Marco W. Schilham

Internally Controlled Real-Time PCR Monitoring of Adenovirus DNA Load in Serum or Plasma of Transplant Recipients

ABSTRACT Adenoviruses have been recognized as important pathogens in immunocompromised hosts. Particularly in pediatric allogeneic stem cell transplant recipients, the morbidity of the patients and mortality in those patients with disseminated infections have been found to increase over the last few years. Severe infections are predominantly but not exclusively caused by subgroup C adenoviruses. A multiplex real-time PCR assay using molecular beacons as probes was developed to enable monitoring of adenovirus DNA in those patients with simultaneous identification of subgroups. An internal control was coamplified in the multiplex PCR to check for the DNA isolation procedure as well as the presence of inhibitors in the clinical samples. The assay has been applied retrospectively in patient groups with different clinical outcomes of infection. In fatal cases, significantly higher adenovirus loads developed, exceeding even 1011 copies/ml of serum or plasma. Patients with viral loads over 106 copies/ml appear to have an increased risk for fatal complications. This quantitative real-time PCR assay has been prospectively used clinically since 2002 to study the course of adenovirus infection. In addition, the assay provides objective start and end points of therapeutic interventions, including the clinically important evaluation of antiviral drugs.

High Levels of Adenovirus DNA in Serum Correlate with Fatal Outcome of Adenovirus Infection in Children after Allogeneic Stem-Cell Transplantation

An increase in the incidence of adenovirus (AdV) infection leading to death among children who have undergone allogeneic stem-cell transplantation has made it necessary to find new ways to monitor AdV infection. In this retrospective study, levels of AdV DNA in serum samples obtained from 36 transplant recipients with stool cultures positive for AdV were measured by polymerase chain reaction (PCR) semiquantitatively by analyzing serial dilutions of the DNA template. Six (86%) of 7 children who died of AdV infection, compared with only 2 (7%) of 29 other patients, had high serum levels of AdV DNA (detectable by PCR at a > or =100-fold dilution of the DNA template; P<.0001). High serum levels of AdV DNA were reached a mean of 18 days before death (range, 6-29 days). Quantification of adenoviral DNA in serum may prove to be a valuable tool to diagnose and monitor AdV infection and disease in immunocompromised children.

Immune Reconstitution and Clearance of Human Adenovirus Viremia in Pediatric Stem-Cell Recipients

BACKGROUND Human adenovirus (HAdV) infections are increasingly frequent complications of allogeneic stem-cell transplantation (SCT), especially in children. Only a few data on the correlation between immune recovery and the course of HAdV infection are available, and data on HAdV-specific responses are lacking. METHODS In a prospective study, we determined the correlation between the HAdV DNA load in plasma and lymphocyte reconstitution in 48 children after allogeneic SCT. Additionally, HAdV-specific humoral and cellular immune responses were investigated. RESULTS HAdV infection occurred in 21 patients (44%), and, in 6 of these patients, the infection progressed to viremia, as demonstrated by the presence of HAdV DNA in plasma. Low lymphocyte counts at the onset of infection were predictive of HAdV viremia. Survival of patients with HAdV viremia was associated with an increase in lymphocyte counts during the first weeks after infection. In these patients, HAdV-specific CD4+ T cell responses, as well as increases in titers of neutralizing antibody, were detected after clearance of HAdV DNA from plasma. CONCLUSIONS Lymphocyte reconstitution appears to play a crucial role in clearance of HAdV viremia and survival of the host, warranting further development of therapeutic interventions aimed at improving immune recovery.

Effect of Ribavirin on the Plasma Viral DNA Load in Patients with Disseminating Adenovirus Infection

Adenovirus (AdV) infections are an increasingly frequent and potentially fatal complication in allogeneic stem cell transplant recipients. To determine the antiviral potential of ribavirin in an unbiased way, 4 patients without immune recovery were prospectively analyzed by quantitative measurement of plasma AdV DNA load. Administration of ribavirin at the first signs of AdV dissemination was not accompanied by a decrease in the plasma AdV DNA load in any of these patients, and an increase in the AdV load was even documented in 3. These observations question the potential of ribavirin to improve the outcome for patients with disseminating AdV infection and support a critical evaluation of antiviral treatments for AdV infection that involves the kinetics of virus DNA load as an objective parameter of viral replication.

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

INTRODUCTION Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. METHODS AND RESULTS All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. CONCLUSION These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.

Level of minimal residual disease prior to haematopoietic stem cell transplantation predicts prognosis in paediatric patients with acute lymphoblastic leukaemia: a report of the Pre-BMT MRD Study Group

Level of minimal residual disease prior to haematopoietic stem cell transplantation predicts prognosis in paediatric patients with acute lymphoblastic leukaemia: a report of the Pre-BMT MRD Study Group

Extensive Cross-Reactivity of CD4+ Adenovirus-Specific T Cells: Implications for Immunotherapy and Gene Therapy

ABSTRACT Adenovirus (Ad)-specific T-cell responses in healthy adult donors were investigated. Ad5, inactivated by methylene blue plus visible light, induced proliferation and gamma interferon (IFN-γ) production in peripheral blood mononuclear cells of the majority of donors. Responding T cells were CD4+ and produced IFN-γ upon restimulation with infectious Ad5 and Ads of different subgroups. T-cell clones showed distinct cross-reactivity patterns recognizing Ad serotypes from either one subgroup (C), two subgroups (B and C), or three subgroups (A, B, and C). This cross-reactivity of Ad-specific T cells has relevance both for Ad-based gene therapy protocols, as well as for the feasibility of T-cell-mediated adoptive immunotherapy in recipients of an allogeneic stem cell transplantation.

Minimal residual disease prior to stem cell transplant for childhood acute lymphoblastic leukaemia

Summary. Allogeneic stem cell transplantation (SCT) is a highly effective therapy for childhood acute lymphoblastic leukaemia (ALL). Concerns about unnecessary toxicity and expense mean that SCT is currently largely reserved for children who cannot be cured with chemotherapy. Not surprisingly, many such children also fail SCT. Retrospective studies have shown that a single analysis of minimal residual disease (MRD) pre‐SCT identified those at highest risk of relapse. It is now appropriate to call for the universal incorporation of standardized MRD testing into SCT protocols as the next step to maximize the clinical impact of this technology in ALL.

Pro-inflammatory chemokine-chemokine receptor interactions within the Ewing sarcoma microenvironment determine CD8(+) T-lymphocyte infiltration and affect tumour progression.

Ewing sarcoma is an aggressive round cell sarcoma with poor patient prognosis, particularly in cases of advanced‐stage disease. Dynamic tumor‐host immune interations within the tumor microenvironment may polarize in situ immune responses and shape tumor development and/or progression. To gain insight into the nature of tumour–host immune interactions within the Ewing sarcoma microenvironment, the presence and spatial distribution of infiltrating CD8+/CD4+ T‐lymphocytes were evaluated in therapy‐naive Ewing sarcoma. Expression profiling of 40 different chemokines and several chemokine receptors was performed in therapy‐naive tumours and cell lines by qPCR, immunohistochemistry, and flow cytometry. Considerable inter‐tumour variation was observed regarding density, type, and distribution of infiltrating T‐lymphocytes. Tumour‐infiltrating T‐cells contained significantly higher percentages of CD8+ T‐lymphocytes as compared to stroma‐infiltrating cells, suggesting preferential migration of this T‐cell type into tumour areas. Gene expression levels of several type 1‐associated, pro‐inflammatory chemokines (CXCR3‐ and CCR5‐ligands CXCL9, CXCL10, and CCL5) correlated positively with infiltrating (CD8+) T‐lymphocyte numbers expressing corresponding chemokine receptors. Survival analyses demonstrated an impact of tumour‐infiltrating, and not stroma‐infiltrating, CD8+ T‐lymphocytes on tumour progression. At protein level, both tumour and stromal cells expressed the IFNγ‐inducible chemokines CXCL9 and CXCL10. CCR5‐ligand CCL5 was exclusively expressed by non‐tumoural stromal/infiltrating cells. Together, our results indicate that an inflammatory immune microenvironment with high expression of type 1‐associated chemokines may be critical for the recruitment of (CD8+) T‐lymphocytes expressing corresponding chemokine receptors. The observed impact of tumour‐infiltrating (CD8+) T‐lymphocytes is consistent with a role for adaptive anti‐tumour immunity in the prevention of Ewing sarcoma progression. Recognition of the merits and exploitation/induction of an inflammatory microenvironment may improve the efficacy of natural immune responses against, and (adoptive) immunotherapeutic approaches for, Ewing sarcoma. Copyright

Interference with T cell receptor–HLA-DR interactions by Epstein–Barr virus gp42 results in reduced T helper cell recognition

Epstein–Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)–class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Vα-domains as visualized by superimposing the crystal structures for gp42–HLA-DR1 and TCR–MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.