ALBERT

Description: To study the complex pathophysiology of aGvHD in allogeneic hematopoietic cell transplantation (HCT) we transplanted transgenic luciferase expressing T cell populations into lethally irradiated HCT recipients (murine MHC major mismatch model, H-2q into H-2d). Tracking of light emitting donor T cells in living animals and detailed studies by multi color immunofluorescence microscopy (IFM) and FACS revealed the tight links of spatial and temporal evolution in this complex immune process. Donor derived T cells migrate to T cell areas in lymphoid tissues within a period of 12 hours. In the initial periods donor CD4+ T cells appear first with CD8+ T cell infiltration at later time points. Donor T cells start proliferating in lymphatic tissues on day 2 after transfer, as observed by BrdU stainings. Although alloreactive T cells are similarly activated in all lymphoid organs, they only up-regulate gut homing molecules after more than 5 cell divisions (CFSE proliferation analysis by FACS) in certain lymphoid organs (Peyer’s patches, mesenteric LN and spleen). Abruptly on day 4 after HCT, T cells migrate into intestinal sites. These findings strongly suggested, that specific priming sites are required for alloreactive T cells to induce a distinct type of tissue tropism in GvHD. In contrast to previous reports peformed without host conditioning, depletion of certain lymphoid organs (e.g. Peyer’s patches) before HCT or antibody blocking experiments did not control aGVHD. BLI showed, that anti-L-selectin or anti-MAdCAM-1 antibody treatment alone or in combination was effective in blocking donor T cell migration to lymph nodes and Peyer’s patches, while redirecting these cells to liver and spleen. Subsequently cells proliferated predominantly in the spleen until day 3 after HCT. Surprisingly we observed a full picture of gut infiltration on day 4 and skin involvement on day 5–6, similar in dynamics and strength to the aGvHD isotype control group. These findings demonstrated, that other lymphoid organs can functionally compensate for inducing gut and skin homing of alloreactive T cells. Of importance, we demonstrated that T cells that lacked homing molecules for secondary lymphoid organs had alloreactive properties in vitro, yet did not cause aGVHD in vivo. In summary, the activation of alloreactive T cells in specific sites throughout the body is complex and involves the acquisition of homing molecule expression. Transplantation of T cells with defined homing properties therefore, appears to be a promising alternative in conferring protective immunity early after HCT without the risk of aGvHD.

Description: 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.

Description: 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.

Description: Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (TEM) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

Description: Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (〉120 d, p

Description: 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-2q, Thy1.1+) splenocytes into conditioned (2×400rad) Balb/c recipients (H-2d, 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.

Description: © The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chapman, A. S. A., Beaulieu, S. E., Colaco, A., Gebruk, A. V., Hilario, A., Kihara, T. C., Ramirez-Llodra, E., Sarrazin, J., Tunnicliffe, V., Amon, D. J., Baker, M. C., Boschen-Rose, R. E., Chen, C., Cooper, I. J., Copley, J. T., Corbari, L., Cordes, E. E., Cuvelier, D., Duperron, S., Du Preez, C., Gollner, S., Horton, T., Hourdez, S., Krylova, E. M., Linse, K., LokaBharathi, P. A., Marsh, L., Matabos, M., Mills, S. W., Mullineaux, L. S., Rapp, H. T., Reid, W. D. K., Rybakova (Goroslavskaya), E., Thomas, T. R. A., Southgate, S. J., Stohr, S., Turner, P. J., Watanabe, H. K., Yasuhara, M., & Bates, A. E. sFDvent: a global trait database for deep-sea hydrothermal-vent fauna. Global Ecology and Biogeography, 28(11), (2019): 1538-1551, doi: 10.1111/geb.12975.

Description: Motivation Traits are increasingly being used to quantify global biodiversity patterns, with trait databases growing in size and number, across diverse taxa. Despite growing interest in a trait‐based approach to the biodiversity of the deep sea, where the impacts of human activities (including seabed mining) accelerate, there is no single repository for species traits for deep‐sea chemosynthesis‐based ecosystems, including hydrothermal vents. Using an international, collaborative approach, we have compiled the first global‐scale trait database for deep‐sea hydrothermal‐vent fauna – sFDvent (sDiv‐funded trait database for the Functional Diversity of vents). We formed a funded working group to select traits appropriate to: (a) capture the performance of vent species and their influence on ecosystem processes, and (b) compare trait‐based diversity in different ecosystems. Forty contributors, representing expertise across most known hydrothermal‐vent systems and taxa, scored species traits using online collaborative tools and shared workspaces. Here, we characterise the sFDvent database, describe our approach, and evaluate its scope. Finally, we compare the sFDvent database to similar databases from shallow‐marine and terrestrial ecosystems to highlight how the sFDvent database can inform cross‐ecosystem comparisons. We also make the sFDvent database publicly available online by assigning a persistent, unique DOI. Main types of variable contained Six hundred and forty‐six vent species names, associated location information (33 regions), and scores for 13 traits (in categories: community structure, generalist/specialist, geographic distribution, habitat use, life history, mobility, species associations, symbiont, and trophic structure). Contributor IDs, certainty scores, and references are also provided. Spatial location and grain Global coverage (grain size: ocean basin), spanning eight ocean basins, including vents on 12 mid‐ocean ridges and 6 back‐arc spreading centres. Time period and grain sFDvent includes information on deep‐sea vent species, and associated taxonomic updates, since they were first discovered in 1977. Time is not recorded. The database will be updated every 5 years. Major taxa and level of measurement Deep‐sea hydrothermal‐vent fauna with species‐level identification present or in progress. Software format .csv and MS Excel (.xlsx).

Description: We would like to thank the following experts, who are not authors on this publication but made contributions to the sFDvent database: Anna Metaxas, Alexander Mironov, Jianwen Qiu (seep species contributions, to be added to a future version of the database) and Anders Warén. We would also like to thank Robert Cooke for his advice, time, and assistance in processing the raw data contributions to the sFDvent database using R. Thanks also to members of iDiv and its synthesis centre – sDiv – for much‐valued advice, support, and assistance during working‐group meetings: Doreen Brückner, Jes Hines, Borja Jiménez‐Alfaro, Ingolf Kühn and Marten Winter. We would also like to thank the following supporters of the database who contributed indirectly via early design meetings or members of their research groups: Malcolm Clark, Charles Fisher, Adrian Glover, Ashley Rowden and Cindy Lee Van Dover. Finally, thanks to the families of sFDvent working group members for their support while they were participating in meetings at iDiv in Germany. Financial support for sFDvent working group meetings was gratefully received from sDiv, the Synthesis Centre of iDiv (DFG FZT 118). ASAC was a PhD candidate funded by the SPITFIRE Doctoral Training Partnership (supported by the Natural Environmental Research Council, grant number: NE/L002531/1) and the University of Southampton at the time of submission. ASAC also thanks Dominic, Lesley, Lettice and Simon Chapman for their support throughout this project. AEB and VT are sponsored through the Canada Research Chair Programme. SEB received support from National Science Foundation Division of Environmental Biology Award #1558904 and The Joint Initiative Awards Fund from the Andrew W. Mellon Foundation. AC is supported by Program Investigador (IF/00029/2014/CP1230/CT0002) from Fundação para a Ciência e a Tecnologia (FCT). This study also had the support of Fundação para a Ciência e a Tecnologia, through the strategic project UID/MAR/04292/2013 granted to marine environmental sciences centre. Data compiled by AVG and EG were supported by Russian science foundation Grant 14‐50‐00095. AH was supported by the grant BPD/UI88/5805/2017 awarded by CESAM (UID/AMB/50017), which is financed by FCT/Ministério da Educação through national funds and co‐funded by fundo Europeu de desenvolvimento regional, within the PT2020 Partnership Agreement and Compete 2020. ERLL was partially supported by the MarMine project (247626/O30). JS was supported by Ifremer. Data on vent fauna from the East Scotia Ridge, Mid‐Cayman Spreading Centre, and Southwest Indian Ridge were obtained by UK natural environment research council Grants NE/D01249X/1, NE/F017774/1 and NE/H012087/1, respectively. REBR's contribution was supported by a Postdoctoral Fellowship at the University of Victoria, funded by the Canadian Healthy Oceans Network II Strategic Research Program (CHONe II). DC is supported by a post‐doctoral scholarship (SFRH/BPD/110278/2015) from FCT. HTR was supported by the Research Council of Norway through project number 70184227 and the KG Jebsen Centre for Deep Sea Research (University of Bergen). MY was partially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (project codes: HKU 17306014, HKU 17311316).