Metabolomic identification of α-ketoglutaric acid elevation in pediatric chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is the most common cause for non-relapse mortality postallogeneic hematopoietic stem cell transplant (HSCT). However, there are no well-defined biomarkers for cGVHD or late acute GVHD (aGVHD). This study is a longitudinal evaluation of metabolomic patterns of cGVHD and late aGVHD in pediatric HSCT recipients. A quantitative analysis of plasma metabolites was performed on 222 evaluable pediatric subjects from the ABLE/PBMTC1202 study. We performed a risk-assignment analysis at day + 100 (D100) on subjects who later developed either cGVHD or late aGVHD after day 114 to non-cGVHD controls. A second analysis at diagnosis used fixed and mixed multiple regression to compare cGVHD at onset to time-matched non-cGVHD controls. A metabolomic biomarker was considered biologically relevant only if it met all 3 selection criteria: (1) P ≤ .05; (2) effect ratio of ≥1.3 or ≤0.75; and (3) receiver operator characteristic AUC ≥0.60. We found a consistent elevation in plasma α-ketoglutaric acid before (D100) and at the onset of cGVHD, not impacted by cGVHD severity, pubertal status, or previous aGVHD. In addition, late aGVHD had a unique metabolomic pattern at D100 compared with cGVHD. Additional metabolomic correlation patterns were seen with the clinical presentation of pulmonary, de novo, and progressive cGVHD. α-ketoglutaric acid emerged as the single most significant metabolite associated with cGVHD, both in the D100 risk-assignment and later diagnostic onset analysis. These distinctive metabolic patterns may lead to improved subclassification of cGVHD. Future validation of these exploratory results is needed. This trial was registered at www.clinicaltrials.gov as #NCT02067832.

Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

BACKGROUND: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large patient cohorts, we identified increased numbers of CD56brightPerforin- regulatory-like NK cells (NKreg-like) associated with cGvHD suppression. Thus, we hypothesized that NKreg-like cells may be a potential candidate for cGvHD cell therapy. AIM: To expand NKreg-like cells while maintaining regulatory phenotype and function. METHODS: Total NK cells were first expanded with IL-2, which was then combined with rapamycin, Transforming Growth Factor Beta 1 (TGF-ß1), NECA (Adenosine A2A receptor (A2AR) agonist), metformin, or dexamethasone, to prevent change in cell phenotype/function. The functional characteristics were evaluated via T cell suppression assays and the phenotype was measured using flow cytometry. The optimal expansion protocol was compared in terms of function and metabolism for three NK expansion media, and cells from cord vs. peripheral blood. Further, expanded NKreg-like cell gene expression was characterized using bulk RNA sequencing. Finally, NKreg-like cells were differentiated from CD34+ hematopoietic stem and progenitor cells (HSPCs) and compared in terms of proliferation and function. RESULTS: The expansion of total NK cells found the addition of TGF-ß1 and/or NECA with the pulsing of rapamycin in IL-2 containing media to prevent NKreg-like differentiation (up to 200-fold expansion). Expanded NKreg-like cells maintained a phenotype, transcriptome, and T cell suppression similar to freshly isolated NKreg-like cells. NKreg-like expansion was greatest in the Immunocult media (up to 300-fold), and NKreg-like cells from peripheral blood demonstrated significantly greater proliferation than cells isolated from cord blood (65-fold). The metabolic profile of NKreg-like and cytolytic NK cells appeared similar at baseline, though rapamycin induced a shift to oxidative over glycolytic metabolism. Further, we demonstrated that suppressive NKreg-like cells may alternatively be expanded from CD34+ cells isolated from cord blood, reaching an average 340-fold expansion. CONCLUSIONS: In conclusion, our studies have optimized two alternative expansion approaches for deriving functional NKreg-like cells. Additionally, evaluating the transcriptomic and metabolic characteristics provides useful information regarding NKreg-like cell function and differentiation. With further optimization and in vivo validation, we may work towards preparing these cells as a therapy for cGvHD.

CD56bright CD16- natural killer cells as an important regulatory mechanism in chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity after hematopoietic stem cell transplantation (HSCT). In large patient populations, we have shown a CD56bright natural killer (NK) population to strongly associate with a lack of cGvHD and we hypothesize that these cells function to suppress cGvHD. We aimed to isolate and define the characteristics of regulatory NK (NKreg) cells associated with suppression of cGvHD. Immunophenotypic evaluation of a large pediatric population found the CD56bright NK population associated with a lack of cGvHD to be perforin-, Granzyme B-, and CD335+. Transcriptome analysis of a small patient cohort of CD56bright compared to CD56dim NK cells found the NKreg cells to also overexpress Granzyme K, IL-7R, GPR183, RANK, GM-CSFR, TCF7, and IL23A. Further analysis of this CD56bright NKreg population found a subpopulation that overexpressed IRF1, and TNF. We also found that viable NKreg cells may be isolated by sorting on CD56+ and CD16- NK cells, and this population can suppress allogeneic CD4+ T cells, but not Treg cells or CD8+ T cells through a non-cytolytic, cell-cell contact dependent mechanism. Suppression was not reliant upon the NKp44, NKp46, or GPR183 receptors. Additionally, NKreg cells do not kill leukemic cells. Moreover, this is the first paper to clearly establish that a CD56brightCD3-CD16-perforin- NKreg population associates with a lack of cGvHD and has several unique characteristics, including the suppression of helper T-cell function in vitro. With further investigation we may decipher the mechanism of NKreg suppression and operationalize expansion of NKreg cells associated with cGvHD suppression.

Immune profile differences between chronic GVHD and late acute GVHD: results of the ABLE/PBMTC 1202 studies.

Human graft-versus-host disease (GVHD) biology beyond 3 months after hematopoietic stem cell transplantation (HSCT) is complex. The Applied Biomarker in Late Effects of Childhood Cancer study (ABLE/PBMTC1202, NCT02067832) evaluated the immune profiles in chronic GVHD (cGVHD) and late acute GVHD (L-aGVHD). Peripheral blood immune cell and plasma markers were analyzed at day 100 post-HSCT and correlated with GVHD diagnosed according to the National Institutes of Health consensus criteria (NIH-CC) for cGVHD. Of 302 children enrolled, 241 were evaluable as L-aGVHD, cGVHD, active L-aGVHD or cGVHD, and no cGVHD/L-aGVHD. Significant marker differences, adjusted for major clinical factors, were defined as meeting all 3 criteria: receiver-operating characteristic area under the curve ≥0.60, P ≤ .05, and effect ratio ≥1.3 or ≤0.75. Patients with only distinctive features but determined as cGVHD by the adjudication committee (non-NIH-CC) had immune profiles similar to NIH-CC. Both cGVHD and L-aGVHD had decreased transitional B cells and increased cytolytic natural killer (NK) cells. cGVHD had additional abnormalities, with increased activated T cells, naive helper T (Th) and cytotoxic T cells, loss of CD56bright regulatory NK cells, and increased ST2 and soluble CD13. Active L-aGVHD before day 114 had additional abnormalities in naive Th, naive regulatory T (Treg) cell populations, and cytokines, and active cGVHD had an increase in PD-1- and a decrease in PD-1+ memory Treg cells. Unsupervised analysis appeared to show a progression of immune abnormalities from no cGVHD/L-aGVHD to L-aGVHD, with the most complex pattern in cGVHD. Comprehensive immune profiling will allow us to better understand how to minimize L-aGVHD and cGVHD. Further confirmation in adult and pediatric cohorts is needed.

Circulating cell-free mitochondrial DNA as a diagnostic and prognostic biomarker in chronic and late acute graft-versus-host disease in children.

In an earlier study, we found that mitochondrial DNA (mtDNA) concentration is elevated in adults with chronic graft-versus-host disease (cGvHD), acting as an endogenous source of TLR9 agonists to augment B-cell responses. To validate this in children, we evaluated mtDNA plasma expression in a large pediatric cohort (ABLE/PBMTC 1202 study). Plasma cell-free mtDNA (cf-mtDNA) copy numbers were measured in 202 pediatric patients using quantitative Droplet Digital polymerase chain reaction (ddPCR). Two evaluations were performed: 1) before the onset of cGvHD or late acute graft-versus-host disease (aGvHD) at day 100 ± 14 days and 2) at the time of cGvHD onset compared with time-matched non-cGvHD controls. We found that cf-mtDNA copy numbers were not affected by immune reconstitution post-hematopoietic stem cell transplantation but were higher on day 100 before the onset of late aGvHD and at the onset of cGvHD. We found that cf-mtDNA was not impacted by previous aGvHD, but correlated with the early onset, NIH moderate/severe cGvHD, and did not correlate with other immune cell populations, cytokines, or chemokines but did with the metabolites spermine and taurine. Similar to adults, children have elevated plasma cf-mtDNA concentrations at the early onset of cGvHD, especially in NIH moderate/severe cGvHD, elevation with late aGvHD, and associated with metabolites involved in mitochondrial function.

A diagnostic classifier for pediatric chronic graft-versus-host disease: results of the ABLE/PBMTC 1202 study.

The National Institutes of Health Consensus criteria for chronic graft-versus-host disease (cGVHD) diagnosis can be challenging to apply in children, making pediatric cGVHD diagnosis difficult. We aimed to identify diagnostic pediatric cGVHD biomarkers that would complement the current clinical criteria and help differentiate cGVHD from non-cGVHD. The Applied Biomarkers of Late Effects of Childhood Cancer (ABLE) study, open at 27 transplant centers, prospectively evaluated 302 pediatric patients after hematopoietic cell transplant (234 evaluable). Forty-four patients developed cGVHD. Mixed and fixed effect regression analyses were performed on diagnostic cGVHD onset blood samples for cellular and plasma biomarkers, with individual markers declared relevant if they met 3 criteria: an effect ratio ≥1.3 or ≤0.75; an area under the curve (AUC) of ≥0.60; and a P value <5.814 × 10-4 (Bonferroni correction) (mixed effect) or <.05 (fixed effect). To address the complexity of cGVHD diagnosis in children, we built a machine learning-based classifier that combined multiple cellular and plasma biomarkers with clinical factors. Decreases in regulatory natural killer cells, naïve CD4 T helper cells, and naïve regulatory T cells, and elevated levels of CXCL9, CXCL10, CXCL11, ST2, ICAM-1, and soluble CD13 (sCD13) characterize the onset of cGVHD. Evaluation of the time dependence revealed that sCD13, ST2, and ICAM-1 levels varied with the timing of cGVHD onset. The cGVHD diagnostic classifier achieved an AUC of 0.89, with a positive predictive value of 82% and a negative predictive value of 80% for diagnosing cGVHD. Our polyomic approach to building a diagnostic classifier could help improve the diagnosis of cGVHD in children but requires validation in future prospective studies. This trial was registered at www.clinicaltrials.gov as #NCT02067832.

Chordoma and chondrosarcoma gene profile: implications for immunotherapy.

Chordoma and chondrosarcoma are malignant bone tumors characterized by the abundant production of extracellular matrix. The resistance of these tumors to conventional therapeutic modalities has prompted us to delineate the gene expression profile of these two tumor types, with the expectation to identify potential molecular therapeutic targets. Furthermore the transcriptional profile of chordomas and chrondrosarcomas was compared to a wide variety of sarcomas as well as to that of normal tissues of similar lineage, to determine whether they express unique gene signatures among other tumors of mesenchymal origin, and to identify changes associated with malignant transformation. A HG-U133A Affymetrix Chip platform was used to determine the gene expression signature in 6 chordoma and 14 chondrosarcoma lesions. Validation of selected genes was performed by qPCR and immunohistochemistry (IHC) on an extended subset of tumors. By unsupervised clustering, chordoma and chondrosarcoma tumors grouped together in a genomic cluster distinct from that of other sarcoma types. They shared overexpression of many extracellular matrix genes including aggrecan, type II & X collagen, fibronectin, matrillin 3, high molecular weight-melanoma associated antigen (HMW-MAA), matrix metalloproteinase MMP-9, and MMP-19. In contrast, T Brachyury and CD24 were selectively expressed in chordomas, as were Keratin 8,13,15,18 and 19. Chondrosarcomas are distinguished by high expression of type IX and XI collagen. Because of its potential usefulness as a target for immunotherapy, the expression of HMW-MAA was analyzed by IHC and was detected in 62% of chordomas and 48% of chondrosarcomas, respectively. Furthermore, western blotting analysis showed that HMW-MAA synthesized by chordoma cell lines has a structure similar to that of the antigen synthesized by melanoma cells. In conclusion, chordomas and chondrosarcomas share a similar gene expression profile of up-regulated extracellular matrix genes. HMW-MAA represents a potential useful target to apply immunotherapy to these tumors.

Asp(344) and Thr(345) are critical for cation exchange mediated by NhaD, Na(+)/H(+) antiporter of Vibrio cholerae.

The Vc-NhaD is an Na(+)/H(+) antiporter from Vibrio cholerae belonging to a new family of bacterial Na(+)/H(+) antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu(100)Ala, Glu(251)Ala, Glu(342)Ala, and Asp(393)Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp(344)Ala, Asp(344)Asn, and Thr(345)Ala caused a complete loss of both Na(+)/H(+) and Li(+)/H(+) antiport activity whereas the Asp(344)Glu variant exhibited reduced Na(+)/H(+) and Li(+)/H(+) antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na(+)/H(+) antiport. We discuss the possible role of Asp(344) and Thr(345) in the functioning of Vc-NhaD.

Identifying mechanisms for therapeutic intervention in chordoma: c-Met oncoprotein.

STUDY DESIGN: A human sacral chordoma cell line, CCL3, was established and in vitro characterization of c-Met oncoprotein in chordoma cells was performed. OBJECTIVE: Determination of whether c-Met plays an important role in chordoma's malignancy. SUMMARY OF BACKGROUND DATA: Chordomas are malignant life-threatening tumors that arise from the remnants of the notochord. c-Met is an oncoprotein that is expressed by a variety of solid tumors, including chordomas, and HGF is its high affinity ligand. In the present study, we investigated c-Met and HGF expression, localization, and function in human chordoma cells. METHODS: SDS-PAGE, Western blotting, immunofluorescence techniques, and cell migration functional assays were used to asses c-Met and HGF expression, localization, and functional activity. RESULTS: Intracellular protein tyrosine phosphorylation was enhanced on HGF binding, and an increase in the amount of 50 kDa alpha-chain of c-Met was detected in HGF-stimulated cells. Immunostaining of c-Met and HGF revealed membrane/cytoplasmic localization in nonstimulated cells, and perinuclear colocalization in HGF-stimulated cells. Positive chemotactic and migration activity in response to HGF was also demonstrated. CONCLUSION: Our data supports our hypothesis that the c-Met oncoprotein plays a leading role in the metastatic process in chordomas, and that a c-Met-HGF pair is involved in chordoma malignancy. Taking into consideration the very limited treatment options and an extremely poor prognosis for the chordoma patients, our results are a valuable and promising addition to the current situation in managing chordomas.

The role of extracellular factors in human metastatic chordoma cell growth in vitro.

STUDY DESIGN: Human metastatic chordoma cells were isolated, and initial in vitro characterization was performed. Biochemical and physiologic changes were observed in response to pH, oxygen, and glucose. OBJECTIVE: The extracellular microenvironment directly affects metastatic chordoma cell phenotype in vitro. SUMMARY OF BACKGROUND DATA: Chordomas are primary bone tumors that usually occur in the spine or skull. Chordomas arise from embryonic notochordal remnants along the axial skeleton, most commonly the sacrum, followed by the base of the skull and the mobile spine. Due to a high degree of resistance to radiation and chemotherapy, chordomas eventually cause death by direct growth or by metastasizing to other organs. METHODS: Extracellular pH, oxygen, and glucose levels in the culture medium were controlled, and cell response was assessed using MTT staining, SDS-PAGE, Western blotting, tandem mass spectrometry, TUNEL, immunofluorescence, and flow cytometry. RESULTS: In this study, we present a new chordoma cell line established from metastatic tissue and novel data characterizing some aspects of chordoma cell phenotype in different conditions in vitro. Chordoma biologic markers were expressed in the new cell line. Alkaline pH dramatically increased intracellular protein tyrosine phosphorylation, metabolic activity, and albumin accumulation in the cells, while acidic pH caused apoptosis. CONCLUSION: The level of proliferation, apoptosis, and tyrosine phosphorylation, as well as the overall protein expression profile, strongly depended on extracellular media pH and oxygen/glucose levels. Chordoma's preferred extracellular microenvironment in vitro was rather alkaline, with an optimum at pH 8.5, and apoptotic changes were induced at acidic pH. We found that bovine serum albumin was accumulated by chordoma cells from the incubation media, and this accumulation depended on extracellular pH, with the highest accumulation at alkaline pH.

Cloning, functional expression in Escherichia coli and primary characterization of a new Na+/H+ antiporter, NhaD, of Vibrio cholerae.

Vibrio cholerae is the infectious agent of the deadly diarrheal disease, cholera. Na+ ion homeostasis is believed to play a key role in both physiology and pathogenicity of this bacterium. However, molecular mechanisms of sodium exchange in V. cholerae are still poorly understood. In the present work a gene encoding an unusual Na+/H+ antiporter, nhaD, was identified in the V. cholerae genome. nhaD was cloned from Vibrio cholerae and expressed in Escherichia coli. The antiporter functioned in an E. coli nhaAnhaB mutant strain to confer resistance to LiCl and NaCl. When assayed in inside-out subbacterial vesicles, V. cholerae NhaD demonstrated high affinity for Na+ ions (1.1 mM Na+ was required for the half-maximal response at the pH-optimum). The most striking feature of Vc-NhaD is a unique pH-profile of its activity with a sharp maximum at pH 8.0, different from that of any bacterial sodium-proton antiporter described so far. The difference is rationalized as being the result of a His to Arg substitution in a putative pH sensing residue.