Cloning and characterization of recombinant rhesus macaque IL-10/Fc(ala-ala) fusion protein: a potential adjunct for tolerance induction strategies.

The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc(ala-ala). RM IL-10/Fc(ala-ala) was purified to approximately 98% homogeneity by affinity chromatography and shown to be endotoxin-free (<0.008 EU/microg protein). The biological activity of IL-10/Fc(ala-ala) was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc(ala-ala) had significantly greater potency than human IL-10/Fc(ala-ala) and exhibited a circulating half-life of approximately 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc(ala-ala) can influence tolerance outcomes in NHP.

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells.

OBJECTIVE: To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS: We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS: CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION: Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.

Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma.

We previously developed a system using murine strome (HESS-5), which could expand umbilical cord blood (UCB) stem and progenitor cells, especially CD34+/38- cells, in the presence of human recombinant cytokines. In this study, the ability of expanded UCB- or bone marrow (BM)-CD34+ cells to differentiate into dendritic cells (DCs) was examined. DCs could be induced either from short or long term cultured CD34+ cells after switching the cytokines from Flk-2/Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO) to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (immature type) plus tumor necrosis factor (TNF)-alpha with stimulation by CD40L transfectant (mature type). Each immature or mature UCB-DCs showed a dextran uptake or a potent allo-T lymphocytes proliferative ability, respectively. Furthermore, those DCs from BM significantly stimulated auto-T lymphocytes in an antigen (varicella zoster virus) specific manner. In conclusion, a novel culture system using HESS-5 is useful to support a rapid and sustained generation of primitive myeloid cells which can develop into functional DCs.

The efficient generation of CD83 positive immunocompetent dendritic cells from CD14 positive acute myelomonocytic or monocytic leukemia cells in vitro.

The ability of leukemic cells to differentiate to mature dendritic cells (DCs) was investigated in six acute myelomonocytic or monocytic leukemia cases. It was found that CD14 positive cells were more efficiently changed to CD83 positive mature typed DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) compared with CD14 negative cells. Such leukemia derived DCs expressed a sufficient level of costimulatory molecules (CD80 and CD86), and were shown to be monoclonal based on an the X-inactivation analysis. They also stimulated not only allo- but auto-T lymphocytes, which thereafter became cytotoxic T lymphocytes (CTLs).

Serum soluble HLA-DR antigens in autoimmune hepatitis.

To investigate the significance of HLA-class II, especially DR antigens, in autoimmune hepatitis (AIH), the serum concentrations of soluble HLA-DR antigen (sDR) were measured in 16 patients with AIH. The expression of HLA-DR antigens in the liver tissues of AIH patients was also studied by immunohistochemistry. AIH at diagnosis showed markedly higher serum sDR levels than controls, in which the liver tissues exhibited positive staining of HLA-DR antigens. Seven patients received corticosteroid therapy, in whom the serum sHLA-DR concentration was reduced dramatically from activated to remission stage. In sequentially follow-up cases, sDR correlated well with the disease activity, and also with the change of surface DR expression in the liver. A single major band with a molecular size of 60 kDa was detected, both in patient's sera and in normal control sera, by Western blotting. In conclusions, serum sHLA-DR level could be a marker reflecting immunological activity of the disease.

Umbilical cord blood T lymphocytes are induced to apoptosis after being allo-primed in vitro.

In this study, the immunity of umbilical cord blood (UCB) T lymphocytes against allo-antigens was investigated by a standard MLC. No significant difference, between the UCB T cells or peripheral blood (PB) mature T cells, was observed in the primary responses (stimulation index (SI), 51.8 +/- 14.8 and 46.5 +/- 15.0, respectively). In contrast, in the secondary response, the SI obtained with the CD4 T cells from UCB decreased dramatically (16.3 +/- 6.4), while it increased with the CD4 T cells from PB (118.5 +/- 21.7). UCB (CD4 and CD8) T cells separately showed much higher frequencies of apoptosis after a primary allo-priming, compared with PB CD4 and CD8 T cells (CD4, UCB 30.5% vs PB 0.8%; CD8, UCB 32% vs PB 1.3%). The higher apoptotic level of the UCB CD4 T cells was confirmed by a second, ELISA-based, Tunel assay (OD values, UCB CD4 1.93 +/- 0.31 vs PB CD4 0.59 0.9; P < 0.01). Those apoptotic steps were not attributed to the amount of cytokine (IL-2, 4 and IFN-gamma) production, which was found to be similar in both cases. In conclusion, UCB lymphocytes are much more likely to be induced to apoptosis by allo-priming than adult lymphocytes. This supports their possible, successful engraftment across barriers of HLA incompatibility.

Delayed hyperacute xenograft rejection in porcine to canine fetal liver transplantation.

Fetal tissues are generally considered to express weaker antigenic cell-surface molecules than adult tissues. We have reported that transplantation of porcine fetal liver tissue (fragments) is useful for acute and chronic hepatic failure in rats. We further investigated, in the present study, whether transplantation of a porcine fetal liver has the advantage of delayed hyperacute xenograft rejection (HAR) in comparison with that of an adult liver. Porcine fetal liver heterotopically transplanted into dogs was compared. Haematoxylin-eosin (HE) and immunohistochemical studies using IgM, C3, IgG antibodies were performed in serial biopsies of the liver grafts. Lectin binding to target antigen epitopes on pig and dog tissues was studied by flow cytometry. Carbohydrate expression on the liver was also studied by immunohistochemistry. The macroscopic and HE section findings indicate that HAR started 15 min postgraft in fetal and adult liver grafts. Thereafter, vascular changes and parenchymal damage progressed more rapidly in the adult grafts. The final HAR time in adult liver transplantation was determined to be 60 min, while it was determined to be 180 min in fetal liver transplantation. IgM, C3 and IgG were deposited more strongly in the adult grafts than in the fetal grafts up until 60 min after xenografting. Phaseolus vulgaris erythroagglutinin lectin competitively blocked dog sera binding to porcine PBLs. The fetal liver expressed oligosaccharide at a significantly lower level than the adult liver. We conclude that porcine fetal liver xenografts had a significantly delayed HAR.

In vitro and in vivo grafting of xeno pig fetal liver fragments using ultrafiltration membrane.

Transplantation of xeno fetal liver fragments (FLF) could be an alternative or supplementary therapy for acute and chronic liver failure not resolved by routine medical therapies. However, the xenografts themselves are rejected by the host immune system. To overcome these problems, immunoisolate capsules with various cutoff points, from 50,000 (YM30) to 500,000 (ZM500) were tested for their protective effects on FLF graft survival. In an in vitro study, the capsule with the smallest cutoff size (YM30) had an excellent protective effect on the grafts it contained, and showed the lowest GOT values in the culture supernatant and the normal histological structure. In an in vivo study using rats, the same capsule enabled a FLF graft to survive as long as 21 days, even with severe IgG deposition on and within the graft. In another in vivo study, which used beagle dog, however, it did not improve the natural course of survival of the graft, which had totally degenerated by day 7. In conclusion, 1) Immunocapsules, especially those with the smallest cutoff values, impeded the infiltration of the (xeno) humoral attacking factor, but the blocking effect was not complete, as shown by the immunoglobulin (IgG) deposit on the grafts they contained. 2) The FLFs with capsules survived longer than those without capsules--only in rats, not in beagles. This difference may be attributable to the difference of the extent of humoral or nutritional response to the xenografts.

The impact of HLA-A matching in corneal transplantation.

Previously, we have reported the results of our retrospective study on the effect of HLA class II allele matching on the outcome of corneal transplant. Here, we demonstrate our findings of the study for HLA class I allele matching in the same study subjects. Eighty transplant recipients were typed for HLA-A, and 79 transplant recipients were typed for HLA-B alleles, by PCR-SSOP. The association between HLA class I allele matching and 1-year rejection-free graft survival was evaluated. When a total of 79 transplant recipients were subdivided into groups with matching (one to four alleles matched) and without matching (no allele matched) for HLA class I (HLA-A and -B), a significantly higher rate of 1-year rejection-free graft survival was detected in transplant recipients with matching, compared with those without matching (p=0.0258). We have found that matching for at least one HLA class I allele was more beneficial especially in high-risk transplant recipients (p=0.0076). Also, an analysis of matching for each locus separately, detected that, HLA-A matching was significantly associated with a higher rate of 1-year rejection-free graft survival. Transplant recipients with HLA-A matching (one or two-alleles matched) had significantly higher rejection-free graft survival compared with those without matching (no allele matched), when high- and low-risk groups were analyzed together (p=0.0099). Furthermore, matching for HLA-A allele was significantly beneficial compared with no matching in high-risk transplant recipients (p=0.0154). Nevertheless, no significant effect of HLA-B matching was detected. We conclude that HLA class I, especially HLA-A matching has a beneficial effect for corneal transplant outcome.

Pancreatic islet isolation variables in non-human primates (rhesus macaques).

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

Increased frequency of CD3/8/56-positive umbilical cord blood T lymphocytes after allo-priming in vitro.

Umbilical cord blood (UCB) or adult peripheral blood mononuclear cells (PBMC) were repeatedly stimulated by HLA-mismatched allogeneic Epstein-Barr virus (EBV)-transformed cell lines, and the bulk responders or single-cloned cells were immunophenotypically analyzed by flow cytometry. One month after the allo-stimulation, not in PBMC but in UCB, the proportion of CD3/8/56 triple positive T lymphocytes significantly increased. Furthermore, UCB clones exhibited those unique CD3/8/56 markers at an extremely higher frequency than PB clones. They showed as much strong killing activity against allo-stimulators as conventional PB CD56-negative, CD8 T-cell clones, whereas they did not kill the other target, Raji cells. UCB CD3/8/56 T cell clones produced a smaller amount of interferon-gamma compared with PB CD4 or PB CD8 T cell clones. We concluded that CD8 T cells coexpressed with CD56 marker expanded after allo-priming in vitro and would become one of the graft-versus-host (GVH) effectors after UCB transplantation.

Inhibition of Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) activity by soluble HLA class I in vitro.

In the present study, the effects of soluble HLA (sHLA) class I molecules against EBV-specific CTL were examined. Two different sources of sHLA class I, either bioengineered spliced form of HLA-B7 (sB7) or natural production from EBV-transformed B cells (natural sHLA), were added during the induction of CTL or incubated with MHC-restricted CD8+ CTL, which were selected by immunobeads just before testing for their cytotoxic activity. Both sB7 and natural sHLA class I blocked the generation of CD8+ CTL and also inhibited the cytotoxic activity of established CTL in a dose-dependent manner. In both ways, natural sHLA class I was effective in 10-fold lower concentrations compared with sB7. The inhibitory effect did not require a sharing of the HLA allotypes between sHLA and the CTL. CTL, after being treated with sHLA, underwent apoptosis, which was considered here as the main mechanism.

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells.

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients.

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-gamma-producing CD8 cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4+ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients.