Onset Of Graft-versus-host Disease Research Articles

Abstract A066: Histone deacetylase 11 is an epigenetic regulator of T-cell pro-inflammatory function and novel target for enhancing T-cell anti-tumor activity

Abstract The efficacy of immunotherapy is dependent on the ability of antigen-specific T-cells to mount a proper anti-tumor response while avoiding a plethora of immunosuppressive mechanisms. Unfortunately, these immunosuppressive mechanisms often overwhelm the T-cell response, resulting in patients failing to respond. Histone deacetylases (HDACs) are a family of epigenetic modifiers named for their ability to remove acetyl groups from lysine residues of histone tails, thereby regulating the chromatin structure and gene expression. Here we report that HDAC11, the most recently discovered HDAC, is a regulator of T-cell pro-inflammatory function. Previous reports have shown HDAC11 expression to be tissue restricted. Consequently, we compared expression levels of HDAC11 using an eGFP reporter mouse, in various immune cells. It was found that T-cells expressed relatively high levels of HDAC11. However, while resting T-cells, both central memory and naïve, had high levels of HDAC11 expression, effector or effector memory T-cells displayed lower levels. Additionally, T-cells experienced rapid down-regulation of HDAC11 upon activation. To expand upon these observations, the function of T-cells from HDAC11KO mice were investigated. Initial experiments revealed no gross changes in the numbers or maturation of T-cells. However, HDAC11KO mice displayed an increased percentage of central memory CD8+ T-cells (p<0.05). Additionally, upon activation these T-cells were more apt to acquire effector status (p<0.001). Upon activation, HDAC11KO T-cells demonstrated a robust increase in IL-2, TNF and IFNγ production (p<0.01), but showed no differences in Th2 or Th17 cytokine production. These T-cells also expressed higher levels of the effector molecules granzyme B and perforin (p<0.01). Moreover, post-activation, CD8+ T-cells lacking HDAC11 also displayed a higher percentage of proliferating cells, and more divisions of proliferating cells (p<0.01). HDAC11KO T-cells displayed higher resistance to proliferative suppression by Tregs and failed to succumb to anergy in a high dose antigen model. Indeed, while OTII T-cells from mice injected with high dose OVA peptide displayed reduced levels of IFNγ production compared to cells from non-injected mice after rechallenge, HDAC11KO/OTII T-cells had an increase in IFNγ production. Mechanistically, T-cells devoid of HDAC11 did not appear to have differences in TCR signaling, but did express higher levels of the transcription factors EOMES and T-bet. Chromatin immunoprecipitation (ChIP) revealed higher basal levels of acetylation of these genes in HDAC11KO mice. ChIP analysis also revealed interactions of HDAC11 at the promoters of both EOMES and T-bet. In a graft vs. host disease (GvHD) model, HDAC11KO T-cells produced a more rapid onset of GvHD, and were able to do so with transfers of cell numbers below that able to induce GvHD using WT T-cells (p<0.01). This GvHD was characterized by greater levels of IFNγ and TNF as well as increased T-cell expansion (p<0.05). In a B16 melanoma model, adoptive transfer of HDAC11KO T-cells resulted in delayed tumor progression (p<0.05) compared to WT T-cells. To begin investigating the clinical potential of these data, the expression levels of HDAC11 were investigated in melanoma tumor sections utilized to grow tumor infiltrating lymphocytes (TIL) for clinical trials of adoptive cell therapy. A reduced level of HDAC11 was found in tumor sections from those patients responding to TIL therapy compared to progressing patients (p<0.01). Collectively, beyond demonstrating HDAC11 as a novel epigenetic regulator of T-cell pro-inflammatory function, these results highlight HDAC11 as a novel target for enhancing the efficacy of immunotherapy. Citation Format: David M. Woods, Andressa L. Sodre, Karrune Woan, Alejandro Villagra, Amod Sarnaik, Jeffrey Weber, Eduardo M. Sotomayor. Histone deacetylase 11 is an epigenetic regulator of T-cell pro-inflammatory function and novel target for enhancing T-cell anti-tumor activity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A066.

Evidence for CD19+ (B) CD8+ (T)-Cell Interactions in Blood and Tissues from Patients with Graft-Versus-Host Disease

Graft-versus -host disease (GVHD), a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), is mediated by cytotoxic T cells (CTL), recruited to target organs by immune-mediated injury. There is growing evidence that B cells play a pathogenic role in GVHD, although the underlying mechanisms are unclear.We previously described the interaction between autologous CD19+ B cells and activated CD8+ CTLs in normal donors (Deola et al, 2008). In vitro antigen-stimulated CTLs formed stable couplets with B cells, independent of antigen presentation. Coupling enhanced CTL survival and proliferation, and stimulated B-cell release of chemokines, such as MIG, IP-10 and I-TAC, which are pivotal for T-cell migration to GVHD target organs.Herein, we retrospectively analyzed a cohort of patients undergoing immune monitoring after allo-HSCT. To determine the role of CD19(B)-CD8(T) coupling in GVHD, we purposely included patients with both acute and chronic GVHD who had at least 5% circulating B cells. The cohort included 292 consecutive blood samples: 176 from GVHD patients (acute, chronic, overlap syndrome), 116 from non-GVHD patients, and 21 from healthy controls. We restricted sample selection by including only patients with GVHD onset within 150 days from sampling (26 patients, 40 samples overall), and we excluded from the non-GVHD sample cohort any patient with previous or suspect GVHD (achieving 36 samples overall, belonging to 15 different patients).B-T couplets were significantly higher in patients with GVHD, compared with both allo-HSCT patients without GVHD and with healthy controls (P =0.048 and 0.0008, respectively). Particularly remarkable was the difference between patients developing GVHD within 30 days from sampling and the controls. B-T couplets were measured in 7 patients where sampling was available both before and after GVHD onset, and the difference was again statistically significant (P =0.016).Nine patients in the entire cohort received donor lymphocyte infusions (DLI) for a molecular relapse of their disease. Interestingly, B-T couplets were significantly higher after DLIs, regardless of subsequent GVHD development.A validation cohort of children with acute or chronic GVHD after allo-HSCT was then analyzed. This consisted of 13 patients with no signs of GVHD and 11 patients with GVHD. Patients with GVHD had a significantly higher number of B-T couplets compared with non-GVHD patients (P =0.029). By focusing the analysis on patients with GVHD onset within 150 days prior to immune monitoring (7 patients), the p-value for the comparison was 0.014. Again, the score of couplets in patients developing GVHD within 30 days from sampling was higher.We finally looked at tissue distribution of B and T cells in 7 skin and gut biopsies from patients with GVHD, obtained 3-13 months after allo-HSCT. Three skin biopsies of allo-HSCT patients without features of GVHD were also included.Immunohistochemistry for CD20 showed that 5 out of 7 biopsies from patients with GVHD hosted B cells (up to 10), focally coupled with CD8+ T cells. The control biopsies from patients without signs of GVHD 4-15 months after allo-HSCT (n=3) stained negatively for B cells. GVHD and control biopsies were also screened with laser scanner microscope, again finding evidence of B-cell/T-cell coupling.Collectively, these data point to the interplay between B cells and CTL both in blood and in tissues from allo-HSCT patients with GVHD, as well as after DLI. Conceivably, B-T cell coupling amplifies the inflammatory signal, both systemically and locally. B cells may attract additional immune cells, thus supporting CTL survival and expansion. DisclosuresNo relevant conflicts of interest to declare.

The Relationship Between Some Cytokines and Graft Versus Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation in Thalassemia Major Patients

Background: Research has shown that the relationship between cytokines and graft versus host disease is close, but there are still a lot of cytokines and graft versus host disease relationships which are not clear. At the same time, thalassemia patients with graft versus host disease may exist in particularity.Objects: To investigate the relationship between some cytokines and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in β-thalassemia major patients.Methods: The study included 16 patients who developed graft-versus-host disease (GVHD) after receiving hematopoietic stem cell transplantation in the Department of Pediatrics in Nanfang Hospital between the time period of 2011 July to 2015 March. We observed the dynamic variation of interleukin 6 (IL-6), interleukin 8(IL-8), interleukin 12(IL-12), tumor necrosis factor-α(TNF- α) and macrophage migration inhibitory factor(MIF) in 16 patients before onset of GVHD, when GVHD occurred, at fourth and seventh day after onset of GVHD, and when GVHD almost disappeared.Results: There was a significant difference in serum levels of interleukin-6, interleukin-12, tumor necrosis factor-α, macrophage migration inhibitory factor at different times. The highest levels of different cytokines appeared when GVHD occurred, followed by those at seventh day after it occurred. There was also a significant difference in serum levels of interleukin-8 at different times, and the highest level appeared at fourth day after GVHD occurred. There is a significant positive correlation between IL-6 and IL-12,IL-12 and TNF-α,IL-12 and MIF,TNF- α and MIF when GVHD occurred. There is a significant positive correlation between IL-12 and TNF-α when GVHD occurred and on the fourth day . There is a significant positive correlation between IL-12 and TNF-α,IL-12 and MIF,TNF-α and MIF when GVHD appeared on the seventh day. There is a significant positive correlation between IL-6 and IL-12,IL-12 and TNF-α,IL-12 and MIF,TNF-α and MIF when GVHD almost disappeared.Conclusion: The dynamic expression of IL-6,IL-8,IL-12,TNF-α and MIF can estimate the immune function of β-thalassemia major patients who develops graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, and can be used as the immunobiology indicators for the early diagnosis of graft-versus-host disease. IL-6, IL-8, IL-12, TNF-α and MIF may have correlation with each other in different time of graft versus host disease. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Regulatory T Cell Reconstitution after Allogeneic Hematopoietic Cell Transplantation: Expansion Potential at 100 Days Predicts De Novo Graft-Versus-Host Disease By Six Months Post-Transplantation

IntroductionRegulatory T cells (CD4+CD25high CD127low Foxp3+) (Tregs) are of increasing interest in the context of allogeneic hematopoietic cell transplantation (HCT), as they may be able to prevent or suppress graft-versus-host disease (GVHD). Relatively little is known about Treg profiles during the post-HCT period. We tested the hypothesis that Treg phenotype and function correlates to GVHD status.MethodsPeripheral blood was drawn at 100 days and six months post-HCT from adult patients enrolled on an IRB-approved protocol. Eight-color flow cytometric analysis of Treg subsets was performed on peripheral blood mononuclear cells. Patients' FACS-sorted CD4+CD25high CD127low CD49dlow Tregs were cultured with their CD3/CD28-stimulated T cells and analyzed for proliferation via 3H-thymidine incorporation and cytokine production in culture supernatants. Clinical GVHD evaluations were extracted from medical records. Age-matched healthy controls were recruited for comparison. Data were analyzed using univariate regression, Mann-Whitney t-tests, ANOVA, and Wilcoxon matched-pairs signed rank tests.ResultsTwenty-eight patients (median age 61 years old) were studied. Total percentages of Tregs were similar at +100d and +6m post-HCT (median 9.3% and 9.15% of total CD4+ T cells, respectively), with similar levels of transcription factor Helios and markers of memory/activation (CD45RO), proliferation (Ki-67), and apoptosis (activated caspase-3). Healthy controls' Tregs had higher caspase-3 expression than patients'; all other markers exhibited similar levels. CD45RO+ and Ki-67+ levels were increased among Helios+ Tregs compared to Helios-Tregs, whereas caspase-3 levels were decreased. The absolute number of circulating Tregs was comparable between +100d and +6m, displaying lower numbers compared to healthy controls attributable to overall lower total T cell numbers.In vitro addition of sorted Tregs to stimulated bulk T cells isolated from patients at +100d post-HCT induced median 41% proliferation reduction (i.e., suppression), reduced levels of pro-inflammatory IL-2, IL-5, GM-CSF, IFNγ, and TNFα, and increased levels of anti-inflammatory IL-10 compared to stimulated bulk T cells alone (Treg:T bulk ratio of 1:2). These changes were of equivalent magnitude in cultures from healthy controls. No net change was noted for IL-17, while IL-35 and IL-12 levels were below detection. Univariate analysis revealed linear relationships between cellular proliferation suppression and IL-2, IL-4, IL-10, IL-13, GM-CSF, IFNγ, and TNFα relative changes.Across all +100d samples, higher degrees of Treg proliferation (Ki-67+) corresponded with increased IFNγ and TNFα suppression in culture supernatants (p=0.0462 and p=0.0276, respectively) and specifically correlated with enhanced suppression of IFNγ-producing CD8+ T cells (p=0.0280). Furthermore, higher Treg expansion potential (measured by Treg Ki-67/caspase-3 ratio) suppressed IFNγ and TNFα more potently (p=0.0479 and p=0.0484, respectively) and was associated with a higher proportion of Tregs comprising total CD4+ T cells (p=0.0266).Higher Treg Ki-67 expression at 100 days post-HCT was associated with increased likelihood of de novo GVHD onset by six months post-HCT (p=0.01208). Similarly, a higher Treg expansion potential profile was associated with GVHD onset by six months (p=0.0078).ConclusionTregs from patients at 100 days and six months post-HCT display similar memory/activation and proliferation profiles and functional suppressive ability as Tregs from healthy controls. Among these patients, a proliferative Treg profile strongly correlates with increased in vitro suppressive function on a cell-per-cell basis and is further associated with reduced apoptotic profile, representing an expansive Treg profile. As these profiles can predict GVHD developing by six months post-HCT, the increased associated cell-per-cell suppressive potential may represent a compensatory yet ultimately inadequate Treg response to early GVHD-associated inflammatory changes. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Outcomes of Grades II-IV Acute Graft-Versus-Host Disease Post-Allogeneic Hematopoietic Stem Cell Transplantation: How Much Progress Was Achieved?

Despite recent reduction in mortality following hematopoietic cell transplant (HCT), graft versus host disease (GVHD) remains a major causes of failure. It is unclear if advances in antiviral, antifungal and antibacterial prophylaxis/treatment, and immunosuppression, have led to better survivals following the onset of acute GVHD (aGVHD). We therefore analyzed within the CIBMTR database outcomes over time of allogeneic HCT recipients who developed grade II-IV aGVHD. The objectives were to determine the change in overall survival (OS), disease-free survival (DFS), treatment-related mortality (TRM) and to identify risk factors.Methods: Between 1999 and 2012, 7295 patients with AML (57%), ALL (29%) or MDS (14%) received sibling (35%) or unrelated donor (65%) blood (65%) or marrow (35%) HCT (1216 for 1999-2001, 2329 for 2002-2005, 3750 for 2006-2010), and 3312 (45%) developed grades II-IV aGVHD. Median follow-up was 141 (3-174), 96 (3-144) and 49 (3-95) months for 1999-2001, 2002-2005, and 2006-2010 periods respectively. Multivariate models were built using the Cox's proportional hazards model. Clinical variables were tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption were adjusted through stratification. Stepwise model building procedure was used in developing models for each outcome with a threshold of 0.05 for both entry and stay in the model. Interactions between the main variable, (i.e. year of transplant) and the adjusted covariates were tested.Results: Univariate analyses showed a decrease in the incidence of grades III-IV aGVHD: 56 % (52-60), 47 %(44-50), and 38% (36-40) for 1999-2001, 2002-2005, and 2006-2010, respectively (p= <0.001). Multivariate analyses showed that age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), disease status at transplant and KPS influenced OS; while age of recipient, organ affected by aGVHD, treatment of aGVHD (steroids vs. other), CMV serostatus matching, disease status at transplant, unrelated donor age and KPS affected OS and TRM. After adjusting for these factors, recipients of tacrolimus-based GVHD prophylaxis who developed grade II aGVHD had better OS after 2006 (HR 0.78, p=0.02), while a trend for lower TRM (HR 0.79, p=0.056) and better OS (HR 0.83, p=0.08) were observed for those with grades III-IV aGVHD.Conclusions: Incidence of grades III-IV aGVHD has overall decreased and OS and TRM have improved over time for recipients of tacrolimus-based GVHD prophylaxis who developed grades II-IV aGVHD. DisclosuresAlousi:Therakos, Inc: Research Funding.

Gut Microbiome Changes after Stem Cell Transplantation

Introduction:Disruption of the intestinal barrier after stem cell transplantation (SCT), as a consequence the conditioning regimen, may significantly affect the gastrointestinal (GI) microbiome and thus the immune response of gut associated lymphoid tissue. Indeed, dysbiosis of the microbioma has been reported to trigger a variety of human diseases, such as inflammatory bowel disorders and acute infections. In the SCT setting, a correlation has been identified between changes in the microbioma and post-SCT events, such as as graft versus host disease (GvHD), infections and transplant related mortality (TRM).Patients:The aim of our study was to analyze the human gut microbiome composition before and after transplantation and to assess a relationship, if any, between microbiome imbalance and post-transplant outcome. For this purpose, we prospectively studied 11 patient (pts), 8 undergoing an allogeneic (matched sibling, n=7, unrelated n=1) and 3 an autologous SCT. The diagnosis was AML (n=8) or multiple myeloma (n=3); the age ranged between 30 and 60 (median 48). The allogeneic SCT patients were prepared with a myeloablative regimen (n=5) or a reduced intensity regimen (n=3); the myeloma patients received Melphalan 200 mg/m^2. Antibiotic prophylaxis was given to the allogeneic SCT recipient. Stool samples were collected at baseline before starting SCT conditioning in both groups and on day +15. Among the allo-SCT patients, another sample was provided at the time of GvHD onset. Samples were stored at -80°C until they were processed. . DNA was isolated with QIAamp DNA Stool Mini kit. The hypervariable V1-V3 region of the 16s RNA gene was amplified by PCR and sequenced on 454 GS Junior Titanium. Taxonomic composition was defined according to Greengenes database. IBM SPSS Statistics 22 was used for statistical analysis.Results:The baseline microbiome composition in auto-SCT and allo-SCT patients was comparable. In the auto-SCT, day +15 samples showed a reduction of Bacteroidetes and an increase of Proteobacteria (p=0.0071) compared with baseline. In the allo-SCT setting, at day +15 we identified an increase of Bacteroidetes and a reduction of Firmicutes (p=0.0023). At the time of GvHD onset, patients showed a reduction of Bacteroidetes. with an increase of Firmicutes and Proteobacteria (t-test, p=0.0020). Surprisingly, Bacteroidetes were more represented in patients with GvHD-GI involvement than in the others (t-test, p=0.0001). Figure 1.Discussion and conclusion:We identified that, starting from the same baseline microbioma composition, the conditioning regimen promoteschanges in the microbiome, which are different between auto- and allo-SCT. After auto-SCT we documented an increase of Proteobacteria (Klebsiella, Proteus, Acinetobacter, Haemophilus, Pseudomonas, Enterobacteriaceae) and a reduction of Bacteroidetes (Bacteroides, Saprospirae, Prevotella). After allo-SCT, instead, there was an increase of Bacteroidetes and a reduction of Firmicutes (Bacilli, Lactobacilli, Clostridium, Enterococci, Streptococci). Moreover, patients who developed GvHD harboured more Firmicutes and Proteobacteria and less Bacteroidetes than pts without this complication. In patients with gut GvHD, Proteobacteria were more represented than in pts with liver or skin involvement. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Ocular Chronic Graft-versus-Host Disease on Quality of Life

Ocular involvement can be quite symptomatic in patients with chronic graft-versus-host disease (GVHD). The prevalence of and risk factors for ocular GVHD and its impact on quality of life (QOL) in patients with chronic GVHD were studied in a prospective, multicenter, longitudinal, observational study. This study enrolled 342 patients with 1483 follow-up visits after allogeneic hematopoietic cell transplantation. All patients in this analysis were diagnosed with chronic GVHD requiring systemic treatment and enrolled within 3 months of chronic GVHD diagnosis. The symptom burden of ocular GVHD was based on the degree of dry eye symptoms, frequency of artificial tear usage, and impact on activities of daily living. Patients' QOL was measured by self-administered questionnaires. Variables associated with ocular GVHD at enrollment and subsequent new-onset ocular GVHD and the associations with QOL were studied. Of the 284 chronic GVHD patients, 116 (41%) had ocular GVHD within 3 months of chronic GVHD diagnosis (“early ocular GVHD”). Late ocular GVHD (new onset > 3 months after chronic GVHD diagnosis) occurred in 64 patients. Overall cumulative incidence at 2 years was 57%. Female gender (P = .005), higher acute GVHD grade (P = .04), and higher prednisone dose at study entry (P = .04) were associated with early ocular GVHD. For patients who did not have ocular GVHD within 3 months of chronic GVHD diagnosis, presence of prior grades I to IV acute GVHD (HR 1.78, P = .04) was associated with shorter time to late ocular GVHD, whereas female donor–male recipient (HR .53, P = .05) was associated with longer time to late ocular GVHD onset. Using all visit data, patients with ocular GVHD had worse QOL, as measured by Functional Assessment of Cancer Therapy Bone Marrow Transplantation (P = .002), and greater chronic GVHD symptom burden, as measured by the Lee symptom overall score excluding the eye component (P < .001), compared with patients without ocular GVHD. In conclusion, this large, multicenter, prospective study shows that ocular GVHD affects 57% of patients within 2 years of chronic GVHD diagnosis. Women, patients on higher doses of prednisone at study entry, and those with a history of acute GVHD were at higher risk for ocular GVHD. Strong evidence suggests that ocular GVHD is associated with worse overall health-related QOL.

Onset of ocular graft-versus-host disease symptoms after allogeneic hematopoietic stem cell transplantation.

To study the factors affecting the time to onset of ocular graft-versus-host disease (GVHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). A retrospective chart review of 200 patients with ocular GVHD was performed to evaluate the association between various donor-recipient characteristics and the time to onset of ocular GVHD after allo-HSCT. The median time to onset of chronic ocular GVHD after allo-HSCT was 293 days (range, 26-2308 days). Patients receiving fully human leukocyte antigen (HLA)-matched transplants had a delayed onset of ocular GVHD (median, 294 days) compared with mismatched transplants (219 days; P = 0.029). HLA-matched transplants from related donors had delayed onset of ocular GVHD (307 days) compared with HLA-matched (286 days; P = 0.168) and HLA-mismatched (231 days; P = 0.015) transplants from unrelated donors. Ocular GVHD followed systemic GVHD in 76% of patients but preceded systemic disease in 7%, occurred concurrently in 15%, and was not associated with systemic GVHD in 2% of patients. The time elapsed between the occurrence of systemic and ocular GVHD was significantly longer in matched-related transplants (250 days) than in matched-unrelated transplants (120 days; P = 0.004). The onset of ocular GVHD after allo-HSCT is variable and is influenced by donor-recipient matching characteristics. In the majority of patients with GVHD, ocular involvement follows the occurrence of systemic manifestations; however, importantly, it can also precede or develop independently of systemic disease in a minority of patients. Regular ophthalmic follow-up is recommended after allo-HSCT regardless of concurrent systemic GVHD status.

Age influences post-graft-versus-host disease non-relapse mortality in adults with acute graft-versus-host disease of varying severity following allogeneic hematopoietic cell transplant

We retrospectively analyzed 2682 patients who developed grade II–IV acute graft-versus-host disease (GVHD). On analysis with stratification into five age groups (20–29, 30–39, 40–49, 50–59 and ≥ 60), 2-year non-relapse mortality rates (NRM) after the onset of GVHD were 20.7, 26.2, 26.6, 37.0 and 40.4%, respectively (p < 0.001). We found a significant interaction between the patient's age and GVHD severity with respect to NRM (p = 0.004). On multivariate analyses stratified by GVHD severity, the hazard ratio (HR) for NRM in the groups aged 50 years or more (reference: age group 20–29) was about twice as great in patients with grade II acute GVHD when compared with grade III–IV disease (HR in those aged 50–59 years: 2.9 for grade II and 1.5 [p = 0.03 and 0.04] for grades III–IV; HR if ≥ 60 years: 3.3 for grade II and 1.5 for grades III–IV [p < 0.001 for both]).

A prognostic score for acute graft-versus-host disease based on biomarkers: a multicentre study

Graft-versus-host disease (GVHD) is the major cause of non-relapse mortality after allogeneic haemopoietic stem-cell transplantation (SCT). The severity of symptoms at the onset of GVHD does not accurately define risk, and thus most patients are treated alike with high dose systemic corticosteroids. We aimed to define clinically meaningful risk strata for patients with newly diagnosed acute GVHD using plasma biomarkers. Between April 13, 2000, and May 7, 2013, we prospectively collected plasma from 492 SCT patients with newly diagnosed acute GVHD and randomly assigned (2:1) using a random number generator, conditional on the final two datasets having the same median day of onset, into training (n=328) and test (n=164) sets. We used the concentrations of three recently validated biomarkers (TNFR1, ST2, and Reg3α) to create an algorithm that computed the probability of non-relapse mortality 6 months after GVHD onset for individual patients in the training set alone. We rank ordered the probabilities and identified thresholds that created three distinct non-relapse mortality scores. We evaluated the algorithm in the test set, and again in an independent validation set of an additional 300 patients who underwent stem cell transplant and were enrolled on multicentre clinical trials of primary therapy for acute GVHD. In all three datasets (training, test, and validation), the cumulative incidence of 6-month non-relapse mortality significantly increased as the Ann Arbor GVHD score increased. In the multicentre validation set, scores were 8% (95% CI 3-16) for score 1, 27% (20-34) for score 2, and 46% (33-58) for score 3 (p<0·0001). Conversely, the response to primary GVHD treatment within 28 days decreased as the GVHD score increased 86% for score 1, 67% for score 2, and 46% for score 3 in the multicentre validation set, p<0·0001). Biomarker-based scores can be used to guide risk-adapted therapy at the onset of acute GVHD. High risk patients with a score of 3 are candidates for intensive primary therapy, while low risk patients with a score of 1 are candidates for rapid tapers of systemic steroid therapy. The National Cancer Institute, the National Heart, Lung, and Blood Institute, the National Institute of Allergy and Infectious Diseases, the Doris Duke Charitable Fund, the American Cancer Society, and the Judith Devries Fund.

High Number of Memory T Cells Is Associated with Higher Risk of Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The pathophysiology of acute graft-versus-host disease (GVHD) remains poorly understood in humans. Although T cell subsets have been identified to play a major role in disease initiation in rodents, clinical data on the effect of these different subsets are scarce and conflicting. To address this question, immunophenotyping analyses were performed on the graft in 210 patients. The onset of acute GVHD was retrospectively correlated with these subpopulations. In an adjusted analysis, only the absolute count of CD45lo/CD62Llo CD8+ T cells (effector memory T cells) was significantly associated with the onset of grade 2 to 4 acute GVHD. Thus, in contrast to experimental data, we found that the number of effector memory but not of naïve T cells was associated with the onset of GVHD. These results should be kept in mind while clinical trials, which aim to deplete naïve T cells, are underway in several institutions.

A Biomarker Algorithm Defines Onset Grades of Acute Graft-Vs-Host Disease with Distinct Non-Relapse Mortality

The severity of symptoms at the onset of graft versus host disease (GVHD) does not accurately define risk, and thus most patients (pts) are treated alike with high dose systemic steroids. We hypothesized that concentrations of one or more plasma biomarkers at the time of GVHD diagnosis could define distinct non-relapse mortality (NRM) risk grades that could guide treatment in a multicenter setting. We first analyzed plasma that was prospectively collected at acute GVHD onset from 492 HCT pts from 2 centers, which we randomly divided into training (n=328) and validation (n=164) sets; 300 HCT pts who enrolled on multicenter BMT CTN primary GVHD therapy clinical trials provided a second validation set. We measured the concentrations of 3 prognostic biomarkers (TNFR1, REG3α, and ST2) and used competing risks regression to create an algorithm from the training set to compute a predicted probability (p) of 6 mo NRM from GVHD diagnosis where log[-log(1-p)] = -9.169 + 0.598(log2TNFR1) - 0.028(log2REG3α) + 0.189(log2ST2). We then rank ordered p from lowest to highest and identified thresholds that met predetermined criteria for 3 GVHD grades so that NRM would increase 15% on average with each grade. A range of thresholds in the training set met these criteria, and we chose one near each median to demarcate each grade.In the resulting grades, risk of NRM significantly increased with each grade after the onset of GVHD in both the training and validation sets (FIG 1A,B). Most (80%) NRM was due to steroid-refractory GI GVHD, even though surprisingly only half of these pts presented with GI symptoms. We next applied the biomarker algorithm and thresholds to the second multicenter validation set (n=300) and observed similarly significant differences in NRM (FIG 1C). Relapse, which was treated as a competing risk for NRM, did not differ among the three GVHD grades (Figure 1D-F). The differences in NRM thus translated into significantly different overall survival for each GVHD grade (Figure 1G-I). These differences in survival are explained by primary therapy response at day 28, which was highly statistically different for each of Ann Arbor grade (grade 1, 81%; grade 2, 68%; grade 3, 46%; p<0.001 for all comparisons).We performed additional analyses on the multicenter validation set of pts that developed GVHD after treatment with a wide spectrum of supportive care, conditioning and GVHD prophylaxis practices. As expected, the Glucksberg grade at GVHD onset did not correlate with NRM (data not shown). Despite small sample sizes, the same biomarker algorithm and thresholds defined three distinct risk strata for NRM within each Glucksberg grade (FIG 2A-C). Pts with the higher Ann Arbor grades were usually less likely to respond to treatment. Unexpectedly, approximately the same proportion of pts were assigned to each Ann Arbor grade (~25% grade 1, ~55% grade 2, ~20% grade 3) regardless of the Glucksberg grade (FIG 2D-F).Several clinical risk factors, such as donor type, age, conditioning, and HLA-match, can predict treatment response and survival in patients with GVHD. Using Ann Arbor grade 2 as a reference, we found that Ann Arbor grade 1 predicted a lower risk of NRM (range 0.16-0.32) and grade 3 a higher risk of NRM (range 1.4-2.9), whether or not any of these clinical risk factors were present.To directly compare Ann Arbor grades to Glucksberg grades, we fit a multivariate model with simultaneous adjustment for both grades. FIG 3 shows that Ann Arbor grade 3 pts had significantly higher risk for NRM (p=0.005) and Ann Arbor grade 1 pts had significantly less risk for NRM (p=0.002) than pts with Ann Arbor grade 2. By contrast, the confidence intervals for the HRs of the Glucksberg grades encompassed 1.0, demonstrating a lack of statistical significance between grades.In conclusion, we have developed and validated an algorithm of plasma biomarkers that define three grades of GVHD with distinct risks of NRM and treatment failure despite differences in clinical severity at presentation. The biomarkers at GVHD onset appear to reflect GI tract disease activity that does not correlate with GI symptom severity at the time. This algorithm may be useful in clinical trial design. For example, it can exclude pts who are likely to respond to standard therapy despite severe clinical presentations, thus limiting the exposure of low risk pts to investigational agents while also identifying the high risk pts most likely to benefit from investigational approaches. [Display omitted] [Display omitted] [Display omitted] DisclosuresLevine:University of Michigan: GVHD biomarker patent Patents & Royalties. Braun:University of Michigan: GVHD biomarker patent Patents & Royalties. Ferrara:University of Michigan: GVHD biomarker patent Patents & Royalties.

A Radio-Resistant Perforin-Expressing Lymphoid Population Controls Allogeneic T Cell Engraftment, Activation and Onset of Gvhd

IntroductionImmunosuppressive pre-transplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplant (BMT). The role of residual post-conditioning recipient immunity in determining engraftment is poorly understood. Although recipient perforin has previously been shown to modulate myeloid engraftment from mouse bone marrow, and adoptive transfer of natural killer (NK) cells can ameliorate the onset of graft-versus host disease (GVHD), the role of perforin-expressing endogenous recipient NK and NKT cells in modulating donor T cell engraftment has not been described. Using an MHC-mismatched mouse model, we examined the role of perforin-expressing NK cells in regulating both the rate and activation status of donor lymphocyte engraftment after allogeneic transplantation.MethodsAn MHC-disparate model of BMT was established transplanting BALB/c donor BM (H-2Kd), into female 6-14 week old C57BL/6 WT and C57BL/6.perforin KO (H-2Kb) recipients. On day 0, recipient mice were administered a split dose of lethal radiation using a caesium source (2 x 6 gray), and injected i.v. with 5e6 T cell depleted BM (TCD-BM) cells from MHC-mismatched or syngeneic donors. On day 2, recipient mice were injected i.v. with 1e6 BALB/c purified splenic T cells at a 2:1 CD4+:CD8+ T cell ratio. Mice were monitored daily, and killed at selected time points post-transplant (typically day 5-7 for short term engraftment, or day 20 for long term engraftment) to examine donor lymphoid cell engraftment.ResultsAn HLA-mismatched BMT mouse model demonstrated that both the rate and proportion of donor lymphoid cell engraftment, and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5-7 days post BMT in perforin-deficient (pfn-/-) recipients, compared with wild-type (WT). Critically, we found that the absence of perforin resulted in the increased production of pro-inflammatory cytokines, in particular IL-6, from engrafting donor T cells. IL-6 has recently been identified as a primary driver of both mouse model and clinical GVHD. Correlating with pro-inflammatory cytokine secretion, effector memory donor T lymphocytes expanded more rapidly in pfn-/- recipients than in WT mice, possibly arising from more rapid proliferation or selective survival of these donor cells. In WT recipients, donor lymphoid cell engraftment was enhanced to that seen in pfn-/- recipients, by depleting NK cells prior to BMT, demonstrating that a perforin-dependent, NK-mediated host-versus graft effect limits the rate of donor cell engraftment and T cell activation. We found that radiation-resistant NKT cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the host-versus graft effect. Furthermore, reduced pre-transplant irradiation doses in pfn-/- recipients permitted long-term donor lymphoid cell engraftment and improved survival (Figure 1).Figure 1: Reduced total body irradiation in perforin-deficient mice permits rapid donor lymphoid cell engraftment. On day 0, WT or pfn-/- (KO) mice were irradiated with variable doses (12-6 gray), and injected i.v. with 5e6 TCD-BM cells from BALB/c donors. On day 2, recipient mice were injected i.v. with 1e6 splenic BALB/c T cells. On day 7 (A) and day 20 (B) after BMT, the BM cells of WT or KO mice were stained for H-2Kd (donor cells) and engraftment determined as a percentage of donor lymphocytes. (C) Survival of KO mice administered 9 gray (closed circles), 7.5 gray (triangles) and 6 gray (lines) after BMT described above. [Display omitted] [Display omitted] [Display omitted] ConclusionsThe challenge in clinical HSCT is to promote reliable donor engraftment and full immunological reconstitution whilst minimizing pre-transplant conditioning and associated toxicity. Our findings suggest that suppression of perforin activity or selective depletion of recipient NK cells prior to BMT may allow the combined advantage of reduced transplant toxicity whilst maintaining donor engraftment and promoting the graft versus tumour effect. DisclosuresNo relevant conflicts of interest to declare.

CD4+ T-Cell STAT3 Activity Is Increased before Acute Graft-Versus-Host Disease Onset and Its Pharmacologic Inhibition Selectively Controls Alloresponses

STAT3 is a key mediator of inflammation, and is associated with murine auto- and alloreactivity. Germline and somatic activating STAT3 mutations have correlated with human inflammatory disorders, including colitis and polyendocrinopathy. STAT3 phosphorylation is required for pathogenic Th17 differentiation, and antagonizes beneficial Treg development. We show that CD4+ T-cell STAT3 activity is elevated early after allogeneic hematopoietic cell transplantation (HCT) compared with healthy non-HCT controls, and identifies patients at risk of developing grade II-IV acute graft-versus-host disease (GVHD) before the onset of symptoms. Using human in vitro and murine in vivo systems, we demonstrate that STAT3 phosphorylation may be selectively targeted with S3I-201 to suppress the alloresponse while preserving graft-versus-leukemia (GVL). Peripheral blood T-cells were obtained on day +21 from consented HCT recipients lacking any signs of acute GVHD (n=5 non-HCT controls, n=18 HCTs). T-cell STAT3 Y705 activity was stimulated with a pulse of IL-6 (4000 IU/ml, 15 minutes) and measured by flow cytometry. Patients were followed for acute GVHD onset from day +22 to day +100. While resting STAT3 activity was very low in all groups, stimulated CD4+ STAT3 phosphorylation significantly increased among the patients that later developed acute GVHD during the observation period (Fig 1 A: non-HCT control: 8.83%; n=10 grade 0-I: 28.8%; n=8 grade II-IV: 67.6% median pSTAT3+/CD4+; P<.05 and <.001; Fig 1 B: representative histograms show day +21 STAT3 activity in a patient that never acquired GVHD v a patient that later developed grade II GVHD at day +43). The absolute number of pSTAT3+ CD4+ T-cells on day +21 was also increased among the patients that acquired grade II-IV acute GVHD (grade 0-1: 45.5; grade II-IV: 83.5 CD4+ T-cells/ul; P<.05). The total absolute number of CD4+ T-cells, and number and degree of pSTAT3+ CD8+ T-cells, was similar among all HCT groups. We previously demonstrated that S3I-201 suppressed human DC-allostimulated T-cells, selectively polarized STAT5 activity while inhibiting STAT3, abrogated Th17 responses, and permitted the expansion of potent antigen-specific inducible Treg (Betts et al, Journal of Leukocyte Biology, 2014). We now show S3I-201 prevents GVHD in vivo, where Balb/c mice received myeloablative irradiation (850 cGy) followed by an allograft of T-cell depleted or replete (1x10e6) C57BL/6 bone marrow (5x10e6). S3I-201 (2.5mg/kg or DMSO control) was given i.p. from day 0 to +7. S3I-201 significantly delayed GVHD onset and improved survival (Fig 2, 10 mice/group, 0% v 56% alive at day +60, P<.0001, n=2). To determine the effect of S3I-201 on GVL, similarly transplanted Balb/c mice additionally received 2000 luciferase-expressing A20 cells. In this pilot experiment, median survival for the vehicle-treated BM-A20 and T cell-A20 groups was 26 days, 40.5 days for S3I-BM-A20, and not reached for S3I-T cell-A20 (67% alive at day +60). Bioluminescence (IVIS 200) remained undetectable in both the T cell-A20 and S3I-T cell-A20 groups during the experiment. Moreover, as mTOR and STAT3 signaling both influence Th17 differentiation we show that concurrent exposure of human T-cells to rapamycin and S3I-201 optimally suppresses RORgammaT expression and enhances allo-inactivation (Fig 3 A: P<.05, <.001, n=3, Fig 3 B: colorimetric proliferation assay, DC:T 1:30, P<.05, n=4). Ongoing experiments are underway to study the effect of combined rapamycin and S3I-201 on GVHD and GVL in vivo. Our preclinical data implicates STAT3 activation early in the onset of acute GVHD, where its selective inhibition with S3I-201 controls alloreactivity without untoward impairment of Treg or GVL. Future investigation of this approach will incorporate specific STAT3 inhibition with rapamycin to develop novel GVHD prevention strategies, and validate the association of STAT3 activity with acute GVHD risk in a large cohort of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BK Virus Hemorrhagic Cystitis Is Associated with Graft Versus Host Disease and Increased Health Care Resource Utilization in Patients Undergoing Allogeneic Stem Cell Transplantation

Background BK virus (BKV) is a polyomavirus that carries up to a 90% sero-prevalence in the general population. After infection the virus enters a latent phase in the renourinary epithelium. In hematopoietic stem cell transplant patients, BKV reactivation may lead to hemorrhagic cystitis with an incidence of 7%-70%. Aside from BKV, hemorrhagic cystitis (&gt;72 h) has been linked to patient age, conditioning regimen, graft versus host disease (GVHD), and unrelated/mismatch donor. Symptoms range from dysuria to severe hemorrhage leading to clot formation, urinary obstruction and renal impairment. These complications have been shown to impact length of hospitalization, cost of care, and mortality. The incidence and impact of hemorrhagic cystitis in patients receiving fludarabine (Flu), busulfan (Bu)and anti-thymocyte globulin (ATG) conditioning is unknown. We carried out this study to determine the incidence and its effect on patient outcomes including overall survival, relapse, GVHD, and health care resource use. Patient Selection and Methods We reviewed the medical records of 661 consecutive patients undergoing allogeneic stem cell transplant from January 1, 2003 to January 1, 2013. Hemorrhagic cystitis was diagnosed with symptoms (cystitis with microscopic or macroscopic hematuria, in the absence of other causes such as bacterial/fungal infection or nephrolithiasis) of at least 1 week following stem cell transplant. Microbiological confirmation was obtained routinely after 2005. Controls were matched to cases in a 2:1 ratio in the following priority: age (+/- 10 years), donor type, disease type, GVHD prophylaxis and disease status. All cases received FluBuATG +/- total body irradiation (400 cGy), and all except two received methotrexate (MTX) and cyclosporine (CsA) for GVHD prophylaxis (only received CsA). Results Hemorrhagic cystitis was identified in 117/661 transplant recipients over ten years, for an incidence of 17.7%. After removing cases with a previous transplant, unsuitable matched controls, and incomplete data, 94 evaluable cases were matched to 188 controls. The cohort median age was 50.25 years. Diagnoses included AML (38.3%), ALL (21.3%), MDS (11.7%), CLL (13.8%), NHL (6.4%) and myeloproliferative syndromes (6.4%). Disease statuses included CR1 (31.9%, CR2 (19.1%), untreated (14.9%), PR (13%) and primary refractory (4.3%). Of the cases, 77 of 83 (92.8%) tested positive for BKV in urine by PCR. Median onset was 45 days (range 12 – 279 days). Median BK viral copies was 2242.5 x 106copies/mL (range 0.285 to 4530000). Cases had higher proportions of significant (Grade 2-4) acute GVHD (43.6% vs 27.0%, P=0.0052) and chronic GVHD (cGVHD) requiring steroids (34.9% vs 18.6%, P=0.004). The median onset of significant acute GVHD (aGVHD) was 27 days (range 11 – 103 days), and for cGVHD was 108 days (range 29 – 236 days). Out of 41 aGVHD cases, 8 occurred after BKV hemorrhagic cystitis diagnosis while 23 occurred prior. In 30 cGVHD cases, 23 occurred after BKV hemorrhagic cystitis diagnosis while 7 occurred prior. Cases also had longer hospital stays in the 6 months following transplant (65.5 days vs 40.5 days, P&lt;0.0001). There were no differences in regards to overall survival (HR=1.088, 95% CI 0.7519-1.580), or relapse-free survival (HR=1.057, 95% CI 0.7364-1.519). Evaluating all patients with significant aGVHD, there was no difference in regards to overall survival between controls and cases (HR=0.8042, 95% CI 0.5205-1.205). This was similar for with patients with cGVHD (HR=0.9949, 95% CI 0.6020-1.643) Conclusions This retrospective study shows that BKV hemorrhagic cystitis is a common complication of allogeneic stem cell transplant. While it does not impact overall survival even in ATG-conditioned patients, it is associated with GVHD and longer hospitalizations. Future studies are warranted to determine how BKV hemorrhagic cystitis is influenced by GVHD. Additionally, studies into the pathophysiology of BK virus reactivation with the eventual goal of developing prophylaxis, preemptive therapy or treatment are also warranted. Disclosures No relevant conflicts of interest to declare.

A Phase I/II Trial Evaluating the Use of a Histone Deacetylase Inhibitor Panobinostat (LBH589) in Addition to Glucocorticoids in Patients with Acute Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) remains the principal obstacle to successful outcomes in allogeneic hematopoietic stem cell transplant (HCT). Glucocorticoids are the current standard initial treatment for acute GVHD with variable complete responses rates (30% to 60%). New immunosuppressive strategies are required to improve survival and to decrease immunosuppressive toxicities. Vorinostast, a histone deacetylase inhibitor (HDACi), have shown efficacy for acute GVHD prevention in MRD HCT. Panobinostat is a potent inhibitor of deacetylases and HSP90 belonging to a structurally novel class of the cinnamic hydroxamic acid class and is one of most potent pan-HDACi. This protocol tested the safety and efficacy of Panobinostat (LBH589) as initial adjunct treatment for acute GVHD, administered within 72 hours of the first high dose glucocorticoid (methylprednisolone 0.8 mg/Kg/day IV or equivalent PO for 14 days and then taper per MD discretion). We have enrolled 19 subjects, median age 53 years (range, 34-76), male (n=12)/female (n=7), white(n=14)/hispanic(n=5); with diagnosis of CLL (n=2), MDS (n=2), Myeloma (n=1), Follicular NHL (n=1), CML(n=1), Myelofibrois (n=3), AML (n=5), MDS/CMML (n=3) or ALL(n=1). Conditioning regimens included Busulfan(BU)/fludarabine(FLU) AUC 5300 (n=10) or AUC 3500 (n=3), FLU/Melphalan (n=4) or Pentostatin/BU (n=2); and GVHD prophylaxis for MUD 8/8 (n=11) or MRD (n=5) HCT with TAC/MTX (n=6), TAC/rapamycin(n=7), TAC/MMF(n=3) and for mismatched transplants with either TAC/RAPA/ATG (n=2) or TAC/MTX/ATG (n=1). Median day of acute GVHD (n=16) onset was day + 37 post HCT (26 -109 days) with overall grade GVHD II (n=13) or III (n=6); and median day of acute symptoms in overlap GVHD patients (n=3) was day + 712 (528-981). All Patients were treated with voriconazole (n=15) or micafungin (n=4) for fungal prophylaxis. For the first four patients Panobinostat was administered intravenously (IV) weekly x 4 at 2.5MG/M2 (n=3) or 5MG/M2 IV (n=1) with all 4 achieving either CR (n=3) or PR (n=1) GVHD responses by day +15 of Panobinostat. Due to manufacturer discontinuation of IV formulation, the protocol was amended to use PO Panobinostat. Using 10mg PO TIW 3 doses q week x 4 weeks, we treated 2 subjects which were both discontinued from study drug due to presumed GHVD progression within 7 days of Panobinostat (after 3-4 doses). First subject had grade II GVHD (skin stage 3, gut stage 1 and liver stage 0) that progress in gut and skin; second subject with grade II GVHD (skin stage 3, gut stage 1, liver stage 1) with LFTs worsening ultimately evolving into VOD. Due to safety concerns next subjects were treated with 5 mg PO TIW 3 doses q week x 4 weeks, dose that was determined to be the maximal tolerated dose (MTD) after 6 patients completed therapy in phase I. Currently we are enrolling in phase II portion (n=7). GVHD response rate among MTD treated was complete in 85% (n=11), partial in 7.6% (n=1) or progressive in 7.6% (n=1) by day +36 after Panobinostat with majority achieving responses by day +21. Chronic GVHD at day +365 in evaluable patients (n=6) was none (n=3) or mild (n=3) and steroid was discontinued at a median of 3 months (3-6). Hematological toxicities in evaluable patients (n=13) were mild with worsening of prior thrombocytopenia (n=7/10), anemia (n=3/10) and leukopenia (n=3/10) and returned to baseline within 1-2 weeks; LFTs deterioration (n=1) within 1 week of Panobinostat in a GVHD stages 3 liver/3 skin patient; pericarditis/cardiogenic shock CTCAE 5 of unclear etiology (n=1); worsening thyroid function (n=1) and hypercholesterolemia (n=1). Preliminary correlative studies in MTD treated patients showed that CD4 and CD8 numbers remained stable during treatment. T regulatory cells numbers decreased at day +8 after Panobinostat and recovered by days +15 and +29 of treatment. Level of T regs inducing cytokines (TGFB and IL-10) increased, possibly contributing to an immune-modulatory environment. There is evidence of an increased in acetylation of histone 3 in CD4, CD8 and monocytes subsets over time. We are encouraged with tolerability of level -1 Panobinostat dose and the high GVHD response rate of 85% which may compare favorably to the historical GVHD response rate. These results suggest a potential role for Panobinostat as a tool to improve success of glucocorticoids for acute GVHD treatment. DisclosuresNo relevant conflicts of interest to declare.

Low Percent of Granzyme B Positive T-Regulatory Cells (CD4+CD25high) As a Predictor of Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

Introduction. Granzyme B is a serine protease commonly found in the granules of cytotoxic lymphocytes and natural killer cells. It is secreted with the pore forming protein perforin and mediates apoptosis in target cells. Granzyme B – mediated cytolysis is one of the regulatory mechanisms (together with IL-2 receptors (CD25)) by which T-regulatory cells (Tregs) influence on T-effectors cells. It is a well-known fact that Tregs participate in a pathogenesis of aGVHD and their amount after allo-HSCT inversely correlates with the probability of aGVHD incidence. No clinical data exist regarding the Granzyme B positive Tregs in this setting.Patients and methods. Peripheral blood samples were collected in EDTA-tubes at day +30, +60, +90 after allo-HSCT and at day of acute GVHD onset from 27 patients with hematological malignancies (AML=12, ALL n=12, LPD=2, MDS =1; 6 with active disease, 21 - in CR) after allo-HSCT (from matched unrelated donor n=23, from matched related donor n=4; MAC = 10, RIC=17).The anti-CD4-APC-Cy7, anti-CD25-FITC and anti-Granzyme B-PE (Becton Dickinson, USA) antibodies were used to determine T-regulatory cells population as CD4+CD25high. We did not include anti-FoxP3 antibodies as FoxP3 is not so specific in humans and particularly after allo-HSCT due to technical difficulties, several isoforms, FoxP3-positivity on activated nonregulatory CD4+ cells. CD4- lymphocytes (CD8+ and NK-cells certainly containing granzyme B) were used as internal positive control to define the percent of CD4+CD25highGranzymeB+ cells and gMFI (geometric mean fluorescence intensity) among the mononuclear cell fraction. 50000 of CD4+ cells were analyzed on a BD FACSCanto II (Becton Dickinson, USA).Results. 11 patients (42%) have developed aGVHD (II-IV) at a median time of +26 day (8 - 88) after HSCT. The percent of CD4+CD25highGranzymeB+ cells among CD4+cells at day +30 after allo-HSCT in patients who never developed aGVHD was statistically higher (9,49±2,79; p=0.003) than in patients at day of aGVHD onset (3,8±1,78). It’s worth to note that in 4 patients who did not have signs of a GVHD on day +30 but developed it later (on day +40, +45, +74, +88), the percent of CD4+CD25highGranzymeB+ cells at day +30 was as low (3,38±1,47) as at day of aGVHD onset and significantly lower (p=0.003) than in patients who never developed aGVHD, thus predicting the occurrence of aGVHD in the future.Conclusion Despite the fact that the analyzed group is small, we suggest that low relative amount of CD4+CD25highGranzymeB+ cells after allo-HSCT may contribute to the development aGVHD and even predict it onset. This fact, of course, needs further investigation. DisclosuresNo relevant conflicts of interest to declare.

Changes in circulating endothelial cells count could become a valuable tool in the diagnostic definition of acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is burdened by life-threatening complications, with graft-versus-host disease (GvHD) being the major cause of morbility and mortality. Recently, clinical and physiopathologic evidences showed that vascular endothelium can be a target of GvHD in the early phase and circulating endothelial cells (CECs) represent surrogate markers of endothelial damage. Using the CellSearch System (Veridex LLC, Raritan, NJ), CECs were counted before (T1), after conditioning regimen (T2), at engraftment (T3), at GvHD onset (T4), and after steroid treatment (T5) in 40 patients (7 Hodgkin's Disease, 13 Acute Myeloblastic Leukemia, 5 Acute Lymphoblastic Leukemia, 8 Multiple Myeloma, 3 Chronic Lymphocytic Leukemia, 1 Non-Hodgkin Lymphoma, 1 Chronic Myeloid Leukemia, 2 Severe Aplastic Anemia) undergoing allo-HSCT. The median CEC per milliliter at T1 was 20 (n=33, range 4-718), in comparison to a value of 2 (range, 1-14) in controls (P<0.001). At T3, CEC per milliliter were 47 (range, 16-148) in GvHD patients and 92 (range, 23-276) in patients without GvHD (P=0.006). This difference remained significant in multivariate analysis (odds ratio, 0.97; 95% confidence interval, 0.96-0.99; P=0.02). At GvHD onset, the relative increase of CEC counts from time of engraftment (T4 vs. T3) was 44% (range, -43% to 569%) in GvHD patients versus 0% (range, -49% to 2%) in patients without GvHD (P=0.003), being confirmed as significant in multivariate analysis (odds ratio, 1.04; 95% confidence interval, 1.0-1.08; P=0.04). Changes in CEC count can represent a promising marker to monitor endothelial damage in patients undergoing allo-HSCT and could become a valuable tool in the diagnostic definition of GvHD.

AFM detection of biophysical characteristics of specific regulatory T Cells after allogeneic hematopoietic stem cell transplantation

Based on the dynamics of antigen recognition from T cell receptor complex and T cell activation, different function of T cells may present different morphology feature, at least at molecular level. Regulatory T (Treg) cells play a critic role in regulation the onset of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Little is known about their morphology feature which may relate to the regulating function in GVHD. In this study, we detected the biophysical architectural changes of specific Treg cells subsets after allo-HSCT using biological atomic force microscopy to characterize their biological characteristics and improve our understanding of cell structure-function relationships in GVHD. There were dramatic overall shape and surface membrane deformations of the Treg cells associated with patients at GVHD onset or without GVHD. The Treg cells at GVHD onset or without GVHD could be distinguished by the morphologic parameters of morphology, membrane nanostructure, and membrane pore. These AFM parameters of Treg cells morphology differed obviously between GVHD and without GVHD. The multiple irregular microspikes could be observed on the surface of Treg cells without GVHD. The remarkable biological morphology changes in the cell membrane structure of Treg cells after allo-HSCT might be related to the function of Treg cells which could inhibit the occurrence and development of GVHD.

Validation of National Institutes of Health Global Scoring System for Chronic Graft-Versus-Host Disease (GVHD) According to Overall and GVHD-Specific Survival

A new severity grading system for graft-versus-host disease (GVHD) was established by the National Institutes of Health (NIH) consensus criteria (NCC). However, its prognostic value still needs to be validated. Four hundred twenty-five consecutive patients who survived beyond 100 days after allogeneic stem cell transplantation were reviewed and reclassified using NCC. GVHD-specific survival (GSS) and cumulative incidence of relapse were compared according to the NIH global score at the onset and peak of chronic GVHD (cGVHD). Of 346 patients with cGVHD diagnosed by the Revised Seattle Criteria, 317 patients were reclassified according to the NCC as classic cGVHD (n = 144) and overlap syndrome (n = 173). The NIH global scores at onset were mild (43.2%), moderate (42.3%), and severe (14.5%), whereas more moderate (55.5%) and severe (31.6%) cGVHD was observed at the peak of cGVHD. With a median follow-up duration of 34 months, the 5-year GSS was significantly worse for the severe group than the moderate/mild groups at onset and at peak: 50.9% ± 7.8% versus 89.7% ± 3.2% versus 93.5% ± 2.4% at onset (P < .001) and 69.1% ± 5.2% versus 93.2% ± 2.1% versus 97.3% ± 2.7% at peak (P < .001). Severe NIH global score at onset and peak were confirmed as a poor prognostic factor for GSS in multivariate analysis. The cumulative incidence of relapse did not differ among the severity groups at onset or peak. In conclusion, the new NIH global scoring system was shown to differentiate a high-risk group of patients (with severe grade cGVHD) in terms of long-term transplant outcomes.