Hybrid Macromolecular Constructs as a Platform for Spectral Nanoprobes for Advanced Cellular Barcoding in Flow Cytometry.

Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers.

A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.

Frequent issues and lessons learned from EuroFlow QA.

EuroFlow Quality Assessment was designed to provide a feedback on the quality of the standardization effort in executing the EuroFlow protocols for sample preparation and instrument setup. It was first beta-tested by the members of the EuroFlow consortium internally (2010-2013) and opened to the external participants from 2015 onwards. The goal of participation in the EuroFlow QA is to evaluate whether the technical quality of the data generated by the laboratory is comparable to the data of the EuroFlow members and thus if a non-EuroFlow member participant can use the EuroFlow reference sample database for his own patient evaluation. Also it assesses whether data are sufficiently standardized for automated population gating and alarm notification. By spring 2018, a total 87 laboratories from 32 countries on five continents have registered for the EuroFlow QA program. We evaluated 163 results of 2015-2016 QA rounds, where we noted clear improvement in the score of first-time participants (median score of 91% correct) when they participated second time or later (median score of 94% correct, p = 0,017), which was comparable to EuroFlow member scores (median score of 97% correct). Among frequent mistakes, we found non-adherence to the EuroFlow protocols (improper reagent used), improper gating and some compensation issues. In summary, we show that EuroFlow QA has a positive impact on improvement of standardized data quality of non-member laboratories adhering to the EuroFlow standard operating procedures and reagent panels.

AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells.

AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1-/- mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.

CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs.

Naive lymphocytes continually enter and exit lymphoid organs in a recirculation process that is essential for immune surveillance. During immune responses, the egress process can be shut down transiently. When this occurs locally it increases lymphocyte numbers in the responding lymphoid organ; when it occurs systemically it can lead to immunosuppression as a result of the depletion of recirculating lymphocytes. Several mediators of the innate immune system are known to cause shutdown, including interferon alpha/beta (IFN-alpha/beta) and tumour necrosis factor, but the mechanism has been unclear. Here we show that treatment with the IFN-alpha/beta inducer polyinosine polycytidylic acid (hereafter 'poly(I:C)') inhibited egress by a mechanism that was partly lymphocyte-intrinsic. The transmembrane C-type lectin CD69 was rapidly induced and CD69-/- cells were poorly retained in lymphoid tissues after treatment with poly(I:C) or infection with lymphocytic choriomeningitis virus. Lymphocyte egress requires sphingosine 1-phosphate receptor-1 (S1P1), and IFN-alpha/beta was found to inhibit lymphocyte responsiveness to S1P. By contrast, CD69-/- cells retained S1P1 function after exposure to IFN-alpha/beta. In coexpression experiments, CD69 inhibited S1P1 chemotactic function and led to downmodulation of S1P1. In a reporter assay, S1P1 crosslinking led to co-crosslinking and activation of a CD69-CD3zeta chimaera. CD69 co-immunoprecipitated with S1P1 but not the related receptor, S1P3. These observations indicate that CD69 forms a complex with and negatively regulates S1P1 and that it functions downstream of IFN-alpha/beta, and possibly other activating stimuli, to promote lymphocyte retention in lymphoid organs.

LIME: a new membrane Raft-associated adaptor protein involved in CD4 and CD8 coreceptor signaling.

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.