Evidence for B cell exhaustion in chronic graft-versus-host disease

Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). A number of studies support a role for B cells in the pathogenesis of cGvHD. In this study, we report the presence of an expanded population of CD19+CD21− B cells with features of exhaustion in the peripheral blood of patients with cGvHD. CD21− B cells were significantly increased in patients with active cGvHD compared to patients without cGvHD and healthy controls (median 12.2 versus 2.12 versus 3%, respectively; p < 0.01). Compared with naïve (CD27−CD21+) and classical memory (CD27+CD21+) B cells, CD19+CD21− B cells in cGvHD were CD10 negative, CD27 negative and CD20hi, and exhibited features of exhaustion, including increased expression of multiple inhibitory receptors such as FCRL4, CD22, CD85J, and altered expression of chemokine and adhesion molecules such as CD11c, CXCR3, CCR7, and CD62L. Moreover, CD21− B cells in cGvHD patients were functionally exhausted and displayed poor proliferative response and calcium mobilization in response to B-cell receptor triggering and CD40 ligation. Finally, the frequencies of circulating CD21− B cells correlated with cGvHD severity in patients after HSCT. Our study further characterizes B cells in chronic cGVHD and supports the use of CD21−CD27−CD10− B cell frequencies as a biomarker of disease severity

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Aquatic Toxicology 159 (2015): 198-207, doi:10.1016/j.aquatox.2014.12.010.Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ~400 times higher, and the levels of non-dioxin-like PCBs ~3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased expression of hepatic PXR, CYP3A and Pgp genes upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.This study was supported by grants from FORMAS (216-2007-468) and University of Gothenburg to MCC, and by the Superfund Research Program at Boston University, NIH grant P42ES007381 to JJS, MEH, and SIK. Data interpretation was aided by reference to a preliminary draft of the Fundulus heteroclitus genome sequence, which was supported by funding from the National Science Foundation (collaborative research grants DEB-1120512, DEB-1265282, DEB-1120013, DEB-1120263, DEB-1120333, DEB-1120398). This study was also supported by NOAA Grant No. NA16RG2273 (WHOI Sea Grant Project No. R/P-70 to SIK and MEH) and by funding from Adlerbertska Forskningsstiftelsen, Helge Ax:son Johnsons Stiftelse and Wilhelm och Martina Lundgrens Vetenskapsfond to BW and JG

Proceedings from the Second Haploidentical Stem Cell Transplantation Symposium-Haplo2014, San Francisco, California, December 4, 2014

Significant progress has been made over the past decade in haploidentical transplantation, with the development of novel methods to control intense alloreactive reactions generated in the major HLA-mismatched setting. Application of post-transplantation cyclophosphamide has gained worldwide acceptance as an effective and low-cost way to perform this type of transplantation, with outcomes now similar to those from HLA-matched unrelated donors. These advances have resulted in improved treatment-related mortality, whereas disease relapse has emerged as the most common cause of treatment failure. In addition, improvements in immunologic reconstitution after transplantation are much needed, not only in haploidentical transplantation but in all forms of stem cell transplantation. This symposium has focused on some of the most promising methods to control alloreactivity in this form of transplantation and application of cellular therapy to prevent disease relapse after transplantation, as well as understanding immunologic reconstitution and foreseeable approaches to improve immune recovery after transplantation

Risk Factors for Bronchiolitis Obliterans Syndrome after Initial Detection of Pulmonary Impairment after Hematopoietic Cell Transplantation

Pulmonary chronic graft-versus-host-disease (cGVHD), or bronchiolitis obliterans syndrome (BOS), is a highly morbid complication of hematopoietic cell transplantation (HCT). The clinical significance of a single instance of pulmonary decline not meeting the criteria for BOS is unclear. We conducted a retrospective analysis in a cohort of patients who had an initial post-HCT decline in the absolute value of forced expiratory volume in 1 second (FEV1) of ≥10% or mid-expiratory flow rate of ≥25% but not meeting the criteria for BOS (pre-BOS). We examined the impact of clinical variables in patients with pre-BOS on the risk for subsequent BOS. Pre-BOS developed in 1325 of 3170 patients (42%), of whom 72 (5%) later developed BOS. Eighty-four patients developed BOS without detection of pre-BOS by routine screening. Among patients with pre-BOS, after adjusting for other significant variables, airflow obstruction (hazard ratio [HR], 2.0; 95% confidence interval [CI], 1.1 to 3.7; P = .02), percent-predicted FEV1 on decline (HR, .98; 95% CI, .97 to 1.0; P = .02), active cGVHD (HR, 7.7; 95% CI, 3.1 to 19.3; P \u3c .001), peripheral blood stem cell source (HR, 3.8; 95% CI, 1.7 to 8.6; P = .001), and myeloablative conditioning (HR, 2.0; 95% CI, 1.1 to 3.5; P = .02) were associated with subsequent BOS. The absence of airflow obstruction and cGVHD had a negative predictive value of 100% at 6 months for subsequent BOS, but the positive predictive value of both factors was low (cGVHD, 3%; any obstruction, 4%; combined, 6%). Several clinical factors at the time of pre-BOS, particularly active cGVHD and airflow obstruction, increase the risk for subsequent BOS. These factors merit consideration to be included in screening practices to improve the detection of BOS, with the caveat that the predictive utility of these factors is limited by the overall low incidence of BOS among patients with pre-BOS

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

AbstractDetection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT

Deletion of the Short Arm of Chromosome 1 (del 1p) is a Strong Predictor of Poor Outcome in Myeloma Patients Undergoing an Autotransplant

AbstractSeveral chromosomal abnormalities detected by conventional cytogenetic analysis have an adverse impact on the outcome in myeloma patients. A wide spectrum of abnormalities involving chromosomes 1, 13, 14, and 17 has been described. We analyzed the outcome of 83 patients with clonal cytogenetic abnormalities, who underwent high-dose therapy and autologous stem cell transplantation for multiple myeloma at our institution. Clonal abnormalities were detected at diagnosis by conventional cytogenetic analysis in 83 patients. Patients underwent a single autologous transplant between April 2000 and May 2005. Preparative regimen was high-dose melphalan alone (73), or a combination of topotecan, melphalan, and cyclophosphamide (TMC = 10). The most commonly observed chromosomal abnormalities were deletion of chromosome 13 (32%), hyperdiploidy (21%), deletion of chromosome 1p (18%), and t (11; 14) in 7% patients. Median follow-up among surviving patients was 25.5 months. Median interval from diagnosis to autotransplant was 7.7 months (range: 2.5-52). Median progression-free survival (PFS) for the entire group was 19 months and the median overall survival (OS) was 52 months. On univariate analysis, both PFS and OS were significantly shorter in patients with deletion 1p (P = .001 and <.0001, respectively). Thirty-two patients whose cytogenetic abnormalities returned to normal prior to autotransplant had longer PFS and OS than patients with persistent abnormalities (P = .02 and .08, respectively). Deletion 1p is associated with a significantly shorter remission and survival in patients undergoing high-dose therapy and a single autologous transplant for myeloma

Clinical Relevance of Myc/BCL2 Expression and Cell of Origin in Patients With Diffuse Large B-Cell Lymphoma Treated With Autologous Transplant

Dual expression of MYC and BCL2 proteins (double-expressor lymphoma [DEL]) as well as cell of origin (COO) are important prognostic factors in patients with diffuse large B-cell lymphoma (DLBCL) after conventional chemotherapy. We studied the prognostic impact of DEL and COO in patients with relapsed DLBCL treated with autologous stem cell transplant (ASCT). Three-hundred and three patients with stored tissue samples were identified. Classification was successful in 267 patients: 161 (60%) were DEL/non-double hit (DHL), 98 (37%) were non-DEL/non-DHL, and 8 (3%) were DEL/DHL. Compared to non-DEL/non-DHL, DEL/DHL had worse overall survival while DEL/non-DHL did not significantly differ in overall survival. On multivariable analysis, DEL/DHL, age \u3e60 years, and \u3e2 prior therapies, but not COO, were important prognostic factors for overall survival. When we explored the interaction of COO and BCL2 expression, patients with germinal center B-cell (GCB)/BCL2 (+) had inferior progression-free survival (PFS) compared to GCB/BCL2 (-) patients (HR, 4.97; P = 0.027). We conclude that the DEL/non-DHL and non-DEL/non-DHL subtypes of DLBCL have similar survival after ASCT. The negative impact of GCB/BCL2 (+) on PFS warrants future trials targeting BCL2 after ASCT. The inferior outcomes in DEL/DHL need to be verified in a larger number of patients

A Nasal Inflammatory Cytokine Signature Is Associated with Early Graft-versus-Host Disease of the Lung after Allogeneic Hematopoietic Cell Transplantation: Proof of Concept

Respiratory inflammation in bronchiolitis obliterans syndrome (BOS) after hematopoietic cell transplantation (HCT) is poorly understood. Clinical criteria for early-stage BOS (stage 0p) often capture HCT recipients without BOS. Measuring respiratory tract inflammation may help identify BOS, particularly early BOS. We conducted a prospective observational study in HCT recipients with new-onset BOS (n = 14), BOS stage 0p (n = 10), and recipients without lung impairment with (n = 3) or without (n = 8) chronic graft-versus-host disease and measured nasal inflammation using nasosorption at enrollment and then every 3 mo for 1 y. We divided BOS stage 0p into impairment that did not return to baseline values (preBOS, n = 6), or transient impairment (n = 4). We tested eluted nasal mucosal lining fluid from nasosorption matrices for inflammatory chemokines and cytokines using multiplex magnetic bead immunoassays. We analyzed between-group differences using the Kruskal-Wallis method, adjusting for multiple comparisons. We found increased nasal inflammation in preBOS and therefore directly compared patients with preBOS to those with transient impairment, as this would be of greatest diagnostic relevance. After adjusting for multiple corrections, we found significant increases in growth factors (FGF2, TGF-α, GM-CSF, VEGF), macrophage activation (CCL4, TNF-α, IL-6), neutrophil activation (CXCL2, IL-8), T cell activation (CD40 ligand, IL-2, IL-12p70, IL-15), type 2 inflammation (eotaxin, IL-4, IL-13), type 17 inflammation (IL-17A), dendritic maturation (FLT3 ligand, IL-7), and counterregulatory molecules (PD-L1, IL-1 receptor antagonist, IL-10) in preBOS patients compared to transient impairment. These differences waned over time. In conclusion, a transient multifaceted nasal inflammatory response is associated with preBOS. Our findings require validation in larger longitudinal cohorts