Graft-versus-host Disease Patients Research Articles

High Readmission Rates Are Associated with a Significant Economic Burden and Poor Outcome in Patients with Grade 3/4 Acute GvHD

Abstract 2061The impact of graft versus host disease (GvHD) on readmission rates following allogeneic stem cell transplantation (SCT) has not been reported. We hypothesised that patients with GvHD would have a higher readmission rate than patients without GvHD. To test this hypothesis a retrospective review was undertaken of 187 consecutive patients who underwent allogeneic SCT from 1/1/2006 to 30/4/2009 at the Royal Marsden Hospital, UK. Data were collected from the electronic patient record. The study was approved by the hospital audit committee.Patient characteristics: male 113(60%), median age 47 yrs (17–70), diagnosis: acute leukaemia 123 (66%), chronic leukaemia 22 (12%), lymphoma 23 (12%), myeloma 12 (6%), other 7 (4%), donor: sibling 60 (32%), unrelated 115 (61.5%), cord 12 (6.5%), stem cell source: PBSC: 158 (84.5%), BM 17 (9%), cord 12 (6.5%); donor sex: M 111(59.5%), F 64 (34%), cord 12 (6.5%); match: full 130 (69.5%), mismatch 45 (24%), cord 12 (6.5%), conditioning intensity: full 73 (39%), reduced 114 (61%); use of alemtuzumab 113 (60%); GvHD prophylaxis: cyclosporine 116 (62%), mycophenolate 32 (17%), cyclosporine and methotrexate 26 (14%), other 13 (7%).Median follow up was 3.8 (2.2–5.5) yrs from transplant. 118/187 (63%) of patients developed GvHD. There was no significant difference between patients with or without GvHD except for increased use of alemtuzumab in the non-GvHD group (71% v 54%, p=0.024). GvHD was diagnosed clinically at the transplant centre. Biopsies were undertaken if the diagnosis was unclear. Glucksberg criteria were used to stage acute GvHD (aGvHD). NIH criteria were used to diagnose chronic GvHD (cGvHD). 45/118 (38%) had biopsy-proven GvHD. 88/187 (47%) had aGvHD (grades: 1 – 17 (9%), 2 – 43 (23%), 3 – 15 (8%), 4 –13 (7%)). 58/187 (31%) of patients had cGvHD. 36/187 (19%) had both acute and chronic GvHD (52 (29%) had aGvHD alone, 22 (12%) had cGvHD alone). 8 developed GvHD following donor lymphocyte infusion. 104/118 (88%) required steroid treatment. 61/118 (52%) commenced steroids within 100 days of transplant. 61/118 (52%) were steroid refractory. 5 patients had received a second allogeneic transplant for relapse to induce GvHD and graft versus malignancy effect.The median duration of initial transplant admission was 31 days (20–138) in GvHD group compared to 32 days (16–103) in non GvHD group (p=NS). 14 patients died during initial admission (5 in GvHD group, 9 in non-GvHD group) and were excluded from readmission analysis. The overall readmission rate was higher in GvHD patients (89% (101/113) v 41/60 (68%), p=0.001). In the first 100 days post transplant, 42/56 (75%) of patients who had started steroids were readmitted compared to 60/117 (51%) in patients who started steroids after day 100 or had no GvHD (p=0.003).Critical care unit admission was higher in the GvHD group (38/113 (34%) v 7/60 (12%),p=0.002). The mean total number of admission days was higher in the GvHD group (42 days v 18 days, p<0.001). 337/455 (74%) of readmission episodes and 231/305 (76%) of infection-related admissions were in the GvHD group. Patients with grade 3/4 GvHD had a higher readmission rate (96%) than grade 1/2 GvHD patients (88%) or patients with no aGvHD (74%). The mean total number of admission days was 57 days in grade 3/4 GvHD compared to 37 days in grade 1/2 GvHD. (p=0.054).Costs were calculated based on the 2010 hospital tariff (€600/inpatient day and €2295/CCU day). The mean cost of readmission was higher in GvHD patients (€32217) than in non-GvHD patients (€15622) (p=0.003). Mean cost was higher in grade3/4 GvHD (€44535) than in grade 1/2 GvHD (€27001) (p=0.032). These figures do not include drug or procedural costs.Overall survival was 42% at 2 years. Survival was higher in the GvHD group (48%) than in the non GvHD group (30%) (p=0.006). Survival was higher in those with grade 1/2 GvHD (55%) compared to those with grade 3 or 4 GvHD (14%) (p<0.001). There was no significant difference in survival in those with grade 1/2 GvHD (55%) and those with no GvHD (43%) (p=0.15).In conclusion, patients with GvHD had significantly higher readmission rates and longer duration of admission than those without GvHD. Despite the high readmission rate, patients with grades 1/2 GvHD had improved survival. Patients with grade 3/4 GvHD had a very high level of readmission and poor outcome. Improving strategies for managing GvHD may help to reduce post transplant readmission rates and the associated burden on the healthcare system. Disclosures:Dignan:Therakos, a Johnson and Johnson company: Honoraria, Research Funding. Shaw:Therakos, a Johnson and Johnson company: Honoraria, Speakers Bureau.

Biomolecular Characteristics of Specific Regulartory T Cells of Graft-Versus-Disease Based on T-Cell Receptor Analysis,

Abstract 4067Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important therapetic option for a number of malignant and refractory haematological diseases. Graft versus host disease (GVHD) is the main complications following allo-HSCT. Recent data indicated that regulartory T (Treg) cells might participate in mediating GVHD and graft-versus-leukemia (GVL) effect after allo-HSCT. However, the distribution and clonality of T cell receptor (TCR) repertoire of specific Treg cells in GVHD remains unclear. To further characterize this feature, we analyzed the distribution and clonality of TCR repertoire of Treg cells at GVHD onset to further understand the biological characteristics of specific Treg cells subsets of GVHD.The complementarity-determining region 3 (CDR3) sizes of TCR repertoire (TCR Vα, Vβ, Vδ and Vγ subfamilies) were analyzed in the Treg cells of recipients at GVHD onset, using RT-PCR and genescan technique. To determine the expression levels and expression pattern of TCR Vγ I-III subfamily genes, the expression levels and expression pattern of CD3 γ, δ, ε, ζ genes, and the expression levels of regulatory genes of Treg cells (FOXP3 and GATA-3 genes), we performed quantitative analysis by real-time PCR.The frequency of TCR Vβ subfamilies of Treg cells at GVHD onset was significant lower than that in the Treg cells without GVHD. Oligoclonality were detected in TCR Vα 15, Vα 23, Vβ 14 and Vδ 3 subfamilies of the Treg cells from GVHD patients, and oligoclonality were detected in TCR Vα 4, Vα 21, Vβ 16 and Vβ 19 subfamilies of the Treg cells from patients without GVHD. The TCR Vγ I-III subfamilies expression levels in Treg cells were decreased significantly after allo-HSCT. The expression pattern of TCR Vγ subfamilies of Treg cells at GVHD onset was TCR Vγ III>TCR Vγ I>TCR Vγ II, and different with the expression pattern of Treg cells without GVHD (TCR Vγ I>TCR Vγ II>TCR Vγ III). The expression pattern of CD3 γ, δ, ε, ζ genes of Treg cells at GVHD onset was also changed, and GVHD might mainly influence the expression levels of CD3 δ and CD3 ε genes of Treg cells. The expression levels of GATA-3 gene had a positive correlation with the expression levels of FOXP3 gene in Treg cells with GVHD. The expression levels of GATA-3 gene in Treg cells at GVHD onset were significant higher than that in the Treg cells without GVHD, but the expression levels of FOXP3 gene in Treg cells at GVHD onset were significant lower than that in the Treg cells without GVHD.In conclusion, oligoclonality of TCR Vα 15, Vα 23, Vβ 14 and Vδ 3 subfamilies of Treg cells might related with GVHD. The expression pattern of high TCR Vγ III gene expression and low TCR Vγ II gene expression in Treg cells might correlate with GVHD. GVHD might mainly influence the expression levels of CD3 δ and CD3 ε genes in TCR signal transduction of Treg cells. The high expression levels of GATA-3 gene in Treg cells might play a role in mediating GVHD, and GATA-3 could be a novel treatment target of GVHD immunotherapy. Disclosures:Wu:Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (No.200902332, No.20080440776): Research Funding. Liu:Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (No.200902332, No.20080440776): Research Funding.

Chronic Graft-Versus-Host Disease and Systemic Sclerosis: The Same Pathophysiology?,

Abstract 4076Due to their clinical similarities, controversy has existed as to whether the pathophysiology of chronic graft-versus-host disease (GVHD) and systemic sclerosis (SSc) is the same. The principal cellular effector mechanism in SSc is clonal autoreactive CD4 T lymphocytes that activate fibroblasts, resulting in increased collagen production. We have previously reported that the principal cellular effector mechanism in histocompatible murine chronic GVHD is autoreactive CD4 T lymphocytes, which also stimulated fibroblast collagen production. More recently defects in naturally occurring regulatory T lymphocytes (Treg) have been reported in adult chronic GVHD patients. Using the Miyana classification (Immunity 30:899, 2009), which discriminates between resting Treg lymphocytes, activated Treg lymphocytes, and conventional T lymphocytes (Tcon), we assessed whole blood from pediatric patients, who were more than 1 year following allogeneic hematopoietic stem cell transplantation (HSCT), or patients with active juvenile SSc (jSSc), for their total number of Treg lymphocytes and their relative proportion of resting and activated Treg lymphocytes (r/a ratio).Normal individuals had an average of 15,300 Treg lymphocytes/mL with 10,890 resting Treg/mL and 4,410 activated Tregs/mL or a r/a ratio of 2.5. The resting Treg lymphocytes were 17.1% of the CD4+, CD25+, CD127- T lymphocytes while the activated Treg lymphocytes were 6.9%. Allogeneic HSCT recipients without a history of either acute or chronic GVHD had elevated numbers of total Treg lymphocytes (25,979/mL) (P=.05) with 19,199 resting and 5,780 activated Treg lymphocytes or a r/a ratio of 3.3, secondary to the relative increase in the proportion of resting Treg lymphocytes. Recipients, who had had only acute GVHD (Grade 2–4) but no history of chronic GVHD, also had an increased number of total Treg lymphocytes with a normal r/a ratio.Recipients, who had active chronic GVHD, had reduced numbers of total Treg lymphocytes (10,040/mL) (P=.02) with reduced numbers of both resting (7,631/mL) and activated (2,408/mL) Treg lymphocytes. Half of the recipients with active chronic GVHD had more activated Treg lymphocytes than resting T lymphocytes, resulting in an inverted r/a ratio of <1.0. HSCT recipients, who had resolved chronic GVHD, had increased numbers of total Treg lymphocytes (39,342/mL) with increases in both resting (33,325/mL) and activated (6,016/mL) Treg lymphocytes with a r/a ratio of 5.5 due to the relative increase in the proportion of resting Treg lymphocytes.Patients with jSSc had Treg lymphocyte patterns similar to HSCT recipients with active chronic GVHD; their total number of Treg lymphocytes was reduced (843/mL) (P=.01) with reduced resting (532/mL) and activated (661/mL) Treg lymphocytes and an inverted r/a ratio of 0.8 due to the relative excess of activated Treg lymphocytes. Thus, pediatric patients with either active chronic GVHD or jSSc have reduced total numbers of Treg lymphocytes with a relative increase in the proportion of activated Treg lymphocytes, suggesting that the abnormalities in Treg lymphocytes are central to the pathogenesis of both clinical syndromes. HSCT recipients with a history of resolved chronic GVHD had increased numbers of Treg lymphocytes, indicating that the resolution of chronic GVHD requires increased numbers of Treg lymphocytes with a normal ratio of resting to activated Treg lymphocytes. Disclosures:No relevant conflicts of interest to declare.

Deficiency of CD73/Ecto-5'-Nucleotidase Enhances Acute Graft-Versus-Host Disease

Abstract 312Acute graft-versus-host disease (GvHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT). We have recently shown that extracellular adenosine triphosphate (ATP) enhances GvHD by activation of the purinergic receptor P2X7R. The abundance of extracellular ATP is regulated by ecto-nucleotidases such as CD39, which dephosphorylate ATP to ADP and AMP. Ecto-5'-nucleotidase (CD73) degrades AMP to adenosine, which can itself exert tolerogenic functions via activation of A2A and/or A2B adenosine receptors (AR). Extracellular ATP and adenosine have immunoregulatory roles under different inflammatory conditions. Deficiency of CD73, the enzyme that metabolizes AMP to adenosine, could therefore have pro- or antiinflammatory activity by reducing adenosine levels in the microenvironment. Currently, the roles of CD73 and adenosine in GvHD are unclear.By using cd73−/− donor or recipient mice and a specific CD73/ecto-5’-nucleotidase inhibitor we observed enhanced GvHD severity when CD73 was genetically deleted or pharmacologically blocked. CD73 was upregulated on CD4+ and CD11c+ cells after irradiation, suggesting an inducible rescue mechanism along with CD39 to counteract ATP accumulation resulting from tissue damage due to the preconditioning treatment. CD73 deficiency led to enhanced T cell expansion and IFN-γ and IL-6 production in GvHD mice. Migratory capacity of cd73−/− T cells was increased, compatible with the ability of adenosine to inhibit transendothelial migration. A2A receptor deficiency led to increased numbers of proliferating allogeneic T cells in allo-HCT recipient mice, suggesting that signaling through the A2A receptor via CD73-generated adenosine is a significant part of the mechanism by which CD73 limits the severity of GvHD. Furthermore, pharmacological blockade of CD73 improved graft-versus-tumor activity in an in vivo B cell leukemia model.In conclusion, we demonstrate that CD73 plays a protective role in GvHD with enhanced disease severity in its absence, while blocking CD73 led to increased graft-versus-tumor effect. These data have potential clinical implications as the alloimmune response could be enhanced by CD73 blocking in patients with residual tumor burden after transplantation. Conversely, immune modulation by addition of CD73 enzyme or an adenosine receptor agonist could improve the outcome of GvHD patients. Disclosures:No relevant conflicts of interest to declare.

Hyperbilirubinemia after hematopoietic stem cell transplantation: comparison of clinical and pathologic findings in 41 autopsied cases

Hyperbilirubinemia is often associated with morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Diagnosis of its etiology is usually made clinically among various possible causes, and analysis of histological findings as compared with the clinical diagnosis has not been performed sufficiently. We retrospectively analyzed clinical and pathological findings in 41 autopsied patients who died with hyperbilirubinemia (>2 mg/dL). Overall, liver graft-versus-host disease (GVHD) showed the most prominent discordance between clinical and pathological diagnoses. Only 11 of the 22 patients, considered to have liver GVHD clinically, had GVHD findings at autopsy. Serum gamma-glutamyl transpeptidase (GGT), GGT/aspartate aminotransferase (AST) ratio, and alkaline phosphatase (ALP)/AST ratio in GVHD patients were significantly higher compared with those without GVHD (p = 0.02, <0.01, and 0.03, respectively), which was useful in clinical diagnosis of liver GVHD. Other major findings include liver invasion of the primary malignancies in 8 patients, post-transplant lymphoproliferative disorder of the liver in two patients, and disseminated liver invasion by fungus or varicella-zoster virus in one patient, respectively. Although analysis of clinical data is useful for narrowing diagnosis, histological analysis by liver biopsy is crucially important, especially in cases suspected of having GVHD.

Impaired Aortic Elastic Properties in Patients with Chronic Graft-versus-Host Disease

Graft-versus-host disease (GVHD), which develops as a result of the immunologic response that donor T-lymphocytes generate against host tissue following hematopoietic stem cell transplantation (HSCT), is the leading cause of morbidity and mortality in these patients. The aim of this study is the investigate relation between aortic wall stiffness and duration of the disease in patients with chronic GVHD. The study population included 32 patients (18 men; mean age, 36.9±12.5 years, and mean disease duration=14.7±2.9 months) who received HSCT and was diagnosed with GVHD and 44 patients (23 men; mean age, 35.2±9.6 years, and mean disease duration=13.5±2.4 months) who did not develop GVHD following HSCT. All patients underwent baseline echocardiography before HSCT and were followed. After approximately 10-14 months following HSCT, these patients were divided into two groups based on whether they had developed chronic GVHD, and were compared to aortic stiffness parameters and cardiac functions. There was no change in basal characteristics, laboratory and echocardiographic findings, and aortic stiffness parameters in both groups before HSCT (P>0.05). After HSCT, the mean aortic strain and distensibility values of the chronic GVHD patients were significantly lower, compared with the non-GVHD patients (9.8±3.2% vs. 12.9±5.0%, P=0.002 and 4.1±1.5×10(-6) cm2/dyn vs. 5.3±2.1×10(-6) cm2/dyn; P=0.005, respectively). In addition, aortic stiffness index was increased in the chronic GVHD group compared with non-GVHD group (2.7±1.7 vs. 2.0±0.8, P=0.03). Aortic stiffness measurements were significantly different in chronic GVHD group compared to non-GVHD group and these findings suggested useful explanation for the potential mechanism about the development of disease.

Platelet Lysate Mucohadesive Formulation to Treat Oral Mucositis in Graft Versus Host Disease Patients: A New Therapeutic Approach

Optimal treatment of oral mucositis (OM) due to graft versus host disease (GvHD) is currently not available. Platelet-derived growth factors (PDGFs) have high capability for tissue healing and may play a role in repairing the mucosal barrier. The aim of the present work was to develop a mucoadhesive formulation to administer platelet lysate to oral cavity prolonging contact time of platelet lysate with oral mucosa. The mucoadhesive formulation was characterized for in vitro properties (PDGF-AB concentration, mucoadhesive properties, cytotoxicity, fibroblast proliferation, wound healing). Moreover, a preliminary clinical study on seven GvHD patients with OM refractory to other therapies was conducted, to evaluate feasibility, safety, and efficacy. GVPL (mucoadhesive gel vehicle mixed with platelet lysate)showed good mucoadhesive properties; additionally, it was characterized by good biocompatibility in vitro on fibroblasts and it was able to enhance fibroblast proliferation and wound healing, maintaining the efficacy for up to 14 days following storage at 2-8°C. In vivo, clinical response was good-to-complete in five, fair in one, none in the remaining one. The in vitro results indicate that GVPL has optimal mucoadhesive and healing enhancer properties, maintained over time (up to 14 days); preliminary clinical results suggest that oral application of platelet lysate-loaded mucoadhesive formulation is feasible, safe, well tolerated, and effective. A larger controlled randomized study is needed.

Dry Eye Disease in Chronic Graft-Versus-Host Disease: Results From a Spanish Retrospective Cohort Study

We describe the incidence, clinical features, and final outcome of dry eye disease (DED) associated with chronic graft-versus-host disease (GVHD) over a 5-year period. We reviewed 109 clinical charts from patients undergoing hematopoietic stem cell transplantation (HSCT) between January 2000 and December 2005, abstracting data from the 57 patients who developed chronic GVHD and survived at least 180 days after allogenic HSCT. DED occurred in 22 (40%) patients at an average of 16.5 months after HSCT. Photophobia, irritation, and foreign body sensation were the most frequent symptoms, while punctate keratitis was the most common sign. DED tended to improve over time, with fewer than 5% of patients requiring more than two topical medications at the end of follow-up. Our study represented a formal evaluation of DED incidence among Spanish chronic GVHD patients. It is of utmost importance to assure patients will undergo a comprehensive ophthalmologic evaluation throughout their follow-up.

Decreased pro‐inflammatory cytokines and increased CCR7 expression on T‐lymphocyte subsets are predictive of response to extracorporeal photopheresis in patients with GvHD

Decreased pro‐inflammatory cytokines and increased CCR7 expression on T‐lymphocyte subsets are predictive of response to extracorporeal photopheresis in patients with GvHD

Use of mycophenolate mofetil in patients received allogeneic hematopoietic stem cell transplantation in Japan

We evaluated the use of mycophenolate mofetil (MMF) after hematopoietic stem cell transplantation (HSCT) in Japan from 1999 to 2008. MMF was administered to 301 patients, including 157 for the prevention of graft-versus-host disease (GVHD), 94 for the treatment of acute GVHD and 50 for the treatment of chronic GVHD. The three most common doses were 500mg twice daily, 250mg three times daily and 1,000mg twice daily, given to 63, 54 and 45 patients, respectively. The incidence of grade II-IV acute GVHD was 30.0% and grade III-IV was 20.0% in the GVHD prevention group. Among treated patients, disappearance or improvement of subjective symptoms occurred in 57.0% of acute GVHD patients and in 52.0% of chronic GVHD patients. With regard to safety, the following major adverse events (grade 3 or more) were recorded: 31 infections, 31 neutropenia, 28 thrombocytopenia, 25 diarrhea and 1 renal disorder. A total of 116 patients developed grade 3 or 4 adverse events, but 79 were successfully treated with supportive treatment. Thus, our findings suggest that MMF is safe and effective for the prevention and treatment of GVHD in patients who have received an allogeneic stem cell transplant.

The role of B cells in acute graft-versus-host disease

TO THE EDITOR: Mounting evidences implicates B cells in the pathogenesis of chronic graft-versus-host disease (GVHD). The body of evidence includes findings such as correlation between chronic GVHD and antibody production against Y chromosome-encoded minor histocompatabilty antigens (mHA) generated after sex-mismatched allogeneic stem cell transplantation (SCT), the clinical response of steroid-refractory chronic GVHD to rituximab, and association between high serum B cell-activating factor (BAFF) levels and chronic GVHD [1]. However, the role of B cells in acute GVHD remains controversial. Some studies that showed the association of high B cell count in the grafts in SCT patients with acute GVHD and beneficial effects of rituximab (used for B cell lymphoma treatment before transplantation) in acute GVHD patients, have suggested the possible role of B cells in the development of acute GVHD. Kim et al. reported that the BAFF level/absolute lymphocyte count (ALC) ratio or that of APRIL (a proliferation-inducing ligand) level/ALC at the time of acute GVHD diagnosis was associated with disease severity [2]. In their study, patients with grade III-IV acute GVHD (6) had higher BAFF level/ALC or APRIL level/ALC ratio than the corresponding ratio observed in patients with grade II acute GVHD (9). These findings suggest that BAFF level/ALC or APRIL level/ALC ratio can help determine the severity of acute GVHD and need to be confirmed in large prospective studies. However, it would be somewhat unreasonable to conclude that B cells may play an important role in the development of acute GVHD on the basis of these findings alone. First, counting the number of B cells should be performed along with measurement of the BAFF or APRIL levels because higher serum BAFF levels may reflect relative B lymphopenia depending on the period after transplantation. Furthermore, comparison of the findings for patients with and without acute GVHD for the same period after allogeneic SCT is essential for investigating the relationship between B cells and acute GVHD. BAFF has a complex, dichotomous role in immunity, which is mediated by the differential regulation of T cell- and B cell-dependent immune responses [3]. BAFF, a critical regulator of normal B cell homeostasis in mice and humans, also promotes T cell activation and survival [4]; these T cells play a pivotal role in acute GVHD pathogenesis. However, recent studies have also demonstrated the negative regulatory role of BAFF in T cell function [3, 5]. Walters et al. found that BAFF-transgenic mice accepted islet allografts and showed delayed rejection of skin allografts. On the basis of these results, they proposed that BAFF plays an anti-inflammatory role in T cell biology by promoting the expansion of regulatory T cells [3]. We studied the potential protective effect of BAFF against acute GVHD during the peritransplantation period in the setting of allogeneic SCT in humans [5]. Thus, further experimental and clinical studies are needed to elucidate the role of B cells in the development of acute GVHD and gain more insight into the pathogenesis of acute GVHD by studying the association of BAFF with B cells and T cells.

Photodepletion differentially affects CD4+ Tregs versus CD4+ effector T cells from patients with chronic graft-versus-host disease

AbstractEven the most potent immunosuppressive drugs often fail to control graft-versus-host disease (GVHD), the most frequent and deleterious posttransplantation complication. We previously reported that photodepletion using dibromorhodamine (TH9402) eliminates T cells from healthy donors activated against major histocompatibility complex–incompatible cells and spares resting T cells. In the present study, we identified photodepletion conditions selectively eradicating endogenous proliferating T cells from chronic GVHD patients, with the concomittant sparing and expansion of CD4+CD25+ forkhead box protein 3–positive T cells. The regulatory T-cell (Treg) nature and function of these photodepletion-resistant cells was demonstrated in coculture and depletion/repletion experiments. The mechanism by which Tregs escape photodepletion involves active P-glycoprotein–mediated drug efflux. This Treg-inhibitory activity is attributable to interleukin-10 secretion, requires cell-cell contact, and implies binding with cytotoxic T-lymphocyte antigen 4 (CTLA-4). Preventing CTLA-4 ligation abrogated the in vitro generation of Tregs, thus identifying CTLA-4–mediated cell-cell contact as a crucial priming event for Treg function. Moreover, the frequency of circulating Tregs increased in chronic GVHD patients treated with TH9402 photodepleted cells. In conclusion, these results identify a novel approach to both preserve and expand Tregs while selectively eliminating CD4+ effector T cells. They also uncover effector pathways that could be used advantageously for the treatment of patients with refractory GVHD.

Novel Photodepletion Strategy to Preserve and Expand Tregs While Eliminating CD4+ Effector T Cells From Patients with Chronic Graft-Versus-Host Disease

AbstractAbstract 353Even the most potent immunosuppressive drugs often fail to control graft-versus-host disease (GVHD), the most frequent and deleterious post-transplantation adverse condition. The development of a strategy to eliminate alloreactive T cells and spare regulatory T cells (Tregs) would provide a direly needed therapeutic option for patients with refractory GVHD. We previously reported that photodepletion using dibromorhodamine (TH9402) eliminates T cells from healthy donors activated against major histocompatibility complex (MHC)-incompatible cells and spares resting T cells. In the present study, we identified novel photodepletion conditions (1.32 μM TH9402, 45 minutes incubation, and 5 J/cm2 light delivery at 514 nm) selectively eradicating 60–90% of endogenous proliferating host T cells as measured by 3H-thymidine incorporation, and CD4+CD25+FOXP3- effector memory phenotype cells (p<0.001) from chronic GVHD patient cells, with concomitant sparing of the majority of CD4+CD25+FOXP3+ Tregs.All GVHD patients tested (n=10) had severe extensive chronic disease and were refractory to at least 2 immunosuppressive agents. Preservation of Treg function after photodepletion was confirmed by demonstrating reduction in the proliferation of GVHD T lymphocytes by 66.6±7.1% to 75.8±7.0% (mean±SEM; p<0.001, n=6) after their exposure to photodepletion lymphocytes at ratios of 8:1 to 1:1, respectively. The fact that immunomagnetic depletion of CD4+CD25+ T cells from lymphocytes exposed to photodepletion abrogated the inhibitory activity of both GVHD patient and healthy donor cells indicated that these Tregs harbored a CD4+CD25+ phenotype. Moreover, Treg activity was recovered upon CD4+CD25+ cell repletion. Treg survival to photodepletion relied on P-glycoprotein induced efflux of TH9402 since inhibition of this membrane transporter by verapamil (50 μM) induced TH9402 accumulation and Treg demise upon illumination. We found that IL-10 secretion increased when photodepletion cells were cultured with GVHD untreated cells (p<0.01) and anti-IL-10 monoclonal antibodies (mAbs) blocked their suppressive activity. TGF-β was not found to be implicated in the suppression of proliferation. Allowing cell-cell contact between photodepletion exposed lymphocytes and cGVHD cells in 3- to 6-day cultures led to doubling of Treg numbers (p<0.01, n=5) while separating photodepletion treated and untreated GVHD cells abrogated both IL-10 secretion and Treg suppressive activity. Additionally, blocking CTLA-4 ligation with anti-CTLA-4 mAbs abrogated Treg function and prevented the generation of Tregs ex vivo (p<0.05), thus identifying CTLA-4-mediated cell-cell contact as a crucial priming event for Treg function.This increase in Tregs was not caused by increased proliferation as measured by EdU labelling. Exposing isolated CD4+CD25- cells to TH9402 photodepletion itself also failed to increase the number of FOXP3 expressing cells. However Treg numbers increased when CD4+CD25- cells were incubated with CD4+CD25+ cells previously exposed to photodepletion, suggesting that photodepletion cells promote the acquisition of Treg features in CD4+CD25- T cells.This photodepletion strategy was then evaluated in 5 patients suffering from refractory chronic GVHD. The patients had their cells collected by lymphopheresis, exposed to photodepletion and reinfused back into them on a weekly basis. Interestingly, the frequency of circulating Tregs in cGVHD patients undergoing TH9402 photodepletion increased in comparison to healthy donors (p<0.05), with a doubling in their numbers in comparison to pre-treatment levels (p<0.05) as early as 2 weeks after initiating treatment and these patients' conditions improved. In conclusion, these results identify a new approach to both preserve and expand Tregs while selectively eliminating CD4+ effector T cells. They also uncover effector pathways that could be used advantageously for the treatment of patients with refractory GVHD. Disclosures:Egeler:Kiadis Pharma: Employment. Roy:Kiadis Pharma: Research Funding.

Analysis of Clinical Outcome and Survival in Pediatric Patients Undergoing Extracorporeal Photopheresis for the Treatment of Steroid-refractory GVHD

Extracorporeal photopheresis (ECP) has been used for treatment of steroid-refractory graft-versus-host disease (GVHD) with encouraging results. However, only a few pediatric centers have experienced with ECP, mainly by technical difficulties of leukapheresis in children. We retrospectively analyzed 27 patients treated during 5 years with ECP for steroid-refractory acute and chronic GVHD. We performed an "off-line" approach using a continuous flow cell separator (Cobe Spectra) and a Ultraviolet A irradiator (UVAMATIC). Among acute GVHD patients, 11 of 21 obtained a complete remission (CR) and another 8 reached partial remission. In the chronic GVHD group, 3 patients obtained CR and another 2 were in partial remission. Disease-free survival probability was 43±9% in the whole group. The only variable that impacts on disease-free survival on multivariate analysis was achieving CR with ECP. We also found a better CD4/CD8 ratio after ECP. In detail, among CD4 lymphocyte population, an increase in the effector memory and T-regulatory population was found. We may conclude that ECP is a safe and effective steroid-refractory GVHD treatment using the ex-vivo collection method, even in small children. Clinical response favorably impacts on long-term survival and seems to be associated with peripheral blood lymphocyte subset changes generating peripheral tolerance.

Extracorporeal photochemotherapy induces arginase 1 in patients with graft versus host disease

The benefits of extracorporeal photochemotherapy (ECP; psoralen and UVA exposure of blood mononuclear cells) in graft-versus-host-disease (GVHD) are well-recognized, but the mechanisms of action remain elusive. As the metabolism of l-arginine in immune cells is known to play a role in immune tolerance, we investigated the effect of ECP on arginine metabolism, and the influence of extracellular l -arginine concentration on the response to ECP in cells from patients on therapy by ECP for a GVHD and healthy donors cultured before and after ECP in the presence of different concentrations of arginine (0, 50, 100, 200 and 1000μmol/l). At baseline arginine was not metabolized through the same pathway in patients and donors. When cells were exposed to ECP, the production of ornithine but not NO° was enhanced, while mRNA of arginase 1 was up-regulated but not INOS. In GVHD patients, increasing arginine concentration resulted in down-regulation of IFNγ and TNFα mRNA expression, whereas IL10 was up-regulated especially at physiological plasma levels (between 0 and 100μM). Overall, our study shows that ECP orients the metabolism of arginine toward the arginase pathway together with shifting the cytokine profile toward IL-10, providing new insights into the enigmatic mechanism of action of ECP.

Acute but not chronic graft-versus-host disease is associated with a reduction of circulating CD4+CD25highCD127low/− regulatory T cells

Defects in central and peripheral tolerance are thought to contribute to life-threatening graft-versus-host disease (GvHD), a severe complication following allogeneic stem cell transplantation (SCT). Recent investigations have demonstrated regulatory T cells (Tregs) to suppress allogeneic immune reactions. Therefore, SCT patients with no or critically low numbers of Tregs may have an increased risk of GvHD. To address this hypothesis, we analyzed the recovery of CD4(+)CD25(high)CD127(low/-) Tregs in the peripheral blood of patients who have never developed GvHD (n = 6), patients who developed acute/chronic GvHD (n = 18), and patients who developed chronic GvHD without an earlier acute manifestation (n = 5) every 30 days for the first 6 months after peripheral blood SCT (PBSCT). The number of Tregs continuously improved in acute/chronic GvHD patients, but always remained lower than Tregs quantified in patients who never developed a GvHD. In contrast, chronic GvHD patients who did not develop acute GvHD earlier displayed significantly increased Treg cell numbers at the timepoint of chronic inflammation. These results indicate that numerically deficient Tregs following PBSCT are associated with the development of acute but not chronic GvHD.

Mesenchymal stromal cells for steroid-refractory acute graft-versus-host disease: a report of two cases

Severe graft-versus-host disease (GVHD) is a lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, there have been only a few limited therapeutic options for the treatment of GVHD. Here we have reported two cases of the patients with acute steroid-refractory GVHD who underwent allogeneic HSCT. These patients received infusions of third-party clonal mesenchymal stem cells (cMSCs), which were isolated by a subfractionation culturing method (SCM) developed recently by our group, and showed marked improvement of the disease. MSCs represent a new potential therapeutic option in the treatment of steroid-refractory acute GVHD. They showed safety and improved clinical findings of the acute GVHD patients. However, further investigations are needed to understand the accurate mechanisms of MSCs and larger well-designed human clinical trials are necessary to prove further the safety and efficacy of the cMSCs for the treatment of acute GVHD patients.

Gastric contents in pediatric patients following bone marrow transplantation

Graft versus host disease (GVHD) of the gut is thought to delay gastric emptying and so may increase the risk of aspirating retained contents while under anesthesia. Knowing that gastric emptying is delayed in patients with GVHD might lead one to choose to intubate the trachea for all patients with suspected GVHD, who present for diagnostic esophagogastricduodenoscopy (EGD). We are not aware of published data that gives specific guidance as to the need for intubation in the pediatric bone marrow or stem cell transplantation (BMT) population. This review was intended to evaluate the gastric contents (pH and volume) in this group of patients, to provide anesthesiologists with data that would inform their decisions about airway management for these patients. Retrospective chart review of patients <or=19 years of age undergoing EGD between 2004 and 2006. Gastric content volume and pH were measured in addition to underlying disease state and treatment. We compared BMT patients with suspected GVHD to nontransplant patients with other underlying gastrointestinal conditions. Data were obtained for 77 patients post-BMT undergoing EGD, including 40 patients whose biopsies and endoscopic findings were positive for GVHD, and 37 patients with no demonstrable GVHD. Records of 144 non-BMT patients undergoing EGD within the same study period were also reviewed. Patients in the BMT group overall did not have higher volumes when compared to non-BMT patients. A secondary comparison of BMT patients who were found to have GVHD vs BMT patients without GVHD suggests that gastric content volume may be elevated with GVHD. Patients in the BMT group had statistically significantly higher gastric pH than patients in the non-BMT group. It is possible that the higher gastric volume in the GVHD-positive group could put them at slightly higher risk for aspiration, but the severity of any pneumonitis, should aspiration occur, might be mitigated, by the tendency toward a higher gastric pH in the BMT patients.

Basement membrane antibodies in sera of haematopoietic cell recipients are associated with graft‐versus‐host disease

Graft-versus-host disease (GvHD) occurs frequently after haematopoietic cell transplantation (HCT). Mucocutaneous lesions of GvHD may mimic bullous autoimmune dermatoses, and 10 cases of concurrent GvHD and a bullous autoimmune disease have been reported in the literature. To determine the frequency of circulating antibodies to the cutaneous basement membrane zone (BMZ) in HCT patients with GvHD in comparison with HCT patients without GvHD, psoriasis patients and healthy controls. We examined 42 patients with chronic GvHD, 18 HCT patients without GvHD, 11 psoriasis patients and 40 healthy controls, prospectively. Sera were tested by indirect immunofluorescence (IIF) on salt-split skin, NC16a-ELISA and immunoblot using keratinocyte extracts. Univariate statistical analyses and logistic regression were performed to assess possible correlations of graft and patient characteristics with the presence of BMZ antibodies. Circulating basement membrane zone (BMZ) antibodies were detected in 10/42 (24%) GvHD sera by immunoblot, but not in any of the HCT sera from patients without GvHD (0/18; 0%). The antibodies targeted collagen VII, BP230, collagen XVII/BP180 or p200/laminin gamma1. Clinically manifest bullous autoimmune dermatoses (bullous pemphigoid or epidermolysis bullosa acquisita) were found in two GvHD patients. 1/11 (9%) psoriasis sera and 1/40 (2.4%) healthy control sera reacted with collagen XVII or BP230, respectively. Circulating BMZ antibodies are significantly associated with chronic GvHD in contrast to uncomplicated HCT. Recurrent mucocutaneous lesions in chronic inflammatory skin disorders may liberate antigens, which may lead to production of BMZ antibodies, particularly in the context of GvHD-mediated reduced self-tolerance.

Chemokine Receptor CCR5 Mediates AlloImmune Responses in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD.