ALBERT

Beschreibung: Abstract 4542 Immunosuppressive treatment is widely used, especially after allogeneic stem cell transplantation (HSCT) to prevent or treat graft versus host disease (GvHD). Common drugs are Ciclosporin A or Tacrolimus in combination with steroids. However, immunosuppressive treatment and the underlying conditions are associated with an increased risk of viral reactivations with persistent pathogens like cytomegalovirus or adenovirus. In the absence of a protective immune response, virus infection remains a life-threatening complication after HSCT. Here we investigated the antiviral T-cell response (n=12) after ex vivo exposition with Ciclosporin A, Tacrolimus, Prednisolone or Mycophenolate at time of immunosuppression, 24 hours and 72 hours later. Analysis has been done with IFNgamma Elispot assays, confirmed by intracellular cytokine staining in flow cytometry and analysis of T-cell proliferation detected by CFSE. The antiviral T-cell response is suppressed after 24 hours using normal serum concentrations (100-200ng/ml) of cyclosporine A. T-cell annergy, induced by cyclosporine, could be reversible, after 72 without immunosuppression. Tacrolimus has a stronger immunosuppressive effect on T-cell activation within the same time and even low levels of 1ng/ml induce T-cell suppression after 72 hours. Peak levels of calcineurin inhibitors even suppressed the T-cell response to superantigens like staphylococcal antigen B or PHA. As expected, Prednisolone had a short and dose dependend effect on T-cell activation. Mycophenolate has a mild effect on the activation of virus-specific T-cells. However, all three drugs induced a significant reduction in Ag-specific T-cell proliferation. In conclusion, Interferonγ detection in virus-specific T-cells is a good diagnostic tool for clinicians to monitor the risk of viral complications in immunosuppressed patients. Tacrolimus, Cyclosporin A, Prednisolone and Mycophenolate induce an activation defect in Ag-specific T-cells with decreasing severity. The effect is reversible and corresponds to high or low serum levels. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Key Points Adoptive transfer of TH-1 cells is a safe and effective treatment of refractory AdV infection after stem cell transplantation. AdV-related mortality was 9.5% in patients with a response to ACT (overall survival 71%) compared with 100% mortality in nonresponders.

Beschreibung: Abstract 3047 Allogeneic stem cell transplantation (SCT) can expose patients to a transient but marked immunosuppression, during which viral infections are an important cause of morbidity and mortality. The control of these infections will ultimately depend on the restoration of adequate T-cell immunity. Most viral infections after SCT are caused by endogenous reactivation of persistent pathogens such as cytomegalovirus (CMV), adenovirus (ADV) and Epstein-Barr-virus (EBV). Risk of viral complications is even higher under GvHD treatment or prophylaxis like calcineurin inhibitors and steroids. Post transplant often the immunosuppression needs to be reduced to improve viral complications with the risk of GvHD. The virus-specific T-cell responses in peripheral blood have been shown to be a good marker of immunological protection, but has not been used for clinical decision making and the guidance of drug plasma levels. Therefore, we performed a prospective clinical trial in 33 adult and pediatric patients after allogeneic stem cell transplantation receiving pharmacologic immunosuppression with steroids, Cyclosporin A, Tacrolimus, Everolimus or Mycophenolate. Median Age was 16 years. T-cell responses were analyzed ex vivo against Cytomegalovirus (pp65), Adenovirus (hexon antigen) and Epstein-Barr Virus (EBNA, LMP) using intracellular cytokine staining. In addition in vitro analysis of the proliferation responses using CFSE were performed. Responses were compared to healthy donors. The T-cell responses in vitro under low, high and supraphysiologic plasma concentrations of the respective drugs were investigated. Under the direct influence of steroids, activated, virus-specific T-cells underwent apoptosis. Among the Calcineurin inhibitors, Tacrolimus had the strongest inhibition on virus-specific T-cell immunity, followed by Cyclosporin A. But, under low therapeutic levels, Virus speciffic T-cell responses have been able to develop in PBMCs. Mycophenolate had only in high concentrations a strong effect on the T-cell response against viral pathogens. Relevant differences in the frequency of virus-specific T-cells secreting IFN-g could be detected within the CD4 compartment in correlation to the level of immunosuppression. In conclusion we could show that detection of virus-specific T-cells could be used to guide the level of immunosuppression in case of viral complications after allogeneic stem cell transplantation, since emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 2342 Reactivation of Epstein-Barr-Virus (EBV) after allogeneic stem cell transplantation (SCT) is responsible for significant morbidity and mortality. EBV is also assotiated with the development of some malingancies, such as Burkitt-Lymphoma or nasopharyngeal carcinoma (NPC). In particular, the EBV-induced lymphoproliferative disorder (PTLD) is a rare but severe condition after SCT. PTLD is often associated with insufficient immune responses against EBV in transplant recipients. There is no effective antiviral drug treatment against EBV by now. Given that T-cell immunity is crucial for protection against infection or reactivation of EBV, cellular immunotherapy is a promising therapeutic option. The Epstein-Barr-Virus Nuclear Antigen 1 (EBNA-1) has been shown to contain immunodominant T-cell epitopes with T-cell responses in the majority of the healthy population. Here we report adoptive EBNA-1-specific T-cell transfer in seven pediatric and adult patients with chemorefractory EBV-reactivation after allogenic SCT. Four patients had PTLD and one had metastatic relapse of a NPC. EBNA-1-specific T-cells were isolated from the SCT-donor by using an IFNγ-capture technique. These small T-cell populations were immediately infused to the patient without in vitro expansion steps. The adoptive T-cell transfer contained both, CD4+ T-helper cells and CD8+ cytotoxic T-cells. The patients with a mean age of 20 years were treated with antigen specific T-cells from haploidentical, matched unrelated or matched sibling donor SCT between day 72 and 410 post SCT. The T-cell dose varied from 150–7750 T-cells/ kg. No acute toxicity was observed. In vivo T-cell responses before adoptive T-cell transfer were absent and were detectable in all of the patients within the first weeks after adoptive transfer, associated with a partial clinical and/or virological response to the adoptive T-cell transfer. In three of the patients a second specific T-cell administration was needed to achieve an improvement of the EBV-related condition. PTLD or EBV-infection was not a cause of death in any of the other six patients. In conclusion we could show that adoptive T-cell-immunotherapy is safe, feasible and a promising therapeutic option in patients with EBV- infection and/or PTLD, having the advantage of not being immunosuppressive compared to chemotherapy against PTLD. Infusion of small IFNγ producing EBNA-1-specific T-cell populations resulted in an in vivo expansion of specific T-cells. Emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Cytomegalovirus (CMV) disease and infection refractory to antiviral treatment after allogeneic stem cell transplantation (allo-SCT) is associated with a high mortality. Adoptive transfer of CMV-specific T cells could reconstitute viral im-munity after SCT and could protect from CMV-related complications. However, logistics of producing virus-specific T-cell grafts limited the clinical application. We treated 18 patients after allo-SCT from human leukocyte antigen–mismatched/haploidentical or human leukocyte antigen–matched unrelated donors with polyclonal CMV-specific T cells generated by ex vivo stimulation with pp65, followed by isolation of interferon-γ–producing cells. Patients with CMV disease or viremia refractory to antiviral chemotherapy or both were eligible for adoptive T-cell transfer and received a mean of 21 × 103/kg pp65-specific T cells. In 83% of cases CMV infection was cleared or viral burden was significantly reduced, even in cases of CMV encephalitis (n = 2). Viral control was associated with in vivo expansion of CMV-specific T lymphocytes in 12 of 16 evaluable cases, resulting in reconstitution of antiviral T-cell responses, without graft-versus-host disease induction or acute side effects. Our findings indicate that the infusion of low numbers of CMV-specific T cells is safe, feasible, and effective as a treatment on demand for refractory CMV infection and CMV disease after allo-SCT.

Beschreibung: Abstract 796 In pediatric patients human adenovirus (HAdV) was identified as a common viral pathogen responsible for significant morbidity and mortality post allo SCT. Antiviral chemotherapy is often insufficient. Given that T-cell immunity is crucial for protection against adenoviral infection/reactivation, cellular immunotherapy is a promising therapeutic option. The capsid protein Hexon has been shown to contain immunodominant T-cell epitopes, with T-cell responses in the majority of the healthy population. Therefore a prospective phase I/II clinical study was performed analysing safety and feasibility of adoptive Hexon-specific T-cell transfer in patients after allogeneic SCT and HAdV infection refractory to Cidofovir treatment. Hexon-specific T-cells were isolated from the SCT-donor by using the IFNγ secretion system and small T-cell populations were immediately infused, without in vitro expansion steps. Fourty pediatric and adult patients with a mean age of 15 years were treated according to the study protocol after haploidentical, matched unrelated and matched sibling donor SCT between day 11 and 327 post SCT. The T-cell dose varied from 300-25000 T-cells/kg. No acute toxicitiy was observed. In two patients GvHD °I-°II of the skin occured within two weeks after administration of specific T-cells, one patient also developed GvHD of the gut. In vivo T-cell responses were absent in all patients before adoptive T-cell transfer and detectable in 70% of evaluable patients within the first weeks after adoptive transfer, associated with a clinical and/or virological response to the adoptive T-cell transfer. However, in patients with adenoviral disease response rate was lower and 6 of 14 evaluable patients succumbed with the infection within few days, in spite of adoptive immunotherapy. This lead to the assumption, that adoptive treatment in patients with severe infection related morbidity was to late during the course of infection. In conclusion we could show that adoptive immunotherapy is safe, feasible and a promising therapeutic option in patients with HAdV infection. Infusion of small IFNγ producing Hexon-specific T-cells populations resulted in an in vivo expansion of specific T-cells in the majority of cases. Emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load. Disclosures: No relevant conflicts of interest to declare.