Publications by authors named "Matthias Wölfl"

49 Publications

A T-Cell Surface Marker Panel Predicts Murine Acute Graft-Versus-Host Disease.

Front Immunol 2020 29;11:593321. Epub 2021 Jan 29.

Department of Medicine II, University Hospital of Würzburg, Würzburg, Germany.

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8 T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.
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http://dx.doi.org/10.3389/fimmu.2020.593321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880247PMC
January 2021

Neonatal Acute Lymphoblastic Leukemia with t(9;11) Translocation Presenting as Blueberry Muffin Baby: Successful Treatment by ALL-BFM Induction Therapy, Allogeneic Stem Cell Transplantation from an Unrelated Donor, and PCR-MRD-Guided Post-Transplant Follow-Up.

Am J Case Rep 2020 Oct 27;21:e927153. Epub 2020 Oct 27.

Department of Pediatrics, Section of Pediatric Hematology and Oncology, Stem Cell Transplantation (SCT), University Hospital Würzburg, Würzburg, Germany.

BACKGROUND Neonatal acute leukemia is a rare condition. Little is known about its incidence and outcomes, and treatment options have not been standardized. CASE REPORT A 3-day old, apparently healthy male newborn was referred to the pediatric intensive care unit with multiple violaceous macules and a few papules on his face and upper trunk. After initial spontaneous regression, the lesions reappeared. Skin biopsy and bone marrow aspirate revealed a diagnosis of acute lymphoblastic leukemia (ALL). ALL induction therapy was initiated on day 24, resulting in morphological remission at the end of induction therapy. ALL chemotherapy was guided by sequential PCR-based monitoring of minimal residual disease (MRD). The patient received a transplant from an unrelated HLA high-resolution matched (10/10 loci) permissive donor. He was followed-up after transplant conducted by sequential PCR-based measurements of MRD in bone marrow. CONCLUSIONS Neonatal leukemia often presents as congenital skin lesions known as blueberry muffin rash. ALL induction therapy was started at the end of the neonatal period. Treatment was well-tolerated and effective. Early donor search and PCR-MRD guided treatment surveillance can help to achieve and maintain molecular remission.
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http://dx.doi.org/10.12659/AJCR.927153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603802PMC
October 2020

Prostaglandin E in a TLR3- and 7/8-agonist-based DC maturation cocktail generates mature, cytokine-producing, migratory DCs but impairs antigen cross-presentation to CD8 T cells.

Cancer Immunol Immunother 2020 Jun 25;69(6):1029-1042. Epub 2020 Feb 25.

Laboratory for Stem Cell Processing and Cellular TherapyUniversity Medical Center, Children's Hospital, Würzburg, Germany.

Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E (PGE) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8 T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8 T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8 T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE during DC maturation and reproducible with several responder T-cell lines. In conclusion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides.
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http://dx.doi.org/10.1007/s00262-019-02470-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223547PMC
June 2020

Disease risk and GVHD biomarkers can stratify patients for risk of relapse and nonrelapse mortality post hematopoietic cell transplant.

Leukemia 2020 07 4;34(7):1898-1906. Epub 2020 Feb 4.

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The graft-versus-leukemia (GVL) effect after allogeneic hematopoietic cell transplant (HCT) can prevent relapse but the risk of severe graft-versus-host disease (GVHD) leads to prolonged intensive immunosuppression and possible blunting of the GVL effect. Strategies to reduce immunosuppression in order to prevent relapse have been offset by increases in severe GVHD and nonrelapse mortality (NRM). We recently validated the MAGIC algorithm probability (MAP) that predicts the risk for severe GVHD and NRM in asymptomatic patients using serum biomarkers. In this study we tested whether the MAP could identify patients whose risk for relapse is higher than their risk for severe GVHD and NRM. The multicenter study population (n = 1604) was divided into two cohorts: historical (2006-2015, n = 702) and current (2015-2017, n = 902) with similar NRM, relapse, and survival. On day 28 post-HCT, patients who had not developed GVHD (75% of the population) and who possessed a low MAP were at much higher risk for relapse (24%) than severe GVHD and NRM (16 and 9%); this difference was even more pronounced in patients with a high disease risk index (relapse 33%, NRM 9%). Such patients are good candidates to test relapse prevention strategies that might enhance GVL.
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http://dx.doi.org/10.1038/s41375-020-0726-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332389PMC
July 2020

Viral reactivations following hematopoietic stem cell transplantation in pediatric patients - A single center 11-year analysis.

PLoS One 2020 4;15(2):e0228451. Epub 2020 Feb 4.

Department of Oncology, Hematology and Stem Cell Transplantation, University Children's Hospital Würzburg, Würzburg, Germany.

Viral reactivation occurs frequently in the context of immunodeficiency and immunosuppression after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and can cause severe complications. The aim of this single-center retrospective analysis was to characterize viral infections in the first year after HSCT, to investigate risk factors and to study the impact of viral infections on transplantation outcome. This will facilitate the identification of at-risk patients and the development of new preventive strategies. 107 pediatric allo-HSCT from January 2005 through December 2015 were analyzed for infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), adenovirus (ADV), herpes simplex virus (HSV) and varicella zoster virus (VZV). Viral infections were detected after 68.2% of transplantations. The viruses most commonly encountered were HHV-6 (36/107) and EBV (30/107). Severe viral disease was rare (7/107) and none of the patients died as result of viral reactivation. Important risk factors for viral infections were higher age at HSCT, donor type and occurrence of acute graft-versus-host disease (aGvHD). Especially for EBV, transplant from an unrelated donor and in-vivo T-cell depletion (TCD) had a significant effect on infection rates, whereas for CMV the strongest effect was seen by donor and recipient serostatus with recipient seropositivity most predictive for reactivation. The occurrence of severe aGvHD was associated with EBV and ADV infections. For HSV, the recipient serostatus was identified as prognostic factor for HSV infections, while we found higher age at time of HSCT as risk factor for VZV infections. The overall survival of patients with or without viral infections did not differ significantly. Interestingly, when looking at the 85 patients in our cohort who had received an HSCT for a malignant disease, a tendency towards lower relapse rates was seen in patients affected by viral infections (HR 0.51, 95% CI 0.25 - 1.06, p = 0.072). Viral reactivations are common after pediatric allo-HSCT, though severe complications were rare in our collective. Determining risk factors for viral reactivations may help to identify patients in need of intensified monitoring and to individualize preventive strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228451PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999888PMC
April 2020

The MAGIC algorithm probability is a validated response biomarker of treatment of acute graft-versus-host disease.

Blood Adv 2019 12;3(23):4034-4042

Tisch Cancer Institute and.

The Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm probability (MAP), derived from 2 serum biomarkers, measures damage to crypts in the gastrointestinal tract during graft-versus-host disease (GVHD). We hypothesized that changes in MAP after treatment could validate it as a response biomarker. We prospectively collected serum samples and clinical stages of acute GVHD from 615 patients receiving hematopoietic cell transplantation in 20 centers at initiation of first-line systemic treatment and 4 weeks later. We computed MAPs and clinical responses and compared their abilities to predict 6-month nonrelapse mortality (NRM) in the validation cohort (n = 367). After 4 weeks of treatment, MAPs predicted NRM better than the change in clinical symptoms in all patients and identified 2 groups with significantly different NRM in both clinical responders (40% vs 12%, P < .0001) and nonresponders (65% vs 25%, P < .0001). MAPs successfully reclassified patients for NRM risk within every clinical grade of acute GVHD after 4 weeks of treatment. At the beginning of treatment, patients with a low MAP that rose above the threshold of 0.290 after 4 weeks of treatment had a significant increase in NRM, whereas patients with a high MAP at onset that fell below that threshold after treatment had a striking decrease in NRM that translated into clear differences in overall survival. We conclude that a MAP measured before and after treatment of acute GVHD is a response biomarker that predicts long-term outcomes more accurately than change in clinical symptoms. MAPs have the potential to guide therapy for acute GVHD and may function as a useful end point in clinical trials.
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http://dx.doi.org/10.1182/bloodadvances.2019000791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963240PMC
December 2019

On-target restoration of a split T cell-engaging antibody for precision immunotherapy.

Nat Commun 2019 11 26;10(1):5387. Epub 2019 Nov 26.

University Clinic Würzburg, Department of Internal Medicine II, Hematology and Oncology, Würzburg, Germany.

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (V) or variable heavy (V) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
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http://dx.doi.org/10.1038/s41467-019-13196-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879491PMC
November 2019

Re-expression of CD14 in Response to a Combined IL-10/TLR Stimulus Defines Monocyte-Derived Cells With an Immunoregulatory Phenotype.

Front Immunol 2019 28;10:1484. Epub 2019 Jun 28.

Department of Hematology, Oncology and Stem Cell Transplantation, University Children's Hospital, University of Würzburg, Würzburg, Germany.

Interleukin 10 is a central regulator of the antigen-presenting function of myeloid cells. It exerts immunomodulatory effects and induces a regulatory phenotype in monocyte-derived cells . We analyzed phenotype and function of monocytic cells in relation to the cytokine milieu and the timing of TLR-based activation. In GM-CSF/IL-4 cultured human monocytic cells, we identified two, mutually exclusive cell populations arising from undifferentiated cells: CD83 fully activated dendritic cells and CD14 macrophage like cells. Re-expression of CD14 occurs primarily after a sequential trigger with a TLR signal following IL-10 preincubation. This cell population with re-expressed CD14 greatly differs in phenotype and function from the CD83 cells. Detailed analysis of individual subpopulations reveals that exogenous IL-10 is critical for inducing the shift toward the CD14+ population, but does not affect individual changes in marker expression or cell function in most cases. Thus, plasticity of CD14 expression, defining a subset of immunoregulatory cells, is highly relevant for the composition of cellular products (such as DC vaccines) as it affects the function of the total product.
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http://dx.doi.org/10.3389/fimmu.2019.01484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611188PMC
October 2020

Role of B cells in chronic graft-versus-host disease after allogeneic stem cell transplantation in children and adolescents.

Br J Haematol 2019 09 28;186(5):e133-e137. Epub 2019 May 28.

Department of Paediatric Haematology/Oncology, University Children's Hospital Würzburg, Würzburg, Germany.

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http://dx.doi.org/10.1111/bjh.15977DOI Listing
September 2019

Cognate Nonlytic Interactions between CD8 T Cells and Breast Cancer Cells Induce Cancer Stem Cell-like Properties.

Cancer Res 2019 04 28;79(7):1507-1519. Epub 2019 Jan 28.

Department of Obstetrics and Gynecology, Würzburg University Hospital, University of Würzburg, Würzburg, Germany.

Targeting of tumor immune escape mechanisms holds enormous therapeutic potential. Still, most patients progress under immune checkpoint blockade and some even become hyperprogressors. To investigate how cancer cells respond to activated but ineffective T cells, we challenged peptide-loaded MCF-7 breast cancer cells with antigen-specific CD8 T cells in which lytic granules had been destroyed by pretreatment with Concanamycin A. Gene expression analysis after coculture revealed simultaneous induction of PD-L1, IDO1, CEACAM1, and further immunoregulatory checkpoints in breast cancer cells. Strikingly, we further observed gene signatures characteristic for dedifferentiation and acquisition of pluripotency markers including Yamanaka factors. Cognate interaction with nonlytic CD8 T cells also increased the proportion of stem cell-like cancer cells in a cell-to-cell contact- or (at least) proximity-dependent manner in various cell lines and in primary breast cancer cell cultures; this induction of stem cell-like properties was confirmed by enhanced tumor-forming capacity in immunodeficient mice. Resulting tumors were characterized by enhanced cell density, higher proliferation rates, and increased propensity for lymphoid metastasis. These findings describe a widely underappreciated pathway for immune escape, namely immune-mediated dedifferentiation of breast cancer cells, which is associated with profound changes in gene expression and cellular behavior. As the enhanced malignant potential of cancer cells after nonlytic cognate interactions with CD8 T cells enables increased tumor growth and metastasis in BALB/c mice, the described mechanism may provide a possible explanation for the clinical phenomenon of hyperprogression in response to unsuccessful immunotherapy. SIGNIFICANCE: This study shows that ineffective immune responses not only fail to clear a malignancy, but can also activate pathways in cancer cells that promote stemness and tumor-seeding capacity.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0387DOI Listing
April 2019

Early and late complications following hematopoietic stem cell transplantation in pediatric patients - A retrospective analysis over 11 years.

PLoS One 2018 16;13(10):e0204914. Epub 2018 Oct 16.

University Hospital Wuerzburg, Children's Department of Oncology, Hematology and Stem Cell Transplantation, Wuerzburg, Germany.

Hematopoietic stem cell transplantation (HSCT) has been an effective method for treating a wide range of malignant or non-malignant disorders. In case of an autologous HSCT, patients receive their own stem cells after myeloablation before extraction. Allogeneic HSCT uses stem cells derived from a donor. Despite being associated with a high risk of early and long-term complications, it is often the last curative option. 229 pediatric patients, who between 1 January 2005 and 31 December 2015 received an HSCT at the University Children's Hospital Wuerzburg, were studied. Correlations between two groups were calculated with the Chi square test or with a 2x2-contingency table. To calculate metric variables, the Mann-Whitney-U-test was used. Survival curves were calculated according to Kaplan and Meier. Significance was assumed for results with a p-value <0.05 (CI (Confident Interval) 95%). We retrospectively analyzed 229 pediatric patients (105 females, 124 males) for early and late complications of allogeneic and autologous hematopoietic stem cell transplantation. Median age at HSCT was seven years. Underlying diseases were leukemia (n = 73), lymphoma (n = 22), solid tumor (n = 65), CNS (central nervous system)- tumor (n = 41), and "other diseases" (n = 28). Survival times, overall survival, and event-free survival were calculated. Of all patients, 80.8% experienced complications of some degree, including mild and transient complications. Allo-HSCT (allogeneic HSCT) carried a significantly higher risk of complications than auto-HSCT (autologous HSCT) (n = 118 vs. n = 67; p = < .001) and the remission rate after allo-HSCT was also higher (58.7% vs. 44,7%; p = .032). Especially infection rates and pulmonary complications are different between auto- and allo-HSCT. Leukemia patients had the highest risk of early and late complications (95,0%; p < .001). Complications within HSCT are major risk factors following morbidity and mortality. In order to detect complications and risk factors early, strict recordings are needed to reduce the rate of complication by recognition and prevention of triggering factors. In the future, these factors should receive greater attention in the planning of HSCT post-transplantation care in order to improve the results of the transplantation and establish protocols to prevent their occurrence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204914PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191171PMC
March 2019

High-grade glioma associated immunosuppression does not prevent immune responses induced by therapeutic vaccines in combination with T depletion.

Cancer Immunol Immunother 2018 Oct 27;67(10):1545-1558. Epub 2018 Jul 27.

Department of Pediatric Oncology, University Children's Hospital Würzburg, Josef-Schneider-Strasse 2, 97080, Würzburg, Germany.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4/CD8 T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (T). In parallel to T-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8VLA-4 T-cells with CNS-homing properties, but not of CD4 VLA-4 T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.
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http://dx.doi.org/10.1007/s00262-018-2214-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182405PMC
October 2018

Spontaneous reversion of a lineage switch following an initial blinatumomab-induced ALL-to-AML switch in -rearranged infant ALL.

Blood Adv 2018 06;2(12):1382-1385

Pediatric Hematology, Oncology and Stem Cell Transplantation, Children's Hospital, University of Würzburg, Würzburg, Germany; and.

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http://dx.doi.org/10.1182/bloodadvances.2018018093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020817PMC
June 2018

MAGIC biomarkers predict long-term outcomes for steroid-resistant acute GVHD.

Blood 2018 06 15;131(25):2846-2855. Epub 2018 Mar 15.

Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY.

Acute graft-versus-host disease (GVHD) is treated with systemic corticosteroid immunosuppression. Clinical response after 1 week of therapy often guides further treatment decisions, but long-term outcomes vary widely among centers, and more accurate predictive tests are urgently needed. We analyzed clinical data and blood samples taken 1 week after systemic treatment of GVHD from 507 patients from 17 centers of the Mount Sinai Acute GVHD International Consortium (MAGIC), dividing them into a test cohort (n = 236) and 2 validation cohorts separated in time (n = 142 and n = 129). Initial response to systemic steroids correlated with response at 4 weeks, 1-year nonrelapse mortality (NRM), and overall survival (OS). A previously validated algorithm of 2 MAGIC biomarkers (ST2 and REG3α) consistently separated steroid-resistant patients into 2 groups with dramatically different NRM and OS ( < .001 for all 3 cohorts). High biomarker probability, resistance to steroids, and GVHD severity (Minnesota risk) were all significant predictors of NRM in multivariate analysis. A direct comparison of receiver operating characteristic curves showed that the area under the curve for biomarker probability (0.82) was significantly greater than that for steroid response (0.68, = .004) and for Minnesota risk (0.72, = .005). In conclusion, MAGIC biomarker probabilities generated after 1 week of systemic treatment of GVHD predict long-term outcomes in steroid-resistant GVHD better than clinical criteria and should prove useful in developing better treatment strategies.
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http://dx.doi.org/10.1182/blood-2018-01-822957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014357PMC
June 2018

Characterization of a novel OTX2-driven stem cell program in Group 3 and Group 4 medulloblastoma.

Mol Oncol 2018 04 1;12(4):495-513. Epub 2018 Mar 1.

Regenerative Medicine Program, Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada.

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self-renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof-of-principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self-renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.
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http://dx.doi.org/10.1002/1878-0261.12177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891039PMC
April 2018

Double Peripheral Venous and Arterial Cannulation for Extracorporeal Membrane Oxygenation in Combined Septic and Cardiogenic Shock.

Am J Case Rep 2017 Jun 28;18:723-727. Epub 2017 Jun 28.

Department of Anaesthesia and Critical Care, University Hospital of Würzburg, Würzburg, Germany.

BACKGROUND The use of venoarterial extracorporeal membrane oxygenation (va-ECMO) via peripheral cannulation for septic shock is limited by blood flow and increased afterload for the left ventricle. CASE REPORT A 15-year-old girl with acute myelogenous leukemia, suffering from severe septic and cardiogenic shock, was treated by venoarterial extracorporeal membrane oxygenation (va-ECMO). Sufficient extracorporeal blood flow matching the required oxygen demand could only be achieved by peripheral cannulation of both femoral arteries. Venous drainage was performed with a bicaval cannula inserted via the left V. femoralis. To accomplish left ventricular unloading, an additional drainage cannula was placed in the left atrium via percutaneous atrioseptostomy (va-va-ECMO). Cardiac function recovered and the girl was weaned from the ECMO on day 6. Successful allogenic stem cell transplantation took place 2 months later. CONCLUSIONS In patients with vasoplegic septic shock and impaired cardiac contractility, double peripheral venoarterial extracorporeal membrane oxygenation (va-va-ECMO) with transseptal left atrial venting can by a lifesaving option.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499631PMC
http://dx.doi.org/10.12659/ajcr.902485DOI Listing
June 2017

An early-biomarker algorithm predicts lethal graft-versus-host disease and survival.

JCI Insight 2017 02 9;2(3):e89798. Epub 2017 Feb 9.

Tisch Cancer Institute, the Icahn School of Medicine at Mount Sinai.

No laboratory test can predict the risk of nonrelapse mortality (NRM) or severe graft-versus-host disease (GVHD) after hematopoietic cellular transplantation (HCT) prior to the onset of GVHD symptoms. Patient blood samples on day 7 after HCT were obtained from a multicenter set of 1,287 patients, and 620 samples were assigned to a training set. We measured the concentrations of 4 GVHD biomarkers (ST2, REG3α, TNFR1, and IL-2Rα) and used them to model 6-month NRM using rigorous cross-validation strategies to identify the best algorithm that defined 2 distinct risk groups. We then applied the final algorithm in an independent test set ( = 309) and validation set ( = 358). A 2-biomarker model using ST2 and REG3α concentrations identified patients with a cumulative incidence of 6-month NRM of 28% in the high-risk group and 7% in the low-risk group ( < 0.001). The algorithm performed equally well in the test set (33% vs. 7%, < 0.001) and the multicenter validation set (26% vs. 10%, < 0.001). Sixteen percent, 17%, and 20% of patients were at high risk in the training, test, and validation sets, respectively. GVHD-related mortality was greater in high-risk patients (18% vs. 4%, < 0.001), as was severe gastrointestinal GVHD (17% vs. 8%, < 0.001). The same algorithm can be successfully adapted to define 3 distinct risk groups at GVHD onset. A biomarker algorithm based on a blood sample taken 7 days after HCT can consistently identify a group of patients at high risk for lethal GVHD and NRM. The National Cancer Institute, American Cancer Society, and the Doris Duke Charitable Foundation.
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http://dx.doi.org/10.1172/jci.insight.89798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291735PMC
February 2017

Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients.

J Clin Microbiol 2017 01 28;55(1):101-109. Epub 2016 Dec 28.

Kinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany.

Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-Aspergillus ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance.
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http://dx.doi.org/10.1128/JCM.01682-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228221PMC
January 2017

IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

Oncoimmunology 2016 Jul 19;5(7):e1188245. Epub 2016 May 19.

Children's Hospital, Pediatric Hematology, Oncology and Stem Cell Transplantation, University of Würzburg, Würzburg, Germany; Clinical Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany.

Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1.
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http://dx.doi.org/10.1080/2162402X.2016.1188245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006926PMC
July 2016

Pillars Article: Induction of Tolerance by IL-10-Treated Dendritic Cells. J. Immunol. 1997. 159: 4772-4780.

J Immunol 2016 09;197(5):1547-55

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September 2016

MB3W1 is an orthotopic xenograft model for anaplastic medulloblastoma displaying cancer stem cell- and Group 3-properties.

BMC Cancer 2016 Feb 17;16:115. Epub 2016 Feb 17.

University Children's Hospital, Pediatric Oncology, Hematology and Stem Cell Transplantation, University of Würzburg, Würzburg, Germany.

Background: Medulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.

Methods: We established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.

Results: Besides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.

Conclusions: This model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.
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http://dx.doi.org/10.1186/s12885-016-2170-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756501PMC
February 2016

International, Multicenter Standardization of Acute Graft-versus-Host Disease Clinical Data Collection: A Report from the Mount Sinai Acute GVHD International Consortium.

Biol Blood Marrow Transplant 2016 Jan 16;22(1):4-10. Epub 2015 Sep 16.

Blood and Marrow Transplantation Program, University of Michigan, Ann Arbor, Michigan; Blood and Marrow Transplantation Program, The Icahn School of Medicine at Mount Sinai Hospital, New York, New York. Electronic address:

Acute graft-versus-host disease (GVHD) remains a leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. The clinical staging of GVHD varies greatly between transplant centers and is frequently not agreed on by independent reviewers. The lack of standardized approaches to handle common sources of discrepancy in GVHD grading likely contributes to why promising GVHD treatments reported from single centers have failed to show benefit in randomized multicenter clinical trials. We developed guidelines through international expert consensus opinion to standardize the diagnosis and clinical staging of GVHD for use in a large international GVHD research consortium. During the first year of use, the guidance followed discussion of complex clinical phenotypes by experienced transplant physicians and data managers. These guidelines increase the uniformity of GVHD symptom capture, which may improve the reproducibility of GVHD clinical trials after further prospective validation.
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http://dx.doi.org/10.1016/j.bbmt.2015.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706482PMC
January 2016

Fulminant skin GvHD with a cytokine pattern resemblant of cytokine release syndrome successfully treated with multimodal immunosuppression including tocilizumab.

Pediatr Blood Cancer 2015 Nov 7;62(11):2033-5. Epub 2015 Jul 7.

University Hospital of Würzburg, Children's Hospital, Pediatric Stem Cell Transplantation, Josef-Schneider-Strasse 2, 97080, Würzburg, Germany.

Acute Graft-versus-Host-Disease (GvHD) is a potentially life-threatening complication after allogeneic stem cell transplantation. If not treated early and adequately, the complex immunological mechanisms may lead to a self-perpetuating cycle of alloreactivity, which is then associated with a high mortality. Here we assessed the cytokine profile on a daily basis in a patient with grade 4 skin GvHD, demonstrating a signature resembling cytokine-release-syndrome. After multimodal immunosuppressive intervention, including treatment with the IL6 receptor-blocking antibody tocilizumab, the severe clinical symptoms unexpectedly resolved within 48 hr.
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http://dx.doi.org/10.1002/pbc.25595DOI Listing
November 2015

MRI demyelination pattern and clinical course in a child with cerebral X-linked adrenoleukodystrophy (X-ALD).

Acta Radiol Open 2015 Apr 1;4(4):2047981615573655. Epub 2015 Apr 1.

Departments of Radiology and Neuroradiology, University Hospital of Würzburg, Würzburg, Germany.

The clinical spectrum in boys with X-linked adrenoleukodystrophy (X-ALD) ranges from isolated adrenocortical insufficiency and slowly progressive myelopathy to devastating cerebral demyelination. In the individual case, the disease course still remains unpredictable. Research findings suggest an important role of brain magnetic resonance imaging (MRI) lesion patterns as prognostic markers for X-ALD. Hence, familiarity with imaging features of childhood X-ALD in combination with clinical manifestation is required in order to stratify affected patients for therapy. We report on MRI findings and clinical course of cerebral X-ALD in a young boy with a rare subtype of white matter demyelination.
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http://dx.doi.org/10.1177/2047981615573655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385045PMC
April 2015

The TGF-β-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

J Leukoc Biol 2014 Oct 16;96(4):633-45. Epub 2014 Jul 16.

Department of Obstetrics and Gynecology, Interdisciplinary Center for Clinical Research,

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-β are well established, we wondered whether TGF-β could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-β promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-β up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-β type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-β signaling.
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http://dx.doi.org/10.1189/jlb.3A0114-025RDOI Listing
October 2014

Impact of sequence variation in a dominant HLA-A*02-restricted epitope in hepatitis C virus on priming and cross-reactivity of CD8+ T cells.

J Virol 2014 Oct 9;88(19):11080-90. Epub 2014 Jul 9.

Institute of Virology, University of Duisburg-Essen, University Hospital, Essen, Germany

Unlabelled: CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8+ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS31406-1415 epitope on in vitro priming of naive CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8+ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants.

Importance: The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.
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http://dx.doi.org/10.1128/JVI.01590-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178828PMC
October 2014

Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells.

Cytotherapy 2014 Jul 13;16(7):946-64. Epub 2014 May 13.

Laboratory of Pediatric Immunology, Department of Microbiology and Immunology, KU Leuven, Belgium (on behalf of the HGG-Immuno Network).

Background And Aims: One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol.

Methods: All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated.

Results: Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 μg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter.

Conclusions: Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.
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http://dx.doi.org/10.1016/j.jcyt.2014.02.017DOI Listing
July 2014

Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells.

Nat Protoc 2014 Apr 27;9(4):950-66. Epub 2014 Mar 27.

1] Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. [2] Department of Immunology, University of Washington, Seattle, Washington, USA. [3] Department of Medicine, University of Washington, Seattle, Washington, USA.

Antigen-specific priming of human, naive T cells has been difficult to assess. Owing to the low initial frequency in the naive cell pool of specific T cell precursors, such an analysis has been obscured by the requirements for repeated stimulations and prolonged culture time. In this protocol, we describe how to evaluate antigen-specific priming of CD8(+) cells 10 d after a single specific stimulation. The assay provides reference conditions, which result in the expansion of a substantial population of antigen-specific T cells from the naive repertoire. Various conditions and modifications during the priming process (e.g., testing new cytokines, co-stimulators and so on) can now be directly compared with the reference conditions. Factors relevant to achieving effective priming include the dendritic cell preparation, the T cell preparation, the cell ratio at the time of priming, the serum source used for the experiment and the timing of addition and concentration of the cytokines used for expansion. This protocol is relevant for human immunology, vaccine biology and drug development.
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http://dx.doi.org/10.1038/nprot.2014.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312138PMC
April 2014