TRPM2 channels do not impact survival of activated CD4⁺ T cells under altered redix conditions.

<p>Suvival of activated bead-stimulated CD4⁺ T cells (bead:cell ratio of 1∶4) from wildtype and trpm2-deficient mice under various concentrations of H₂O₂ (0.001–10 mM) using the amount of ATP relased following cell lysis as a parameter of cell viability (n = 3; ns  =  not significant).</p

TRPM2 cation channels critically determine proliferation and proinflammatory cytokine secretion following various strengths of polyclonal T cell receptor triggering.

<p>(<b>A–D</b>) Proliferation as determined by ³[H]-thymidine uptake (<b>A</b>; bead:cell ratio of 1∶4) and secretion of proinflammatory IL-2 (<b>B</b>), IFN-γ (<b>C</b>) and IL-17 (<b>D</b>) determined by ELISA from the supernatants were significantly reduced in spleenocytes from trpm2-deficient as compared to wildtype mice following anti-CD3/CD28 bead-stimulation for 3 days at various bead:cell ratios (1∶1 to 1∶16; n = 3 for all experiments). P-values ≤0.05 were considered significant (*). P-values ≤0.01 and ≤0.001 were considered highly significant (** and ***, respectively).</p

Average SOA_max (ms) for the control group (N = 8) and the patients (N = 6), depending on the eye to which the target was presented.

<p>The mask was always presented to the opposite eye. Error bars depict the 95% confidence interval of the mean. Each connected pair of circles represents SOA_max-values from a single subject / patient.</p

Normalized visibility ratings observed in the control experiment as a function of SOA for four subjects.

<p>The control experiment featured only dichoptic conditions, in which targets and masks were presented to different eyes. The two subjects depicted in the left half of the figure, the left eye received blurred stimuli to mimic loss of visual acuity. The two subjects depicted on the right, the right eye received blurred stimuli. The bar charts show the temporal position of the individual minima (SOAmax).</p

Illustration of stimulus setup and procedure.

<p>(a) The display was viewed through a mirror stereoscope. The gray squares were constantly presented to ensure binocular fusion. (b) Example of a trial sequence, here with a target presented to the right eye, and a mask to the left eye, both above the fixation point. Negative SOAs refer to trials where the mask preceded the target. (c) Illustration of the assignment of keys to perceived target contrast. The same figure (with German labels) was part of the on-screen instruction.</p

Stimuli used in the control experiment.

<p>Stimuli depicted in a) and b) are identical to the stimuli used in the main experiment. Stimuli depicted in c) and d) were created by blurring the contours with a Gaussian kernel while keeping overall stimulus energy (i.e. the average brightness of the display) constant.</p

Individual Fit results–Main Experiment.

<p>Individual Fit results–Main Experiment.</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

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^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