The α4β7 blockade with vedolizumab is effective in prevention of graft <i>vs</i>. host disease using a xenogeneic mouse model
Authors: Senthilnathan Palaniyandi, Reena Kumari, Ethan Strattan, Timothy Huang, Jing Du, Natalya Hakim, Melissa V. Kesler, Gerhard C. Hildebrandt
Journal: American Journal of Hematology (2019)

Acute graft vs host disease (aGVHD) leads to significant morbidity and mortality after allogeneic hematopoietic cell transplant (allo-HCT). A critical step during GVHD development involves donor T-cells migrating to Peyer's patches, leading to generation of host-reactive cytotoxic T lymphocytes, (Peyer's patch is the essential gut site in initiating murine acute and lethal graft-vs-host reaction). Conditioning regimen-related tissue damage leads to the release of inflammatory cytokines, augmenting interactions between host antigen-presenting cells (APCs) and donor T-cells. Activated alloreactive donor T-cells then proliferate and mature to effector T-cells, contributing to tissue injury through inflammatory cytokine release. This includes TNF and IFN-γ, or through direct-cellular cytotoxic mechanisms such as TRAIL, Fas/FasL, Perforin/Granzyme inducing epithelial apoptosis, Paneth cell loss, and impaired mucosal barrier integrity,1 subsequently accelerating systemic GVHD. Targeting donor T-cell migration by interrupting the interactions of the chemokine receptors CXCR3 and CCR5 with their respective ligands has been shown to be efficient in decreasing intestinal injury.2 However, expression of these receptors is not restricted to the GI tract. Therefore, targeting a more GI-specific pathway involved in recruiting donor T-cells would potentially be more effective and associated with less systemic side effects. Reports from earlier studies demonstrated the importance of α4β7 in allo-reactive T-cells in the pathogenesis of intestinal GVHD and overall GVHD morbidity. This was shown when either the donor T cells lack expression of α4β7, or with the use of a blocking antibody against the α4β7 receptor, mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), which is predominantly expressed in the GI tract.3 Vedolizumab is a humanized antibody against α4β7 integrin on T cells, and blocks their binding to MAdCAM-1 on endothelial cells, thereby preventing their extravasation into gastrointestinal submucosa. Blockade of this interaction is efficacious in animal models of IBD.4 Of interest, patients suffering from intestinal GVHD have a higher percentage of α4β7 integrin expressing memory T-cells.5 Clinically, VDZ has been successfully used in the treatment of steroid refractory GVHD of the gut,6 and we hypothesized that α4β7 blockade with VDZ may assist in preventing GVHD before further treatment is needed. We therefore studied the benefits of VDZ in the prevention of intestinal GVHD using a humanized mouse model, demonstrating for the first time that preventive VDZ is efficacious to reduce this deleterious complication after allo HCT. Humanized mouse model of xenogeneic gvhd: Experiments with human blood and mice were approved by the institutional review board (IRB) and the Institutional Animal Care and Use Committee (IACUC) Human and Animal Ethics Committees at the University of Kentucky. Human peripheral blood mononuclear cells (huPBMCs) were isolated by density centrifugation using Ficoll-Paque (FISHER), and were injected through the tail vein (30 million/mouse) into sub-lethally (350 cGy) total body irradiated female RAG2−/− γc−/− mice.7 Non-GVHD control groups were irradiated but did not receive huPBMCs. Vedolizumab or a control antibody was given intraperitoneally at a dose of 4 mg/kg body weight on day −1 (early preventive approach) or day +8 (late preventive approach), and followed at every 2 weeks. Mice were monitored up to 8 weeks for signs of clinical GVHD using a scoring system previously described or analyzed at day +28. Histological, immunohistochemical and image analysis: The GVHD target organs (small and large bowel, liver, and lung) were formalin-preserved and paraffin-embedded. Hematoxylin/eosin stained sections were scored using a semi-quantitative GVHD scoring system by pathologists who were blinded as to the sample grouping (JD, NH, MK).8 Tissue sections were processed for immunohistochemistry with anti-huCD3mAb (Dako). All IHC slides were digitally scanned by an Aperio ScanScope XT system and quantified by Positive Pixel Count V9 algorithm. Cytokine bead array: Th1/Th2/Th17 cytokines were determined in serum samples through the Human Cytometric Bead Array kit (BD Biosciences, San Jose, CA). Bead fluorescence was measured using the BD Biosciences LSRII flow cytometer and analyzed with the accompanying software. Immunoprecipitation and blotting: Tissue homogenate was immune-precipitated with the anti-human-α4 mAb (Abcam) at 4 °C, overnight. Immobilized Protein A/G resin slurry was added to the reaction mixture and further incubated for 2-4 hours at 4 °C. Eluted immune complex was separated on SDS polyacrylamide gel. Western blots were stained with monoclonal anti-mouse MAdCAM-1 (Biolegend) and anti-human-α4 (Abcam). The results were quantified using ImageJ software. Statistics: Experimental data are expressed as means ± SD. Differences between groups were analyzed by the two-tailed t test. Survival data was analyzed using the Log-rank test. Graphs were prepared using GraphPad Prism software (GraphPad, San Diego, CA). Vdz improved survival and reduced gvhd severity: To induce xenogeneic GVHD, we transferred huPBMCs into sub-lethally irradiated RAG2−/− γc−/− mice.7 Vedolizumab or control antibody was given intraperitoneally as an early preventive or a late preventive approach. Time points were chosen in order to start α4β7 blockade prior to allogeneic donor T-cell priming, and migration to the intestinal tract and Peyer's patches (early preventive approach). The late preventive approach was used mainly to target GVHD symptoms secondary to effector T-cell migration to intestine. The CD45+ cells in spleen on day +28 after infusion demonstrated strong human leukocyte engraftment with more than 80% of spleen cells being huCD45+. Early administration of VDZ significantly improved survival (Figure 1A) and decreased clinical GVHD symptoms (Figure 1B) after transplantation. In addition, we observed improved survival (Figure 1C) and decreased clinical GVHD scores (Figure 1D), using the late preventive method as well. All recipients, who did not receive huPBMCs but were treated with VDZ survived, indicating VDZ safety in this model. Improved survival and lessened clinical GVHD in VDZ treated mice correlated with significantly decreased GVHD gut pathology (Figure 1E-H) on day +28 after transplantation using either treatment schedule. No differences in pathology were seen in liver or lung at this time point. Serum cytokine analysis on day +28 revealed marked decreases in TNF after both early and later administration of VDZ, and a decrease in IFN-γ in VDZ late preventively treated recipients (Figure 1I-L). Vdz reduces t-cell infiltration in intestine through blocking the binding of Α4Β7 to madcam-1: Note, MAdCAM-1 is expressed in the digestive tract endothelium. It is involved in the recruitment of lymphocytes expressing integrin α4β7, through binding to β7 subunit of integrin. Vedolizumab binds exclusively to the gut-tropic α4β7 integrin,9 blocking its binding with MAdCAM-1 and reducing the T-cell trafficking.10 In an effort to verify this mechanism of action for VDZ in our model, we performed immunoprecipitation with anti-human alpha4 antibody to pool the protein complexes of alpha4. Following separation from beads, immunoprecipitation eluent was subjected to blotting with anti-mouse MAdCAM-1 antibody to quantify the released MAdCAM-1 from alpha4complexes. Intensity of the MAdCAM-1 was measured and normalized with the alpha4 intensity. We observed significant decrease in MAdCAM-1 intensity in ileum samples of VDZ treated recipients compared to control recipients in both approach (Figure S1 A-D). This indicates that VDZ blocks the binding of MAdCAM-1 to α4β7. Further, histologic analysis of T-cell infiltration of small intestine and colon by immunohistochemistry for CD3 (Figure S1 E-K) revealed significantly lower infiltration of human T-cells in VDZ treated recipients (Figure S1 E-F). In conclusion, our data expands prior reports on the importance of T cell migration into the intestinal tract for GVHD development and propagation3 and shows that gut specific α4β7 blockade on donor T cells using VDZ is efficacious, warranting further clinical trial investigation such as current protocol NCT03657160. This study was funded by TAKEDA Pharmaceuticals U.S.A through IISR grant (SP and GCH). This research was supported by the Biospecimen Procurement & Translational Pathology Shared Resource Facility and Flow Cytometry and Cell Sorting Core Facility: Supported by the Markey Cancer Center and an NCI Center Core Support Grant (P30CA177558). 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