Defects in hematopoietic differentiation in NZB and NZC mice.

Hematopoietic stem cell activity in inbred NZB and NZC mice has been determined by transplantation and endogenous spleen colony assays. Whereas NZB mice show normal colony-forming unit (CFU) activity in the transplantation assay, they show markedly elevated endogenous CFU. NZC mice also show this markedly elevated endogenous CFU activity, but in the transplantation assay show only about 5-10% of normal CFU counts. When NZC stem cells are tested for CFU activity in irradiated recipients of the H-2(d) type, almost normal colony numbers occur. NZB stem cells however also cannot form colonies in NZC mice. These results suggest that NZC mice have a defect in the micro-environment of the spleen which renders them incapable of allowing transplanted CFU to form colonies. Genetic analysis of both the NZC defect as a CFU recipient, and the elevated endogenous count in NZB and NZC, shows that both are controlled by single recessive genes which are not linked to either coat color, agouti, H-2 or Ig loci. Of even more relevance is the finding that these hematopoietic abnormalities are not linked to the genes involved in controlling autoantibody formation to red cells in the NZB mice. These mice therefore appear to show two distinct hematopoietic abnormalities, the analysis of which may be of considerable value in understanding the detailed events of hematopoietic stem cell differentiation.

Cellular requirements for the rejection of skin allografts in rats.

The role of bone marrow-derived cells in the rejection of skin allografts in rats was investigated. Lewis rats, rendered tolerant of BN antigens and bearing healthy grafts, were thymectomized, irradiated with 900 rad, and injected with varying doses of either normal isologous bone marrow, normal lymph node cells, and/or lymph node cells presensitized to BN antigens. In some experiments rats were also adoptively sensitized to tuberculin. Results showed that, although necessary for the elicitation of tuberculin skin reactions, bone marrow cells are not needed for the rejection of previously tolerated skin allografts. Rats receiving lymph node cells alone rejected their grafts in about 6-7 days. In addition, rats injected with bone marrow alone also rejected their grafts, although significantly later than did lymph node cell recipients, indicating that rat marrow contains a population of cells capable of reacting to transplantation antigens. These cells were found capable of reacting to major transplantation antigens but not minor as they were ineffective in causing the rejection of Ag-B compatible Fischer skin grafts. From experiments utilizing bone marrow from neonatally thymectomized donors and cells treated with an antiserum to rat T cells, these competent cells in the marrow were shown to be thymus derived.

Survival of transplanted neural progenitor cells enhanced by brain irradiation.

OBJECT: Authors of previous studies have reported that adult transplanted neural progenitor cells (NPCs) are suitable for brain cell replacement or gene delivery. In this study, the authors evaluated survival and integration of adult rat-derived NPCs after transplantation and explored the potential impact on transplant survival of various mechanical and biological factors of clinical importance. METHODS: Adult female Fischer 344 rats were used both as a source and recipient of transplanted NPCs. Both 9L and RG2 rat glioma cells were used to generate in vivo brain tumor models. On the 5th day after tumor implantation, NPCs expressing green fluorescent protein (GFP) were administered either intravenously (3.5 x 10(7) cells) or by stereotactic injection (1 x 10(4)-1 x 10(6) cells) into normal or tumor-bearing brain. The authors evaluated the effect of delivery method (sharp compared with blunt needles, normal compared with zero-volume needles, phosphate-buffered saline compared with medium as vehicle), delivery sites (intravenous compared with intratumoral compared with intraparenchymal), and pretreatment with an immunosuppressive agent (cyclosporin) or brain irradiation (20-40 Gy) on survival and integration of transplanted NPCs. RESULTS: Very few cells survived when less than 10(5) cells were transplanted. When 10(5) cells or more were transplanted, only previously administered brain irradiation significantly affected survival and integration of NPCs. Although GFP-containing NPCs could be readily detected 1 day after injection, few cells survived 4 days to 1 week unless preceded by whole-brain radiation (20 or 40 Gy in a single fraction), which increased the number of GFP-containing NPCs within the tissue more than fivefold. CONCLUSIONS: The authors' findings indicate that most NPCs, including those from a syngeneic autologous source, do not survive at the site of implantation, but that brain irradiation can facilitate subsequent survival in both normal and tumor-bearing brain. An understanding of the mechanisms of this effect could lead to improved survival and clinical utility of transplanted NPCs.

Dog leukocyte antigen-haploidentical stem cell allografts after anti-CD44 therapy and reduced-intensity conditioning in a preclinical canine model.

OBJECTIVE: We previously described a nonmyeloablative hematopoietic stem cell transplantation regimen in dog leukocyte antigen (DLA)-identical littermate recipients consisting of low-dose total body irradiation (TBI) before and mycophenolate mofetil (MMF)/cyclosporine (CSP) given after transplant to control both graft-vs-host and residual host-vs-graft reactions. In this study, we sought to develop a reduced-intensity regimen to achieve engraftment across major histocompatibility complex barriers in DLA-haploidentical littermate recipients. MATERIALS AND METHODS: We tested a regimen of 450-cGy TBI with or without postgrafting MMF/CSP for 28 and 35 days, respectively, and with the administration of monoclonal antibody (mAb) S5 (anti-CD44), at a dose of 0.2 mg/kg/day from days -7 through -2, prior to receiving TBI. RESULTS: One of six dogs conditioned with 450-cGy TBI alone achieved engraftment of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells. Three of six dogs achieved sustained donor cell engraftment using 450-cGy TBI and posttransplantation MMF/CSP. None of three dogs given mAb S5 followed by 450-cGy TBI showed signs of donor cell engraftment. However, when S5 mAb pretreatment was added to 450-cGy TBI and postgrafting MMF/CSP, 10 of 12 dogs achieved sustained engraftment (p = 0.008 or 0.007 vs 450-cGy alone or to S5 + 450-cGy TBI without MMF/CSP, respectively), with only three dogs developing severe graft-vs-host disease on this short regimen of immunosuppression. CONCLUSION: These results show that engraftment across a DLA haplotype-mismatched barrier can be achieved after reduced-intensity conditioning when mAb S5 directed at CD44 is added to this regimen.

Interstitial pneumonitis following total body irradiation for bone marrow transplantation using two different dose rates.

A total of 22 patients with leukemia (10 ALL, 11 AML, 1 CML) have undergone allogeneic bone marrow transplantation (BMT) by the Quebec Co-operative Group for Marrow Transplantation from 1980 to 1982. All patients received 900 cGy total body irradiation (TBI), in a single fraction, on the day preceding BMT. The first 11 patients were treated on a cobalt unit at a constant dose rate of 4.7 to 6.3 cGy/min. Six of these patients developed interstitial pneumonitis (IP). The clinical course of three patients, two with idiopathic and one with drug-induced pneumonitis, was mild and recovery was complete in all. The other three patients developed severe infectious IP and two died. The next 11 patients were treated with a sweeping beam technique on a 4 MV linear accelerator delivering a total tumor dose of 900 cGy at an average dose rate of 6.0 to 6.5 cGy/min but an instantaneous dose rate of 21.0 to 23.5 cGy/min. Eight patients developed severe IP. Five of these were idiopathic and four died. Three were infectious and all died. The fatality of interstitial pneumonitis appeared to be greater in the group treated with the sweeping beam technique.

Growth pattern of tumor xenografts in Wistar rats after treatment with cyclophosphamide, total lymphoid irradiation and/or cyclosporin A.

Wistar rats treated with cyclophosphamide, total lymphoid irradiation (TLI), and/or cyclosporin A (CSA) develop a state of immune suppression permitting the growth of tumor xenografts. Experiments were carried out on this newly developed model to investigate the growth patterns of a mouse osteosarcoma and a human colon adenocarcinoma. The combination of cyclophosphamide and CSA permitted a limited period of growth of the mouse osteosarcoma with a tumor take rate of 66%. No takes were observed with the human adenocarcinoma. The combination of cyclophosphamide and TLI resulted in a period of immunosuppression followed by recovery of the immune status. During the period of immunosuppression, tumor xenografts showed a 100% take rate. The most efficient immunosuppression was achieved by a combination of cyclophosphamide, TLI and CSA administered on alternate days. Wistar rats subjected to this treatment showed prolonged tolerance to mouse osteosarcoma and human adenocarcinoma xenografts. There was no alteration in the tumor doubling time or histological morphology of the xenografts in the adapted host as compared with those in the donor tumors. The tumor growth curve showed a pattern of initial growth, a period of stagnation, followed by a steady but slower growth phase. The significance of the results and the advantages of the rat model described in this paper for human tumor xenotransplantation are discussed.

Ultraviolet irradiation in transplantation biology. Manipulation of immunity and immunogenicity.

Ultraviolet irradiation, particularly in the UVB range, has profound effects on immunological mechanisms. Optimum and tolerable doses of exposure vary from species to species, and from organ to organ. As a result of limited depth penetration and possibly significant energy absorption in nontargeted cells, every model requires diligent determination of an effective nontoxic approach. Nevertheless, it is clear that UVB and UVC irradiation can abolish proliferative and stimulatory ability as well as accessory/antigen-presenting ability of leukocytes in vitro. UV treatment alters cell-surface properties, calcium mobilization, cytokine production and release, and other subcellular processes. Preliminary data suggest that these manipulations also suppress immunity and reduce immunogenicity in vivo. Exposure of solid organs and of large volumes of blood is difficult due to technical problems--in particular poor depth penetration and absorption of UV energy in generally available transfusion bags.

Characteristics of histocompatibility barriers in congenic strains of mice. III. Passive enhancement of skin allografts in X-irradiated hosts.

Passive immunological enhancement of skin allografts was investigated in three donor-host combinations of congenic mice disparate at non-H-2 loci. Serum against the graft donor was derived from mice that had received donor strain lymphoid cells as neonates, and thereby were rendered specifically tolerant of a skin allograft. We refer to this serum as "allograft-tolerant" serum. Each strain combination was chosen to provide only two non-H-2 histoincompatibilities present in the donor and absent in the host. The differences are categorized as immunogenetically strong, moderate, or weak, on the basis of skin allograft survival times. With passively administered allograft-tolerant serum significantly prolonged graft survivals were noted for the weakest combination only. Combined treatment with sublethal X-irradiation and allograft-tolerant serum signigicantly prolonged graft survivals were noted for the weakest combination only. Combined treatment with sublethal X-irradiation and allograft-tolerant serum significantly prolonged graft survival in both the moderate and weak combinations, with the largest effect present in the weakest disparity. A hyperimmune alloantiserum (produced in adults) directed against the graft donor prolonged allograft survival in the strongest disparity when given in combination with irradiation. In this combination, graft survival time was increased in hosts exposed to X-ray alone, but joint treatment with X-ray and the alloantiserum gave the largest increment. In contrast, combined treatment with the serum and an antithymocyte alloantiserum did not affect graft survival times. Treatment with both radiation and antithymocyte serum did not prolong graft survival beyond that in mice given only X-radiation. Immunological enhancement with central inhibition is assumed as the mechanism underlying prolonged graft survival, and it is suggested that a population of thymus-derived "killer" cells, sensitive to X-irradiation, is required for normal graft rejection.

The loss of Ia+ Langerhans' cells during graft-versus-host disease in rats.

The effects of graft-versus-host-disease (GVHD) on different tissues and cells were studied. High and low lymphocyte doses were employed for the induction of GVHD, and the results were compared. It was shown that ear skin was more susceptible to attack than abdominal skin. It is speculated that this could be due to the different vascular networks in the two areas. Weight loss, skin erythema, splenomegaly, histology, and Langerhans' cell density were used to assess GVHD. The Langerhans' cell density was assessed using the Ia antigen cell marker. It is shown that Langerhans' cell density is a sensitive index for confirming GVHD. Weight loss is the least sensitive indicator. The features of GVHD occurred earlier and were more severe in animals receiving the higher dose of lymphocytes, with Ia antigen appearing on the keratinocytes in the later stages of the disease. We conclude that Langerhans' cells are sensitive to the effects of GVHD and that they can provide a diagnostically useful indicator of the disease.

Immune response to ultraviolet-induced tumors. I. Transplantation immunity developing in syngeneic mice in response to progressor ultraviolet-induced tumors.

Ultraviolet-light-induced murine skin tumors were analyzed for the ability to induce transplantation immunity and cytotoxic lymphocytes in syngeneic mice. A correlation was found between tumor regression and the induction of cytotoxic T cells with specificity for a unique tumor-associated antigen. Processing tumors possessed tumor-associated transplantation antigens (TATA), which could be demonstrated by transplantation in hyperimmunized mice. Progression correlated with a lack of splenic cytotoxic T cell reactivity. High levels of in situ cytotoxic reactivity could be induced by presenting the tumor-specific antigen on nongrowing tumor cells. Tumor-bearer hosts were shown to be sensitized to TATA because cultured tumor-bearer T cells adoptively transferred protection against tumor outgrowth. Mechanisms of the in vivo suppression of antitumor immunity are discussed.

Mechanisms of allograft rejection of corneal endothelium.

The local intraocular graft-vs.-host (GVH) reaction, involving the destruction of the corneal endothelial cells of the rabbit host by sensitized donor lymphoid cells, has been used to study the mechanism of corneal allograft rejection. Pretreatment of donor cells with a specific mouse monoclonal hybridoma anti-T cell antibody and complement suppresses the destructive reaction, suggesting that a cellular-immune mechanism is primarily involved. Pretreatment of donor cells with mitomycin-C completely abolishes the local GVH reaction, indicating that the effector lymphocytes must undergo mitosis within the eye before they can engage in target cell destruction. Finally, studies of the local GVH reaction in irradiated leukopenic recipients or in preinflamed rabbit eyes suggest that host leukocytes may contribute nonspecifically to enhance the destructive process. These studies show that the local ocular GVH reaction may provide a useful model for the study of the mechanisms involved in the rejection of corneal allografts.

Kidney allograft tolerance in diabetic patients after total lymphoid irradiation (TLI).

The value of total lymphoid irradiation (TLI) combined with low dose prednisone as sole immunosuppressive regimen in renal allograft transplantation in humans has been investigated. Seventeen patients with end-stage diabetic nephropathy received TLI to a cumulative dose of 20-30 Gy in fractions of 1 Gy. Cadaver kidneys were grafted as soon as they were available after completion of TLI. Low dose prednisone was given after transplantation. Profound and long-term immunosuppression has been achieved in all patients. Six patients live already more than one year (greater than 2 years in 3 of them) and 7 for less than one year with a functioning kidney graft. Two out of these 13 patients had repeated rejection episodes necessitating a supplementary immunosuppressive treatment with cyclosporine A. One patient returned to chronic hemodialysis 11 months after transplantation and died of pericardial tamponade one month later. One patient had severe acute rejection for which cyclosporine A was administered; he died of septic shock as a consequence of immune deficiency a month later. The other two patients succumbed to other causes (myocardial infarction and hyperglycemia). The amount of steroids and azathioprine administered to these patients was substantially lower than in the case of conventional immunosuppression. The preliminary results are thus encouraging. However, the treatment schedule as used in the present study can not yet be considered as optimal since the majority of patients still had one or more rejection episodes. Further investigations are warranted. The optimal dose of radiation, the importance of the interval between TLI and organ transplantation, the influence of splenectomy on the immunity, etc., are still to be assessed.

Canine small bowel transplantation. A study of the immunological responses.

A canine small bowel allograft model was used to determine the effects of radiation to the graft in modifying the immunological effects of the passenger leukocytes. When untreated allografts were transplanted, death of the recipient animals occurred at a mean of nine days. The allograft was well-preserved and showed no signs of rejection. The reasons for attributing death to graft-versus-host (GVH) disease are discussed. When allografts were treated with 150 rads prior to transplantation, allograft rejection occurred, with death of the recipient animals at a mean of 9.2 days. This was the only group in which cell-mediated immunity developed. When allografts were treated with 50 rads, prolonged survival of the recipients to a mean of 28 days was noted. It is postulated that in this group a balance was struck between the allograft rejection reaction and GVH disease, with prolongation of allograft survival.

Effect of H-2 complex on the growth of embryo-derived teratomas in mice.

Seven-day-old embryos of several H-2 congenic strains were transplanted under the kidney capsules of syngeneic adult recipients to determine the genetic factors(s) governing the in vivo growth of embryo-derived teratomas. A.TH(H-2t2) and A.TL(H-2t1) strains showed significantly greater tumor weights than A.BY(H-2b) and A.SW(H-2s) strains. The A(H-2a) strain was intermediate in tumor size. A comparison of the genic constitution of the H-2 complex in each congenic strain suggested that the H-2D locus and/or its distal regions affected the growth of embryo-derived teratomas. The teratoma induced in the B10.A(H-2a) strain was smaller than that in the A(H-2a) strain, indicating that the genetic background of the A strain is favorable for teratoma growth. Histological observations demonstrated that the existence of embryonal carcinoma cells was necessary for the growth of teratomas. A radiation-sensitive immunological factor in the recipient probably plays a role in stimulating teratoma growth.

Reduction of interferon-gamma as a critical mechanism by which ultraviolet radiation prevents tumor rejection.

Mice irradiated with UVB, unlike nonirradiated mice, are highly susceptible to syngeneic, immunogenic tumors induced by UVB irradiation or by chemicals. We postulated that UV induced susceptibility to immunogenic tumors results from a reduction in host capacity to generate an interferon (IFN)-gamma immune response to tumor antigens. Shaved BALB/c mice were exposed to 6 x 10(5) J m-2 of UVB radiation delivered intermittently over 12 weeks. The UVB-irradiated and nonirradiated mice received intradermal injections of UVM12 or BP2 tumor cells. After 0, 1.5, 3, 7 or 21 days, draining lymph nodes were excised. Lymph node cells were incubated with UVM12 or BP2 cells that had received 2.5 Gy of gamma-radiation. After 48 h in culture, supernatants were analyzed for IFN-gamma content by enzyme-linked immunosorbent assay and cellular RNA was extracted for mRNA detection by reverse transcriptase-polymerase chain reaction analysis. At 7 days after tumor injection, draining lymph node cells from nonirradiated control mice secreted significant levels of IFN-gamma and contained at least 0.0729 amol of IFN-gamma mRNA/microgram cDNA upon in vitro exposure to gamma-irradiated tumor cells. Draining lymph node cells removed from UV-irradiated mice contained only 18% as much IFN-gamma mRNA and secreted little or no IFN-gamma when exposed to gamma-irradiated tumor cells. A single injection of antibody directed against murine IFN-gamma rendered normal mice as susceptible as UV-irradiated mice to BP2 tumor cells. Thus, chronic UV irradiation leads to an inability of host tumor draining lymph node cells to mount an IFN-gamma response to tumor antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional lymph node x-radiation suppresses allograft rejection.

We studied the effect of a single dose of x-radiation given to the regional lymph nodes on allograft rejection. Our model was an A-strain tumor, transplanted to C57B1/6, DBA/2, and C57B1/6 x CBA hosts. Irradiation (1,000 rad) of the regional lymph nodes inhibited graft rejection, as judged by increased tumor growth, slower rejection, and, in the case of DBA/2 mice, by fewer regressions; irradiation of the contralateral lymph nodes did not have this effect. The mechanism of immunosuppression appeared to be B-cell independent, since immunosuppression could be induced by regional x-radiation as readily in B-cell deficient mice as in normal mice. The suppression was critically time-dependent, viz, irradiation before, or 6 or more days after, tumor graft was without significant effect. Treatment of the allogeneic host with cyclophosphamide several days before grafting, so as to reduce T-suppressor cells, attenuated the immunosuppression of regional x-radiation.

Potential of 109Pd-labeled lymphocytes for selective lymphatic ablation.

The biodistribution of lymphocytes labeled with 109Pd was investigated in Lewis rats to determine if they might be useful for selective lymphoid ablation. 109Pd-labeled lymphocytes demonstrated significant lymphoid localization. However, there was a fall in the accumulation of radiolabeled lymphocytes in lymphoid tissue when the 108Pd carrier dose or the 109Pd radioactive dose incorporated per 10(8) lymphocytes was increased from 0.12 mg to 0.20 mg and from 21.3 microCi to 54.6 microCi, respectively (P less than 0.001). 109Pd-labeled syngeneic and allogeneic lymphocytes demonstrated similar tissue distribution patterns. These results raise the possibility of using 109Pd-labeled lymphocytes for selective lymphoid ablation, but emphasize the need for using high specific activity 109Pd and large amounts of lymphocytes for labeling. This will minimize cell damage and allow maximum therapeutic results to be obtained. The use of large numbers of cells might best be accomplished by using donor lymphocytes.

Cell-mediated immunity in the gecko, Tarentola annularis.

The gecko Tarentola annularis, rejects skin allografts in a chronic manner, whereas the cutaneous response to tuberculin has the characteristics of mammalian delayed hypersensitivity reaction. Moreover, inoculation of gecko's spleen lymphocytes into skin of intact or whole-body--heavily irradiated allogeneic hosts, readily elicits a severe, local reaction (normal lymphocyte transfer reaction). The data indicate that slow skin graft rejection in T. annularis cannot be ascribed to either defective cellular immune reactivity or to the absence of strong antigenic disparity in the species.

Significance of the length of time interval between lethal irradiation and transplantation of haemopoietic cells.

It could be shown that the efficiency of haemopoietic cell transplantation into lethally irradiated mice depended upon the degree of histocompatibility between the donor and the host as well as on the number of transplanted cells. With a prolongation of the time interval between irradiation and transplantation a proportionally higher number of transplanted haemopoietic cells was required to maintain the curative effect of transplantation. The haemopoietic microenvironment in lethally exposed mice remained unchanged until the tenth day after irradiation.

Gamma-irradiation reduces the allogenicity of donor corneas.

PURPOSE: To evaluate the utility and allogenicity of gamma-irradiated corneal allografts. METHODS: Corneal buttons were harvested from C57BL/6 mice and decellularized with gamma irradiation. Cell viability was assessed using TUNEL and viability/cytotoxicity assays. Orthotopic penetrating keratoplasty was performed using irradiated or nonirradiated (freshly excised) C57BL/6 donor grafts and BALB/c or C57BL/6 recipients. Graft opacity was assessed over an 8-week period and graft survival was evaluated using Kaplan-Meier survival curves. Mixed-lymphocyte reactions and delayed-type hypersensitivity assays were performed to evaluate T-cell alloreactivity. Real-time PCR was used to investigate the corneal expression of potentially pathogenic T-helper 1, 2, and 17 cell-associated cytokines. RESULTS: Corneal cells were devitalized by gamma irradiation as evidenced by widespread cellular apoptosis and plasma membrane disruption. Nonirradiated allograft and isograft rates of survival were superior to irradiated allograft and isograft rates of survival (P < 0.001). Mixed lymphocyte reactions demonstrated that T-cells from irradiated allograft recipients did not exhibit a secondary alloimmune response (P < 0.001). Delayed-type hypersensitivity assays demonstrated that irradiated allografts did not elicit an alloreactive delayed-type hypersensitivity response in graft recipients (P ≤ 0.01). The corneal expression of T-helper 1, 2, and 17 cell-associated cytokines was significantly lower in failed irradiated allografts than rejected nonirradiated allografts (P ≤ 0.001). CONCLUSIONS: Gamma-irradiated corneas failed to remain optically clear following murine penetrating keratoplasty; however, gamma irradiation reduced the allogenicity of these corneas, potentially supporting their use in procedures such as anterior lamellar keratoplasty or keratoprosthesis implantation.