Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD³⁺ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.
Animals ; Antibody-Dependent Cell Cytotoxicity ; Carcinoma, Hepatocellular ; Cell Survival ; Cell- and Tissue-Based Therapy ; Cryopreservation* ; Freezing ; Heterografts ; Humans* ; Interleukin-2 ; Killer Cells, Natural* ; Methods ; Mice ; Mice, SCID ; Muromonab-CD3 ; Phenotype ; Rituximab

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.
Animals ; CD28 Antigens ; genetics ; immunology ; Electroporation ; Humans ; Immunity, Cellular ; Interleukin-2 ; immunology ; K562 Cells ; Mice ; Muromonab-CD3 ; immunology ; Neoplasms, Experimental ; genetics ; immunology ; pathology ; RNA, Messenger ; genetics ; immunology ; T-Lymphocytes ; immunology ; Tumor Necrosis Factor Receptor Superfamily, Member 9 ; genetics ; immunology

BACKGROUND: Steroid pulse therapy has been used for patients with acute rejection after kidney transplantation. The ABCB1 gene codes for P-glycoprotein, a transporter that is involved in the metabolism of steroids. However, the role of ABCB1 polymorphisms has not been investigated in patients with acute rejection after kidney transplantation. METHODS: Among 763 patients that received kidney or simultaneous pancreas-kidney transplantation at Seoul National University Hospital between May 1996 and July 2009, 684 patients agreed to genetic sampling for polymorphisms. Acute rejection was defined as biopsy-proven, acute cellular rejection with increased serum creatinine, or in the context of delayed or slow graft function. Steroid-resistance was defined as no improvement in serum creatinine, need for additional OKT3 or ATG treatment, or repeated acute rejection within 30 days. Three polymorphisms of ABCB1 gene (C1236T, C3435T, G2677T/A) were assessed. RESULTS: C allele frequency of C3435T was 59.3% and of C1236T 40.1%. Patients who were steroid-resistant (n=37) had higher serum creatinine at kidney biopsy compared to those who were steroid-sensitive (n=49, P<0.001). The frequency of ABCB1 gene polymorphisms (C1236T and C3435T) did not differ significantly between patients who were steroid-sensitive and those who were resistant. An association with G2677T/A could not be analyzed due to a high failure rate of genotyping. CONCLUSIONS: ABCB1 gene polymorphisms (C1236T and C3435T) were not associated with steroid resistance in patients with acute cellular rejection after kidney transplantation.
Biopsy ; Creatinine ; Gene Frequency ; Humans ; Kidney ; Kidney Transplantation ; Muromonab-CD3 ; P-Glycoprotein ; Rejection (Psychology) ; Steroids ; Transplants

Establishing Epstein-Barr virus(EBV)-specific cytolytic T lymphocytes(EBV-CTLs) from peripheral blood mononuclear cells(PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC). In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3), recombinant human interleukin(IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.
Biopsy ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; Cells, Cultured ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-4 ; metabolism ; Lymphocytes, Tumor-Infiltrating ; immunology ; virology ; Monocytes ; pathology ; Muromonab-CD3 ; pharmacology ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; virology ; Receptors, IgG ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: CD25 monoclonal antibody (basiliximab, BA) has been reported an excellent effect on induction immunosuppression than ALG, ATG, OKT3 in renal transplantation. Since we can use BA only in the group of high risk kidney recipient, we want to evaluate the effect of BA treated group by comparing with conventional 3 drugs combination group (calcineurin inhibitor/cyclosporine or tacrolimus, mycophenolate mofetil and prednisolon). METHODS: Total 103 recipients were treated by BA and conventional 3 drugs from March 2000 through February 2006, and these cases were compared with 122 recipients without BA. Government guidelines for using BA were in cadaveric transplantation, more than 4 HLA mismatching, zero DR antigen matching, retransplantation and more than 80% positive PRA. The episode of acute rejection (AR), steriod resistant acute rejection, change of serum creatinine, maintaining dosage of calcineurin inhibitor (CNI) and other side effects were compared between groups. RESULTS: The rate of AR within 12 months after transplantation was 11.7% in BA group while 9.0% in control group (P=0.451). The steroid resistant acute rejection was 16.6% in BA group and 27.3% in control group (P=0.119). Rejection free graft survival adjusted various clinical risk factors by Cox-regression test were no significance between groups. Serum creatinine level at one year after transplantation was 1.61+/-.1 and 1.46+/-.7 mg/dL in BA and control group, and recipients number whose SCr over 1.5 mg/dL were 39.0% and 32.7% (P=0.326). Cyclosporin dosage at one year in BA and control group were 4.21 and 3.62 mg/kg (P=0.202) and 0.11 and 0.17 mg/kg in tacrolimus group (P=0.291). Incidence of PTDM and viral infection were all no difference statistically between groups. CONCLUSION: In conclusion, BA induction immunosuppression with CNI, mycophenolate mofetil and prednisolon used in high risk kidney recipient effectively control the acute rejection and steroid resistant acute rejection for one year at least the same as control group. But since there was no more beneficial effect in BA added to Tac group than conventional Tac based immunosuppression, this 4 drug combination is better avoided if possible.
Cadaver ; Calcineurin ; Creatinine ; Cyclosporine ; Graft Survival ; Immunosuppression ; Incidence ; Kidney Transplantation ; Kidney* ; Muromonab-CD3 ; Risk Factors ; Tacrolimus ; Transplantation

Adult ; Graft Rejection ; drug therapy ; Humans ; Immunosuppressive Agents ; therapeutic use ; Liver Transplantation ; Male ; Middle Aged ; Muromonab-CD3 ; therapeutic use

OBJECTIVETo detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation.

METHODSThe novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay.

RESULTSA sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control.

CONCLUSIONIt is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.

Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; immunology ; Cell Proliferation ; Cells, Cultured ; Cytokines ; secretion ; Graft vs Host Disease ; immunology ; Humans ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Culture Test, Mixed ; Muromonab-CD3 ; pharmacology ; T-Lymphocytes ; cytology ; immunology

Purpose: OKT3 is a very powerful immunosuppressive drug in acute allograft rejection treatment but its side-effects such as fever, nausea, vomiting, and pulmonary edema are strongly linked with its inducing cytokines such as tumor necrosis factor alphaTNF-alpha, interleukin-6(IL-6), and interferon-gammaIFN-gamma. Interleukin-10(IL-10) inhibits proinflammatory cytokines which are produced by activated monocyte/ macrophages and prevents production of cytokines in acute inflammatory states. The purpose of this study is to determine the effect of exogenous administration of the anti- inflammatory cytokine, IL-10 on TNF-alpha IL-6, and IFN-gammaproduction and pulmonary injury after OKT3 injection. METHODS: For experiment, Sprague-Dawley rat weighed 300~400 gm was injected either OKT3(0.6mg/kg i.v.) only or recombinant IL-10(0.5microgram/rat i.p. one hour before the injection of OKT3(IL-10/OKT3). The rats were divided into three groups; control group(n=5); normal saline injected group(1.0ml/rat i.v.), OKT3 group(n=5); OKT3 only injected group, and IL-10/OKT3 group; IL-10 plus OKT3 injected group. After two hours of injection, all animals were sacrificed and submitted for a study of serologic and histologic changes. Student t-test was used for statistical analysis. To evaluate the cytokine production the serum levels of TNF-alpha, IL-6, and IFN-gamma level were measured. The serum levels of TNF-alpha, IL-6, and IFN-gamma were also significantly decreased in IL-10/ OKT3 group(109.6+/-38.0, 65.2+/-14.1, 96.2+/-17.3pg/ml) compared with OKT3 group(399.8+/-71.4, 155.4+/-45.1, 297+/-87.0pg/ml)(p<0.05). To determine the pulmonary injury, wet/dry ratio and microscopic findings for the lung tissue were analyzed. RESULTS: The wet/dry ratio of the lung tissue was significantly decreased in IL-10 /OKT3 group (3.52+/-0.31) compared with OKT3 group(4.16+/-0.48)(p<0.05). Microscopic findings of lung tissue revealed severe neutrophilic infiltration and microvascular congestion in the OKT3 group, but in IL-10/ OKT3 group, neutrophilic infiltration and microvascular congestion were decreased. CONCLUSION: In this study the inhibitory effect of IL-10 on the production of proinflammatory cytokines by OKT3 treatment was significant. This results suggest that the exogenous IL-10 injection may decrease the complications associated with OKT3 treatment of allograft rejection in organ transplantation.
Allografts ; Animals ; Cytokines ; Estrogens, Conjugated (USP) ; Fever ; Humans ; Interleukin-10* ; Interleukin-6 ; Lung ; Lung Injury ; Macrophages ; Muromonab-CD3 ; Nausea ; Neutrophils ; Organ Transplantation ; Pulmonary Edema ; Rats ; Rats, Sprague-Dawley ; Transplants ; Tumor Necrosis Factor-alpha ; Vomiting

OBJECTIVETo explore methods of preventing and reversing rejection after simultaneous pancreas-kidney (SPK) transplantation.

METHODSSeventeen patients underwent SPK transplantation from September 1999 to September 2003 were reviewed retrospectively. Immunosuppression was achieved by a triple drug regimen consisting of cyclosporine, mycophenolate mofteil (MMF), and steroids. Three patients were treated with anti-CD3 monoclone antibody (OKT3, 5 mg x d(-1)) for induction therapy for a mean period of 5-7 days. One patients received IL-2 receptor antibodies (daclizumab) in a dose of 1 mg x kg(-1) on the day of transplant and the 5th day posttransplant. One patient was treated with both OKT3 and daclizumab for induction.

RESULTSNo primary non-functionality of either kidney or pancreas occurred in this series of transplantations. Function of all the kidney grafts recovered within 2 to 4 days after transplantation. The level of serum creatinine was 94 +/- 11 micromol/L on the 7th day posttransplant. One patient experienced the accelerated rejection, resulting in the resection of the pancreas and kidney grafts because of the failure of conservative therapy. The incidence of the first rejection episodes at 3 months was 47.1% (8/17). Only the kidney was involved in 35.3% (6/17); and both the pancreas and kidney were involved in 11.8% (2/17). All these patients received a high-dose pulse of methylprednisone (0.5 g x d(-1)) for 3 days. OKT3 (0.5 mg x d(-1)) was administered for 7-10 days in two patients with both renal and pancreas rejection. All the grafts were successfully rescued.

CONCLUSIONRejection, particularly acute rejection, is the major cause influencing graft function in SPK transplantation. Monitoring renal function and pancreas exocrine secretion, and reasonable application of immunosuppressants play important roles in the diagnosis and treatment of rejection.

Adult ; Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; Creatinine ; blood ; Diabetes Mellitus, Type 1 ; surgery ; Diabetes Mellitus, Type 2 ; surgery ; Female ; Follow-Up Studies ; Graft Rejection ; drug therapy ; Humans ; Immunoglobulin G ; therapeutic use ; Immunosuppressive Agents ; therapeutic use ; Kidney Transplantation ; Male ; Middle Aged ; Muromonab-CD3 ; therapeutic use ; Pancreas Transplantation ; Prednisone ; analogs & derivatives ; therapeutic use ; Retrospective Studies

PURPOSE: Antibody mediated rejection (AMR), although less common than acute cellular rejection (ACR), may be recalcitrant to conventional rescue therapy. AMR is caused by de novo B-cell mediated production of immunoglobulin G antibody (IgG) targeted against specific allograft antigen in a presensitized recipient. Rituximab is a chimeric murine- human anti-CD20 monoclonal antibody which targets CD-20 positive B-cells for elimination. Rituximab has been described to improve allograft salvage for refractory AMR. METHODS: From January 2002 to May 2004, 11 patients were diagnosed with AMR. The first 5 patients (non-rituximab group: NRG) were treated with high dose steroids, plasmapheresis followed by IVIG (500 mg/kg/dose) in addition to OKT3 and/or rabbit antithymocyte globulin. The latter 6 patients (rituximab group: RG) were given Rituximab (375 mg/m2) with IVIG following plasmapheresis. All patients had biopsy proven AMR. RESULTS: Four patients received allografts from living donors and one patient from cadaveric donor in NRG. Each three patients received allografts from living or cadaveric donors in RG. One patient of RG had a positive anti-HLA B-cell crossmatch by CDC (complement dependent cytotoxicity). The anti-donor antibody was reduced to zero with negative CDC and flowcytometry through a desensitization protocol prior to transplantation. The time to diagnosis of AMR in both groups were 17.8+/-18.17 days (NRG); 11+/-2.5 days (RG). ACR was identified in conjunction with AMR in 2 (40%: NRG), 4 patients (66.7%: RG), respectively. All patients had biopsies with classic features of AMR on light microscopy, including C4d staining. Three (50%) patients of RG had positive post-transplantation CDC and donor-specific antibody (DSA) identified. Mean serum creatinine (SCr) upon diagnosis of AMR were 4.3+/-1.71 mg/dL (NRG); 5.77+/-2.65 mg/dL (RG). The rescue rate of RG was superior than NRG (83% vs. 40%, P>0.05). The time to rescue from AMR in both groups were 40.5 +/-28.99 days (NRG); 48+/-54.67 days (RG). Mean SCr of the rescued patients were 1.65+/-0.07 mg/dL (NRG); 2.2+/-1.4 (RG) with median follow up of 120 days (range 33~319 days). Allograft nephrectomies were performed in 3 patients of NRG. CONCLUSION: Rescue therapy with Rituximab improves allograft salvage after AMR and should be considered early in the treatment of biopsy proven AMR.
Allografts ; Antilymphocyte Serum ; B-Lymphocytes ; Biopsy ; Cadaver ; Centers for Disease Control and Prevention (U.S.) ; Creatinine ; Diagnosis ; Follow-Up Studies ; Humans ; Immunoglobulin G ; Immunoglobulins, Intravenous ; Kidney Transplantation* ; Kidney* ; Living Donors ; Microscopy ; Muromonab-CD3 ; Nephrectomy ; Plasmapheresis ; Rituximab ; Steroids ; Tissue Donors