Umbilical cord blood T cells can be isolated and enriched by CD62L selection for use in 'off the shelf' chimeric antigen receptor T-cell therapies to widen transplant options.

Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCReeeT cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCReeeT cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed antileukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.

Prospective Analysis of Radiation-Induced Contrast Enhancement and Health-Related Quality of Life After Proton Therapy for Central Nervous System and Skull Base Tumors.

PURPOSE: Intracerebral radiation-induced contrast enhancement (RICE) can occur after photon as well as proton beam therapy (PBT). This study evaluated the incidence, characteristics, and risk factors of RICE after PBT delivered to, or in direct proximity to, the brain and its effect on health-related quality of life (HRQoL). METHODS AND MATERIALS: Four hundred twenty-one patients treated with pencil beam scanning PBT between 2017 and 2021 were included. Follow-up included clinical evaluation and contrast-enhanced magnetic resonance imaging at 3, 6, and 12 months after treatment completion and annually thereafter. RICE was graded according to Common Terminology Criteria for Adverse Events version 4, and HRQoL parameters were assessed via European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ)-C30 questionnaires. RESULTS: The median follow-up was 24 months (range, 6-54), and median dose to 1% relative volume of noninvolved central nervous system (D1%CNS) was 54.3 Gy relative biologic effectiveness (RBE; range, 30-76 Gy RBE). The cumulative RICE incidence was 15% (n = 63), of which 10.5% (n = 44) were grade 1, 3.1% (n = 13) were grade 2, and 1.4% (n = 6) were grade 3. No grade 4 or 5 events were observed. Twenty-six of 63 RICE (41.3%) had resolved at the latest follow-up. The median onset after PBT and duration of RICE in patients in whom the lesions resolved were 11.8 and 9.0 months, respectively. On multivariable analysis, D1%CNS > 57.6 Gy RBE, previous in-field radiation, and diabetes mellitus were identified as significant risk factors for RICE development. Previous radiation was the only factor influencing the risk of symptomatic RICE. After PBT, general HRQoL parameters were not compromised. In a matched cohort analysis of 54/50 patients with and without RICE, no differences in global health score or functional and symptom scales were seen. CONCLUSIONS: The overall incidence of clinically relevant RICE after PBT is very low and has no significant negative effect on long-term patient QoL.

Proton or Carbon Ion Therapy for Skull Base Chordoma: Rationale and First Analysis of a Mono-Institutional Experience.

BACKGROUND: Skull base chordomas are radio-resistant tumors that require high-dose, high-precision radiotherapy, as can be delivered by particle therapy (protons and carbon ions). We performed a first clinical outcome analysis of particle therapy based on the initial 4-years of operation. METHODS: Between August 2017 and October 2021, 44 patients were treated with proton (89%) or carbon ion therapy (11%). Prior gross total resection had been performed in 21% of lesions, subtotal resection in 57%, biopsy in 12% and decompression in 10%. The average prescription dose was 75.2 Gy RBE in 37 fractions for protons and 66 Gy RBE in 22 fractions for carbon ions. RESULTS: At a median follow-up of 34.3 months (range: 1-55), 2-, and 3-year actuarial local control rates were 95.5% and 90.9%, respectively. The 2-, and 3-year overall and progression-free survival rates were 97.7%, 93.2%, 95.5% and 90.9%, respectively. The tumor volume at the time of particle therapy was highly predictive of local failure (p < 0.01), and currently, there is 100% local control in patients with tumors < 49 cc. No grade ≥3 toxicities were observed. There was no significant difference in outcome or side effect profile seen for proton versus carbon ion therapy. Five patients (11.4%) experienced transient grade ≤2 radiation-induced brain changes. CONCLUSIONS: The first analysis suggests the safety and efficacy of proton and carbon ion therapy at our center. The excellent control of small to mid-size chordomas underlines the effectiveness of particle therapy and importance of upfront maximum debulking of large lesions.

Normofractionated and moderately hypofractionated proton therapy: comparison of acute toxicity and early quality of life outcomes.

Aim: Data on the safety of moderately hypofractionated proton beam therapy (PBT) are limited. The aim of this study is to compare the acute toxicity and early quality of life (QoL) outcomes of normofractionated (nPBT) and hypofractionated PBT (hPBT). Material and methods: We prospectively compared acute toxicity and QoL between patients treated with nPBT (dose per fraction 1.8-2.3 Gy, n = 90) and hPBT (dose per fraction 2.5-3.1 Gy, n = 49) in following locations: head and neck (H&N, n = 85), abdomen and pelvis (A&P, n = 43), and other soft tissue (ST, n = 11). The toxicities were grouped into categories-mucosal, skin, and other sites-and evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 at baseline, treatment completion, and 3 months after PBT completion. QoL was evaluated with the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ)-C30 scale for all locations and additionally with EORTC QLQ-HN35 for H&N patients. Results: Overall, the highest toxicity grades of G0, G1, G2, and G3 were observed in 7 (5%), 40 (28.8%), 78 (56.1%), and 15 (10.8%) patients, respectively. According to organ and site, no statistically significant differences were detected in the majority of toxicity comparisons (66.7%). For A&P, hPBT showed a more favorable toxicity profile as compared to nPBT with a higher frequency of G0 and G1 and a lower frequency of G2 and G3 events (p = 0.04), more patients with improvement (95.7% vs 70%, p = 0.023), and full resolution of toxicities (87% vs 50%, p = 0.008). Skin toxicity was unanimously milder for hPBT compared to nPBT in A&P and ST locations (p = 0.018 and p = 0.025, respectively). No significant differences in QoL were observed in 97% of comparisons for QLQ-C30 scale except for loss of appetite in H&N patients (+33.3 for nPBT and 0 for hPBT, p = 0.02) and role functioning for A&P patients (0 for nPBT vs +16.7 hPBT, p = 0.003). For QLQ-HN35, 97.9% of comparisons did not reveal significant differences, with pain as the only scale varying between the groups (-8.33 vs -25, p = 0.016). Conclusion: Hypofractionated proton therapy offers non-inferior early safety and QoL as compared to normofractionated irradiation and warrants further clinical investigation.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation.

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

TCRαß/CD3 disruption enables CD3-specific antileukemic T cell immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαß/CD3 complex. Only transduction after disrupting assembly of TCRαß/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR-CD3- T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.

Union of light ion therapy centers in Europe (ULICE EC FP7) - Objectives and achievements of joint research activities.

Under the umbrella of the European Network for Light Ion Therapy (ENLIGHT), the project on Union of Light Ion Centers in Europe (ULICE), which was funded by the European Commission (EC/FP7), was carried out from 2009 to 2014. Besides the two pillars on Transnational Access (TNA) and Networking Activities (NA), six work packages formed the pillar on Joint Research Activities (JRA). The current manuscript focuses on the objectives and results achieved within these research work packages: "Clinical Research Infrastructure", "Biologically Based Expert System for Individualized Patient Allocation", "Ion Therapy for Intra-Fractional Moving Targets", "Adaptive Treatment Planning for Ion Radiotherapy", "Carbon Ion Gantry", "Common Database and Grid Infrastructures for Improving Access to Research Infrastructures". The objectives and main achievements are summarized. References to either publications or open access deliverables from the five year project work are given. Overall, carbon ion radiotherapy is still not as mature as photon or proton radiotherapy. Achieved results and open questions are reflected and discussed in the context of the current status of carbon ion therapy and particle and photon beam therapy. Most research topics covered in the ULICE JRA pillar are topical. Future research activities can build upon these ULICE results. Together with the continuous increase in the number of particle therapy centers in the last years ULICE results and proposals may contribute to the further growth of the overall particle therapy field as foreseen with ENLIGHT and new joint initiatives such as the European Particle Therapy Network (EPTN) within the overall radiotherapy community.

Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option.

Immune checkpoints are promising targets in cancer therapy. Recently, poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2) have been identified as novel immune checkpoints. In this investigation we show that acute myeloid leukemia (AML) cell lines and AML patient samples highly express the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ligands PVR and PVRL2. Using two independent patient cohorts, we could demonstrate that high PVR and PVRL2 expression correlates with poor outcome in AML. We show for the first time that antibody blockade of PVR or PVRL2 on AML cell lines or primary AML cells or TIGIT blockade on immune cells increases the anti-leukemic effects mediated by PBMCs or purified CD3+ cells in vitro. The cytolytic activity of the BiTE® antibody construct AMG 330 against leukemic cells could be further enhanced by blockade of the TIGIT-PVR/PVRL2 axis. This increased immune reactivity is paralleled by augmented secretion of Granzyme B by immune cells. Employing CRISPR/Cas9-mediated knockout of PVR and PVRL2 in MV4-11 cells, the cytotoxic effects of antibody blockade could be recapitulated in vitro. In NSG mice reconstituted with human T cells and transplanted with either MV4-11 PVR/PVRL2 knockout or wildtype cells, prolonged survival was observed for the knockout cells. This survival benefit could be further extended by treating the mice with AMG 330. Therefore, targeting the TIGIT-PVR/PVRL2 axis with blocking antibodies might represent a promising future therapeutic option in AML.

Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers.

CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.

Gene Modified T Cell Therapies for Hematological Malignancies.

This article focuses on clinical applications of T cells transduced to express recombinant T cell receptor and chimeric antigen receptor constructs directed toward hematological malignancies, and considers newer strategies incorporating gene-editing technologies to address GvHD and host-mediated rejection. Recent data from clinical trials are reviewed, and an overview is provided of current and emerging manufacturing processes; consideration is also given to new developments in the pipeline.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases.

TAL-effector nucleases (TALENs) are attractive tools for sequence-specific genome modifications, but their delivery still remains problematic. It is well known that the presence of multiple sequence repeats in TALEN genes hampers the use of lentiviral vectors. We report that lentiviral vectors readily package full-length vector mRNAs encoding TALENs, but recombination during reverse transcription prevents successful delivery. We reasoned that preventing reverse transcription of lentiviral-vector RNA would allow transfer of TALENs as mRNA. We demonstrate that lentiviral particles containing genetically inactivated reverse transcriptase (RT) mediated efficient transduction of cultured cells and supported transient transgene expression. For proof-of-principle, we transferred CCR5- and TCR-specific TALEN pairs for efficient targeted genome editing and abrogated expression for each of the receptor proteins in different cell lines. Combining the high specificity of TALENs with efficient lentiviral gene delivery should advance genome editing in vitro and potentially in vivo, and RT-deficient lentiviral vectors may be useful for transient expression of various other genes-of-interest.

Assessment of improved organ at risk sparing for meningioma: light ion beam therapy as boost versus sole treatment option.

PURPOSE: To compare photons, protons and carbon ions and their combinations for treatment of atypical and anaplastical skull base meningioma. MATERIAL AND METHODS: Two planning target volumes (PTVinitial/PTVboost) were delineated for 10 patients (prescribed doses 50 Gy(RBE) and 10 Gy(RBE)). Plans for intensity modulated photon (IMXT), proton (IMPT) and carbon ion therapy ((12)C) were generated assuming a non-gantry scenario for particles. The following combinations were compared: IMXT+IMXT/IMPT/(12)C; IMPT+IMPT/(12)C; and (12)C+(12)C. Plan quality was evaluated by target conformity and homogeneity (CI, HI), V95%, D2% and D50% and dose-volume-histogram (DVH) parameters for organs-at-risk (OAR). If dose escalation was possible, it was performed until OAR tolerance levels were reached. RESULTS: CI was worst for IMXT. HI<0.05±0.01 for (12)C was significantly better than for IMXT. For all treatment options dose escalation above 60 Gy(RBE) was possible for four patients, but impossible for six patients. Compared to IMXT+IMXT, ion beam therapy showed an improved sparing for most OARs, e.g. using protons and carbon ions D50% was reduced by more than 50% for the ipsilateral eye and the brainstem. CONCLUSION: Highly conformal IMPT and (12)C plans could be generated with a non-gantry scenario. Improved OAR sparing favors both sole (12)C and/or IMPT plans.

Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector.

Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.

Efficient lentiviral transduction and transgene expression in primary human B cells.

Primary human B cells are an attractive target for gene-therapeutic applications, but have been found to be relatively resistant toward transduction with lentiviral vectors (LVVs), even though a number of different envelope pseudotypes were tested. Moreover, low transgene expression in primary human B cells has impeded the use of LVVs for this target cell. We investigated the transduction potential of gibbon-ape leukemia virus (GALV) Env-pseudotyped LVVs for primary human B cells. By establishing optimized transduction kinetics and multiplicities of infection, we were able to regularly obtain transduction efficiencies of more than 50% in CD40L-activated B cells. Noteworthy, with the use of GALV-pseudotyped LVVs we could achieve a more than 10-fold higher yield of transduced activated B cells in direct comparison with LVVs pseudotyped with measles virus glycoproteins. Phenotyping of transduced primary B cells revealed a majority of memory B cells, a long-lived phenotype, presumed to be well suited for enduring therapeutic interventions. Finally, by combining the enhancer (Eµ) and the matrix/scaffold-attachment regions (MARs) of the human immunoglobulin heavy chain with the promoter of spleen focus-forming virus (SFFV) we aimed at generating a novel LVV particularly suitable for B cell transgenesis. We show that the optimized vector facilitated significantly higher transgene expression in various B cell lines and, more importantly, primary human B cells (mean factor of three). In summary, we have established a novel protocol for the efficient lentiviral transduction of primary human B cells and have improved transgene expression in B cells by a specific vector modification.

SOCS3 promotes interleukin-17 expression of human T cells.

SOCS3 is a feedback regulator of cytokine signaling that affects T-cell polarization. Human tuberculosis is accompanied by increased SOCS3 expression in T cells, and this may influence susceptibility against Mycobacterium tuberculosis. Because the role of SOCS3 in human T-cell function is not well defined, we characterized cytokine expression and proliferation of human T cells with differential SOCS3 expression in the present study. We established a flow cytometry-based method for SOCS3 protein quantification and detected higher SOCS3 levels induced by M tuberculosis specific T-cell activation and a transient decrease of SOCS3 expression in the presence of mycobacteria-infected macrophages. Notably increased SOCS3 expression was detected in IL-17-expressing T-cell clones and in CD161(+) T helper type 17 cells ex vivo. Ectopic SOCS3 expression in primary CD4(+) T cells by lentiviral transduction induced increased IL-17 production but diminished proliferation and viability. Recombinant IL-7 inhibited SOCS3 expression and reduced IL-17-expressing T-cell proportions. We concluded that higher SOCS3 expression in human T cells favors T helper type 17 cells. Therefore, increased SOCS3 expression in human tuberculosis may reflect polarization toward IL-17-expressing T cells as well as T-cell exhaustion marked by reduced proliferation.

Investigations on parotid gland recovery after IMRT in head and neck tumor patients.

PURPOSE: in recent years, the role of intensity-modulated radiotherapy (IMRT) for head and neck irradiation has increased. The main motivation is sparing the parotid gland and reduction of xerostomia. Generally, relative parotid volumes have been evaluated for treatment outcome and planning constraints, neglecting that absolute parotid volumes can vary significantly. The aim of the present study was to investigate changes in parotid gland function and set this in relation to absolute volumes. MATERIAL AND METHODS: 46 head and neck patients were treated by sparing at least the contralateral parotid gland. The mean dose to the contralateral gland was limited to 26 Gy. Parotid function was measured with scintigraphy before and at 3, 6, 9, and 12 months after radiotherapy. Gland recovery was correlated with absolute parotid gland volumes and mean dose. Finally the dose-effect relationship was investigated. RESULTS: the dose-volume histograms (DVHs) for the ipsi- and contralateral glands were significantly different. A correlation between absolute volumes receiving certain doses and the function loss after 3, 6, 9, and 12 months was found. The most significant correlation was found for the absolute volume that received at least 40 Gy (aV40). ED50 values of 23-38 Gy were observed for more than 50% function loss and and 52-68 Gy afor more than 75% function loss. CONCLUSION: the mean dose, aV40 or aV26, revealed similar correlations with the excretion rate and with recovery. Hence, also absolute volumes can be used for treatment planning. Longer recovery times show higher ED50 values indicating partial regeneration of gland functions.