Anti-TRBC1 antibody-based flow cytometric detection of T-cell clonality: standardization of sample preparation and diagnostic implementation

Simple Summary The anti-TRBC1 antibody JOVI-1 has recently been identified as a flow cytometry marker potentially useful for assessment of T-cell clonality. The aim of this study was to optimize a flow cytometric method for routine use of anti-TRBC1 to assess T-cell clonality and validate it in a large series of normal and pathological samples. Our results showed that the best resolution to accurately identify TRBC1(+) cells was achieved by adding the CD3 antibody either simultaneously or after TRBC1. In addition, TRBC1(+)/TRBC1(-) ratios within different T alpha beta-cell subsets are provided as expected reference ranges for polyclonal T-cells. Based on the optimized approach here proposed, we detected monoclonal T alpha beta-cell populations with high specificity (96%) and a high analytical sensitivity/level of detection (<= 10(-4)), when clonal T-cells exhibited immunophenotypic aberrancies. These findings further support and extend previous observations about the utility of TRBC1 for the diagnostic screening and monitoring of clonal T alpha beta-cell populations.A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor beta-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess T alpha beta-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal T alpha beta-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1(+) and TRBC1(-) monoclonal and polyclonal T alpha beta-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1(+)/TRBC1(-) ratios within different T alpha beta-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRV beta families, whereas highly-variable TRBC1(+)/TRBC1(-) ratios were observed in more mature vs. naive T alpha beta-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal T alpha beta-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal T alpha beta-cells of <= 10(-4) in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature T alpha beta(+) leukemia/lymphoma patients.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Correlation of gene expression and protein production rate - a system wide study

<p>Abstract</p> <p>Background</p> <p>Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on <it>Saccharomyces cerevisiae </it>chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.</p> <p>Results</p> <p>We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of <it>T. reesei</it>. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.</p> <p>Conclusions</p> <p>Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).</p

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in T alpha beta-Large Granular Lymphocytic Leukemia

Simple Summary: TRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing T alpha beta-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal T alpha beta large granular lymphocytes (T alpha beta-LGL) show broader TRBC1(+)/TRBC1(-) ratios vs. total T alpha beta cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1(+) T alpha beta cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in T alpha beta CD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRV beta families is required.& nbsp;Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor beta chain (TRBC1) expression for assessing T alpha beta-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature T alpha beta cells (i.e., T-LGL normal-counterpart) show broader TRBC1(+)/TRBC1(-) ratios vs. total T alpha beta cells. We compared the distribution and absolute counts of TRBC1(+) and TRBC1(-) T alpha beta-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1(+) or TRBC1(-) T alpha beta-LGL ranged between 0.36 and 571 cells/mu L (3.2-91% TRBC1(+) cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/mu L (96% TRBC1(+) cells). Among the distinct TCRV beta families, the CD28(-) effector-memory and terminal-effector polyclonal T alpha beta cells ranged between 0 and 25 TRBC1(+) or TRBC1(-) cells/mu L and between 0 and 100% TRBC1(+) cells, while clonal LGL ranged between 32 and 5515 TRBC1(+) or TRBC1(-) cells/mu L, representing 98% TRBC1(+) cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of T alpha beta-LGL suspected of T-LGLL based on altered percentages of TRBC1(+) T alpha beta cells. However, in the absence of lymphocytosis or in the case of T alpha beta CD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of T alpha beta-LGL-expressing individual TCRV beta families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.Stemcel biology/Regenerative medicine (incl. bloodtransfusion

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in T alpha beta-Large Granular Lymphocytic Leukemia

Simple Summary: TRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing T alpha beta-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal T alpha beta large granular lymphocytes (T alpha beta-LGL) show broader TRBC1(+)/TRBC1(-) ratios vs. total T alpha beta cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1(+) T alpha beta cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in T alpha beta CD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRV beta families is required.& nbsp;Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor beta chain (TRBC1) expression for assessing T alpha beta-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature T alpha beta cells (i.e., T-LGL normal-counterpart) show broader TRBC1(+)/TRBC1(-) ratios vs. total T alpha beta cells. We compared the distribution and absolute counts of TRBC1(+) and TRBC1(-) T alpha beta-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1(+) or TRBC1(-) T alpha beta-LGL ranged between 0.36 and 571 cells/mu L (3.2-91% TRBC1(+) cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/mu L (96% TRBC1(+) cells). Among the distinct TCRV beta families, the CD28(-) effector-memory and terminal-effector polyclonal T alpha beta cells ranged between 0 and 25 TRBC1(+) or TRBC1(-) cells/mu L and between 0 and 100% TRBC1(+) cells, while clonal LGL ranged between 32 and 5515 TRBC1(+) or TRBC1(-) cells/mu L, representing 98% TRBC1(+) cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of T alpha beta-LGL suspected of T-LGLL based on altered percentages of TRBC1(+) T alpha beta cells. However, in the absence of lymphocytosis or in the case of T alpha beta CD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of T alpha beta-LGL-expressing individual TCRV beta families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations