Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Motivation: Primary data analysis methods are of critical importance in second generation DNA sequencing. Improved methods have the potential to increase yield and reduce the error rates. Openly documented analysis tools enable the user to understand the primary data, this is important for the optimization and validity of their scientific work.Results: In this article, we describe Swift, a new tool for performing primary data analysis on the Illumina Solexa Sequencing Platform. Swift is the first tool, outside of the vendors own software, which completes the full analysis process, from raw images through to base calls. As such it provides an alternative to, and independent validation of, the vendor supplied tool. Our results show that Swift is able to increase yield by 13.8%, at comparable error rate.Availability and Implementation: Swift is implemented in C++and supported under Linux. It is supplied under an open source license (LGPL3), allowing researchers to build upon the platform. Swift is available from http://swiftng.sourceforge.net.Contact: new@sgenomics.org; nava.whiteford@nanoporetech.comSupplementary information: Supplementary data are available at Bioinformatics online.

Key Points T-cell clones identified in the GI tract of patients with steroid-refractory acute GI GVHD expand in the blood with disease progression. The T-cell repertoire in the GI tract of steroid-refractory patients at the time of diagnosis is more similar than for responsive patients.

AimsThe intraportal pancreatic islet transplantation (IPIT) model of diabetic rats is an insulin mediated model of hepatocarcinogenesis characterized by the induction of clear cell foci (CCF) of altered hepatocytes, which are pre-neoplastic lesions excessively storing glycogen (glycogenosis) and exhibiting activation of the AKT/mTOR protooncogenic pathway. In this study, we transferred the IPIT model to the mouse and combined it with the knockout of the transcription factor carbohydrate responsive element binding protein (chREBP).MethodsC57BL/6J Wild-type (WT) and chREBP-knockout (chREBP-KO) mice (n = 297) were matched to 16 groups (WT/ chREBP-KO, experimental/control, streptozotocine-induced diabetic/not diabetic, one/four weeks). Experimental groups received the intraportal transplantation of 70 pancreatic islets. Liver and pancreatic tissue was examined using histology, morphometry, enzyme- and immunohistochemistry and electron microscopy.ResultsCCF emerged in the liver acini downstream of the transplanted islets. In comparison to WT lesions, CCF of chREBP-KO mice displayed more glycogen accumulation, reduced activity of the gluconeogenic enzyme glucose-6-phosphatase, decreased glycolysis, lipogenesis and reduced levels of the AKT/mTOR cascade members. Proliferative activity of CCF was ∼two folds higher in WT mice than in chREBP-KO mice.ConclusionsThe IPIT model is applicable to mice, as murine CCF resemble preneoplastic liver lesions from this hepatocarcinogenesis model in the rat in terms of morphological, metabolic and molecular alterations and proliferative activity, which is diminished after chREBP knockout. chREBP appears to be an essential component of AKT/mTOR mediated cell proliferation and the metabolic switch from a glycogenotic to lipogenic phenotype in precursor lesions of hepatocarcinogenesis.

228 Donor T cell alloreactivity in hematopoietic stem cell transplantation (HSCT) can result in acute graft-versus-host disease (GVHD) which is a major cause of morbidity and mortality that limits the clinical use of HSCT. Despite the potential large diversity in possible T cells (1015) it is thought that within each individual patient undergoing HSCT, a tractable and defined number of T cell clones mediate GVHD. We hypothesized that the identification of the most frequent T cell clones in the gastrointestinal (GI) tract of human patients with GVHD and subsequent tracking of these clones in the peripheral blood may provide a way to bracket and study disease related clones. To investigate T cell clonal dynamics in human GVHD, we collected endoscopic GI tract biopsy samples and matched blood specimens for twelve patients undergoing myeloblative HSCT with suspected GI GVHID prior to or within one day of corticosteroid therapy. We also collected peripheral blood thirty days after biopsy. Five of these patients were negative for GVHD or had mild grade I/II GI GVHD, whereas seven had severe and fatal grade III/IV GI GVHD. These patients had heterogenous indications, conditioning regimens and GVHD prophylaxis which included cyclosporine, tacrolimus and methotrexate. None of the patients had cytomegolovirus (CMV) colitis or other identifiable GI or significant systemic infectious complications, although four patients had low-level blood CMV positivity by PCR. As a control, we compared endoscopic GI biopsies from patients with inflammatory bowel disease as well as normal healthy screening subjects. We extracted genomic DNA and performed T cell receptor (TCR) β CDR3 repertoire sequencing at Stanford and with GigaGen GigaMune Rep-Seq™, both using the Illumina next generation sequencing MiSeq™ and HiSeq™ platforms. Results: For each tissue sample from each patient, between 1,000–4,000 unique T cell sequences were observed in endoscopic tissue samples (2–6 samples per patient). We sorted the most frequent clones in the GI tissue samples by rank order and fractionated the top 100 clones for analysis. Many of these top clones were found in the matched peripheral blood at the time of diagnosis with most of these occurring at frequencies well below those in the GI tissue. For the patients with negative or mild GVHD at the time of diagnosis, the GI-identified clones that were found in the peripheral blood had a mean frequency and standard deviation of 0.001369 +/− 0.0016. This is indistinguishable from peripheral blood samples from patients with severe GVHD which showed a mean frequency and standard deviation of 0.0013 +/− 0.0009 (P=0.36, 2 tailed t-test). We next tracked the GI-identified clones in the peripheral blood to the obtain fold change of the clones at thirty days after diagnosis to the time of diagnosis. The patients with negative or mild GVHD had a mean, median, and standard deviation in fold change of 1.9, 1.7, and 0.9 respectively. The patient with severe GVHD patients had a mean, median and standard deviation in fold change of 32.2, 30.7, and 17.3. A two tailed t-test comparison of the two groups showed they were statistically distinguishable with p-values of 0.0003 for examination of the mean fold change and 0.0005 for the median fold change. For patients with severe GVHD, the peripheral blood at 30 days after diagnosis showed a highly oligoclonal expansion of individually private GI-identified clones. Many of these clones were identified in their respective donor T cell pool. For those patients evaluated with upper and lower endoscopy, many clones were shared between the colon and duodenum or gastrum, however, the dominant clones between these sites appeared to be different. Conclusion: We found that a comparison of the fold change in the peripheral blood frequencies of the top one hundred clones identified in the GI tract biopsies can statistically distinguish five negative or mild GVHD patients from seven severe and fatal cases. These results support the use of T cell repertoire sequencing and associated approaches in human patients to both clarify the pathophysiology of the disease and eventually perhaps to provide an independent immune biomarker that could guide therapy. A major challenge will be to further phenotype and examine the T cell clones identified in the GI tissue and to investigate ways to determine which clones are highly associated or even causal to GVHD. Disclosures: Meyer: GigaGen Inc: Consultancy, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Johnson:GigaGen Inc.: Employment, Equity Ownership.

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Rabbit anti-thymocyte globulin (ATG) given with conditioning for allogeneic haematopoietic stem cell transplantation (alloHSCT) is effective in reducing the risk of chronic graft-versus-host disease (cGVHD). Whether conventional risk factors for cGVHD apply to ATG-conditioned alloHSCT is not known. Between the years 2004 and 2011, 356 adults (median age 48) from 3 centres received 4.5 mg/kg of Thymoglobulin prior to alloHSCT for haematologic malignancy (acute leukaemia 58%, chronic myeloid leukaemia 6%, other myeloid malignancy 15%, other lymphoid malignancy 21%). Donors were unrelated in 64%. Conditioning was myeloablative in 94%. Peripheral blood grafts were used in 97%. At 3 years, overall survival was 61.0% (95% CI 55.3-67.3%), cumulative incidence of relapse was 32.6% (95% CI 26.2-38.5%) and transplant-related mortality was 18.8% (13.2-24.1%). 342 patients were evaluable for the primary endpoint of cGVHD requiring systemic immunosuppression (cGVHD-IS). The cumulative incidence of cGVHD-IS at 3 years was 37.2% (95% CI 31.1-42.7%). On multivariate analysis, only prior grade 2-4 aGVHD (HR 3.08, 95% CI 2.10-4.52, P < .001) was associated with a significant increase in risk of cumulative incidence of cGVHD-IS. Recipient age of greater than 40 years was associated with significantly less cGVHD-IS (HR 0.66, 95% CI 0.45-0.97, P ¼ .03) in univariate but not multivariate analysis. The use of unrelated donors, donor age over 40, female donor/male recipient gender combination and recipient CMV seropositivity were not associated with increased cGVHD-IS. There was insufficient power to determine the effect of graft type (peripheral blood vs bone marrow) on cGVHD-IS, but the incidence did not appear higher in peripheral blood graft recipients. In summary, in this cohort, traditional pre-transplant risk factors for cGVHD were not predictive. In patients undergoing in vivo T-cell depleted alloHSCT, novel predictors of cGVHD may be needed.Chronic graft versus host disease (cGVHD) is a common complication of allogeneic stem cell transplantation (ASCT). Tacrolimus prophylaxis has been proven to be instrumental in the prevention of acute graft versus host disease (aGVHD) a common precursor to developing cGVHD. Our institution uses oral tacrolimus prior to ASCT because we perform ASCT in the outpatient setting and increases ease of administration. We retrospectively reviewed our experience in 94 consecutive patients (pts) who received ASCT from 10 /10 HLA matched donors, 47 were matched sibling donors (MSD) and 47matched unrelated donors (MUD) with a median age of 50 and 52 respectively. All patients received GVHD prophylaxis with tacrolimus started orally between day -3 and day -1 at a dose of 0.06mg/kg per day divided twice daily. Standard short course methotrexate (MTX) was prescribed for all pts (D1 dose 15mg/m2, D3, 6, and 11 doses at 10mg/ m2). Pts who had sub-therapeutic tacrolimus levels (less than 5ug/L) on the day of transplant (D0) were compared to pts who had therapeutic tacrolimus levels (greater than 5ug/ L) for inc...