Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure.

Introduction: Aspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application. Methods: To facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix). Results: For the fast CSA-based approach we detected IFN-γ+ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4+ helper T cells with a central-memory phenotype were expanded while CD8+ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ. Discussion: For patients with IA, the immediate adoptive transfer of IFN-γ+ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

The landscape of tumor cell states and spatial organization in H3-K27M mutant diffuse midline glioma across age and location.

Histone 3 lysine27-to-methionine (H3-K27M) mutations most frequently occur in diffuse midline gliomas (DMGs) of the childhood pons but are also increasingly recognized in adults. Their potential heterogeneity at different ages and midline locations is vastly understudied. Here, through dissecting the single-cell transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs, we delineate how age and anatomical location shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We show that stem-like oligodendroglial precursor-like cells, present across all clinico-anatomical groups, display varying levels of maturation dependent on location. We reveal a previously underappreciated relationship between mesenchymal cancer cell states and age, linked to age-dependent differences in the immune microenvironment. Further, we resolve the spatial organization of H3-K27M DMG cell populations and identify a mitotic oligodendroglial-lineage niche. Collectively, our study provides a powerful framework for rational modeling and therapeutic interventions.

Directed Evolution of Stabilized Monomeric CD19 for Monovalent CAR Interaction Studies and Monitoring of CAR-T Cell Patients.

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Value of Autoantibody Expression During Long-Term Follow-Up in Paediatric ALL Patients After Allogeneic Haematopoietic Stem Cell Transplantation.

Objectives: Chronic graft-versus-host disease (cGvHD) following haematopoietic stem cell transplantation (HSCT) shares many similarities with de novo autoimmune disorders, being associated with the presence of autoantibodies. However, data on the implication of autoantibodies in paediatric HSCT recipients are scarce. In this single-centre study of paediatric patients with acute lymphoblastic leukaemia (ALL) surviving longer than 3 months, our objectives were to evaluate autoantibody expression and investigate the correlation with cGvHD and immune reconstitution using serially monitored parameters. Methods: We investigated circulating autoantibodies together with cellular and humoral parameters [including major T- and B-cell subsets, natural killer (NK) cells, and immunoglobulin levels] in 440 samples from 74 patients (median age 10.9 years, range 2.7-22.2 years) serially during long-term follow-up of median 8 years (range 0.4-19.3 years). Evaluations comprised of patient and transplant characteristics, precisely reviewed details of National Institute of Health (NIH)-defined cGvHD, and outcome data such as relapse, overall survival (OS) and mortality. Analysis of these clinical parameters was performed to identify possible associations. Results: Autoantibodies were detected in 65% (48/74) of patients. Anti-nuclear antibodies were the most common, occurring in 75% (36/48) of patients with autoantibodies. When comparing demographic data and transplant characteristics, there were no significant differences between patients with and without autoantibody expression; 5-year OS was excellent, at 96.4 and 95.8%, respectively. Neither the expression of autoantibodies nor the occurrence of cGvHD correlated with significantly worse OS or relapse rate. Furthermore, there was no significant association between autoantibody profiles and the incidence, overall severity or organ involvement of cGvHD. Patients with autoantibodies showed significantly better immune reconstitution, with overall higher numbers of T cells, B cells, and serum immunoglobulins. In autoantibody-positive patients with cGvHD, autoantibody production positively correlated with the expansion of CD56+ NK cells (236.1 vs. 165.6 × 103 cells/mL, respectively; p = 0.023) and with signs of B-cell perturbation, such as higher CD21low B cells (23.8 vs. 11.8 × 103 cells/mL, respectively; p = 0.044) and a higher ratio of CD21low B cells/CD27+ memory B cells (1.7 vs. 0.4, respectively; p = 0.006) in comparison to autoantibody-positive patients without cGvHD. Furthermore, when assessing the correlation between autoantibody positivity and the activity of cGvHD at time of analysis, indicators of aberrant B-cell homeostasis were substantiated by a lower proportion of CD27+ memory B cells (9.1 vs. 14.9%, respectively; p = 0.028), a higher ratio of class-switched CD27+IgD-/CD27+ memory B cells (3.5 vs. 5.1%, respectively; p = 0.013), significantly elevated numbers of CD21low B cells (36.8 vs. 11.8 × 103 cells/mL, respectively; p = 0.013) and a higher ratio of CD21lowB cells/CD27+ memory B cells (2.4 vs. 0.4, respectively; p = 0.034) in the active vs. the no cGvHD group. We then assessed the potential role of autoantibody expression in the context of elevated CD19+CD21low B cells (cutoff >7%), a well-known marker of cGvHD. Surprisingly we found a significant higher proportion of those cases where elevated CD21low B cells correlated with active cGvHD in samples from the autoantibody-negative group vs. the antibody-positive group (82 vs. 47%, respectively; p = 0.0053). When comparing immune parameters of the large proportion of survivors (89%) with the small proportion of non-survivors (11%), data revealed normalisation within the B-cell compartment of survivors: there were increased numbers of CD27+ memory B cells (54.9 vs. 30.6 × 103 cells/mL, respectively; p = 0.05), class-switched CD27+IgD- B cells (21.2 vs. 5.0 × 103 cells/mL, respectively; p < 0.0001), and immunoglobulin G4 (40.9 vs. 19.4 mg/dL, respectively; p < 0.0001). Overall mortality was significantly associated with an elevated proportion of CD21low B cells (13.4 vs. 8.8%, respectively; p = 0.039) and CD56+ NK cells (238.8 vs. 314.1 × 103 cells/mL, respectively; p = 0.019). In multivariate analysis, better OS was significantly associated with lower numbers of CD56+ NK cells [hazard ratio (HR) 0.98, p = 0.041] and higher numbers of CD27+ memory B cells [(HR) 1.62, p = 0.014]. Conclusion: Our data shows that autoantibody profiles are not suitable biomarkers for diagnosing cGvHD in children or for predicting cGvHD severity, disease course and outcome. We identified a number of indicators of aberrant immune homeostasis associated with active cGvHD in paediatric ALL patients after HSCT. These findings confirm published results and suggest that candidate B cell subpopulations may serve as a surrogate measure for characterisation of cGvHD in paediatric HSCT for malignant diseases, and warrants confirmation in larger, multicentre studies.

Single-Cell RNA-Seq Reveals Cellular Hierarchies and Impaired Developmental Trajectories in Pediatric Ependymoma.

Ependymoma is a heterogeneous entity of central nervous system tumors with well-established molecular groups. Here, we apply single-cell RNA sequencing to analyze ependymomas across molecular groups and anatomic locations to investigate their intratumoral heterogeneity and developmental origins. Ependymomas are composed of a cellular hierarchy initiating from undifferentiated populations, which undergo impaired differentiation toward three lineages of neuronal-glial fate specification. While prognostically favorable groups of ependymoma predominantly harbor differentiated cells, aggressive groups are enriched for undifferentiated cell populations. The delineated transcriptomic signatures correlate with patient survival and define molecular dependencies for targeted treatment approaches. Taken together, our analyses reveal a developmental hierarchy underlying ependymomas relevant to biological and clinical behavior.

Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis.

Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.

Transfer and loss of allergen-specific responses via stem cell transplantation: A prospective observational study.

BACKGROUND: Currently, no estimates can be made on the impact of hematopoietic stem cell transplantation on allergy transfer or cure of the disease. By using component-resolved diagnosis, we prospectively investigated 50 donor-recipient pairs undergoing allogeneic stem cell transplantation. This allowed calculating the rate of transfer or maintenance of allergen-specific responses in the context of stem cell transplantation. METHODS: Allergen-specific IgE and IgG to 156 allergens was measured pretransplantation in 50 donors and recipients and at 6, 12 and 24 months in recipients post-transplantation by allergen microarray. Based on a mixed effects model, we determined risks of transfer of allergen-specific IgE or IgG responses 24 months post-transplantation. RESULTS: After undergoing stem cell transplantation, 94% of allergen-specific IgE responses were lost. Two years post-transplantation, recipients' allergen-specific IgE was significantly linked to the pretransplantation donor or recipient status. The estimated risk to transfer and maintain individual IgE responses to allergens by stem cell transplantation was 1.7% and 2.3%, respectively. Allergen-specific IgG, which served as a surrogate marker of maintaining protective IgG responses, was highly associated with the donor's (31.6%) or the recipient's (28%) pretransplantation response. CONCLUSION: Hematopoietic stem cell transplantation profoundly reduces allergen-specific IgE responses but also comes with a considerable risk to transfer allergen-specific immune responses. These findings facilitate clinical decision-making regarding allergic diseases in the context of hematopoietic stem cell transplantation. In addition, it provides prospective data to estimate the risk of transmitting allergen-specific responses via hematopoietic stem cell transplantation.

Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms.

Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.

An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Diagnostic Parameters of Adenoviremia in Pediatric Stem Cell Transplant Recipients.

Despite recent progress in the diagnostic risk assessment of human adenovirus (HAdV) infections in immunocompromised patients, clinical complications mediated by these viruses continue contributing to significant morbidity and mortality, particularly in the pediatric hematopoietic allogeneic stem cell transplant (HSCT) setting. Current data highlight the importance of monitoring stool samples to assess the risk of invasive HAdV infections in children undergoing HSCT. The advent of novel, more effective antiviral treatment options might permit successful virus control even at the stage of systemic infection, thus increasing the interest in optimized HAdV monitoring in peripheral blood (PB). We have screened over 300 pediatric HCST recipients by serial monitoring of stool and PB specimens, and identified 31 cases of invasive HAdV infection by quantitative pan-adenovirus RQ-PCR analysis of consecutive PB specimens. The diagnostic parameters assessed included HAdV peak levels (PL) and the time-averaged area under the curve (AAUC) of virus copy numbers. The predictive value for patient outcome reflected by non-relapse and HAdV-related mortality was determined. The patients were assigned to quartiles based on their PL and AAUC, and the readouts were highly correlated (p < 0.0001). Non-relapse mortality in patients by AAUC quartile (lowest to highest) was 26, 50, 75, and 86%, respectively, and AAUC was strongly correlated with non-relapse mortality (p < 0.0001), while the association between PL and non-relapse mortality was less pronounced (p = 0.013). HAdV-related mortality was absent or very low in patients within the two lower quartiles of both PL and AAUC, and increased to ≥70% in the upper two quartiles. Despite the significant correlation of PL and AAUC with patient outcome, it is necessary to consider that the risk of non-relapse mortality even within the lowest quartile was still relatively high, and it might be difficult therefore to translate the results into differential treatment approaches. By contrast, the correlation with HAdV-related mortality might permit the identification of a low-risk patient subset. Nevertheless, the well-established correlation of HAdV shedding into the stool and intestinal expansion of the virus with the risk of invasive infection will expectedly remain an essential diagnostic parameter in the pediatric HSCT setting.

Post-transplant Replication of Torque Teno Virus in Granulocytes.

Torque Teno virus (TTV) in humans is characterized by ubiquitous occurrence in peripheral blood (PB), without any related disease described to date. Several studies reported a significant increase of TTV plasma DNA levels in allogeneic transplant recipients, and suggested a correlation of elevated virus titers with immunosuppression and transplant-related complications. However, the site of viral replication in this setting has remained unclear. We have studied TTV in serial plasma specimens derived from 43 pediatric allogeneic hematopoietic stem cell transplantation (HSCT) recipients by RQ-PCR, and found increasing TTV-DNA levels in all patients post-transplant, with a peak around day +100 and maximum virus copy numbers reaching 4 × 10E9/ml. To assess whether the virus replicates in PB-cells, leukocyte subsets including granulocytes, monocytes, NK-cells, T- and B-lymphocytes were serially isolated by flow-sorting for TTV analysis in 19 patients. The virus was undetectable in most cell types, but was identified in granulocytes in all instances, revealing a median DNA copy number increase of 1.8 logs between days +30-100 post-transplant. Our data therefore provide evidence for TTV replication in granulocytes in this setting. In a control cohort of immunocompetent children and in HSCT recipients before day +30, TTV positivity in granulocytes was less common (33%), and the copy numbers were considerably lower. However, rising TTV replication about 2 weeks after granulocyte engraftment (>500 cells/µl) was observed suggesting that granulocyte recovery might be required for TTV expansion in severely immunosuppressed transplant recipients.

CD28 Blockade Ex Vivo Induces Alloantigen-Specific Immune Tolerance but Preserves T-Cell Pathogen Reactivity.

Donor T-cells contribute to reconstitution of protective immunity after allogeneic hematopoietic stem cell transplantation (HSCT) but must acquire specific tolerance against recipient alloantigens to avoid life-threatening graft-versus-host disease (GvHD). Systemic immunosuppressive drugs may abrogate severe GvHD, but this also impedes memory responses to invading pathogens. Here, we tested whether ex vivo blockade of CD28 co-stimulation can enable selective T-cell tolerization to alloantigens by facilitating CD80/86-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) signaling. Treatment of human allogeneic dendritic cell/T-cell co-cultures with a human CD28 blocking antibody fragment (α-huCD28) significantly abrogated subsequent allospecific immune responses, seen by decreased T-cell proliferation and of type 1 cytokine (IFN-γ and IL-2) expression. Allo-tolerization persisted after discontinuation of CD28 blockade and secondary alloantigen stimulation, as confirmed by enhanced CTLA-4 and PD-1 immune checkpoint signaling. However, T-cells retained reactivity to pathogens, supported by clonotyping of neo-primed and cross-reactive T-cells specific for Candida albicans or third-party antigens using deep sequencing analysis. In an MHC-mismatched murine model, we tolerized C57BL/6 T-cells by ex vivo exposure to a murine single chain Fv specific for CD28 (α-muCD28). Infusion of these cells, after α-muCD28 washout, into bone marrow-transplanted BALB/c mice caused allo-tolerance and did not induce GvHD-associated hepatic pathology. We conclude that selective CD28 blockade ex vivo can allow the generation of stably allo-tolerized T-cells that in turn do not induce graft-versus-host reactions while maintaining pathogen reactivity. Hence, CD28 co-stimulation blockade of donor T-cells may be a useful therapeutic approach to support the immune system after HSCT.

Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection.

BACKGROUND: Human adenovirus (HAdV) infections remain a significant cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Efficient antiviral T-cell responses are necessary to clear infection, which is hampered by delayed immune reconstitution and medical immunosuppression after HSCT. Protective immunity may be conferred by adoptive transfer of HAdV-specific T cells. For identification of patients at risk and monitoring of treatment responses diligent assessment of anti-HAdV cellular immune responses is crucial. The HAdV-derived protein hexon has been recognized as a major immunodominant target across HAdV species. We aimed at identifying further targets of protective anti-HAdV immune response and characterizing immunogenic epitopes. METHODS: Nineteen candidate nonamers from hexon and penton proteins were identified by epitope binding prediction. Peptides were synthesized and tested for in vivo immunogenicity by screening peripheral blood mononuclear cells from healthy volunteers (n = 64) and HAdV-infected stem cell recipients (n = 26) for memory T cells recognizing the candidate epitopes in the context of most common HLA alleles. RESULTS: Functional CD8+ T cells recognizing seven epitopes were identified, among them four penton-derived and two hexon-derived peptides. The HLA-A*01-restricted penton-derived peptide STDVASLNY (A01PentonSTDV) and HLA-A*02-restricted hexon-derived peptide TLLYVLFEV (A02HexonTLLY) were recognized by more than half of the persons carrying the respective HLA-type. CONCLUSIONS: Thus, the HAdV-derived penton protein is a novel major target of the anti-HAdV immune response. Identification of new immunodominant epitopes will facilitate and broaden immune assessment strategies to identify patients suitable for T-cell transfer. Knowledge of additional target structures may increase T-cell recovery in manufacturing processes.

Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

Risk assessment of relapse by lineage-specific monitoring of chimerism in children undergoing allogeneic stem cell transplantation for acute lymphoblastic leukemia.

UNLABELLED: Allogeneic hematopoietic stem cell transplantation is required as rescue therapy in about 20% of pediatric patients with acute lymphoblastic leukemia. However, the relapse rates are considerable, and relapse confers a poor outcome. Early assessment of the risk of relapse is therefore of paramount importance for the development of appropriate measures. We used the EuroChimerism approach to investigate the potential impact of lineage-specific chimerism testing for relapse-risk analysis in 162 pediatric patients with acute lymphoblastic leukemia after allogeneic stem cell transplantation in a multicenter study based on standardized transplantation protocols. Within a median observation time of 4.5 years, relapses have occurred in 41/162 patients at a median of 0.6 years after transplantation (range, 0.13-5.7 years). Prospective screening at defined consecutive time points revealed that reappearance of recipient-derived cells within the CD34(+) and CD8(+) cell subsets display the most significant association with the occurrence of relapses with hazard ratios of 5.2 (P=0.003) and 2.8 (P=0.008), respectively. The appearance of recipient cells after a period of pure donor chimerism in the CD34(+) and CD8(+) leukocyte subsets revealed dynamics indicative of a significantly elevated risk of relapse or imminent disease recurrence. Assessment of chimerism within these lineages can therefore provide complementary information for further diagnostic and, potentially, therapeutic purposes aiming at the prevention of overt relapse. This study was registered at clinical. TRIALS: gov with the number NC01423747.

Selection of adenovirus-specific and Epstein-Barr virus-specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)-compliant conditions.

BACKGROUND AIMS: Despite antiviral drug therapies, human adenovirus (HAdV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections still contribute substantially to transplant-related death of patients after hematopoietic stem cell transplantation. Earlier clinical studies demonstrated successful adoptive transfer of magnetically selected CMV-specific T cells via removable, and thus Good Manufacturing Practice-compliant, major histocompatibility class I streptamers. Thus, the primary focus of the present study was the selection of HAdV-streptamer+ T cells, although in three experiments, EBV-streptamer+ T cells were also selected. METHODS: Cells from leukaphereses of healthy donors were prepared in large (1-6 × 10(9)) and small (25 × 10(6)) cell batches. Whereas the larger batch was directly labeled with streptamers to select HAdV- and/or EBV-specific T cells (large-scale), the smaller batch was used to generate in vitro virus-specific T-cell lines before streptamer labeling for streptamer selection (small-scale). Isolation of HAdV- and/or EBV-specific T cells was performed with the use of the CliniMACS device. RESULTS: The purity of HAdV- and EBV-streptamer+ T cells among CD3+ cells, obtained from large-scale selection, was up to 6.7% and 44%, respectively. If HAdV- and EBV-streptamers were applied simultaneously, the purity of antigen-specific T cells reached up to 50.7%. A further increase in purity reaching up to 98% was achieved by small-scale selection of HAdV-specific T cells. All final products fulfilled the microbiological and chemical release criteria. Interferon-γ-response indicating functional activity was seen in 6 of 9 HAdV and 2 of 3 EBV large-scale selections and in 2 of 3 HAdV small-scale selections. CONCLUSIONS: HAdV-streptamers were shown to be clinically feasible for few patients after the large-scale approach but for larger patient numbers if combined with EBV-streptamers or after the small-scale approach.

Human adenovirus-specific γ/δ and CD8+ T cells generated by T-cell receptor transfection to treat adenovirus infection after allogeneic stem cell transplantation.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/ß T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/ß T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/ß T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/ß T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.