Detection of the T cell antigens CD3 and CD4 on CD20⁺ lymphocytes by flow cytometry.

<p>The appearance of both CD3 (A) and CD4 antigens (B) on the surface of CD20⁺ lymphocytes is shown for one representative patient. Freshly isolated lymphocytes (left) were compared to lymphocytes analyzed after overnight (oN) storage of whole blood samples at 4°C (right). The statistically significant difference in the number of CD3-expressing CD20⁺ (CD3^lowCD20⁺) lymphocytes between two independent groups of donors (fresh <i>versus</i> oN/4°C) was determined by the two-sided Mann-Whitney-U-Test. Outliers are depicted as circles.</p

Numbers of CD3^lowCD20⁺ B cells are time- and temperature-dependent.

<p>The increase in the number of CD3^lowCD20⁺ B cells was time-dependent, but independent of storage conditions (4°C <i>versus</i> room temperature (RT) <i>versus</i> humidified atmosphere at 37°C, 5% CO₂). CD3^lowCD20⁺ B cells were detectable at earlier time points and more pronounced at 4°C storage compared to RT and 37°C incubation, respectively. Shown are the results of two independent experiments performed in the same patient.</p

CD3-Positive B Cells: A Storage-Dependent Phenomenon

<div><p>The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before <i>in vitro</i>/<i>ex vivo</i> testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after <i>ex vivo</i> storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that <i>ex vivo</i> cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the <i>in vivo</i> situation, it is suggested to minimize times of <i>ex vivo</i> blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.</p></div

Flotillin-2 associates with core proteins of the adherens junction.

<p>(A) MCF10A cells were grown confluent for three days and stained for flotillin-2, E-cadherin γ-catenin and desmoglein-3 using specific antibodies and fluorochrome coupled secondary antibodies. Scale bar: 20 µm. (B & C) Coimmunoprecipitation of adhesion proteins with flotillins. Flotillins were precipitated with antibodies against flotillin-2 or flotillin-1 from MCF10A cells grown confluent for 5 days (B) or 10 days (C). (D) Coimmunoprecipitation of flotillins with an antibody against γ-catenin. For each experiment, 750 µg total protein was used.</p

Detection of CD3 on B cell surfaces is a result of cell number- dependent T-B cell interactions.

<p>Semiquantitative RT-PCR for CD3 was performed using RNA isolated from 7×10⁵ MACS-purified CD20⁺ B cells and a CD3-expressing T cell line (TCL). (A) Endogenous expression of CD3 was only found in the TCL, which served as a positive control, but neither in CD3^-CD20⁺ nor in CD3^lowCD20⁺ B cells. β-actin was used as loading control. (B) The appearance of CD3^lowCD20⁺ B cells was demonstrated after co-culture of MACS-purified CD4⁺ T cells and CD20⁺ B cells in different ratios overnight (oN) at 4°C. Storage of CD20⁺ B cells alone under the same conditions was not associated with an induction of the CD3^lowCD20⁺ B cell population, pointing at the need for T cells. Furthermore, the quantity of CD3^lowCD20⁺ B cells was dependent on the number of CD4⁺ T cells, i.e. increasing T-B cell ratios caused elevated numbers of CD3^lowCD20⁺ B cells. Shown are the results of up to three independent experiments.</p

Mapping of the interaction domain of γ-catenin with flotillins.

<p>(A) Prediction of ARM domains in the amino acid sequence of human junction plakoglobin (SwissProt: NP_002221.1) with the SMART software. (B) Scheme of the generated γ-catenin constructs. NT = N-terminus, CT = C-terminus, FL = full length, ARM = Armadillo domains. (C) Indirect GST pulldown from MCF10A lysate with 5 µg of purified γ-catenin FL-GST (1), NT-GST (2), NT+ARM1-8-GST (3), ARM1-6-GST (5), ARM1-12-GST (4), ARM1-12+CT-GST (9) and CT-GST (10). (D) Indirect GST pulldown from MCF10A lysates with 5 µg of purified γ-catenin FL-GST (1), ARM1-6-GST (5), ARM6-12-GST (6), ARM8-12-GST (7), ARM1-12-GST (4), ARM6-8-GST (8). Control: GST (pGEX4T1). For each indirect pulldown, 1.25 mg of total protein was used. Asterisks mark purified full-length proteins used in the experiment.</p

Flotillin-2 knockdown increases raft association of E-cadherin and γ-catenin in MCF10A cells.

<p>(A) Lipid rafts were isolated using detergent extraction from MCF10A control and flotillin-2 knockdown (<i>sh</i>-F2-A/B) cells after 10 days of confluent growth. Fractions 1–12 were stained with antibodies against E-cadherin, γ-catenin, flotillin-2, flotillin-1, GAPDH and transferrin receptor (TfnR). Lipid raft fractions were detected with an HRP-coupled cholera toxin subunit B. (B–C) Densitometric quantification of E-cadherin (B) and γ-catenin (C) distribution in the fractions. The signals were normalized to fraction number 12. Data points represent the mean ± SD of three independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison test. *, p<0.05; **, p<0.01; ***, p<0.001.</p

CD3^lowCD20⁺ cells belong to the B lymphocyte compartment.

<p>Phenotyping of CD3^lowCD20⁺ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3^lowCD20⁺ cells to the population of B lymphocytes. B cells showing characteristic features of an activated (CD80, CD86, CD5) and memory (IgG) phenotype are elevated in CD3^lowCD20⁺ B cells compared to the CD3^-CD20⁺ B cell subset. Additionally, expression of the specific T cell markers αβ- and γδ-TCR were only marginally found on the cell surface of CD3^lowCD20⁺ and CD3^-CD20⁺ B cells, respectively, excluding that the CD3^lowCD20⁺ cells are T-B-cell doublets. Extreme values are illustrated as asterisks, outliers as circles. Shown are the results from at least 5 individual patients. Comparative statistical evaluation of different B cell subset regarding cell surface antigen expression was not performed due to the small sample size.</p

Localization of flotillin-2 and γ-catenin in various human cell lines.

<p>Staining of flotillin-2 (left column) and γ-catenin (middle column) with specific antibodies in human non-tumorigenic epithelial MCF10A cells, human breast adenocarcinoma MCF7 cells, human HaCaT keratinocytes, human cervical cancer HeLa cells and human epidermoid carcinoma A431 cells. Scale bar: 20 µm.</p

Localization of E-cadherin and γ-catenin is altered in flotillin-2 depleted MCF10A cells.

<p>(A) The expression level of various cell-cell contact proteins is not altered in MCF10A cells depleted of flotillin-1 (<i>sh</i>-F1-A/B) or flotillin-2 (<i>sh</i>-F2-A/B). Flotillins were stably knocked down in MCF10A cells by means of lentiviruses carrying respective <i>sh</i>RNAs. Expression of indicated proteins was studied in cell lysates by menas of Western blot. (B) MCF10A cells depleted of flotillin-1 (<i>sh</i>-F1-A) or flotillin-2 (<i>sh</i>-F2-A) were grown for 3 days on coverslips, fixed and stained with antibodies against flotillin-2, E-cadherin and γ-catenin. Scale bar: 20 µm. (C) and (D) Quantification of relative cell border distribution of E-cadherin and γ-catenin in MCF10A cells depleted of flotillin-1 (<i>sh</i>-F1-A/B) or flotillin-2 (<i>sh</i>-F2-A/B). Data points represent the mean ± SD of three independent experiments in which 20 cell-cell borders were measured. One-way ANOVA with Bonferroni’s multiple comparison test. *, p<0.05; **, p<0.01; ***, p<0.001.</p