RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection.

Acute cellular rejection (ACR) is the adverse response of the recipient's immune system against the allogeneic graft. Using human surveillance endomyocardial biopsies (EMBs) manifesting ACR and murine allogeneic grafts, we profiled implicated microRNAs (miRs) and mRNAs. MiR profiling showed that miR-21, -142-3p, -142-5p, -146a, -146b, -155, -222, -223, and -494 increased during ACR in humans and mice, whereas miR-149-5p decreased. mRNA profiling revealed 70 common differentially regulated transcripts, all involved in immune signaling and immune-related diseases. Interestingly, 33 of 70 transcripts function downstream of IL-6 and its transcription factor spleen focus forming virus proviral integration oncogene (SPI1), an established target of miR-155, the most upregulated miR in human EMBs manifesting rejection. In a mouse model of cardiac transplantation, miR-155 absence and pharmacological inhibition attenuated ACR, demonstrating the causal involvement and therapeutic potential of miRs. Finally, we corroborated our miR signature in acute cellular renal allograft rejection, suggesting a nonorgan specific signature of acute rejection. We concluded that miR and mRNA profiling in human and murine ACR revealed the shared significant dysregulation of immune genes. Inflammatory miRs, for example miR-155, and transcripts, in particular those related to the IL-6 pathway, are promising therapeutic targets to prevent acute allograft rejection.

The Leuven Immunomodulatory Protocol Promotes T-Regulatory Cells and Substantially Prolongs Survival After First Intestinal Transplantation.

Intestinal transplantation (ITx) remains challenged by frequent/severe rejections and immunosuppression-related complications (infections/malignancies/drug toxicity). We developed the Leuven Immunomodulatory Protocol (LIP) in the lab and translated it to the clinics. LIP consists of experimentally proven maneuvers, destined to promote T-regulatory (Tregs)-dependent graft-protective mechanisms: donor-specific blood transfusion (DSBT); avoiding high-dose steroids/calcineurin-inhibitors; and minimizing reperfusion injury and endotoxin translocation. LIP was tested in 13 consecutive ITx from deceased donors (2000-2014) (observational cohort study). Recipient age was 37 years (2.8-57 years). Five-year graft/patient survival was 92%. One patient died at 9 months due to aspergillosis, another at 12 years due to nonsteroidal anti-inflammatory drug-induced enteropathy. Early acute rejection (AR) developed in two (15%); late AR in three (23%); all were reversible. No chronic rejection (CR) occurred. No malignancies developed and estimated glomerular filtration rate remained stable post-Tx. At last follow-up (3.5 years [0.5-12.5 years]), no donor-specific antibodies were detected and 11 survivors were total parenteral nutrition free with a Karnofsky score >90% in 8 recipients (follow-up >1 years). A high frequency of circulating CD4+ CD45RA- Foxp3hi memory Tregs was found (1.8% [1.39-2.21]), comparable to tolerant kidney transplant (KTx) recipients and superior to stable immunosuppression (IS)-KTx, KTx with CR, and healthy volunteers. In this ITx cohort we show that DSBT in a low-inflammatory/pro-regulatory environment activates Tregs at levels similar to tolerant-KTx, without causing sensitization. LIP limits rejection under reduced IS and thereby prolongs long-term survival to an extent not previously attained after ITx.

Induction of specific transplantation tolerance across xenogeneic barriers in the T-independent immune compartment.

After transplantation of primarily vascularized xenografts (Xgs), T-independent mechanisms may lead to Xg rejection before T-cell activation even takes place. The possibility of achieving T-independent xenotolerance was evaluated in nude rats that normally reject hamster cardiac Xgs within 4 days by non-T cell-mediated mechanisms. After donor antigen infusion, temporary NK-cell depletion and a 4-week administration of Leflunomide, hamster heart grafts survived even after withdrawal of immunosuppression. Tolerant rats accepted second hamster hearts, but promptly rejected mouse heart Xgs. In vivo immunization and in vitro cytotoxicity assays indicated that this species-specific tolerance was based on B-lymphocyte and NK-cell tolerance respectively.

Xenotransplantation: where are we in 2008?

Xenotransplantation holds promise to solve the ever increasing shortage of donor organs for allotransplantation. In the last 2 decades, major progress has been made in understanding the immunobiology of pig-into-(non)human primate transplantation and today we are on the threshold of the first clinical trials. Hyperacute rejection, which is mediated by pre-existing anti-alpha Gal xenoreactive antibodies, can in non-human primates be overcome by complement- and/or antibody-modifying interventions. A major step forward was the development of genetically engineered pigs, either transgenic for human complement regulatory proteins or deficient in the alpha1,3-galactosyltranferase enzyme. However, several other immunologic and nonimmunologic hurdles remain. Acute vascular xenograft rejection is mediated by humoral and cellular mechanisms. Elicited xenoreactive antibodies play a key role. In addition to providing B cell help, xenoreactive T cells may directly contribute to xenograft rejection. Long-term survival of porcine kidney- and heart xenografts in non-human primates has been obtained but required severe T and B cell immunosuppression. Induction of xenotolerance, e.g. through mixed hematopoietic chimerism, may represent the preferred approach, but although proof of principle has been delivered in rodents, induction of pig-to-non-human primate chimerism remains problematic. Finally, it is now clear that innate immune cells, in particular macrophages and natural killer cells, can mediate xenograft destruction, the determinants of which are being elucidated. Chronic xenograft rejection is not well understood, but recent studies indicate that non-immunological problems, such as incompatibilities between human procoagulant and pig anticoagulant components may play an important role. Here, we give a comprehensive overview of the currently known obstacles to xenografting: immune and non-immune problems are discussed, as well as the possible strategies that are under development to overcome these hurdles.

CTLA-4 blockade in murine bone marrow chimeras induces a host-derived antileukemic effect without graft-versus-host disease.

We studied the effect of CTLA-4 blockade on graft-versus-leukemia and graft-versus-host responses in a mouse model of minor histocompatibility-mismatched bone marrow transplantation. Early CTLA-4 blockade induced acute graft-versus-host disease. Delayed CTLA-4 blockade resulted in a lethal condition with lymphosplenomegaly, but with stable mixed T-cell chimerism, unchanged alloreactive T-cell frequencies and absent anti-host reactivity in vitro. In contrast, multiorgan lymphoproliferative disease with autoimmune hepatitis and circulating anti-DNA auto-antibodies were documented. Splenic lymphocytes exhibited ex vivo spontaneous proliferation and a marked proliferative response against host-type dendritic cells pulsed with syngeneic (host-type) tissue-peptides. Both phenomena were exclusively mediated by host and not donor T cells, supporting an autoimmune pathogenesis. Selectively host-derived T-cell immune reactivity was equally documented against leukemia-peptide-pulsed dendritic cells, and this was paralleled by a strong in vivo antileukemic effect in anti-CTLA-4-treated and subsequently leukemia-challenged chimeras. In conclusion, delayed CTLA-4 blockade induced a host-derived antileukemic effect, occurring in the context of an autoimmune syndrome and strictly separated from graft-versus-host disease. Both antileukemic and autoimmune responses depended on the allogeneic component, as neither effect was seen after syngeneic bone marrow transplantation. Our findings reveal the potential of using CTLA-4 blockade to establish antileukemic effects after allogeneic hematopoietic stem cell transplantation, provided autoimmunity can be controlled.

Intestinal transplantation: from the laboratory to the clinics.

The intestine has long been seen as a "forbidden" organ to transplant. This is because the first attempts at Intestinal Transplantation (ITx) were defeated by rejection, technical problems, infection and graft versus host disease. Results of ITx have improved in the short-term (70 to 80% 1-year patient survival) but remain inferior to other solid organ transplants in the long-term (5 years patient survival of 50% or less). Reasons for this difference between intestine and other organ transplants are reviewed. Development of immunomodulatory protocols--e.g. protocols aiming at reducing the rejection response and facilitating engraftment--are described. Our center experience with a consecutive series of five intestinal transplants utilizing a new protolerogenic protocol and low immunosuppression is described. At time of writing, these five patients are rejection-free, nutritionally independent and lead a normal life.

1,25-Dihydroxyvitamin D3 prevents insulitis in NOD mice.

The active form of vitamin D, 1,25(OH)2D3, can prevent various forms of experimentally induced autoimmune disorders. The aim of this study was to confirm these findings in NOD mice that spontaneously develop an autoimmune type of diabetes mellitus. Therefore, the effect of a long-term 1,25(OH)2D3 treatment on the incidence of insulitis, the histological lesion preceding diabetes, was studied. Forty-three NOD mice were treated with 1,25(OH)2D3 (5 micrograms/kg) i.p. every other day from age 21 days on, when no insulitis was present yet. At day 100, 16 control mice receiving the treatment vehicle (arachis oil) had an incidence of insulitis of 75%, whereas only 41% of the 1,25(OH)2D3-treated animals developed insulitis (P < 0.025). Calcemia, determined 24 h after the last 1,25(OH)2D3 injection was 2.5 +/- 0.1 mM, which was higher than in control animals (2.3 +/- 0.1 mM), but was well tolerated. Cellular immunity, as assessed with the mixed lymphocyte reaction performed at day 100, was not impaired significantly. This study demonstrates that long-term treatment with high doses of 1,25(OH)2D3 is able to decrease the incidence of insulitis in spontaneous autoimmune diabetes without major side effects.

Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-beta mRNA in the grafts.

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.

Sex difference in resistance to dexamethasone-induced apoptosis in NOD mice: treatment with 1,25(OH)2D3 restores defect.

The NOD mouse, a model for type 1 diabetes, is characterized by resistance to apoptosis in immunocytes. The aim of this study was to investigate a link between apoptosis in NOD thymocytes and autoimmunity. First, we demonstrated that the sexual dimorphism in diabetes incidence in NOD mice (females are more diabetes-prone than males) is reflected by differences in apoptosis. Apoptosis in NOD thymocytes, 24 h after dexamethasone, was decreased in both sexes compared with C57B1/6, but it was lower in female mice (26 +/- 2%) than in male mice (50 +/- 3%, P < 0.001). Further, we demonstrated that sex hormones themselves play a central role in this difference, since castration of NOD male mice, which increases diabetes incidence, decreased apoptosis levels (32 +/- 2%), while treatment of NOD female mice with dihydrotestosterone, which protects against diabetes, restored apoptosis to male levels (42 +/- 1.5%). Finally, we demonstrated that 1,25-dihydroxyvitamin D3, a steroid hormone that prevents diabetes in NOD mice, restored apoptosis levels to C57B1/6 reference levels. This improved apoptosis was seen in male (68 +/- 1 vs. 50 +/- 3% in untreated NOD mice, P < 0.001) but especially in female NOD mice (51 +/- 5 vs. 26 +/- 2% in untreated NOD mice, P < 0.001). Fluorescence-activated cell sorter analysis of thymocyte subsets revealed marked differences, especially in CD4+CD8+ and CD4+ cells. We conclude that the sexual dimorphism in diabetes incidence in NOD mice is paralleled by a dimorphism in resistance to apoptotic signals in NOD thymocytes. This resistance to apoptosis is driven by sex hormones and is corrected by 1,25-dihydroxyvitamin D3.

1alpha,25-dihydroxyvitamin D3 induces an autoantigen-specific T-helper 1/T-helper 2 immune shift in NOD mice immunized with GAD65 (p524-543).

Prevention of type 1 diabetes in NOD mice by 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is accompanied by a T-helper (Th) 1/Th2 cytokine shift in the pancreas. The aim of this study was to investigate whether this immune shift also occurs outside of the pancreas and whether it is limited to autoantigen-specific immune responses. NOD mice treated with 1alpha,25(OH)2D3 (5 microg/kg every 2 days) or control vehicle were immunized with GAD65 (p524-543) or ovalbumin (OVA) in the rear footpads. First, we examined T-cell proliferation and cytokine production (via enzyme-linked immunosorbent assay) of draining lymph node cells in vitro with or without peptide rechallenge. Although no differences in proliferation were measured between control and 1alpha,25(OH)2D3-treated mice after in vitro GAD65 rechallenge, a marked shift in cytokine secretion profile was seen in 1alpha,25(OH)2D3-treated mice: interleukin-4 was increased (37 +/- 5 vs. 21 +/- 12 pg/ml in controls, P < 0.005), whereas gamma-interferon levels were decreased (6 +/- 3 vs. 9 +/- 3 ng/ml in controls, P < 0.05). This shift was absent in OVA-primed mice. Second, we measured cytokine profiles by reverse transcriptase-polymerase chain reaction in popliteal lymph nodes at different time points after priming with GAD65 or OVA in vivo. A marked Th1/Th2 shift occurred in 1alpha,25(OH)2D3-treated mice after in vivo priming with GAD65. Again, this shift was absent after OVA immunization. Finally, we measured cytokine profiles after rechallenge with a panel of autoantigens (GAD65, heat shock protein 65, insulin B-chain) and control antigens (OVA, keyhole limpet hemocyanine, myelin proteolipid protein, tetanus toxin) and confirmed the Th1/Th2 shift in autoantigen-injected mice but not in control antigen-injected mice. In conclusion, the immune deviation induced by 1alpha,25(OH)2D3 in NOD mice can also be induced in the peripheral immune system but is limited to pancreatic autoantigens.

Role of CD4+ and CD8+ T cells in the rejection of heart or islet xenografts in recipients with xenotolerance in the innate immune compartment.

UNLABELLED: To further study the interactions between innate and adaptive immunity in xenotransplantation, we explored the relative contribution of T-cell subsets in vascularized (heart) and cellular (islets) xenografts in a model with established xeno-non-reactivity of the innate system. MATERIALS: Specific innate xenotolerance was induced in xenoheart (hamster) recipients (nude rats) by a tolerizing regimen (TR), consisting of donor antigen infusion, temporary natural killer (NK)-cell depletion and a 4-week administration of leflunomide. Hamster pancreatic islets were transplanted either 1 week after heart transplantation or alone and syngeneic T-cell adoptive transfer was performed 10 days later. Purified CD3(+), CD4(+), and CD8(+) T cells were given 2 weeks after withdrawal of all drugs. At the day of rejection, xenografts were removed for histology. Serum was taken and IgM and IgG xenoantibody titers were measured by flow cytometry. RESULTS: Both heart and islet grafts were rejected after CD4(+) reconstitution. After CD8(+) T-cell adoptive transfer, cellular grafts were not rejected but vascularized grafts were rejected, although only after several months. Rejection in CD4(+) reconstituted nude rats was accompanied by the generation of predominantly IgG xenoantibodies. CONCLUSION: CD4(+) T lymphocytes are able to rapidly initiate the rejection of islet xenografts in the presence of a xenotolerant innate immune system either by breaking the "innate tolerance" (e.g., by activating macrophages and NK-cells) or through a mechanism without any involvement of the innate tolerance (e.g., T-dependent IgG antibody production). In contrast, CD8(+) T cells provoke a late rejection of only xenoheart grafts.

Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060.

Pharmacological amounts of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have potent immunoregulatory activity, but with marked effects on calcium and bone metabolism. In this study we demonstrate that nonhypercalcemia-inducing nondemineralizing doses of an analog of 1,25-(OH)2D3, 1 alpha,25-(OH)2-20-epi-22-oxa-24,26,27-trishomo-vitamin D (KH1060), can prevent type I diabetes. Female NOD mice received 1,25-(OH)2D3 (5 micrograms/kg), KH1060 (0.4 or 0.2 micrograms/kg), or the treatment vehicle ip every 2 days from 21-200 days of age. The incidence of diabetes in controls was 17 of 31 (55%), whereas 7 of 38 (18%) 1,25-(OH)2D3-treated mice, 3 of 27 (11%) KH1060 (0.4 micrograms/kg)-treated mice, and 6 of 27 (22%) KH1060 (0.2 micrograms/kg)-treated mice developed diabetes (P < 0.025 vs. controls). Protection was achieved with the low KH1060 dose without effects on calcium or bone metabolism, as evaluated by serum calcium (9.5 +/- 0.4 vs. 9.4 +/- 0.3 mg/dl in controls; P = NS), serum osteocalcin (82 +/- 17 vs. 83 +/- 20 ng/ml; P = NS), bone calcium content (6.8 +/- 0.7 vs. 6.4 +/- 0.5 mg/tibia; P = NS), urinary calcium (21 +/- 4 vs. 21 +/- 4 mg/dl; P = NS), pyridinoline excretion, and duodenal calbindin-D9K concentration. The proposed mechanism of action is a restoration of suppressor cell activity, as demonstrated in vitro (suppressor cell assay) and in vivo (cell transfer experiments). This study demonstrates that an analog of 1,25-(OH)2D3 prevents type I diabetes in NOD mice without significant effects on calcium or bone metabolism.

Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short induction course of cyclosporin A.

In nonobese diabetic (NOD) mice, type I diabetes can be prevented without generalized immunosuppression by nonhypercalcemic analogs of vitamin D3 when treatment is started early, i.e. before the autoimmune attack, reflected by insulitis, occurs. The aim of this study was to investigate whether these substances can arrest progression to clinically overt diabetes when administered in a more advanced disease stage, namely when the autoimmune attack is ongoing, reflecting the situation in prediabetic subjects in whom immune intervention is being considered. We, therefore, evaluated the protective potential of MC1288 (20-epi-1,25-dihydroxyvitamin D3) a nonhypercalcemic analog of 1,25-dihydroxyvitamin D3, both alone and in combination with a short induction course of cyclosporin A, in NOD mice that already have insulitis, as demonstrated in pancreatic biopsies performed 15 days before the start oftherapy. Subsequently, mice were randomized into a control group, receiving the treatment vehicle (n = 26), and three treatment groups, receiving, respectively, 7.5 mg/kg x day cyclosporin A (CyA) from days 85-105 (n = 19), 0.1 microg/kg x 2 days MC1288 from days 85-200 (n = 20), or the combination of these two regimens (n = 20). At the time of the pancreatic biopsy (day 70), insulitis was evenly distributed in all groups, and 27.7% of the islets scored showed signs of destructive insulitis. Diabetes outcome by 200 days was 74% (14 of 19) in the CyA-treated group, comparable to the diabetes incidence in control mice (65%; 17 of 26; P = NS). Treatment with MC1288 alone could not reduce disease incidence (70%; 14 of 20), but the combination therapy reduced diabetes incidence to 35% (7 of 20; P < 0.05 vs. untreated; P < 0.01 vs. CyA group; P < 0.025 vs. MC1288). All treatments were well tolerated, without major side-effects on calcium or bone metabolism and without signs of generalized immunosuppression. Cotransfer experiments could not reveal the induction of suppressor cells. Reverse transcription-PCR on pancreatic tissue revealed significantly lower levels of interferon-gamma and higher levels of interleukin-4 in the combination group. In conclusion, nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 administered to NOD mice when the autoimmune disease is already active can prevent clinical diabetes when this therapy is combined with a short induction course of an immunosuppressant such as CyA.

Changes of lymphocyte subsets after local irradiation for early stage breast cancer and seminoma testis: long-term increase of activated (HLA-DR+) T cells and decrease of "naive" (CD4-CD45R) T lymphocytes.

Blood lymphocyte subsets of early breast cancer patients and of men with stage I seminoma of the testis were studied up to 6 years after radiotherapy. Similar results were obtained in the two patient groups. After a temporary decrease, the CD4-w29 or "memory" T cells recovered completely, while the CD4-45R or "naive" T cells remained decreased up to 6 years after irradiation. The number of CD8 T lymphocytes did not change during or after treatment. Because of the decrease of a subset of CD4 cells, and the unchanged values of CD8 cells, the CD4/CD8 ratio decreased significantly after irradiation, and remained lower than before treatment up to 5-6 years after radiotherapy. The number of both HLA-DR positive CD4 and HLA-DR positive CD8 T cells ("activated" T cells) increased significantly after irradiation. The natural killer (NK) cells were not affected by treatment. We propose that the recovery of the CD4 cells is limited to the CD4-w29 ("memory") population because of thymic dysfunction in older humans. The impact of the observed immune modulation on the low susceptibility for infections after local irradiation, and on putative antitumour immune responses is discussed.

Determination of mixed chimerism by a simple flow cytometry method.

A simple, sensitive and accurate method was developed to determine the level of lymphoid chimerism in bone marrow-transplanted rodents. The method is based on flow cytometry using polyclonal alloantisera and labeled second step anti-IgG antibodies. Using mixtures of spleen cells from different mouse strains, it was demonstrated that low levels of chimeric cells (less than 1%) could easily be detected. Moreover, using two-color fluorescence analysis, the level of chimerism could also be determined in subpopulations of lymphoid cells, e.g., CD4 or CD8 cells and was found to be identical to the results obtained in unseparated lymphoid populations. The method was compared to the complement dependent cytotoxicity assay (CDCA) and to the flow cytometric determination of chimerism using labeled monoclonal antibodies against specific MHC antigens. CDCA was found to be more labor intensive and could only estimate the composition of the cell mixtures without detecting low levels of chimerism (less than 5%). The results of flow cytometry, using directly labeled monoclonal antibodies or polyclonal antibodies with second step reagents, were identical. It is concluded that, due to its simplicity and high sensitivity, the method described permits reliable determination of the level of mixed chimerism in rodents and is an excellent alternative when no anti-MHC mAbs are available.

Measurement of alloantibody by flow cytometry.

A new method based on flow cytometry has been developed to determine IgG alloantibodies in the serum of immunized rodents. The method utilizes target lymphoid cells, diluted serum and labeled anti-mouse or anti-rat IgG antibodies. In serum from highly immunized animals alloantibodies could be demonstrated up to a dilution of (3 x 10(4))-1 which makes the test approximately 300 times more sensitive than a simultaneously performed complement-dependent cytotoxicity assay (CDCA). Moreover a linear relationship between the amount of alloantibody and the log value of the mean fluorescence of the target cells was found. This linearity permits the comparison of alloantibody production between individual samples by comparing the mean fluorescence values. The reproducibility of the assay was excellent since the coefficients of variation for all dilutions were less than 15%. By using two-color fluorescence the method can discriminate alloantibodies directed against class I and class II MHC antigens. Finally, in addition to its high sensitivity and good reproducibility, the method was found to be at least twice as fast as CDCA.

Immunologic changes after loco-regional radiotherapy and fractionated total body irradiation (TBI) in mice.

The immunologic effects of fractionated irradiation to both hind limbs and the tail of adult (2.5-3 months old) male Balb/c mice were investigated. A dose of 34 Gy given in 17 fractions of 2 Gy, 1 fraction per day, 5 days per week, was delivered with a 60Co source. A significant decrease of the total splenocyte count (29% of control value) and of the PHA(phytohemagglutinin)-induced proliferation of T cells (22% of control value) was found immediately after irradiation. Both parameters normalized within 30 days after irradiation. Immediately after irradiation, the MLC (mixed lymphocyte culture) was supranormal (126% of control value), dropped to 45% 1 week later, and normalized within 1 month after radiotherapy. The NK (natural killer) activity was significantly decreased only the first week after loco-regional irradiation, while the LAK (lymphokine activated killer) activity was not altered at all. The percentage of goat-anti-mouse+ cells (mainly B lymphocytes) was not changed immediately after loco-regional irradiation, but rose to supranormal values (175% of control level) 3 months after irradiation. A persistent decrease of the percentage and the absolute numbers of the Lyt2+ cells (= CD8+ cells, suppressor/cytotoxic phenotype) was observed up to 3 months after irradiation, while the percentage of L3T4+ cells (= CD4+ cells, helper phenotype) remained normal for the total follow-up. No differences in allogeneic skin graft survival could be demonstrated between irradiated and control animals. The observed immunological effects could not be explained by the scatter irradiation to the whole body as total body irradiation (TBI) administered in a dose and dose rate similar to the scatter dose did not result in persistent immunologic changes. No dose-rate effect could be demonstrated in a low dose fractionated total body irradiation schedule. A total body irradiation similar to the scatter dose in humans did not result in significant immunologic changes.

Influence of overall treatment time in a fractionated total lymphoid irradiation as an immunosuppressive therapy in allogeneic bone marrow transplantation in mice.

Three groups of C57/BL/Ka mice received total lymphoid irradiation (TLI) in a total dose of 34 Gy in three different fractionation schedules. In the first group daily fractions of 2 Gy were given during 3 1/2 weeks. In the second group 4 to 5 fractions with 3 1/2 hr interval were given each day, thus delivering 17 fractions in 4 days. In the third group three fractions were given daily for two consecutive days and was repeated two times after 8 or 9 days interval, resulting in a total treatment time of 3 1/2 weeks. The tolerance of all different schedules was excellent. No difference in the peripheral white blood cell and lymphocyte counts nor the degree of immunosuppression as measured by phytohaemaglutinin or concanavalin A induced blastogenesis and mixed lymphocyte reaction were observed at the end of the treatment and up to 200 days. When bone marrow transplantation was performed one day after the end of each schedule, chimerism without signs of graft versus host disease was induced in all the groups. However, from the results in a limited number of animals it seems that concentrated schedules were less effective for chimerism induction. It has been demonstrated that it is possible to reduce drastically the overall treatment time for TLI before bone marrow transplantation. Further investigations are necessary in order to determine the optimal time-dose-fractionation factors and the different parameters involved in the transplantation.

The influence of neonatal thymectomy on the development of radiation myelopathy in rats.

To investigate the possible contribution of cellular immunity in the development of radiation injury of the central nervous system, Wag/Rij rats were thymectomized at birth and irradiated to the cervical spinal cord at the age of 3 months. At the time of paralysis or at the end of the follow-up period (when rats were 1-year-old) the animals were sacrificed and the mediastinum was examined histologically. In 95% of the neonatally thymectomized animals no thymus was left. These rats showed a firm impairment of the cellular immunity, as they had a 40% reduction of the T-lymphocytes in the spleen, and a 70% reduction of the mixed lymphocyte reaction, compared to age-matched controls. Both single dose and two-fraction irradiation experiments were performed. No modification of the latency time to develop paralysis was observed comparing thymectomized and age-matched controls. The incidence of foreleg paralysis after cervical spine irradiation (single dose or two-fraction) was identically distributed in the follow-up period for both neonatally thymectomized and control Wag/Rij rats. The ED50 value derived in the single dose experiments was 20.3 Gy for the control animals, and 20.9 Gy for thymectomized rats, and in the two fraction experiments 29 Gy for controls and 29.6 Gy for thymectomized rats. None of these differences are significant. It appears that neonatal thymectomy, in spite of its firm suppression of the cellular immunity, has no major influence on the development of radiation myelopathy in rats.

Graft-versus-host reactivity and graft-versus-leukemia effect in murine allogeneic bone marrow chimeras conditioned with total body irradiation or total lymphoid irradiation.

To investigate whether graft-versus-host reactivity (GVHR) and the graft-versus-leukemia (GVL) effect can be differentiated, C3H-->AKR mixed bone marrow (BM) chimeras were prepared using two different conditioning regimens. Total body irradiation (TBI) chimeras were induced by infusing 5 x 1O(6) T cell depleted syngeneic AKR BM cells together with 15 x 1O(6) non T cell depleted allogeneic C3H BM cells 1 day after a single fraction of 10.5 Gy TBI. Total lymphoid irradiation (TLI) chimeras were prepared by injecting only 15 x 10(6) non T cell depleted C3H BM cells after 10 daily fractions of 2 Gy TLI Both groups of chimeras were healthy, without clinical or histological signs of graft-versus-host disease (GVHD). In both groups, clonal deletion of antihost-reactive donor type T lymphocytes was found. Whereas TBI chimeras did resist rejection by host type splenocytes injected 2 months after transplantation, TLI chimeras did not. As the latter phenomenon is believed to reflect remaining GVHR, TLI chimeras did have a lower remaining GVHD capacity than TBI chimeras. Nevertheless, TLI chimeras survived significantly longer after host type leukemia challenge (injection of 6 x 10(6) AKR lymphoma cells). The better survival of the TLI chimeras was not due to the radiation regimen, because TLI or TBI conditioned syngeneic AKR-->AKR BM recipients did succumb equally rapidly after AKR lymphoma injection. Donor type (CM) lymphokine-activated killer cell activity, however, was higher in TLI chimeras and may explain the better GVL activity in TLI mice. This model thus illustrates that GVHD and GVL effects can be dissociated and are differentially influenced by the conditioning regimen used for BM transplantation.