Immunoglobulin and T Cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-Transplantation Relapse and Survival

AbstractMinimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10−4 after induction predicts subsequent relapse. Likewise, MRD ≥ 10−4 in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL. Consensus-primed immunoglobulin (Ig), T cell receptor (TCR) amplification and high-throughput sequencing (HTS) permit use of a standardized algorithm for all patients and can detect leukemia at 10−6 or lower. We applied the LymphoSIGHT HTS platform (Sequenta Inc., South San Francisco, CA) to quantification of MRD in 237 samples from 29 adult B cell ALL patients before and after allo-HCT. Using primers for the IGH-VDJ, IGH-DJ, IGK, TCRB, TCRD, and TCRG loci, MRD could be quantified in 93% of patients. Leukemia-associated clonotypes at these loci were identified in 52%, 28%, 10%, 35%, 28%, and 41% of patients, respectively. MRD ≥ 10−4 before HCT conditioning predicted post-HCT relapse (hazard ratio [HR], 7.7; 95% confidence interval [CI], 2.0 to 30; P = .003). In post-HCT blood samples, MRD ≥10−6 had 100% positive predictive value for relapse with median lead time of 89 days (HR, 14; 95% CI, 4.7 to 44, P < .0001). The use of HTS-based MRD quantification in adults with ALL offers a standardized approach with sufficient sensitivity to quantify leukemia MRD in peripheral blood. Use of this approach may identify a window for clinical intervention before overt relapse

The Slow-Releasing Hydrogen Sulfide Donor, GYY4137, Exhibits Novel Anti-Cancer Effects In Vitro and In Vivo

The slow-releasing hydrogen sulfide (H2S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H2S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H2S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H2S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G2/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H2S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H2S donors should be investigated further as potential anti-cancer agents

Use of estradiol-BSA conjugates to characterize membrane estrogen binding sites in brain cells: Biochemical and physiological studies

Rapid nongenomic actions of estrogen probably involve one or several membrane estrogen receptors/binding proteins, which are different from classical estrogen receptor (ER). Specific membrane binding sites for estrogen (mEBS) in female rat brain were demonstrated by a novel and sensitive radioligand binding assay using 17$\beta$-estradiol covalently linked with (\sp{125}I) -labeled bovine serum albumin (17$\beta$-E-6- (\sp{125}I) BSA conjugate), as a ligand. These membrane binding sites (dissociation constants of 3-34 nM and total binding sites of 3-40 pmol/mg protein) in crude synaptosomal fractions (P2) from hypothalamus, olfactory bulb, and cerebellum show distinct binding specificity from classical ER. To establish if the mEBS identified by 17$\beta$-E-6- (\sp{125}I) BSA represent putative membrane estrogen binding proteins, a ligand blotting technique was used. Three major binding proteins with molecular weights of about 23, 28, and 32 kDa, and three additional proteins (18, 40, and 130 kDa), were specifically labeled. The 23 and 40 kDa proteins were concentrated in mitochondrial fractions (mP2) whereas 18, 28, and 32 kDa proteins were enriched in microsomal fractions (P3). One of these membrane estrogen binding proteins (23 kDa) was purified from digitonin-solubilized P2 fractions by affinity columns coupled with 17$\beta$-E-6-BSA and was identified as the oligomycin-sensitivity conferring protein (OSCP), a subunit of the F\sb0F\sb1 ATP-synthase/ATPase required for the coupling of a proton gradient across the F\sb0 sector of the mitochondrial membrane to ATP synthesis in the F\sb1 sector. OSCP, therefore, represents an estrogen target site in addition to ER in the cells and may play an important role in estrogen-induced changes in cell energy metabolism. The unlabeled ligand, 17$\beta$-E-6-BSA (1-100 nM), was biologically active in rapidly facilitating the release of dopamine from corpus striatum, an important estrogen target site that lacks classical ER. This rapid action of estrogen is consistent with the presence of mEBS in the plasma membranes of these cells and future work is ongoing to purify this putative mEBS and/or clone its cDNA.U of I OnlyETDs are only available to UIUC Users without author permissio

Combining Next-Generation Sequencing and Immune Assays: A Novel Method for Identification of Antigen-Specific T Cells

<div><p>In this study, we combined a novel sequencing method, which can identify individual clonotypes based on their unique T cell receptor (TCR) rearrangement, with existing immune assays to characterize antigen-specific T cell responses. We validated this approach using three types of assays routinely used to measure antigen-specific responses: pentamers which enable identification of T cells bearing specific TCRs, activation marker expression following antigen stimulation and antigen-induced proliferation to identify cytomegalovirus (CMV) specific clonotypes. In one individual, 8 clonotypes were identified using a pentamer reagent derived from the CMV pp65 protein. The same 8 clonotypes were also identified following sequencing of cells that upregulated an activation marker following incubation with an identical peptide derived from pp65. These 8 and an additional 8 clonotypes were identified using a more sensitive CFSE-based proliferation assay. We found clear sequence homology among some of the clonotypes identified, and the CDR3 region in one clonotype was identical to a previously published pp65-specific clonotype sequence. Many of these CMV-specific clonotypes were present at frequencies below 10⁻⁵ which are undetectable using standard flow-cytometric methods. These studies suggest that an immune response is comprised of a diverse set of clones, many of which are present at very low frequencies. Thus, the combination of immune assays and sequencing depicts the richness and diversity of an immune response at a level that is not possible using standard immune assays alone. The methods articulated in this work provide an enhanced understanding of T cell-mediated immune responses at the clonal level.</p></div

miR-10a in Peripheral Blood Mononuclear Cells Is a Biomarker for Sepsis and Has Anti-Inflammatory Function

Background. Recent literature has reported the use of circulating microRNAs (miRNAs) as biomarkers for sepsis. Immune cells play an essential role in the pathophysiology of sepsis. The aim of this prospective study was to identify miRNAs in peripheral blood mononuclear cells (PBMC) that could differentiate between sepsis and infection based on Sepsis-3 definition. Methods. A total of 62 patients (41 with sepsis and 21 with infection suffering from pneumonia but without sepsis) and 20 healthy controls were enrolled into the study. PBMC at admission were examined for a panel of 4 miRNAs (miR-10a, miR-17, miR-27a, and miR-125b), which have been documented to participate in inflammatory response in immune cells, via qRT-PCR. Data were validated in a mouse model of sepsis induced via cecal ligation and puncture (CLP) and THP-1 monocytes. Results. miR-10a levels in PBMC at admission were significantly lower in sepsis patients compared with patients with infection and healthy controls. miR-10a levels were negatively correlated with disease severity scores as well as levels for c-reactive protein and procalcitonin. In addition, low miR-10a expression had a diagnostic value for sepsis and a prognostic value for 28-day mortality in receiving operating characteristic analysis. Compared with infection patients and healthy controls, PBMC from sepsis patients also had higher levels of mitogen-activated kinase kinase kinase 7 (MAP3K7), a known target protein of miR-10a and an activator of the NF-κB pathway. In the mouse model of CLP-induced sepsis, miR-10a levels in PBMC were significantly decreased as early as 8 h after CLP. Overexpression of miR-10a in THP-1 cells significantly reduced the expression of MAP3K7 and proinflammatory cytokines including IL-6, TNF-α, and MCP-1. Conclusions. PBMC miR-10a levels are decreased in sepsis and negatively correlated with the disease severity. Levels of miR-10a could distinguish between sepsis and infection and predict 28-day mortality. miR-10a plays an anti-inflammatory role in the pathogenesis of sepsis

Assay schema.

<p>Assay schema.</p

Identification of CMV pp65₄₉₅-specific T cell clonotypes from sorted responding cells following peptide incubation.

<p>Clonotype frequencies from sorted responding CD137⁺ cells following CMV pp65₄₉₅ peptide incubation versus either sorted non-responding CD137⁻ cells (<b>A</b>) or unsorted PBMCs (<b>B</b>). The 9 red dots in A indicate clonotypes greater than 10-fold enriched and exceeding a 20-cell equivalent minimum frequency threshold in the sorted (CD137⁺) population. Red dots in B indicate those clonotypes identified in A. Clonotypes identified in A are not enriched in sorted CD137⁺ cells versus CD137⁻ T cells (<b>C</b>) following incubation without peptide.</p

Overlap between clonotypes identified in pentamer and CD137 assays.

<p>(<b>A</b>) Plot shows clonotype frequencies of the 8 clonotypes (red) identified in the pentamer analyses (outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074231#pone-0074231-g002" target="_blank">Figure 2A</a>) in the clonotype profiles of CD137⁺ responding cells following CMV pp65₄₉₅ peptide incubation versus sorted non-responding CD137⁻ cells. (<b>B</b>) Plot shows clonotype frequencies of the 9 clonotypes (red) identified in the CD137 assay analyses (outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074231#pone-0074231-g003" target="_blank">Figure 3A</a>) in the clonotype profiles of sorted CMV pp65₄₉₅ pentamer⁺ cells versus pentamer⁻ cells. 8/9 of these clonotypes are overlapping with those identified in A.</p

Summary of CMV pp65₄₉₅-specific CD8⁺ T cell clonotypes identified in each immune assay.

<p>The table lists all 16 clonotype sequences and corresponding frequencies in each of the sorted populations for each of the four immune assays as well as clonotype frequencies in fresh unsorted PBMCs. Frequencies in bold font indicate the clonotypes that met the selection criteria for that assay. Frequencies in normal font did not meet the selection criteria for that assay.</p

Identification of CMV pp65₄₉₅-specific T cell clonotypes from sorted pentamer⁺ T cells.

<p>(<b>A</b>) Clonotype frequencies from CMV pp65₄₉₅ pentamer⁺ versus pentamer⁻ CD8⁺ T cells from a characterized CMV responder (outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074231#s2" target="_blank">Methods</a> section). The 8 red dots indicate clonotypes greater than 10-fold enriched and exceeding a 20-cell equivalent minimum frequency threshold in the sorted (pentamer⁺) population. (<b>B</b>) All 8 clonotypes identified in A are enriched in (unsorted) PBMCs from the same individual. Red dots indicate clonotypes identified in A.</p