RESUMEN
Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV- HIV- PCNSL and 2 EBV- HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV- PCNSL. As with prior studies, EBV- HIV- PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV- PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV- HIV- PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.
Asunto(s)
Neoplasias del Sistema Nervioso Central/etiología , Neoplasias del Sistema Nervioso Central/inmunología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Linfoma/virología , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Femenino , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Tolerancia Inmunológica , Linfoma/etiología , Masculino , Persona de Mediana Edad , Mutación , Transcriptoma , Microambiente TumoralRESUMEN
In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígeno HLA-A2/inmunología , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/aislamiento & purificación , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/virología , Proteínas de la Matriz Viral/inmunología , Adolescente , Adulto , Anciano , Presentación de Antígeno , Linfocitos T CD8-positivos/virología , Femenino , Genes MHC Clase I , Antígeno HLA-A2/genética , Herpesvirus Humano 4/genética , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Proteínas de la Matriz Viral/genética , Adulto JovenRESUMEN
Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio-process to purify CMRF-56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF-56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/trasplante , Mieloma Múltiple/terapia , Anticuerpos Monoclonales/aislamiento & purificación , Presentación de Antígeno/inmunología , Antígenos de Diferenciación/inmunología , Antígenos de Neoplasias/inmunología , Biotinilación , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Separación Inmunomagnética/métodos , Leucaféresis , Mieloma Múltiple/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación/métodosRESUMEN
Understanding the biology of the DC and its pivotal role in immune response initiation and regulation has enabled new effective anti-cancer therapies to be developed for treatment of a wide range of tumor types. Many studies on DC-based cancer vaccine development have focused on the development of methods that can effectively deliver tumor Ags to DCs. Numerous methods have been developed or evaluated for the delivery of defined and undefined tumor Ags to DCs for processing and presentation to T lymphocytes. This review provides a brief summary of these methods, the techniques that are used in each, how they have -- or could -- be applied clinically, as well as the advantages and disadvantages of each method.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Neoplasias/terapia , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Antígenos CD/inmunología , Antígenos de Neoplasias/genética , Vacunas contra el Cáncer/inmunología , Técnicas de Cultivo de Célula/métodos , Citocinas/inmunología , ADN de Neoplasias/genética , ADN de Neoplasias/inmunología , Antígenos de Histocompatibilidad/inmunología , Humanos , Activación de Linfocitos/inmunología , Ratones , Neoplasias/inmunología , Péptidos/inmunología , Proteínas/inmunología , ARN Neoplásico/inmunología , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , VacunaciónRESUMEN
A synthetic peptide conjugate based on the N-terminal sequence of a 10 000 MW immunosuppressive serum protein (reOLT 4) was used to immunize female Lewis rats prior to mating, in order to determine whether blocking this protein had an effect on pregnancy. The N-terminal sequence of (reOLT 4) has close sequence homology to the beta-chain of rat haemoglobin so a peptide conjugate based on the N-terminal sequence of this protein was also used to immunize female Lewis rats. Controls included animals that were not immunized and animals that received the peptide carrier, diphtheria toxoid (DT). No statistical differences were found in gestation time or litter sizes in these groups. Differences were, however, evident between these groups and animals that received DT-(reOLT 4) (group 4) or the DT-beta-chain haemoglobin (group 5). There were no statistical differences in litter size or gestation time for group 4 when compared with group 5. Enzyme-linked immunosorbent assay and dot-blot analysis revealed that rats from both groups also had strong responses against DT, the peptide conjugate they were immunized with and the corresponding full-length protein. In both cases, animals from group 4 and group 5 had weak responses to the peptide that they did not receive, together with lower erythrocyte counts and haematocrits, and elevated heart to body weight ratios. Additionally, antibody purified on a (reOLT 4) immunoaffinity column was capable of binding to rat erythrocytes. A second investigation comparing anaemia prior to fertilization and maintained anaemia over the gestation period revealed that only the latter was capable of decreasing litter size to the same degree as obtained for groups 4 and 5. We conclude that for groups 4 and 5 it is the autoimmune effect of continual anaemia over the gestation period, mediated by autoantibodies, which results in the observed lower litter size.
Asunto(s)
Proteínas Sanguíneas/inmunología , Globinas/inmunología , Inmunosupresores/inmunología , Tamaño de la Camada/inmunología , Fragmentos de Péptidos/inmunología , Vacunas Sintéticas/inmunología , Animales , Femenino , Globinas/química , Rechazo de Injerto/inmunología , Hematócrito , Inmunohistoquímica , Masculino , Embarazo , Ratas , Ratas Endogámicas Lew , Vacunación/efectos adversos , Vacunas Conjugadas/inmunologíaAsunto(s)
Rechazo de Injerto/prevención & control , Inmunosupresores/uso terapéutico , Trasplante de Hígado/inmunología , Secuencia de Aminoácidos , Animales , Supervivencia de Injerto , Trasplante de Corazón/inmunología , Proteínas , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Factores de Tiempo , Trasplante HomólogoAsunto(s)
Citocinas/genética , Trasplante de Hígado/inmunología , Animales , Citocinas/análisis , Citocinas/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Interferón gamma/genética , Interleucina-2/genética , Trasplante de Hígado/patología , Masculino , Ratas , Ratas Endogámicas , Transcripción Genética , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/genéticaAsunto(s)
Rechazo de Injerto/prevención & control , Supervivencia de Injerto/fisiología , Trasplante de Corazón/inmunología , Inmunosupresores/uso terapéutico , Trasplante de Hígado/inmunología , Secuencia de Aminoácidos , Animales , Supervivencia de Injerto/efectos de los fármacos , Humanos , Inmunosupresores/química , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Proteínas , Ratas , Ratas Endogámicas , Trasplante HomólogoRESUMEN
Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation was performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 micrograms single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days; n = 7 vs 6.5 days; n = 5, respectively, P = 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days; n = 7) or those which received only Intralipos (median = 10.5 days; n = 5, P = 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum.
Asunto(s)
Inmunosupresores/sangre , Trasplante de Hígado/fisiología , Animales , Anticuerpos/inmunología , Cromatografía de Afinidad , Dermatoglifia del ADN , Electroforesis en Gel de Poliacrilamida , Trasplante de Corazón/fisiología , Inmunosupresores/química , Inmunosupresores/inmunología , Masculino , Proteínas , Ratas , Ratas Endogámicas , Homología de Secuencia de AminoácidoRESUMEN
The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1a) livers into PVG (RT1c) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1(1)) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.
Asunto(s)
Movimiento Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Tolerancia Inmunológica , Leucocitos/inmunología , Trasplante de Hígado/inmunología , Animales , Médula Ósea/inmunología , Médula Ósea/patología , Inmunidad Innata , Cinética , Trasplante de Hígado/patología , Pulmón/inmunología , Pulmón/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Mesenterio , Ratas , Ratas Endogámicas Lew , Bazo/inmunología , Bazo/patología , Timo/inmunología , Timo/patologíaRESUMEN
The effect of serum from orthotopic liver retransplanted rats (re-OLT serum) on graft-versus-host disease (GVHD) was studied in rats. In the re-transplantation model of rat liver, orthotopic liver transplantation (OLT) was carried out in the DA (RT1a) into PVG (RT1c) combination; two days later the DA liver was removed and a new PVG liver implanted into the same recipient (re-OLT). In the in vivo GVHD model, male PVG rats were sublethally irradiated and injected intravenously with 3 x 10(8) DA or BN (RT1n) spleen through the penial vein. Within 1 h of the inoculation, rats of the experimental group were injected with 1 ml of re-OLT serum taken at postoperative day (POD) 7. Rats in the control group received 1 ml of normal PVG serum or syngeneic re-OLT serum (PVG-PVG, PVG-PVG). All PVG rats in the control groups died of GVHD within 21 days after the inoculation of DA or BN spleen lymphocytes. However, when the animals were treated with re-OLT serum, 100% (6/6) of the rats survived more than 60 days, following inoculation with DA lymphocytes but not with BN lymphocytes. The POD 7 re-OLT serum showed a strong inhibition against DA anti-PVG mixed lymphocyte reaction (MLR), although re-OLT serum did not contain soluble DA class I antigens, anti-DA class I or II antibody. The potential GVHD inhibitory factors in re-OLT serum may be two unique immunosuppressive proteins, which have been detected by SDS PAGE and reported previously. We conclude that re-OLT serum has immunosuppressive factors, which, at least in part, prevented the induction of GVHD in rats.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Inmunosupresores/sangre , Trasplante de Hígado/inmunología , Animales , Enfermedad Injerto contra Huésped/sangre , Masculino , Ratas , Ratas Endogámicas BN , Reoperación , Trasplante HomólogoAsunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Terapia de Inmunosupresión/métodos , Trasplante de Hígado/inmunología , Animales , Sangre/inmunología , Rayos gamma , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Hígado/métodos , Prueba de Cultivo Mixto de Linfocitos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas , Reoperación , Trasplante HomólogoRESUMEN
The tolerance induced by orthotopic liver transplantation [DA (RT1a) rats to PVG (RT1c) rats] can be prevented by total body irradiation of the donor rat. Reconstitution of the irradiated donor with DA splenic leukocytes reintroduces this tolerance. To investigate the major histocompatibility complex (MHC) specificity of passenger leukocytes, irradiated DA donors were reconstituted by third-party BN (RT1n) splenic leukocytes. The reconstitution with BN splenocytes re-established DA-specific tolerance in PVG recipients, as confirmed by subsequent DA cardiac allografting, while BN hearts were rejected with second-set tempo. To determine which cell components play an important role in re-establishing liver graft tolerance, DA splenic leukocytes were further purified into three types: T, B, and adherent cells. Only "T-cell-enriched" preparations restored liver graft tolerance in three out of five PVG recipients. These results suggest that passenger leukocytes of differing MHC types can help to induce liver-specific tolerance and that T cells in the liver graft may be essential to regulate tolerance induction.
Asunto(s)
Leucocitos/inmunología , Trasplante de Hígado/inmunología , Animales , Trasplante de Corazón/inmunología , Tolerancia Inmunológica , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratas , Bazo/citologíaRESUMEN
In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart graft in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination, DA (RT1a) donor into PVG (RT1c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosuppressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 micrograms of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1n) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 5/6 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the injection of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.
Asunto(s)
Supervivencia de Injerto/efectos de los fármacos , Tolerancia Inmunológica/fisiología , Inmunosupresores/uso terapéutico , Trasplante de Hígado/inmunología , Animales , Rechazo de Injerto/etiología , Trasplante de Corazón/inmunología , Antígenos de Histocompatibilidad/inmunología , Inmunosupresores/sangre , Inmunosupresores/aislamiento & purificación , Inmunosupresores/farmacología , Isoanticuerpos/farmacología , Masculino , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Proteínas , Conejos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas , Trasplante Heterotópico , Trasplante Homólogo/inmunologíaAsunto(s)
Transfusión de Componentes Sanguíneos , Enfermedad Injerto contra Huésped/prevención & control , Intestino Delgado/trasplante , Trasplante de Hígado/fisiología , Trasplante Homólogo/fisiología , Animales , Peso Corporal , Supervivencia de Injerto , Terapia de Inmunosupresión/métodos , Trasplante de Hígado/inmunología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas , Trasplante Homólogo/inmunologíaRESUMEN
Liver grafts between certain rat strain combinations, such as DA (RT1a)-into-PVG (RT1c), are accepted without the use of immunosuppressive agents. To explore the nature and role of serum proteins in liver-induced immunosuppression, we have developed a retransplantation model of rat liver grafting. In this procedure, orthotopic liver transplantation (OLT) is carried out in the DA-into-PVG combination; two days later the DA liver is removed and a new PVG liver implanted into the same recipient (re-OLT). Serum from re-OLT rats was immunosuppressive when tested in mixed lymphocyte reactions (MLR). Three novel proteins were detected in re-OLT serum by SDS-PAGE, with sizes of 180 kD, 87 kD, and 10 kD. The N-terminal sequences of these were distinct and did not match protein sequences in the computer databases, although there was some homology between the 10 kD sequence and the beta-chain of rat hemoglobin. Purified 87 kD and 10 kD proteins were immunosuppressive in MLR; in both cases suppression was dose-dependent and nonstimulator-specific. Production of the 180 kD and 87 kD molecules required the presence of the recipient spleen. We conclude that re-OLT serum contains novel immunosuppressive proteins, which may be products of immune recognition and associated with the immediate termination of graft rejection.
Asunto(s)
Inmunosupresores/aislamiento & purificación , Trasplante de Hígado , Secuencia de Aminoácidos , Animales , Inmunosupresores/sangre , Masculino , Datos de Secuencia Molecular , Peso Molecular , Ratas , Ratas Endogámicas , Análisis de SecuenciaRESUMEN
High sensitivity and low background, which are the significant features of the procedure of Johansson and Skoog (J. Biochem. Biophys. Methods. 1987, 14, (Suppl.) 33) for silver staining of nucleic acids in polyacrylamide gels, have been improved by excluding ethanol from all solutions and increasing the length of washing steps after sensitisation and staining. Including a fixation step before sensitisation increased the contrast of the bands even at high concentration of nucleic acid, producing a shift in the tone of colour from black to grey instead of gold to yellow bands. This technique is superior in terms of sensitivity and background to a number of other silver staining protocols, three which utilise silver diammine and three which utilise silver nitrate ions.
Asunto(s)
Amoníaco , Electroforesis en Gel de Poliacrilamida , Ácidos Nucleicos/análisis , Compuestos de Plata , Tinción con Nitrato de Plata/métodos , Biomarcadores/química , Peso Molecular , Sensibilidad y EspecificidadRESUMEN
The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5 x 10(8) splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.