Signs Of Graft-versus-host Disease Research Articles

Transplantation of Donor-Recipient Chimeric Cells Restores Peripheral Blood Cell Populations and Increases Survival after Total Body Irradiation-Induced Injury in a Rat Experimental Model.

Current therapies for acute radiation syndrome (ARS) involve bone marrow transplantation (BMT), leading to graft-versus-host disease (GvHD). To address this challenge, we have developed a novel donor-recipient chimeric cell (DRCC) therapy to increase survival and prevent GvHD following total body irradiation (TBI)-induced hematopoietic injury without the need for immunosuppression. In this study, 20 Lewis rats were exposed to 7 Gy TBI to induce ARS, and we assessed the efficacy of various cellular therapies following systemic intraosseous administration. Twenty Lewis rats were randomly divided into four experimental groups (n = 5/group): saline control, allogeneic bone marrow transplantation (alloBMT), DRCC, and alloBMT + DRCC. DRCC were created by polyethylene glycol-mediated fusion of bone marrow cells from 24 ACI (RT1a) and 24 Lewis (RT11) rat donors. Fusion feasibility was confirmed by flow cytometry and confocal microscopy. The impact of different therapies on post-irradiation peripheral blood cell recovery was evaluated through complete blood count, while GvHD signs were monitored clinically and histopathologically. The chimeric state of DRCC was confirmed. Post-alloBMT mortality was 60%, whereas DRCC and alloBMT + DRCC therapies achieved 100% survival. DRCC therapy also led to the highest white blood cell counts and minimal GvHD changes in kidney and skin samples, in contrast to alloBMT treatment. In this study, transplantation of DRCC promoted the recovery of peripheral blood cell populations after TBI without the development of GVHD. This study introduces a novel and promising DRCC-based bridging therapy for treating ARS and extending survival without GvHD.

Tocilizumab increases regulatory T cells, reduces natural killer cells and delays graft-versus-host disease development in humanized mice treated with post-transplant cyclophosphamide.

Graft-versus-host disease (GVHD) is a life-threatening complication following donor hematopoietic stem cell transplantation, where donor T cells damage host tissues. This study investigated the effect of tocilizumab (TOC) combined with post-transplant cyclophosphamide (PTCy) on immune cell engraftment and GVHD development in a humanized mouse model. NOD-scid-IL2Rγnull (NSG) mice were injected intraperitoneally with 2 × 107 human (h) peripheral blood mononuclear cells and cyclophosphamide (33 mg kg-1 ) or saline on days 3 and 4, then TOC or control antibody (0.5 mg mouse-1 ) twice weekly for 28 days. Mice were monitored for clinical signs of GVHD for either 28 or 70 days. Spleens and livers were assessed for human leukocyte subsets, and serum cytokines and tissue histology were analyzed. In the short-term model (day 28), liver and lung damage were reduced in PTCy + TOC compared with control mice. All groups showed similar splenic hCD45+ leukocyte engraftment (55-60%); however, PTCy + TOC mice demonstrated significantly increased (1.5-2-fold) splenic regulatory T cells. Serum human interferon gamma was significantly reduced in PTCy + TOC compared with control mice. Long-term (day 70), prolonged survival was similar in PTCy + TOC (median survival time, > 70 days) and PTCy mice (median survival time, 56 days). GVHD onset was significantly delayed in PTCy + TOC, compared with TOC or control mice. Notably, natural killer cells were reduced (77.5%) in TOC and PTCy + TOC mice. Overall, combining PTCy with TOC increases regulatory T cells and reduces clinical signs of early GVHD, but does not improve long-term survival compared with PTCy alone.

Allogeneic, donor-derived, second-generation, CD19-directed CAR-T cells for the treatment of pediatric relapsed/refractory BCP-ALL

AbstractAutologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T cells transduced with a second-generation (4.1BB) CD19-directed CAR for treatment of patients with BCP-ALL in a hospital-exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR–Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy-based manufacturing process (CD19-CAR–Lenti_ALLO). Thirteen children/young adults received ALLO–CAR-T cells between March 2021 and October 2022. Doses ranged between 1.0 × 106 and 3.0 × 106 CAR-T cells per kg. The toxicity profile was comparable with that of autologous CAR-T cells, characterized mainly by cytopenia, cytokine release syndrome (maximum grade 1), and grade 2 immune-effector cell–associated neurotoxicity syndrome. One case of acute graft-versus-host disease (GVHD) occurred and was rapidly controlled with steroids and ruxolitinib. None of the other patients, including 3 given ALLO–CAR-T cells from an HLA-haploidentical donor, experienced GVHD. Two patients received ALLO–CAR-T cells before HSCT and showed a significant expansion of CAR-T cells without any sign of GVHD. All patients obtained complete remission (CR) with absence of minimal residual disease in the bone marrow. With a median follow-up of 12 months (range, 5-21), 8 of 13 patients maintained CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly refractory BCP-ALL relapsing after allo-HSCT without showing increased toxicity as compared with autologous CAR-T cells.

Round-Robin test for the histological diagnosis of acute colonic Graft-versus-Host disease validating established histological criteria and grading systems

Histomorpholgy is one of the mainstays of acute Graft-versus-host disease (GvHD) diagnosis. However, concerns about reproducibility and the most appropriate grading system question its usefulness. Our aim was to assess histomorphological parameters and previously reported grading systems for GvHD regarding reproducibility and validity. Moreover, we propose that sum scores, derived by combining separately scored morphological parameters into a total score, might provide a simplified but equally effective means to grade GvHD. A total of 123 colon biopsies were assessed across four pathologists for intestinal GvHD using a Round-Robin test and results were correlated with clinical findings. Interobserver reproducibility was high for histological parameters that were evaluated as indicators of acute GvHD. Published grading systems were moderately reproducible (ICC 0.679–0.769) while simplified sum scores, in comparison, showed better interrater reliability (ICC 0.818–0.896). All grading systems and sum scores were associated with clinical signs of GvHD and in part with therapy response and survival. However, they were not able to stratify patients according to the clinical severity of GvHD. In a hot-spot analysis 1 crypt apoptotic body (CAB) in 10 crypts was a reasonable cut-off value for minimal diagnostic criteria of GvHD. In conclusion, histology can contribute to the diagnosis of GvHD and is reproducible. Published grading systems are able to reflect clinical findings as are simplified sum scores, which showed improved reproducibility and might be easier to handle as they are based on adding up histological parameters rather than transferring histological findings into a separate grading system. Sum scores will have to be further tested in a prospective setting.

NSCLC patient-derived xenografts perpetuating tumor-associated, tumor-reactive T cells that respond to immunotherapy but do not cause GVHD in host mice

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of malignancy-related deaths worldwide. We wished to overcome a current major limitation of PDX modeling, by providing autologous human T cells suitable for immunotherapy responses, but notably, without graft versus host disease (GVHD). We have developed PDXs from NSCLC by implanting surgically resected patient tumors subcutaneously in NOD scid gamma (NSG) mice. We used a metastatic PDX model to test potential therapeutic efficacy of a novel T cell molecule, anti-human-CD3ɛ monovalent Fab fragment (Mono-OKT3-Fab) and compared tumor growth results with anti-human PD-L1 therapy. Overall engraftment rate of unique, primary patient tumors was 52%. Over multiple passages in vivo, PDX tumors retained expression of primary tumor-matched NSCLC-diagnostic pathological markers. Moreover, primary-tumor-associated T cells were present and passaged in mice in vivo, without producing any signs of GVHD. Immunotherapy showed significant response from a PDL-1+ tumor, consistent with functional tumor-specific T cell passaging in our system. In contrast, control experiments using patient-matched human PBMCs showed vigorous GVHD in the presence or absence of co-transplanted PDX tumor; this demonstrated that non-tumor-associated lymphocytes from these patients were capable of xenoreaction, even though tumor-associated T cells were not. We observed that PDX-NSG mice treated with either modality showed significantly reduced tumor burden and increased survival over control-Ig-treated mice. Overall, our study shows that PDXs implanted in NSG mice from surgically resected-NSCLC retain T cells sufficient for preclinical evaluation of novel T cell-targeting immunotherapies. NIH, National Cancer Institute, IOTN(U01 CA244314) NIH, NIAID, R01 AI097187 Mizzou Start-up funds

Histological diagnosis of acute graft-versus-host disease in different sites of the upper gastrointestinal tract with correlation to endoscopic findings

Graft-versus-host disease (GvHD) involving the intestine is a threat to patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). We evaluated biopsies from different sites of the upper gastrointestinal tract (GIT) of 97 patients after alloHSCT. Forty-six patients with clinical symptoms consistent with upper GI GvHD revealed histological features of GvHD in the esophagus, stomach, and/or duodenum. Biopsies of the duodenum and esophagus were significantly more sensitive for signs of GvHD than those of the gastric antrum or corpus. The histological features of GvHD were significantly correlated with the endoscopic findings of ulcers, erosion, atrophy, and white plaques; however, the sensitivity and specificity of the latter were low. In univariate analysis, overall mortality was significantly associated with histological GvHD signs in all four sites. Nonrelapse mortality was associated with histologic GvHD features in the antrum only. Regarding GvHD diagnosis, biopsies of the upper gastrointestinal tract should include the duodenum and/or esophagus.

S2946 Chronic Graft versus Host Disease Masquerading CMV Colitis After Liver Transplantation: A Clinical Challenge

Introduction: Graft versus host disease (GVHD) after solid organ transplantation is a rare but serious complication with high mortality that typically occurs 1-8 weeks post liver transplantation (LT). Steroids are first line treatment and data on second line treatment are less widely available. Case Description/Methods: 54-years-old woman with history of alcohol-related cirrhosis went under LT with a positive cytomegalovirus (CMV) and HCV male donor. She was discharged home without any major complications. Immune suppression included tacrolimus, mycophenolate mofetil, (MMF) and prednisone taper. She was admitted to hospital 3 months later with diarrhea and acute kidney injury. C. difficile PCR was negative, CMV PCR was positive, and biopsies obtained during colonoscopy revealed severe CMV colitis. She was started on ganciclovir. Tacrolimus was reduced and MMF was discontinued. Fever and diarrhea were continued despite CMV treatment and later she developed a diffuse skin rash. This initially thought to be due to ganciclovir, but skin biopsy was concerning for GVHD. Gastric and colon biopsies and peripheral blood chimerism (donor T cell 81.3%; normal < 1%) confirmed GVHD. She was started on steroid therapy with minimal improvement in diarrhea as well as minimal improvement after the addition of topical budesonide and octreotide. Her course was complicated by posterior reversible encephalopathy syndrome and status epilepticus and was intubated. Tacrolimus was stopped. She then developed pancytopenia with severe neutropenia despite discontinuation of ganciclovir. Bone marrow biopsy showed 10.7% donor alleles. Infliximab was initiated. Stool output decreased and neutropenia resolved after 1 week. Seizures were controlled and she was extubated. Chimerism analysis of peripheral blood after 11 days showed significant reduction of donor T cells (58.5%). However, after 2 weeks, diarrhea worsened and donor T cells increased to 70.9% in blood. Ruxolitinib and cyclosporin were started in addition to infliximab but she developed septic shock and expired (Figure). Discussion: Persistent diarrhea and skin rash after liver transplantation may be signs of GVHD but these symptoms can also be seen in more common conditions such as C. difficile infection, drug reaction, and CMV colitis. Biopsy and chimerism analysis might both be needed for diagnosis of GVHD in patients with low pretest probability. Post LT GVHD that is refractory to steroid therapy may respond to infliximab. Sepsis is the most common cause of mortality in LT GVHD.Figure 1.: Sample micrographs from short tandem repeat (STR) testing (chimerism analysis) on isolated T cells from peripheral blood.

Autologous CD7-targeted CAR T-cell therapy for refractory or relapsed T-cell acute lymphoblastic leukemia/lymphoma.

7035 Background: Refractory or relapsed (r/r) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) has a poor prognosis. Our previous report showed that donor-derived CD7 CAR-T cell therapy could induce remission of r/r T-ALL, but graft-versus-host disease (GVHD) occurred in some patients (Pan et al. J Clin Oncol 2021;39:3340-3351). We recently conducted a phase I trial to evaluate autologous CD7 CAR-T therapy in r/r T-ALL/LBL, which may avoid GVHD while showing the efficacy; here we report the early result with the approval of the Data and Safety Monitoring Committee and Institutional Review Board (IRB). Methods: The patients who had CD7+ r/r T-ALL/LBL and no leukemia cells in peripheral blood were eligible for this phase I trial (NCT04840875). The CD7 CAR construct included an endoplasmic reticulum anchor domain fused to a CD7 binding domain to prevent CD7 expression on cell surface, which contributed to minimize CAR T cell fratricide. CAR T product was checked to ensure lack of tumor contamination before infusion. The trial followed a “3+3” dose escalation process. Adverse events and efficacy were primary and secondary endpoints. Results: A total of 5 patients (Pt1-5), with a median age of 3.8 years (range, 1.9-13), were enrolled between Sept 2021 and Jan 2022. At enrollment, Pt1 had mediastinal mass and blasts in pleural fluid; Pt4 was in central nervous system (CNS)-3 status; Pt2, 3 and 5 had marrow disease with a median burden of 1.35% (range, 0.07%-7.31%). Pt1, 2 and 3 received 5×105 (±20%) cells/kg; Pt5 received 1×106 (±20%) cells/kg; Pt4 received cells below the target dose. Three patients had cytokine release syndrome (CRS), including one grade 3; the median onset was on day 5 (range, 1-9) with the median duration of 4 days (range, 3-14). No patient had neurotoxicity, GVHD or infection. Grade 3-4 hematological toxicity occurred in all five patients, which recovered to grade 2 within 30 days. On one month post-infusion, four patients achieved complete remission, and one patient (Pt4) still had leukemia cells in the cerebrospinal fluid. With the median follow-up time of 62 days (range, 35-136), one patient (Pt1) underwent stem cell transplantation (SCT) on 2.9 months post-infusion, and had a CD7- relapse on 1.4 months post-SCT; the other three responders remained in MRD- CR. In the four patients who received target dose, the median peak CAR T cell count in peripheral blood was 4.27×102/uL (range, 2.49-5.61) by flow cytometry, and all five patients had detectable CAR transgene by PCR on their last visits. There was a decrease of CD7-positive normal T cells and an increase of CD7-negative counterparts in all responders. Conclusions: Autologous CD7 CAR T cell therapy was safe and effective in the induction of remission in r/r T-ALL/LBL patients, without signs of GVHD. Longer follow-up time of more cases will be used to further evaluate this therapy. Clinical trial information: NCT04840875.

Humanized Mouse Model to Study the P2X7 Receptor in Graft-Versus-Host Disease.

Humanized mouse models of graft-versus-host disease (GVHD), where human immune cells are injected into immune deficient mice, are well established and provide opportunities to investigate pathways involved in GVHD development. This chapter provides an overview of human immune cell isolation, injection of these cells into immune deficient mice, monitoring of mice for signs of GVHD, and assessment of human cell engraftment using flow cytometry. Further, this chapter focuses on the P2X7 signaling pathway involved in GVHD, and describes a strategy to block the P2X7 receptor and examine the effect of this on GVHD development.

Graft-versus-host disease in the female genital tract: a prospective cohort study

Graft-versus-host disease (GVHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD in the female genital tract can cause sinusorrhagia, dyspareunia, synechia, and even complete vagina occlusion. This prospective study aimed to evaluate the clinical characteristics and effects of preventive and prompt treatment for genital GVHD in females undergoing HSCT (n = 40). Genital GVHD was diagnosed in 11 of 40 patients (27.5%), and the most common complaint was vaginal dryness (54.6%). The majority of patients (63.6%) presented mild genital GVHD (clinical score 1), with interlabial fissures and lichen-like lesions, while a minority of patients (9.1%) presented advanced genital GVHD (clinical score 3) with the fusion of the small and large lips. The median time of onset of genital GVHD signs was 10months after HSCT, concomitant with GVHD in the skin and oral cavity. Personalized and topical therapy was effective in most cases (81.8%), and no patient required surgical intervention. We confirmed that female genital GVHD affects approximately one-third of females undergoing HSCT, highlighting the importance of periodic gynecological monitoring for early detection and treatment to improve care for these females.

Cyclosporine A but Not Corticosteroids Support Efficacy of Ex Vivo Expanded, Adoptively Transferred Human Tregs in GvHD.

Reshaping the immune balance by adoptive transfer of regulatory T-cells (Tregs) has emerged as a promising strategy to combat undesired immune reactions, including in Graft-versus-Host Disease (GvHD), which is the most lethal non-relapse complication of allogeneic hematopoietic stem cell transplantation. Currently however, little is known about the potentially inhibitory in vivo effects of conventional immunosuppressive drugs, which are routinely used to treat GvHD, on adoptively transferred Tregs. Here we demonstrate drug-specific effects of the conventional immunosuppressive drugs Cyclosporine A, Mycophenolate mofetil and methylprednisolone on adoptively transferred Tregs in a humanized NOD/SCID/IL2Rgamma-/- GvHD mouse model. The clinical course of GvHD and postmortem organ histology, including cellular organ infiltration, showed that co-administration of Cyclosporine A and Tregs is highly beneficial as it enhanced Treg accumulation at inflammatory sites like lung and liver. Similarly, co-administration of Mycophenolate mofetil and Tregs improved clinical signs of GvHD. In contrast, co-administration of methylprednisolone and Tregs resulted in reduced Treg recruitment to inflammatory sites and the fast deterioration of some animals. Consequently, when clinical trials investigating safety and efficacy of adjunctive Treg therapy in GvHD are designed, we suggest co-administering Cyclosporine A, whereas high doses of glucocorticosteroids should be avoided.

Early Endothelial Activation in a Mouse Model of Graft vs Host Disease Following Chemotherapy.

Allogenic hematopoietic stem cell transplant (allo-HSCT) can lead to sinusoidal obstruction syndrome (SOS) and graft-versus-host disease (GvHD) in some individuals. GvHD is characterised by an immune triggered response that arises due to donor T cells recognizing the recipient tissue as “foreign”. SOS results in impaired liver function due to microvascular thrombosis and consequent obstruction of liver sinusoids. Endothelial damage occurs following chemotherapy and allo-HSCT and is strongly associated with GvHD onset as well as hepatic SOS. Animal models of GvHD are rarely clinically relevant, and endothelial dysfunction remains uncharacterised. Here we established and characterised a clinically relevant model of GvHD wherein Balb/C mice were subjected to myeloablative chemotherapy followed by transplantation of bone marrow (BM) cells± splenic T-cells from C57Bl6 mice, resulting in a mismatch of major histocompatibility complexes (MHC). Onset of disease indicated by weight loss and apoptosis in the liver and intestine was discovered at day 6 post-transplant in mice receiving BM+T-cells, with established GvHD detectable by histology of the liver within 3 weeks. Together with significant increases in pro-inflammatory cytokine gene expression in the liver and intestine, histopathological signs of GvHD and a significant increase in CD4+ and CD8+ effector and memory T-cells were seen. Endothelial activation including upregulation of vascular cell adhesion molecule (VCAM)- 1 and downregulation of endothelial nitric oxide synthase (eNOS) as well as thrombosis in the liver indicated concomitant hepatic SOS. Our findings confirm that endothelial activation is an early sign of acute GvHD and SOS in a clinically relevant mouse model of GvHD based on myeloablative chemotherapy. Preventing endothelial activation may be a viable therapeutic strategy to prevent GvHD.

LPSlow-Macrophages Alleviate the Outcome of Graft-Versus-Host Disease Without Aggravating Lymphoma Growth in Mice.

Despite significant therapeutic advances, graft-versus-host disease (GvHD) remains the main life-threatening complication following allogeneic hematopoietic stem cell transplantation. The pathogenesis of GvHD is dominated by a dysregulated allogeneic immune response that drives fibrosis and autoimmunity in chronic forms. A multitude of cell therapy approaches, including infusion of myeloid cells, has been proposed to prevent GvHD through tolerance induction but yielded variable results. Myeloid cells like macrophages can be reprogrammed to develop adaptive-like features following antigenic challenge to reinforce or inhibit a subsequent immune response; a phenomenon termed ‘trained immunity’. Here we report that, whereas LPSlow-trained macrophages elicit a suppressor effect on allogeneic T cell proliferation and function in vitro in an IL-10-dependent manner, Bacille Calmette et Guérin (BCG)-trained macrophages exert an opposite effect. In a murine model of sclerodermatous chronic GvHD, LPSlow-trained macrophages attenuate clinical signs of GvHD with significant effects on T cell phenotype and function, autoantibodies production, and tissue fibrosis. Furthermore, infusion of LPSlow-macrophages significantly improves survival in mice with acute GvHD. Importantly, we also provide evidence that LPSlow-macrophages do not accelerate A20-lymphoma tumor growth, which is significantly reduced upon transfer of BCG-macrophages. Collectively, these data indicate that macrophages can be trained to significantly inhibit in vitro and in vivo allo-reactive T cell proliferation without exhibiting pro-tumoral effect, thereby opening the way to promising clinical applications.

Abstract 1514: Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells

Abstract The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [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 1514.

NOTCH Inhibition Separates GVL from GVHD Via Modulating Host Dendritic Cell and Donor T-Cell Activation

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eradicate chemorefractory leukemia and reduce the relapse rate of high risk cases through the graft versus-leukemia (GVL) activity. However, graft-versus host disease (GVHD) limited the overall clinical benefit of allo-HSCT. Efforts are made to separate GVL from GVHD but few approaches are confirmed effective. Notch is a highly conserved signaling system important for lymphocyte effector and memory differentiation. Previous studies have shown that inhibition of Notch signaling could reduce aGVHD ex vivo and in vivo by downregulating both activated antigen presenting cells (APCs) and CD8+ T cells. It is less established whether Notch inhibition could also maintain GVL activity while reducing GVHD. The aim of the current work was to further study whether GVL activity could be maintained or enhanced by Notch inhibition.Methods: Murine models of GVHD and GVL were established (C57BL/6→BALB/c). γ-secretase inhibitor N-S-phenyl-glycine-t-butyl ester (DAPT) was used to block Notch1 signaling. The recipients were monitored daily for survival, clinical signs of GVHD and tumor size. GVHD histopathology were systematically examined and evaluated based on the scoring system. In order to study the mechanism of Notch inhibition on both GVHD and GVL, phenotypes of APCs and T cells in recipients were analyzed by flow cytometry. Expression of FasL by PCR, levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by ELISA and cytotoxicity of T cells were compared in recipients with or without Notch inhibition.Results: Reduced GVHD was observed in Notch inhibited recipients, which is similar to the results of previous study. However, in GVL model, both the median time when tumor volume is over 0.5cm3 and the change of tumor volume had no difference with or without Notch inhibition. Suppressed differentiation of CD8+ cytotoxic T cells (CTL) and decreased levels of TNF-α did not result in attenuated GVL activity. Further mechanism studies showed that Notch inhibition up-regulates CD11c+CD80+ DCs and central memory T cells (CD4+CD62L+ and CD8+CD62L+ T cells) while suppressing CD8+ CTL differentiation. We then compared levels of TNF-α and IFN-γ which are important in mediating aGVHD. Levels of TNF-α were decreased but IFN-γ were increased rather than decreased after Notch inhibition. We hypothesized that the contradictory results may ascribe to the difference between TNF-α and IFN-γ in inducing GVHD and GVL. Reduced TNF-α and increased IFN-γ levels could be the causes for down-regulated donor CTL activity. Moreover, FasR-FasL interaction plays a critical role in GVL instead of aGVHD. FasL expression were similar in in DAPT groups and the controls, suggesting similar GVL activity in recipients with or without Notch inhibition.Conclusions: Notch inhibition reduces GVHD and does not impair GVL effect. Increased number of central memory T cells and higher levels of interferon-γ (IFN-γ) could be the causes for enhanced GVL. DisclosuresLi:Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding; Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding.

Targeting T Cell Bioenergetics By Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells to Prevent Graft-Versus-Host Disease While Maintaining Antitumor Immunity

Immune effector cell therapy is a potent form of cancer treatment with a high curative potential for certain human malignancies. Yet, the success of current approaches is offset by associated toxicities, including acute graft-versus-host-disease (GVHD). In the allogeneic setting, GVHD is caused by an attack on the recipient's tissues by donor T cells. Ex vivo application of phototherapy to selectively deplete GVHD-causing cells prior to transplant may prevent GVHD. However, none of the photosensitive agents in use today have demonstrated selectivity without significant toxicity occurring in resting cells. We have designed the first-in-class photosensitizer 2-Se-Cl with the ability to accumulate in alloreactive T cells in proportion to degree of oxidative phosphorylation. Unique to 2-Se-Cl is the ability to potently stimulate P-glycoprotein (P-pg). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria, and protects resting lymphocytes essential for antipathogen and antitumor responses. We have previous demonstrated the ex vivo photodepletion of alloreactive T cell using 2-Se-Cl successfully prevented GVHD in a lethal model of the disease with full retention of antipathogen immunity and 3rd party responses. To evaluate for the retention of antitumor immunity after photodepletion with 2-Se-Cl, a well-established complete MHC antigen-mismatched murine model of hematopoietic stem cell transplant (HSCT) was employed. To prepare the cells for photodepletion, donor C57BL/6 splenocytes were cocultured for 4 days with irradiated (20 Gy) Balb/c splenocytes, and then photodepleted (PD) with 2-Se-Cl. On the day of HSCT, recipient Balb/c mice were irradiated with 8.5Gy, and received 7.5 x106 T cell-depleted (TCD) C57BL/6-derived bone marrow cells accompanied by 2.5 x106 PD - treated cells, or 105 recipient-derived (A20, Balb/c-derived) leukemia cells (engineered to express firefly luciferase), or both. To evaluate for the retention of 3rd party responses, C3H/HeJ were irradiated (9.5 Gy) and received a combination of 7.5 x106 TCD C57BL/6-derived bone marrow cells accompanied by 2.5 x106 PD - treated cells. All mice were monitored for signs of GVHD according to a well-established mouse GVHD grading system. Three animals underwent HSCT in each group in 3 independent experiments. Recipient Balb/c mice that received PD-treated splenocytes survived > 45 days without evidence of GVHD or leukemia, equivalent to controls (recipients of TCD bone marrow cells only). In contrast, mice that were inoculated with leukemia at time of HSCT without the addition of PD-treated splenocytes died of leukemia progression (see figure). Additionally, all third-party C3H/HeJ mice that received the same number of PD-treated cells died of lethal GVHD, consistent with the selective depletion of the alloimmune response towards BalB/c by 2-Se-Cl. These results demonstrate that ex vivo application of 2-Se-Cl protects against lethal GVHD while maintaining antitumor immunity. The high selectivity of this novel photosensitizer may have broad applications, including the rapid production of immune effector cell products with potent antitumor activity and a low potential for toxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. GVHD is one of the most severe and frequently lethal hematological complications after VT procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. Our study included both clinical and experimental data. A total of 108 patients were divided into 3 groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into 2 groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. The patients with native spleen preservation showed a lower rate of GVHD ( P <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

RUNX inhibitor suppresses graft-versus-host disease through targeting RUNX-NFATC2 axis.

Patients with refractory graft‐versus‐host disease (GVHD) have a dismal prognosis. Therefore, novel therapeutic targets are still needed to be identified. Runt‐related transcriptional factor (RUNX) family transcription factors are essential transcription factors that mediate the essential roles in effector T cells. However, whether RUNX targeting can suppress, and GVHD is yet unknown. Here, we showed that RUNX family members have a redundant role in directly transactivating NFATC2 expression in T cells. We also found that our novel RUNX inhibitor, Chb‐M’, which is the inhibitor that switches off the entire RUNX family by alkylating agent–conjugated pyrrole‐imidazole (PI) polyamides, inhibited T‐cell receptor mediated T cell proliferation and allogenic T cell response. These were designed to specifically bind to consensus RUNX‐binding sequences (TGTGGT). Chb‐M’ also suppressed the expression of NFATC2 and pro‐inflammatory cytokine genes in vitro. Using xenogeneic GVHD model, mice injected by Chb‐M’ showed almost no sign of GVHD. Especially, the CD4 T cell was decreased and GVHD‐associated cytokines including tissue necrosis factor‐α and granulocyte‐macrophage colony‐stimulating factor were reduced in the peripheral blood of Chb‐M’ injected mice. Taken together, our data demonstrates that RUNX family transcriptionally upregulates NFATC2 in T cells, and RUNX‐NFATC2 axis can be a novel therapeutic target against GVHD.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

Improved engraftment of human peripheral blood mononuclear cells in NOG MHC double knockout mice generated using CRISPR/Cas9

Humanized mice are widely used to study the human immune system in vivo and develop therapies for various human diseases. Human peripheral blood mononuclear cells (PBMC)-engrafted NOD/Shi-scid IL2rγnull (NOG) mice are useful models for characterization of human T cells. However, the development of graft-versus-host disease (GVHD) limits the use of NOG PBMC models. We previously established a NOG-major histocompatibility complex class I/II double knockout (dKO) mouse model. Although humanized dKO mice do not develop severe GVHD, they have impaired reproductive performance and reduced chimerism of human cells. In this study, we established a novel beta-2 microglobulin (B2m) KO mouse model using CRISPR/Cas9. By crossing B2m KO mice with I-Ab KO mice, we established a modified dKO (dKO-em) mouse model. Reproductivity was slightly improved in dKO-em mice, compared with conventional dKO (dKO-tm) mice. dKO-em mice showed no signs of GVHD after the transfer of human PBMCs; they also exhibited high engraftment efficiency. Engrafted human PBMCs survived significantly longer in the peripheral blood and spleens of dKO-em mice, compared with dKO-tm mice. In conclusion, dKO-em mice might constitute a promising PBMC-based humanized mouse model for the development and preclinical testing of novel therapeutics for human diseases.