Matthias Eyrich

A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen‐disparate haematopoietic stem cells from parental donors

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)‐disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA‐matched donor. Success against major obstacles such as graft‐versus‐host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long‐term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34+ haematopoietic stem cells after rigorous T‐cell depletion were prospectively monitored for their immune reconstitution during the first post‐transplant year. Natural killer (NK) cells showed a marked increase on du2003+30. T and B cells began to reconstitute on du2003+72 and +68 respectively. During extended follow‐up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T‐cell receptor (TCR)‐repertoire complexity. Naive T cells emerged 6u2003months post transplantation, paralleled by an increase in TCR‐repertoire diversity. All patients self‐maintained sufficient immunoglobulin levels after du2003+200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T‐, B‐ and NK‐cell compartments within the first post‐transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA‐matched donor.

Use of CD137 to study the full repertoire of CD8+ T cells without the need to know epitope specificities†

CD137 (4‐1BB) is a member of the TNFR‐family with costimulatory function, triggering prosurvival signals in activated T‐cells. Upregulation of CD137 upon stimulation allows identifying and isolating live, human antigen‐specific CD8+ T‐cells of all phenotypes, and therefore provides a comprehensive detection method. Furthermore responses against antigen mixtures can be easily detected, enabling antigen discovery in a stepwise deconvoluting approach. In this article, we will discuss various aspects of this methodology, including potential pitfalls as well as a variety of applications, as illustrated by examples from our laboratory.

A prospective comparison of immune reconstitution in pediatric recipients of positively selected CD34+ peripheral blood stem cells from unrelated donors vs recipients of unmanipulated bone marrow from related donors.

Summary:Positively selected CD34+ hematopoietic stem cells from unrelated donors (UD-HSCT) have been successfully transplanted, but little is known about immune reconstitution in this setting. Here we report a prospective comparison of immune reconstitution in recipients of UD-HSCT and of unmanipulated bone marrow from matched sibling donors (MSD-BMT). T-cell reconstitution occurred more than 100 days later in the UD-HSCT than in the MSD-BMT group. The first T cells after UD-HSCT were almost exclusively CD45RO+ HLA-DR+, whereas early-emerging T cells after MSD-BMT more frequently expressed CD62L, CD28, and CD25. In both groups, numbers of CD45RA+ naive T cells increased after 180 days. After UD-HSCT, the T-cell-receptor (TCR)-repertoire was severely skewed and showed significantly reduced diversity during the first year, but only minor abnormalities were seen after MSD-BMT. TCR-diversity increased simultaneously with the number of naive T cells. In both groups, we observed transient expansions of γδ T cells. B cells were reconstituted more rapidly in UD-HSCT than in MSD-BMT recipients, whereas the rapidity of NK-cell reconstitution was similar in the two groups. In summary, T-cell reconstitution was slower after UD-HSCT than after MSD-BMT because of the delayed recovery of early memory-type T cells with reduced TCR-diversity, whereas naive T-, NK-, and B cells were reconstituted similarly in the two groups.

Primed tumor-reactive multifunctional CD62L+ human CD8+T-cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8+ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L+, CCR7+, CD28+). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.

Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis

BackgroundInfection with varicella-zoster virus (VZV) contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZV-immunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses.MethodsIn this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZV-infections in our center and compare them to published data. Furthermore, we report three instructive cases.ResultsHospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroid-dependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h.ConclusionOur data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.

Transplantation of highly purified peripheral-blood CD34 + progenitor cells from related and unrelated donors in children with nonmalignant diseases

Summary:Transplantation of allogeneic stem cells is currently the only curative treatment for some nonmalignant pediatric diseases. We investigated whether transplantation of purified CD34+ stem cells prevents acute and chronic GvHD and reduces transplant-related mortality. A total of 25 pediatric patients with nonmalignant diseases underwent allogeneic transplantation from 26 donors (matched related n=4, matched or partially matched unrelated n=14, mismatched related n=8). All grafts were purified peripheral-blood CD34+ stem cells mobilized with G-CSF. Patients received a median of 12.9 × 106 CD34+ progenitor cells with a median of 6.1 × 103 contaminating T-lymphocytes per kilogram of body weight. No post transplant immunosuppressive drugs were given for prophylaxis of GvHD. Engraftment was seen in 21 patients. Three patients engrafted after a second transplant and one patient failed to engraft. Two patients had autologous reconstitution 1.5 years post transplant and one of them was successfully retransplanted. No acute GvHD >grade II was seen, and only two patients developed limited, chronic GvHD. In all, 22 patients (88%) are alive with a median follow-up of 3.7 years. In total, 19 patients (76%) are free of disease or of progression. Transplantation of highly purified peripheral-blood CD34+ stem cells is associated with low toxicity in patients with nonmalignant diseases.

OKT‐3‐based reconditioning regimen for early graft failure in HLA‐non‐identical stem cell transplants

Primary non‐engraftment or early rejection after transplantation of haematopoietic stem cells represent life‐threatening complications of allogeneic stem cell transplantation. Management of early graft failure has been problematic, as the risk of fatal infectious complications increases with the time of pancytopenia and as a second transplant preceded by a conventional myeloablative conditioning regimen has been associated with high rates of cumulative organ toxicity. For paediatric patients with early graft failure following the transplantation of highly purified major histocompatibility complex (MHC)‐disparate haematopoietic stem cells, we have evaluated an immunosuppressive OKT‐3/methylprednisolone‐based reconditioning regimen with low toxicity in preparation for a secondary transplant of purified haematopoietic stem cells from the same donor. This report presents the results from a 4‐year pilot study including six patients with early graft failure. The results demonstrate that this antibody‐based regimen can be used effectively to prepare patients for secondary transplantation. Successful engraftment after a second transplant procedure was achieved in five of these six high‐risk patients. The median interval between first and second transplant was 27u2003d (range 22–51u2003d), and the median time for engraftment was 10u2003d (range 9–13u2003d). Chimaerism analysis of microsatellite regions by polymerase chain reaction (PCR) demonstrated complete donor chimaerism in four of these patients within the first month after secondary transplant and revealed mixed chimaerism in one patient who converted to complete chimaerism after T‐cell add‐back.

Pediatric high grade glioma of the spinal cord: results of the HIT-GBM database

Little is known about pediatric spinal cord high grade gliomas (SCHGG) beyond their dismal prognosis. Here, we analyzed the HIT-GBM® database for the influence of surgical resection on survival. Between 1991 and 2010 the HIT-GBM group collected data from European children diagnosed with high grade glioma. Patients with the following inclusion criteria were analyzed in this study: astrocytic histology, WHO grade III or IV, age at diagnosis <18xa0years, and tumor localized to the spinal cord. 28 patients (mean age 11.28xa0years, 14 male) with primary SCHGG were identified. The tumor sizes were measured by the span across adjacent vertebrae and varied greatly (range: 1–20, median: 4). Histology was classified as WHO grade III in 15 and grade IV in 13 tumors. Of note, the four largest tumors identified were WHO grade III. Surgery was classified as complete resection (nxa0=xa06), subtotal resection (STR) (nxa0=xa07), partial resection (nxa0=xa012) or biopsy only (nxa0=xa03). 27 patients received chemotherapy, 22 of which also received radiation. With the mean follow-up time of 2.88 (SDxa0±xa02.95) years, 14 patients were still alive resulting in a median overall survival of 2.5xa0years (SExa0±xa01.6). The positive prognostic indicators for overall survival were: age younger than 5xa0years (Pxa0=xa00.047), WHO grade III (Pxa0=xa00.046), absence of necrosis (Pxa0=xa00.025) and gross total resection (GTR) (Pxa0=xa00.012). The prognosis of SCHGG might not be as miserable as generally assumed. GTR is of benefit. Larger data sets and meta-analysis are necessary to identify patient sub-groups.

TH1 predominance is associated with improved survival in pediatric medulloblastoma patients

Medulloblastoma, a primitive neuro-ectodermal tumor that arises in the posterior fossa, is the most common malignant brain tumor occurring in childhood. Even though 60–70% of children with medulloblastoma will be cured with intensive multimodal therapy, including surgery, radiotherapy, and chemotherapy, a significant proportion of surviving patients may suffer from long-term treatment-related sequelae. Therapeutic success is limited especially in younger children by radiotherapy-induced neurocognitive longterm deficits. In order to avoid or delay craniospinal radiotherapy, high-dose chemotherapy followed by autologous stem cell transplantation (HSCT) has become an established treatment modality. Data on the host immunologic environment in medulloblastoma patients are rare, notably data on cytokine expression and immune reconstitution in patients with medulloblastoma undergoing HSCT are lacking. In this present study, we therefore decided to prospectively assess immune function following 24 consecutive autologous HSCT in 17 children with medulloblastoma treated according to the German-Austrian-Swiss HIT-2000-protocol. TH1 predominance was found to be the most important factor for probability of survival. Already before HSCT, survivors showed higher IFNγ levels in sera as well as higher numbers of IFNγ-positive T-cells. After transplantation, this effect was even more pronounced. Patients with higher numbers of IFNγ- and TNFα-positive T-cells had a more favorable outcome at all analyzed time points. In addition, patients in complete remission (CR) before transplantation, known to have a better prognosis a priori, showed higher expression of IFNγ in T-cells. Taken together, this is the first report to demonstrate that high expression of IFNγ and TNFα in T-cells of medulloblastoma patients in the early post-transplant period correlates with a better prognosis. Our data point toward a potentially important influence of TH1-cytokine expression before and after transplantation on the survival of pediatric medulloblastoma patients.

Dasatinib medication causing profound immunosuppression in a patient after haploidentical SCT: functional assays from whole blood as diagnostic clues.

Dasatinib medication causing profound immunosuppression in a patient after haploidentical SCT: functional assays from whole blood as diagnostic clues