The greatest source of progress in automated theorem proving in the last 30 years has been the development of better search heuristics, usually based on developer experience and empirical evaluation, but increasingly also using automated optimization techniques. Despite this progress, we still know very little about proof search. We have mostly failed to identify good features for characterizing homogeneous problem classes, or for identifying interesting and relevant clauses and formulas.I propose the challenge of bringing together inductive techniques (generalization and learning) and deductive techniques to attack this problem. Hardware and software have finally evolved to a point that we can reasonably represent and analyze large proof searches and search decisions, and where we can hope to achieve order-of-magnitude improvements in the efficiency of the proof search.

s of the talks March 25 – 30, 2018, Aussois, France

GOALS:We evaluated the serum levels of eosinophil cationic protein (ECP) and mast cell tryptase (MCT) as surrogate markers for response to treatment in adults with eosinophilic esophagitis (EoE) under topical steroid therapy with fluticasone.BACKGROUND:EoE is a chronic disease characterized histologically by eosinophilic inflammation of the esophagus. Esophageal mastocytosis and mast cell activation have been implicated in EoE pathogenesis.STUDY:Fifteen patients with EoE completed this prospective observational study. Before and after 3 months of therapy with fluticasone, eosinophilic and mast cell counts were analyzed from histologic samples of the esophagus and were correlated with serum markers ECP and MCT.RESULTS:Fluticasone-therapy significantly decreased mean eosinophils [from 42.2 to 16.2 eosinophils/high-power field (hpf); P=0.004] and mast cells (from 13.9 to 5.1 mast cells/hpf; P=0.001) in the esophageal epithelium. There was a significant decrease of mean ECP (from 15.6 to 5.5 μg/L; P=0.024) and MCT-serum-values (from 4.7 to 3.8 μg/L; P=0.029) under therapy. Serum-ECP correlated significantly with histologic eosinophilic counts after fluticasone-therapy (r=0.54; P=0.038) in contrast to serum-MCT.CONCLUSIONS:Serum-ECP but not serum-MCT could be a promising noninvasive biomarker to assess response to topical corticosteroid therapy in EoE. These findings should be confirmed by larger studies; ClincialTrials.gov number, NCT01624129.

BACKGROUND:Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention.METHODS:Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles.RESULTS:We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD.CONCLUSIONS:Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.

Automated theorem provers (ATPs) consist of a number of complicated algorithms, that can be parameterized and combined together in different ways. Examples of such parameterizations are clause weighting and selection schemes, term orderings, sets of inference and reduction rules used, etc. E [8] (as some other ATPs) has a language for packaging such useful combinations of parameterizations into strategies. Over 200 strategies have been named and are part of the E source code. Such a large number of strategies can be used to experiment with data-driven methods that try to estimate how to solve a new problem, by considering a large database of previously solved problems and their suitable characterization. E is probably the first ATP that has applied machine learning to strategy selection. There are different ways how to do this, and how to optimize a large set of strategies in general. We will consider some of them and report some results obtained.

Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.

Programmed death 1 (PD-1) is known as an important factor for the development of tolerogenicity. This has been proven in chronic viral infections and different tumor models. To address the role of PD-1 and its ligand programmed death ligand 1 (PD-L1) in different stages of malignant melanoma, we investigated peripheral blood and tumor tissues in regard to overall survival (OS) and prognostic relevance. One hundred samples of peripheral blood mononuclear cells from HLA-A2+ patients with malignant melanoma (Stages IIV) were analyzed in seven color FACS combined with multimer analyses for the immunodominant epitope of Melan-A (peptide A2/Melan-Ap26-35mod). Corresponding formalin-fixed paraffin-embedded tissues of primary tumor and distant organ metastases from 37 cases were analyzed by immunohistochemistry for Melan-A, PD-L1 and PD-1 expression. Compared to the total CD8+ T cell population, PD-1 expression by A2/Melan-A+ CD8+ T cells was over-represented in melanoma stages III and IV (p < 0.001). Although elevation of PD-1+ Melan-A+ CD8+ T cells had no significant influence on OS, a positive correlation was observed between PD-L1 expression on melanoma cells and OS (p = 0.05). Correlation of advanced tumor stage with increased A2/Melan-A-multimer+ PD-1+ T cells in the peripheral blood suggest that blocking of PD-1 could have therapeutic potential in advanced stage melanoma.

Abstract Abstract 3746 Acute graft-versus-host disease (aGvHD) is an immune syndrome after allogeneic hematopoietic cell transplantation (allo-HCT) caused by alloreactive donor T cells that attack the gastrointestinal tract, liver and skin. Thus, early T cell migration patterns to these organs could provide first cues for the onset of aGvHD. Hence, a unique surface marker profile of donor T cells at early time points after allo-HCT may be an indicator for patients at risk of aGVHD. Therefore, we analyzed the course of donor T cell activation, proliferation and homing in a clinical relevant murine MHC minor mismatch (miHAg) allo-HCT model to define critical time points and marker profiles for the detection of alloreactive T cells. Luciferase-labeled C57Bl/6 (H-2b) T cells plus bone marrow cells were transplanted into conditioned (8 Gy) MHC major mismatched Balb/c (H-2d) or miHAg Balb/b (H-2b) recipients. Donor T cell migration was visualized by in vivo bioluminescence imaging (BLI) and cells were characterized by multiparameter flow cytometry for 30 consecutive days after allo-HCT. GVHD scoring was performed by histopathology. Donor T cells proliferated exclusively in secondary lymphoid organs until day+3 (initiation phase) before migrating via the peripheral blood into target organs (effector phase). This occured in both models, MHC major mismatch and miHAg allo-HCT, which resulted in hyper-acute (starting at day+6) or acute GVHD (starting at day+21), respectively. In the hyper-acute scenario one wave of T cell migration starting at day+4 sufficed to cause lethal aGVHD. We detected a 4000-fold increase in CD4 and a 1500-fold increase in CD8 donor T cell numbers in the peripheral blood between day+3 and day+6 in this model. In contrast, in the more clinical relevant miHAg allo-HCT model we found 3 waves of T cell migration with peaks at days +6, +11 and +15 after allo-HCT. In the peripheral blood CD4 T cells increased 20-fold, CD8 T cells 50-fold between day+3 and day+6, but more than 40-fold (CD4) and 400-fold (CD8) between day+3 and day+11. After the third peak on day+15 a period followed when we could only detect very few migrating donor T cells in the peripheral blood before aGvHD became clinically apparent on day+21. Next, we asked whether we could identify alloreactive T cells by testing a large panel of surface markers at the defined migration peaks. Indeed, allogeneic T cells upregulated certain homing receptors at these peaks (e.g. at day+11: α4β7 integrin: 27% of CD4 T cells, 3.4×104/ml, 60% of CD8 T cells, 1.6×105/ml; P-selectin ligand: 28% of CD4 T cells, 3.5×104/ml, 35% of CD8 T cells, 9.1×104/ml). In contrast, syngeneic transplanted mice only showed a constant low expression level of those receptors (e.g. at day+11: α4β7 integrin: 20% of CD4 T cells, 9.6×103/ml, 5% of CD8 T cells, 3.1×103/ml; P-selectin ligand: 17% of CD4 T cells, 8.5×103/ml, 10% of CD8 T cells, 6.6×103/ml). However, other markers such as CD44 could be found on more than 80% of all donor T cells in allogeneic or syngeneic recipients. Our results in this clinical relevant mouse model show accelerating waves of T cell migration consistent with an enhancing feedback loop model of aGvHD pathogenesis. The homing receptor expression profile of donor T cells correlated with critical migration waves and clearly differed between mice with or without aGvHD. The assessment of critical time points frame a diagnostic window for a potential predictive test based on the dynamic change of the T cell homing receptor profile after allo-HCT. This preclinical study now awaits to be evaluated in patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 61 In vivo models of complex immune processes like stem cell engraftment, host-pathogen interactions or anti-tumor responses face the challenge to either provide dynamic information in low resolution (e.g. in vivo bioluminescence Imaging, BLI) or provide high resolution information with a limited field of view (e.g. multi-photon laser scanning microscopy/confocal microscopy). To overcome these limitations we applied a novel selective plane illumination microscopy (SPIM) technique (also termed ultramicroscopy), which enabled us to visualize structural and cellular changes in intact organs in high resolution. Here we investigated dynamic shifts at sites of graft-versus-host disease (GVHD) initiation in mice after allogenic hematopoietic cell transplantation (allo-HCT). For in vivo and ex vivo imaging we transplanted 1,2×106 luciferase+ DsRed+ transgenic C57Bl/6 T cells (H-2b, Thy1.1+) plus 5×106 bone marrow (BM) cells (H-2b, Thy1.2+) into myeloablative conditioned allogeneic Balb/c recipients (H-2d, Thy1.2+, 8 Gy) to induce aGVHD. Utilizing BLI we followed the development of GVHD in vivo. At the transition from GVHD initiation phase to effector phase (day+3 and day+4) we prepared the small bowel and Peyer‘s Patches (PPs) of allo-HCT recipients for whole organ microscopy and compared these to organs of untreated mice. Staining for T cell populations (CD4) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) allowed us to visualize MAdCAM-1 expression in relation to T cell areas in whole PPs and surrounding intestinal mucosa (> 8mm3) in a high throughput format by creating approx. 1000 optical sections using multiple lasers and a sensitive CCD camera within 10 minutes. Adding another color channel revealed structural details through measurement of intrinsic autofluorescence. The individual color stacks were overlaid via computational image processing for three dimensional tissue reconstruction, volume measurements and quantification of protein expression. Ultramicroscopy exposed microanatomical structures like intestinal villi, crypts and PPs with its subepithelial dome regions and follicles by giving detailed information about MAdCAM-1 expressing blood vessels and T cell areas. The 3D reconstruction of small intestines in non-conditioned mice revealed that the MAdCAM-1 expression averaged at 1-2% of the entire PP and was predominantly restricted to the high endothelial venules (HEV). During the transition from the initiation to the effector phase of acute GVHD (day3-4) the overall MAdCAM-1 expression in PPs increased by 50fold. At this point MAdCAM-1 expression was also found in parafollicular and subepithelial cell populations, which still need to be characterized further concerning their lineage differentiation. MAdCAM-1 is well known as a vascular addressin molecule. Because of its importance in homing of α4β7+ T and B cell into the gastrointestinal tract we investigated the dynamic expression of MAdCAM-1 during acute GVHD within intact PPs. It is well established that MAdCAM-1 is an important regulator in the homing of α4β7+ T cell to PPs via HEVs. Additionally our data lead to the hypothesis that MAdCAM-1 might contribute to retain proliferating α4β7+ T cells during the initiation of an adaptive immune response and might support a synchronized exit of alloreactive T cells in the aGVHD effector phase. Conventional histological sections are labor intensive, time consuming and often restricted to a very limited region of interest and therefore, can result in missing rare events and under- or overestimation of biological processes. Ultramicroscopy helped us to overcome these limitations and enabled us to visualize immunological processes with subcellular resolution in whole organs. CB and MF, GSH and AB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2451 Poster Board II-428 Hematopoietic cell transplantation (HCT) is a curative therapy for a variety of malignancies. HCT provides disease eradication through both the high-dose conditioning regimen and an allogeneic graft versus tumor effect (GVT), however graft-versus-host disease (GVHD) remains a major obstacle. In a murine aHCT model of bioluminescence imaging (BLI) we have previously demonstrated that acute GVHD can be separated to a GVHD initiation phase confined to secondary lymphoid organs and a subsequent GVHD effector phase in peripheral target tissues. It has been proposed that host conditioning may not only be crucial in the activation of alloreactive T cells but also determine acute GVHD organ manifestation in the effector phase. Here we wanted to investigate how the host conditioning regimen affects the host target tissues in terms of inflammatory cytokines and their role in donor T cell recruitment. We compared lethally irradiated (8Gy) vs. non-irradiated Balb/c wild type or Balb/c Rag-/-cGC-/- (H-2d) -DKO mice that received allogeneic luciferase+ FVB/N T cells (H-2q). Surprisingly, we did not observe marked differences in the donor T cell proliferation (BLI, CFSE), acquisition of activation markers (CD25, CD44, CD69) and homing receptors (a4b7, aEb7, P-selectin ligand, E-selecting ligand) in conditioned, non-conditioned Balb/c Rag-/-cGC-/-. Despite the upregulation of these homing receptors on donor T cells, infiltration of target tissues (intestinal tract, liver and skin) was significantly accelerated in conditioned and delayed in non-conditioned hosts. As T cell recruitment may have occurred as a result of alterations of the milieu inflammatory cytokines in GVHD target tissues, we compared the cytokine profile in conditioned vs. non-conditioned recipients. At days 3 and 6 after transplantation tissues were harvested and cytokines from the target tissues; liver, large bowel, small bowel, peripheral blood and a non target tissue: kidney were analyzed for a TH1/TH2/Th17a cytokines. At day 3 high levels of INF-γ and TNF were detected in the Balb/c WT conditioned host compared to the non-conditioned host in all target tissues (SB, LB, and liver) and most markedly in peripheral blood and the large bowel. More importantly the Balb/c Rag-/-cGC-/- conditioned host displayed about 5 times higher levels of both inflammatory cytokines compared to the non conditioned DKO hosts and to the Balb/c WT. Similar results with a lesser levels were observed both for IL-2 and IL17a. By day 6 similar results are seen but with a much reduced expression of the cytokines, indicating that the cytokine storm peak was maybe at day 3. In summary host conditioning is not a requirement for alloreactive T cell activation rather induced inflammatory cytokines such as TNF and INF-γ are the determinant factors for effector T cell recruitment to GVHD target tissues. JB and AB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Abstract 3559 Poster Board III-496 Acute graft-versus-host disease (GVHD) can be spatio-temporally separated in an initiation phase confined to secondary lymphoid organs (SLO) followed by the GVHD effector phase in the intestinal tract (GIT), liver and skin. Employing non-invasive bioluminescence imaging, fluorescence microscopy and flow cytometry in a murine allogeneic hematopoietic cell transplantation (allo-HCT) model [luciferase + FVB/N (H-2 q ) or luciferase + C57Bl/6 (H-2 b ) splenocytes plus wild type bone marrow into Balb/c-Rag −/− cgChain −/− recipients (H-2 d )] we observed that the proliferation, activation and acquisition of homing receptors by alloreactive T cells occurred in SLO independently whether allogeneic recipients were conditioned (8 Gy) or not conditioned before allo-HCT. However, fewer alloreactive T cells infiltrated target tissues in non-conditioned recipients resulting in significantly delayed GVHD onset as compared to conditioned hosts. We concluded that inflammatory recruitment by GVHD target tissues (GIT, liver and skin) drives the characteristic GVHD organ infiltration. To explore the signals potentially responsible for recruitment of alloreactive T cells into GVHD target tissues we compared the expression of cytokine induced endothelial adhesion molecules of GVHD target versus non-target tissues of conditioned and non-conditioned allo-HCT recipients. We found strong endothelial up-regulation of VCAM-1and MAdCAM-1 in target organs of conditioned recipients. As alloreactive effector T cells upregulate homing receptors (a4b7, a4b1, P-lig, E-lig) for vascular endothelial ligands we asked whether interference with these receptor-ligand interactions would prevent GVHD target infiltration. Blocking of either VCAM-1 or MAdCAM-1 alone or VCAM-1 and MAdCAM-1 combined did not reduce the alloreactive T cell infiltration of the target organs. However, when we simultaneously blocked VCAM-1, MAdCAM-1 and CD62P in allo-HCT recipients we could abrogate GVHD target infiltration by alloreactive T cells in the liver, the skin and small intestines (Grade 0), whereas the colon showed clear signs of acute GVHD (Grade 2). In summary, inflammatory recruitment via endothelial adhesion molecules expressed in GVHD target tissues during the effector phase is essential for GVHD manifestation. Efficient interference of alloreactive T cell homing requires abrogating simultaneously redundant homing receptor-ligand interactions. Application of a combined antibody regimen against VCAM-1, MAdCAM-1 and CD62P appears as a promising strategy to protect from small bowel GVHD. The optimal combination for preventing alloreactive T cell recruitment to the colon still remains to be determined. Late but selective GVHD intervention through combinatorial short-term blockade of effector T cell trafficking may provide an attractive future clinical application to abrogate GVHD and to enhance the graft-versus-leukemia effect. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4497 Patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are at high risk to develop acute graft-versus-host disease (aGVHD) which is caused by alloreactive donor T cells. Early diagnosis of aGVHD remains difficult: there are no efficient methods to identify patients at risk of aGVHD, which could improve disease prevention. Therefore, to potentially predict aGVHD, we asked whether it is possible to detect in vivo activated alloreactive T cells in the peripheral blood immediately before entering aGVHD target tissues. To address this question, we used the CD107a/b degranulation assay (Betts et al., J Immunol Methods 2003 281:65) to measure cytotoxicity responses of alloractive T cells in a mouse model of aGVHD that allows us to trace alloreactive T cells in different phases of aGVHD by flow cytometry. Utilizing bioluminescence imaging in this model we have previously observed two distinct phases in aGVHD pathophysiology (Blood 2005;106:1113): During the initiation phase until day+3 alloreactive T cells are activated, proliferate in secondary lymphoid organs and acquire appropriate homing receptors to migrate into target tissues during the effector phase. In preparation for transplantation experiments, we tested T cell receptor transgenic (OT-1) T cells, which recognize the SIINFEKL-H-2b complex as a positive control. We observed degranulation of in vitro activated OT-1 T cells (84.40 ± 1.10%) against SIINFEKL-H-2b+ targets in contrast to C57Bl/6 wildtype targets (53.17 ± 2.65%). Unstimulated OT-1 T cells degranulated to a lower extend (22.93 ± 1.05%) against SIINFEKL-H-2b+ targets but less against SIINFEKL negative targets (9.47 ± 0.56%) (t-tests p<0.0001). We then transplanted 1,2×106 C57Bl/6 CD4+ and CD8+ T cells (H-2b, Thy1.1+) plus 5×106 C57Bl/6 bone marrow (BM) cells (H-2b, Thy1.2+) into myeloablative conditioned allogeneic Balb/c (H-2d, Thy1.2+, 8 Gy) recipients. As syngeneic controls we used C57Bl/6 (H-2b, Thy1.2+, 9 Gy) recipients. To prove specificity of the functional assay we transplanted 1,2×106 TCR transgenic OT-1 T cells (H-2b, Thy1.2+) plus 5×106 C57Bl/6 BM into conditioned C57Bl/6 (H-2b, Thy1.2+, 9 Gy) mice that expressed the SIINFEKL-H-2b+ complex ubiquitously. On day+2 and day+5 after HCT we analyzed the peripherial blood of the transplanted mice. As expected we did not detect donor T cells in the peripheral blood on day+2 during the GVHD initiation phase. However, by day+5 at the transition to the aGVHD effector phase large numbers of T cells had entered the circulation. Employing the degranulation assay revealed that peripheral blood CD8+ T cells from allogeneic recipients (C57Bl/6 □ Balb/c) degranulated against allogeneic targets (29.08 ± 4.46%), antigen specific CD8+ OT-1 T cells against SIINFEKL expressing targets (43.10 ± 3.78%) but less against SIINFEKL negative targets (9.78 ± 2.64%) (ANOVA p<0.0001). Reactive T cells in syngeneic controls were negligible (< 3%). Importantly, subsequent histopathological analysis of the same allogeneic recipients (where alloreactive T cells had degranulated) revealed aGVHD of the intestinal tract (grade 2-3) and the liver (0-2). No signs of GVHD were observed in mice where T cells had not degranulated (syngeneic controls). These preclinical data from our in vivo experiments encourage translation of this predictive test to patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

Oncolytic virotherapy is a promising strategy for safe and effective treatment of malignancy. We have reported previously that recombinant vesicular stomatitis virus (VSV) vectors are effective oncolytic agents that can be safely administered via the hepatic artery in immunocompetent rats to treat multifocal hepatocellular carcinoma (HCC), resulting in tumor necrosis and prolonged survival. Though the results were encouraging, complete tumor regression was not observed, which led us to explore alternative approaches to further enhance the efficacy of VSV treatment. Transarterial embolization techniques have been shown to improve the efficiency and tumor selectivity of anticancer treatments. Degradable starch microspheres (DSM) are one such embolic agent that provides transient embolization of the therapeautic agent before being degraded by serum amylases. Here we demonstrate via dynamic contrast-enhanced magnetic resonance imaging that in our rat model of multifocal HCC, DSM injection into the hepatic artery results in a substantial reduction in tumor perfusion of systemically applied contrast agent. VSV, when administered in combination with DSM, results in enhanced tumor necrosis and synergistically prolongs survival when compared with VSV or DSM monotherapy. Conclusion: This regimen of viroembolization represents an innovative therapeutic modality that can augment the future development of transarterial oncolytic virus therapy for patients with advanced HCC. (HEPATOLOGY 2008;48: 1864-1873.)

In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

Acute graft-versus-host disease (aGVHD) is caused by alloreactive effector T cells attacking the gastrointestinal tract, liver and skin after allogeneic hematopoietic cell transplantation (aHCT). The mechanism by which alloreactive T cells target these organs and not others remains elusive. Recently, we reported that different secondary lymphoid organs (SLOs), as alloreactive priming sites, can imprint distinct homing phenotypes on evolving alloreactive effector cells in vivo. However, preventing access to selected lymphoid organs (via the use of blocking antibodies or recipient mice lacking Peyer’s patches (PP), PP and lymph nodes (LN) or spleens) did not alter the aGVHD organ manifestation. These findings not only suggested a high redundancy of SLOs as induction sites of aGVHD, but also questioned whether homing instruction of alloreactive T cells by these sites can explain the mechanism of aGVHD target organ manifestation. To test the homing instruction model we transplanted transgenic luciferase + (luc + ) FVB/N (H-2 q , Thy1.1 + ) splenocytes into conditioned (2×400rad) Balb/c recipients (H-2 d , Thy1.2 + ). On day+3 we isolated luc + donor lymphocytes from peripheral LN, mesenteric LN, or spleens and transferred them into conditioned secondary allogeneic recipients. 16 hours later, bioluminescence imaging revealed that allogeneic luc + T cells irrespective of their original priming site targeted the intestinal tract and liver. Subsequently, we compared aHCT of conditioned with non-conditioned secondary Balb/cRag −/− cγ-Chain −/− recipients. Surprisingly, we found allogeneic luc + T cells accumulating in SLOs in non-conditioned recipients in contrast to intestinal and hepatic tissues in conditioned recipients. These in vivo findings establish that alloreactive effector cells migrate to aGVHD target tissues because of attraction to these sites rather than specific instruction by SLOs. Therefore, we propose a signal hierarchy model of alloreactive cell trafficking whereby inflammatory signal/ligand interactions dominate over organ-specific homing receptor/ligand interactions.

CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.

Acute graft-versus-host disease (aGVHD) still results in high morbidity and mortality in patients undergoing allogeneic hematopoietic cell transplantation (aHCT). Early diagnosis of aGVHD remains difficult and is made based on clinical symptoms and histological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestations. Therefore, it is important to develop predictive assays to identify patients at risk of developing aGVHD for improved disease prevention. Using bioluminescence imaging (BLI) we have recently demonstrated that aGVHD pathogenesis is tightly spatially and temporally regulated in a murine model across major histocompatibility barriers. Therefore, we asked whether insights in the timing of aGVHD initiation and effector phase could allow for the development of a diagnostic test whereby specific cell surface profiles can predict the onset of aGVHD. FVB/N (H-2 q , Thy1.1 + , 4x10 6 ) or C57Bl/6 (H-2 b , Thy1.1 + , 4x10 6 ) splenocytes plus bone marrow (BM) cells (5x10 6 ) were transplanted into conditioned (2x400rad) allogeneic Balb/c (H-2 d , Thy1.2 + ) or syngeneic (2x450rad) C57Bl/6 (H-2 b , Thy1.2 + ) recipients. Allogeneic recipients developed the first clinical signs of aGVHD starting at day+6 which progressed rapidly to animal death typically by day+14. However, BLI revealed that aGVHD target organ infiltration had already occurred days earlier. On day+3 after aHCT, dividing (BrdU + ) donor derived allogeneic T cells (Thy1.1 + ) were still confined to the T cell areas in secondary lymphoid organs. Between day+3 and day+4 T cells appeared in the red pulp of the spleen, indicating likely entry into the blood circulation. Thereafter, increasing aGVHD target organ infiltration became apparent. These findings prompted us to analyze peripheral blood (PB) samples from allogeneic vs. syngeneic recipients (day+1 to +6). Until day+3 after aHCT, no significant PB T cell numbers were detectable. However, by day+4 we found a dramatic increase of PB T cells (mostly CD4 + , by day+5 mostly CD8 + ) that were CD44 hi and expressed the homing receptors α4β7 integrin, αEβ7 integrin, CCR9, E-selectin ligand, P-selectin ligand, CCR5, and CXCR3 in allogeneic recipients, but not in syngeneic or BM controls. These T cell populations could be verified as clonally expanded (CFSE lo ) donor-derived alloreactive T cell subsets (Thy1.1 + ). In summary, alloreactive T cells could be identified by the timed up-regulation of a panel of distinct homing receptors. Furthermore, the transition between aGVHD initiation and effector phase emerged as an early diagnostic window for the detection of alloreactive T cells in the peripheral blood days before aGVHD onset. Taken together, this approach could predict aGVHD in order to tailor immunosuppressive therapy for individual patients.

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria still represents a difficult task both in microbiology and pathology. The aim of this study was to determine the frequency of mycobacterial DNA detectable by PCR in formalin-fixed paraffin-embedded tissues showing suspicious granulomatous lesions. A total of 190 archival specimens were analyzed, using a nested PCR protocol, which amplifies a fragment of the mycobacterial 65-kDa heat-shock protein gene. Restriction fragment-length polymorphisms and sequencing were utilized to further analyze the obtained PCR products. Corresponding microbiological culture results were available for 41 cases. We detected mycobacterial DNA in 119 cases (63%), of which 71 (60%) were positive for Mycobacterium tuberculosis complex DNA and 41 (34%) for DNA of nontuberculous mycobacteria. Seven cases (6%) could not be subtyped for technical reasons. The largest group of nontuberculous mycobacteria comprised 29 cases (25% of the 119 positive cases), which were assigned to Mycobacterium fortuitum complex. Mycobacterium avium–intracellulare complex was detected in eight (7%) cases, Mycobacterium gordonae in three (2.5%) and Mycobacterium rhodesiae in a single case (0.8%). All cases of Mycobacterium tuberculosis were unequivocally identified by restriction fragment-length polymorphism analysis. In contrast, sequencing provided a gain of information over restriction fragment-length polymorphism analysis in 37% of the nontuberculous mycobacteria cases (15 of 41). Alignment studies on DNA of nontuberculous mycobacteria showed frequent sequence variations, supporting the existence of sequevars. Comparison of molecular data to available results of microbiological culture assays showed a good concordance of 83%. In conclusion, amplification and sequencing of the mycobacterial 65-kDa heat-shock protein gene is an excellent tool for species identification of mycobacteria, especially nontuberculous mycobacteria, in formalin-fixed paraffin-embedded tissues.