Britta Maecker-Kolhoff

Characteristics of Early and Late Ptld Development in Pediatric Solid Organ Transplant Recipients

Background Posttransplantation lymphoproliferative disorders (PTLD) present a major cause of mortality and morbidity after solid organ transplantation. The purpose of this study was to identify the factors associated with the development of early- and late-onset PTLD in pediatric solid organ transplant recipients. Methods We examined the medical history, laboratory parameters, and pathology of 127 children with PTLD who were registered in the German multicenter pediatric PTLD registry. Data were collected retrospectively from 1991 to 2003 and prospectively from 2004 onward. We compared early (<1 year) and late (>1 year) PTLD using survival analysis. Results The median time to PTLD was 3.00 (95% confidence interval, 2.12–3.26) years. Forty-two patients developed PTLD within the first year after transplantation (early PTLD) and 85 patients developed PTLD after 1 year (late PTLD). Early PTLD development was associated with younger age (P=0.0016), extranodal disease (P=0.019), graft organ involvement (P=0.0065), and immunosuppressive regimens including tacrolimus (P=0.001) or mycophenolate (P=0.0025). Most early PTLD patients experienced graft rejection before PTLD diagnosis (P=0.0081). Early PTLD was often of B-cell lymphoma histology (P=0.024) and tended to be Epstein-Barr virus positive (P=0.052). In contrast, Burkitt’s lymphoma (P=0.0047) and Hodgkin’s disease (P=0.016) were only observed in late PTLDs, which are more likely to present with nodal disease (P=0.019). Overall survival and event-free survival were not significantly different between early and late PTLD. Conclusion Early and late childhood PTLD have distinct characteristics. Whereas early PTLD appears mainly as an Epstein-Barr virus–driven disease especially favored by insufficient immunosurveillance, late PTLD often resembles tumors with distinct pathogenetic alterations and nodal appearance.

Patient, Virus, and Treatment-Related Risk Factors in Pediatric Adenovirus Infection after Stem Cell Transplantation: Results of a Routine Monitoring Program

Human adenovirus (HAdV) infection after hematopoietic stem cell transplantation (HSCT) is associated with significant morbidity and mortality in children. The optimal surveillance and treatment strategies are under discussion. Here, we present data from 238 consecutive pediatric allogeneic HSCT recipients who underwent transplantation in a single center who were included in a prospective, weekly HAdV DNAemia monitoring program by quantitative PCR. HAdV loads >1000 copies/mL were detected in 15.5% of all patients. Despite a low mortality directly attributed to HAdV infection (2 patients, 0.84%), blood HAdV loads >10,000 copies/mL (6.7% of all patients) were significant and independent risk factors for poor survival. We searched for patient, virus, and treatment-related risk factors of HAdV DNAemia and disease. Detection of HAdV in blood before day 50 post transplantation was a major independent risk factor for the development of blood HAdV loads >10,000 copies/mL. HAdV typing revealed A31, C1, and C2 as the predominant pathogens among several other HAdV strains with type C species detected in most patients with severe HAdV disease. Stool HAdV loads were prospectively monitored in 111 patients and correlated with but did not significantly precede detection in blood. Treatment with cidofovir led to stable or reduced viral load in 70% of patients with blood HAdV loads >1000 copies/mL. Thus, early occurrence of HAdV-DNA in blood of pediatric HSCT recipients predisposes for development of high viral loads. Control of HAdV infections was attempted by preemptive cidofovir treatment of patients with high blood HAdV loads or with symptomatic organ infections and correlated with low HAdV-attributed mortality.

Molecular and Clinicopathological Analysis of Epstein-Barr Virus-Associated Posttransplant Smooth Muscle Tumors

Epstein‐Barr virus (EBV)–associated posttransplant smooth muscle tumors (PTSMT) are very rare complications. We aimed to provide a clinicopathological characterization which is based on our own case series (n = 5) as well as previously reported PTSMT cases (n = 63). Meta‐analysis of PTSMT and molecular analysis of tumor cells from our cohort was performed. Most PTSMT developed in kidney‐transplanted patients (n = 41/68, 60%). Liver/transplant liver was the main site of manifestation (n = 38/68, 56%). Tumors occurred after a median interval of 48 months (range 5–348) and developed earlier in children than in adults. Most tumors showed no marked cellular atypia, low mitosis rate and no tumor necrosis. Gene expression analysis of 20 EBV‐related genes, including two microRNAs, revealed overexpression of MYC (p = 0.0357). Therapy was mainly based on surgical resection or reduced immunosuppression but no significant differences in overall survival were evident. Lower overall survival was associated with multiorgan involvement (n = 33/68, 48.5%) and particularly with intracranial PTSMT manifestation (n = 7/68, 10%; p < 0.02), but not transplant involvement (n = 11/68, 16%). In summary, PTSMT differ from conventional leiomyosarcomas by their lack of marked atypia, unusual sites of involvement and defining EBV association. Surgery and reduced immunosuppression show comparable clinical results and prognosis is associated with intracranial manifestation.

Posttransplant Lymphoproliferative Disease after Pediatric Solid Organ Transplantation

Patients after solid organ transplantation (SOT) carry a substantially increased risk to develop malignant lymphomas. This is in part due to the immunosuppression required to maintain the function of the organ graft. Depending on the transplanted organ, up to 15% of pediatric transplant recipients acquire posttransplant lymphoproliferative disease (PTLD), and eventually 20% of those succumb to the disease. Early diagnosis of PTLD is often hampered by the unspecific symptoms and the difficult differential diagnosis, which includes atypical infections as well as graft rejection. Treatment of PTLD is limited by the high vulnerability towards antineoplastic chemotherapy in transplanted children. However, new treatment strategies and especially the introduction of the monoclonal anti-CD20 antibody rituximab have dramatically improved outcomes of PTLD. This review discusses risk factors for the development of PTLD in children, summarizes current approaches to therapy, and gives an outlook on developing new treatment modalities like targeted therapy with virus-specific T cells. Finally, monitoring strategies are evaluated.

Risk Factors and Prognosis in T-Cell Posttransplantation Lymphoproliferative Diseases: Reevaluation of 163 Cases

Background Posttransplantation lymphoproliferative diseases (PTLD) are mainly Epstein-Barr virus (EBV)–associated disorders of B-cell origin. Due to the rarity of monomorphic T-cell PTLD (T-PTLD), knowledge about pathogenesis, risk factors, therapy, and prognosis relies predominantly on case reports and small series. Therefore, we aimed to provide an overview and a retrospective analysis of this rare PTLD subtype. Methods We analyzed all available articles on T-PTLD in the PubMed database as well as in our own databases (Institute of Pathology/Department of Paediatric Haematology and Oncology, Hannover Medical School) from 1988 to 2010. Reevaluated parameters were gender, age, transplanted organ, immunosuppressant regimen, time between transplantation and T-PTLD manifestation, T-PTLD subtype, virus positivity, localization, therapy, and follow-up. Results A total of 163 cases were evaluated. We found that hematopoietic stem cell transplantation was associated with early-onset T-PTLD, whereas late onset occurred after immunosuppression with steroids and azathioprine without administration of calcineurin inhibitors. The major independent favorable prognostic factors were T-PTLD of the large granular lymphocytic leukemia subtype, young age, and a combination of radiotherapy/radiochemotherapy and reduced immunosuppression, whereas the hepatosplenic T-cell lymphoma subtype and cases with involvement of bone marrow, the central nervous system, or graft had an adverse prognosis. Conclusion T-PTLD is a heterogeneous group of different aberrant T-cell proliferations and represents a significant complication following transplantation, showing a uniformly poor prognosis.

Adoptive T-cell immunotherapy from third-party donors: characterization of donors and set up of a T-cell donor registry

Infection with and reactivation of human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) are frequent and severe complications in immunocompromised recipients after hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). These serious adverse events are associated with significant morbidity and mortality. Donor lymphocyte infusions (DLIs) are often used to treat both viral infections and leukemia relapses after transplantation but are associated with potentially life-threatening graft-versus-host disease (GvHD). Adoptive immunotherapy with virus-specific cytotoxic effector T cells (CTLs) derived from seropositive donors can rapidly reconstitute antiviral immunity after HSCT and organ transplantation. Therefore, it can effectively prevent the clinical manifestation of these viruses with no significant acute toxicity or increased risk of GvHD. In conditions, where patients receiving an allogeneic cord blood (CB) transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients, allogeneic third party T-cell donors would offer an alternative option. Recent studies showed that during granulocyte colony-stimulating factor (G-CSF) mobilization, the functional activity of antiviral memory T cells is impaired for a long period. This finding suggests that even stem cell donors may not be the best source of T cells. Under these circumstances, partially human leukocyte antigen (HLA)-matched virus-specific CTLs from healthy seropositive individuals may be a promising option. Therefore, frequency assessments of virus-specific memory T cells in HLA-typed healthy donors as well as in HSCT/SOT donors using a high throughput T-cell assay were performed over a period of 4 years at Hannover Medical School. This chapter will address the relevance and potential of a third-party T-cell donor registry and will discuss its clinical implication for adoptive T-cell immunotherapy.

CXCL13 as a Novel Marker for Diagnosis and Disease Monitoring in Pediatric PTLD

Posttransplant lymphoproliferative disease (PTLD) is a severe complication of immunosuppressive treatment in organ‐grafted children. Early diagnosis of PTLD is hampered by both unspecific clinical symptoms and lack of easy accessible markers. The homeostatic chemokine CXCL13, which plays a crucial role in B‐cell homing and lymphoid organ development, is expressed in some lymphomatous diseases. This study aims to investigate whether serum CXCL13 (sCXCL13) levels correlate with occurrence and regression of PTLD in pediatric solid‐organ graft recipients. Serum samples from PTLD patients (n = 21), patients with Epstein–Barr virus (EBV) reactivation (n = 18), and healthy age‐matched controls (n = 19) were tested for CXCL13 using a commercially available ELISA kit. sCXCL13 levels were significantly higher in PTLD patients than in healthy children. PTLD patients had also higher sCXCL13 values than pediatric solid‐organ recipients with EBV reactivation. An increase in sCXCL13 levels was observed from EBV reactivation to PTLD diagnosis in most cases. Elevated sCXCL13 levels were detected up to 2 years prior to PTLD diagnosis and correlated well with response to cytoreductive treatment in individual patients. sCXCL13, thus, may be a readily available surrogate marker for the diagnosis of PTLD and for monitoring of response to treatment in patients with initially elevated sCXCL13 levels.

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells. CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry. G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF. To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.

Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein-Barr virus-specific T cells as targets of interest in immunotherapeutic approaches.

Adoptive immunotherapy with donor-derived antiviral T cells can prevent viral complications such as with cytomegalovirus (CMV) and Epstein-Barr virus (EBV). In this context accurate monitoring of cellular immunity is essential and requires suitable quantitative and qualitative assays for high-throughput screening. We comparatively analyzed 57 HLA-typed healthy donors for memory T-cell responses to CMV- and EBV-derived proteins, peptide pools and single HLA-restricted peptides by five commonly used immunoassays in parallel: enzyme-linked immunospot (ELISPOT), cytokine secretion assay (CSA), intracellular cytokine staining (ICS), enzyme-linked immunosorbent assay (ELISA) and pMHC multimer staining. T-cell responses varied greatly between the different target antigens in the investigated assays. IFN-γ ELISPOT consistently detected the highest T-cell response levels against CMV and EBV. CMV-specific T cells were detected in 100% of CMV-seropositive donors tested using CMVpp65 protein and/or overlapping CMVpp65 peptide pool. CMV-specific T cells in HLA-A*02:01-positive/CMV-seropositive donors were identified directly by HLA-A02/CMVpp65 (A02pp65) multimer staining and, after short in vitro stimulation with HLA-A*02:01-restricted pp65 peptide, by ELISPOT, ELISA, ICS and CSA. A peptide-specific T-cell response was detected in only 4 HLA-A*02:01-positive donors (50%). Despite A02pp65 peptide negativity, T-cell responses to CMVpp65 protein and/or overlapping peptide pool were detected. Comparing the specific immune response against EBV antigens in healthy donors overall, BZLF1-specific T cells (<92.9% peptides, <56.3% peptide pool) were more frequent than EBNA-specific T cells (<64.3% peptides, <46.9% peptide pool) with higher percentage of positive findings for single HLA-restricted EBV peptides. T-cell response against HLA-B*08 peptide epitopes was predominant (multimer staining: EBNA3A: 9/14 and BZLF1: 7/14, IFN-γ ELISPOT: EBNA3A: 13/14 and BZLF1: 11/14). The fact that responses to EBV-specific antigens were not detected in every single EBV-seropositive donor as well as that the T-cell frequencies in response to the investigated EBV antigens differed strongly in the donor cohort indicates that these epitopes are less immunodominant than CMVpp65. Taken together, precise monitoring of T-cell immunity against infectious agents in potential T-cell donors and post-transplant recipients requires individual selection of antigens and immunoassays for the efficient detection and generation of clinically relevant T cells. Due to its lower detection limit and direct visualization of each IFN-γ-secreting cell we identified ELISPOT analysis to be preferable for high-throughput pre-screening. CSA was found to be advantageous for a more detailed analysis of antigen-specific T-cell subsets.

Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies.

BackgroundHeat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8+ and CD4+ T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65495-503-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for ex vivo expansion of CMV-specific CTLs.MethodsCMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65495-503 peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65495-503 complex.ResultsAbout 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65495-503 peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8+ T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC.ConclusionThis study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.