Occurrence Of Graft-versus-host Disease Research Articles

Post-haematopoietic cell transplantation outcomes: why ST2 became a 'golden nugget' biomarker.

Immunotherapies have emerged as highly promising approaches to treat cancer patients. Allogeneic haematopoietic cell transplantation (HCT) is the most validated tumour immunotherapy available to date but its clinical efficacy is limited by toxicities, such as graft-versus-host disease (GVHD) and treatment resistance leading to relapse. The problems with new cellular therapies and checkpoint inhibitors are similar. However, development of biomarkers post-HCT, particularly for toxicities, has taken off in the last decade and has expanded greatly. Thanks to the advances in genomics, transcriptomics, proteomics and cytomics technologies, blood biomarkers have been identified and validated in promising diagnostic tests, prognostic tests stratifying for future occurrence of GVHD, and predictive tests for responsiveness to GVHD therapy and non-relapse mortality. These biomarkers may facilitate timely and selective therapeutic intervention. This review outlines a path from biomarker discovery to first clinical correlation, focusing on soluble STimulation-2 (sST2) - the interleukin (IL)-33-decoy receptor - which is the most validated biomarker.

NFKB1 gene single-nucleotide polymorphisms: implications for graft-versus-host disease in allogeneic hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) represents a significant cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). NF-kB system is a master regulator of innate immunity responses. It controls the expression of various cytokines and chemokines many of which are involved in GVHD pathogenesis. Chemo(radio) therapy administered during conditioning induces DNA damage and activates DNA damage response (DDR) signaling resulting in irreversible cell cycle arrest - cellular senescence which has been described to be associated with robust pro-inflammatory secretion mostly controlled by NF-kB. The NFKB1 gene encodes the DNA-binding subunit of the NF-kB complex. Using the candidate gene approach, we analyzed possible association of two single-nucleotide polymorphisms (SNPs) rs3774937 C/T and rs3774959 A/G of the NFKB1 gene with GVHD and transplant-related mortality (TRM) occurrence in 109 recipients allografted from HLA-identical donor. Both SNPs in recipients were found to be strongly associated with acute GVHD. Nevertheless, no significant association with chronic GVHD and TRM was found. Presented pilot results contribute to pre-clinical observations and suggest that NF-kB may be an important regulator of HSCT-related inflammatory reactions such as acute GVHD. Novel pathogenic mechanisms of GVHD may arise from perspectives of DDR and cellular senescence where NF-kB plays an essential role.

Age-related modifications of macrophages influenced by “inflammageing” in graft vs. host disease

Most studies focus on the adaptive immune cells in the GVHD pathogenesis, while little is known about innate immune cells in GVHD occurrence and development, especially macrophages. Meanwhile, a higher incidence of graft versus host disease (GVHD) is also found in the elderly patients. Though advances have been made in the modification of macrophages influenced by the inflamm-ageing, there is still no review on the role of macrophages in GVHD and the association between GVHD and the altered macrophages by inflamm-ageing. In this review, we focus on the potential age-related modifications of macrophage in GVHD, which contributes to the change of morbidity and mortality of GVHD. Via literature review, we found that the infiltration of macrophages is associated with GVHD and macrophages are modified in inflamm-ageing state, including the proliferation, migration, phagocytosis, antigen presentation, interaction with other immune cells, and pro-fibrosis. We suppose that altered macrophage functions in inflamm-ageing state contribute to GVHD in elderly patients.

Thymic activity in immune recovery after allogeneic hematopoietic stem cell transplantation in children.

Thymic output was studied prospectively in 52 children who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Thymic activity was assessed by quantification of recent thymic emigrants (RTE) discriminated from the rest of naive T cells by immunophenotype CD3+/CD4+/CD31+/CD45RA+. Thymic output was analyzed in correlation with the kinetics of immune recovery and in relation to other potential risk factors that may influence thymopoiesis: underlying disease, type of HSCT, source of stem cells, age of recipient and donor, type of conditioning, implemented graft versus host disease (GvHD) prophylaxis, viral reactivations (herpes viruses cytomegalovirus – CMV, Epstein-Barr virus – EBV, adenovirus – ADV, BK virus – BKV), occurrence and grade of both acute and chronic graft versus host disease (aGvHD, cGvHD) and number of transplanted CD34 cells/kg. The absolute count of RTE in peripheral blood was evaluated at 6 time points: before the conditioning and on days +15, +30, +60 , +90 and +180 after HSCT. Occurrence of grade II-IV aGvHD was the most important factor associated with low RTE counts after HSCT. History of malignant disease, and transplantation from matched unrelated donor were risk factors for lower thymic output. We found a weak inverse correlation between the age of the recipient and thymic output on post-HSCT day +180. Source of stem cells, type of conditioning, viral reactivations, occurrence of chronic GvHD, age of the donor and the number of transplanted CD34 cells/kg did not affect thymopoiesis in our study group. These preliminary findings and identification of risk factors for deterioration of thymic activity may in the future help in selecting candidates for thymus rejuvenation strategies.

Alterations in NKG2A and NKG2C Subsets of Natural Killer Cells Following Epstein–Barr Virus Reactivation in CTLA4Ig-based Haploidentical Transplantation Is Associated With Increased Chronic Graft-Versus-Host Disease

The impact of newer approaches to haploidentical transplantation on Epstein-Barr virus (EBV) is largely unknown. We prospectively evaluated the incidence of EBV reactivation and its impact on transplantation outcomes in 71 patients undergoing haploidentical transplantation with posttransplantation cyclophosphamide in combination with CTLA4Ig-based T-costimulation blockade. Eight patients developed EBV reactivation at a median of 96 days with no incidence of lymphoproliferative disorder. There was no impact of EBV reactivation on acute graft-versus-host disease (GVHD), nonrelapse mortality, progression-free, or overall survival. Despite an overall incidence of 19%, there was a significant increase in chronic GVHD following EBV reactivation (62.5% versus 8%; P = 0.01). NKG2A subset of CD56 natural killer cells increased substantially and persisted following EBV reactivation and chronic GVHD, with a reciprocal decrease in NKG2C subset, whereas the reverse was witnessed in those without chronic GVHD (P < 0.01). Increase in NKG2C subset and a decrease in the NKG2A subset were witnessed within 3 months of subsidence of chronic GVHD. Thus, CTLA4Ig-based haploidentical transplantation was associated with a low incidence of EBV reactivation without EBV-lymphoproliferative disorder. However, EBV reactivation was associated with a sustained alteration in NKG2A and NKG2C subsets of CD56 natural killer cells which might have a pathogenic role in chronic GVHD.

Mogamulizumab within 55 Days of Allogeneic Transplant: A Report of 3 Cases

Introduction: Mogamulizumab is a humanized monoclonal antibody against CCR4 (CC chemokine receptor 4), which is frequently expressed on certain T-cell malignancies such as adult T-cell leukemia-lymphoma (ATL), peripheral T-cell lymphoma (PTCL), and cutaneous T-cell lymphoma (CTCL), and in addition, on normal immune T-cells such as regulatory T-cells (Tregs). Mogamulizumab is approved as monotherapy in Japan for relapsed or refractory CCR4-positive ATL, PTCL, and CTCL, and in the United States for adult patients (pts) with relapsed or refractory mycosis fungoides (MF) and Sézary syndrome (SS) - two subtypes of CTCL - after ≥1 prior systemic therapy. Based on retrospective analysis of ATL pts in Japan, evidence suggests that a shorter interval (&lt;50 days) between the last dose of mogamulizumab and allogeneic hematopoietic stem cell transplant (allo-HSCT) might increase the risk of graft versus host disease (GVHD)-related complications and negatively impact clinical outcomes compared to a longer interval (Fuji S et al, J Clin Oncol 2016). Outcomes of allo-HSCT within 50 days of mogamulizumab treatment outside of Japan have not been reported. A retrospective review of Western clinical trials of mogamulizumab in T-cell malignancies identified 3 non-ATL pts who went on to receive allo-HSCT within approximately 50 days of their last dose of mogamulizumab. Here, we report safety and outcome information for these cases. Methods: A review of clinical trial pts in the West identified two pts from the open-label, phase 3, randomized controlled MAVORIC trial (ClinicalTrials.gov number: NCT01728805), which compared treatment with mogamulizumab to vorinostat in patients with relapsed or refractory MF/SS; crossover was allowed from the vorinostat to the mogamulizumab treatment arm upon progressive disease or intolerable toxicity. A third pt was identified from an open-label, single-arm phase 2 trial for previously treated PTCL (NCT01611142) in patients with relapsed or refractory CCR4-positive PTCL. In both studies, pts received mogamulizumab 1.0 mg/kg intravenously (IV) on a weekly basis for the first 28-day cycle, then on days 1 and 15 of subsequent cycles. Results: Pts were 49, 61, and 22 years of age with MF, MF/SS, and PTCL, respectively (Table). All pts had received at least 1 prior systemic treatment regimen for their disease. The last dose of mogamulizumab was administered 54, 17, and 45 days prior to allo-HSCT, respectively. Post-transplant, all three pts experienced at least one occurrence of GVHD, both steroid refractory and responsive, with grades ranging from 1 to 4 in the skin, abdomen, colon, and eye. The pts with MF and SS were alive at 1399 days and 1187 days post-transplant, respectively. The pt with PTCL died on day 330 post-transplant. Conclusions: This is the first published case study report to describe three pts with non-ATL T-cell malignancies who received mogamulizumab approximately 50 days or less prior to allo-HSCT and who experienced varying types and severity of GVHD. Two pts with MF/SS-type CTCL, who received mogamulizumab within 55 days of transplant, survived ≥1187 days. Further research is needed to understand the efficacy, safety, and appropriate timing of mogamulizumab prior to allo-HSCT in pts with T-cell malignancies. Disclosures Lechowicz: Kyowa Kirin Inc: Consultancy; Spectrum: Consultancy. Leoni:Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Tabachri:Kyowa Kirin Pharmaceutical Development, Inc.: Employment. Herr:Kyowa Kirin, Inc.: Employment. Lamar:Kyowa: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy.

Impact of ABO Blood Group Incompatibility on Outcomes after Single-Unit Umbilical Cord Blood Transplantation for Malignant Hematological Disease

Objective In contrast to solid organ transplantation, ABO blood group incompatibility was acceptable in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, reports of the effect of donor-recipient ABO incompatibility on long-time survival, graft-versus-host disease (GVHD), and relapse after allo-HSCT were controversial. Relatively few reports existed on the effects of ABO incompatibility after umbilical cord blood transplantation (UCBT). The aim of this study was to investigate the role of major ABO incompatibility on RBC transfusion burden, hematologic recovery, GVHD, transplant-related mortality (TRM), relapse, and overall survival (OS) in UCBT for malignant disease. Methods This retrospective study included 587 malignant hematonosis patients who received myeloablative single-unit unrelated donor UCBT at our center between May 2008 and June 2018. Median follow-up time of the patients alive was 40.7 months (range: 12.0-134.6 months). A total of 230 (39.2%) patients received an ABO-identical transplant, and 357 (60.8%) received ABO-mismatched transplants, including 161 (27.4%) minor, 141 (24.0%) major, and 55 (9.4%) bidirectional ABO-incompatible UCBTs. All patients received myeloablative conditioning regimens and cyclosporine A (CsA) combined with mycophenolate mofetil (MMF) as a GVHD prophylaxis. Results A comparison of ABO compatibility and incompatibility demonstrated no significant differences (P&gt;0.05) in the cumulative incidence of neutrophil, platelet, and red blood cell engraftment . There was no significant difference in the cumulative incidence of grades Ⅱ to Ⅳ aGVHD (P= .527) and Ⅲ to Ⅳ aGVHD (P= .949) among the 4 groups (Figure A , B). In univariate analysis, ABO blood group incompatibility was not associated with cumulative incidence of 180d TRM (Figure C, P= .602). The overall 3-year survival had no statistically significant differences among the 4 groups (Figure D; P= .384). Further, 11 patients were excluded from the analysis of post-UCBT RBC transfusion burden because of missing data and non-red blood cell engraftment. Of the remaining 576 patients, the median number of RBC transfusions during transplant days 0 to 60 was 4 (range, 0 to 106). There was no significant difference in the transfusion burden among all ABO blood type mismatch groups (Table 1, P = .069). Furthermore, none of the patients developed pure red aplastic anemia (PRCA) after UCBT. Conclusion The results showed that ABO blood group incompatibility had no significant impact on hematologic engraftment, the occurrence of GVHD, and the survival of malignant hemoblastosis. Patients with myeloablative single-unit UCBT may not develop PRCA; Donor-recipient ABO incompatibility may not be the major consideration in the selection of umbilical cord blood. Disclosures No relevant conflicts of interest to declare.

Decitabine As Part of a Modified Bu-Cy Conditioning Regimen Significantly Improved the Outcome of High-Risk AML Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Background :Relapse remains a major cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially in patients with high-risk acute myeloid leukemia (AML). Therefore, how to eradicate the minimal residual disease and prevent of relapse are of great importance to improve the outcome of allo-HSCT for these high risk AML patients. M ethods :We retrospectively investigated decitabine (DAC) as part of a modified Bu-Cy regimen for high-risk AML patientsundergoing allo-HSCT. Fifty-nine patients received DAC (20 mg/m2/d,i.v.) for 5 days, followed by modified Bu-Cy (DAC group). A control (CON) of 177 patients (pair-matched 1:3) received modified Bu-Cy only. Results:Median follow-up was 402 (85-1504) days. There was no difference in graft versus-host disease occurrence. Treatment-related mortality (day 100) was 0% (DAC) and 6.2% (CON). The DAC group had better 2-year overall survival (OS, 77% versus 64%, P=0.018) and leukemia-free survival (LFS, 73% versus 56%, P=0.007). The differences were more substantial among patients with active disease: OS, 83% (DAC) versus 47% (CON), P=0.01 and LFS, 77% (DAC) versus 34% (CON), P=0.002. Median time to relapse was 187 days (DAC) versus 86 days (CON) and two-year relapse rates were 10% (DAC) and 49% (CON) for patients with active disease. Patients with cytogenetic monosomy 7 or 7q- abnormality responded better. Conclusions: Our data indicate that novel conditioning regimen containing DAC may be an effective and well-tolerated strategy and confer a subgroup-specific survival advantage in high-risk AML patients undergoing allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Fast CMV and EBV Reactivations after Ruxolitinib for Graft-Versus-Host Disease Treatment

Introduction: Corticosteroid-refractory graft-versus-host disease (GVHD) remains a serious complication of hematopoietic stem cell transplantation (HSCT) with high morbidity and mortality rates. Unfortunately, no standard therapy exists for this setting. Ruxolitinib (ruxo), an oral selective Janus-associated kinase (JAK) inhibitor, achieved good results for corticoresistant acute and chronic GVHD in preclinical and clinical studies, with 80% overall response rates. Recent studies showed an increased risk of infections in patients treated with ruxo, especially Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivations. Patients and methods: In order to assess the efficacy and the safety of ruxo, we reviewed here the outcome of 57 patients who received ruxo for corticosteroid-refractory GVHD (in case of digestive tract involvement or in the absence of available ongoing clinical trials) or as a steroids-sparing medication. EBV and CMV reactivation risks were also assessed in the 137 consecutive patients received HSCT between January 1, 2012 and December 31, 2017 and who presented acute or chonic GVHD (57 of whom received ruxo). For this purpose, each reactivation were analyzed separately as a competing risk with death in a cause specific Cox model of survival after the first GVHD occurrence and the onset of ruxo therapy was coded as time dependent covariate. Results: The median age of 57 patients with ruxo was 55 years (range, 49 to 61). Indication for HSCT was acute myeloid leukemia for 25% patients, lymphoma for 30%, acute lymphoblastic leukemia for 11%, myeloproliferative neoplasm for 14% and myelodysplastic syndrome for 7%. Only 9% of this patients received ruxo before HSCT. Unrelated donor was used for 60% patients and main source of hematopoietic stem cells was peripheral stem cells (93%). T cell depletion with polyclonal anti-thymocyte globulin was performed for 89% of patients. Conditioning with high doses cyclophosphamide was used for 21% patients. A lymphopenia &lt;1G/L persisted at day 100 for 42/51 patients evaluated. In these 57 patients, ruxo was given for 62 episode of GVHD (acute: 21 [which 95% for an acute grade III-IV GVHD]; chronic: 36; overlap syndrome: 5). Response rates to ruxo were 48% for acute GVHD, 60% for overlap syndrom and 58% for chronic GVHD. The overall GVHD related-death rate was 33% (19% for acute GVHD and 14% for chronic GVHD). Our median follow-up was 30 months (range, 9 to 42) after HSCT. Main non-infectious adverse events were cytopenias (17/57) and hepatic cytolysis (6/57) leading to discontinuation or tapering of ruxo in 12 patients. EBV reactivation (&gt; 4 log or increasing viral charge of 0.5 log) occurred in 19 patients after ruxo with a 6-weeks cumulative incidence (6WCuI) of 22% (95CI [95% confidence interval]: 15-34). It was the first reactivation in 13 patients. CMV reactivation (&gt; 3 log) occurred in 8 patients after ruxo with a 6WCuI of 4% (95CI: 7-25). It was the first reactivation in 3 patients (Figure 1). The distribution of first reactivation before ruxo and in the remaining 80 patients is shown in Table 1. Thus, 6WCuI of first EBV and CMV reactivation after the first episode of GVHD was 24% (95CI: 19-31) and 20% (95CI: 15-25) respectively. Finally, onset of ruxo coded as time dependent covariates retained a significant adverse prognostic value for the competing risks of death and first episode of EBV reactivation (HR [Hazard Ratio]: 2,657, p&lt;0,05) as well as first episode of CMV reactivation (HR: 1,747, p&lt;0,05) after first episode of GVHD. Discussion /Conclusion: Ruxo initiation coded as a time-dependent covariate was significantly associated with the overall risk of viral reactivation after the first episode of GVHD and the viral reactivations incidences after ruxo were similar with the incidence of reactivation at the onset of GVHD. Thus, it might be linked to the immuno-compromised state induced by both HSCT and GVHD. Furthermore, as we used ruxo for serious gastro intestinal GVHD, we could have selected patients with a more severe GVHD, requiring multiple immunosuppressive therapy, worsening immune reconstitution. To our knowledge it is the first study to assess the competing risk of CMV and EBV reactivation during ruxo treatment for GVHD. Given its effectiveness in corticoresistant GVHD, ruxo use must not be limited by the fear of viral reactivation at the light of our data, conditioned upon a close monitoring of viral loads in the first weeks. Disclosures No relevant conflicts of interest to declare.

Ptcy + ATG Vs Ptcy Alone As Gvhd Prophylaxis for Peripheral Blood Stem Cells Haplo-Transplants: Comparison of NK and T Cell Effector Reconstitution

Backgound: A higher incidence of graft-versus-host disease (GVHD) has been observed after haplo-transplant of hematopoietic stem cells with post-transplant cyclophosphamide (PTCY) using peripheral blood stem cells (PBSC) instead of bone marrow as source of graft. Combining PTCY with anti-thymoglobulin (ATG) may help to reduce GVHD incidence. Here, early immune reconstitution, especially of NK and T cell compartments, was compared after both types of transplant (PTCY vs PTCY+ATG) investigating their influence on patients (pts) outcomes. Methods: This retrospective study included 58 adults who received a reduced-intensity conditioning (RIC) PBSC haplo-transplant with ciclosporine and mycophenolate mofetyl + PTCY (n=32, period: 08/2014-05-2017) or PTCY+ATG (N=26, period: 05/017-03/2019) as GVHD prophylaxis. The PTCY group received a Baltimore-based RIC regimen with fludarabine (n=10) or clofarabine (n=22). The PTCY+ATG group received a CloB2A1 regimen consisting of clofarabine 30 mg/m²/day (d), d-6 to -2, busulfan 3,4 mg/kg d-3 and d-2, ATG 2,5 mg/kg d-1. Both groups shared similar characteristics except for the median number of CD3+ T cells infused, significantly higher for CloB2A1 pts (27.5x107/kg vs 22.3, p=0.01). Samples from all pts were collected three times a week from d0 until d30, then at d60 and d90/100 in order to evaluate NK and T cells reconstitution by flow cytometry (FCM). All pts provided informed consent for data and samples collection before graft. Results: The median follow-up was 32 and 10,5 months for alive pts in PTCY and PTCY+ATG groups, respectively. Primary graft failure (PGF) was observed in 5 pts in the PTCY+ATG group (19%) vs none in the PTCY group (p=0.03). The PTCY and PTCY+ATG groups shared similar survivals at 1-year with PTCY OS of 71.5% vs PTCY+ATG 60,2% (p=0.73), DFS of 62,5% vs 58.6% (p=0,8) and GRFS of 46.8% vs 5.7% (p=0.47). Incidences of relapse and death were also comparable. Conversely, considering engrafted patients, the incidence of grade 2-4 acute GVHD was significantly lower in the CloB2A1 sub-group (23.8% vs 59%, p=0.02) despite higher numbers of CD3+ T cells infused in this group. The incidence of grade 3-4 acute GVHD was also lower in the CloB2A1 sub-group (4.7% vs 18.7%, p=0.29). Immune reconstitution of the PTCY group has been already reported (Willem, J Immunol 2019). Considering the PTCY+ATG group, T cell reconstitution did not influence the occurrence of acute post-transplant GVHD. Yet, in the group of pts with myeloid diseases (n=20), a higher frequency of CD56- T cells at d20 was associated with a higher relapse incidence (39% vs 18%, p=0.04). The NK cells frequency was lower at d+30 for patients with PGF (52.5% vs 19.5%, p=0.01). Pts who did not relapse were documented with higher d30 frequency of NKG2A+ NK cells (94.4% vs 72.1% of all lymphocytes, p=0.04). Specifically, these pts showed higher NKG2A+ KIR2DL2/3/S2- NK cell levels at d30 (74.1% vs 56.3%, p=0.05) and d60 (74.1% vs 55.5%, p=0.03). Inhibitory KIR/HLA incompatibilities, documented in 4 donor/recipient pairs, were associated with lower NK cell frequency at d60 (15% vs 69.2% of all lymphocytes, p= 0.01) and d100 (9% vs 61.5%, p=0.01) Comparing the PTCY vs the PTCY+ATG groups, the frequency of CD56- T cells was found to be significantly lower during the first month when using PTCy+ATG: d5 (34.3% vs 75.7% of all lymphocytes, p&lt;0.0001), d20 (26.4% vs 54.8%, p&lt;0.0001), d25 (35.9% vs 55.7%, p=0.01) and d30 (29.1% vs 48.1%, p=0.008). However, higher d5 (p=0.0001) and d30 (p=0.003) KIR2DL2/3/S2+ T cell levels were observed in this group. However, this proportion strongly decreased in comparison to the PTCY group at d100 (p&gt;0.0001). NK cell levels were higher at d25 (34.9% vs 18.4% of all lymphocytes, p=0.03) and d30 (42.7% vs 27.9%, p=0.04) in the PTCY+ATG group. Specifically, faster reconstitutions at d20 of mature NKp46+ 2B4- (p=0.002), NKG2A+ (p=0.01) and NKG2A+ KIR2DL2/3/S2- (p=0.0004) cell subsets were found in this group. Conclusion: These results show that PTCy+ATG vs PTCy alone as GVHD prophylaxis limits acute grade 2-4 GVHD occurrence after RIC PBSC haplo-transplant, perhaps because of the combined effect of T and NK cell effector reconstitution. Indeed, the slower T cell reconstitution with PTCy+ATG may limit GvHD occurrence. In parallel, the quicker reconstitution of some NK cells sub-types may limit relapse occurrence. Larger prospective studies are needed to confirm these preliminary results. Disclosures Peterlin: Daiichi-Sankyo: Consultancy; Jazz Pharma: Consultancy; AbbVie Inc: Consultancy; Astellas: Consultancy. Chevallier:Jazz Pharmaceuticals: Honoraria; Daiichi Sankyo: Honoraria; Incyte: Consultancy, Honoraria.

Low Levels of Plasmacytoid Dendritic Cells at the Time of Engraftment Are a Valuable Severe aGVHD Indicator in Children Undergoing Allogeneic Stem Cell Transplantation

Graft-versus-host-disease (GVHD) remains a leading cause of transplant-related mortality (TRM) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients who do not respond to GVHD therapy are at high risk for death without relapse. Strategies that can predict the development of severe GVHD in transplant patients may improve risk stratification for steroids treatment refractory GVHD and TRM. Dendritic cells (DCs) play nonredundant roles in regulating both immunity and tolerance. Clinical and pre-clinical studies suggest that GVHD causes dysregulated development of functional DCs and impaired reconstitution of donor DCs is associated with increased risk of severe GVHD. We demonstrate that donor plasmacytoid DC (pDC) reconstitution around the engraftment time is a useful predictor for the development of severe GVHD in children after allo-HSCT. We examined reconstitution of donor DCs, including total DCs (lineage-negative (LIN-)HLADR+), pDCs (LIN-HLADR+CD123+CD1c-) and CD1c+DCs (LIN-HLADR+CD123-CD1c+), in peripheral blood of patients undergoing allo-HSCT, using age-matched healthy children as controls. The median time for engraftment was 14 days (range, 13.0~17 days), whereas clinical signs of GVHD occurred approximately 10 days later (median time, 24 days;range, 18~35 days). Thus, measuring the number and frequency of circulating blood DCs early at the engraftment time allowed us to evaluate the subsequent occurrence of GVHD. Transplant patients were followed up for 6 months after allo-HSCT. Of evaluable 45 transplant patients, 27 patients had no GVHD (grade 0) or mild GVHD (grade I), and 18 patients developed severe GVHD (grade II-IV GVHD). Patients who developed grade II-IV GVHD had no significant difference in frequency ( median, 0.2%; range, 0.04~0.35%) and number (median, 1.6 cell/ml; range, 1~3.8 cell/ml) of total DCs than patients with grade 0-I GVHD (median frequency, 0.41%; range, 0.15~0.63%, P&lt;0.05; and median number, 2.6 cell/ml , range, 1~6.7 cell/ml,Figure1A,1B). Both pDCs and CD1c+DCs represent the major DC populations in human peripheral blood. Patients with grade II-IV GVHD showed significantly lower frequency and number of pDCs (median frequency, 3.4%; range, 0.64~14%, median number, 0.06/ml, range, 0.02~0.25 cell/ml) than patients with grade 0-I GVHD median frequency, 29.1%, range, 20.3%~39.2%, P&lt;0.01; mediate number, 0.5 cell/ml, range, 0.2~1.9 cell /ml, P&lt;0.01Figure1A,1B). In contrast, there was no significant difference in frequency and number of CD1c+DC between the two groups. These data suggest that the reconstitution of donor pDCs is linked to the occurrence of severe GVHD. Using Receiver operating characteristic (ROC) curve analysis, we explored thresholds of pDCs as a discriminator for grade II-IV GVHD patients versus grade 0-I GVHD patients. The median pDC level in all tested patients was 0.25 cell/ml (range, 0.1~0.9 cell/ml,Figure1C) at the engraftment time. We chose the threshold of pDC level at 0.3 cell/ml to evaluate the difference in patients with high (above the threshold) versus low (below the threshold) pDC levels. Of these 45 patients, 21 (46.7%) patients had high pDCs and 24 (53.3%) were low pDCs. Patients with low pDCs had a significantly higher probability of developing grade II-IV GVHD (p&lt;0.01). The sensitivity of discriminating severe GVHD versus no/mild GVHD was 75% and the specificity was 94%. In addition, low engraftment pDC patients had higher TRM compared to high pDC patients (12.5% versus 0%) for median follow-up time 300 days (range, 210-465 days) after transplantation. However, there was no significant difference in overall survival rate between high pDC- and low pDC-group. Furthermore, the serum level of REG3α and ST2 at the engraftment time was not associated with the incidence of severe GVHD in these patients. Collectively, our findings identify that quantitation of pDCs at the engraftment time is a valuable prognostic predicator for severe GVHD in patients undergoing allo-HSCT. Given the importance of pDCs in modulating GVH reactions, it will be important to further determine whether impaired reconstitution of donor DCs may contribute to dysregulated immune tolerance against host tissues after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Graft-Versus-Host Disease Causes the Failure of Donor Hematopoietic Progenitor Cells to Reconstitute Plasmacytoid Dendritic Cells That Promote Tolerance of Donor T Cells Against the Host

Promoting donor T cell tolerance to host non-hematopoietic tissues remains the ultimate therapeutic goal in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dendritic cells (DCs) play dual functions in regulating alloimmunity. DCs can elicit alloreactive T cell responses to mediate graft-versus-host disease (GVHD), but are also implicated in reducing GVHD. In patients, the depletion of plasmacytoid DCs (pDCs) from donor BM grafts resulted in GVHD acceleration. On the other hand, acute GVHD causes complete failure of donor pDC reconstitution after allo-HSCT, and low levels of donor pDC correlate with significantly increased GVHD severity. Thus, the impairment of pDC reconstitution by GVHD may be responsible for the dysfunctional immune regulation. Delineation of the mechanism involved may allow therapeutic intervention to reduce GVHD and improve the efficacy of allo-HSCT. In this study, we demonstrate that alloreactive T cells produce GM-CSF to impair reconstitution of donor pDCs by inhibiting Flt3 expression and its-regulated transcription programs in DC progenitor cells. Using murine GVHD model, we confirmed GVHD severely impaired reconstitution of both donor pDCs and conventional DCs (cDCs). Adoptive transfer of donor-type pDCs rather than cDCs prevented the occurrence of severe GVHD in mice, suggesting donor pDC reconstitution is important to restore tolerance of donor T cells against host tissues. Flt3 is required to induce pDC production through a successive differentiation pathway: HSC → multiple potential progenitors (MPP) → common DC progenitors (CDP) → precursor DCs (pre-DCs). GVHD mice produced significantly less MPP, CDP and pre-DCs compared to normal donor mice and allogeneic mice receiving T cell-depleted BM. Ex vivo culture with Flt3 ligand (Flt3L) showed that those MPP and CDP derived from GVHD mice dramatically decreased the capacity to produce pDCs. Thus, GVHD not only causes decreased numbers of MPP and CDP but also their intrinsic defect in producing pDCs. While both MPP and CDP gave rise to similar numbers of pDCs within 3 days of culture with Flt3L, MPP produced 40-fold more pDCs than CDP by day 9 of culture. This indicates the impairment in GVHD MPP may have much more profound long-term impact on pDC reconstitution than that in CDP. Based on surface expression of Flt3, normal MPP contained two subsets: CD135high MPP and CD135mod MPP. CD135high MPP produced 4-fold more pDCs than CD135mod MPP. As compared to CD135mod MPP, CD135high MPP expressed lower levels of Ink4 family genes, which are cyclin-dependent inhibitors restraining cell proliferation and survival, suggesting that CD135high MPP represent earlier stage differentiated progenitors with greater proliferative capacity. Intriguingly, although GVHD mice generated similar amount of CD135mod MPP as did normal mice, they failed to reconstitute highly proliferative CD135high MPP. Thus, the failure of donor pDC reconstitution may largely result from GVHD-mediated inhibition of CD135high MPP. Alloreactive T cells are known to produce high levels of effector molecules, such as IFN-γ, TNF-α, GM-CSF and other cytolytic molecules. We observed that GVHD effector T cells significantly reduced the production of pDCs from Flt3L-induced normal MPP. Blocking GM-CSF using neutralizing antibody but not other effector molecules markedly inhibited this repressive effect of GVHD T cells on pDC production. GM-CSF dose-dependently decreased the expression of Flt3 and its-regulated transcription factors Irf8 and Tcf4, which are important for development of functional pDCs. However, GM-CSF failed to inhibit the conversion of SiglecH+ pre-pDCs into pDCs. These data suggest that alloreactive T cells produce GM-CSF to block pDC reconstitution by targeting DC progenitors (e.g., MPP and CDP). Building on these findings, we established a novel optimized culture system to produce adequate numbers of SiglecH+ pre-pDCs. Adoptive transfer of these pre-pDCs prevented GVHD, leading to significantly improved overall survival of mice undergoing allo-HSCT. Our findings identify for the first time that selective restoration of donor pDCs early after allo-HSCT may represent an effective cellular therapy to prevent GVHD. Further delineation of the molecular pathway(s) involved in GVHD inhibition of DC progenitors may allow the development of novel approaches to circumvent mortality and morbidity associated with GVHD. Disclosures Zheng: Pfizer: Research Funding.

IL12 and IFNγ secretion by donor mononuclear cells in response to host antigens may predict acute GVHD after HSCT

One persistent problem of allogeneic hematopoietic stem cell transplantation (HSCT) is acute graft versus host disease (GVHD). The role of cytokines in the pathogenesis of GVHD has been acknowledged.We aimed, in the current study, to investigate the possibility of prediction of acute GVHD through investigating the pattern of interleukin 12 (IL12) and interferon gamma (IFNγ) production of both patients' origin and donors' origin. A total of 45 patients, receiving allogeneic peripheral blood (PB) stem cells from an identical sibling, were included in the study. Patients' plasma was collected after conditioning, during aplastic phase (representing patients' origin) and after engraftment (representing donors' origin). In addition an aliquot from the graft was used as responders in mixed lymphocyte culture (MLC) for 3 days with patients' mitomycin-treated mononuclear cells as stimulators. Culture supernatant was used for detection of IL12 and IFNγ of donors' origin. Fourteen patients developed acute GVHD. In culture supernatant, IL12 was detectable in 7/14 cases with and in none of 31 cases without acute GVHD (p= <0.001). The corresponding figures for IFNγ were 10/14 and 3/31 with significantly higher IFNγ level in cases with than in cases without acute GVHD (p = 0.001). At engraftment the corresponding figures were 7/14 and 5/31 for IL12 and 11/14 and 7/31 for IFNγ with significantly higher cytokine levels in cases with acute GVHD (p = 0.008 and p = 0.001 respectively). At a cutoff of 0.89 pg/ml, IL12 in culture supernatant may predict acute GVHD with absolute specificity of 100% and a sensitivity of 50%. In conclusion, IL12 and IFNγ of donors' origin not of patients' origin may predict the occurrence of acute GVHD. The MLC model may allow prediction of acute GVHD upfront before conditioning of the patient or mobilization of the donor.

Allogeneic stem-cell transplantation in HIV-1-infected patients with high-risk hematological disorders.

Although a number of patients with HIV infection and hematological disease have successfully undergone allogeneic hematopoietic stem-cell transplantation (HSCT), short and long-term outcomes remain not well known. We report the largest Spanish experience treating HIV-infected adult patients with high-risk hematological malignancies with allogeneic HSCT. We retrospectively reviewed 22 HIV-positive patients who received allogeneic HSCT in five centers in Spain. A total of 22 patients with high-risk hematological malignancies were transplanted between 1999 and 2018. Median age was 44 years. With a median follow-up of 65 months (8-112), overall survival and event-free survival were 46%. Nonrelapse mortality was 14% at 12 months and relapse was 24% at 24 months. Grade II-IV acute graft-versus-host disease (GVHD) rate was 44%, and moderate/severe chronic GVHD rate was 41% at 24 months. All patients received combination antiretroviral therapy. Two patients showed severe toxicity related to drug interaction with antiretroviral therapy. 68% of patients showed infectious complications with viral infections as the most frequent cause. Two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. All survivors except one maintained undetectable HIV load at last follow-up after HSCT. Allogeneic HSCT is an effective therapy for high-risk hematological malignancies in patients with HIV infection, and long-term HIV suppression with combination antiretroviral therapy is feasible. However, drug interactions with antiretroviral agents, occurrence of GVHD, and frequent infectious complications account for a complex procedure in this population. Selected HIV-infected patients with hematologic malignancies should be considered for allo-HSCT when indicated, in experienced centers.

P3.60: Native spleen preservation attenuate graft versus host disease in an experimental model of modified multivisceral transplantation.

Introduction: Native spleen used to be removed in patients undergoing modified multivisceral transplantation (MMVT) increasing the risk of sepsis and graft-versus-host disease (GVHD). The mechanistic basis of these effects including mixed chimerism level is poorly understood. Based on a novel experimental procedure of MMVT that triggers GVHD, we aimed to evaluate the effects of native spleen preservation in this model in order to gain insight into the mechanisms that may mediate this phenomenon. Material and Methods: Heterotopic MMVT from Lewis to Brown Norway rats was performed. MMVT graft consisted of stomach, duodenum, pancreas, spleen and the small bowel of the donor. The native spleen was removed (MMVT-S) in half of the MMVT recipients (N=5), while the remaining preserved their own spleen (MMVT+NSP) (N=5). After MMVT mixed hematopoietic chimerism was determined in native spleen and peripheral blood samples by flow cytometry using strain-specific HLA antibodies. Also, histopathological (using Pintar score) and clinical signs of GVHD such as skin rash were evaluated. Results: all animals in the group MMVT-S presented clinical signs of GVHD such as skin rash, weight loss, and diarrhea, among others, between 7–10 days after transplantation. Skin rash was particularly remarkable in the ears and periocular area. Clinical signs of GVHD were less frequent in MMVT+NSP recipients (40%; p<0.05). Also, significant differences between groups were observed in the histopathological study and mixed chimerism in peripheral blood 3 days after MMVT (36.6 and 23.62 % in MMVT-S and MMVT+NSP respectively) (Figure 1). Also, native spleen showed an 18±5 % of chimerism (CD3+ donor cells) 1 week after transplantation. After day 10 post-MMVT, when the signs of transient GVHD disappeared, both groups presented graft rejection. Conclusions: Native spleen preservation in MMVT recipients attenuated the occurrence of GVHD and reduced the level of chimerism compared to recipients that underwent spleen removal during MMVT. Despite more studies are necessary, our preliminary results suggest that native spleen preservation increase the efficacy in removal of anti-recipient reactive clones that would explain the protective effect of native spleen preservation against GVHD.

Early Cytomegalovirus Reactivation and Expansion of CD56brightCD16dim/-DNAM1+ Natural Killer Cells Are Associated with Antileukemia Effect after Haploidentical Stem Cell Transplantation in Acute Leukemia.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation but is suggested to exert a strong antileukemia effect in part due to alterations in the composition of natural killer (NK) cells. We evaluated the impact of early CMV reactivation and changes in NK cell subset recovery on relapse rate and survival after haploidentical stem cell transplantation (haploSCT) for acute leukemia. Fifty patients with acute leukemia who received haploSCT were analyzed. Expression of T cells and specific receptors (NKG2A, NKG2D, DNAM1, and CD57) on circulating NK cells (CD56brightCD16dim/- or CD56dimCD16+ cells) was serially measured using multiparametric flow cytometry. CMV reactivation during the first 100 days was observed in 41 patients (82%) at a median of 23 days after haploSCT. The incidence of acute graft-versus-host disease (GVHD) and chronic GVHD tended to be higher in patients with CMV reactivation, although this difference was not statistically significant. Multivariate analysis showed that CMV reactivation (P = .011) and a dose of infused T cells > 3.2 × 108/kg (P = .027) were independent predictors of a reduced relapse risk and only CMV reactivation (P = .029) was an independent predictor of improved leukemia-free survival. CD56brightCD16dim/-DNAM1+NK cell counts increased from day 30 to 90 in patients with CMV reactivation but decreased after day 30 in patients without CMV reactivation. An increase in CD56brightCD16dim/-DNAM1+ NK cells was not associated with the occurrence of chronic GVHD but was associated with a reduced cumulative relapse rate (16.4% versus 58.0%, P = .019). Multivariate analysis indicates that an increase in the CD56brightCD16dim/-DNAM1+NK cell count was an independent predictor of reduced relapse risk. Our study demonstrates a significant correlation between low relapse rates and CMV reactivation as well as the recovery of CD56brightCD16dim/-DNAM1+ NK cells, providing valuable information for understanding the plausible immunologic mechanism of the graft-versus-leukemia effect.

Decreased CD4+-Th/CD8+-Tc ratio in Bone marrow at diagnosis of acute myeloid leukemia Predicts Increased Risk of Graft-Versus-Host-Disease in Transplant Patients

Abstract Introduction Acute myeloid leukemia (AML) is an aggressive disease with high morbidity and mortality. Transplant is one of the most important treatments for AML. However, graft-versus-host-disease (GVHD) remains one of major problems in transplant patients. Since immune repertoire plays an important role in the GVHD occurrence, we investigate the relationship between lymphocyte subsets and the risk of GVHD. Methods 60 initial diagnostic flow data of AML transplant patients were reviewed (Oregon Health and Science University 2010 to 2016, 30 females and 30 males, with a median age of 51.417 years, range 13–72). 36 patients with GVHD including acute and chronic GVHD while 24 patients without GVHD. The lymphocyte subsets were analyzed including CD3+T-cells, CD3+CD4+Th, CD3+CD8+Tc, CD4/CD8 ratio, CD19+CD20+B, CD19+CD5+B1, CD3+CD56+NKT, CD3−CD56+NK, and three NK subsets (CD56brightNK, CD56normalNK, CD56dimNK). Results As to the overall survival (OS), there’s no statistic difference between age&amp;lt;60 years old or age ≥60 years. Compared with non-GVHD group, decreased percentage (%) of Th, increased % of Tc &amp; low Th/Tc ratio were observed in GVHD group (P=0.0281, P=0.0394, P=0.0340 accordingly). Receiver operating characteristic (ROC) analysis indicates that low Th/Tc ratio predicts increased risk of GVHD (P=0.0231, AUC=0.663, 95% Confidence Interval is 0.530 to 0.780). There’re no statistic differences between GVHD and non-GVHD for all other lymphocyte subsets percentage (P&amp;gt;0.05). Conclusions AML patients with GVHD post transplant have a lower Th cell percentage and higher Tc cell percentage at diagnosis. This is to say, decreased Th/Tc ratio in BM of initial diagnosed AML patients may predict a high risk of GVHD post transplant.

G-CSF administration prior to donor lymphocyte apheresis promotes anti-leukaemic effects in allogeneic HCT patients.

Donor lymphocyte infusion (DLI) is an effective method to establish full donor chimerism or to prevent and treat relapse after allogeneic haematopoietic cell transplantation (allo-HCT). Usually, DLIs are collected from naïve donors as steady-state lymphocytes. When donor lymphocytes are collected during stem cell apheresis, donors are pre-treated with granulocyte colony-stimulating factor (G-CSF). However, the impact of G-CSF stimulation and the resulting composition of DLIs on beneficial anti-leukaemic responses and survival remain elusive. Therefore, we performed a retrospective analysis to evaluate the role of G-CSF-DLIs: 44 patients received either steady-state DLIs or G-CSF-DLIs to prevent and treat relapse or establish full donor chimerism after allo-HCT. The G-CSF-DLI patient cohort showed an improved conversion to full donor chimerism and a lower cumulative incidence of relapse or disease progression without a significantly increased cumulative incidence of graft-versus-host disease (GVHD). CD34+ cells, monocytic myeloid-derived suppressor cells and monocytes as well as donor age and the subsequent occurrence of chronic GVHD were identified as risk factors that significantly improve overall survival after DLI administration. In conclusion, our data suggest that administration of G-CSF-DLIs results in graft-versus-leukaemia effects without exacerbating GVHD, therefore, improving survival after DLIs.

Research progress in mechanisms of mesenchymal stem cells on graft versus host disease

Mesenchymal stem cells (MSC) are non-hematopoietic stem cells with self-renewal and multi-directional differentiation potential, and have low immunogenicity and immune regulation. After years of experiments and preclinical and clinical research experience, MSC are gradually becoming a treatment for preventing and alleviating graft-versus-host disease (GVHD). The characteristics, differentiation potential and surface markers of MSC have been studied in the past, and relevant clinical reports have also been proved that simultaneous infusion of MSC and hematopoietic stem cells (HSC) could promote HSC implantation and support hematopoiesis, as well as prevent and alleviate the occurrence of GVHD. However, the mechanisms are still unclear, so this article reviews the research progress of mechanisms of MSC in GVHD. In order to provide new ideas for the prevention, diagnosis and treatment of GVHD. Key words: Hematopoietic stem cell transplantation; Mesenchymal stem cells; Mesenchymal stem cell transplantation; Graft vs host disease; Immunotherapy; Immunomodulation; Allogeneic hematopoietic stem cell transplantation; Prevention

Fecal Calprotectin as a Diagnostic and Prognostic Biomarker for Gastrointestinal Graft Versus Host Disease: A Systematic Review of Literature.

The current practice for diagnosing graft versus host disease (GVHD) includes clinical or endoscopic evaluation of the patient. Clinical diagnosis is limited by an overlapping symptomatic spectrum with infectious causes, a common scenario in the post-transplant setting where an invasive procedure, such as endoscopy, is often impractical. We, therefore, evaluated the role of fecal calprotectin as a diagnostic as well as a prognostic biomarker for gastrointestinal GVHD (GI-GVHD) occurrence and severity in the post-hematopoietic transplant population. Following Prisma guidelines, we performed a systematic search of articles published after 2004 using the PubMed, Embase, Cochrane Library, and Web of Science databases. After a detailed screening, 10 studies involving a total of 494 patients were included. In the cohorts comparing median fecal calprotectin (mFC) level in GI-GVHD vs. non-GI-GVHD patients, the results indicated an increase in the mFC level in patients with GI-GVHD when compared to non-GI-GVHD patients. Similarly, an increase in the mFC level was seen in accordance with the severity of the disease. Moreover, corticosteroid-resistant patients had a higher mFC level as compared to corticosteroid-sensitive patients. Our study indicates that the mFC level can be used for diagnosing as well as predicting the treatment response to GI-GVHD. However, future randomized prospective trials involving larger populations are needed to further explore its significance.