GVHD In Mouse Model Research Articles

Single-cell RNA-sequencing reveals the transcriptional landscape of lacrimal gland in GVHD mouse model

PurposeTo investigate the global transcriptional landscape of lacrimal gland cell populations in the GVHD mouse model. MethodsSingle-cell RNA sequencing and further bioinformatic analysis of dissociated lacrimal gland (LG) cells from the mouse model were performed. Parts of transcriptional results were confirmed by immunofluorescence staining. ResultsWe identified 23 cell populations belonging to 11 cell types. In GVHD LG, the proportion of acinar cells, myoepithelial cells, and endothelial cells was remarkably decreased, while T cells and macrophages were significantly expanded. Gene expression analysis indicated decreased secretion function, extracellular matrix (ECM) synthesis, and increased chemokines of myoepithelial cells. A newly described epithelial population named Lrg1high epithelial cells, expressing distinct gene signatures, was exclusively identified in GVHD LG. The fibroblasts exhibited an inflammation gene pattern. The gene pattern of endothelial cells suggested an increased ability to recruit immune cells and damaged cell-cell junctions. T cells were mainly comprised of Th2 cells and effective memory CD8+ T cells. GVHD macrophages exhibited a Th2 cell-linked pattern. ConclusionsThis single-cell atlas uncovered alterations of proportion and gene expression patterns of cell populations and constructed cell-cell communication networks of GVHD LG. These data may provide some new insight into understanding the development of ocular GVHD.

A Novel High-Throughput Method for Identifying T-Cell Receptor: Minor Histocompatibility Antigen Interactions in Mouse Models of Graft-Vs-Host Disease

Graft-vs-host disease is a complication of allogeneic stem cell transplant (alloSCT) in which donor T cells target alloantigens expressed by healthy recipient tissues. In major-histocompatibility-matched alloSCT, CD8-mediated GVHD is caused by T cell receptor (TCR) allorecognition of class-I presented minor histocompatibility antigens (miHAs). The vast majority of miHAs remain unknown between any given donor-recipient pairing. However, determining the identities of these miHAs and their cognate TCRs would prove useful towards the development of allografts that minimize GVHD yet preserve the therapeutic benefits of alloSCT. Here, we implement a high-throughput TCR cloning method, TCXpress, to screen alloreactive TCRs from a mouse model of MHC-matched alloSCT against putative miHA targets determined informatically. 129 (H-2 b) mice, which express the immunodominant miHA H60, were lethally irradiated and reconstituted with bone marrow and CD4 + and CD8 + T cells from B6 (H-2 b) donors. At day +10, recipient spleens were harvested and CD44 +PD-1 + donor CD8 + cells were single-cell sorted according to H-2Kb:H60 tetramer (Tet H60) staining into unknown miHA-reactive (Tet H60-) and control H60-reactive (Tet H60+) cohorts for TCR cloning using the TCXpress platform (BlueSphere Bio). Clonotyping was performed by sequencing the CDR3 region of TCRs. Putative miHAs were identified informatically by filtering non-synonymous variants between B6 and 129 reference exomes on predicted class I binding affinities. To interrogate TCR target specificity, Jurkat reporter cells, which express GFP and CD69 upon TCR activation, were transduced with selected alloreactive TCRs and reacted in a multiplexed fashion against a panel of B6WT3 antigen-presenting cells (H-2 b) transduced with tandem minigenes (TMG) of predicted miHAs. To test TCR reactivity against hematopoietically-expressed miHAs independent of our calling strategy, Jurkat reporters were also reacted against bone-marrow derived dendritic cells (BMDCs) from control B6 and 129 mice. From 3 recipient mice, 966 alloreactive TCRs were cloned and sequenced (735 Tet H60- and 231 Tet H60+) corresponding to 553 unique clonotypes (437 Tet H60- and 116 Tet H60+). Sixty-seven percent (N=668) of TCRs sequenced had clonotype occurrences of ≥ 2, indicating clonal expansion post-alloSCT, whereas there were no repeated clonotypes among donor B6 CD8 + cells pre-transplant (N=173 TCRs). A total of 47 Tet H60- TCR clones and 3 Tet H60+ control TCR clones were selected for transduction into Jurkat reporters and screened against a limited TMG library of 158 putative B6→129 miHAs, as well as H60. Expectedly, all TCR clonotypes isolated from the Tet H60+ sort gate reacted against H60-expressing B6WT3 cells and 129 BMDCs. From the Tet H60- TCRs, one clone was validated against another documented 129 miHA, H4, while an additional 4 clones were found to react against novel B6→129 miHAs (Serpinb8 and Cdt1). Interestingly, 2 highly over-represented clones were autoreactive against B6 tissues (B6WT3 cells and BMDCs), but not 129 BMDCs. In summary, we demonstrate the capacity of a novel TCR cloning platform to validate informatically-predicted miHAs and their cognate TCRs in a mouse model of GVHD. This approach may be readily adapted to develop new TCR:miHA therapeutics and gain a deeper understanding of GVH responses targeting miHAs.

Enterococcus Induces MHC-II Expression in the Colonic Epithelium to Aggravate Graft-Versus-Host Disease

Intestinal domination by Enterococcus, a genus of Gram-positive facultative bacteria, has been associated with an increased risk of GVHD mortality in allo-HCT patients and in preclinical models. However, the immunological mechanisms underlying this association remain unclear. Several preclinical studies have demonstrated that MHC-II expression by intestinal epithelial cells (IECs) is involved in allo-antigen presentation and GVHD and can be modulated by the intestinal microbiota. MHC-II genes are constitutively expressed by professional antigen-presenting cells (APCs) but can be induced in non-professional APCs by IFN-γ. Here we reported that Enterococcus could potentially aggravate GVHD through upregulation of MHC-II expression by IECs in an IFN-γ-dependent manner. Using an MHC-disparate mouse model of GVHD (C57BL/6J into BALB/cJ), we profiled the fecal microbiome of GVHD mice by 16S rRNA sequencing and the intestinal epithelium by flow cytometry on day 7 post-transplant. We found that endogenous Enterococcus relative abundance was positively correlated with MHC-II expression by colonic IECs (CD45- CD31- EpCAM+) during GVHD ( Fig. 1a; p=0.0004; 2 replicates; n=19). To determine whether this phenotype occurs in the absence of systemic inflammation, we utilized gnotobiotic mice treated with either E. faecalis, B. producta (a bacteria genus previously associated with improved GVHD outcomes)or PBS via oral gavage. Monocolonization with E. faecalis was sufficient to upregulate MHC-II expression by colonic IECs in non-transplanted (steady-state) animals compared to PBS-treated germfree controls ( Fig. 1b; p=0.0027; 3 replicates; n=8-12/group). On the contrary, monocolonization by B. producta did not induce MHC-II expression by colonic IECs compared to germfree controls ( p=0.96). We also observed that MHC-II upregulation was promoted by specific species within the genus Enterococcus, such that compared to PBS-treated germfree controls, only E. faecalis ( p=0.0007; 3 replicates; n=8-12/group) was able to induce MHC-II expression by colonic IECs, but not E. durans ( p=0.06) , E. gallinarium ( p=0.98) and E. hirae ( p=0.99). Using a Villin-Cre IFN- γ R fl/flmouse model with genetic ablation of the IFN-γ receptor specifically in epithelial cells, we investigated whether MHC-II expression induced by Enterococcus was dependent on IFN-γ signaling. We colonized Villin-Cre pos and Villin-Cre neg littermate controls with E. faecalis via oral gavage after antibiotics decontamination (ampicillin/enrofloxacin) and profiled the intestinal epithelium. We observed that Villin-Cre posmice exhibited a significantly lower MHC-II expression in the small intestine compared to Villin-Cre negmice on day 3 post- Enterococcus colonization ( p=0.012; 2 replicates; n=7/group), suggesting that IFN-γ signaling is necessary to mediate Enterococcus interaction with the intestinal epithelium. Finally, to explore a potential intervention to reduce intestinal inflammation upon Enterococcus domination, we treated GVHD mice with either butyrate-producing B. producta or E. faecalis. Colonization with B. producta reduced fecal Enterococcus burden on day 7 post-transplant ( p=0.007; 2 replicates; n=8/group) and improved survival in GVHD mice, compared to the E. faecalis-colonized group ( p=0.002; 2 replicates; n=18-19/group). We and others have previously shown that butyrate administration can alleviate GVHD through its effects on epithelial homeostasis. To determine whether butyrate could regulate allo-antigen presentation by the intestinal epithelium, we employed the human colorectal adenocarcinoma HT-29 cell culture. We pre-treated the cells with IFNγ (100U/ml) to induce MHC-II expression, then added butyrate (5mM) to the culture for 24 hours. Butyrate treatment was sufficient to suppress MHC-II expression by HT-29 cells, compared to DMSO-treated controls ( p=0.006; 3 replicates; n=6/group). Altogether, our results suggest that one mechanism by which Enterococcus can aggravate GVHD involves the upregulation of MHC-II expression by colonic IECs, and treatment with butyrate might serve as an intervention to prevent intestinal inflammation. Further identification of the upstream mediators involved will provide additional mechanistic insight into the interaction between Enterococcus and the intestinal epithelium at steady state and during GVHD.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Prevent Acute Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation Via Exosome-Mediated MicroRNA and Its Mechanism

To explore molecular mechanisms by which umbilical cord-derived mesenchymal stem cells suppress the development of GVHD after bone marrow hematopoietic stem cell transplantation. A mouse model of aGVHD was constructed after bone marrow hematopoietic stem cell transplantation, and the umbilical cord-derived mesenchymal stem cells were cultured, and then injected into the aGVHD mouse model, so as to investigate its prophylactic efficacy. Prophylactic effect of the exosomes isolated from umbilical cord-derived mesenchymal stem cells on aGVHD mice was assessed. Sequencing analysis of miRNA from exosomes was performed. aGVHD model was successfully constructed after hematopoietic stem cell transplantation. By injecting umbilical cord-derived mesenchymal stem cells into the GVHD mouse model, it was found that the treatment significantly prolonged survival time of mice compared to the untreated group. Injection exosomes derived from umbilical cord-derived mesenchymal stem cells into the GVHD mouse model significantly prolonged the survival time of mice compared to the untreated group. High-throughput sequencing data showed that microRNA such as miR-21 in exosomes isolated from umbilical cord-derived mesenchymal stem cells, which mainly affected the signaling pathways such as cell adhesion, RNA degradation. The umbilical cord-derived mesenchymal stem cells can prevent the occurrence of aGVHD after HSCT, which is mediate by MicroRNA in the exosomes derived from umbilical cord-derived mesenchymal stem cells.

Donor peritoneal-derived cells can attenuate graft-versus-host disease after MHC-incompatible bone marrow transplantation in mice

BackgroundGraft-versus-host disease (GVHD) remains one of the most important barriers to bone marrow transplantation (BMT) and quality of life. Peritoneal-derived cells (PCs) can regulate immune function. MethodsThe GVHD mouse model, which was established by injection with T-cell depleted bone marrow cells and spleen cells, was treated with donor PCs. The survival time, weight, GVHD score, and pathological organ damage were used to assess the efficacy of PCs. Various cytokines (e.g., TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10, and TGF-β) and immunocytes (e.g., CD4+, CD8+, Treg, and NK cells) were employed to investigate the preliminary mechanism. The cytokine levels were tested via Luminex liquid chips, real-time PCR, and Western blotting, and the proportion of each T-cell subpopulation was detected via flow cytometry. One-way Analysis of Variance (ANOVA) followed by Tukey's post-test was employed to perform multiple comparisons, and Kaplan–Meier method was used to perform survival analysis. ResultsCompared to the saline group, the PC group harbored a significant reduction in pathological damage to the colon and small intestine, a longer survival time, and a lower GVHD score. The mice in the PC group had significantly higher levels of TGF-β, IL-10, and IL-4 and lower levels of IFN-γ, TNF-α, and IL-15 than those in the saline group. In the PC group, CD8+ and Th1 ratios decreased, while CD4+, Th2, NK, and Treg ratios increased. ConclusionsDonor PCs can attenuate GVHD caused by MHC-incompatible BMT by regulating cytokine levels and immunocyte ratios.

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic effect in the treatment of GvHD. For this purpose, we studied the effect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without affecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efficacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia effect. This beneficial effect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

Abstract 2981: Establishment and validation of humanized murine modeling platforms to facilitate immuno-oncology research

Abstract Immuno-oncology (I/O) space is proven to be one of the fast-moving areas with numeral blockbuster drugs delivered to the market. However, lacking of more clinically relevant preclinical models appeared to hinder this process as the I/O field is getting more established and the therapeutic modalities becomes more complex. The traditional testing systems such as transplantable xenograft tumor models can no longer meet the requirements of I/O novel drug evaluation and the mouse models with tumor-immune microenvironment interactions are urgently needed. At Simcere, in order to overcome those technical barriers and to accommodate our fast-paced pipeline development, we attempted to establish our own I/O in vivo testing platforms. These platforms were tailored to Simcere's dynamic progress of the research programs. To date, a variety of humanized mouse model-based in vivo modalities have been established in house. These testing systems fall into 4 categories: the human immune system reconstitution mouse tumor model (either using hPBMC or hCD34 stem cell approach), the hTreg focused humanized mouse model, the hTreg interfered GVHD mouse model, and the transgenic mouse model with modified immune targets (I/O targeted model; co-developed with a patterner). Each of these model systems were characterized to gain a comprehensive understanding before they were applied to any programs. The model characterization data showed that, 1) A large population of hCD4 and hCD8 T cells was maintained in peripheral blood of the hPBMC reconstructed mice,and significant percentage of human T,B and other type of immune cells were maintained in the human stem cell reconstituted mice.Anti-PD-L1 or anti-TIGIT antibodies showed significant anti-tumor efficacy in these humanized tumor models; 2) In the hTreg focused humanized mice axis, the proportion and functions of hTregs in the spleen of the hTreg reconstituted mice treated with the Treg modulated drugs were significantly amended, indicating that the system can be used to dissect out drugs with Treg modulating functions; 3) A hTreg interfered mouse GVHD model was proven to be a useful tool to assess effects of exogenous or endogenous Tregs on progression of the disease in a chronic setting; 4) Collaborating with a business patterner, we generated genetically modified mouse models in which selected murine immune targets were replaced with human counterparts. One of these models have been used to evaluate our IND candidate molecules, where significant anti-tumor effects and meaningful PD modulation were observed. To conclude, we have developed four types of I/O in vivo platforms to facilitate development process of immune modulatory and cancer immunotherapy agents. A majority of these modalities have been validated and utilized to drive our internal I/O programs. Optimization and expansion of these platforms are currently ongoing. Citation Format: Yang Liu, Hai Huang, Danni Hao, Ran Pang, Mi Ye, Guojing Wu, Wenjing Li, Liting Xue, Zhenchuan Miao, Ming Yin, Wenqing Yang. Establishment and validation of humanized murine modeling platforms to facilitate immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2981.

Novel Variant Ig Domain (vIgD) Proteins Generated Via Directed Evolution of Igsf Domains Have Therapeutic Efficacy in Animal Models of Graft Versus Host Disease

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™ (vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro for their ability to either agonize or antagonize T cell responses.We successfully engineered the IgSF protein ICOSL, which binds to the costimulatory molecule ICOS on T cells and triggers T cell activation. We used our directed evolution platform to engineer a first-in-class single vIgD domain with higher affinity for ICOS and high affinity binding to the costimulatory molecule CD28, which ICOSL does not normally bind. Following the creation of dual ICOS/CD28 antagonism, the resulting domains were used to generate individual constructs encoding Fc-fusion proteins. These enhanced ICOS/CD28 dual antagonist Fc-fusion proteins were transfected into HEK-293 cells and protein produced by these cells was purified by protein A affinity chromatography. The resultant purified proteins were then tested in a FACS based binding assay on cells transfected with each costimulatory receptor showing up to a 2-3-fold increase in ICOS binding and high affinity binding to CD28. Various ICOS/CD28 dual antagonist Fc-fusion proteins were then tested in soluble format over a range of concentrations for functional activity in mixed lymphocyte reaction cultures (MLR) where allogeneic dendritic cells (DC) were used to stimulate purified human T cells. Most Variants showed significant ability to inhibit the MLR response as read out measuring proliferation and cytokine production. The potency of the Fc-fusion proteins varied somewhat depending on their relative affinities for CD28 and ICOS, with all being superior to wild type ICOSL protein and many showing superiority to the CD28 blocking reagent belatacept (CTLA4-Fc). The level of inhibition correlated well with binding of the Fc-fusion proteins to the T cells in culture.To assess the function of the ICOS/CD28 dual antagonist Fc-fusion proteins in vivo, a subset were tested in the human PBMC-NSG™ GvHD mouse model. This model monitors graft versus host disease generated by xenogeneic responses of T cells in human PBMCs transferred into NSG™. Administration of the dual ICOS/CD28 antagonist Fc-fusion proteins significantly protected mice from the effects of xenogeneic T cell activation in vivo with treated animals showing dramatically enhanced survival and greatly reduced disease scores. Interestingly, the level of protection correlated with the potency of the molecules in the in vitro MLR assay with less potent molecules in MLR assays failing to protect while more potent molecules protected well. Belatacept was also effective in protecting animals from GvHD, but not as well as the ICOS/CD28 dual antagonists produced by our vIgD platform. Collectively, these data demonstrate the vIgD directed evolution platform can generate IgSF proteins with altered affinities and improved binding properties. Variants engineered for desired immunological properties can effectively perturb T cell costimulation and affect T cell responses both in vitro and in vivo with the in vitro potency of the proteins correlating to in vivo potency. The dual ICOS/CD28 antagonists produced with the vIgD platform can be used therapeutically in disease settings where attenuation of T cell responses is beneficial, including GvHD and potentially other autoimmune diseases. Phase I enabling studies are in progress. [Display omitted] DisclosuresLevin:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Rickel:Alpine Immune Sciences: Employment, Equity Ownership. Wolfson:Alpine Immune Sciences: Employment, Equity Ownership. Dillon:Alpine Immune Sciences: Employment, Equity Ownership. Kornacker:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Swanson:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Peng:Alpine Immune Sciences: Employment, Equity Ownership.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

Glycolytic metabolism of pathogenic T cells enables early detection of GVHD by 13C-MRI

AbstractGraft-versus-host disease (GVHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (AHSCT). Definitive diagnosis of GVHD is invasive, and biopsies of involved tissues pose a high risk of bleeding and infection. T cells are central to GVHD pathogenesis, and our previous studies in a chronic GVHD mouse model showed that alloreactive CD4+ T cells traffic to the target organs ahead of overt symptoms. Because increased glycolysis is an early feature of T-cell activation, we hypothesized that in vivo metabolic imaging of glycolysis would allow noninvasive detection of liver GVHD as activated CD4+ T cells traffic into the organ. Indeed, hyperpolarized 13C-pyruvate magnetic resonance imaging detected high rates of conversion of pyruvate to lactate in the liver ahead of animals becoming symptomatic, but not during subsequent overt chronic GVHD. Concomitantly, CD4+ T effector memory cells, the predominant pathogenic CD4+ T-cell subset, were confirmed to be highly glycolytic by transcriptomic, protein, metabolite, and ex vivo metabolic activity analyses. Preliminary data from single-cell sequencing of circulating T cells in patients undergoing AHSCT also suggested that increased glycolysis may be a feature of incipient acute GVHD. Metabolic imaging is being increasingly used in the clinic and may be useful in the post-AHSCT setting for noninvasive early detection of GVHD.

HLA-A*02:01-directed chimeric antigen receptor/forkhead box P3-engineered CD4+ T cells adopt a regulatory phenotype and suppress established graft-versus-host disease

Background aimsTo investigate the feasibility of using CD4 + T cells genetically modified to express an allo-HLA directed CAR and FOXP3 to suppress T cell proliferation and cytokine secretion in GvHD. Methods: Human CD4+ T cells from A*02:01 negative donors were transduced to express A*02 CAR and FOXP3 and co-cultured in mixed lymphocyte reaction assays to demonstrate T cell suppression. A*02- CAR/FOXP CD4+ T cells were then injected into mice engrafted with allogeneic T cells in a GvHD mouse model. ResultsCD4+ T cells genetically modified to express allo-HLA-directed CAR and FOXP3 proliferate rapidly, downregulate CD127 and interferon-γ, express high CD25 and Helios and convert to a stable antigen-dependent suppressive phenotype. In mixed lymphocyte reaction assays, these cells potently suppressed T-cell proliferation and secreted IL-10. In a graft-versus-host disease model, A*02-CAR/FOXP3 CD4+ T cells outperformed polyclonal Tregs by reducing liver and lung inflammation, inhibiting pro-inflammatory cytokine production and limiting grafted CD3+ T-cell expansion. ConclusionsCD4 + T cells expressing allo-antigen directed HLA-specific CAR and FOXP3 act as potent, specific and stable suppressors of inflammation that out-perform their Treg counterparts both in vitro and in vivo.

Visualization of Activated T Cells by OX40-ImmunoPET as a Strategy for Diagnosis of Acute Graft-versus-Host Disease.

Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT), mediated primarily by donor T cells that become activated and attack host tissues. Noninvasive strategies detecting T-cell activation would allow for early diagnosis and possibly more effective management of HCT recipients. PET imaging is a sensitive and clinically relevant modality ideal for GvHD diagnosis, and there is a strong rationale for the use of PET tracers that can monitor T-cell activation and expansion with high specificity. The TNF receptor superfamily member OX40 (CD134) is a cell surface marker that is highly specific for activated T cells, is upregulated during GvHD, and mediates disease pathogenesis. We recently reported the development of an antibody-based activated T-cell imaging agent targeting OX40. In the present study, we visualize the dynamics of OX40 expression in an MHC-mismatch mouse model of acute GvHD using OX40-immunoPET. This approach enabled visualization of T-cell activation at early stages of disease, prior to overt clinical symptoms with high sensitivity and specificity. This study highlights the potential utility of the OX40 PET imaging as a new strategy for GvHD diagnosis and therapy monitoring. SIGNIFICANCE: OX40-immunoPET imaging is a promising noninvasive strategy for early detection of GvHD, capable of detecting signs of GvHD pathology even prior to the development of overt clinical symptoms.

Phase I Clinical Trial of CK0801 (cord blood regulatory T cells) in Patients with Bone Marrow Failure Syndrome (BMF) Including Aplastic Anemia, Myelodysplasia and Myelofibrosis

Background: Aplastic anemia is an autoimmune disease of the bone marrow (BM) characterized by defective and decreased regulatory T cells (Tregs) which results in unchecked cytotoxic T cell mediated bone marrow suppression [1]. In a xenogeneic model of induce BM aplasia, adoptive therapy with cord blood (CB) Tregs reverses such a defect (Fig 1A). Furthermore, as compared to peripheral blood (PB), CB Tregs show a higher expression of Helios, a marker of thymic derived Tregs (Fig 1B) and CB Tregs exert a superior suppression of proliferating conventional T-cells in CFSE assay (Fig 1C). In a xenogeneic mouse model of GVHD, CB Tregs can exert suppression across HLA barrier resulting in survival benefit (Fig 1D); and in another xenogenic lupus model, a single dose of CB Tregs results in significant decrease in circulating effector T cells (Fig 1E) up to 8 weeks. Furthermore, under stressful conditions of continued culture in the presence of inflammatory cytokines including IL15 and IL23, CB Tregs do not secrete IL-17 or express RORγt, a concern of plasticity that plagues PB Tregs (Fig 1F). In fact, CB Tregs increase their secretion of the suppressor cytokine, IL-10 in response to inflammatory stress, which supports our hypothesis, that CB Tregs based adoptive therapy can have wide application and can be used to treat inflammatory disorders. Therefore, we hypothesized that adoptive therapy with CB Tregs can be utilized as treatment for the inflammatory bone marrow failure (BMF) disorders including aplastic anemia, hypoplastic myelodysplasia and primary myelofibrosis. Study Design and Methods: We have now launched a phase I clinical trial examining the role of single infusion of CK0801, an allogeneic, fresh CB Treg product, utilizing novel process development that consist of well-defined qualification criteria for the starting material (cord blood units), parameters for manufacturing and culture-expansion; and well-defined analytic testing and lot release criteria, for the treatment of BMF, where 3 doses are examined: i) dose level I: 1x106 cells/kg; ii) dose level II: 3x106 cells/kg and iii) dose level III: 10x106 cells/kg (NCT03773393). The study follows 3+3 phase I design where 3 patients will be treated at dose level I. If no DLT is observed, then the dose will be escalated to dose level II and if no DLT is observed, then the dose will be escalated to dose level III. If 1 DLT is observed at a dose level, then 3 additional patients will be treated at that level. If no additional DLTs, then that dose level will be defined as MTD. If ≥2DLTs are observed at dose level II or III, then prior dose level is defined as MTD. If ≥2DLTs at dose level I, then DSMB will review and evaluate for study continuation. MTD will be decided when 6 patients are treated at a dose level with < 2 DLTs. The eligibility criteria include: i) diagnosis of aplastic anemia, myelodysplastic syndrome or myelofibrosis, ii) Age > 18 years, iii) cord blood unit matched at HLA 3 out of 6 with the patient for generation of CK0801. The primary endpoint includes dose limiting toxicity (DLT) as defined by i) severe (grade 3 or 4) infusion toxicity within 24 hours; ii) regimen related death within 30 days or iii) severe (grade 3 or 4) cytokine release syndrome within 30 days. The secondary endpoints include i) disease-specific response including complete remission, transfusion independence and hematologic improvement and ii) duration of response. The exploratory endpoints include assessment of PB and BM immune reconstitution and inflammatory cytokines at baseline and scheduled follow ups of Days 0, +1, +3, +7, +14, +21, +30, +60, +90, +180 and +365 in the post-treatment setting. These studies will include T cell compartment analysis: Treg, Effector T cells, T-cell anti-viral activity. Serum will be analyzed for inflammatory cytokines: IL1, IL2, IL4, IL6, IL7, IL8, IL10, IL17, IFN-gamma, ST2, REG3a, OPN, Follistatin, Elafin, TGF-beta. Optional exploratory cytokines: SCF, G-CSF, GM-CSF, HGF, VEGF, SDF1a, MCP1, MCP2, TARC, MIP3a, TECK, CTACK, CCL28, FGF, PDGF, EGF, TGF-α, TLR. The first patient is already consented with a planned total of 9 patients.

Tracking T Cell Activation By OX40 Immuno-PET: A Novel Strategy for Imaging of Graft Versus Host Disease

BACKGROUNDGraft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior non-invasive imaging strategies specifically detecting early GvHD are critically needed to improve clinical care of HCT recipients. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F-FDG) have largely been confounding, mainly due to their low specificity. Monitoring T cell activation and expansion using T-cell targeted PET tracers seems a more promising approach (Ronald et al., Cancer Res 77(11) 2893, 2017). We recently reported a novel immuno-PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive imaging of activated murine T cells expressing the cell surface activation marker OX40 (Alam et al., JCI 128(6) 2569, 2018). In the present work, we evaluated the utility of this immuno-PET strategy to image activated T cells in a major MHC-mismatch mouse model of acute GvHD.METHODSBalb/C (H-2Kd) recipients were irradiated with 8.8 Gy and on the same day received intravenously (i.v.) 5 x 10e6 T-cell depleted bone marrow (BM) cells with or without 1 x 10e6 CD4 and CD8 T cells positively selected from C57BL/6 (H-2Kb) mice. Severity of GvHD was assessed by clinical GvHD scoring. Flow cytometry of lymphoid organs from BM control and GvHD mice was performed at day 7 after HCT to determine OX40 protein expression on immune cells. For imaging studies, anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (an average ratio of 1.4 DOTAs per mAb was obtained) and subsequently radiolabeled with 64CuCl2 (final specific activity 10-15μCi/μg and radiochemical purity >99%). Mice were tail-vein injected with 64Cu-DOTA-mAbOX40 (100 µCi, i.v.) at day 7 after HCT and PET-CT imaging performed 24 hours after injection. Immediately following PET-CT mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter.RESULTSFlow cytometry analysis of OX40 expression in lymphoid organs isolated at day 7 after HCT revealed significantly higher proportions and absolute numbers of OX40 expressing cells in the spleen and cervical lymph nodes (LN) isolated from mice that received BM + T cells (GvHD group) compared with mice having received BM cells alone (p<0.05). In vivo OX40-ImmunoPET performed at day 8 after HCT revealed increased radiotracer uptake in spleen (p < 0.0001), mesenteric LN (p < 0.01) and the abdominal region (p < 0.001) of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64Cu-DOTA-mAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Biodistribution analysis using gamma counting of tissues confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01) and GvHD target organs e.g. small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). Importantly, outcome analysis of GvHD mice receiving tracer doses of OX40 mAb at day 7 after HCT did not reveal any significant worsening of GvHD in terms of survival, body weight loss or GvHD score, compared with mice receiving the appropriate isotype control, supporting the safety of this OX40-targeted imaging approach.CONCLUSIONThe OX40 immuno-PET tracer enabled specific imaging of alloreactive OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64Cu-DOTA-mAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting.FIGURE 1. 64Cu-DOTA-AbOX40 PET-CT imaging in a mouse model of acute GvHD. (A) Representative day 8 64Cu-DOTA-AbOX40 PET-CT images in BM controls or GvHD group. H, heart (including cardiac muscle and blood); Li, liver; Sp, spleen; Bl, blood vessels and venous sinuses; BM, bone marrow; Ab, abdomen. (B) Quantitative region of interest PET image analysis of indicated organs in BM controls (n=12, blue filled boxes) or GvHD mice (n=12, red filled boxes). Outliers are represented as dots. [Mann-Whitney test , ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05]. [Display omitted] DisclosuresGambhir:CellSight Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Establishment and Evaluation of Gvhd Mouse Model for Allogeneic Hematopoietic Stem Cell Transplantation

Objective:To study the feasibility of using cyclophosphamide alone without total body irradiation (TBI) as conditioning regimen for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and establish a graft-versus-host disease (GVHD) mouse model.Methods:Single cell suspension of spleen and bone marrow were prepared from donor C57/BL/6 mice (B6 mice) . The recipient B6D2F1 mice (F1 mice) were signed into 4 groups with 12 mice per each group. Groups I~III mice were conditioned with peritoneal injection of cyclophosphamide (300 mg/m2/d) on day -5, -4 and -3 of allo-HSCT, and group IV mice were used as control with saline injection. On day 0, group I mice were injected through tail vein with a mixture of 2×107 spleen cells, 5×106 bone marrow cells and 1×104 P815 cells; group II with 5×106 bone marrow cells and 1×104 P815 cells; group III with 1×104 P815 cells and group IV with RPMI1640 culture medium. The blood leukocyte counts were analyzed, GVHD score and the pathological leukemia infiltration of skin, liver and colon were evaluated and compared on day 30 after allo-HSCT.Results:(1) The GVHD scores of group I mice were 2 to 4 on day 14 post allo-HSCT, and mice started to die on day 15, eight (8) mice survived and the GVHD scores of them were 1 to 6 with a survival rate of 66.7% on day 30. Group II mice began to die on day 6 and only one survived on day 30 with a survival rate of 8.3%. All of group III mice died on day 18. Group IV mice survived (Figure 1).(2) The leukocyte counts in group I and group II were significantly increased on day 7 post allo-HSCT(p<0.05). There was also a significant difference in total leukocyte counts between groups I/II and group IV (p<0.05). The leukocyte counts in mice of groups I~III were significantly decreased, and there were a significant difference between group I and groups II/III/IV respectively (p<0.05) on day 14 post allo-HSCT. On day 21 after allo-HSCT, the leukocyte count of Group I and II mice were ≥1×109/L, which means that allo-HSCT were successful. The leukocyte counts in group I mice were again increased on day 28 post allo-HSCT. There was no significant difference between Group I and Group IV (p>0.05).(3) Pathological observation: The cell atrophy and necrosis in the hepatic portal area and slight infiltration of the leukemia cells in central vein were observed in group I recipient mice. The number of hepatocytes of the mice decreased, and the infiltration of the leukemia cells in the liver sinus were observed in group II. The liver was heavily infiltrated by leukemic cells in group III mice. The mouse livers were normal in group IV mice.Conclusion:It is feasible to establish GVHD model of the leukemia mice with cyclophosphamide conditioning regimen of allo-HSCT, and simple to operate. This is less harmful to people, animals and the environment.Discussion:The traditional animal model of GVHD is based on TBI conditioning regimen prior to allo-HSCT. However, the process can lead to systemic adverse reactions such as myelosuppression, radiation gastroenteritis, endocrine disorders and systemic function disorders, etc. in both the operators and animals. Therefore, the radiotherapy doses have to be low. At the same time, it is difficult to establish animal models of GVHD due to the uneven distribution of the doses. Cyclophosphamide was selected for the conditioning regimen of allo-HSCT in the study. Cyclophosphamide had the effect direct on killing tumor cells, induced recipients to tolerate the immune of donors, enabled to implant donor cells into recipients smoothly. The stable chimeras were obtained. There are a large number of T and B lymphocytes in donor's spleen cells. By injecting donor spleen cells and active immune cells derived from proliferation and differentiation of hematopoietic stem cells, the effects of graft-anti-leukemia are availably activated in recipients.AcknowledgmentsWe thank Dr Zhifu Xiang very much for his great helpful revisions. DisclosuresNo relevant conflicts of interest to declare.

Abstract B13: The prevention of lymphoproliferative lesions arising in patient-derived cancer xenografts by anti-graft-versus-host-disease agents

Abstract Xenografts derived from engrafting fresh surgical specimens directly onto immunodeficient mice have enabled the development of more relevant in vivo models for human cancers. Such patient-derived xenograft (PDX) models retain similar morphology, heterogeneities, and molecular signatures with the original cancers. We reported the rapid and efficient establishment of PDXs using super-immunodeficient NOG mice (PDX/NOG models). During the establishment process, a transplantable lymphoproliferative lesion (LPL) that replaces the original tumor cells sometimes occurs. The occurrence of LPL is the most problematic aspect of the establishment of PDX. This phenomenon may arise because of the Epstein-Barr virus due to the severely immunodeficient nature of the animal model; however, the mechanisms underlying LPL have not yet been elucidated in detail. In the present study, we histologically analyzed and tried to prevent the occurrence of LPL in PDX/NOG. Over 100 lines of cancer xenografts were established in our previous studies. Throughout these processes, we also established 10 LPL-PDX lines. Some LPL-PDX lines consist of B cells, T cells, and differentiated plasma cells. These LPL lymphocytes exhibited negligible proliferative potency in vitro. The invasions of LPL in the liver, lung, and pancreas were observed immunohistochemically. NOG mice bearing LPL-PDX (LPL-PDX/NOG) were treated with the anti-graft-versus-host-disease (anti-GVHD) drugs FK506 (1.0 mg/kg/day) and mycophenolate mofetil (MMF, 120 mg/kg/day) for 4 weeks using intraperitoneal injections. The LPL proliferations were significantly suppressed with FK506 (p=0.011) and MMF (p=0.014) compared with the control. The occurrence of LPL during establishment of PDX may be based on the GVHD-like mechanism. This phenomenon can be prevented by using anti-GVHD agents in the severely immunodeficient mice. The LPL-PDX/NOG models are also proposed for the utilization of GVHD mouse models with organic damage, particularly chronic GVHD mouse models. Citation Format: Tsuyoshi Chijiwa, Akira Noguchi, Daisuke Komura, Makoto Katayama, Mizuha Haraguchi, Haruo Hashimoto, Hiroshi Suemizu, Yoshiyasu Nakamura, Daisuke Furukawa, Takayuki Isagawa, Hiroto Katoh, Takashi Moriya, Shumpei Ishikawa, Masato Nakamura, Yohei Miyagi. The prevention of lymphoproliferative lesions arising in patient-derived cancer xenografts by anti-graft-versus-host-disease agents [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B13.

Immune modulation by apoptotic dental pulp stem cells in vivo.

Mesenchymal stem cells (MSCs) show considerable promise as a cellular immunotherapy for the treatment of a number of autoimmune and inflammatory disorders. However, the precise physiologically and therapeutically relevant mechanism(s) by which MSCs mediate immune modulation remains elusive. Dental pulp stem cells are a readily available source of MSCs that have been reported to show similar immune modulation in vitro as bone marrow MSCs. To test their potential in vivo, we used a clinically relevant humanized mouse model of GvHD in which only human T cells engraft. In this model, we found no effects on either T-cell proliferation, T-cell phenotype or disease progression. To determine if this lack of efficacy was related to a failure of engraftment or persistence of the cells, we used viability dependent radioactive cell tracking and showed that no cells were detectable after 24-h postinjection. Given the apparent failure of MSC to survive following intravenous injection, we hypothesized that their apoptosis may account for the widely reported therapeutic effect in numerous experimental models in vivo. To address this, we employed a well-established model of allergic airway inflammation to compare the efficacy of live and apoptotic MSCs in a fully immunocompetent model. In this model, both live and apoptotic dental pulp MSCs induced a robust immune suppressive reaction that was substantially greater with apoptotic cells. We propose that the mechanism of immune modulation following systemic application of MSCs is a result of cell entrapment and apoptosis occurring in the lungs.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Novel immunomodulatory proteins generated via directed evolution of variant IgSF domains

Abstract The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins expressed on immune cells that have been extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies that bind a single receptor. Moreover, wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain (vIgD™) platform using directed evolution and yeast display to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains were selected for enhanced or altered affinity to specific recombinant proteins. Fc fusion proteins incorporating these evolved immunomodulatory IgSF domains were then tested in vitro for their ability to either agonize or antagonize T cell responses. Multiple novel vIgD™ fusion proteins have been generated that significantly attenuate T cell activation in vitro as assessed by proliferation and cytokine production. Lead molecules also exhibited in vivo efficacy in the human PBMC-NSG™ GVHD mouse model. Efficacy in vitro and in vivo was superior to wild-type IgSF domains due to the induced alterations in affinity for cognate ligand and through specifically directed changes in their ability to bind additional counter-structures. Our results demonstrate that vIgDs™ can be developed to acquire unique biochemical properties that significantly enhance therapeutic utility as immunomodulatory agents. This vIgD™ therapeutic platform has broad potential to enhance the activity of biologics in treatment of both autoimmune disease and cancer.

Successful Treatment of Animal Models of Acute Graft-Versus-Host Disease with Small-Molecule TNF Inhibitor

Tumor necrosis factors(TNF, including TNFα and TNFβ) have now been shown to be two of the major mediators of inflammation and can induce other inflammatory cytokines and proteases that orchestrate inflammatory responses. Recently, many reports have indicated that overexpression of TNF involved in the development of acute graft-versus-host disease(aGVHD), and TNF inhibitors have tremendous potential for the treatment of this disease. To date, 5 of the current biomacromolecular TNF inhibitors, including infliximab (IFX), etanercept(ETN), adalimumab, golimumab, and certolizumab pegol, are available for the part of the routine treatment of patients with rheumatoid arthritis, Crohn’s disease, aGVHD and other inflammatory diseases. However, over the past years, several severe limitations of those biomacromolecular TNF inhibitors have also emerged, such as poor stability, exclusion from blood/brain barrier and inducing the development of antidrug antibodies (ADA). For this reason, small-molecule chemical compounds have been designed and developed as appropriate alternatives for overcoming those limitations associated with biomacromolecular inhibitors.We previously reported a small molecular TNFα inhibitor C87, a lead chemical compound composed of (E)-4-(2-(4-chloro-3-nitrophenyl) which could directly binds to TNF-α, was designed by computer-aided drug design(CADD) and identified by virtual screening and cell-based assays in vitro, and it could attenuated mouse acute hepatitis mediated by TNFα and improve the mouse survival in vivo. As the docking template for virtual screening of above was based on the crystal structure of TNFβ·TNFR1 complex, C87 was supposed to has the potential to be a small molecule inhibitor of TNFβ which few reports to date have indicated . Therefore, in this study, the inhibition of C87 on TNFβ-induced cytotoxicity will be elucidated in detail in vitro, and more importantly, whether C87 could effectively blocked the development of aGVHD in animal model will also be further investigated.To address those issues, we measured the inhibitory activity of C87 on TNF-mediated cytotoxicity in vitro or in vivo, the results indicated that C87, dissolved in a optimal clinical solvent, could potently and specifically inhibit the TNFβ-induced cytotoxicity in L929 cells in a dose-dependent fashion(IC50=1.59μM). C87 could effectively block TNFβ-induced the activation of Caspase3 and Caspase8 in L929 cells( cultured for 24 hours) and maintain the rate of cell survival up to 80%. In addition, C87 could also suppress the up-regulation of IL-1β and TNFβ itself induced by TNFβ, which was comparable with the TNFβ-neutralizing antibody treatment. Strikingly, C87 could directly bind to TNFβ with high affinity indicated by SPR assay(KD=750 nM). Most importantly, by using a acute GVHD mouse model, low dose C87 (2mg/kg), as single drug treatment for GVHD, could prevent the development of aGVHD in experimental mice efficiently by intraperitoneal administered. The survival rate of low dose C87 groups were 100%(control, 20%), and Low dose C87 also significantly attenuated the aGVHD severity in the damaged organs (such as skin and liver) by using histopathological analysis. And the results of further identification of inflammatory factors in the serum of the treated mice, comparing to the control mice, indicated that more than five kind of cytokines, including IL-2, IL-6, IL-9, IL-18 and GRO-α, were markedly decreased, similar potency to the CSA.Taken together, we demonstrated that small molecular inhibitor C87 could inhibit both TNFα and TNFβ-induced cytotoxicity and effectively block TNF-triggered signaling activation. Importantly, in addition to its efficacy in vitro, C87 showed the exciting treatment effect in aGVHD mouse model at low dose. Thus, our results extended previous work and suggested that C87 is one of the first effective small-molecule inhibitors of TNF identified to date in the treatment for aGVHD in animal model and has the potential to be a novel small molecular agent in treatment for TNF- mediated inflammatory disease in the future. DisclosuresNo relevant conflicts of interest to declare.