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Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.ResultsSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.ConclusionsNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve theSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention andSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparableNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches.

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During aGvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that in absence of host hematopoietic APCs, aGvHD cannot be prevented, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012, Koyama et al., Nat Med 2012). However, the exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses remains controversial and needs to be proven in vivo. Fibroblastic reticular cells (FRCs) have shown to provide the crucial delta-like notch ligand to alloreactive T cells (Chung et al., JCI 2017) in aGvHD, therefore we investigated the role of FRCs MHC class II in aGvHD and their potential role as non-hematopoietic APCs in MHC class II dependent manner. In vitro cultured FRCs cell line as well as FRCs from lethally irradiated mice upregulate MHCII and co-stimulatory molecules. Moreover, FACS sorted FRCs (CD45-CD24-CD31-gp38+) were able to process DQ-OVA via MHC class II machinery, indicating that FRCs have the potential to activate CD4+ T cells. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation initially in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB/NàC57Bl/6). We generated MHCIIΔCcl19 mice with a Ccl19-intrinsic deletion of MHC class II on all Ccl19 expressing reticular lineage cells by crossing mice with floxed H2-Ab1 gene (H2-Ab1fl) with a mouse expressing Cre recombinase under the control of the Ccl19 promoter (Ccl19Cre). On day+3 after allo-HCT, CD4+ T cells activation (CD44 and CD25 expression) and proliferation (Ki67 expression and CFSE dilution) did not differ in the MHCIIΔCcl19 mice from H2-Ab1fl wildtype littermates. To further elucidate FRCs MHC class II in aGvHD milieu, we utilized iFABP-tOVA transgenic model in which OVA is expressed by intestinal epithelial cells as well as ectopically by FRCs of the lymphoid organs. OT-II cells transferred from RagΔ background mice failed to proliferate in the mLNs of lethally irradiated iFABP-tOVA, whereas excessive proliferation was observed in CD11c.DOG mice (where OVA is presented by CD11c-expressing cells). Taken together these results indicate that MHCII on FRCs does not play a role in direct antigen presentation and CD4+ T cell activation. Next, we asked whether MHCII on FRCs influences alloreactivity of CD4+ T cells in the symptomatic phase of aGvHD. Indeed, in MHCIIΔCcl19 mice, CD4+ T cells expressed higher levels of effector molecules: CD44 and CD127 as well as the proliferation marker Ki67 on day +30 of allo-HCT. Furthermore, the proportion of donor CD90.1+CD4+FoxP3+ regulatory T cells (Tregs) were reduced in MHCIIΔCcl19 mice as compared to H2-Ab1fl wild-type littermates. This led to overall poor survival of MHCIIΔCcl19 mice by day+60 after allo-HCT. At this time point in MHCIIΔCcl19 mice CD4+ T cells displayed higher levels of CD44, CD127 and Ki67 and down-regulated PD-1 and Lag3. To further elucidate the effect of FRCs MHC class II on CD4+FoxP3+ donor Tregs, we transplanted CD90.1+CD4+CD25hi Tregs, TCD BM from FVB mice along with naïve luc+ CD90.1+CD4+ T cells from FVB.L2G85 mice. Tregs protected against aGvHD in H2-Ab1fl littermate controls whereas Tregs could not protect MHCIIΔCcl19 recipients rendering them susceptible to aGvHD and to poor overall survival. Conclusively, these results indicate for the first time that MHC class II on FRCs assists to maintain donor Tregs in the SLOs after allo-HCT. Conclusively, we propose a model in which FRCs promote T cell alloreactivity by providing notch ligands (Chung et al., JCI 2017) in the initiation phase and mitigate aGvHD by maintenance of Tregs via MHC class II in the aGvHD-effector phase. Disclosures Einsele: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During acute GvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that absence of host hematopoietic APCs does not prevent acute GvHD, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012). Furthermore, it was even suggested that in the absence of professional APCs allogeneic CD4+ T cells can be activated in the lamina propria by MHC class II expressing myofibroblasts (Koyama et al., Nat Med 2012). As exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses are essential to dissect the priming of GvHD-inducing vs. GvL-mediating allogeneic T cells, we investigated the role of lymph node stromal cells (LNSCs) in the initiation phase of aGvHD and their potential role as non-hematopoietic APCs. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation first in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB→B6). Under these conditions, CD4+ and CD8+ T cells activation and proliferation occurred exclusively in secondary lymphoid organs (SLOs) and not in the intestinal lamina propria early after allo-HCT within the first three days after allo-HCT. To study non-hematopoietic antigen presentation early after allo-HCT, we generated bone marrow B6.MHCIIΔ/Δ→B6.WT chimeras that lacked MHC II expression in the host hematopoietic compartment. Subsequent allo-HCT of these chimeras revealed activation and expansion of allogenic donor CD4+ T-cells exclusively in SLOs and not the lamina propria in the first three days after transplantation. Next, we generated recipient mice that selectively lacked MHCII expression either in CD11c expressing cells or under the control of the Vav1-promoter in all hematopoietic cells. In both type of recipients, allogenic donor CD4+ T-cells were activated within 3 days after allo-HCT in SLOs and no other tissues. After irradiation of B6.WT mice we observed LNSCs upregulate co-stimulatory receptors early after irradiation (24 and 72 hours), in vitro and in vivo suggesting that they may act as active non-hematopoietic APCs. Next, we performed mixed lymphocyte reactions (MLRs) of irradiated APCs derived from TgVav1-Cre+MHCIIΔ/Δmice with alloreactive CD4+ T cells from FVB mice. Here, we observed in the total absence of hematopoietic MHCII antigen presentation reduced but still pronounced activation of alloreactive CD4+ T cells. Therefore, in these transgenic allo-HCT models, hematopoietic cells in the SLOs were dispensable for alloreactive CD4+ T cell activation in the initiation phase of aGvHD. To ascertain that SLOs are the exclusive priming sites of alloreactive CD4+ T cells, we transplanted mesenteric lymph nodes (mLNs) from a B6.CD11c.DTR mice, depleted of CD11c+ cells into B6.MHCIIΔ/Δmice. After successful engraftment of donor mLNs in these mice, allo-HCT revealed these as unique priming sites in MHCII deficient hosts for alloreactive CD4+ T cells, which differentiated into CD44hiCD62Llow effector T cells that were detectable in the transplanted mLNs. Conclusively, these results indicate that specialized non-hematopoietic lymph node stromal cells prime alloreactive CD4+ T cell within the SLOs early after allo-HCT. Pinpointing the molecular pathways in these non-hematopoietic APCs within SLOs that trigger alloreactive T cell responses may prove fruitful for selective therapeutic intervention after allo-HCT. Disclosures No relevant conflicts of interest to declare.

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.