Judith Feucht

Adoptive T-cell therapy with hexon-specific Th1 cells as a treatment of refractory adenovirus infection after HSCT

Hematopoietic stem cell transplantation (HSCT) has improved over the last few decades. However, viral infections are often refractory to pharmacologic treatment and require alternative treatment strategies such as immunotherapy. Adenovirus (AdV) is th predominant disease-causing pathogen in pediatric HSCT. In a clinical trial, we analyzed safety and efficacy of ex vivo adoptive T-cell transfer (ACT) with hexon-specific T cells, predominantly of the T-helper cell 1 (Th1) phenotype, in 30 patients with AdV disease or viremia. ACT was feasible with no acute toxicities or significant onset of graft-versus-host disease. ACT led to in vivo antiviral immunity for up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific T-cell responses. After ACT and a follow-up of 6 months, overall survival was markedly increased in responders (mean, 122 days; 15 survivors) compared with nonresponders who all died shortly after ACT (mean, 24 days; no survivors). AdV-related mortality was 100% in nonresponders compared with 9.5% in responders (≥1 log reduction of DNA copies per milliliter after ACT). In summary, ex vivo ACT of AdV-specific Th1 cells was well tolerated and led to successful and sustained restoration of T-cell immunity correlated with virologic response and protection from virus-related mortality. This cellular immunotherapy is a short-term available and broadly applicable treatment. The study is registered at European Union Clinical Trials Register as 2005-001092-35.

Adoptive T-cell Transfer for Refractory Viral Infections with Cytomegalovirus, Epstein-Barr Virus or Adenovirus after Allogeneic Stem Cell Transplantation

Viral infections after allogeneic stem cell transplantation (SCT) are an important cause of morbidity and mortality as SCT can expose patients to a transient state of combined immunodeficieny. Most viral infections after SCT are caused by the endogenous reactivation of persistent pathogens such as adenovirus (ADV), cytomegalovirus (CMV), and Epstein-Barr-virus (EBV). The control of these infections will ultimately depend on the restoration of adequate T-cell immunity.

Granulocytic myeloid-derived suppressor cells from human cord blood modulate T-helper cell response towards an anti-inflammatory phenotype

Infections are a leading cause of perinatal morbidity and mortality. The outstandingly high susceptibility to infections early in life is mainly attributable to the compromised state of the neonatal immune system. One important difference to the adult immune system is a bias towards T helper type 2 (Th2) responses in newborns. However, mechanisms regulating neonatal T‐cell responses are incompletely understood. Granulocytic myeloid‐derived suppressor cells (GR‐MDSC) are myeloid cells with a granulocytic phenotype that suppress various functions of other immune cells and accumulate under physiological conditions during pregnancy in maternal and fetal blood. Although it has been hypothesized that GR‐MDSC accumulation during fetal life could be important for the maintenance of maternal–fetal tolerance, the influence of GR‐MDSC on the immunological phenotype of neonates is still unclear. Here, we investigated the impact of GR‐MDSC isolated from cord blood (CB‐MDSC) on the polarization of Th cells. We demonstrate that CB‐MDSC inhibit Th1 responses and induced Th2 responses and regulatory T (Treg) cells. Th1 inhibition was cell‐contact dependent and occurred independent of other cell types, while Th2 induction was mediated independently of cell contact through expression of ArgI and reactive oxygen species by CB‐MDSC and partially needed the presence of monocytes. Treg cell induction by CB‐MDSC also occurred cell‐contact independently but was partially mediated through inducible nitric oxide synthase. These results point towards a role of MDSC in regulating neonatal immune responses. Targeting MDSC function in neonates could be a therapeutic opportunity to improve neonatal host defence.

Rapid generation of NY-ESO-1-specific CD4(+) THELPER1 cells for adoptive T-cell therapy.

Tumor-associated antigens such as NY-ESO-1 are expressed in a variety of solid tumors but absent in mature healthy tissues with the exception of germline cells. The immune system anti-cancer attack is mediated by cell lysis or induction of growth arrest through paralysis of tumor cells, the latter of which can be achieved by tumor-specific CD4+, IFNγ-producing THelper type 1 (TH1) cells. Translation of these immune-mediated mechanisms into clinical application has been limited by availability of immune effectors, as well as the need for complex in vitro protocols and regulatory hurdles. Here, we report a procedure to generate cancer-testis antigen NY-ESO-1-targeting CD4+ TH1 cells in vitro for cancer immunotherapy in the clinic. After in vitro sensitization by stimulating T cells with protein-spanning, overlapping peptide pools of NY-ESO-1 in combination with IL-7 and low dose IL-2, antigen-specific T cells were isolated using IFNγ capture technique and subsequently expanded with IL-2, IL-7 and IL-15. Large numbers of NY-ESO-1-specific CD4+ T cells with a TH1 cytokine profile and lower numbers of cytokine-secreting CD8+ T cells could be generated from healthy donors with a high specificity and expansion potential. Manufactured CD4+ T cells showed strong specific TH1-responses with IFNγ+, TNFα+, IL-2+ and induced cell cycle arrest and apoptosis in tumor cells. The protocol is GMP-grade and approved by the regulatory authorities. The tumor-antigen specific CD4+ TH1 lymphocytes can be adoptively transferred as a T-cell therapy to boost anticancer immunity and this novel cancer treatment approach is applicable to both T cells from healthy allogeneic donors as well as to autologous T cells derived from cancer patients.

Induction of Thelper1-driven antiviral T-cell lines for adoptive immunotherapy is determined by differential expression of IFN-γ and T-cell activation markers.

Viral infections with cytomegalovirus (CMV) or human adenovirus (HAdV) after stem cell transplantation are still associated with a high morbidity and mortality. Transfer of T-cell immunity from a healthy individual to a stem cell transplant recipient, known as adoptive T-cell transfer, has been shown to be effective to prevent viral complications. Treatment efficacy will depend on the availability of functional T-cell lines with a strong Thelper1 response. Ex vivo isolation of antigen-specific T cells could be performed on the basis of the cytokine capture technique or antigen-induced expression of activation markers. In this study, we compare the specificity, expansion/differentiation potential, and Thelper1 response against CMV and HAdV after different isolation strategies. Antigen-specific T cells from healthy donors were isolated by antigen-induced expression of IFN-&ggr; and/or CD137 after stimulation with the viral antigens hexon (HAdV) or pp65 (CMV). Isolation of antigen-specific T cells based on the expression of activation markers is feasible and less time consuming, but in contrast to isolation based on IFN-&ggr; secretion, it leads to a reduction of Thelper1 cells. Both isolated CD137+ and isolated IFN-&ggr;+ T cells mainly consist of CD4+ TCentralMemory and TEffectorMemory cells with high expansion potential and effective cytokine production. CD154+ is mainly expressed on CD4+ T cells and shows coexpression with IFN-&ggr; on activated T cells, which cannot be found for CD137+ cells. In conclusion, T-cell lines could be easily generated on the basis of IFN-&ggr;+ and/or expression of the activation marker CD137 but both approaches result in different T-cell populations, which may lead to divergent T-cell responses in vivo.

Posaconazole plasma concentrations in pediatric patients receiving antifungal prophylaxis during neutropenia

Abstract Invasive fungal infections are one of the major complications in pediatric patients during prolonged neutropenia after chemotherapy. Evaluation of the efficacy and safety of the triazole posaconazole in these patients is missing. This multicenter survey analyzed trough concentrations of 33 pediatric patients with a median age of 8 years during 108 neutropenic episodes who received prophylactic posaconazole oral suspension. A total of 172 posaconazole trough levels were determined to median 438 ng/ml (range 111‐2011 ng/ml; mean 468 ± 244 ng/ml). Age and gender had no influence on posaconazole plasma levels. Posaconazole was not discontinued due to adverse events in any of the patients. Only hepatic parameters significantly increased beyond the upper normal limit to median values of ALT of 87 U/l (P < .0001), and AST of 67 U/l (P < .0001). One patient with a median posaconazole trough concentration of 306 ng/ml experienced an invasive fungal infection. In conclusion, posaconazole was effective, safe and feasible in 33 pediatric patients with neutropenia ≥5 days after chemotherapy. Median posaconazole plasma concentrations were approximately 1.6‐fold lower than the recommended plasma level of 700 ng/ml. Larger patient cohorts are needed to evaluate these findings.

Differential expression of THELPER1 cytokines upon antigen stimulation predicts ex vivo proliferative potential and cytokine production of virus‐specific T cells following re‐stimulation

Cytomegalovirus (CMV) and human adenovirus (ADV) infections are causes of morbidity after stem cell transplantation. Antigen (Ag)‐specific T cells are essential for the control of viral infections. However, in vivo expansion potential of T‐cell subpopulations is hardly predictable in humans. Furthermore, ex vivo identification of human T cells with repopulating capacity for adoptive T‐cell transfer has been difficult.