Human Graft-versus-host Disease Research Articles

Targeting T cells with tetravalent bispecific antibodies for the treatment of graft-versus-host disease

AbstractAllogeneic hematopoietic stem cell transplantation is an established treatment for hematological malignancies and some genetic diseases. Acute graft-versus-host disease (GVHD) is the most common and debilitating side effect with poor survival rates of 5% to 30% for severe cases. In this manuscript, we describe a tetravalent T-cell–engaging bispecific antibody (BsAb) based on the immunoglobulin G-[L]-single-chain variable fragment (IgG-[L]-scFv) platform, with all 4 binding domains specific for CD3. In vitro, picomolar concentrations of the CD3×CD3 BsAb induced potent lysis of activated CD4 and CD8 T cells. In immunodeficient mice, in which human T cells induced xenogeneic GVHD, administration of 0.1 μg BsAb per dose depleted the majority of T cells from the peripheral blood, and 10 μg per dose completely reversed established GVHD and achieved a 100% survival rate. In mice bearing NALM6-luc xenografts, treatment with CD3×CD19 BsAb and activated human T cells induced complete remission of the leukemia, and all treated mice developed GVHD by 50 days after treatment. CD3×CD3 BsAb (3-30 μg doses) reversed clinical signs of GVHD, allowing long term follow-up beyond 250 days. T cells were undetectable by polymerase chain reaction in 4 of 5 mice in the 30 μg CD3×CD3 BsAb group 180 days after leukemia injection, and complete necropsies on day 259 revealed no evidence of human T cells or leukemia cells. Curing GVHD allows for long-term follow-up of tumor response heretofore impossible in humanized mouse models. Further studies are warranted to determine whether the CD3×CD3 BsAb has potential for treating clinical GVHD and other autoimmune diseases in humans.

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Inflammatory Change of FoxP3 + Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 + regulatory T (Treg) cells are a subset of CD4 + T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells. We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 hiCD127 loFoxP3 + Treg cells among CD4 + T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA -FoxP3 hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA -FoxP3 lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD. Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA -FoxP3 lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD. We further checked the suppressive activity of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients. TNF-α + Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α - counterpart. Similarly, the percentage of CD39 + cells was lower in TNF-α + Treg cells than in the TNF-α - counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 + T cells or CD8 + T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells in acute GvHD patients. The frequency of TNF-α + cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α + Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD. Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties

BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine–cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Lack of IFN-γ Receptor Signaling Inhibits Graft-versus-Host Disease by Potentiating Regulatory T Cell Expansion and Conversion.

IFN-γ is a pleiotropic cytokine that plays a controversial role in regulatory T cell (Treg) activity. In this study, we sought to understand how IFN-γ receptor (IFN-γR) signaling affects donor Tregs following allogeneic hematopoietic cell transplant (allo-HCT), a potentially curative therapy for leukemia. We show that IFN-γR signaling inhibits Treg expansion and conversion of conventional T cells (Tcons) to peripheral Tregs in both mice and humans. Mice receiving IFN-γR-deficient allo-HCT showed markedly reduced graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects, a trend associated with increased frequencies of Tregs, compared with recipients of wild-type allo-HCT. In mice receiving Treg-depleted allo-HCT, IFN-γR deficiency-induced peripheral Treg conversion was effective in preventing persistent GVHD while minimally affecting GVL effects. Thus, impairing IFN-γR signaling in Tcons may offer a promising strategy for achieving GVL effects without refractory GVHD. Similarly, in a human PBMC-induced xenogeneic GVHD model, significant inhibition of GVHD and an increase in donor Tregs were observed in mice cotransferred with human CD4 T cells that were deleted of IFN-γR1 by CRISPR/Cas9 technology, providing proof-of-concept support for using IFN-γR-deficient T cells in clinical allo-HCT.

Comparative transcriptome profile of mouse macrophages treated with the RhoA/Rock pathway inhibitors Y27632, Fingolimod (Gilenya), and Rezurock (Belumosudil, SLx-2119).

Macrophages play a crucial role in, the currently uncurable, chronic rejection of transplants. In rodent transplantation models, inhibition of the RhoA/Rock pathway disrupts actin-related functions of macrophages, preventing them from entering the graft, and reducing vessel occlusion, fibrosis, and chronic rejection. Among RhoA/Rock inhibitors that inhibit chronic rejection in mouse transplantation are Y27632, Fingolimod, and Rezurock. In a mouse model, Rezurok is more effective in preventing fibrosis and less effective in preventing vessel occlusion than Y27632 or Fingolimod. Fingolimod is FDA-approved for treating multiple sclerosis (MS) and Rezurock for chronic graft versus host disease (GVHD). Still, none had been tested for chronic rejection in humans. To explain the differences in the anti-chronic rejection properties of Y27632, Fingolimod, and Rezurock, we compared the transcriptome profile of mouse macrophages treated with these compounds separately. Treatment with Y27632 or Fingolimod downregulated GTPase and actin pathways involved in cell migration. Rezurock downregulated genes related to fibrosis, such as PTX3, CCR2, CCL2, cell cycle, DNA replication, adaptive immune response, and organelle assembly, while Fingolimod also specifically downregulated NOTCH1 at mRNA . The result of this study not only uncovers which pathways are shared or specific for these drugs but will help in the development of macrophage pathway-targeted therapies in human transplantation, MS, and GVHD. Because macrophages are the major players in immune response, tissue regeneration, renewal, and homeostasis, and development of many diseases, including cancer, the data compiled here will help in designing novel or improved therapies in many clinical applications.

Abstract 5195: BNDG B2m KO plus mouse model is a powerful tool for immunotherapy

Abstract The B-NDG (Biocyotgen; NOD; DNAPK null; IL2rg knockout) mouse model was developed to be an ideal testing platform for anti-tumor therapies in Cell-Line and Patient Derived Xenograft Models (CDX and PDX respectively). B-NDG mice exhibit several notable advantages over classical NOD-scid mice, including increased life span, NK cell deletion, and significantly improved xenograft efficiency. However, like the NOD-scid mice, the utility of B-NDG mice in long-term studies is severely limited by a high incidence of severe and early-onset GvHD (Graft versus Host Disease) - limiting experimental durations to as little as 30 days. To reduce the incidence and severity of GvHD in B-NDG mice, Biocytogen developed the B-NDG B2m KO plus mice. B-NDG B2m KO plus mice was generated using Cas9 approach. Specifically, endogenous mouse B2m was deleted, while B2m cDNA was fused with Fcgrt. MHC Class I and Class II expressions, and immune markers were verified by flow cytometry. Concentration of i.v. administered human IgG was quantified by ELISA. CDX models were generated, and antitumor therapies were administered using established protocols described below. Compared with NOD-scid and B-NDG mice, B-NDG B2m KO plus mice showed undetectable MHC Class I expression in all tested tissues, without compromising MHC Class II expression. Moreover, no significant change in immune profiles or IgG turnover was detected, demonstrating highly targeted disruption of MHC Class I expression in B-NDG B2m KO plus mice. In the human PBMC-induced GvHD model, B-NDG B2m KO plus mice showed significantly improved survival, delayed onset and reduced clinical severity throughout the course of the model. Compared with B-NDG mice, B-NDG B2m KO plus mice exhibits significantly prolonged usefulness, demonstrating utility for up to 50 days after engraftment. To demonstrate the advantages of our B-NDG B2m KO plus mouse model, we conducted two proof-of concept studies that are often limited by GvHD. In the first study, B-NDG B2m KO plus mice were engrafted with human PBMCs and tumorigenic RKO cells on Day 0 and 14 respectively, treated with either vehicle or pembrolizumab and ipilimumab on Day 20, and observed until Day 48. Pembro/ipi treatment significantly inhibited tumor growth. In the second study, B-NDG B2m KO plus mice were engrafted with tumorigenic human NCI-H226 cells on Day 0, treated with either vehicle or CAR-T cells (5E5) once tumor size reached ~150 mm3 (around Day 20), and observed until Day 56. CAR-T treatment significantly suppressed tumor growth. Together, these studies demonstrated that the B-NDG B2m KO plus mouse model is a powerful tool for extended term anti-tumor studies, especially for studies involving immuno- and CAR-T therapies. Citation Format: Hongmei Jiang, Jianqiu Xiao, Travis Rothrock. BNDG B2m KO plus mouse model is a powerful tool for immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5195.

Spatial Transcriptomics of Tissue-Based Immune Response to Acute Graft-Versus-Host Disease of the Lower Gastrointestinal Tract

Background: Corticosteroid resistant acute graft-versus-host disease (GVHD) of the lower gastrointestinal (GI) tract is a leading cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. We hypothesized that distinct gene expression profiles exist within affected GI tissue at GVHD diagnosis and may be associated with treatment outcomes. Methods: Unstained tissue sections from 33 patients with biopsy-confirmed acute lower GI GVHD were used for spatial transcriptomics using the NanoString GeoMxTM Digital Spatial Profiler (DSP) platform. Fluorescent morphology markers (PanCK, CD45 and CD31) were used to visualize the epithelial, immune and endothelial compartments and guide capture of RNA expression profiles using the GeoMxTM Human Whole Transcriptome Atlas (18,676 genes). A total of 739 areas of interest (AOIs) were sampled across the 33 patients with a total of 57,960 immune, 37,610 epithelial, and 2298 vascular endothelial cells analyzed (average of 1756.4 immune, 1139.7 epithelial and 69.6 endothelial cells per case). The gene specific oligos were quantified using Illumina's i5 x i7 dual-indexing system and sequenced on the Illumina NextSeq 500 using a 2 x 75 MidOutput v2.5 flow cell. The FASTQ files were processed to generate target probe-specific count files and saved in DCC format. Gene expression files were normalized per NanoString protocol. All unsupervised clustering and differential expression were done in Bioconductor in the R statistical computing space. Results: The gene expression data for each AOI of immune, epithelial, and endothelial cells were normalized and the top 500 most variable genes were used for unsupervised hierarchical clustering to obtain an unbiased classification of different subtypes. There were two clear major AOI clusters for each of the cellular compartments with immune clustering shown in Figure 1A. The left immune cluster (IC1) and the right immune cluster (IC2) were then evaluated for association with clinical GVHD outcomes. IC1 had an over-representation of patients with steroid sensitive GVHD (Fisher exact p value = <0.0001) and AOIs with low grade pathology (Fisher exact p value = 0.031). To obtain gene level differences between IC1 and IC2, differential expression was performed using a Wilcoxon Rank Sum test and volcano plots were generated to illustrate the most differentially expressed genes across the visual AOI-based clusters (Figure 1B). Genes with an adjusted p-value of 0.05 for association with the particular arm of the volcano plot and at least a 2-fold change over the other cluster were considered significant. Gene set enrichment analysis (GSEA) using curated datasets from MSigDB was performed and identified that IC1 genes were enriched for IFNγ response (q value = 2.99e-5), TNFα signaling (q value = 2.99e-5) and inflammatory response (q value = 0.0063). These IC1 differentially expressed genes included CD74 (835 vs. 361.6 mean normalized gene transcripts), LYZ, β2-microglobulin, C1QB and CD68. IC2, on the other hand, had a prominent exhausted/immunosuppressive signature significantly enriched for Th17 cell genes (q value = 0.027) by GSEA. Notable genes that were statistically significant were FCF1 (152.3 vs. 74 mean normalized transcripts) and USP17L11 (78.3 vs. 36.4 mean normalized transcripts). Extension of this analysis to the epithelial (EC) and vascular endothelial (VC) cell compartments also identified distinct clustering with both having an inflamed (EC1 and VC1) and a basal-like state (EC2 and VC2). Interestingly, the inflamed epithelial cells (EC2) associated with immunosuppressive IC2 immune cells (Fisher exact p-value = 0.006), but inflamed vascular endothelium (VC1) associated with the pro-inflammatory IC1 immune cells (Fisher exact p-value = 0.0005). Conclusion: Spatial transcriptomics provides high resolution gene expression profiles in distinct cellular compartments in human acute GI GVHD samples and provides a better understanding of the relationship of the immune response with epithelial and vascular endothelial cell phenotypes. Initial analysis demonstrates that an inflammatory immune signature is associated with steroid responsive acute GI GVHD and an exhausted phenotype with steroid resistance. Future studies are needed to confirm the predictive value of specific gene expression profiles as biomarkers of corticosteroid response at diagnostic biopsy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Acute graft-versus-host disease (GVHD) causes apoptosis of intestinal stem cells (ISCs) that are key targets in the gastrointestinal (GI) tract (Zeiser and Blazar, NEJM 2917; 377:2167). The receptor interactive protein kinase 1 (RIP1) is an important mediator of apoptosis and we investigated the role of the (RIP1) pathway in GI tract and ISC damage in both human and mouse GVHD models. Immunohistochemical staining of human GI biopsies from GVHD patients showed strong phospho-RIP1 staining in biopsies with Lerner grades 2/3 compared to grades 0/1 (12/17 v 0/8, p < 0.01). We then established in vitro models of ISC apoptosis (at least 50% loss) by adding 10 ng/ml of both IFNg and TNFa to organoid cultures from both ileum and colon (organoids cultures with either IFNg or TNFa alone showed no change). Addition to cultures of 0.1 mcg/ml GNE684, a well-characterized small molecule RIP inhibitor (Patel et al, Cell Death Diff 2020; 27:161), dramatically reversed ISC loss in both human and mouse ileal organoids as measured by cross sectional area (Fig 1). WE observed an identical pattern in colon cultures where GNE684 reversed loss of human ISCs (70.0 + 7.7 % v 29.3 + 2.5 %, p < 0.001) and mouse ISCs (100 + 6.6 % v 41 + 6.6 % p < 0.001). In vivo, 14 daily ip injections of GNE684 prevented mortality in a GVHD mouse model (C3H.SW → B6) in a dose dependent fashion (Fig 2). Systemic GNE administration also improved survival in a second model (Balb/c → B6: day 30 survival 7/10 v 0/10, p < 0.001). In the C3H.SW → B6 model there were dramatic reductions in CD4+ lamina propria effector lymphocytes secreting either IFNg (56 + 11 v 9 + 2, p < 0.001) or IL-17 ( 22 + 10 v 7 + 4, p < 0.01) and in CD8+ effector cells secreting IFNg (39 + 11 v 8 + 3, p < 0.001) or IL-17 ( 19 + 8 v 7 + 2, p < 0.001) on day 10 after HCT. Importantly, GVHD was dramatically reduced when administration of GNE684 was used as treatment for GVHD beginning on day +7 after GVHD was established (day 40 survival: 8/12 v 2/12, p = 0.02). Additionally, survival dramatically improved in RIP1 kinase-dead recipients compared to wild-type B6 controls in both the C3H.SW → B6 model (13/16 v 4/16, p < 0.001) and in the Balb/c →B6 model (13/20 v 1/20, p < 0.001). IFNg secreting CD4+ and CD8+ effector lymphocytes that infiltrated the lamina propria were significantly reduced in RIP1 kinase-dead recipients compared to wild-type controls (58 + 13 v 11 + 7 and 39 + 11 v 14 + 4, respectively [p < 0.001 for both]). Infiltrating lymphocytes secreting IL-17 were also reduced four fold. Importantly, administration of GNE684 preserved beneficial GVL effects when lethal doses of myeloid tumor C1498 cells that caused 100% mortality by day 28 in syngeneic BMT recipients were added to the donor inoculum (day 42 survival: 7/10 v 4/13, p = 0.03). We conclude that RIP1 is a key mediator of GI damage during GVHD. In vitro RIP1 inhibition prevents ISC apoptosis in both mouse and human models of GI GVHD. RIP1 inhibition, either through genetic alteration of the RIP1 kinase domain in transplant recipients or through systemic administration of the RIP1 inhibitor GNE684, protects the GI tract from acute GVHD damage as measured both clinically and histologically in preclinical models while preserving beneficial GVL effects. These data suggest that GNE684 a clinical trial of GNE684 is warranted in patients with gastrointestinal GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Collection of peripheral blood mononucleated cells for chronic graft-versus-host disease immunology research: safety and effectiveness of leukapheresis in 132 patients

BackgroundChronic graft-versus-host disease (GVHD) is a major cause of late morbidity and non-relapse mortality in recipients of allogeneic hematopoietic cell transplantation (HCT). Its biology, however, remains poorly understood, making the studies of its biology and immunomodulatory therapies a difficult task. Such research is often hampered by lymphopenia which is common in these patients and precludes studies of critical cellular subsets across the spectrum of severity of disease. This study explores the potential of leukapheresis to safely acquire and efficiently store immune cells for immunology research in chronic GVHD.MethodsThis is a cross-sectional study in which 132 consecutively accrued patients undergo optional research leukapheresis and a one-week comprehensive outpatient evaluation. Baseline clinical and laboratory data and efficiency of the procedure were reported.ResultsNinety-four of 132 patients (71%) achieved the goal collection of 2 × 10^9 PBMNCs with a mean volume processed of 4.6 L. Only mild decreases in hemoglobin, platelet, lymphocyte and monocytes were observed. All adverse events were mild (grade 1) and had resolved by the time of discharge from the apheresis unit.ConclusionThis study demonstrates feasibility, safety, and efficiency of research leukapheresis in a frail patient population. Results presented promote leukapheresis as a standard research practice option in studies of chronic GVHD in humans which may expedite advances in our understanding of this complex multisystem disease.

S230: HUMAN ΒETA-DEFENSIN 2 TREATMENT MODULATES THE INTESTINAL MICROBIOME AND THE ALLOGENEIC T CELL RESPONSE TO LIMIT ACUTE GRAFT-VERSUS-HOST DISEASE

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy option for patients with hematological malignancies. Acute graft-versus-host disease (GVHD) is primarily driven by allogeneic donor T cells and remains a serious cause of morbidity and mortality post allo-HCT. In particular, acute GVHD of the gastrointestinal tract (GI GVHD) is associated with a high mortality rate and treatment options are limited. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide, which is induced by inflammatory stimuli and possesses both antimicrobial and immunomodulatory functions. Aims: In this study, we aimed to investigate the expression of beta defensins in human and murine GI GVHD and the therapeutic effect of recombinant hBD-2 on acute GVHD development in mouse models. Methods: To investigate the functional role of hBD-2 post allo-HCT, we employed established murine GVHD and graft-versus-leukemia (GVL) models and analyzed the allogeneic T cell response using transcriptome and kinome profiling. Shotgun metagenomic sequencing was used to examine the effect of hBD-2 on the intestinal microbiome. Furthermore, we studied beta-defensin expression in two independent acute GVHD patient cohorts and in mice with acute GVHD. Results: We found that expression of murine beta-defensin 4 (mBD-4), the murine orthologue for hBD-2, was reduced in the colon and ileum of mice developing acute GVHD. Oral treatment of mice with recombinant hBD-2 post allo-HCT reduced weight loss and acute GVHD severity and mortality. Furthermore, hBD-2 treatment affected the intestinal microbial composition, including a shift towards higher abundance of Bacteroides species in hBD-2 treated compared to vehicle treated mice. The changes in the microbiome resulted in reduced neutrophil infiltration of the ileum during acute GVHD induction in hBD-2 treated mice. Additionally, hBD-2 dampened pro-inflammatory Th1 cytokine production (TNF, IFN-gamma) by allogeneic T cells in vivo, while preserving the beneficial GVL effect in two different leukemia models. Mechanistically, oral hBD-2 treatment decreased alloreactive T cell infiltration and the expression of genes involved in T cell receptor (TCR) signaling in the ileum of mice with acute GVHD. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation and metabolism by reducing proximal TCR signaling. In patients with acute GI GVHD, intestinal hBD-2 expression was inadequately induced in response to inflammation at the mRNA and protein level when compared to healthy subjects and patients with ulcerative colitis. Summary/Conclusion: In conclusion, our study demonstrates that hBD-2 reduces acute GVHD severity, likely through its effects in shaping the intestinal microbiota, reducing neutrophil infiltration in the ileum and dampening allogeneic T cell responses. Both human and murine acute GVHD are characterized by a lack of intestinal beta-defensin induction and recombinant hBD-2 represents a potential novel therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Gut microbiome in allogeneic hematopoietic stem cell transplantation and specific changes associated with acute graft vs host disease

Allogeneic hematopoietic stem cell transplantation (aHSCT) is a standard validated therapy for patients suffering from malignant and nonmalignant hematological diseases. However, aHSCT procedures are limited by potentially life-threatening complications, and one of the most serious complications is acute graft-versus-host disease (GVHD). During the last decades, DNA sequencing technologies were used to investigate relationship between composition or function of the gut microbiome and disease states. Even if it remains unclear whether these microbiome alterations are causative or secondary to the presence of the disease, they may be useful for diagnosis, prevention and therapy in aHSCT recipients. Here, we summarized the most recent findings of the association between human gut microbiome changes and acute GVHD in patients receiving aHSCT.

Pharmacological MEK inhibition promotes polyclonal T-cell reconstitution and suppresses xenogeneic GVHD

Rapid immune reconstitution without developing graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation. Here, we analyzed the effects of pharmacological MEK inhibition on human polyclonal T-cell reconstitution in a humanized mouse GVHD model utilizing deep sequencing-based T-cell receptor (TCR) repertoire analysis. GVHD mice exhibited a skewed TCR repertoire with a common clone within target organs. The MEK inhibitor trametinib ameliorated GVHD and enabled engraftment of diverse T-cell clones. Furthermore, trametinib also ameliorated GVHD sparing diverse T cell repertoire, even when it was given from day 15 through 28. Although tacrolimus also reduced development of GVHD, it disturbed diverse T cell reconstitution and resulted in skewed TCR repertoire. Thus, trametinib not only suppresses GVHD-inducing T cells but also promotes human T cell reconstitution in vivo, providing a novel rationale for translational studies targeting human GVHD.

Abstract 2945: Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC

Abstract Severely immunodeficient mouse strains, such as NOD.Cg-Prkdcscid Il2rg-/-, have been used to study the immune response in humans. These mice lack T, B and natural killer (NK) cells and allow for engraftment of human peripheral blood mononuclear cells (PBMCs). However, hPBMC engraftment may lead to severe acute xenogeneic graft versus host disease (GvHD) which arises due to a response from T cells against human leukocyte antigens (HLA) that are encoded by major histocompatibility complex (MHC) class I and II. GvHD shortens animal life span and results in only a brief window of evaluation of human immune cell functions in such mouse models. Efforts have been undertaken to reduce human PBMC-induced GvHD and to subsequently extend the window of human immunity assessment by eliminating mouse MHC I and MHC II. While these beta-2-microglobulin (B2M) knockout mice which are MHC deficient showed fewer symptoms of GvHD, an unintended result was the shorter half life of the antibodies which lead to its rapid clearance. This occurred because B2M is also associated with neonatal Fc receptor (FcRn) and is important to FcRn-mediated in vivo antibody recycling and retention. This is a pharmacokinetic limitation because it renders antibody efficacy assessment and makes these mice unsuitable for studying immune responses. To overcome this limitation, we engineered B-NDG B2M knockout mice plus (NOD.Cg-Prkdcscid Il2rg-/-/Bcgen) that is deficient in MHC I expression but expresses the B2M gene fused in the FcRn gene. In the present study we demonstrate the phenotypic changes in the immune system of the engineered mice using flow cytometry and validate immune tolerance developed over the several weeks of post engraftment by looking at the homeostatic balance between CD8/Treg ratio in the partially reconstituted humanized immune systems. In a parallel study, we used corticosteroid treatment in hPBMC induced B-NDG mice GvHD model. Corticosteroids are the fine line of therapy for GvHD and the phenotypic response generated in B-NDG mice confirms this finding in our GvHD model. Citation Format: Lan Gao, Shruti Sharma, Eugene Lin, Xiaoen Wang, Chengzhang Shang, Yuting Hu, W. Frank An, Mehma Kaur Chawla, Kaiqi Li, Qingcong Lin, Zhaoxue (Luke) Yu, Yujie Liu. Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC [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 2945.

Genetically Engineered Regulatory T Cells for Treatment of Graft-Versus-Host-Disease

Graft-versus-host-disease (GVHD) undermines survival and complicates survivorship of recipients of allogeneic hematopoietic stem-cell transplantation (HSCT). Adoptive transfer of natural T regulatory cells (nTregs) expressing the lineage-specific transcription factor FOXP3 has been successfully used to prevent or treat GVHD in humans and mice. However, human applications of propagated nTregs have been hampered by the need for extensive ex vivo expansion to obtain sufficient numbers. Furthermore, such nTregs could lose expression of FOXP3 compromising their suppressive function after adoptive transfer. Therefore, we have developed a method to generate large numbers of engineered Tregs (eTregs) that stably express FOXP3 and show potent immune suppressive functions in vitro and in a mouse model of GVHD.Primary human CD4+ T cells were transduced with a lentiviral construct to co-express FOXP3 and truncated low-affinity nerve growth factor receptor (dLNGFR). The genetically modified T cells were purified using dLNGFR-specific magnetic beads and propagated by cross-linking CD3/CD28 in the presence of IL-2 for two weeks, resulting in a substantial increase in cell number while maintaining stable expression of FOXP3. Flow cytometry demonstrated that ≥90% purified eTregs were CD4+CD25+CD127neg FOXP3+, reminiscent of bona fide Tregs. In addition, the propagated eTregs maintained TCR Vβ diversity and expressed markers associated with Treg function, including CTLA-4, GITR and CD39.The eTregs vigorously suppressed the proliferation of TCR-stimulated allogeneic CD4+ and CD8+ effector T cells in vitro in a dose-dependent fashion. The suppression was superior to FOXP3+ nTregs isolated from peripheral blood mononuclear cells (PBMC) and numerically expanded by cross-linking CD3/CD28 in the presence of IL-2. We stimulated eTregs with phorbol 12-myristate 13-acetate and a Ca2+ ionophore to determine intracellular IL-2 and IFN-γ by flow cytometry. The production of these cytokines was significantly reduced as compared to stimulated control effector T cells. These results indicated that the eTregs were hyporesponsive or anergic, which is consistent with the findings from nTregs by us and others.To determine the therapeutic potential of eTregs, we used a xenogeneic model of GVHD (xGVHD), induced in sublethally irradiated immunocompromised (NSG) mice. We co-infused allogeneic human PBMC with graded numbers of eTregs and nTregs and demonstrated that the eTregs could significantly protect mice from xGVHD-induced weight loss as well as mortality, as compared to untreated control mice (Figure). Of note, fewer numbers of eTregs were needed compared to nTregs to achieve these biological endpoints. Analysis of peripheral blood from the mice demonstrated the engraftment of eTregs with maintained TCR Vβ diversity and histological analyses of tissues showed substantially reduced infiltrations of human CD45+ cells in the colon, liver and bone marrow in eTreg-treated mice as compared to untreated controls. In addition, cytokines such as human TNF-α and IFN-γ, associated with the pathogenesis of human GVHD, were markedly decreased in the plasma of eTreg-treated mice.In summary, we demonstrated that lentivirus-mediated gene transfer of FOXP3/dLNGFR into primary human CD4+ T cells enabled the generation of potent eTregs that were capable of controlling xGVHD in mice. Our results warrant further study in patients after allogeneic HSCT with GVHD. [Display omitted] DisclosuresTu:Intrexon Corporation: Employment, Equity Ownership. Fox:Intrexon Corporation: Employment, Equity Ownership. Ly:Intrexon Corporation: Employment, Equity Ownership. Steinhart:Intrexon Corporation: Employment, Equity Ownership. Laporte:Intrexon Corporation: Employment. Brennan:Intrexon Corporation: Employment, Equity Ownership. Bolinger:Intrexon Corporation: Employment, Equity Ownership. Cooper:Argos Therapeutics: Equity Ownership; AmpliPhi Biosciences: Equity Ownership; Intrexon Corporation: Equity Ownership, Patents & Royalties; Procter & Gamble: Equity Ownership; Targazyme, Inc.: Equity Ownership; Sangamo Biosciences: Patents & Royalties; Immatics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Miltenyi Biotec: Honoraria; Organovo Holdings: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Research Funding; Ferring: Consultancy. Waldner:Intrexon Corporation: Employment, Equity Ownership.

Toll-like Receptor 4 Gene Polymorphisms in Chinese Population After Allogeneic Hematopoietic Stem Cell Transplantation

Objectives:: Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT) and remains to be a major cause of mortality. Activation of toll-like receptor 4 (TLR-4) by lipopolysaccharide induces the NF-κB signaling pathway to release critical proinflammatory cytokines and increases the recipient response to GVHD. In order to clarify the role of TLR-4 in the occurrence of acute GVHD after HSCT, we collected 208 samples from HSCT recipients and their human lecucyte antigen identical donors to test the hypothesis that TLR-4polymorphism in the recipients or donors influence the risk of acute GVHD in allogeneic HSCT recipients. Methods:: TLR-4 Asp299Gly and Thr399Ile polymorphisms of each sample were examined by using DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Results:: No homozygous or heterozygous variant alleles of the Asp299Gly or Thr339Ile polymorphism were detected in any samples in our study. Our results demonstrate that TLR-4 Asp299Gly and Thr399Ile polymorphisms might be very rare in the Chinese population (Eastern China and Taiwan region). Conclusion:: The results of this study cannot confirm the role of TLR-4 mutations in the pathogenesis of GVHD in humans, yet we reach a definite conclusion by a TLR-4 knockout murine GVHD model in our ongoing project.

Machine Learning-Based Approach to Predict Survival after Allogeneic Hematopoietic Cell Transplantation in Hematologic Malignancies

Background Allogeneic hematopoietic cell transplantation (HCT) has been more widely applicable to the patients with hematologic malignancies owing to the increased donor availability, advances in conditioning regimen, prevention of transplantation-related toxicities, and general supportive care. However, there is no comprehensive and uniform approach for decision making which incorporates transplantation-related factors including patients and donor selection, conditioning intensity, or prevention of graft-versus-host disease (GVHD). In this regard, we aimed to establish and validate a machine learning-based predictive model for survival after allogeneic HCT in hematologic malignancies. Method Data from 2,011 patients with hematologic malignancies (1,464 acute leukemia, 296 myelodysplastic syndrome, 100 chronic myeloid leukemia, 45 myeloproliferative neoplasm, 85 lymphoma, and 21 multiple myeloma) who received allogeneic HCT between December 1993 and December 2019 at the Asan Medical Center were retrospectively analyzed. Results The median overall and event-free survival of total patients were 4.2 year (95% confidence interval [CI], 2.9-5.4) and 1.5 year (95% CI, 1.1-1.8), respectively. To predict post-transplantation survival, the patients were classified into "survived more than 5 years" and "died before 5 years". Among four major machine learning models (random forest [RF], support vector machine, logistic regression, and feed forward neural network), we selected RF method according to the predictive power of each algorithm. Using the RF machine learning algorithm, we developed a post-transplantation survival predicting model with the training cohort of 1,408 patients (70%) and tested it with the validation cohort of 603 patients (30%). Of &amp;gt;200 variables, 33 were selected using recursive feature elimination, and the estimated area under the receiver operator characteristic curve and accuracy of the model was 0.812 and 0.73, respectively. We then evaluated the robustness of predictive power of the model using 10-fold cross-validation in validation cohort. In addition, risk scores were calculated from each patient in the validation cohort, and there was an agreement between the estimated predicted risk and observed risk. Conclusion In conclusion, the machine learning-based prediction model seems feasible assuming post-transplantation survival outcomes in hematologic malignancies. Our findings could be helpful for clinicians to select more appropriate donor in terms of age or type of human leukocyte antigen mismatch, conditioning regimen, and GVHD prophylaxis. Disclosures Lee: Astellas: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Lee:Hanmi: Research Funding.

Abstract 5049: Development of huPBMC-NCG mice to study xenogenic graft versus host disease (GVHD)

Abstract Graft versus host disease (GVHD) is a severe complication of allogeneic stem cell transplantation for leukemia treatment. NCG mice lacking functional murine T, B, NK cells as well as decreasing functional macrophage and dendritic cells could be used as recipients of human peripheral blood mononuclear cells (huPBMC) for the studies of human GVHD. In this study, huPBMC-NCG mice were developed by the engraftment of huPBMC into unconditioned or irradiated NCG mice. The engraftment kinetics, irradiation effect, and preclinical treatment were explored in these mice. After huPBMC engraftment, NCG mice developed GVHD symptoms, including body weight loss, fur loss, reduced activity, hunched posture and severe ruffling, and eventually led to mortality. We also observed human T cell trafficking to target organs including gut, heart, liver, skin, and lung by flow cytometry and immunohistochemistry. Interestingly, irradiation could significantly accelerate the onset of GVHD following huPBMC engraftment. Meanwhile, irradiated huPBMC-NCG mice presented less variability in the kinetics of GVHD and the frequency of this disease in comparison with unconditioned counterparts, thus providing a more stable model for human GVHD studies. For preclinical studies, we found immunomodulatory drugs including Abatacept (CTLA4-Ig) targeting APC cells or anti-Ly6G antibody targeting neutrophils could alleviate GVHD-associated symptoms along with improving overall survival of huPBMC-NCG mice. Taken together, this study established stable huPBMC-NCG mice to mimic the onset and development of human GVHD, which was helpful for the development of therapeutic intervention. Citation Format: Cunxiang Ju, Hongyan Sun, Dingyu Wang, Shiying Guo, Mingkun Zhang, Hainan Wu, Shuai Li, Chao Ju, Jing Zhao, Xiang Gao. Development of huPBMC-NCG mice to study xenogenic graft versus host disease (GVHD) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5049.

Abstract 432: Development of a novel engineered B2M mouse strain on B-NDG background that specifically eliminates MHC I expression while retaining normal FcRn-mediated antibody retention

Abstract Severely immunodeficient mouse strains, such as NOD.Cg-Prkdcscid Il2rg-/-, have been widely used to evaluate human immune responses, including applications in immuno-oncology in recent years. However, when human peripheral blood mononuclear cells (PBMCs) are engrafted in these mice, severe acute xenogeneic graft versus host disease (GvHD) arises due to engagement of human T cell receptors (TCRs) and murine major histocompatibility complex (MHC) I and II. GvHD shortens animal life span and results in only a brief window of evaluation of human immune cell functions in such mouse models. Efforts have been undertaken to reduce human PBMC-induced GvHD and to subsequently extend the window of human immunity assessment by eliminating mouse MHC I and MHC II. While these MHC deficient mice show few symptoms of GvHD, the use of beta-2-microglobulin (B2M) null allele in order to eliminate expression of all MHC I molecules resulted in an unintended consequence in that these mice had rapid antibody clearance. This is because B2M is also associated with neonatal Fc receptor (FcRn) and is important to FcRn-mediated in vivo antibody recycling and retention. Low half-lives of antibodies in B2M null mice render them unsuitable for antibody efficacy assessment. To overcome this undesired pharmacokinetic (PK) limitation of B2M null allele, while retaining its desired effect on eliminating MHC I expression and alleviating GvHD, we engineered a B2M knock-out strain in the background of B-NDG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/Bcgen) that was deficient in MHC I expression but not antibody retention. In present study, we describe genetic, phenotypic, pharmacokinetic, and functional characterizations of the resulting mouse strain, which we believe will be a valuable addition to the collection of severely immunodeficient mouse models that permit extended and appropriate interrogation of antibody therapeutics on human immune cells. Citation Format: W. Frank An, Lan Gao, Xiaoen Wang, Shruti Sharma, Kaiqi Li, Xiqiang Gao, Chengzhang Shang, Yuting Hu. Development of a novel engineered B2M mouse strain on B-NDG background that specifically eliminates MHC I expression while retaining normal FcRn-mediated antibody retention [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 432.

Alternative mechanisms that mediate graft-versus-host disease in allogeneic hematopoietic cell transplants.

Allogeneic hematopoietic cell transplantation (alloHCT) benefits increasing numbers of patients with otherwise lethal diseases. Graft-versus-host disease (GVHD), however, remains one of the most potentially life-threatening complications due to its own comorbidities and the side effects of its treatment. In this issue of the JCI, two groups have turned dogma on its head by providing evidence for alternative mechanisms of acute GVHD (aGVHD) in humans. The principle of donor T cell reactivity elicited by host antigen-presenting cells (APCs) expressing MHC-encoded major HLA disparities or expressing minor histocompatibility antigen (miHA) differences presented by identical HLA molecules remains intact. These reports, however, demonstrate that GVHD can additionally result from peripheral host T cells resident in skin and gut being stimulated against donor APCs in the form of monocyte-derived macrophages. Moreover, these donor monocyte-derived macrophages can themselves mediate cytopathic effects against resident host T cells in skin explants and against a keratinocyte-derived cell line.