients during the first postoperative weeks. To this aim we have applied immunocytological as well as flow cytometric analyses using monoclonal antibodies against polymorphic HLA class I antigens that differed between donor and recipient as well as antibodies against cell differentiation markers. The results demonstrate that donor cells can be detected in the circulation of all lung transplanted patients but there is a considerable interindividual variability between 0.9% and 17.5% (mean 5.1%) on postoperative day 3. Cells were usually detectable for 2–4 weeks and had disappeared in all patients after 1 month. The circulating donor cells consisted exclusively of lymphocytes. T cells were the predominant population, most of which seemed to be CD45R0 +, but B and NK (natural killer) cells were also present. Probably due to the small numbers of patients studied no correlation between clinical parameters and the extent of donor lymphocyte persistence; there were no clinical graft-versus-host reactions. The findings demonstrate the regular existence of a transient (macro)chimerism due to passenger lymphocytes in the early phase after lung transplantation. The immunological function and the relation between this phenomenon and the long-term microchimerism which frequently develops after solid organ transplantation remain unclear.

UV-B modulation of haplo-identical bone marrow cells in the prevention of GVHD and induction of specific transplantation tolerance to intestinal allografts

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis × Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 10 8 BMC and 5 × 10 6 spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100–300 J/m 2 UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m 2 of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94–98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow.

Experimental studies have shown that administration of antilymphocyte serum combined with donor bone marrow cells can induce tolerance to allograft tissue. We have initially reported application of these protocols in clinical studies of cadaveric renal allograft recipients who were treated with MALG and donor-specific bone marrow cells. To evaluate the effectiveness of the donor marrow cells in the production of chimerism, a detection method based on 32P-incorporated PCR was established. The 32P PCR was utilized with primers specific for the HLA class II, VNTR (D17S5 and D1S111), and/or Y-chromosome genes to detect the presence of allogeneic chimerism in the recipients. Immediately posttransplant, 26.4% of marrow recipients demonstrated the presence of allogeneic chimerism prior to the marrow transfusion as did 18% in the untransfused controls. In transfused patients, chimerism was detected most frequently during the 1-3-month interval after marrow transfusion (65%), and then diminished to 50-56% at 3-12 months posttransfusion. In the control group the frequency of allogeneic chimerism was gradually decreased and was undetectable in the majority of the patients beyond 3 months posttransplant while marrow-transfused recipients were more likely to have chimeric cells detected consistently beyond 3 months. Rejection episodes were significantly effected by the presence of chimerism in the recipients. Of the transfused patients, 91.3% who demonstrated allogeneic chimerism were rejection-free as compared with 8.7% who experienced at least one rejection episode (P = 0.01). While the presence of allogeneic chimerism in the control group was correlated with rejection-free graft survival, this difference did not reach statistical significance.

Transplantation in utero of fetal human hematopoietic stem cells into mice results in hematopoietic chimerism.

Allogeneic and xenogeneic hematolymphoid chimerism has been achieved in large and small animals using varied techniques to circumvent immune mediated graft rejection by the recipient. We show here the establishment of long-term chimerism in normal mice transplanted in utero with human fetal hematopoietic stem cells (HSC). HSCs from fetal (13-20 weeks' gestation) human livers were injected into fetal mouse peritoneal cavities on days 11-13 of gestation. Histologic examination demonstrated human chimerism in 29% of 38 live born mice using fluorescein conjugated antibodies to both the CD45 and CD14 antigens present on human peripheral blood (PB) cells. Further investigation using flow cytometric analysis of cells from 70 mice transplanted in utero revealed 28% of mice greater than 16 weeks of age contained human cells in at least one organ at the following frequencies: 14% PB, 8% bone marrow, 8% spleen and 12% thymus. These data indicate that human fetal HSC can be engrafted into mouse fetuses. Additionally, the identification of circulating human cells 18 months following transplantation supports the engraftment and proliferation of a primitive hematopoietic progenitor.

Organ-specific unresponsiveness induced by intrathymic injection of donor bone marrow cells and a short course of immunosuppression in the rat heart transplantation model

Donor- and organ-specific unresponsiveness to Brown Norway (BN) heart allografts was achieved in Lewis (LEW) rats by giving intrathymic donor bone marrow cells (ITBMC) and immunosuppression at the time of transplantation. Antilymphocyte serum (ALS) (1 mlo, days 0,2,4) extended graft survial to a median survival time (MST) of 29.5 days ( n = 6), while ALS + ITBMC extended survival to over 120 days ( n = 6). FK506 (1 MG/KG, DAYS 0,2,4,6,8) too prolonged survival in the FK + ITBMC ( n = 6 MST > 140 days) and FK ( n = 5; MST > 140 days) groups. BN skin grafting provoked the rejection of long-surviving BN heart grafts in the FK group ( n = 5; MST = 14 days), but did not do so in either the ALS + ITBMC ( n = 2; MST > 100 days) or the FK + ITBMC ( n = 4; MST > 93 days) groups. In the FK + ITBMC group, two of the rats which rejected BN skin grafts received a second BN heart, resulting in the graft being accepted indefinitely (> 100 days) without causing the rejection of the first BN heart grafts. These facts suggest that ITBMC and concurrent T cell depletion are decisive for induction of unresponsiveness by this protocol. BN and WF (Wistar Furth) skin grafts were eventually rejected in the LEW rats which accepted BN heart grafts. Persistent allogeneic chimerism was demonstrated in the graft and recipient spleen, suggesting that chimerism may be one of the possible mechanisms of unresponsiveness. The procedure reported here deserves further evaluation, but appears to be a possible clinical strategy for the induction of unresponsiveness.

Induction of donor-specific transplantation tolerance to skin and cardiac allografts using mixed chimerism in (A + B-->A) in rats.

Mixed allogeneic chimerism (A + B-->A) was induced in rats by reconstitution of lethally irradiated LEW recipients with a mixture of T-cell depleted (TCD) syngeneic and TCD allogeneic ACI bone marrow. Thirty-seven percent of animals repopulated as stable mixed lymphopoietic chimeras, while the remainder had no detectable allogeneic chimerism. When evaluated for evidence of donor-specific transplantation tolerance, only those recipients with detectable allogeneic lymphoid chimerism exhibited acceptance of donor-specific skin and cardiac allografts. Despite transplantation over a major histocompatibility complex (MHC)- and minor-disparate barrier, animals accepted donor-specific ACI skin and primarily vascularized cardiac allografts permanently, while rejecting third party Brown Norway (BN) grafts. The tolerance induced was also donor-specific in vitro as evidenced by specific hyporeactivity to the allogeneic donor lymphoid elements, yet normal reactivity to MHC-disparate third party rat lymphoid cells. This model for mixed chimerism in the rat will be advantageous to investigate specific transplantation tolerance to primarily vascularized solid organ grafts that can be performed with relative ease in the rat, but not in the mouse, and may provide a method to study the potential existence of organ- or tissue-specific alloantigens in primarily vascularized solid organ allografts.

CD4+ T CELLS, BUT NOT CD8+ T CELLS, MEDIATE THE BREAKING OF TOLERANCE IN MIXED ALLOGENEIC CHIMERAS (B10 + B10.BR ± B10)

Reconstitution of mouse recipients with a mixture of syngeneic plus allogeneic bone marrow (A+B-->A) results in stable mixed lymphohematopoietic chimerism and donor-specific transplantation tolerance. Previously, it was reported that administration of large numbers of unmanipulated host-type splenocytes to neonatal or adult radiation bone marrow chimeras resulted in a loss of chimerism and donor-specific transplantation tolerance. To characterize the phenotype(s) of cells that were responsible for this loss of chimerism, we performed depletion of various subsets of unmanipulated B10 splenocytes prior to infusion into mixed allogeneic chimeras (B10 + B10.BR-->B10). Recipients were followed serially to identify changes in the level of donor chimerism and by in vitro functional assays of tolerance. We report here that CD4+ T cells, but not CD8+ T cells, were sufficient to mediate the loss of donor chimerism. In all recipients in which allogeneic chimerism became undetectable, there was a simultaneous loss of donor-specific transplantation tolerance.

The requirement for allogeneic chimerism for second transfer of tolerance from mixed allogeneic chimeras (A+B-->A) to secondary recipients.

We have applied the model of mixed allogeneic chimerism (A+B-->A), in which stem cells from both allogeneic and syngeneic donor engraft, to determine the in vivo cellular requirements for transfer of tolerance from mixed chimeras to secondary recipients. Using two approaches, we have demonstrated that the persistence of donor-specific transplantation tolerance is dependent on the presence of bone-marrow-derived cells. When untreated bone marrow from mixed chimeras was transferred to irradiated secondary recipient mice, most of the secondary recipients were rescued, but only 48% were demonstrably chimeric. This pattern of repopulation, therefore, allowed us to examine whether chimerism was required to maintain transplantation tolerance. In all of our studies, the presence of allogeneic chimerism was required for successful transfer of tolerance from mixed allogeneic chimeras to irradiated secondary recipients. Only those secondary recipients which repopulated with demonstrable allogeneic chimerism exhibited in vivo and in vitro evidence for transfer of donor-specific transplantation tolerance. These results were confirmed by using transfer of bone marrow from mixed chimeras depleted of allogeneic class I elements. In an attempt to identify a putative population of suppressor cells, second transfer of splenic lymphoid cells from mixed allogeneic chimeras, containing approximately 6 times more T-lymphocytes that were functionally tolerant to donor alloantigens, was also performed with similar results. These data suggest that the in vivo maintenance of tolerance to MHC transplantation alloantigens requires persistence of donor bone marrow-derived alloantigens.

Allogeneic chimerism in scid mice after neonatal transfer of bone marrow.

Allogeneic chimeras are valuable tools for studies of complex immune cell interactions in vivo. Mice with severe combined immune deficiency (scid) should be ideal hosts for chimerism with allogeneic bone marrow cells as these animals lack mature T and B lymphocytes capable of reacting against donor alloantigens. However, it has been difficult to achieve full reconstitution of adult scid mice even using coisogenic bone marrow grafts without prior irradiation of the recipient. We explored ways to generate complete reconstitution of scid mice with allogeneic bone marrow. Unirradiated adult scid recipients of allogeneic bone marrow were only marginally reconstituted. Adult scid mice pretreated with 250 R were reconstituted with allogeneic bone marrow as measured by serum IgM concentration, peripheral lymphoid cellularity, and mitogen responses, but a potentially important immunologic deficit was found in these mice: 250 R caused a 70% loss of scid macrophages and dendritic cells which persisted at least 5 months. By contrast, when scid mice were injected i.p. with allogeneic bone marrow within the first 24 h after birth, rapid and complete reconstitution of both T and B cell lineages was achieved, and the animals had APC that were both donor and host in origin. Considering the extent and duration of engraftment (43 wk) by allogeneic cells in neonatally transplanted scid mice, it was anticipated that their bone marrow would be chimeric. However, the bone marrow contained very few donor-derived cells, suggesting that lymphopoiesis may be taking place in other organs in these chimeras.

MIXED ALLOGENEIC RECONSTITUTION (A+B→A) TO INDUCE DONOR-SPECIFIC TRANSPLANTATION TOLERANCE

Mixed allogeneic reconstitution, in which a mixture of T-cell-depleted bone marrow of syngeneic host and allogeneic donor type is transplanted into a lethally irradiated recipient (A+B----A), results in mixed lymphopoietic chimerism with engraftment of a mixture of both host and donor bone marrow elements. Recipients are specifically tolerant to donor both in vitro and in vivo. Donor-specific skin grafts survive indefinitely when they are placed after full bone marrow repopulation at 28 days, while third-party grafts are rapidly rejected. To determine whether a delay of a month or more for full bone marrow repopulation is required before a donor-specific graft can be placed, we have now examined whether tolerance induction can be achieved if a graft is placed at the time of bone marrow transplantation. Permanent acceptance of donor-specific B10.BR skin grafts occurred when mixed allogeneic chimerism (B10+B10.BR----B10) was induced and a simultaneous allogeneic donor graft placed. In vitro, mixed reconstituted recipients were specifically tolerant to the B10.BR donor lymphoid cells but fully reactive to MHC-disparate third-party (BALB/c; H-2dd) when assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. These data therefore indicate that a donor-specific graft placed at the time of mixed allogeneic reconstitution is permanently accepted without rejection. To determine whether an allogeneic skin graft alone without allogeneic bone marrow would be sufficient to induce tolerance, syngeneic reconstitution (B10----B10) was carried out, and a simultaneous B10.BR allogeneic skin graft placed. Although skin grafts were prolonged in all recipients, all grafts rejected when full lymphopoietic repopulation occurred at 28 days. Taken together, these data suggest that allogeneic donor bone marrow elements are required for the induction and maintenance of donor-specific transplantation tolerance and that allogeneic skin grafts alone are not sufficient for tolerance induction.

Studies of allogeneic bone marrow and spleen cell transplantation in a murine model using ultraviolet-B light

Ultraviolet irradiation inhibits alloreactive and mitogen-induced responses and might reduce both graft-versus-host and host-versus-graft reactions after bone marrow transplantation (BMT). We have studied proliferative responses to mitogens and reactivity in mixed lymphocyte culture after irradiation with ultraviolet (UV)-B light using splenocytes from Balb/c (H-2d) and CBA (H-2k) mice. Response to mitogens and in MLC was strongly inhibited by 20 J/m2 and abolished at 50 J/m2. Clonogenic cell recovery (CFU-GM; CFU-S) after UV-B irradiation was also reduced. When bone marrow and spleen cells were transplanted from parent (Balb/c) animals into F1 hybrid (Balb/c X CBA) recipients, all animals died with features indicative of graft-versus- host disease (GVHD) in 34 days. If the grafts were first irradiated with 100 J/m2 of UV-B at a mean wavelength of 310 nm, then 76% survived to day 80 when they were killed and shown to have normal marrow cellularity. The remainder died in marrow aplasia or of GVHD. H-2 typing in a group of surviving recipients showed either donor hematopoiesis only (8 of 15), mixed allogeneic chimerism (5 of 15), or recipient type hematopoiesis (2 of 15). Higher doses (200 to 300 J/m2) were detrimental to survival with 88% of recipients dying in marrow aplasia. Syngeneic BMT in Balb/c mice showed slower hematopoietic reconstitution when the grafts were first irradiated with 100 J/m2. After BMT from Balb/c to CBA mice all recipients of unirradiated grafts died within 54 days. By contrast, after graft irradiation with 100 J/m2 survival of recipient animals to day 80 was 59%. If these grafts were treated with 50 J/m2 survival was only 26% with an increase in deaths due to GVHD. Hematopoiesis at day 80 in a group of survivors studied by Ig heavy chain allotyping indicated donor type hematopoiesis in 6 of 10 (50 J/m2) and 2 of 9 (100 J/m2). These data indicate that UV-B irradiation inhibits lymphocyte reactivity and can prevent GVHD. However, there is clear in vitro and in vivo evidence of stem cell damage, such that autologous marrow recovery was demonstrated in a proportion of recipients. In parent----F1 UV-irradiated transplants, sustained hematopoietic recovery was effected in the majority by donor stem cells.

Studies of Allogeneic Bone Marrow and Spleen Cell Transplantation in a Murine Model Using Ultraviolet-B Light

Ultraviolet irradiation inhibits alloreactive and mitogen-induced responses and might reduce both graft-versus-host and host-versus-graft reactions after bone marrow transplantation (BMT). We have studied proliferative responses to mitogens and reactivity in mixed lymphocyte culture after irradiation with ultraviolet (UV)-B light using splenocytes from Balb/c (H-2d) and CBA (H-2k) mice. Response to mitogens and in MLC was strongly inhibited by 20 J/m2 and abolished at 50 J/m2. Clonogenic cell recovery (CFU-GM; CFU-S) after UV-B irradiation was also reduced. When bone marrow and spleen cells were transplanted from parent (Balb/c) animals into F1 hybrid (Balb/c X CBA) recipients, all animals died with features indicative of graft-versus-host disease (GVHD) in 34 days. If the grafts were first irradiated with 100 J/m2 of UV-B at a mean wavelength of 310 nm, then 76% survived to day 80 when they were killed and shown to have normal marrow cellularity. The remainder died in marrow aplasia or of GVHD. H-2 typing in a group of surviving recipients showed either donor hematopoiesis only (8 of 15), mixed allogeneic chimerism (5 of 15), or recipient type hematopoiesis (2 of 15). Higher doses (200 to 300 J/m2) were detrimental to survival with 88% of recipients dying in marrow aplasia. Syngeneic BMT in Balb/c mice showed slower hematopoietic reconstitution when the grafts were first irradiated with 100 J/m2. After BMT from Balb/c to CBA mice all recipients of unirradiated grafts died within 54 days. By contrast, after graft irradiation with 100 J/m2 survival of recipient animals to day 80 was 59%. If these grafts were treated with 50 J/m2 survival was only 26% with an increase in deaths due to GVHD. Hematopoiesis at day 80 in a group of survivors studied by Ig heavy chain allotyping indicated donor type hematopoiesis in 6 of 10 (50 J/m2) and 2 of 9 (100 J/m2). These data indicate that UV-B irradiation inhibits lymphocyte reactivity and can prevent GVHD. However, there is clear in vitro and in vivo evidence of stem cell damage, such that autologous marrow recovery was demonstrated in a proportion of recipients. In parent----F1 UV-irradiated transplants, sustained hematopoietic recovery was effected in the majority by donor stem cells.

Mixed chimerism to induce tolerance for solid organ transplantation

Chimerism, or the coexistence of tissue elements from more than one genetically different strain or species in an organism, is the only experimental state that results in the induction of donor-specific transplantation tolerance. Transplantation of a mixture of T-cell-depleted syngeneic (host-type) plus T-cell-depleted allogeneic (donor) bone marrow into a normal adult recipient mouse (A + B → A) results in mixed allogeneic chimerism. Recipient mice exhibit donorspecific transplantation tolerance, yet have full immunocompetence to recognize and respond to third-party transplantation antigens. After complete hematolymphopoietic repopulation at 28 days, animals accept a donor-specific skin graft but reject major histocompatibility complex (MHC) locus-disparate third-party grafts. We now report that permanent graft acceptance can also be achieved when the graft is placed at the time of bone marrow transplantation. Histologically, grafts were viable and had only minimal inflammatory changes. This model may have potential future clinical application for the induction of donor-specific transplantation tolerance.

Iron carrier proteins facilitate engraftment of allogeneic bone marrow and enduring hemopoietic chimerism in the lethally irradiated host

Cell-free supernatants of rabbit bone marrow were fractionated, separated, and purified by Ultrogel and Superose chromatography. A single fraction promoted engraftment of allogeneic bone marrow and enduring hemopoietic chimerism across the H-2 barrier in lethally irradiated mice. This “bio-active” fraction, analyzed by reducing SDS-PAGE electrophoresis, and transblotted on PVDF membrane, and purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main prealbumin band that when examined for primary structure by Edman degradation, proved to be rabbit transferrin. This was also attested by highly specific precipitation of the prealbumin band with polyclonal antibodies to rabbit transferrin. Iron-saturated human transferrin, lactotransferrin, and egg transferrin (conalbumin) were assayed in irradiated C57BL/6 mice infused with bone marrow from histoincompatible BALB/c donors. Mice treated with iron-loaded transferrins survive and develop enduring allogeneic chimerism with no discernible signs of graft-versus-host disease. Iron carrier proteins thus provide an unique means of achieving successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations.

Mhc allograft immunogenicity Its Role in Bone Marrow Transplantation and Its Constant Downregulation

Since 1972 our laboratory has performed a large variety of studies in the rat and mouse models. These have shown that it is possible to achieve MHC fully allogeneic hematopoietic chimerism with complete restitution of immunological reactivity but lasting immunotolerance of donor type cells following transplantation of lymphocyte-purged bone marrow into lethally irradiated recipients. The clinical application of this obvious immunobiological potential is, however, still in its infancy. This is largely due to the belief that graft-versus-host reactions and host-versus-graft reactions balance each other out--a view that cannot be upheld. A decisive element in the network of immunoregulation following BMT is the immunogenic strength of the BM. In this context the following bits of information are given: (1) Bone marrow that has been completely purged of lymphocytes loses its allograft immunogenicity to a great extent and may even allow induction of tolerance, whereas unpurged BM is strongly immunogenic. (2) This reduction of alloimmunogenicity is only partially determined by lymphocytes and other MHC class II-expressing cells. (3) By incubating bone marrow with our new cytotoxic monoclonal antibody K31, directed against an epitope located an all human lymphocytes and defined cells of the monocytic lineage, both the alloimmunogenicity and the accessory cell potential of the bone marrow can be greatly reduced. (4) Recent analyses with macrophage cell clones rather than heterogeneous mixtures of BM cells indicate that oncogene transfections may allow us to completely down-regulate their alloimmunogenicity, in spite of unaltered high MHC expression, by genetically downregulating Il-1. In conclusion, it is suggested that MHC allogeneic BMT will become feasible in the human, as it is well-established in preclinical models.

Effects of T cell depletion in radiation bone marrow chimeras. III. Characterization of allogeneic bone marrow cell populations that increase allogeneic chimerism independently of graft-vs-host disease in mixed marrow recipients.

The opposing problems of graft-vs-host disease vs failure of alloengraftment severely limit the success of allogeneic bone marrow transplantation as a therapeutic modality. We have recently used a murine bone marrow transplantation model involving reconstitution of lethally irradiated mice with mixtures of allogeneic and syngeneic marrow to demonstrate that an allogeneic bone marrow subpopulation, removed by T cell depletion with rabbit anti-mouse brain serum and complement (RAMB/C), is capable of increasing levels of allogeneic chimerism. This effect was observed in an F1 into parent genetic combination lacking the potential for graft-vs-host disease, and radiation protection studies suggested that it was not due to depletion of stem cells by RAMB/C. We have now attempted to characterize the cell population responsible for increasing allogeneic chimerism in this model. The results indicate that neither mature T cells nor NK cells are responsible for this activity. However, an assay involving mixed marrow reconstitution in an Ly-5 congenic strain combination was found to be more sensitive to small degrees of stem cell depletion than radiation protection assays using three-fold titrations of bone marrow cells. Using this assay, we were able to detect some degree of stem cell depletion by treatment with RAMB/C, but not with anti-T cell mAb. Nevertheless, if the effects of alloresistance observed in this model are considered, the degree of stem cell depletion detected by such mixing studies in insufficient to account for the effects of RAMB/C depletion on levels of allogeneic chimerism, suggesting that another cell population with this property remains to be identified.

Lessons from past experience in cancer immutiotherapy and their application to cancer and AIDS treatment and prophylaxis

Passive immunotherapy which we attempted in 1957 using polyclonal antibodies from immunized donors aggravated tumor evolution: we suspected that blocking by Ig, tumor cell antigen epitopes which are necessary to enhance or even maintain the T-lymphocyte effector role in anti-cancer immunity could be the reason for this tumor aggravation by "specific" antibodies. Today the very limited results of monoclonal antibodies in cancer treatment favors this hypothesis. The aggravation of HIV by (some) antibodies suggests that the same phenomenon may also happen in this condition. In 1957, on the other hand, we described the therapeutically beneficial cytostatic targetting effect of polyclonal antibodies, an effect which has often been quoted and widely confirmed. Does the reason for the poor results of monoclonal antibody targetting in tumor treatment reside in cancer antigen heterogeneity? This is what an experiment we conducted with Olsson on AkR leukemia indicated, suggesting that several monoclonals should be simultaneously combined for most patients, to be determined by the antigenic study not only of each tumor but of each localisation. We shifted our cancer immunotherapy approach to an adoptive form in 1959; a), after establishing partial and transient, then total and permanent allogeneic marrow graft and chimerism in man; b), after describing the graft versus leukemia (GvL) action of the graft versus host (GvH) effect in leukaemic mice; c), and the same phenomenon in humans. Our observations on human allogeneic grafted bone marrow GvH and GvL were statistically confirmed in 1979 by the Seattle group. In the 1970s we attempted to induce strong GvL with weak GvH by replacing allogeneic bone marrow graft by allogeneic lymphocyte transfusions without host conditioning. Remarkable results were registered in mice, and in patients with acute leukaemia--5 complete remissions out of 7 patients who developed moderate GvH, and only 3 out of 70 in those who did not develop GvH. As we now know that CD4-, CD8+ lymphocytes are not all MHCl-restricted, we consider that the allogeneic CTL approach is worth trying as reinforcement treatment of (at least) severe hematopoietic malignancies. If it is indeed worthwhile in some severe neoplasias to include the adoptive form of immunotherapy in the multi-treatment program, the replacement of the patient's bone marrow by engrafted allogeneic stem cells has not been proven necessary, as the persistence in "ALL cured" chemotherapy patients of abnormally differentiated cells does not reduce the expectancy of cure.(ABSTRACT TRUNCATED AT 400 WORDS)