Expression Of Certain Antigens Research Articles

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

Immunohistochemical Heterogeneity of the Endothelium of Blood and Lymphatic Vessels in the Developing Human Liver and in Adulthood

The endothelium of liver sinusoids in relation to the endothelium of other blood vessels has specific antigen expression similar to the endothelium of lymphatic vessels. Bearing in mind that there is no consensus as to the period or intensity of the expression of certain antigens in the endothelium of blood and lymphatic vessels in the liver, the aim of our study was to immunohistochemically investigate the dynamic patterns of the expression of CD31, CD34, D2-40, and LYVE-1 antigens during liver development and in adulthood on paraffin tissue sections of human livers of 4 embryos, 38 fetuses, 6 neonates, and 6 adults. The results show that, in a histologically immature liver at the end of the embryonic period, CD34 molecules are expressed only on vein endothelium localized in developing portal areas, whereby the difference between portal venous branches and CD34-negative central veins belongs to the collecting venous system. In the fetal period, with aging, expression of CD34 and CD31 molecules on the endothelium of central veins and blood vessels of the portal areas increases. Sinusoidal endothelium shows light and sporadic CD34 immunoreactivity in the late embryonic and fetal periods, and is lost in the neonatal and adult periods, unlike CD31 immunoreactivity, which is poorly expressed in the fetal and neonatal periods but is present in adults. The endothelium of sinusoids and lymphatic vessels express LYVE-1, and the endothelium of lymphatic vessels express LYVE-1 and D2-40 but not CD34. Similarity between the sinusoidal and lymphatic endothelium includes the fact that both types are LYVE-1 positive and CD34 negative.

Abstract 2811: Allogeneic cell cased vaccine development by shared antigen screening

Abstract The fundamental premise of allogeneic vaccines for cancer relies upon the presence of common antigens that are shared between established cell lines and a large proportion of patients with histologically related tumors. Recent results from The Cancer Genome Atlas have clearly demonstrated that somatic mutations in exon region DNA, while potentially immunogenic, do not result in the translation of antigenic proteins that are similar from patient to patient. Instead, these data reinforce the concept that dysregulation of common pathways contributes to the general property of de-differentiation in tumor cells and the acquisition of immature phenotypes as compared to untransformed precursor cells. This phenomenon leads to expression of mucinous and embryonal antigens, collectively known as cancer testis antigens, that are otherwise rare in mature tissues and to which central tolerance remains incomplete. We have developed allogeneic vaccines based on parallel screening of non-small cell lung, bladder, triple-negative breast, ovarian and melanoma tumor cell lines and patient tumor samples. The baseline expression of certain antigens, including SSX2, SSX4, CT10, MAGE-A1, MAGE-A10, LAGE-1 and NY-ESO-1 was found to vary widely both between tumor samples and non-transformed tissue controls. Among all antigens screened, MAGE-A3 was most commonly upregulated across transformed tissue samples, however other antigens including BAGE-1, XAGE-1b and MAGE-A10 were found to be most dramatically upregulated. This analysis provides a screening tool with which to appropriately profile and prioritize individual cell lines for vaccine development in histologically diverse tumor types. Citation Format: Vadim V. Deyev, Neal Schilling, Taylor H. Schreiber. Allogeneic cell cased vaccine development by shared antigen screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2811. doi:10.1158/1538-7445.AM2014-2811

Immunophenotypic features of acute myeloid leukemia with inv(3)(q21q26.2)/t(3;3)(q21;q26.2)

Immunophenotypic identification of myeloid specific antigens is an important diagnostic tool in the management of patients with acute myeloid leukemia (AML). These antigens allow determination of cell of origin and degree of differentiation of leukemia blasts. AML with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is a relatively rare subtype of AML. The immunophenotypic characteristics of inv(3) AML patients are somewhat limited. We identified 14 new cases of hematological disorders with increased myeloid blasts carrying inv(3)(q21q26.2)/t(3;3)(q21;q26.2). Also, we identified another 13 cases previously published in the literature, where the immunophenotype of inv(3)(q21q26.2) was documented. As a group, patients with AML with inv(3)(q21q26.2) had high levels of early myeloid (CD13, CD33, CD117 and MPO) and uncommitted markers (CD34, HLA-DR and CD56) and a high rate of monosomy 7 in addition to the inv(3)(q21q26.2). Differential karyotype and expression of certain antigens were noted in patients with de novo AML with inv(3)(q21q26.2) vs. those with inv(3)(q21q26.2)-containing blasts.

Differential stimulation of monocytic cells results in distinct populations of microparticles.

Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

Isotype controls in phenotyping and quantification of microparticles: A major source of error and how to evade it

Background The characterisation and quantification of cell-derived microparticles (MPs) using flow cytometry are often complicated by a low staining intensity and a non-discrete signal pattern of many cell surface antigens. Fluorescence-labelled isotype controls (ICs) are commonly used to set limits for the discrimination of antigen positive vs. negative events. Objectives The influence of different ICs on the characterisation and quantification of MPs was studied. Antigen negative MPs stained with an antibody of interest were evaluated as an alternative control. Methods MPs were prepared from platelets, endothelial cell lines and leucemic cell lines and stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Results are given as the mean fluorescence intensity (MFI) or percentage of “false-positive” events above a fluorescence intensity > 1. Results Using identical instrument settings, seven different ICs (FITC-conjugates N = 3, PE-conjugates N = 4) resulted in a wide range of MFI and percentage of positive events with a mean coefficient of variation (CV) of 0.77. Instead, NMPs showed less variability with a mean CV of 0.50 and allowed a reliable and reproducible quantification of MPs when set as controls with < 2% false-positive events above an FI > 1. As a result, the expression of certain antigens (e.g. CD62P) was lower compared to previous reports in the literature. Conclusions Diversity in the staining intensity of isotype controls is a potential source of error in the characterisation and quantification of MPs by flow cytometry. The use of antigen negative MPs to adjust instrument settings is suggested.

Endothelial Cell and Stromal Antigens in Human Periapical Granulation Tissue

Periapical granulation tissue consists of vasculature of varying sizes and types, infiltrating cells, and other stromal elements. We examined the differential expression of endothelial and stroma antigens in this tissue to determine their tissue distribution in order to obtain hints on their functions. Some of the antigens examined were present only in the endothelial lining of vasculature, including high endothelial venules (e.g. CD31 and CD105), whereas others were more widely expressed by both vascular and stromal elements (e.g. CD29, CD63, CD44, and CD151). Immunohistochemical analysis using monoclonal antibodies specific to certain tissue compartments revealed the tissue architecture more precisely and the expression of certain antigens in the tissue suggested special roles for these antigens. Tissue distribution of CD63, CD143, CD147, and CD151 in periapical granulation tissue is first reported in the present study.

Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: A study of five cases controlled by genetic methods

We recently investigated samples of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and normal bone marrow (BM). We found that leukemic blasts, compared to their physiologic counterpart cells, frequently display aberrant phenotypes with respect to levels of expression of certain antigens. Using multiparameter flow cytometry, these differences enabled us to trace leukemic cells admixed to normal BM, which suggested that this approach might be a useful strategy for minimal residual disease detection. In the present study, we used the same multiparameter approach (“comparative phenotype mapping”) to prove that such quantitative phenotypic differences really exist between malignant and normal BCP when simultaneously present in the BM. We demonstrate this by five exemplary follow-up BM samples from patients with BCP-ALL, all of which showed phenotypically aberrant cells according to levels of expression of CD10, CD11a, CD19, CD34, CD44, or CD45RA, as well as according to altered orthogonal light scattering properties. We confirmed the leukemic nature of these cells by polymerase chain reaction-based detection of bcr1/abl transcripts, and of leukemia clone-specific immunoglobulin heavy chain rearrangements in only the suspicious cells when sorted by flow cytometry, but not in normal BCP or non-B cells. Comparative phenotype mapping thus allows one to distinguish between normal and leukemic cells, and we show that it may enable rapid, specific, and quantitative detection of residual/resurgent leukemia in BCP-ALL.

Assessment of endothelial immunophenotype — limitation of flow cytometric analysis

Flow cytometry is generally utilized to quantify antigen expression by cells in suspension. To detect antigens on endothelium, which grows as a monolayer, either the creation of a suspension of endothelial cells for flow cytometry, or the use of alternative techniques (such as immunoperoxidase staining) is required. We demonstrate here that creating suspensions of endothelial cells for flow cytometry underestimates the expression of certain antigens. In addition, morphological information regarding certain antigens (serpins and fibronectin) is only discernible by immune microscopy, a subjective procedure. We would recommend caution in using flow cytometry for the estimation of endothelial antigens. Using computerized estimates of microscopic immunostaining (e.g., with video image analysis) it may be possible to overcome some of the subjective limitations of immune microscopy.

Immunophenotypic differences between normal glia, astrocytomas and malignant gliomas: correlations with karyotype, natural history and survival

The karyotypic and antigenic phenotypes of early passage normal and malignant glial cultures were correlated in vitro. Astrocytomas (4) were distinguished from the normal glia (8) by a mixed near-diploid karyotype and anchorage-independent growth. Malignant gliomas (41) demonstrated cytogenetic abnormalities ranging from mixed normal G- and Q-banded and near-diploid culture, through mixed near-diploid/hyperdiploid to predominantly hyperdiploid stem-lines. This correlated with the differential expression of certain antigens and established qualitative antigenic differences from normal glia.Associations were found between histopathologic grade of glial neoplasm and the expression of antigens 5.1H11 ( p = 0.0002), CNT/11 ( p = 0.001), CNT/10 ( p = 0.004), CAT301 ( p = 0.014), M111 ( p = 0.024), and L101 ( p = 0.044). An ominous association was demonstrated between the duration of clinical survival and the expression of antigens 5.1H11 ( p = 0.0007), CNT/10 ( p = 0.027) and B2.6 ( p = 0.038). Correcting for diagnosis and age, multivariate analysis demonstrated that HLA-DR ( p = 0.050) and 5.1H11 ( p = 0.069) were unfavorably correlated with patient survival. This suggests the application of the in vitro immunophenotype for its predictive utility, as well as a novel method of selection of tumor-associated antigens for monoclonal antibody-mediated immunotherapy.

Cell membrane perturbation of resting T cells and thymocytes causes display of activation antigens.

Three human lymphocyte antigens recognized by monoclonal antibodies OKIa1, OKT9, and OKT10 were found to be minimally represented on resting peripheral T cells (all three) and thymocytes (OKIa1 and OKT9). These antigens, which are present on "activated" T cells, were promptly displayed on "resting" T cells or thymocytes following cross-linking of surface-bound monoclonal antibody by horse alpha-mouse IgG. These experiments suggested that membrane perturbations may induce the expression of certain antigens that are normally present in an unexpressed form in resting cells.