GVHD In Mice 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.

BLIMP-1 and CEACAM1 cooperate to regulate human regulatory T cell proliferation and function

Abstract BLIMP-1, encoded by the PRDM1 gene, is a major transcriptional hub distinct from Foxp3 in human Tregs, but its mechanisms of action have not been fully determined. Here, using CRISPR/Cas9-mediated disruption of PRDM1, we show that BLIMP-1 limits Treg proliferation while supports Treg functional activity. PRDM1KO human Tregs are less suppressive and exhibit increased expansion in a humanized mouse model of xenogeneic GVHD. BLIMP-1 restrains Treg proliferation by lowering IL-2R signaling through downregulation of CD25 and by inhibiting responsiveness to TCR/CD28 signaling and mTOR activation through upregulation of CEACAM1, an immune checkpoint. Expression of CEACAM1 is induced by IL-2 in human, but not mouse, Tregs. Prolonged IL-2R signaling leads to optimal BLIMP-1 expression and chromatin opening of human CEACAM1, leading to high cell surface CEACAM1 through a 3’ regulatory region containing motifs for binding PRDM1 and STATs. Correspondingly, CEACAM1 is highly induced on Tregs from autoimmune patients undergoing low-dose IL-2 therapy, and these Tregs showed reduced proliferation. In vivo studies using xenogeneic GVHD mouse model show that CEACAM1KO human Tregs have higher suppressive activities, indicating a potential role of CEACAM1 in Treg function. Collectively, our findings uncover a novel IL-2-dependent regulatory loop whereby BLIMP-1 and CEACAM1 act to limit human Treg proliferation and contribute to Treg suppressive function.

GenSci120, a humanized PD-1 agonistic monoclonal antibody, enhances the binding of both PD-L1 and PD-L2 to PD-1, and mitigates the symptoms of GVHD in a mouse model

Abstract Background: Autoimmune diseases impose tremendous health and economic burden to the individuals and families they affect. The fundamental mechanism of autoimmune diseases is a break in immune tolerance and homeostasis. PD-1 plays an important role in maintaining immune tolerance by antagonizing CD28/TCR signaling in autoreactive T cells. Stimulating the PD-1 co-inhibitory pathway by an agonistic antibody would suppress pathogenic immune responses and restore immune homeostasis. Methods: A PD-1 agonistic monoclonal antibody, GenSci120, was generated by Beacon single plasma cell cloning and murine antibody humanization technologies. In vitro T cell inhibition, ADCC and ligand blocking activities of GenSci120 were characterized. In vivo efficacy in a mouse GVHD model and PK in the human FcRn transgenic mice were evaluated. Results: GenSci120 exhibited potent T cell inhibition activity in a luciferase reporter assay and a primary T cell proliferation assay. GenSci120 also showed ADCC activity in an LDH release assay. Remarkably, GenSci120 enhanced the binding of both PD-L1 and PD-L2 to PD-1 in a flow cytometry assay. In a mouse model, GenSci120 significantly alleviated the symptoms of GVHD. Furthermore, GenSci120 demonstrated longer half-life than competitors’ antibodies in the human FcRn transgenic mice. Conclusion: These data suggest the potential of GenSci120 as a novel therapy to treat autoimmune disorders and support the evaluation of this molecule in clinical studies.

Recombinant Human Thrombopoietin Reduces the Risk of Acute Graft-Versus-Host-Disease and Its Mechanisms

Objective: Acute graft-versus-host-disease (aGVHD) is an important complication that is observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to evaluate the effects of recombinant human thrombopoietin (TPO; rhTPO) on aGVHD using clinical data, mice models, and peripheral blood mononuclear cells (PBMCs). Methods: From 2016 to 2018, 162 cases of allo-HSCT in our hospital were recruited in this study. The correlation between TPO use and aGVHD was analyzed. After a GVHD model was constructed in Balb-C mice, HE staining was performed to compare the pathophysiological changes of the liver, intestine, lung, kidney, and other organs of the mice. The levels of lymphocyte subsets and cytokines in the mice spleens in the different groups were detected. PBMCs were isolated from the peripheral blood of healthy persons and patients with allo-HSCT via Ficoll gradient centrifugation and treated with different concentrations of rhTPO. The proliferation of PBMCs was detected via the CCK8 method, and the differentiation of PBMCs into lymphocytes was analyzed via flow cytometry. Results: 1) The occurrence of aGVHD was observed in 10 patients (47.6%) in the control group and 27 (23.1%) in the treatment group. The difference between the two groups was statistically significant (χ2=5.465, P=0.019). 2) The results of the aGVHD mice proved that rhTPO decreased the damage to the mouse liver, intestine, lung, and kidney tissues. In addition, splenic cell subpopulations CD4+cells, CD8+cells, T cells, and NK cells increased in the GVHD group but decreased due to rhTPO (P<0.05). After rhTPO treament, T cell subpopulations Th2 and Th17 cells decreased in the GVHD mice (P<0.05). Cytokines CD8+IL-17A+ and CD8+IL-4+IFNγ showed no difference between the four groups (P>0.05). Compared to GVHD mice, CD8+IL-4+ decreased in TPO-treated GVHD mice, and the effect of TPO at a high dose was more significant (P<0.05). 3) The results of the PMBCs suggested that a low dose of rhTPO could promote the proliferation of PMBCs, while a high dose could inhibit the proliferation. rhTPO promoted the differentiation of PBMCs into the CD3-PC5 subset. Conclusions: Altogether, this study indicates that rhTPO reduces the risk of aGVHD and is a protective factor against the occurrence of aGVHD.

Coexpression of Tim-3 and PD-1 of T Lymphocytes Prevents Acute Graft-Versus-Host Disease

T lymphocytes are critical players in acute graft-versus-host disease (aGVHD). TIM3expression on the T cell surface was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, their clinical potential is greatly limited by their low cell number in peripheral blood (PB). We already introduced a novel method to obtain a high number of PD1 +TIM3 + lymphocytes that culturing CD3 + cells isolated from G-CSF mobilized peripheral blood stem cells (CD3 +G-PBSC) with human serum albumin (HSA). We also confirmed that cultured PD1 +TIM3 + lymphocytes are the most potent subset of the CD3 + cells that inhibit T cell proliferation. In this study, we investigate the immunosuppressive effects of PD1 +TIM3 + lymphocytes after a 2-day culture with HSA (D2-CD3 +G-PBSC) or after a 4-day culture with HSA (D4-CD3 +G-PBSC). Also, we evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes in the xenograft mouse aGVHD model. Methods. The healthy donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator, and CD3 + cells were isolated by positive selection by magnetic-activated cell sorting (MACS) using CD3 Dynabeads™. CD3 + G-PBSC were cultured with 5% HSA for 2 days or 4 days (1x10 5 cells/mL). PD1 and TIM3 expressions were analyzed using flow cytometry (FACS). Sorted T lymphocytes were cultured with irradiated allo-MNCs for 3 days (Mixed Lymphocyte Reaction; MLR). 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure T cell proliferation. aGVHD was induced by intraperitoneal injection of human PB MNCs to the NOD-SCID IL-2Rγ null mouse after exposure of 3Gy irradiation. The treatment efficacy of PD1 +TIM3 + lymphocytes was analyzed by determining the survival rate after tail vein injection of PD1 +TIM3 + lymphocytes (1x10 7/kg). Results. Normally, very few cells (0.2±0.2%) were PD1 +TIM3 + lymphocytesin PB. The numbers of PD1 +TIM3 + lymphocytes markedly increased by 74-fold in D2-CD3 +G-PBSC and 370-fold in D4-CD3 +G-PBSC, respectively. CD3 + G-PBSC were composed of three types of cells such as PD1 -TIM3 -, PD1 +TIM3 -, and PD1 +TIM3 + lymphocytes. To check the origin of PD1 +TIM3 + lymphocytes after HSA treatment, PD1 -Tim3 - and PD1 +Tim3 - lymphocytes from CD3 +G-PBSC were sorted and cultured with HSA. It was confirmed that PD1 +TIM3 + lymphocytes originated from PD1 -TIM3 - lymphocytes. To assess the immunosuppressive effects of cultured T lymphocytes, we used CSFE-MLR. D2-CD3 +G-PBSC significantly increased the levels of unstimulated T cells compared to D4-CD3 +G-PBSC (% of unstimulated T cells in D2-CD3 +G-PBSC vs. in D4-CD3 +G-PBSC; 38.9±3.0% vs. 13.9±6.6%). Also, both PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated significantly enhanced levels of immunosuppression (% of unstimulated T cells PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 31.6±5.5 and 36.6±13.3, respectively). Lastly, we tested the effects of anti-PD1 or anti-TIM3 blocking mAb on the PD1 +TIM3 + lymphocytes. The immunosuppressive effects of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC were significantly decreased by anti-PD1 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 21.1±4.5 from 31.6±5.5 and 18.4±7.8 from 36.6±13.3, respectively). However, the immunosuppression of PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC was not decrease by anti-TIM3 mAb (% of unstimulated T cells in PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC; 28.7±3.3 from 31.6±5.5 and 22.7±5.6 from 36.6±13.3, respectively). These data indicated that PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC have the same phenotype as PD1 +TIM3 + lymphocytes from D4-CD3 +G-PBSC, however, their functions are different. Next, we used the xenograft mouse aGVHD model to evaluate the treatment efficacy of PD1 +TIM3 + lymphocytes for preventing aGVHD. In the control group, 14% of the mice survived at 100 days. Mice that were treated with PD1 +TIM3 + lymphocytes from D2-CD3 +G-PBSC and D4-CD3 +G-PBSC demonstrated 100% and 71% survival rate at 100 days, respectively. Conclusion. Ex vivo HSA treatment of CD3 + G-PBSCs for 2 days shows promising therapeutic potential for the treating patients with aGVHD.

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.

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

Pharmacological targeting EZH2 to modulate chronic graft-versus-host disease

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.

Dendritic cell-based amelioration of graft versus host disease in humanized mice

Abstract Graft versus host disease (GVHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pre-transplantation therapy, up to 60% of these patients develop aGVHD, and require additional immunosuppressive intervention. Previously we showed that treatment of mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, significantly increased survival of GVHD mice compared to control PBS-treated mice (70% vs 10%, respectively), with EPS-treated mice surviving more than 100 days after aGVHD induction. To test if EPS can also ameliorate GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of HLA-A2neg human PBMCs. After deriving dendritic cells (DC) from CD34+ cord blood cells, we pretreated them with EPS or PBS, and injected them together with PMBCs into the NSG-HLA-A2 mice. We found that EPS-treated DC significantly prolonged the survival of these mice, compared to PBS-treated DC. We also found that EPS-treated DC downregulated expression of activation markers (CD80, CD86), upregulated expression of inhibitory molecules (PD-L1, PD-L2), and inhibited activation of alloreactive T cells in MLR. We conclude that EPS induces tolerogenic human DC, which ameliorate GVHD in humanized NSG-HLA-A2 mice, likely by inhibiting alloreactive T cell response. Supported by NIH R41 AI155281

BET-bromodomain and EZH2 inhibitor–treated chronic GVHD mice have blunted germinal centers with distinct transcriptomes

AbstractDespite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

Dual Inhibition of the MEK/ERK and PI3K/AKT Pathways Prevents Pulmonary Graft-Versus-Host Disease through Suppression of Arteriovenous Inflammation and Bronchiolitis

IntroductionDespite clinical development of novel immunosuppressants, the prognosis of pulmonary graft-versus-host disease (pGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still poor. It is clinically classified into obstructive and constrictive subtypes, but its pathophysiology is not fully elucidated due to difficulty in collecting human lung specimens. We have shown that the MEK inhibitors that target the MAP kinase pathway suppress gastrointestinal and cutaneous GVHD in mice. Therefore, other kinase inhibitors may have potentials to suppress pGVHD. We here explored key molecules in pGVHD patients who underwent lung transplantation and verified the potency of dual inhibition of the MEK/ERK and PI3K/AKT pathways.MethodsFor human histopathological analysis, we reviewed 45 lung specimens from the allo-HSCT recipients who underwent lung transplantation at our institute for pGVHD from 2008 through 2018. Single-cell level imaging mass cytometry of human pGVHD specimens was performed to visualize interactions of lymphocytes and monocytes around bronchioles. As for a murine pGVHD model, eight-week-old B10.BR (H2K k) mice were pretreated with 240 mg/kg cyclophosphamide and 7 Gy total body irradiation, followed by infusion of C57BL/6J (H2K b) bone marrow cells and splenocytes. Vehicles, the MEK inhibitors trametinib (tra) 0.1 mg/kg/day, cobimetinib (cobi) 0.1 mg/kg/day, the PI3K inhibitor taselisib (tase) 5 mg/kg/day and tacrolimus (tac) 1 mg/kg were given from day 0 through 28. Evaluation was made at day 42 following transplant: primary endpoint was survival rate and secondary endpoints included development of bronchiolitis and values of airway resistance and lung compliance. Immunosuppressive effects of those reagents for human cells were evaluated in vitro: CSFE dilution of T cells stimulated with allogeneic dendritic cells; expression of CD23/CD69 on B cells activated with IL-4/CD40L; TNF-α production of monocytes stimulated with lipopolysaccharide.ResultsThree pathological findings characterize human pGVHD: bronchiolitis obliterans, lymphocyte infiltration and wall thickening of bronchioles; pleuroparenchymal fibroelastosis (PPFE), proliferation of subpleural elastic fibers; peribronchitis, macrophage aggregation around the bronchi. Most cases with PPFE had subpleural arteriovenous inflammation, and immunostaining revealed that B cells and macrophages were abundantly infiltrated around the bronchioles. These results indicated that suppressing B cells and macrophages may be crucial to prevent pGVHD through avoiding vascular inflammation and bronchiolitis.In the murine pGVHD, cobi but not tra/tac improved survival rate (vehicle 31%; cobi 62%, tra 7%, tac 10% at day 42; p < 0.01). Cobi attenuated development of bronchiolitis, venulitis and peribronchial macrophage aggregation. Cobi improved respiratory functions, such as airway resistance (vehicle 1.65 ± 0.19, cobi 2.86 ± 0.75 cmH 2O.s/mL, p < 0.001) and lung compliance (vehicle 13.14 ± 0.71 cobi 8.99 ± 1.08 μL/cmH 2O, p < 0.05). Notably, T cell infiltration around bronchioles was inhibited by cobi and tra, but B cell infiltration was inhibited only by cobi.Whereas cobi inhibited phosphorylation of AKT, tra compensatory upregulated it. Cobi inhibited B cell activation more than tra (%CD23 +CD69 + B cells: 32.8% with cobi 1μM, 53.0% with tra 1μM, p < 0.05). In addition, cobi suppressed TNF-α production by monocytes more strongly than tra (%TNF-α + monocytes: 53.1% with cobi 1μM vs 71.6% with tra 1μM, p < 0.05). Dual inhibition of the MEK/ERK and PI3K/AKT pathways with combination of tra and tase strongly suppressed B cells in vitro, and improved survival rate compared with vehicle and monotherapy of tra or tase in mice (Figure). Finally, imaging mass cytometry of human pGVHD specimens revealed that lymphocytes around bronchioles and venules were positive for phospho-ERK1/2 and phospho-AKT.ConclusionspGVHD is associated with vascular inflammation and bronchiolitis in humans and mice. Furthermore, phosphorylation of ERK1/2 and AKT is upregulated in human pGVHD. Given that dual inhibition of MEK/ERK and PI3K/AKT signaling suppresses vascular inflammation and bronchiolitis thorough inhibition of B cells and monocytes in mice, this strategy may provide a novel treatment option against human pGVHD. [Display omitted] DisclosuresTakaori-Kondo: ONO PHARMACEUTICAL CO., LTD.: Research Funding; Bristol-Myers K.K.: Honoraria; Celgene: Research Funding.

Differentiation of a CD4/CD8αβ Double Positive T Cell Population from the CD8 Pool Is Both Predictive and Sufficient to Mediate Graft-Vs-Host Disease

Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology.To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation.To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8.Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. [Display omitted] DisclosuresCapitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

Donor bone marrow–derived macrophage MHC II drives neuroinflammation and altered behavior during chronic GVHD in mice

AbstractGraft-versus-host disease (GVHD) remains the leading cause of nonrelapse mortality after allogeneic stem cell transplantation for hematological malignancies. Manifestations of GVHD in the central nervous system (CNS) present as neurocognitive dysfunction in up to 60% of patients; however, the mechanisms driving chronic GVHD (cGVHD) in the CNS are yet to be elucidated. Our studies of murine cGVHD revealed behavioral deficits associated with broad neuroinflammation and persistent Ifng upregulation. By flow cytometry, we observed a proportional shift in the donor-derived T-cell population in the cGVHD brain from early CD8 dominance to later CD4 sequestration. RNA sequencing of the hippocampus identified perturbations to structural and functional synapse-related gene expression, together with the upregulation of genes associated with interferon-γ responses and antigen presentation. Neuroinflammation in the cortex of mice and humans during acute GVHD was recently shown to be mediated by resident microglia-derived tumor necrosis factor. In contrast, infiltration of proinflammatory major histocompatibility complex (MHC) class II+ donor bone marrow (BM)–derived macrophages (BMDMs) was identified as a distinguishing feature of CNS cGVHD. Donor BMDMs, which composed up to 50% of the CNS myeloid population, exhibited a transcriptional signature distinct from resident microglia. Recipients of MHC class II knockout BM grafts exhibited attenuated neuroinflammation and behavior comparable to controls, suggestive of a critical role of donor BMDM MHC class II expression in CNS cGVHD. Our identification of disease mediators distinct from those in the acute phase indicates the necessity to pursue alternative therapeutic targets for late-stage neurological manifestations.

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.

The immunoregulatory functions of exosome derived from mesenchymal stem cells that are genetically modified by adeno-associated virus

目的验证天然状态间充质干细胞来源的外泌体（MSC-exosome）治疗小鼠急性移植物抗宿主病（GVHD）的效果和可能机制，探索并建立定向基因修饰MSC-exosome的方法并验证修饰后MSC-exosome的功能。方法构建小鼠急性GVHD模型，观察比较不同剂量MSC-exosome组和MSC组的生理指标评分、生存和体重减低程度；进而通过体外T细胞活化实验和体内OVA抗原特异性T细胞活化实验检测比较组间活化T细胞的增殖水平。构建重组表达载体，获得携带PD-L1和PD-L1-ITGB1的AAV2YF3突变体，进而感染人MSC，分离获得其外泌体。检测比较天然状态和修饰后MSC-exosome在体外和体内对活化T细胞的增殖水平和调节性T细胞（Treg）比例的影响。结果①MSC-exosome（300 µg×3次）和小鼠MSC（1×106×3次）均能够有效改善急性GVHD小鼠的生理指标评分、生存和体重减低程度。②相比IL-2对照组，10、25、50 µg人MSC-exosome和1×106人MSC体外共孵育处理均能够抑制活化T细胞的增殖，增殖比例分别为86.0％（IL-2）、40.0％、39.6％、42.8％和41.0％；相比PBS对照组，50、100、200 µg小鼠MSC-exosome和1×106小鼠MSC在体内均能够抑制抗原特异性活化的OT-1细胞的增殖，增殖比例分别为42.6％、33.1％、14.2％、10.6％和14.6％。③携带PD-L1和PD-L1-ITGB1的AAV2YF3突变体定向修饰人MSC-exosome的表达率分别超过40％和60％。④相比天然状态，PD-L1和PD-L1-ITGB1定向修饰后MSC-exosome在体内对抗原特异性活化的OT-1细胞具有更好的增殖抑制效果；在体外亦能明显抑制活化T细胞的增殖，并诱导提高Treg的比例。结论MSC-exosome具有与MSC相似的免疫调节作用。经过PD-L1和PD-L1-ITGB1修饰后的MSC-exosome能够有效抑制活化T细胞的增殖，并且能够诱导提高Treg的比例。

Hybrid cytokine IL233 renders protection in murine acute graft vs host disease (aGVHD)

Previously, we generated IL233, a hybrid cytokine composed of interleukin (IL)-2 and IL-33, with better therapeutic potential than either cytokine in multiple inflammatory diseases, in part through promoting T-regulatory cells (Tregs). Here we test the potential of IL233 pretreatment in a murine model of excessive Th1 activation, the parent-into-F1 model of acute GVHD (aGVHD). Five days of IL233 pretreatment of the recipients blocked or delayed the aGVHD-linked loss of B cells as seen in either the peripheral blood (day-11) or lymph nodes (day-14). IL233 pretreatment also prevented the expansion of donor CD8 T-cells in blood and LN at day-14 and significantly reduced day-14 serum IFNγ and TNFα compared to saline treated GVHD mice although, the level of Tregs did not statistically differ between saline and IL233-treated mice. Overall, the current study provides support for the use of IL233 as a therapeutic option in excessive Th1/CD8-driven conditions.

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.