Clonotype analysis of cytomegalovirus-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.

Short-term anti-CD4 plus anti-TNF-alpha receptor treatment in allogeneic small bowel transplantation results in long-term survival.

BACKGROUND: Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. METHODS: Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. RESULTS: Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. CONCLUSIONS: Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.

Evidence for genetic susceptibility towards development of posttransplant lymphoproliferative disorder in solid organ recipients.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a life-threatening complication after organ transplantation. The identification of risk factors for PTLD development is important for disease management. It has been shown that cytokine gene polymorphisms are associated with lymphoma and Epstein-Barr virus (EBV)-associated diseases in nonimmunosuppressed patients. In the present case-control study, we analyzed the impact of -1082 interleukin (IL)-10, -308 tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1 (codon 10, 25), and +874 interferon (IFN)-gamma gene single-nucleotide polymorphisms on the late onset EBV-associated PTLD. METHODS: Out of 1,765 solid organ recipients, 38 patients with late-onset EBV-associated PTLD and 408 matched solid organ recipients were selected and enrolled in the study. Single nucleotide polymorphisms (SNPs) for -1082IL-10, -308TNF-alpha, TGF-beta1 (codon 10, 25), and +874IFN-gamma genes were analyzed by a sequence specific primer polymerase chain reaction and were related to the PTLD development, and the disease course and outcome. RESULTS: The TGF-beta1 (codon 25) GG genotype was detected more frequently in controls than in PTLD patients (odds ratio=0.34, 95% confidence interval: 0.17-0.69, P=0.0022). The frequency of -1082 IL-10 GG genotype was also significantly higher in controls than in PTLD patients (odds ratio=0.5, 95% confidence interval: 0.25-1.0, P=0.044). There were no associations between -308TNF-alpha, TGF-beta1 codon 10, and +874IFN-gamma SNPs and PTLD. Disease course and outcome were not associated with any cytokine SNPs. CONCLUSIONS: Polymorphisms in two key anti-inflammatory cytokines, IL-10 and TGF-beta, are associated with susceptibility to EBV-associated PTLD, suggesting that a shift in pro-/anti-inflammatory response is involved in the pathogenesis of PTLD.

Predictive value of cytokine gene polymorphisms for the development of end-stage renal disease.

BACKGROUND: Cytokines play a crucial role in different immunopathological conditions. Cytokine secretion is reported to be determined by polymorphisms in the cytokine genes. Since TNF-alfa and IL-10 are involved in regulation of inflammation, and TGF-beta 1 can induce fibrosis and renal insufficiency - dominant features of end-stage renal disease (ESRD), we explored the hypothesis that polymorphisms of these cytokine genes may be possible genetic susceptibility factors for the progression of renal failure. METHODS: We studied the IL-10 (-1082), TNF-alfa (-308), TGF-beta 1 (codon 10;25) gene single nucleotide polymorphisms in 118 healthy donors and 103 patients with ESRD (44 hemodialysis patients with diabetic nephropathy and 59 hemodialysis patients with glomerulonephritis) using PCR-SSP. RESULTS: Significant associations of ESRD with the TGF-beta 1 (codon 10) TT (odds ratio [OR] = 5.31; 95% confidence interval [95% CI], 3.77-7.02; p<0.001) and IL-10 (-1082) GG (OR=2.35; 95% CI, 1.67-3.15; p<0.01) genotypes were found. Statistical analysis of genotype frequencies made separately for the underlying renal disease (diabetes or glomerulo-nephritis) revealed the same linkage trend: TGF-beta 1 (codon 10) TT and IL-10 (-1082) GG were associated with type 2 diabetic nephropathy (p<0.001 and p<0.05, respectively) and chronic glomerulonephritis (p<0.001 and p<0.01, respectively). No significant differences in the TNF-alfa , TGF-beta 1 (codon 25) genotype distribution between healthy controls and patients with diabetic nephropathy- or glomerulonephritis-associated ESRD were detected. CONCLUSIONS: Carriage of the TGF-beta 1 (codon 10) TT and IL-10 (-1082) GG genotypes may increase susceptibility to ESRD in German patients with type 2 diabetes or glomerulonephritis.

Generation of HCMV-specific T-cell lines from seropositive solid-organ-transplant recipients for adoptive T-cell therapy.

Chronically immunosuppressed patients, like solid-organ-transplant (SOT) recipients, are at increased risk for severe human cytomegalovirus (HCMV) infection. Despite the availability of effective antiviral drugs, lasting control of remaining viruses is dependent on an effective T-cell immunity. So in some patients conventional antiviral therapy cannot control the infection and prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Selective reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells derived from healthy donors is a safe and effective approach in hematopoietic stem cell transplant recipients. The aim of this study was to determine whether functional HCMV-specific T cells can also be generated from chronically immunosuppressed patients. Autologous CD4+/8+ T-cell lines directed against the HCMV protein IE-1 were generated from the peripheral blood of SOT patients using a recently developed modular protocol easily applicable to good-manufacturing-practice conditions. T-cell lines from SOT patients showed similar features as cells from healthy donors regarding phenotype, functionality (HCMV-specific killing, gene expression pattern, and cytokine secretion), IE-1 epitope recognition, and dominance of distinct T-cell receptor V beta families. Most importantly, this protocol also allowed the generation of T-cell lines from immunosuppressed patients with HCMV infection/chronic HCMV disease. Our data suggest the potential of this autologous approach for the treatment of SOT recipients suffering from HCMV infection/disease poorly responding to virostatic therapy.

Generation of EBV-specific T cells for adoptive immunotherapy: a novel protocol using formalin-fixed stimulator cells to increase biosafety.

Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.

Identification of dialysis patients with panel-reactive memory T cells before kidney transplantation using an allogeneic cell bank.

Donor-reactive cellular sensitization does not routinely suggest humoral sensitization and vice versa, but both predict poor kidney transplant outcome. Irrespective of donor reactivity, panel-reactive antibody (PRA) screening identifies patients who are at enhanced risk. Therefore, it was hypothesized that panel-reactive memory T cell reactivity (PRT) might be an additional risk assessment factor of dialysis patients who are on the transplant waiting list. IFN-gamma-enzyme-linked immunosorbent spot memory T cell frequencies were determined in 10 healthy volunteers and 41 hemodialysis patients using for stimulation an allogeneic cell bank (ACB) from 17 healthy individuals who represented the most frequent white HLA antigens. Positive responses to ACB were analogous to PRA defined as percentage of positive assays of the ACB sets. Hemodialysis patients expressed higher PRT levels compared with healthy volunteers. Five of 10 PRT++ patients were PRA negative, and only four of 10 PRA++ patients exhibited PRT reactivity, suggesting independence of humoral and cellular sensitization. Pretransplantation PRT testing of recipients might improve individual risk assessment to make individualized therapy decisions.

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines.

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.

Allo-specific T-cells encoding for viral IL-10 exert strong immunomodulatory effects in vitro but fail to prevent graft rejection.

Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy.

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

Antigen-dependent transgene expression in kidney transplantation: a novel approach using gene-engineered T lymphocytes.

So far, gene therapy in transplantation mainly focuses on the expression of therapeutic proteins in the graft itself. Insufficient transfection and inflammatory responses that are due to the immunogenicity of multiple vector systems are often limiting factors in these approaches. The potential of genetically modified T lymphocytes was investigated as a delivery system for therapeutic transgenes to transplanted organs as a way to circumvent immunogenicity and efficiency problems in a rat transplant model. Gene-engineering of alloantigen-specific rat T cell lines was performed by using a Moloney murine leukemia virus (MoMuLV)-based enhanced green fluorescence protein (EGFP) encoding retroviral transduction system. The ex vivo gene-modified lymphocytes were adoptively transferred into syngeneic rats carrying allogeneic, syngeneic, or third party kidneys. Homing behavior, activation level, and transgene expression of the adoptively given cells were monitored. The T(EGFP) lymphocytes infiltrated the transplanted kidneys in an antigen-specific manner. High numbers of alloantigen-specific T lymphocytes accumulated exclusively in allografts but not in syngeneic or third party grafts. Flow cytometric analysis revealed that only T(EGFP) lymphocytes found in allografts had an activated phenotype that resulted in higher transgene expression. Alloantigen-specific homing, activation, and transgene expression are important prerequisites for the guarantee of localized delivery and expression of transgenic proteins by gene-engineered T lymphocytes. Antigen-specific gene-targeting strategies using ex vivo modified T lymphocytes with donor specificity are a novel approach to the delivery of therapeutic transgenes in transplantation.