Graft-versus-host Disease In Group Research Articles

Early Tapering of Cyclosporine Is Feasible in Haploidentical Stem Cell Transplantation: A Single Center Experience.

Cyclosporine-A (CsA) and post transplantation cyclophosphamide (PTCy) are common agents used for graft versus host disease (GVHD) prophylaxis in Haploidentical hematopoietic cell transplantation (haplo-HCT). However, the impact of CsA cessation timing in the posttransplant setting on clinical outcomes is uncertain. We aimed to investigate the impact of a novel approach that integrated early CsA cessation with PTCy utilization. This study was a single arm retrospective study carried out at a tertiary referral hospital hematology and bone marrow transplantation center between 2009 and 2022. The patients who received haplo-HCT with ATG, PTCy and CsA as GVHD prophylaxis were included. CsA was planned for cessation starting at day 45 to day 60. Acute and chronic GVHD were evaluated and graded. CsA blood concentrations and its impact on acute and chronic GVHD was evaluated. Thirty-one patients composed of 19 (61.3%) male and 12 (38.7%) female patients with a median age of 31 years (20-58). Busulfan and TBI based conditioning regimens were the most utilized regimens. The majority of donors were first degree relatives. Stem cell origin was peripheral blood for all patients. GVHD prophylaxis consisted of ATG, CsA and PTCy. Acute GVHD was observed in 9 (29%) cases, whereas chronic GVHD was seen in 3 (9.7%) cases, with 2 of them having overlapping GVHD. Age, gender, number of chemotherapy lines, transplant characteristics, infused CD34 cell count, and engraftment durations were similar among patients with and without GVHD. Patients with GVHD had similar 1st, 2nd, 3rd and 4th week CsA concentrations compared to patients without GVHD (p>0.05). The presence of GVHD was not associated with worse progression free survival and overall survival (p=0.6, p=0.5, respectively). CMV reactivation was more common in the GVHD group. In the current study, we did not find an impact of CsA concentration on GVHD and post-transplant outcomes in Haplo-HCT setting. Therefore, together with the use of PTCy, early CsA cessation can be an option; further studies are needed to understand all aspects of this approach.

Hspa5 Deficiency Blocks Intestinal Enterocyte Differentiation in Graft-Versus-Host Disease

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective therapy for hematological malignancies. Graft-versus-host disease (GVHD) is a life-threatening complication, and the intestine is one of the major sites affected. Currently, immunosuppressive agents remain the preferred choice for GVHD treatment. However, treatment resistance still occurs in a large population, indicating that some tissue damage may be irreversible. Methods: The murine transplant and GVHD model were established as previously described. To characterize the intestinal damage mediated by radiation and GVHD, murine small intestine tissues were collected at day 14 after transplantation. Primary small intestinal crypts were isolated from each group and cultured for five consecutive days to evaluate their ability to proliferate and differentiate. Single-Cell analysis was performed to characterize the impaired intestinal repair after injury. Results: At day 14, the villus-to-crypt ratio was partially recovered in the BM group, but remained imbalanced in the GVHD group. We observed a significant decrease in the expression of Reg1, a gene expressed by enterocytes (EC), in the GVHD groups compared to the BM group. we further utilized a 3D organoid model to investigate intestinal growth and differentiation. The organoid size from the GVHD group was comparable to the BM and control groups. However, the number of buds per organoid was significantly reduced in the GVHD group compared to the BM and control groups. Immunofluorescence staining confirmed that cells in GVHD-derived organoids exhibited high proliferation (stained by Mki67) but impaired differentiation of enterocyte (stained by Aldob). To gain insights into the cellular mechanisms influencing enterocyte differentiation, we dissociated murine small intestinal tissues from each group into single cells and sequenced them using the 10X Genomics platform. Through pseudochronological analysis, each cell was ordered along the predicted lineages and assigned a pseudotime. We found that enterocytes in GVHD exhibited a long and discontinuous trajectory, suggesting explicit blockage of EC differentiation. Functional enrichment analysis showed that pathways related to cell growth and differentiation, such as MAPK, ERBB, and inositol phosphate metabolism, were highly enriched in M2 of the control and BM groups. In contrast, MAPK was dramatically downregulated at the early stage of ISC differentiation in the GVHD group. Next, we analyzed the differentially expressed genes in EP between the GVHD and BM groups. Endoplasmic reticulum (ER) chaperone-related genes from the heat shock protein (Hsp) 70 family (Hspa1a/1b/5), Hsp90 family (Hsp90aa1/ab1), and Hsp40 family (Dnajb1) were significantly decreased in GVHD compared to BM. Notably, Hspa5 (also known as GRP78 or Bip), which serves as a sensor of glucose levels and misfolded proteins, exhibited reduced protein levels, along with decreased IRE1ɑ phosphorylation (p-IRE1ɑ) and Xbp1s, as well as decreased PERK phosphorylation (p-PERK) and ATF6 expression. To validate our conjecture, we utilized small intestinal organoids cultured for 3 days for further experimentation. RT-qPCR demonstrated that energy restriction downregulated the mRNA expression of Hspa5 and other chaperones. Furthermore, we treated intestinal organoids with HA15, a BIP inhibitor, which resulted in significantly enlarged area but fewer buds. Consistent with our previous findings, Hspa5 was downregulated in the HA15-treated group, along with decreased expression of Hsp family and enterocyte markers. Since Hsp family plays key roles in restoring cellular homeostasis, we wondered if upregulating Hspa5 could protect intestinal cells from outside stimuli such as pro-inflammatory cytokines. When intestinal organoids were cocultured with IFN-g, we found that Bix, the selective Bip inducer, protected organoids from apoptosis by increasing Hsp70 expression. Conclusion: Given that immunosuppression partly resolves GVHD, strategies aimed at restoring intestinal homeostasis should be given sufficient attention. Our study reveals that intestinal enterocyte differentiation is blocked in GVHD, possibly due to starvation and energy restriction. Deficiency in Hspa5 results in impaired intestinal repair, and increasing Hsp levels may enhance cell resistance to cytokine storms.

Single-Cell Transcriptional Survey of the Initiation, Propagation and Maintenance of the Intestinal Graft-Versus-Host Disease

Background: Graft-versus-host disease (GVHD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT). The intestine not only is a primary target of immune attack, but also regulates the severity of systematic GVHD. Although the critical roles of immune cells in GVHD have been documented, the dynamic changes of cell populations, and cell-associated transcriptional signatures during immune attack and epithelial regeneration remain elusive. Methods: To better characterize disease progression, tissues were harvested from murine intestine suffering from GVHD (GVHD_D3, GVHD_D10, GVHD_D21), coupled with irradiated and repaired samples (BM_D3, BM_D10, BM_D21), and healthy control (CON). Each sample mixed with segments of proximal and distal small intestine from three mice. Tissues were dissociated into single-cell suspensions and processed using the 10x Genomics Chromium workflow. After data pre-processing and quality control, we profiled 69242 individual cells from 7 samples. Results: Uniform manifold approximation and projection (UMAP) analysis yielded 17 clusters, including 8 types of epithelial cells, 8 types of immune cells and 1 type of stroma cell. In healthy mice, epithelial cells were highly enriched in murine small intestine, taking up 79.31% of total cells: differentiated enterocytes (EC) (59.31%) represented the largest cohort of epithelial cells, while intestinal stem cells (ISC), trans-amplifying (TA) cells and enterocyte progenitor (EP) only occupied very small portions. In mucosal immune system, the abundance of T cells (9.91%) and B cells (10.43%) was much higher than that of ILC, macrophage and neutrophils. Upon radiation and the development of GVHD, cell populations of murine small intestine dramatically changed. Three days after transplantation, the proportion of ECs dramatically dropped. On the contrast, ɣδ T cells and ILC rapidly accumulated in the damaged intestine. From day 3 to day 10, we observed a steep decrease in ɣδ T cells from samples of BM and GVHD group. At our last observation, BM resorted its epithelial components, while GVHD had a great abundance of T cells (41.73%) and a lower level of differentiated ECs (36.90%). In epithelial components, a total of 44468 epithelial cells were subclustered them into 22 subsets. ISC serves as the production department, which was enriched in mitochondrial genes and genes regulating protein folding. The cluster TA_1 from D21 samples was enriched in pathway related to structure organization and BMP signals, which is required for the initiation of intestinal differentiation and morphogenesis. At day 3, a rapid expansion of subsets of TA_2 and Regenerative progenitors caught our attention. Metabolic pathways such as tryptophan catabolic process, arachidonic acid secretion, methionine metabolic process were highly correlated with wound healing and cell proliferation. EP_1 emerging at day 3 majored in material transportation. EP in samples from control and BM (EP_2) was able to repair DNA damage, while EP_3 that mostly detected in GVHD sample were involved in metabolic disorders, which may explain its deficiency in EC differentiation. T cells and ILC were divided into 16 subsets. In addition to its well-known effects in host defense, several genes that expressed in γδT cells are essential for maintaining intestinal homeostasis. The Tgfb3-Vegfa+ subset regulated microvillus organization and intestinal absorption; the Tgfb3-Vegfa- subset participated in sensory and ciliary movement. At day3, the above clusters undergone a rapid expansion, suggesting an enhanced protective role of γδT cells in the early period of transplantation. We also identified different subtypes of T cells between BM and GVHD samples. Two clusters of cycling CD8+ T cells were more enriched in GVHD samples, which also express high levels of cytotoxic markers. While in BM, we found two clusters of anergic T cells without expression of markers of naïve, memory, effector and exhaustion. The double positive T cells in GVHD may regulate immune infiltration, while the CD4+ Th2-like cell in BM may link to immunoglobin production and Treg differentiation. Conclusions: Single-Cell transcriptional analysis revealed that different cell populations between BM and GVHD. T cells in GVHD were highly-proliferative and cytotoxic, and epithelial cells in GVHD had metabolic disorder and were resistant to enterocyte differentiation.

Analysis of Hospitalization Outcomes in Bone Marrow Transplant Patients with Graft-Versus-Host Disease: A Nationwide Study

Background: Graft-versus-host disease (GVHD) is a well-known complication in patients with bone marrow transplantation. There is a need for additional data on the in-patient outcomes of GVHD in patients who have undergone bone marrow transplantation. Our analysis aims to evaluate the outcomes of GVHD in hospitalized patients with bone marrow transplantation. Method: In this retrospective study, we utilized data from the National Inpatient Sample (NIS) database from 2016 to 2019. Using ICD-10 codes, we identified hospitalizations involving bone marrow transplantation and categorized them into two groups: those with GVHD and those without GVHD. To account for potential confounding variables, we considered a comprehensive set of factors, including age, sex, race, zip code-based income quartile, hospital region, hospital teaching status, hospital division, hospital bed size, insurance status, and the Charlson comorbidity index score. We performed univariable logistic regression analysis to calculate unadjusted odds ratios. Subsequently, we conducted a multivariable logistic regression analysis to account for potential confounding effects and calculated adjusted odds ratios.(see Figure), where the model included only variables that were found to be associated with the outcome of interest in the univariable regression analysis at a significance level of P < 0.2. Proportions were compared using the Fisher exact test for categorical variables, and continuous variables were compared using the Student t-test. All P-values were two-sided, and the significance level was set at P < 0.05, indicating statistical significance. Results: From 2016 to 2019, data were collected from 13,999 hospitalizations involving bone marrow transplants. Among these cases, 836 hospitalizations were linked to GVHD patients. A comparison of patient characteristics showed that individuals with GVHD had a mean age of 51.61 years, while those without GVHD had a slightly higher mean age of 55.55 years. There were slight variations in gender and race distributions between the two groups, with a higher percentage of males and whites observed in the GVHD group compared to the group without GVHD. The analysis of outcomes between the two groups revealed significant differences. Patients with GVHD experienced notably longer mean lengths of stay (41.40 days vs. 21.31 days, P = 0.000) and incurred higher mean total hospital charges ($824,058 vs. $335,765, P = 0.000). Furthermore, after adjusting for confounding factors, GVHD posed a substantial risk to patients undergoing bone marrow transplantation. The adjusted odds ratio (aOR) for mortality in those with GVHD was 7.20 (95% CI: 5.54 to 9.36, P = 0.000) compared to those without GVHD, indicating a considerably higher risk of death. The coefficient for the length of stay was 19.36 days (95% CI: 17.29 to 21.42), and the coefficient for total hospital charges was $453,733 (95% CI: $396,577 to $510,889) in patients with GVHD compared to those without GVHD hospitalizations. Moreover, the presence of GVHD in patients was associated with elevated risks of various medical conditions. The adjusted odds ratios (aORs) for sepsis, pneumonia, acute respiratory failure, intubation and mechanical ventilation, Clostridium difficile infection, and acute kidney injury (AKI) in patients with GVHD were 2.79 (95% CI: 2.28 to 3.41, P = 0.000), 3.30 (95% CI: 2.57 to 4.24, P = 0.000), 5.10 (95% CI: 4.01 to 6.49, P = 0.000), 4.88 (95% CI: 3.75 to 6.34, P = 0.000), 1.45 (95% CI: 1.13 to 1.86, P = 0.003), and 3.57 (95% CI: 2.97 to 4.29, P = 0.000), respectively (see Figure). Conclusion: Patients with GVHD in patients with bone marrow transplantation experience higher mortality rates, extended hospital stays, and increased hospital charges. Additionally, they are at a significantly higher risk of developing severe medical conditions, including sepsis, pneumonia, acute respiratory failure, Clostridium difficile infection, and acute kidney injury (AKI). These findings highlight the importance of closely monitoring and effectively managing GVHD to enhance patient outcomes and alleviate the burden of complications associated with bone marrow transplantation. Nevertheless, further prospective studies are necessary to gain a deeper understanding and more comprehensive evaluation of the outcomes in these hospitalized patients.

Study on the Impact of G-CSF Mobilization on the Level of MDSCs in Donor Peripheral Blood and Its Correlation with Patient Prognosis

Introduction:MDSCs are a newly discovered and heterogeneous population of immature bone marrow cells with immunosuppressive characteristics, capable of inhibiting innate and adaptive immune responses. Granulocyte colony-stimulating factor (G-CSF) is a member of the hematopoietic growth factor cytokine family responsible for the proliferation, maturation, differentiation, and survival of neutrophil lineage. In many hospitals in China, allo-HSCT procedures often involve administering G-CSF continuously for 5 days to healthy donors before collecting the graft, to mobilize white blood cells. This study discusses the impact of G-CSF mobilization on the absolute values and functions of total immunosuppressive myeloid-derived suppressor cells (MDSCs) and their subsets, M-MDSCs and P-MDSCs, in the peripheral blood of healthy donors. Furthermore, it explores the effects of transferring MDSCs from the graft to the patient's body on their prognosis and immune reconstitution. Method：From August 2022 to December 2022, a total of 72 donors and 72 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were selected as the subjects at Lu Daopei Hospital in Beijing. Among the donors, there were 47 males and 25 females, with a median age of 34 years (19, 48). Among the patients, there were 33 males and 39 females, with a median age of 34 years (23, 45). The patients were diagnosed as acute leukemia or MDS in CR status and further categorized into infection group, graft-versus-host disease (GVHD) group, and no-event group based on post-transplantation outcomes. Peripheral blood samples were collected from the donors before and on the 5th day after mobilization, as well as from the graft and the patients. Flow cytometry was employed to determine the absolute counts of MDSCs and their subsets. Result 1. The absolute count of total MDSCs in the donor's peripheral blood before mobilization was 3/μl (2, 8), accounting for 0.1% (0.0, 0.2) of PBMCs, significantly lower than the absolute count of total MDSCs in the donor's peripheral blood after G-CSF mobilization, which was 2283/μl (1091, 5288), accounting for 6.4% (3.2, 9.5) of PBMCs, P<0.01. In the infection group, the absolute count and percentage of total MDSCs, P-MDSCs, and LOX1+P-MDSCs in the donor's peripheral blood after mobilization were higher than those in the donor's peripheral blood corresponding to the no-event group, and significantly higher than those in the donor's peripheral blood corresponding to the GVHD group. P<0.05. Similarly, the absolute count and percentage of total MDSCs, P-MDSCs, and LOX1+P-MDSCs in the correspondingdonor's peripheral blood inthe infection group after mobilization were higher than those in the donor's peripheral blood corresponding to the no-event group, and significantly higher than those in the donor's peripheral blood corresponding to the GVHD group., P<0.05. The absolute count and percentage of M-MDSCs in the infection group were higher than the no-event group and GVHD group, but the differences among the three did not reach statistical significance. Patients who developed infections after transplantation received a higher absolute count of total MDSCs from the graft compared to the no-event group, and significantly higher than the GVHD group, P<0.05. The mortality rate in the infection group was 30% (8/26), in the GVHD group was 14% (3/21), and in the no-event group was 0% (0/25). The ROC curve area under the curve (AUC) for the absolute count of total MDSCs per kilogram of body weight from the donor in the GVHD group was 0.669, with a cutoff of 1.7×107/kg, while in the infection group, the AUC was 0.661, with a cutoff of 4.3×107/kg. Conclusion: G-CSF can induce an increase in the content and enhancement of function of MDSCs in the donor's peripheral blood Maintaining a balance of MDSCs helps ensure immune homeostasis after transplantation.

Risk factors for catheter-related bloodstream infections associated with home parental nutrition in children with intestinal failure: A prospective cohort study

Catheter-related bloodstream infection (CRBSI) is the most common, potentially life-threatening complication of long-term parenteral nutrition (PN). We prospectively assessed the incidence and risk factors for CRBSI in children receiving long-term home PN (HPN) for intestinal failure (IF) in a single IF rehabilitation center. Data regarding episodes and potential risk factors for CRBSI in children on HPN were prospectively recorded. Forty-one of 75 children were diagnosed with CRBSI. The overall CRBSI rate was 1.61 per 1000 catheter days. The indications for HPN were gastrointestinal motility disorders in 35%, short bowel syndrome (SBS) in 28% graft versus host disease (GvHD) post bone marrow transplant in 17%, congenital enteropathy in 15%, and severe neurodevelopmental impairment in 5%. Gastrointestinal motility disorders had significantly higher CRBSI rate compared to other groups (p<0.0005; 2.74 in motility group vs 1.54 in GvHD group vs 0.52 in congenital enteropathies vs 0.36 in SBS group vs 0.67 in severe neurodevelopmental delay). Multivariate analysis revealed that enterocutaneous distal stoma (ileostomy or colostomy) (HR 3.35 [95% CI, 1.63-6.86]; p<0.001), age <2 years (HR 0.28 [95% CI, 0.15-0.53]; p<0.0001), male sex (HR 2.28 [95% CI, 1.51-3.43]; p<0.0001), non-use of taurolidine citrate lock (HR 2.70 [95% CI, 1.72-4.11]; p<0.0001) and gastrointestinal motility disorder (HR 3.02 [95% CI, 1.81-4.91]; p<0.001) were independent risk factors for developing CRBSI. Extra care in managing PN connections and disconnections should be taken in children with an underlying gastrointestinal motility disorder, distal enterocutaneous stoma, male sex and those aged <2 years since they are at a significantly higher risk of CRBSI. Early introduction of taurolidine lock should be considered.

Abstract 643: Correlations of dietary fiber with intestinal microbiome and outcomes in allogeneic hematopoietic cell transplantation

Abstract Background: Allogeneic hematopoietic cell transplantation (allo-HCT) can be a curative therapy for patients with hematological malignancies. However, allo-HCT poses a significant risk for post-transplant mortality due to graft-versus-host disease (GVHD), which is associated with loss of beneficial intestinal microbiota and decrease of microbial metabolites. In addition, dietary fiber can modulate the intestinal microbiome and the production of short-chain fatty acids (SCFAs), which are microbial metabolites associated with intestinal homeostasis. Therefore, we hypothesized that a high-fiber diet is associated with an increase of beneficial commensals, higher concentrations of SCFAs, and decreased risk of GVHD. Methods: We collected dietary data and fecal samples from 73 allo-HCT patients (GVHD n=45, No-GVHD n=28) at Memorial Sloan Kettering Cancer Center, collected longitudinally throughout the allo-HCT course, and performed 16s rRNA, shotgun metagenomic sequencing, and gas chromatography mass spectrometry (n=311). We assessed the α-diversity, microbiome composition, and concentrations of fecal SCFAs in response to fiber intake. We also tested different concentrations of dietary fiber (0%, 6%, 12% and 40%) in a preclinical GVHD model (C57BL/6J into BALB/c, n=30 per group, n=180 total) and assessed GVHD lethality and fecal microbial composition. Results: We found that in allo-HCT patients, fiber intake significantly correlated with microbial α-diversity (R=0.3, p=0.01) and patients with a higher fiber diet had a significantly higher microbial α-diversity when compared to the low fiber cohort (p&amp;lt;0.001). Remarkably, the decrease in dietary fiber post-allo-HCT correlated with a significant reduction in the fecal concentrations of SCFAs in the GVHD group (n=14), butyrate (p=0.003), propionate (p=0.01), and acetate (p=0.004), that was not significant in the No-GVHD cohort (n=28), indicating that low dietary fiber could lead to lower production of bacterial metabolites in GVHD. In addition, mice receiving a fiber-rich diet (12%) had a significant reduction in GVHD lethality (p=0.02) compared to mice receiving lower or higher fiber (0%, 6%, 40%). A 12% fiber diet also resulted in higher microbial α-diversity (p=0.02) and a significant decrease in Enterococcus faecalis (p=0.05), a pathogen associated with worse GVHD mortality in mice and humans. Conclusions: Our study demonstrates that a) higher fiber intake in patients after allo-HCT is associated with an increase in α-diversity and SCFA concentrations, and b) optimal fiber consumption in pre-clinical GVHD models leads to an increase in α-diversity, a decrease in pathogen mono-domination, and more importantly, a decrease in GVHD lethality. These results suggest that dietary fiber could be used in the prevention of GVHD. Citation Format: Jenny Paredes, Peter Adintori, Anqi Dai, Ruben J. Faustino Ramos, Teng Fei, Harold Elias, Marina Burgos da Silva, Romina Ghale, Charlotte Pohl, Justin Cross, Oriana Miltiadous, Marcel van den Brink. Correlations of dietary fiber with intestinal microbiome and outcomes in allogeneic hematopoietic cell transplantation [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 643.

The composition of the intestinal microbiota after allogeneic haematopoietic stem cell transplantation and its association with graft versus host disease as assessed by 16Sribosomal ribonucleic acid.

To observe the evolution of the intestinal microbiota in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and discuss the relationship between the intestinal microbiota and graft-versus-host disease (GVHD). In this study, 11 patients who underwent allo-HSCT in the Aerospace Central Hospital from January 2021 to October 2021 were selected, along with 11 donors. Fecal specimens were collected 7 times: at admission, after pre-treatment, and every 3 weeks after transplantation from patients and once from donors. The composition of the intestinal microbiota and its association with GVHD after allogeneic hematopoietic stem cell transplantation were analyzed by 16S rRNA sequencing. Of the 11 patients, 5 developed GVHD, and 6 did not. The diversity of the intestinal microbiota among GVHD patients first increased and then decreased after transplantation, while that among non-GVHD patients first increased and then tended to be stable. The diversity of the intestinal microbiota among GVHD patients was lower than that among non-GVHD patients before pre-treatment and after transplantation. The taxa diversity of the intestinal microbiota in the non-GVHD group was better than that in the GVHD group before allo-HSCT, and the difference was statistically significant (P<0.05 for OTUs and CHAO1 index). The taxa abundance of Enterococcaceae 2.16% (2.13%, 2.22%) before allo-HSCT was significantly higher than that in the non-GVHD group 1.33% (0.27%, 1.52%), and the difference was statistically significant (P=0.004). There was no significant difference between the GVHD group and the non-GVHD group in the diversity of the intestinal microbiota of donors (P<0.05). The characteristics of the intestinal microbiota in the final sample of patients in the GVHD group were similar to the preoperative structure of the intestinal microbiota. In conclusion: The decrease in the diversity of the intestinal microbiota after HSCT may be a risk factor for the occurrence of GVHD. The presence of Enterococcaceae in the intestinal microbiota may be associated with an increased risk of developing GVHD. The intestinal microbiota reconstitute to be close to the intestinal microbiota composition of the donors in the non-GVHD group.

EPC infusion ameliorates acute graft-versus-host disease-related endothelial injury after allogeneic bone marrow transplantation.

Graft-versus-host disease (GVHD) damages vascular endothelium. Endothelial progenitor cell (EPC) can differentiate to endothelial cell and promote angiogenesis, but its role in endothelial damage in GVHD is unclear. In this study, we intend to assess whether EPC infusion promotes the repair of endothelial injury in GVHD mouse model. Male BALB/c mice were randomly divided into 5 groups: control group, total body irradiation group (TBI group), allogeneic bone marrow transplantation group (Allo-BMT group), acute graft versus host disease group (GVHD group), EPC infusion group (GVHD+EPC group) followed by analysis of mice survival, acute GVHD (aGVHD) score, T cell infiltration by immunofluorescence, as well as continuity of vascular endothelium in liver. Compared with Allo-BMT group, the clinical and pathological score of aGVHD mice were higher. On day 21 after transplantation, a large number of mononuclear cell infiltrations were seen in the target tissues of aGVHD mice and mice died within 30 days. In addition, aGVHD group also presented increased subendothelial infiltration of CD3+ T cells in the liver, decreased VE-cadherin expression and elevated major histocompatibility complex (MHC) II molecule expression in the endothelium. Moreover, expression of MHC-II molecule increased in endothelial cell after irradiation injury and LPS stimulation, indicating abnormally activated endothelial cell with antigen-presenting function. Interestingly, infusion of EPC reduced the clinical and pathological score of aGVHD, decreased infiltration of mononuclear cells, improved survival as well as upregulated VE-cadherin and downregulated MHC-II molecule. EPC infusion can mobilize to affected endothelium to decrease the infiltration of T cells and pathological endothelial activation contributing to ameliorating the damage of endothelium. EPC infusion combined with bone marrow transplantation might be a perspective strategy for the prevention and treatment of aGVHD.

Prevention of Acute Graft-Versus-Host Disease Using an Engineered Mouse Orthogonal IL-2/IL-2Rβ Regulatory T Cells

Regulatory T cells are a critical population for the maintenance of self-tolerance and the use of these cells for therapeutic purpose in transplantation has emerged based on their capacity to control immune activation, exhibiting positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining technical challenges in the clinical practice of Treg administration is the number of Treg required to effectively prevent GvHD. In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus preventing acute -GvHD.For investigation of the suppressive function of Treg orthogonal IL-2/IL-2 receptor system in a murine major mismatch of acute GvHD model, the BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x10 6 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x10 6 C57Bl/6 FoxP3 DRT-GFP-luc+ Treg cells (fTreg) or with 0.2x10 6 and 1x10 6 oTreg (transduced with the construct for murine oIL-2 receptor β chain) on day 0. On day 2, C57Bl/6 Tcon cells (1x10 6) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/daily) for 14 days and then 3 times a week for another 14 days.Using bioluminescence imaging, we observed that ortho IL2Rβ +Treg not only maintains migration ability to the gut, mLN and LN but also exhibited significant expansion ability treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P<0.0001 and P=0.0001, respectively). Furthermore, the group oTreg-oIL2 (1:1) showed a significant increase in the survival curve compared to the fTreg group (P=0.03). When decreasing the number of oTreg administrated (5:1 Tcon:Treg ratio) a significant increase in the survival (Tcon vs Tcon:oTreg (5:1)+oIL2; P<0.0001) in the oIL2 group. Similar survival curves were observed between this group and fTreg group (1:1).oIL-2 protein injection was able to selectively expand the oTreg in vivo, with a significantly increased percentage of Foxp3 GFP+ in CD4 + T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) and spleen after 7 days post-transplant. We also observed a significant decrease in the percentage of effector T cells in the LN, mLN and spleen of the mice transplanted with oTreg and oIL-2 administration after 7 days post transplantation compared with GvHD group. We further demonstrated that ortho-IL2 administration-maintained graft-versus-leukemia (GvL) responses against lymphoma cells (A20).Engineered orthogonal cytokine receptor improves the suppressing abilities of Treg potential in a murine model of acute GvHD with the ability to maintain GvL, representing a novel approach to the utilization of Treg in Allogeneic Hematopoietic Stem Cell Transplant. DisclosuresGarcia: Synthekine: Membership on an entity's Board of Directors or advisory committees. Blazar: Tmunity Therapeutics: Other: Co-founder; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Rheos Medicines: Research Funding; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Dynamic mRNA expression of donor-derived activating KIR genes and their significant effects on clinical outcome after haematopoietic stem cell transplantation.

Numerous reports suggest that activating killer immunoglobulin-like receptors (aKIRs) of natural killer (NK) cells, in addition to inhibitory KIRs (iKIRs), play a prognostic role after allogeneic haematopoietic stem cell transplantation (allo-HSCT). We aimed to investigate the association between the dynamic expression of KIRs on NK cells and the outcomes, particularly regarding graft-versus-host disease (GvHD). This study retrospectively enrolled 260 pairs of donors and recipients who had undergone allo-HSCT without in-vitro T cell depletion. The mRNA transcription level of KIRs was determined by quantitative real-time polymerase chain reaction (RT-qPCR). The levels of aKIR transcripts were decreased more than those of iKIRs during the occurrence of GvHD. The transcription levels of KIR2DS2 and KIR2DS4 in the patients developing GvHD, compared with those who were at a tolerance state, showed the most significant decrease in the month at their peak transcription levels (p=0.03, p=0.002). Significantly decreased expression of KIR2DS1 (p=0.02), KIR2DS3 (p=0.04) and KIR2DS5 (p=0.04) in the GvHD group was observed when the transcription levelreached amaximum. High expression of KIR3DS1 was associated with superior overall survival (OS) (p<0.001). The expression of KIR2DS4 in the KIR genotype Bx group decreased more during GvHD, particularly at 3M (p=0.02). These findings suggest that KIR genes are potential post-HSCT biomarkers and dynamic changes in the KIR transcription levels can be detected to better predict the occurrence and evaluate the treatment of GvHD after transplantation.

Impact of Omitting Post-Transplant Mini-Methotrexate Doses in Allogeneic Hematopoietic Cell Transplantation: A Single-Center Retrospective Study

Background: Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Despite its efficacy in combination with calcineurin inhibitors, methotrexate (MTX) may increase the risk of complications such as mucositis and delayed hematopoiesis and can contribute to hepatic and renal toxicities. As a result, doses are often omitted in the setting of treatment-related toxicities. Herein we report outcomes in the setting of omitting doses of mini-MTX (5 mg/m2 IV days 1, 3, 6, and 11) and describe management when these doses are omitted.Methods: This retrospective study identified patients aged 18 years or older who underwent HCT between 1/2008 and 12/2015 with planned GVHD prophylaxis of mini-MTX for four doses with a calcineurin inhibitor (cyclosporine or tacrolimus). Outcomes were compared between patients who had 1-3 doses omitted (group 1) and those who received all four planned doses of mini-MTX (group 2). Differences in baseline characteristics were determined using Chi-square, Fisher's exact, or Wilcoxon Rank-Sum tests as appropriate. Overall and relapse free survival (OS, RFS, respectively) were estimated using the Kaplan-Meier method with differences assessed with a log-rank test. The cumulative incidence method for competing risks was used in estimating non-relapse mortality (NRM), GVHD, as well as GVHD-related mortality, with differences assessed with Gray's test. Relapse was considered a competing risk for NRM while relapse or death were considered competing risks for GVHD. Non-GVHD related death was considered a competing risk for GVHD related mortality. For all analyses related to the number of MTX doses received, a landmark analysis was conducted at day 11 (scheduled date of 4th dose).Results: Of the 188 consecutive patients that were identified, 29 had 1-3 doses of MTX omitted. Baseline characteristics are summarized in Table 1. Notably, no patient in group 1 had ATG (0% vs 10.7%, p = 0.079). Omission of MTX was most commonly due to mucositis (14/29 patients), followed by hyperbilirubinemia, pleural effusions, acute kidney injury, and anasarca/ascites. When any mini-MTX was omitted, the most common strategies were to initiate mycophenolate mofetil (MMF) at 1g IV Q8H (17/29 patients) or corticosteroids (8/29 patients; both MMF and steroids were used in 4 patients). Overall, there was a non-significant increase in the incidence of acute grade 2-4 GVHD when comparing groups 1 and 2 (100-day incidence: 60.7% vs 50.9% respectively, p = 0.501) as well as more severe, grade 3-4 acute GVHD (100-day incidence 28.6% vs. 17%, p = 0.163). Similarly, there was an absolute, non-significant increase in the cumulative incidence of GVHD-related mortality (Table 2). Overall survival and NRM were negatively impacted when MTX was omitted (OS at 12 months: 55.2% vs 73%, respectively, p = 0.015; NRM at 12 months 32.1% vs. 10.1%, respectively, p = 0.003). The most common cause of NRM in both groups was GVHD (group 1: 6/11 patients vs. group 2: 20/29 patients), but organ toxicity was the second leading cause in group 1 (3/11 patients) compared to zero patients in group 2. Other results are summarized in Table 2.Conclusion: While limited by a small number of patients in group 1, it appears our management strategy of introducing an alternative agent (e.g. MMF ± corticosteroids) may ameliorate some of the increased risk of acute GVHD. The patients in group 1 did have worse OS and NRM compared to group 2. The presence of toxicities that caused the omission of MTX may have contributed to these findings. These results appear consistent with recent reports on outcomes, when the planned course of full-dose MTX as GVHD prophylaxis was not administered (Hamilton BK et al., Blood Cancer J 2015). The impact of omitting MTX doses on chronic GVHD incidence in this population will be reported in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Outcome of Patients Receiving Extracorporeal Photopheresis for Acute and Chronic Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies: Results from an Observational Study on 106 Patients

Graft-versus-host disease (GVHD), and complications of its treatment are the major causes of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT). Even with the use of standard GVHD prophylaxis with classical administration of Anti-thymocyte globulin (ATG), calcineurin inhibitors (CNIs) and methotrexate, the incidence of acute and chronic forms of GVHD remains not negligible. Extracorporeal photopheresis (ECP) has shown very promising results as curative strategy for GVHD but also when used prophylactically.The primary objective of this observational study was to perform an exhaustive description concerning patients receiving ECP after steroid resistance for either acute or chronic GVHD following allo-HCT, secondary objectives were to evaluate the efficacy and long-term outcomes.We included 106 patients who underwent allo-HCT between years 2001 and 2016, 65 (61%) were males, median age at allo-HCT was 52 years (20-67). Major diagnoses were acute leukemias (N=48, 45%), myelodysplastic syndromes (N=18, 17%) and multiple myeloma (N=12, 11%). Before transplantation, 56 (53%) patients were in complete remission or in chronic phase. Donors were identical siblings in 51% of patients, matched unrelated in 24%, mismatched unrelated in 24% and haploidentical in 1%. Cells source was peripheral blood in 65%, bone marrow in 27% and cord blood in 8%. Conditioning regimens were of reduced intensity in 62% and myeloablative in 38%. ATG was used during conditioning in 67% of patients. GVHD prophylaxis consisted on cyclosporine A (CsA) alone in 22 (21%) patients, CsA + mycophenolate mofetil in 29 (27%), CsA + methotrexate in 28 (26%), the rest received other combinations.In acute GVHD, first treatment consisted in methyl-prednisolone 2mg/Kg/day in combination with CNIs; and in chronic GVHD, it was methyl-prednisolone 0.5 - 1 mg/Kg/day + CNIs. ECP was performed using peripheral venous access. The ECP used the CELLEX system from Therakos®. ECP was initiated after transplantation either for acute GVHD [N=25 (24%), 12 grade III and 13 grade IV] affecting skin alone (N=5), gut alone (N=12), gut + liver (N=8); or for chronic GVHD [N=81 (76%), 15 (14%) limited and 66 (62%) extensive]. A total of 3300 ECP sessions were performed, majority (N=91, 85%) were twice a week; the median number of sessions/patient was 22 (min: 2, max: 132) for a median duration of 2.5 months (range: 0.5-48). ECP response evaluation was assessed after 4 weeks of treatment, patients were considered responding to ECP if more than 50% resolution of clinical GVHD manifestation of the organ involved was registered.Among the 25 patients treated for acute GHVD, one was not evaluable because of disease progression and early death, 16/24 (67%) were responders to ECP and 8/24 (33%) were not responders and died later from GVHD associated to other complications. Among the 81 patients with chronic GVHD, 63/81 (78%) were responders and had tapering of ECP, the remaining 18 patients had limited response were associated to infection, had other complications and died later. Of note, in both forms of acute and chronic GVHD, responding patients had more skin or mucosa forms whereas non-responding patients has more organ-involved forms especially liver and gut.After a median follow-up of 21 months, 52 (49%) patients are alive and 54 (51%) died with 39 (24%) from TRM causes (26 of them from GVHD) and 13 (26%) from relapse. Patients with acute GVHD had a median OS of 6 months with a survival probability at 2 years of 35% [95%CI: 14-56], those with chronic GVHD had a median OS of 72 months with a survival probability at 2 years of 68% [95%CI: 56-78]. The cumulative incidence of relapse for the whole population at 2 years was 16.7% (95% CI: 10-24), when stratifying according to the GVHD group, patients with chronic GVHD had lower relapse rate at 2 years with 12.6% (95% CI: 6-21) versus 31% (95% CI: 13-52) for those with acute GVHD, p=0.05.In conclusion, we confirm that ECP is an effective treatment for GVHD with overall response rate of more than 74%. Interestingly, we observed a low relapse incidence in patients receiving ECP for longer time as in the case of chronic GVHD, which could be related to the GVL effect itself but also a possible involvement of the ECP. As patients with advanced phase of GVHD were poor responders, we suggest that an early use of ECP in the acute phase of the inflammation before organ damage, could lead to optimal results. [Display omitted] DisclosuresNicolini:Incyte Biosciences: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; ARIAD: Honoraria, Speakers Bureau.

Conjunctival Matrix Metalloproteinase-9 Clinical Assessment in Early Ocular Graft versus Host Disease.

Purpose Graft versus Host Disease (GVHD) typically affects the ocular surface, with a presentation resembling Dry Eye Disease (DED). Although the etiopathology is not completely known, the conjunctiva might be a key site of T-cell activation. The differential diagnosis might be tricky at early stages, because of the lack of dedicated clinical and laboratory tests. To meet these needs, we evaluated the suitability of ocular surface matrix metalloproteinase-9 (MMP-9) clinical test. Methods Consecutive GVHD patients, referred to IRCCS San Raffaele Scientific Institute, were recruited. DED patients served as controls. MMP-9 was tested through InflammaDry immunoassay kit in both groups; Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity, fluorescein Tear Break-up Time (TBUT), corneal and conjunctival staining, and Schirmer test I were also collected. Parametric and nonparametric statistical tests were used to analyze the intergroup differences; Receiver Operating Characteristics (ROC) curve analysis was carried out to perform sensitivity and specificity evaluations. Results Forty-five GVHD and 40 DED patients were included. MMP-9 expression was significantly higher in GVHD group than in DED (84.4% vs 33%, p ≤ 0.001). Corneal and conjunctival staining scores resulted worse in GVHD than in DED (0.95 ± 1.16 vs 0.40 ± 0.63, p=0.02; 0.77 ± 0.42 vs 0.40 ± 0.49, p=0.0005, respectively). No significant differences regarded the other collected parameters. GVHD group was characterized by positive correlations between MMP-9 and conjunctival staining (rho = 0.55, p=0.0002) and between MMP-9 and OSDI (rho = 0.3, p=0.01); a faint inverse correlation was found between MMP-9 and Schirmer test (rho = −0.25, p=0.04). Conclusion MMP-9 has a role in physiologic cellular remodeling; when a proinflammatory stimulus occurs, MMP-9 molecules are overreleased in the extracellular matrix. The positive expression of MMP-9 in GVHD may be interpreted as the consequence of a T-cell aggression against self-antigens and may be considered a reliable biomarker to detect ocular surface inflammation in GVHD, even in early stages of the disease.

Assessment of ST2 and Reg3a levels in patients with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Background/aim Graft-versus-host disease (GVHD) is a crucial complication leading to significant morbidity and mortality allogeneic hematopoietic stem cell transplantation which occurs in approximately half of the transplant recipients. Suppression of tumorigenicity 2 (ST2) and regenerating islet-derived 3-alpha(Reg3a) might be important biomarkers to predict acute GVHD.Materials and methods In the present study, blood samples were collected from 17 patients with acute GVHD and 12 control patients after allogeneic stem cell transplantation. ST2 and Reg3a were measured in plasma samples compared in patients with acute GVHD and the controls. ResultsMedian age of the study population was 42 years (range 19–49). When compared to controls, the mean ST2 levels was significant higher in acute GVHD (9794 ng/dL vs. 2646 ng/dL, P = 0.008). Mean Reg3a level did not show significant difference between control and acute GVHD group (8848 ng/dL vs. 5632 ng/dL, respectively, P = 0.190).Conclusion The ST2 level might be used as a significant biomarker for predicting acute GVHD.

The Association between Meibomian Gland Atrophy and Corneal Subbasal Nerve Loss in Patients with Chronic Ocular Graft-versus-host Disease

ABSTRACT Purpose: To investigate the association between meibomian gland (MG) loss and corneal subbasal nerve plexus density in patients with chronic graft-versus-host disease (GVHD) related dry eye disease (DED). Materials and Methods: This cross-sectional study included 22 adult patients with severe DED secondary to chronic GVHD. Control group comprised age- and sex-matched 28 healthy subjects with no evidence of ocular disease. All subjects underwent tear breakup time (TBUT), corneal staining, Schirmer I test without anesthesia, quantitative MG drop-out assessment using infrared meibography and corneal subbasal nerve density measurements with in vivo confocal microscopy (IVCM) (ConfoScan4, Nidek, Japan). One eye per patient was included for statistical purposes. Mann–Whitney U test and one-way multivariate ANOVA test were used for comparative analyses. Results: Compared to healthy subjects (mean age = 26.9 ± 13.5 years (range = 20–44 years)), patients with chronic GVHD (mean age = 29.6 ± 12.6 years (range = 19–45 years)) had worse meibography scores (p < .001), reduced corneal subbasal nerve plexus densities (p < .001), lower TBUT scores (p = .012), lower Schirmer I values (p = .001) and higher corneal staining scores (p = 003). Meiboscores in the GVHD and control groups were 2.9 ± 1.1 (range = 1–4) vs. 0.7 ± 0.4 (range = 0–2) for the superior (p < .001), and 3.2 ± 1.2 (range = 2–4) vs. 0.5 ± 0.3 (range = 0–2) for inferior (p < .001) eyelids, respectively. Corneal subbasal nerve densities of patients with GVHD did not reveal a correlation with meiboscores (r = 0.030; p = .709 for the inferior and r = 0.268; p = .075 for the superior eyelids) but showed a weak correlation with Schirmer I test values (r = 0.268; p = .014). Conclusions: Patients with chronic GVHD are at high risk for developing DED and MG dysfunction. In the setting of chronic GVHD-related DED, MG loss does not appear to be a significant factor for corneal subbasal nerve damage.

Pre-Transplant Gene Expression May Predict Risk of Graft Versus Host Disease

Background Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to graft versus tumor effect (GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD have been identified to date. To study the mechanisms of GVHD and GVT and to identify potential GVHD markers we applied a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcriptome profiling. The TFA is a multiplex microfluidics q-PCR based assay, linked to a computational model for functional analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures (Chaussabel et a 2014, Speake et a 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases. Herein we report the discovery of an association between the expression abundance of a set of genes pre-transplant and the development of acute or chronic GVHD. Methods Seventy-eight patients candidate to allo-HCT were enrolled on the study. Patients donated micro-quantities of blood (50-600 microliters) prior to transplant conditioning, weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high-throughput qPCR system. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Here we report the univariate analysis of gene expression data and its association with GVHD in 31 patients for whom data are available until at least day 169 post-transplant (169-672, median 513). SIgnificant genes were tested in a longitudinal analysis with 4 combined linear-mixed models, measuring the parameters: 1) groups (GVHD NO-GVHD) 2) time 3) interaction of groups over time. Results Patients' characteristics are summarized in Fig 1; 26 patients developed GVHD (12 acute GVHD only, 3 late acute GVHD, 1 overlap syndrome, 5 acute and chronic GVHD and 5 chronic GVHD only). By TFA analysis of pre-transplant samples, we identified 3 genes significantly downregulated among patients who developed GVHD (GPSM3, LRG1 and EPHX4), and 2 genes borderline (&amp;lt;1.5 folds) upregulated (IFI35, PLK3). Among these, GPSM3 (an MHC regulated gene) has shown to have alleles protective against severe autoimmune diseases as RA, and LRG1 to facilitate CD34 and myeloid progenitors growth by antagonizing TGF-beta effects, but also to be upregulated in GVHD patients. Several other genes (111) were found to be &amp;gt;1.5 fold upregulated in the GVHD group, but with a non-significant p-value (Volcano-plot Fig.1). Of note, 12 of them were &amp;gt;4 fold upregulated, and their transcriptome pertains to B cells, T lymphocytes, monocytes, IFN, TNF, other cytokines and neutrophil activation. Moreover, among these genes IFN-genes were consistently represented by multiple IFN-modules, emerging as promising candidates. Longitudinal analyses confirmed that EPHX4 and GPSM3 distinguished the cohorts for all the measured parameters in the post-transplant time. EPHX4 increased over time in both groups but significantly more in the NO-GVHD group (with a fixed effect estimate of 2.05); GPSM3 instead decreased over time, again significantly more in the NO-GVHD group (with a fixed effect estimate of -0.95). LRG3 showed significance only for the parameter "time", resulting after transplant more abundant in the GVHD group, thus confirming previous literature data. IFN modules were also tested longitudinally, showing very distinct patterns in the cohorts, and several significant genes (IFI27, NT5C3, CCR1, LBA1, Fig.1) Conclusion Our data demonstrate that TFA may be used to assess expression of immune related genes in transplant patients and to identify specific pathways that may be predictive for GVHD development. Additional work is required to validate our data in a larger cohort and for better understanding of the role of those genes in triggering or protecting against GVHD. Upon completion of the study and the longitudinal analyses, we aim to define in depth transcriptome signatures of GVHD and relapse. Disclosures No relevant conflicts of interest to declare.

Aberrant HLA-DR expression in the conjunctival epithelium after autologous serum treatment in patients with graft-versus-host disease or Sjögren's syndrome.

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.

Prospective validation of the L-index reflecting both the intensity and duration of lymphopenia and its detailed evaluation using a lymphocyte subset analysis after allogeneic hematopoietic stem cell transplantation

We prospectively validated the previously reported L-index, which reflects both the intensity and duration of lymphopenia, and further evaluated it using a lymphocyte subset analysis after allogeneic hematopoietic stem cell transplantation (HSCT) (n = 30). The L-index was defined as the area over the lymphocyte curve during lymphopenia (<700/μl), and calculated from the start of conditioning to day30 (L-index(30)) and day100 (L-index(100)). The lymphocyte subset including CD3, CD4, CD8, CD19 and CD56 was analyzed before and at 14, 21, 28, 42, 56, 70, and 84 days after HSCT. Cytomegalovirus (CMV) antigenemia was detected as >3 cells/2 slides by the C10/11 method in 21 cases (CMV-AG ≥3 group) at a median of 34 days. L-index(30) was significantly higher in the CMV-AG ≥3 group than in the CMV-AG <3 group (median 20,358 vs 17,235, P = .028). Recovery of the CD4+ and CD56+ cell counts between days 14 and 28 after HSCT was impaired in the CMV-AG ≥3 group. Regarding graft-versus-host disease (GVHD), grade II-IV acute GVHD was identified in 14 patients (GVHD group) at a median of 31 days. L-index(30) was significantly lower in the GVHD group (median 19,048 vs 22,256, P = .043). Recovery of CD3+ cells including both CD4+ and CD8+ cells between days 14 and 28 tended to be better in the GVHD group. In conclusion, L-index(30) was significantly associated with CMV reactivation and grade II-IV acute GVHD, but its clinical significance seemed to differ according to the results of a lymphocyte subset analysis.

Sparing of the Lower Gastrointestinal Tract Microbiota Is Associated with Reduced Acute Graft-Versus-Host Disease

Introduction. Clinical graft-versus-host disease (GVHD) phenotypes can selectively involve the upper or lower tract and extraintestinal tissues and organs. High gut microbiota diversity and the abundance of certain bacterial commensals within the Clostridiales order are associated with decreased risk of GVHD-related mortality after allogeneic stem cell transplantation (allo-HCT). The microbiota-derived short-chain fatty acid butyrate has been found beneficial in reducing murine GI aGVHD. Conversely, microbiota disruption is associated with expansions of potentially pathogenic bacteria, including facultative anaerobes, and can worsen allo-HCT outcomes. Since the vast majority of the intestinal microbiota biomass resides within the colon, we hypothesized that certain features of the colonic microbiota confer local protection to the lower GI tract. Methods. We evaluated 216 patients (median age 55 years) who underwent unmodified allo-HCT and had stool samples collected under our institutional fecal microbiome biobank between 01/2011 and 02/2017. Patients were classified in three groups according to the organ involved in aGVHD: Upper GI only (UGI, n = 56), lower GI with or without upper GI involvement (LGI, n =61 ), no GI tract involvement (non-GI, n = 29). A fourth group of 70 control patients with no GVHD whatsoever were selected on the basis of the study inclusion and exclusion criteria and having evaluable samples in our biobank in a temporal distribution that matched onset of GVHD. Microbial diversity was computed using Simpson's reciprocal index. Results. A total of 902 stool specimens were analyzed with an average of 4.2 samples/patient. Samples were grouped into pre-onset (day -20 to day 0) and post-onset (day 1 to day 20) aGVHD. Trends in intestinal microbiota features relative to the day of aGVHD onset showed distinct dynamics among the groups. Prior to aGVHD onset, all groups showed similar microbial diversity, and similar abundance of anaerobic commensals, including members of genus Blautia (Fig 1A-C). After aGVHD onset, microbial diversity and commensal anaerobe abundance in LGI cases were significantly lower than non-GI (p = 0.01 and 0.008, respectively) and UGI (p = 0.03 and 0.03, respectively) groups (Fig. 1A, 1B). The LGI group also had significantly lower abundance of the genus Blautia (p = 0.02, Fig 1C). Conversely, the LGI group had increased prevalence of facultative anaerobes (LGI vs. non-GI, p = 0.03, Fig. 1D). Prior to aGVHD onset, the LGI cases had lower abundance of predicted butyrate-producing bacteria (LGI vs. no-GVHD, p = 0.03), which was maintained in all three GVHD groups post-onset aGVHD (Fig 1E). Conclusions. Patients without GVHD, non-GI and UGI aGVHD phenotypes had higher microbial diversity, abundance of anaerobe commensals including genus Blautia, and predicted butyrate-producing bacteria after aGVHD onset than patients with lower GI tract involvement by aGVHD. In contrast, the LGI cases had features consistent with microbiota dysbiosis including prevalence of facultative anaerobes, and lower abundance of butyrate-producing bacteria before and after aGVHD. These findings are consistent with a model in which a diverse microbiota abundant in potential beneficial commensals exerts a local protective effect in the lower GI tract and have potential practical implications in future prophylactic and therapeutic interventions in aGVHD. Figure 1 Disclosures Gomes: Seres Therapeutics: Other: Part of Salary. Slingerland:Seres Therapeutics: Other: Salary supported by Seres funding. Pamer:Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Seres Therapeutics: Honoraria, Patents &amp; Royalties; MedImmune: Honoraria; Novartis: Honoraria; Ferring Pharmaceuticals: Honoraria. Peled:Seres therapeutics: Consultancy, Honoraria. van den Brink:Acute Leukemia Forum (ALF): Consultancy, Honoraria; Merck &amp; Co, Inc.: Consultancy, Honoraria; Magenta and DKMS Medical Council: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Other: Licensing; Seres Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria; Therakos: Consultancy, Honoraria; Flagship Ventures: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Evelo: Consultancy, Honoraria.