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Background: The medical coding of radiology reports is essential for a good quality of care and correct billing, but at the same time a complex and error-prone task., Objective: To assess the performance of natural language processing (NLP) for ICD-10 coding of German radiology reports using fine tuning of suitable language models., Material and Methods: This retrospective study included all magnetic resonance imaging (MRI) radiology reports acquired at our institution between 2010 and 2020. The codes on discharge ICD-10 were matched to the corresponding reports to construct a dataset for multiclass classification. Fine tuning of GermanBERT and flanT5 was carried out on the total dataset (ds total ) containing 1035 different ICD-10 codes and 2 reduced subsets containing the 100 (ds 100 ) and 50 (ds 50 ) most frequent codes. The performance of the model was assessed using top‑k accuracy for k = 1, 3 and 5. In an ablation study both models were trained on the accompanying metadata and the radiology report alone., Results: The total dataset consisted of 100,672 radiology reports, the reduced subsets ds 100 of 68,103 and ds 50 of 52,293 reports. The performance of the model increased when several of the best predictions of the model were taken into consideration, when the number of target classes was reduced and the metadata were combined with the report. The flanT5 outperformed GermanBERT across all datasets and metrics and was is suited as a medical coding assistant, achieving a top 3 accuracy of nearly 70% in the real-world dataset ds total ., Conclusion: Finely tuned language models can reliably predict ICD-10 codes of German magnetic resonance imaging (MRI) radiology reports across various settings. As a coding assistant flanT5 can guide medical coders to make informed decisions and potentially reduce the workload., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Introduction: Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes., Methods: Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®., Results: We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×10 9 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×10 5 /kg, 1×10 6 /kg, 1×10 7 /kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen., Discussion: In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets., Clinical Trial Registration: https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019., Competing Interests: UK: Consultant and/or speaker fees: AstraZeneca, Affimed, Glycostem, GammaDelta, Zelluna, Miltenyi Biotec and Novartis Pharma GmbH, Bristol-Myers Squibb GmbH & Co. KGaA; MAk, MAp, MAs, IB, AR, PA und CS are employees of Miltenyi Biotec and Miltenyi Biomedicine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Aleksandrova, Leise, Priesner, Aktas, Apel, Assenmacher, Bürger, Richter, Altefrohne, Schubert, Holzinger, Barden, Bezler, von Bergwelt-Baildon, Borchmann, Goudeva, Glienke, Arseniev, Esser, Abken and Koehl.)

Objectives: This randomized clinical trial focused on patients with thin peri-implant soft-tissue height (STH) (≤ 2.5 mm) and investigated the impact of an allogenic collagen scaffold (aCS) on supracrestal tissue height and marginal bone loss (MBL)., Material & Methods: Forty patients received bone level implants and were randomly assigned to the test group with simultaneous tissue thickening with aCS or the control group. After three months, prosthetic restoration occurred. STH measurements were taken at baseline (T0) and reopening surgery (TR), with MBL assessed at 12 months (T1). Descriptive statistics were calculated for continuous variables, and counts for categorical variables (significance level, p = 0.05)., Results: At T1, 37 patients were available. At T0, control and test groups had mean STH values of 2.3 ± 0.3 mm and 2.1 ± 0.4 mm. TR revealed mean STH values of 2.3 ± 0.2 mm (control) and 2.6 ± 0.7 mm (test), with a significant tissue thickening of 0.5 ± 0.6 mm in the test group (p < 0.03). At T1, control and test groups showed MBL mean values of 1.1 ± 0.8 mm and 1.0 ± 0.6 mm, with a moderate but significant correlation with STH thickening (-0.34), implant position (0.43), history of periodontitis (0.39), and smoking status (0.27)., Conclusion: The use of an aCS protocol resulted in soft tissue thickening but did not reach a threshold to reliably reduce MBL compared to the control group within the study's limitations., Clinical Relevance: Peri-implant STH is crucial for maintaining peri-implant marginal bone stability. Marginal bone stability represents a crucial factor in prevention of peri-implantitis development. German register of clinical trial registration number DRKS00033290., (© 2024. The Author(s).)

This study evaluated the brushing efficacy of different interdental brushes around a multibracket appliance in vitro. In four models displaying misaligned and aligned teeth with and without attachment loss, the brushing capacities of three interdental brushes (IDB) were tested: A waist-shaped IDB with a diameter of 9 mm at both ends and 5 mm in the middle (B1), a cylindrical brush with a diameter of 9 mm (B2) and one with 5 mm (B3). Before cleaning, the black teeth in the respective models were stained white with titanium (IV) oxide and the percentage of cleaned surface was planimetrically assessed. In addition, the forces applied to the IDB were also recorded. The effect of brush and model on expected cleaning performance was examined using an analysis of variance (ANOVA). The cleaning performance of the brushes in decreasing order was B2>B3>B1; no significant differences between the different tooth areas and models were found. With regard to force measurements, significant differences were found with the highest and lowest forces IDB (2) and (1), respectively. There was a significant correlation between force and cleaning performance: The higher the force needed the higher was the cleaning performance. In summary, this study showed that cylindrical interdental brushes achieved a better cleaning performance than the waist-shaped IDB. Given some shortcomings of this first laboratory study, more research is still needed, but IDB may represent a valuable yet still clinically underused tools.

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo . Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo . In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β., Competing Interests: Niels Werchau, Bettina Kotter, Elvira Criado-Moronati, Andre Gosselink, Nicole Cordes, Dominik Lock, Simon Lennartz, Carolin Kolbe, Nora Winter, Karin Teppert, Fabian Engert, Brian Webster, Joerg Mittelstaet, Daniel Schaefer, Mario Assenmacher, Thomas Schaser, Pierre Abramowski, Andrew D. Kaiser were employees of Miltenyi Biotec B.V. & Co. KG at the time the study was conducted. Niels Werchau, Bettina Kotter, Joerg Mittelstaet, and Andrew D. Kaiser are coinventors of a patent application focusing on sensing of soluble antigens with adapter CAR technology. Joerg Mittelstaet and Andrew D. Kaiser are coinventors of a patent application focusing on adapter CAR technology. Michael von Bergwelt-Baildon: Astellas Pharma, Bristol-Myers Squibb, Kite Gilead, Miltenyi Biotec, MOLOGEN, MSD, Novartis, Roche (Honoraria, Speakers’ Bureau, Research Funding, Travel, Accomodations, Expenses)., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.

Background: There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition., Methods: We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR., Results: We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1-3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns., Conclusions: We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting., Competing Interests: Competing interests: DL, CB, SL, KT, NW, MA, AK and TS are employees of Miltenyi Biotec. MH is listed as an inventor on patent applications and granted patents that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and the University of Würzburg that are related to CAR technologies and the use of MC DNA for genetransfer into lymphocytes and that have been licensed—in part—to industry. MH is a cofounder and equity owner of T-CURX. MSchm and MSchl are listed as inventors on granted patents of PlasmidFactory that cover the use of transposons in combination with Minicircle technology for cell transfection. No competing financial interests exist for the remaining authors., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future., Competing Interests: N.C., C.K., D.L., T.H., D.S., B.K., S.K., I.B., M.A., T.S., and A.D.K. are employees of Miltenyi Biotec B.V. & Co. KG. N.C., T.S., and A.D.K. have relevant IP to the findings disclosed. No competing financial interests exist for the remaining authors., (© 2021 The Author(s).)

Pedicle clamping (PC) during liver resection for colorectal metastases (CRLM) is used to reduce blood loss and allogeneic blood transfusion (ABT). The effect on long-term oncologic outcomes is still under debate. A retrospective analysis of the impact of PC on ABT-demand regarding overall (OS) and recurrence-free survival (RFS) in 336 patients undergoing curative resection for CRLM was carried out. Survival analysis was performed by both univariate and multivariate methods and propensity-score (PS) matching. PC was employed in 75 patients (22%). No increased postoperative morbidity was monitored. While the overall ABT-rate was comparable (35% vs. 37%, p = 0.786), a reduced demand for more than two ABT-units was observed ( p = 0.046). PC-patients had better median OS (78 vs. 47 months, p = 0.005) and RFS (36 vs. 23 months, p = 0.006). Multivariate analysis revealed PC as an independent prognostic factor for OS (HR = 0.60; p = 0.009) and RFS (HR = 0.67; p = 0.017). For PC-patients, 1:2 PS-matching ( N = 174) showed no differences in the overall ABT-rate compared to no-PC-patients (35% vs. 40%, p = 0.619), but a trend towards reduced transfusion requirement (>2 ABT-units: 9% vs. 21%, p = 0.052; >4 ABT-units: 2% vs. 11%, p = 0.037) and better survival (OS: 78 vs. 44 months, p = 0.088; RFS: 36 vs. 24 months; p = 0.029). Favorable long-term outcomes and lower rates of increased transfusion demand were observed in patients with PC undergoing resection for CRLM. Further prospective evaluation of potential oncologic benefits of PC in these patients may be meaningful.

Exposure-lag-response associations shed light on the duration of pathogenesis for radiation-induced diseases. To investigate such relations for lung cancer mortality in the German uranium miners of the Wismut company, we apply distributed lag non-linear models (DLNMs) which offer a flexible description of the lagged risk response to protracted radon exposure. Exposure-lag functions are implemented with B-Splines in Cox models of proportional hazards. The DLNM approach yielded good agreement of exposure-lag-response surfaces for the German cohort and for the previously studied cohort of American Colorado miners. For both cohorts, a minimum lag of about 2 year for the onset of risk after first exposure explained the data well, but possibly with large uncertainty. Risk estimates from DLNMs were directly compared with estimates from both standard radio-epidemiological models and biologically based mechanistic models. For age > 45 year, all models predict decreasing estimates of the Excess Relative Risk (ERR). However, at younger age, marked differences appear as DLNMs exhibit ERR peaks, which are not detected by the other models. After comparing exposure-responses for biological processes in mechanistic risk models with exposure-responses for hazard ratios in DLNMs, we propose a typical period of 15 year for radon-related lung carcinogenesis. The period covers the onset of radiation-induced inflammation of lung tissue until cancer death. The DLNM framework provides a view on age-risk patterns supplemental to the standard radio-epidemiological approach and to biologically based modeling.

Background: Earlier detection of anastomotic leakage (AL) after colorectal procedures could minimize the detrimental clinical impact of AL and thereby reduce morbidity and mortality., Study Design: We conducted a prospective study with assessment of the diagnostic accuracy of monocytic HLA-DR (mHLA-DR) expression compared with WBCs, C-reactive protein (CRP), and procalcitonin (PCT) in predicting AL in patients undergoing elective colorectal operation with anastomosis., Results: Comparison of the blood marker values on postoperative day (POD) 4 revealed significant differences for all markers, but the difference for mHLA-DR was highly significant (15% expression of monocytes in AL patients vs 34% in patients without AL; p = 0.001). Together with WBC (p = 0.026), mHLA-DR expression was the only test to show significance on day 3 (14% vs 31%; p < 0.001). Receiver operating characteristic analysis revealed that mHLA-DR expression had superior diagnostic accuracy compared with all other diagnostic markers both on POD 3 (mHLA-DR area under the curve [AUC] 0.928; WBC AUC 0.734; CRP AUC 0.707; PCT AUC 0.672) and POD 4 (mHLA-DR AUC 0.887; WBC AUC 0.738; CRP AUC 0.709; PCT AUC 0.696). Monocytic HLA-DR had a negative predictive value of at least 94% on PODs 3 and 4, as well as specificity and positive predictive values of 100% at a threshold of 23% on POD 3 and 24% on POD 4, respectively., Conclusions: Expression of mHLA-DR appears to be a more accurate predictor for AL after colorectal operation compared with WBC, CRP, and PCT. It represents a promising test to precisely monitor the perioperative course of high-risk patients and contribute to safer discharge., (Copyright © 2019 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4 + /CD8 + -separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4 +  = 50%, CD8 +  = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Immunogenicity of biotherapeutics and the elicitation of anti-drug antibodies are a key concern for their efficacy, pharmacokinetics, and safety. A particularly severe consequence of immunogenicity of a biotherapeutic is the rare development of antibody-mediated pure red cell aplasia (PRCA) in anemic patients treated with aggregated forms of recombinant human erythropoietin (rhEPO). Here, we investigated in vitro T-cell responses to experimentally heat-induced rhEPO aggregates, and to tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA. Heat-stressed rhEPO elicited T-cell responses only in blood obtained from healthy individuals identified as responders, whereas nonstressed rhEPO overall did not induce reactions neither in responders nor nonresponders. Tungsten-induced rhEPO aggregates in clinical lots associated with rhEPO-neutralizing antibodies and PRCA could induce in vitro T-cell responses in blood obtained from healthy donors, in contrast to rhEPO from low tungsten syringes. Importantly, ex vivo T-cell recall responses of patients treated with rhEPO without PRCA showed no T-cell responses, whereas T cells of a patient who developed PRCA after treatment with a clinical batch with elevated levels of tungsten and rhEPO aggregates showed a clear response to rhEPO from that clinical batch. To our knowledge, this is the first time that T-cell assays confirm the root cause of increased rhEPO immunogenicity associated with PRCA., Competing Interests: Conflict-of-interest disclosure: M. Kuwana has received a research grant from Novartis; T.R.-S., B.C., S.-D.C., and M. Kammüller are full-time employees of Novartis; T.M.W. has been a former full-time employee of Novartis; and M.A., O.H., F.Z., R.F., V.K., and A.S. are full-time employees of Hexal AG.

Age-related macular degeneration (AMD) is a vision impairing disease of the central retina characterized by early and late forms in individuals older than 50 years of age. However, there is little knowledge to what extent also younger adults are affected. We have thus set out to estimate the prevalence of early AMD features and late AMD in a general adult population by acquiring color fundus images in 2,840 individuals aged 25 to 74 years of the Cooperative Health Research in the Region of Augsburg project (KORA) in South Germany. Among the 2,546 participants with gradable images for each eye, 10.9% (n = 277) had early AMD features (applying the 9-step Age-Related Eye Disease Study Severity Scale), 0.2% (n = 6) had late AMD. Prevalence increased with age, reaching 26.3% for early AMD features and 1.9% for late AMD at the age 70+. However, signs of early AMD were found in subjects as young as 25 years, with the risk for early AMD features increasing linearly by years of age in men, and, less consistent with a linear increase, in women. Risk for early AMD features increased linearly by pack years of smoking in men, not in women, nor was there any association with other lifestyle or metabolic factors. By providing much sought-after prevalence estimates for AMD from Central Europe, our data underscores a substantial proportion of the adult population with signs of early AMD, including individuals younger than 50 years. This supports the notion that early AMD features in the young might be under-acknowledged., Competing Interests: Competing Interests: Author H.H. has received lecture honorarium, travel support and is a member of the advisory board of the companies Bayer AG (Leverkusen, Germany), Allergan (Dublin, Ireland), and Novartis Pharma GmbH (Nürnberg, Germany). All other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background Aims: Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective., Methods: We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137., Results: In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 × 10(9) peripheral blood mononuclear cells, we isolated 27 × 10(3)-318 × 10(3)Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens., Discussion: Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 × 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The Wilms' tumour-1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1-specific T-cell repertoire because self-derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1-specific T cells from the peripheral blood. Remarkably, we detected WT1-specific CD4(+) and CD8(+) T-cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4(+) T-cell compartment, whereas WT1-specific CD8(+) T cells almost universally displayed a naive phenotype. Moreover, memory CD4(+) and naive CD8(+) T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4(+) and CD8(+) T-cell lineages with respect to memory formation in response to a self-derived antigen. Nonetheless, WT1-specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self-reactive T cells for immunotherapeutic purposes., (© 2015 John Wiley & Sons Ltd.)

The recent successes of adoptive T-cell immunotherapy for the treatment of hematologic malignancies have highlighted the need for manufacturing processes that are robust and scalable for product commercialization. Here we review some of the more outstanding issues surrounding commercial scale manufacturing of personalized-adoptive T-cell medicinal products. These include closed system operations, improving process robustness and simplifying work flows, reducing labor intensity by implementing process automation, scalability and cost, as well as appropriate testing and tracking of products, all while maintaining strict adherence to Current Good Manufacturing Practices and regulatory guidelines. A decentralized manufacturing model is proposed, where in the future patients' cells could be processed at the point-of-care in the hospital.

CD4(+) T cells orchestrate immune responses against fungi, such as Aspergillus fumigatus, a major fungal pathogen in humans. The complexity of the fungal genome and lifestyle questions the existence of one or a few immune-dominant Ags and complicates systematic screening for immunogenic Ags useful for immunotherapy or diagnostics. In this study, we used a recently developed flow cytometric assay for the direct ex vivo characterization of A. fumigatus-specific CD4(+) T cells for rapid identification of physiological T cell targets in healthy donors. We show that the T cell response is primarily directed against metabolically active A. fumigatus morphotypes and is stronger against membrane protein fractions compared with cell wall or cytosolic proteins. Further analysis of 15 selected single A. fumigatus proteins revealed a highly diverse reactivity pattern that was donor and protein dependent. Importantly, the parallel assessment of T cell frequency, phenotype, and function allowed us to differentiate between proteins that elicit strong memory T cell responses in vivo versus Ags that induce T cell exhaustion or no reactivity in vivo. The regulatory T cell (Treg) response mirrors the conventional T cell response in terms of numbers and target specificity. Thus, our data reveal that the fungal T cell immunome is complex, but the ex vivo characterization of reactive T cells allows us to classify Ags and to predict potential immunogenic targets. A. fumigatus-specific conventional T cell responses are counterbalanced by a strong Treg response, suggesting that Treg-depletion strategies may be helpful in improving antifungal immunity., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.

The identification and functional characterization of pathogen-specific T cells plays a critical role in immunological research and diagnostics. In addition to the present standard technologies such as intracellular cytokine staining (ICS), enzyme-linked immunospot (ELISPOT) and peptide-major-histocompatibility-complex (MHC) multimer staining, we aimed to develop a multiplex detection assay, which provides fast in vitro functional data for both human CD4 and CD8 T cells with different antigen specificities in one sample. In this study, we have exploited the expression of CD83 on B cells to develop the cell array-based indirect T cell recognition assay (ITRA). In detail, B cells are pulsed with different pathogen peptide pools and fluorescently barcoded. Thereafter the B cells are pooled and co-cultured with autologous T cells. Subsequently each B cell population is analyzed via flow cytometry for CD83 expression, which indicates antigen-specific interaction with CD4 T cells. Moreover, we revealed donor dependent variations of cytotoxic activity of pathogen-specific CD4 T cells and CD8 T cells, evidenced by specific lysis of peptide-pulsed B cells. Taken together, ITRA is a novel antigen presenting cell (APC) array based method to analyze the presence and function of various antigen-specific T cells in one sample. It has the potential to be used in the future for epitope/antigen screening in research and for analysis of anti-tumor, anti-pathogen or autoimmune T cell responses in patient samples., (Copyright © 2013. Published by Elsevier B.V.)

NK cells do not express recombination-dependent antigen-specific receptors and are traditionally defined as cells of the innate immune response. The activation of NK cells was believed to be controlled by the net balance of signals from a multitude of activating and inhibitory receptors irrespectively of antigen specificity. However, murine antigen-specific memory NK cells in liver have been described to mediate hapten or viral specific recall response and are capable of infiltrating to the site of infection. The mechanisms by which NK cells recognize target cells in an antigen-specific manner are largely unclear. Using a novel multiplex killing assay, we screened the NK cell (human) cytotoxic activity of 35 different donors against different virus peptide pools loaded autologous B cells. We have found that human NK cells from some CMV and EBV positive donors can recognize peptide loaded autologous B cells as targets and perform antigen-specific cytotoxic killing. This may provide evidence that NK cells are able to scan the peptide repertoire on the target cell surface and virus-derived peptides may influence the NK cell activation-inhibition balance.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo., From the Clinical Editor: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth., (© 2013.)

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8(+) T(N) cells (CD4(-)CD62L(+)CD45RA(+)) and CD8(+) T(CM) (CD4(-)CD62L(+)CD45RA(-)) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T(N) and T(CM) we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

Ag-specific CD4(+) T cells orchestrating adaptive immune responses are crucial for the development of protective immunity, but also mediate immunopathologies. To date, technical limitations often prevented their direct analysis. In this study, we report a sensitive flow cytometric assay based on magnetic pre-enrichment of CD154(+) T cells to visualize rare Ag-reactive naive and memory Th cells directly from human peripheral blood. The detection limit of ≈ 1 cell within 10(5)-10(6) permitted the direct enumeration and characterization of auto-, tumor-, or neo-Ag-reactive T cells within the naive and even memory CD4(+) T cell repertoire of healthy donors. Furthermore, the analysis of high target cell numbers after pre-enrichment of rare Ag-specific T cells from large blood samples dramatically improved the identification of small subpopulations. As exemplified in this work, the dissection of the Ag-specific memory responses into small cytokine-producing subsets revealed great heterogeneity between pathogens, but also pathogen-related microsignatures refining Th cell subset classification. The possibility to directly analyze CD4(+) T cells reactive against basically any Ag of interest at high resolution within the naive and memory repertoire will open up new avenues to investigate CD4(+) T cell-mediated immune reactions and their use for clinical diagnostics.

T cell lines with defined cytokine profiles are an invaluable tool for assessing the control of immune responses both in vitro and in vivo. Production of such cell lines can be complex and time-consuming. Here we present a powerful technique to assay the cytokines produced by T cells activated polyclonally or with specific antigens. This paper presents a detailed methodology for the identification and isolation of cytokine-producing T cells activated with the artificial superantigen, CytoStim, or viral and fungal antigens. These cells can be analysed for different cytokines simultaneously, or cultured further to rapidly establish T cell lines making known cytokine types. We highlight the enumeration, isolation and phenotype of interleukin-17-producing T cells, and the rapid generation of virus-specific Th1 T cell lines., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37 degrees C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37 degrees C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA-peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 x 10(3)/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4(+) and CD8(+) CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-gamma (IFN-gamma) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 x 10(8) combined CD4(+) and CD8(+) CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-gamma production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4(+) and CD8(+) T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4(+) and CD8(+) T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4(+) and CD8(+) CMV-specific T cells under conditions mimicking good manufacturing practice.

T helper (Th) lymphocytes can develop memory for the expression of particular cytokines, like IL-4 or IL-10, in that reexpression of those cytokines is independent of the original costimulatory signal IL-4 and depends only on T cell receptor stimulation. Here, we show that in the course of Th2 cell differentiation in vitro, IL-4 memory is established during primary activation of naïve Th cells, whereas the establishment of IL-10 memory requires repetitive stimulation of the Th cell with IL-4 and T cell receptor. Likewise, established IL-10 memory, maintained in the absence of further IL-4 signals, was observed in individual IL-10-producing cells generated from in vivo antigen-experienced CD62L(low) Th cells and isolated by using the newly developed cytometric cytokine secretion assay for IL-10. In naïve Th cells undergoing primary activation, the induction of both IL-4 and IL-10 memory requires DNA synthesis, but reexpression of the cytokine genes can occur throughout cell cycle. In in vitro polarized Th2 cell populations, Th cells with IL-4 or IL-10 memory do not differ in proliferative behavior. Populations of Th cells isolated from polarized Th2 cultures according to expression of IL-4 or IL-10 also do not differ in proliferative behavior. Their proliferation mainly depends on IL-2. Thus, effector memory Th lymphocytes with memory for IL-4 or IL-10 expression are not intrinsically impaired in their proliferative potential and can play an essential role in reactive immunological memory and its regulation.