ALBERT

Description: Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3+ regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8+ T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

Description: Abstract 2436 Poster Board II-413 Sensitive in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus–host disease (GVHD) and graft-versus-tumor responses (GVT). Near-infrared (NIF) fluorescent proteins (FP) appear advantageous for deeper tissue penetration due to minimized absorbance by hemoglobin, water and lipids. Therefore we tested whether a recently published NIF FP (FP635, “Katushka”) could serve as a single reporter for whole body and single cell imaging. To compare signal intensities of eGFP and FP635 we generated fluorescent MOSEC cell lines (mouse ovarian cancer), titrated them in vitro and subcutaneously (s.c.) in vivo in Balb/c nu/nu mice. MOSEC FP635 showed twice the signal intensities compared to MOSEC eGFP in vitro by spectral fluorescence imaging (FLI). In vivo the eGFP signal was attenuated 〉60% in contrast to only 20% for FP635 from subcutaneous sites. However, FP635 signals from deep tissue layers were quenched. To address whether reduced signal attenuation of FP635 may allow sensitive visualization of immune processes by FLI and multiphoton-laser-scanning-microscopy (MPM) we generated transgenic mice in the genetic C57Bl/6 (B6) background, expressing FP635 under the ubiquitin promoter. Transgenic founders were selected upon signal intensities of leukocyte populations measured by flow cytometry in the PerCP channel. Combination of FP635 with colors other than red were possible for multiparameter flow cytometry. Next, eGFP, DsRed and FP635 splenocytes from transgenic donors were titrated as described above. In vitro signal intensities of FP635 splenocytes were 〉5 times lower compared to the other two FPs. FP635 signal absorption in vivo was low (30%) which is consistent with MOSEC titration results. In vivo DsRed detection was most sensitive and signals were similarly attenuated as FP635 in contrast to eGFP (60%). Subsequently, we aimed to visualize FP635 in a model of GVHD, where alloreactive T cells undergo massive expansion. Balb/c nu/nu mice were lethally irradiated and transplanted with 5×106 B6.WT bone marrow cells plus either 2×107 B6.DsRed+Luciferase+ or 2×107 B6.FP635 splenocytes. Sensitivity for DsRed cell detection was superior over FP635 cells. FP635 signal was only weakly detectable in lymph nodes (LN) by ex vivo FLI, where DsRed signals were detectable at earlier timepoints and LNs were even visualized by in vivo FLI. DsRed+ Luciferase+ double transgenic splenocytes allowed direct comparison of bioluminescence imaging (BLI) to FLI. Timely in vivo visualization of immune cells in deep tissues was feasible only by BLI. After whole body imaging the suitability of FP635 for MPM was checked by co-injecting eGFP B cells and either DsRed or FP635 T cells intravenously into RAG-/- mice. As FP635 is a NIF FP we expected to achieve deeper tissue penetration in hemoglobin rich organs, such as the spleen, in single cell microscopy. After 6 weeks of adoptive cell transfer we imaged spleens by MPM. Tissue penetration depths of DsRed or FP635 T cells were compared to eGFP B cells. No advantage in penetration depth of FP635 over DsRed was measured. Photobleaching is an important factor for microscopy, especially if cells are to be tracked over long time. FP635 transfected 293T cells bleached faster (t1/2=108 sec) than 293T cells transfected with eGFP (t1/2〉900 sec) or DsRed (t1/2=411 sec). These experiments indicate that very high expression levels of FP635 need to be achieved for imaging. The signal attenuation of FP635 is low which may increase the sensitivity but in our hands DsRed showed comparable characteristics. Yet, the fast photobleaching of FP635 compared to the broadly established FPs DsRed and eGFP may be disadvantageous for long term microscopic tracking of cells. Our data indicate that BLI is by far superior over FLI in sensitivity and tissue penetration for whole body imaging of immune cells. However, FLI of red or near-infrared clonally selectable tumor cell lines may provide a welcome color addition to study immune cell-tumor interactions in combined models of BLI and FLI. Disclosures: No relevant conflicts of interest to declare.

Description: Donor CD4+Foxp3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) while maintaining the anti-tumoral effect of transplanted conventional T cells in preclinical mouse models. Current clinical study protocols with donor Tregs for treatment or prophylaxis of GVHD rely on their ex vivo expansion and infusion in high numbers. Here we present a fundamentally novel strategy for inhibiting GVHD that is based on the in vivo expansion of recipient Tregs prior to allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in Treg biology. To this end we constructed a recombinant nonameric TNFR2-specific agonist, mimicking the activity of murine membrane-bound TNF on TNFR2 without TNFR1 stimulation, thereby avoiding the inflammatory side effects observed with conventional TNF. In vitro, this TNFR2-agonist expanded natural Tregs from wild type but not from TNFR2 KO mice. Accordingly, a human variant of this TNFR2-specific agonist expanded human Tregsin vitro. In vivo treatment of healthy mice with the murine TNFR2-agonist significantly increased Treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute GVHD. Next, we pre-treated recipient mice with this novel TNFR2-agonist to expand host-type radiation resistant Tregs prior to of allo-HCT in two models across MHC barriers (C57BL/6, H-2b-〉Balb/c, H-2d and FVB/N, H-2q-〉C57BL/6, H-2b). TNFR2-agonist pre-treatment resulted in significantly prolonged survival and reduced GVHD severity when compared to TNFR2-deficient recipients or untreated allo-HCT recipients. This was accompanied by reduced donor T cell proliferation and infiltration into GVHD target organs as assessed by in vivo and ex vivo bioluminescence imaging, flow cytometry and immunofluorescence microscopy. While in vivo TNFR2-agonist pre-treatment protected allo-HCT recipients from GVHD, anti-tumor effects of transplanted T cells remained unaffected in two different murine B cell leukemia models. In vivo depletion of host derived Tregs completely abrogated the protective effect of TNFR2-agonist pre-treatment. Our study shows that the expansion of host Tregs by selective in vivo TNFR2-activation significantly improves the outcome after allo-HCT and results in prolonged tumor-free survival. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: 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-2q) or luciferase+ C57Bl/6 (H-2b) splenocytes plus wild type bone marrow into Balb/c-Rag−/−cgChain−/− recipients (H-2d)] 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.

Description: Acute graft versus host disease (aGVHD) remains a major complication in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), the only curative treatment for many malignant hematologic diseases. After initial priming in secondary lymphoid organs, alloreactive donor T cells efficiently migrate to the intestinal tract, liver and skin. We observed that alloreactive effector T cells infiltrating and attacking the lamina propria of the small and large intestines closely interact with intestinal myeloid cells of host origin. Here we asked whether these intimate interactions regulate alloreactive effector T cell responses and how they impact intestinal aGvHD. To address these questions, we employed non-invasive bioluminescence imaging, fluorescence and confocal microscopy, clinical and histopathologic scoring, flow cytometry and single cell RNA sequencing in murine models of myeloablative, MHC-mismatched allo-HSCT. In the intestinal lamina propria, we observed that allogeneic T cells closely interacted with CD11b+CD11c+CD103- radio-resistant host type hematopoietic myeloid antigen presenting cells. Selective depletion of intestinal CD11chi or CX3CR1+CD11chi host cells 3 or 8 days after allo-HSCT accelerated alloreactive T cell infiltration, increased T cell mediated inflammatory cytokine production and exacerbated tissue damage resulting in hyperacute lethal aGvHD. These results suggested a strong immunoregulatory effect of these intestinal host-type myeloid cells. Single cell RNA-Seq analysis and flow cytometry (e.g. MHC II, CD11c, F4/80, CD26, CD64, CCR2, CX3CR1), lineage reporter- and defined knockout mice determined these cells as non-classical monocyte derived macrophages as the development and differentiation of these cells did not depend on Flt3, Zbtb46, and CCR2 but rather on CSF-1R and CX3CR1. Adoptive transfer, bone marrow chimeras and parabiosis experiments revealed that these non-classical monocyte derived macrophages differentiated from non-circulating non-classical monocytic precursors. Finally, PD-L1 expression on these intestinal host non-classical monocyte derived macrophages but not on stroma or other hematopoietic cells regulated alloreactive T cell responses in the intestinal tract. Based on these findings we postulate that a specialized and persistent subpopulation of host non-classical monocyte derived macrophages can potently suppress alloreactive T cells in the lamina propria of the intestinal tract. Fostering the differentiation and function of these tissue resident cells may represent an attractive therapeutic strategy to prevent intestinal aGvHD. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Fn14 activation is involved in intestinal apoptosis after allo-HCT and contributes to gastrointestinal GVHD. Fn14 blockade with an ADCC-defective human immunoglobulin G1 antibody reduces GVHD severity without modulating GVL responses.

Description: 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