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ObjectiveA pilot study suggested that combination therapy with low-dose anti-thymocyte globulin (ATG) and pegylated granulocyte colony-stimulating factor (GCSF) preserves C-peptide in established type 1 diabetes (T1D) (duration 4 months to 2 years). We hypothesized that 1) low-dose ATG/GCSF or 2) low-dose ATG alone would slow the decline of β-cell function in patients with new-onset T1D (duration

Abstract Background Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection. Therefore we set out to investigate several processes that might impact cytokine profiles such as blood collecting tubes, duration of storage, and number of freeze thawing cycles. Results Since under physiological conditions cytokine concentrations normally are low or undetectable we spiked cytokines in the various plasma and serum samples. Overall recoveries ranged between 80-120%. Long time storage showed cytokines are stable for a period up to 2 years of storage at -80°C. After 4 years several cytokines (IL-1α, IL-1β, IL-10, IL-15 and CXCL8) degraded up to 75% or less of baseline values. Furthermore we show that only 2 out of 15 cytokines remained stable after several freeze-thawing cycles. We also demonstrate implementation of an internal control for multiplex cytokine immunoassays. Conclusion All together we show parameters which are essential for measurement of cytokines in the context of clinical trials.

Abstract Background RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene™ and Tempus™ blood collection tubes, and each comes with their own RNA purification method. In both systems the blood is immediately lysed when collected into the tube and RNA stabilized using proprietary reagents. Both systems enable minimal blood handling procedures thus minimizing the risk of inducing changes in gene expression through blood handling or processing. Because the RNA purification steps could influence the total RNA pool, we examined the impact of RNA isolation, using the PAXgene™ or Tempus™ method, on gene expression profiles. Results Using microarrays as readout of RNA from stimulated whole blood we found a common set of expressed transcripts in RNA samples from either PAXgene™ or Tempus™. However, we also found several to be uniquely expressed depending on the type of collection tube, suggesting that RNA purification methods impact results of differential gene expression profiling. Specifically, transcripts for several known PHA-inducible genes, including IFNγ, IL13, IL2, IL3, and IL4 were found to be upregulated in stimulated vs. control samples when RNA was isolated using the ABI Tempus™ method, but not using the PAXgene™ method (p < 0.01, FDR corrected). Sequenom Quantiative Gene Expression (QGE) (SanDiego, CA) measures confirmed IL2, IL4 and IFNγ up-regulation in Tempus™ purified RNA from PHA stimulated cells while only IL2 was up-regulated using PAXgene™ purified (p < 0.05). Conclusion Here, we demonstrate that peripheral blood RNA isolation methods can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological cohorts. A modified method based upon the Tempus™ system was found to provide high yield, good post-hybridization array quality, low variability in expression measures and was shown to produce differential expression results consistent with the predicted immunologic effects of PHA stimulation.

Background: Regorafenib has shown substantial clinical activity in patients with advanced biliary tract cancers (BTCs). Preclinical data suggested that this drug modulates antitumour immunity and is synergistic with immune checkpoint inhibition., Patients and Methods: This is a single-arm, multicentric phase II trial. Regorafenib was given 3 weeks/4, 160 mg quaque die (once a day) (QD); avelumab 10 mg/kg IV was given every two weeks, beginning at C1D15 until progression or unacceptable toxicity. The primary end-point was the confirmed objective response rate under treatment, as per Response Evaluation Criteria in Solid Tumours 1.1. The secondary end-points included the following: 1-year non-progression rate; progression-free survival (PFS) and overall survival; safety and biomarkers studies performed on sequential tumour samples obtained at baseline and at cycle 2 day 1., Results: Thirty-four patients were enrolled in four centres. Twenty-nine patients were assessable for efficacy after central radiological review. The best response was partial response for four patients (13.8%), stable disease for 11 patients (37.9%) and progressive disease for 14 patients (48.3%). The median PFS and overall survival were 2.5 months (95% confidence interval [CI] [1.9-5.5]) and 11.9 months (95%CI [6.2-NA]) respectively. The most common grade 3 or 4 clinical adverse events related to treatment were hypertension (17.6%), fatigue (14.7%) and maculopapular rash (11.8%). High baseline levels of programmed cell death ligand 1 and of indoleamine 2, 3-dioxygénase expression were associated with improved outcomes., Conclusions: Regorafenib combined with avelumab has antitumour activity in a subset of heavily pretreated biliary tract cancer population. Further investigations are needed in patients selected based on tumour microenvironment features., Clinical Trial Registration: NCT03475953., Competing Interests: Conflict of interest statement AB, CR and JPG: employees from Immusmol/Explicyte. AI: research grants MSD, BMS, ROCHE and personal fees: Epizyme, Bayer, Lilly, Roche, Springworks; non-financial support: Merck. All remaining authors have declared no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Only a minority of patients derive long-term clinical benefit from anti-PD1/PD-L1 monoclonal antibodies. The presence of tertiary lymphoid structures (TLS) has been associated with improved survival in several tumor types. Here, using a large-scale retrospective analysis of three independent cohorts of cancer patients treated with anti-PD1/PD-L1 antibodies, we showed that the presence of mature TLS was associated with improved objective response rate, progression-free survival, and overall survival independently of PD-L1 expression status and CD8+ T-cell density. These results pave the way for using TLS detection to select patients who are more likely to benefit from immune checkpoint blockade., Competing Interests: DECLARATION OF INTERESTS LV, MB, SC, SLM, ML, IS, MT, GR, SP, MC, FC, CL, KB, MK, IG, CSF, VV, FC, WHF, and FLL: Nothing to disclose AB, JPG, and CR: Employees of Immusmol/Explicyte EO, AS: Employees of Astra Zeneca AI: Received research grants from Astra Zeneca, Bayer, BMS, Chugai, Merck, MSD, Pharmamar, Novartis, Roche, and received personal fees from Epizyme, Bayer, Lilly, Roche, and Springworks BB: Received grants from AstraZeneca , Pfizer , Eli Lilly , Onxeo , Bristol Myers Squibb , Inivata , Abbvie , Amgen , Blueprint Medicines , Celgene , GlaxoSmithKline , Ignyta , Ipsen , Merck KGaA , MSD Oncology , Nektar , PharmaMar , Sanofi , Spectrum Pharmaceuticals , Takeda , Tiziana Therapeutics , Cristal Therapeutics , Daiichi Sankyo , Janssen Oncology , OSE Immunotherapeutics , BeiGene , Boehringer Ingelheim , Genentech , SERVIER , Tolero Pharmaceuticals YL: Received grants and personal fees from Janssen, during the conduct of the study; personal fees and non-financial support from Astellas, grants and personal fees from Sanofi, personal fees and non-financial support from Roche, personal fees and non-financial support from AstraZeneca, grants, personal fees and non-financial support from MSD, personal fees and non-financial support from BMS, personal fees from Clovis , personal fees and non-financial support from Seattle Genetics, personal fees from Incyte, personal fees from Pfizer. AM: Received research grants from Mersu, Bristol-Myers Squibb, Boehringer Ingelheim, Transgene, MSD and received personal fees from Bristol-Myers Squibb, AstraZeneca, MedImmune, Oncovir, Merieux JCS: Has received consultancy fees from AstraZeneca, Astex, Clovis, GSK, GamaMabs, Lilly, MSD, Mission Therapeutics, Merus, Pfizer, Pharma Mar, Pierre Fabre, Roche/Genentech, Sanofi, Servier, Symphogen, and Takeda.

Cell-based immunotherapies have had remarkable success in the clinic, specifically in the treatment of hematologic malignancies. However, these strategies have had limited efficacy in patients with solid tumors. To better understand the challenges involved, the National Cancer Institute (NCI) convened an initial workshop with immuno-oncology thought leaders in December 2018 and a follow-up workshop in December 2020. The goals of the NCI workshops on cell-based immunotherapy for solid tumors were to discuss the current state of the field of cell-based immunotherapy, obtain insights into critical knowledge gaps, and identify ways in which NCI could facilitate progress. At both meetings, subjects emphasized four main types of challenges in further developing cell-based immunotherapy for patients with solid tumors: scientific, technical, clinical, and regulatory. The scientific barriers include selecting appropriate targets, ensuring adequate trafficking of cell therapy products to tumor sites, overcoming the immunosuppressive tumor microenvironment, and identifying appropriate models for these investigations. While mouse models may provide some useful data, the majority of those that are commonly used are immunodeficient and unable to fully recapitulate the immune response in patients. There is therefore a need for enhanced support of small early-phase human clinical studies, preferably with adaptive trial designs, to provide proof of concept for novel cell therapy approaches. Furthermore, the requirements for manufacturing, shipping, and distributing cell-based therapies present technical challenges and regulatory questions, which many research institutions are not equipped to address. Overall, workshop subjects identified key areas where NCI support might help the research community in driving forward innovation and clinical utility: 1) provide focused research support on topics such as tumor target selection, immune cell fitness and persistence, cell trafficking, and the immunosuppressive tumor microenvironment; 2) support the rapid translation of preclinical findings into proof of concept clinical testing, harmonize clinical trial regimens, and facilitate early trial data sharing (including negative results); 3) expand manufacturing support for cell therapies, including vectors and reagents, and provide training programs for technical staff; and 4) develop and share standard operating procedures for cell handling and analytical assays, and work with the Food and Drug Administration to harmonize product characterization specifications., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Oral therapies have highly modified cancer patient management and changed hospital practises. We introduce a specific Oral Therapy Centre and retrospectively review information prospectively recorded by co-ordination nurses (CNs) (the DICTO programme). We describe the roles played by CNs in the management of oral cancer therapies at Limoges Dupuytren Hospital between May 2015 and June 2018. All cancers, irrespective of stage or whether oral general chemotherapy or targeted therapy was prescribed, are included. We followed up 287 patients of median age 67 years (range 26-89 years). Of these, 76% had metastases and 44% were on first-line therapy. The vast majority (88%) of their first CN contacts occurred just after physician consultation and lasted an average of 60 min. As part of follow-up, the CNs made 2719 calls (average 10 min) to patients to educate them and to verify compliance and drug tolerance. They also received 833 calls from patients (70%) or their relatives or health professionals (30%) seeking advice on management of side effects. In addition to the initial appointments, 1069 non-scheduled follow-up visits were made to assess side effects (49.2%). The CNs devoted 5 h to each patient over 3 months of treatment (i.e. 25 min/day) and, also organised scheduled hospitalisations in the department of oncology for 51% of patients. We show the interest and real-life work in a specific oral therapy centre within oncology department with the role of CNs to facilitate the global health care of the patients.

Sarcoma is rare and heterogenous with various subtypes having a different prognostic. Desmoid is a tumour with a local aggressiveness; GIST with KIT mutation responds massively to target treatment as IMATINIB, whereas soft tissue sarcoma and leiomyosarcoma are very aggressive with poor response to systemic therapies. Interventional radiology plays an important role in the diagnosis of sarcomas with image-guided percutaneous core needle biopsy being the most commonly used biopsy technique in the diagnosis of sarcomas. Biopsy access routes discussed with the surgeon, and skin access is tattooed. Surgery is a mainstay of sarcoma treatment; the resection can be large. Indeed, resection objective is R0 because quality of surgical margins impacts local control and survival. Radiotherapy is possible in neoadjuvant or in adjuvant treatment to improve local control rate. Recently radiotherapy enhancer injected percutaneously in soft tissue sarcoma has proven benefit in increasing the rate of R0 complete surgical resection. Several studies showed better local control rate linked with post-operative radiotherapy. In patients affected by oligometastatic disease, complete surgical resection of all metastatic sites is in fact considered the primary treatment because complete remission is critical for cure. The decision making to use local therapies is complex, depends upon diverse presentations and histologies, and should always be taken in a multidisciplinary discussion. Today, percutaneous image-guided treatments with ablation technologies (radiofrequency ablation, cryotherapy, microwaves ablation) provide high rate of durable local control for small-sized malignant deposit in many organs including lung, liver and bones. Sarcoma must be managed by multimodality treatment in expert reference centres. Such management has a considerable impact on the prognosis.

Background: Soft-tissue sarcomas (STSs) are a group of rare cancers that can occur at any age. Prognostic outcomes of patients with STS are usually established at the time of the patient's initial disease presentation. Conditional survival affords a dynamic prediction of prognosis for patients surviving a given period after diagnosis. Estimates of conditional survival can provide crucial prognostic information for patients and caregivers, guide subsequent cancer follow-up schedules, and impact decisions regarding management. This study aims to estimate conditional survival and prognostic factors in patients with STS according to age at diagnosis (≤75 years and ≥75 years)., Subjects, Materials, and Methods: A total of 6,043 patients with nonmetastatic STS at first diagnosis who underwent complete surgical resection (R0 or R1) were assessed. Cox proportional hazards regression was used to establish prognostic factors of conditional metastasis-free survival and overall survival at 1, 2, and 5 years after diagnosis., Results: Elderly patients have more adverse prognostic features at presentation and tend to receive less aggressive treatment than do younger patients. However, at baseline as well as at each conditional survival time point, the 5-year estimated probability of metastatic relapse decreases in both young and elderly patients and is almost identical in both groups at 2 years and 5 years after initial diagnosis. Prognostic factors for metastatic relapse and death change as patient survival time increases in both young and elderly patients. Grade, the strongest prognostic factor for metastatic relapse and death at baseline, is no longer predictive of metastatic relapse in patients surviving 5 years after initial diagnosis. Leiomyosarcoma is the histological subtype associated with the highest risk of metastatic relapse and death in young patients surviving 5 years after initial diagnosis. The positive impact on the outcome of peri-operative treatments tends to decrease and disappears in patients surviving 5 years after initial diagnosis., Conclusion: Conditional survival estimates show clinically relevant variations according to time since first diagnosis in both young and elderly patients with STS. These results can help STS survivors adjust their view of the future and STS care providers plan patient follow-up., Implications for Practice: For patients with sarcoma who are followed up years after being treated for their disease, a common scenario is for the patient and caregivers to ask practitioners what the longer-term prognosis may be. The question posed to practitioners may be, "Doc, am I now cured? It's been 5 years since we finished treatment." Survival probability changes for patients who survive a given period of time after diagnosis, and their prognosis is more accurately described using conditional survival. By analyzing more than 6,000 sarcoma patients, an overall improvement was found in the risk of relapse as patients conditionally survive. Prognostic factors for metastatic relapse and death change as patient survival time increases in both young and elderly patients., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2019.)

With the increased use of cancer immunotherapy, a number of immune-related adverse events (irAEs) are being identified. These irAEs can be compared with known autoimmune disorders in similar tissues, with important similarities and differences. Understanding the etiology of irAEs may bring to light concepts applicable to immune responses in cancer, autoimmunity, and infectious disease. This immunobiology is especially relevant to cancer patients with preexisting allogeneic transplants or autoimmune disease who are undergoing cancer immunotherapy. To address these facets of cancer immunotherapy, academic leaders from these various disciplines discussed current irAE basic and clinical research, irAE diagnosis and management, and the need for biomarkers and algorithms to identify individuals at risk for irAEs at a conference jointly sponsored by the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of Arthritis and Musculoskeletal and Skin Diseases in Bethesda, MD, on March 22-23, 2018. Mechanisms and models to characterize irAEs, standardize protocols, store biospecimens, and capture and analyze irAE data were also reviewed during the inaugural Cancer, Autoimmunity, and Immunology Conference. This summary highlights cancer immunotherapy-induced irAEs, the challenges ahead, and the opportunities for greater understanding of autoimmune conditions.

Metastatic soft-tissue sarcoma (STS), a devastating disease, has a median overall survival of only 12-18 months. Until recently, therapeutic options were limited and relied primarily on the use of anthracycline-based chemotherapy. Over the past two decades, improvement in the knowledge of the biology of STS has allowed the investigation of new therapeutic strategies including new cytotoxic agents, epigenetic drugs, specific targeted therapies, and immunotherapeutic treatments., (Copyright © 2018. Published by Elsevier Inc.)

The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: β-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5'-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 μg/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzyme-selective chemical inhibitors.

Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.

Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Imidazoles and triazoles represent major classes of antifungal azole derivatives. With respect to UDP-glucuronosyltransferase (UGT) enzymes, the drug metabolism focus has mainly concentrated on their inhibitory effects with little known about azoles as substrates for UGTs. N-Glucuronide metabolites of the imidazole antifungals, tioconazole and croconazole, have been reported, but there are currently no reports of N-glucuronidation of triazole antifungal agents. In this study, evidence for glucuronidation of azole-containing compounds was studied in human liver microsomes (HLM). When a glucuronide metabolite was identified, azoles were incubated in 12 recombinant UGT (rUGT) enzymes, and enzyme kinetics were determined for the UGT with the most intense glucuronide peak. Six imidazole antifungals, three triazoles, and the benzodiazepine alprazolam (triazole) were evaluated in this study. All compounds investigated were identified as substrates of UGT. UGT1A4 was the main enzyme involved in the metabolism of all compounds except for fluconazole, which was mainly metabolized by UGT2B7, probably mediating its O-glucuronide metabolism. UGT1A3 was also found to be involved in the metabolism of all imidazoles but not triazoles. In both HLM and rUGT K(m) values were lower for imidazoles (14.8-144 microM) than for triazoles (158-3037 microM), with the exception of itraconazole (8.4 microM). All of the imidazoles studied inhibited their own metabolism at high substrate concentrations. In terms of UGT1A4 metabolism, itraconazole showed kinetic features characteristic of imidazole rather than triazole antifungals. This behavior is attributed to the physicochemical properties of itraconazole that are similar to those of imidazoles in terms of clogP.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

IL-7 and IL-7Ralpha (IL-7R) form a non-redundant ligand receptor system which plays a crucial role in human T cell immunity. Both IL-7 and IL-7R are multi-exonal genes and exhibit alternative splicing. We measured the relative distribution of IL-7 and IL-7R spliced mRNA from patients with MS and healthy individuals and observed extensive alternative splicing of both genes with marked differences in proportional transcript expression levels. We report here for the first time that the IL-7 transcript, lacking exon 4, and not the full length IL-7 represents the dominant IL-7 RNA transcript in human PBMCs and a novel IL-7R splice variant lacking exons 5, 6 and 7., (Copyright 2010 Elsevier B.V. All rights reserved.)

Between-sample variation in high-throughput flow cytometry data poses a significant challenge for analysis of large-scale data sets, such as those derived from multicenter clinical trials. It is often hard to match biologically relevant cell populations across samples because of technical variation in sample acquisition and instrumentation differences. Thus, normalization of data is a critical step before analysis, particularly in large-scale data sets from clinical trials, where group-specific differences may be subtle and patient-to-patient variation common. We have developed two normalization methods that remove technical between-sample variation by aligning prominent features (landmarks) in the raw data on a per-channel basis. These algorithms were tested on two independent flow cytometry data sets by comparing manually gated data, either individually for each sample or using static gating templates, before and after normalization. Our results show a marked improvement in the overlap between manual and static gating when the data are normalized, thereby facilitating the use of automated analyses on large flow cytometry data sets. Such automated analyses are essential for high-throughput flow cytometry.

Anti-CD3 mAbs may prolong beta cell function up to 2 years in patients with new onset Type 1 diabetes (T1DM). A randomized open label trial of anti-CD3 mAb, Teplizumab, in T1DM was stopped after 10 subjects because of increased adverse events than in a previous trial related with higher dosing of drug. Teplizumab caused transient reduction in circulating T cells, but the recovered cells were not new thymic emigrants because T cell receptor excision circles were not increased. There was a trend for reduced loss of C-peptide over 2 years with drug treatment (p=0.1), and insulin use was lower (p<0.001). In 4 drug-treated subjects followed up to 60 months, C-peptide responses were maintained. We conclude that increased doses of Teplizumab are associated with greater adverse events without improved efficacy. The drug may marginate rather than deplete T cells. C-peptide levels may remain detectable up to 5 years after treatment.

Background: FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes., Methods: FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody., Results: Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS., Conclusions: The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC., (2008 Clinical Cytometry Society.)

A significant skewing of the peripheral T cell repertoire has been shown in relapsing-remitting multiple sclerosis (MS). Most of the studies already performed in this field are cross-sectional and therefore, little is known of the T cell repertoire evolution over time in MS and the correlation of T cell repertoire variation with clinical and MRI parameters. This study was performed on serially harvested frozen PBMC from nine untreated MS patients (27 samples) and 14 healthy individuals. The blood T cell repertoire of each patient was analysed at the complementarity determining region 3 (CDR3) level and compared with a monthly MRI scan performed over a six month period with assessment of T2 lesion load and gadolinium enhancing lesions. A highly significant blood T cell repertoire skewing was observed in MS patients as compared with healthy controls (p<0.01). In addition, the number of altered Vbeta families correlated significantly with both the T2 lesion volume and the number of gadolinium enhancing lesions as assessed by MRI (Spearman correlation tests, r=0.51 and r=0.44, p<0.01 and p<0.05 respectively). Furthermore, the variation of the number of altered Vbeta families over time also correlated with the appearance of new gadolinium enhancing lesions (r=0.36, p=0.05). These findings which need confirmation on larger serial cohorts, suggest an association between the magnitude of TCRBV CDR3 length distribution alterations in the peripheral blood of MS patients and the disease process.

T cell receptors recognize small changes in peptide ligands leading to different T cell responses. Here, we analyzed a panel of HLA-A2-Tax11-19 reactive T cell clones to examine how small allelic variations of MHC molecules could alter the functional outcome of antigen recognition. Similar to the effects induced by antigenic altered peptide ligands, weak or partial agonistic T cell functions were identified in individual T cell clones with the recognition of MHC-altered peptide ligands (MAPLs). Interestingly, one subtype of HLA-A2 molecules induced an unusual type of partial agonistic function; proliferation without cytotoxicity. Modeling of crystallographic data indicated that polymorphic amino acids in the HLA-A2 peptide binding groove, especially the D-pocket, were responsible for this partial agonism. Reciprocal mutations of the Tax peptide side chain engaging the D-pocket indeed restored the agonist functions of the MHC-peptide complex. Whereas early intracellular signaling events were not efficiently induced by these MAPLs, phosphorylated c-Jun slowly accumulated with sustained long-term expression. These data indicate that MAPLs can induce atypical partial agonistic T cell function through structural and biochemical mechanisms similar to altered peptide ligands.

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

The number of cases of the sexually transmitted diseases chlamydia and condylomata acuminata continues to increase. An overview of the diagnosis, treatment, possible disease sequelae, and patient education is presented as an update for the nurse practitioner.