DA Grafts Research Articles

Generation of Suppressive Blood Cells for Induction of Donor- Specific Tolerance in a Rat Heart Transplant Model: A Clinically Relevant Model

Purpose: Our previous studies showed that the chemotherapeutic drug mitomycin c (MMC) renders strongly stimulatory dendritic cells (DCs) suppressive. Donor-derived MMC DCs induced specific tolerance in rat heart allograft recipients. Since in clinical transplantation (Tx) peripheral blood mononuclear cells (PBMCs) are easier available than DCs we tried to replace DCs by PBMCs. Materials and methods: Donor blood (1ml) or PBMCs (108) were incubated for 30 min. with MMC, washed and injected i.v. into recipients 1 week before allogeneic heart Tx (DA to PVG). Blood and spleen cells (SPCs) of tolerant recipients were analyzed by FACS for regulatory cells (Tregs) and adoptively transferred into syngeneic animals transplanted with DA grafts. Grafts were immunohistochemically analyzed for cellular infiltration, vascular lumen narrowing and complement (C4d) deposition. The cytokine profile of recipient blood was determined by multiplex immunoassay. Results: MMC-treated donor blood injected into recipients prolonged allograft survival (34.43 ± 3.95 vs. 8.56 ± 0.27 in untreated and 21 ± 4.16 days in donor-blood transfused recipients). A stronger effect up to tolerance (50% of the animals) was obtained when donor blood was replaced with MMC- PBMCs (64.8 ± 16.8 vs. 8.56 ± 0.27 in untreated and 34.43 ± 3.95 days in PBMC treated recipients). The effect was abrogated by elimination of monocytes from PBMCs. Third-party heart allograft survival showed no prolongation indicating donor-specific tolerance. Tolerated grafts had cellular infiltrates with a significantly increased number of Foxp3+ cells and decreased deposition of CD4d in blood vessels in comparison to rejected grafts. FACS analysis of PBMCs and SPCs of tolerant animals revealed an increased percentage of CD4+CD25+Foxp3+ Tregs when compared to rejecting animals (PBMCs: 6.12 + 0.99 vs. 5.52 + 0.28%, p > 0.05; SPCs: 8.31 + 1.11 vs. 6.74 + 0.13%, p = 0.02). Both, PBMCs and SPCs of tolerant animals prolonged allograft survival up to tolerance by adoptive transfer into syngeneic recipients. Cytokine profile analysis suggests a Th2 deviation early after transplantation. Non-significant reduction of vascular lumen in allografts (as measure for chronic rejection) of tolerant animals in comparison to syngeneic ones was observed. Conclusions: A single pretransplant infusion of MMC-PBMCs is able to induce donor-specific suppression up to tolerance in a heart allotransplant model without concomitant use of immunosuppressants. The cell subpopulation which induces suppression is mainly monocytes. Suppression might be mediated by CD4+CD25+Foxp3+ Tregs, since these cells were found in increased number in blood, spleen and grafts of tolerant animals, and tolerance can be adoptively transferred by PBMCs and SPCs. MMC-PBMCs reduce but do not fully prevent chronic rejection. The described model has clinical relevance.

Mobilization of Host Stem Cells Enables Long-Term Liver Transplant Acceptance in a Strongly Rejecting Rat Strain Combination

Careful examination of liver, kidney and heart transplants in human recipients has revealed small numbers of host bone marrow derived stem cells in the graft. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long-term graft function without immunosuppression. We proposed to "engineer" repopulation after transplant in a strain combination (dark agouti [DA] to Lewis green fluorescent protein+[LEW GFP+]) which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Treatment on days 0, 1, 2, 3 and 7 after transplantation with low-dose (0.1 mg/kg) tacrolimus (T) designed to blunt rejection combined with plerixafor (P) to mobilize host stem cells resulted in greater than 180 days graft survival with extensive albeit spotty conversion of a small (50%) DA graft to the recipient LEW GFP+ genotype. Subsequent skin grafting revealed donor-specific graft prolongation. The T plus P treatment resulted in higher levels of Lin-Thy1+CD34+CD133+ stem cells and Foxp3+ regulatory T cells in the blood and liver at day 7. Thus, pharmacological mobilization of host stem cells sustains liver allografts by two mechanisms: repopulation of injured donor cells and regulation of the immune response.

ES cell-derived renewable and functional midbrain dopaminergic progenitors

During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2(+)Corin(+) cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2(+)Corin(+) cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2(+)Corin(+) cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.

Effects of the novel protein kinase C inhibitor AEB071 (Sotrastaurin) on rat cardiac allograft survival using single agent treatment or combination therapy with cyclosporine, everolimus or FTY720

NVP-AEB071 (AEB, sotrastaurin), an oral inhibitor of protein kinase C (PKC), effectively blocks T-cell activation. The immunosuppressive effects of oral AEB were demonstrated in a rat local graft versus host (GvH) reaction and rat cardiac transplantation models. T-cell activation was suppressed by 95% in blood from AEB-treated rats, with a positive correlation between T-cell inhibition and AEB blood concentration. In GvH studies, AEB inhibited lymph node swelling dose-dependently (3-30 mg/kg). BN and DA cardiac allografts were acutely rejected within 6-10 days post-transplantation in untreated LEW rats. AEB at 10 and 30 mg/kg b.i.d. prolonged BN graft survival to a mean survival time of 15 and >28 days, and DA grafts to 6.5 and 17.5 days, respectively. In the DA to LEW model, combining a nonefficacious dose of AEB (10 mg/kg b.i.d.) with a nonefficacious dose of cyclosporine, everolimus or FTY720 led to prolonged median survival times (26 days, >68 days and >68 days, respectively). Pharmacokinetic monitoring excluded drug-drug interactions, suggesting synergy. In conclusion, these studies are the first to demonstrate that AEB prolongs rat heart allograft survival safely as monotherapy and in combination with nonefficacious doses of cyclosporine, everolimus or FTY720. Thus, AEB may have the potential to offer an alternative to calcineurin inhibitor-based therapies.

Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)

Abstract Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival. NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant. Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095

Walking pattern analysis after unilateral 6-OHDA lesion and transplantation of foetal dopaminergic progenitor cells in rats

Functional sensorimotor recovery after transplantation of mesencephalic dopaminergic (DAergic) neurons has been well documented in the rat 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. However, the functional restoration of more specific gait-related patterns such as skilled walking, balance, and individual limb movements have been insufficiently studied. The purpose of this study was to investigate the behavioural effects of intrastriatal DA grafts on different aspects of normal and skilled walking in rats following unilateral 6-OHDA lesions of the medial forebrain bundle. Rats were subjected to drug-induced rotation, detailed footprint analysis, and assessment of skilled walking in the ladder rung walking test prior and after the transplantation of E14 ventral mesencephalon-derived progenitor cells. Good DAergic graft survival, as revealed by immunohistochemistry, was accompanied by a compensation of drug-induced rotational asymmetries. Interestingly, the analysis of walking patterns displayed a heterogeneous graft-induced response in skilled and non-skilled limb use. Grafted animals made fewer errors with their contralateral limbs in skilled walking than the sham-transplanted rats, and they improved their ipsi- and contralateral limb rotation. However, the parameter distance between feet showed a delayed recovery, and the stride length was not affected by the DA grafts at all. These findings indicate that ectopic intrastriatal transplantation of E14 ventral mesencephalon-derived cells promotes recovery of gait balance and stability, but does not ameliorate the shuffling gait pattern associated with 6-OHDA lesions. A full restoration of locomotor gait pattern might require a more complete and organotypic reconstruction of the mesotelencephalic DAergic pathway.

Immunological Tolerance-Related Genes in a Spontaneous Tolerant Model of Rat Liver Transplantation Explored by Suppression Subtractive Hybridization

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgapl), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.

Focal not widespread grafts induce novel dyskinetic behavior in parkinsonian rats

Dyskinesias are a common consequence of dopaminergic therapy in patients with Parkinson's disease. Little is known about the influence of cellular replacement strategies upon drug-induced dyskinesias. In the current study, we employed parkinsonian rats to test whether the distribution of dopamine neuron grafts could differentially alter striatal circuitry and levodopa-induced dyskinesias. Specifically, we compared behavioral and neurochemical consequences of dopamine reinnervation restricted to a focal region of the striatum to innervation encompassing the majority of the striatum by distributing the same number of cells into single locus or multiple locations. Both the single-site and widespread grafts reduced pregraft dyskinesias and normalized FosB/DeltaFosB in the dorsal two-thirds of the lateral striatum. However, single-site DA graft recipients developed a robust, novel forelimb-facial stereotypy and upregulated FosB/DeltaFosB expression in the ventrolateral striatum, an area associated with movements of tongue and forelimbs. The onset of forelimb-facial stereotypy correlated with measures of increased graft function.

Efficiency of intrathymic tolerance induction in various inbred rat strains: relationship with T H1/T H2 status of the recipient?

Summary The simultaneous transplantation and intrathymic tolerance induction (STITTI) protocol induces a longlasting state of functional tolerance in over 90% of AO (RT1 u ) recipients transplanted with a fully MHC-incompatible PVG (RT1 c ) cardiac allograft. Similar results are obtained when using LEWIS (RT1 1 ) rats as recipients of either PVG or DA (RT1 avl ) grafts. However, when STITTI is performed on PVG and BN (RT1 n ) as recipient animals receiving spleen cells intrathymically and a cardiac allograft from respectively AO and PVG rats, this procedure results in significantly shorter graft survival (MST PVG → BN 25 ± 9 days; AO → PVG 31 ± 8 days) as compared to the combinations using AO (MST PVG → AO > 236 ± 28 days) and LEWIS (MST PVG → LEW > 366 ± 51 days; DA → LEW > 123 ± 33 days) rats as recipients. Since both PVG and BN rats are relatively deficient in their ability to produce IFNγ and intrathymic IFNγ responses are very dominant upon intrathymic injection of alloantigens, it is argued that the inability to effectively induce a longlasting state of functional tolerance in BN and PVG rats using the STITTI protocol may be related to their decreased IFNγ-production potential.

Malononitrilamides and tacrolimus additively prevent acute rejection in rat cardiac allografts

The novel immunosuppressive agents malononitrilamides (MNA) 279 and 715 are derivatives of A77 1726, the primary metabolite of leflunomide. The effects of these agents have been previously demonstrated in rat skin and cardiac allo- and xenotransplant models. The aim of this study was to evaluate the combination of MNA and tacrolimus in a high-responder rat cardiac allotransplant model. Graft survival was evaluated following 10 days of post-transplant oral therapy of MNA or tacrolimus alone and the drugs combined in PVG recipients of DA grafts. An iso-effect curve of single and combined drugs was constructed. Histological changes in grafts were evaluated at 10 days. MNA (20 mg/kg) or tacrolimus (2.4 mg/kg) alone prolonged graft survival with median survival of 14 and 13.5 days, respectively. Combined therapy of MNA (10 mg/kg) and tacrolimus 1.6 mg/kg likewise resulted in a median survival of 13.25 days and an iso-effect curve for these doses was constructed. Another iso-effect curve for median graft survival of 18–19 days, including MNA (10 mg/kg) + tacrolimus (3.2 mg/kg) in combination, MNA (30 mg/kg) alone and tacrolimus (4.8 mg/kg) alone, was constructed and both isoboles showed a straight line, demonstrating additive effects (zero interaction). In addition, histological analysis of grafts confirmed the benefit of the drug combination. No additional toxicity was noted with combined therapy. Optimal doses of MNA or tacrolimus had comparative effects on graft survival and histological changes, and a combination of the two drugs was beneficial with respect to both these parameters. The iso-effect curves verified additives effects of the drug combination.

The cellular basis of cardiac allograft rejection: VIII. Mechanisms underlying delayed allograft rejection in PVG C6-deficient rats.

The delayed allograft rejection in C6-deficient PVG C6- rats compared with normal PVG rats has been attributed to the lack of alloantibody activation of the membrane attack complex of complement. As T cells alone have been shown to effect graft rejection, we examined T-cell responses in PVG C6- rats. The cellular infiltrate and its mRNA for cytokines and effector molecules in DA heart allografts to PVG and PVG C6- rats was compared by immunoperoxidase staining and semiquantitative reverse transcriptase polymerase chain reaction. The ability of pure populations of T cells or alloantibody to mediate DA heart graft rejection in irradiated (750 rads) PVG and PVG C6- rats was also compared. The median rejection time of DA heart allografts was 8 days in PVG rats and 17.5 days in PVG C6-. PVG C6- rats sensitized to DA by two skin grafts rejected DA heart grafts in 5-6 days. CD3+, CD4+, CD8+, interleukin-2 receptor-positive T cell, macrophage, and natural killer cell infiltration, as well as class II major histocompatibility complex and intercellular adhesion molecule-1 up-regulation, in grafts was similar in naive PVG and PVG C6- rats. mRNA for T helper 1 cytokine interleukin-2, interferon-gamma, tumor necrosis factor-beta, macrophage molecules tumor necrosis factor-alpha, and inducible nitric oxide synthase, as well as cytotoxic T-cell effector molecules perforin and granzyme A and B, were found to be the same in the grafts from both naive PVG and naive PVG C6- rats. Thus, there appeared to be no difference in the T-cell effector response between the PVG and PVG C6- groups. There were higher alloantibody titers in PVG C6- rats than in PVG hosts. Irradiation ablated rejection and alloantibody responses and reconstitution with naive T cells alone restored rejection in both PVG and PVG C6- rats. Irradiated rats given serum from PVG rats that had rejected DA grafts did not effect rejection of DA grafts even if given naive T cells. Sensitized T cells restored second set. PVG C6- rats have normal T-cell responses and can mediate allograft rejection in the absence of alloantibody. The failure of PVG C6- to reject allografts rapidly may be a result of the poor clearance of alloantisera leading to enhancement of graft survival rather than a critical role for complement and membrane attack complex in acute rejection.

Single dose anti-CD4 monoclonal antibody for induction of tolerance to cardiac allograft in high- and low-responder rat strain combinations

Repeated adminsitration of monoclonal antibodies (mAb) directed againt the CD4 lymphocyte receptor may induce specific, long-lasting unresponsiveness to fully MHC-mismathed cardiac allografts in rats without additional immunosuppression. We assessed the effect of a single dose of murine anti-rat depleting anti-CD4 mAb (OX-38) on allograft survival in high- and low-responder rat strain combinations. Isogenic strains of DA (RT1 av1), PVG (RT1 c), AUG (RT1 c), and WF (RT1 u) rats were used. Recipients in antibody treated groups were given one dose of 5 mg/kg OX-38 mAb on the day of transplant, a dose which was shown to effectively deplete (or block) circulating CD4 + T cells. Other groups were treated for 10 days with cyclosporin A (CsA) and/or Linomide, a novel immunomodulator, which is the first compound able to fully eliminate the effect of CsA in the rat cardiac allograft model. The DA strain was identified as a low-responder to the allogeneic haplotype RT1 c (PVG or AUG), but not to RT1 u (WF), and developed true tolerance following RT1 c grafting and OX-38 or low-dose CsA (5 mg/kg) induction, as verified by the response to retransplantation of a graft from the same donor strain or a third-party challenge. PVG recipients of DA grafts were characterized by high response and only modest (OX-38; median 9.5 days) or moderate (CsA; 23.5 days) prolongation of graft survival. Contrasting graft survival results were obtained in the low-responder combination, either very early rejection (at 10 days) or permanent graft survival (> 100 days). Linomide challenge affected CsA treatment in the high-responder combination but not tolerance induction in the low-responder combination, or the effect of OX-38. It was concluded that in rat heart transplantation a single-dose anti-CD4 mAb therapy may induce permanent donor-specific unresponsiveness in a low-responder strain combination, and that anti-CD4 mAb seems to be unique among immunosuppressive agents while being resistent to challenge by Linomide.

FINE SPECIFICITY OF PEPTIDE DETERMINANTS FOR INDIRECT T CELL RECOGNITION OF CLASS I MHC ALLOANTIGENS

CD4+ T cells from the LEW (RT1l) rat strain are able to recognize a 24-amino acid peptide from the alpha-helical region of the alpha 1-domain of the DA (RT1avl) RT1.A class I MHC molecule. This response is known to play a role in the effector mechanisms of rejection of DA grafts by LEW recipients. In this study, we demonstrate that the WAG (RT1u) strain is also able to respond to this peptide, but that the PVG (RT1c) strain does not respond. Fine specificity studies using a nested set of 15 mers derived from the 24 mer indicate that the LEW strain recognizes multiple T cell epitopes spanning the length of the peptide, while the WAG strain response is limited to the N-terminal region. With regard to B cell immunity, both the LEW and WAG strains give strong antibody responses when immunized with the free peptide, while the antibody response in the PVG strain is weak. Interestingly, in all 3 strains, the antibodies appear to be directed at the N- and C-terminal regions of the peptide, and to be almost entirely dependent upon the presence of the N- and C-terminal amino acids. These studies are potentially important when considering the specificity of rejection responses, and most particularly when considering the specific suppression of rejection mediated by indirect T cell allorecognition.

FINE SPECIFICITY OF PEPTIDE DETERMINANTS FOR INDIRECT T CELL RECOGNITION OF CLASS I MHC ALLOANTIGENS

CD4+ T cells from the LEW (RT11) rat strain are able to recognize a 24-amino acid peptide from the α-helical region of the α1-domain of the DA (RT1avl) RT1.A class I MHC molecule. This response is known to play a role in the effector mechanisms of rejection of DA grafts by LEW recipients. In this study, we demonstrate that the WAG (RT1u) strain is also able to respond to this peptide, but that the PVG (RT1c) strain does not respond. Fine specificity studies using a nested set of 15 mers derived from the 24 mer indicate that the LEW strain recognizes multiple T cell epitopes spanning the length of the peptide, while the WAG strain response is limited to the N-terminal region. With regard to B cell immunity, both the LEW and WAG strains give strong antibody responses when immunized with the free peptide, while the antibody response in the PVG strain is weak. Interestingly, in all 3 strains, the antibodies appear to be directed at the N- and C-terminal regions of the peptide, and to be almost entirely dependent upon the presence of the N- and C-terminal amino acids. These studies are potentially important when considering the specificity of rejection responses, and most particularly when considering the specific suppression of rejection mediated by indirect T cell allorecognition.

EVIDENCE OF ALLOREACTIVE T SUPPRESSOR CELLS IN THE MAINTENANCE PHASE OF SPONTANEOUS TOLERANCE AFTER ORTHOTOPIC LIVER TRANSPLANTATION IN THE RAT

In the DA-->PVG rat strain combination liver allografts are spontaneously tolerated and induce a state of systemic tolerance towards donor antigens. Thirty long-term (> 100 days)-surviving liver recipients in this strain combination were used to study humoral and cellular mechanisms of tolerance in the maintenance phase after liver transplantation. In the popliteal lymph node assay splenocytes of liver grafted or native PVG rats displayed a similar proliferative response in (DAxPVG)F1 hybrid rats, indicating an unaffected capacity of these cells to mount graft-versus-host reactions. Using adoptive transfer assays T-splenocytes (1x10(7) cells, MRC Ox 19+, MRC Ox12-, CD 5) of long-term-surviving liver recipients were able to suppress the rejection of DA hearts (donor-specific), of DA and AO kidney grafts in irradiated (300 cGy) PVG recipients. Serum of long-term-surviving liver recipients was able to suppress the rejection of semiallogeneic (DAxPVG)F1 heart grafts after double injection of 1 ml serum, while fully allogeneic DA heart as well as skin and pancreaticoduodenal grafts were resistant to passive enhancement. The results confirm the development of spontaneous tolerance after liver grafting in the DA-->PVG strain combination, and the demonstration of T cells with suppressive capacity in adoptive transfer experiments indicates a role for T suppressor cells in the maintenance phase of spontaneous tolerance after liver grafting.

EVIDENCE OF ALLOREACTIVE T SUPPRESSOR CELLS IN THE MAINTENANCE PHASE OF SPONTANEOUS TOLERANCE AFTER ORTHOTOPIC LIVER TRANSPLANTATION IN THE RAT

In the DA→PVG rat strain combination liver allografts are spontaneously tolerated and induce a state of systemic tolerance towards donor antigens. Thirty long-term (>100 days)-surviving liver recipients in this strain combination were used to study humoral and cellular mechanisms of tolerance in the maintenance phase after liver transplantation. In the popliteal lymph node assay splenocytes of liver grafted or native PVG rats displayed a similar prolif-erative response in (DA×PVG)F1 hybrid rats, indicating an unaffected capacity of these cells to mount graft-versus-host reactions. Using adoptive transfer assays T-splenocytes (1×107 cells, MRC O× 19+, MRC O×12-, CD 5) of long-term-surviving liver recipients were able to suppress the rejection of DA hearts (donor-specific), of DA and AO kidney grafts in irradiated (300 cGy) PVG recipients. Serum of long-term-surviving liver recipients was able to suppress the rejection of semiallogeneic (DA×PVG)F1 heart grafts after double injection of 1 ml serum, while fully allogeneic DA heart as well as skin and pancreaticoduodenal grafts were resistant to passive enhancement. The results confirm the development of spontaneous tolerance after liver grafting in the DA×PVG strain combination, and the demonstration of T cells with suppressive capacity in adoptive transfer experiments indicates a role for T suppressor cells in the maintenance phase of spontaneous tolerance after liver grafting.

Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspensions in the rat Parkinson model

A microtransplantation approach has been used in order to achieve more complete reinnervation of the dopamine denervated rat striatum by fetal nigral cell suspensions injected into multiple striatal sites. A total of 450, 000 cells, obtained from the ventral mesencephalon of embryonic day 14 rat fetuses, were implanted either in the conventional way as two 1.8-μl deposits centrally in the head of the caudate-putamen (‘Macro grafts’), or as eighteen 0.2-μl deposits disseminated over six needle penetrations in the same area using a 50–70 μ m glass capillary tip (‘Micro grafts’). Non served lesioned rats served as controls. Dopamine neuron survival (as assessed by tyrosine hydroxylase immunohistochemistry at 4 months after transplantation) was 2.8-fold greater in the Micro grafts as compared to the Macro grafts. Striatal dopamine tissue levels (determined in a separate group of rats) was increased 2.5-fold in the head of the caudate-putamen (from 12.5% of normal in the Macro graft group to 30% of normal in the Micro graft group). Consistent with this, the overall graft-derived tyrosine hydroxylase positive fiber outgrowth was more extensive in the Micro graft group and covered larger areas of the previously denervated caudate-putamen. The results show that distribution of the fetal nigral tissue in multiple small deposits provides for increased dopamine neuron survival, probably because of a closer contact between the implanted cells and the surroundinghost striatal tissue in the small-sized graft deposits. Less bleeding and necrosis at the implantation site may also have contributed to this effect. The present microtransplantation procedure is an efficient means to increase overall dopamine neuron survival and to achieve more complete reinnervation of the denervated striatum in the rat Parkinson model. It also substantially increased the reproducibility of DA graft survival between animals.

SPONTANEOUS ACCEPTANCE OF LIVER ALLOGRAFTS IN THE RAT

In a model of arterialized rat liver transplantation, the biological indicators of liver dysfunction and the phenotype and in vitro function of graft-infiltrating cells have been compared in rejected (DA-to-Lew) and spontaneously accepted (Lew-to-DA) grafts, 2-8 days after grafting. Recipients of rejected and nonrejected allografts had, during this time, similar loss in body weight and plasma levels of transaminase. The markers of cholestasis, however, increased from days 3 and 4 onward in the recipients of rejected grafts, but remained low and similar in the recipients of nonrejected allografts and those of syngeneic grafts. From days 2 to 6 the phenotype, IL-2 responsiveness, and donor-specific cytotoxic potential of the leukocytes infiltrating rejected and nonrejected allografts were comparable. On days 7 and 8, although the proportion of T cell subpopulations was identical in both combinations, activated CD4+ graft-infiltrating cells were reduced in the nonrejected grafts. Also at this time, donor-specific cytotoxic cells were no longer detected in nonrejected grafts, whereas activity had reached a peak in the rejected grafts. These results suggest that liver grafts in both the LEW-->DA (grafts not rejected) and DA-->LEW (grafts rejected) strain combinations undergo tissue damage, but that the type of damage differs between the two combinations. Specifically, cholestasis was only observed in grafts that would subsequently be rejected. The difference in graft damage occurred at a very early time point (3 or 4 days after grafting), at which time neither the intensity nor phenotype of the graft infiltrate, its IL-2 responsiveness, or its cytotoxic potential varied between the two combinations. Thus, a lack of immune reactivity, as assessed by these parameters, does appear not to be responsible for spontaneous acceptance of liver transplants in the LEW-->DA strain combination.

THE ROLE OF T SUPPRESSOR CELLS IN THE MAINTENANCE OF SPONTANEOUSLY ACCEPTED ORTHOTOPIC RAT LIVER ALLOGRAFTS

Orthotopic liver allografts from BN donors to LEW recipients are spontaneously accepted, and the recipients develop donor-specific immunological unresponsiveness. This unresponsiveness may be mediated by suppressor T cells. Immunomagnetically purified splenic T cells from LEW rats bearing BN liver grafts were shown to adoptively transfer suppression of skin, heart, and kidney graft rejection in a donor-specific manner, prolonging the survival of BN but not third-party DA grafts. However, the suppressor T cells were sessile, being resident in the spleen but not present in thoracic duct lymph. The presence of a nonrecirculating suppressor T cell in rats spontaneously accepting liver transplants is strongly suggestive of an important function in the maintenance of donor-specific unresponsiveness, although the contribution of other possible mechanisms of unresponsiveness has not been investigated.

PROLONGATION OF RENAL ALLOGRAFT SURVIVAL IN THE RAT TREATED WITH AMNIOTIC FLUID

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.