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Analysis of anti-human CD22 human mAbs affinity and specificity on human PBMCs. a) Affinity determination of anti-human CD22 mAbs using SPR by flowing various concentration of CD22 antibody over CD22 chip-bound. b) Flow cytometry analysis of CD22 mAbs on human PBMC. Human PBMC were labeled with anti human CD19 (APC) and purified CD22 mAbs (clone γ1λ1, γ3λ3-5, γ23κ5-2 or γ27λ26) coupled with Alexa Fluor 568 fluorochrome or with a commercial mouse mAb anti-human CD22 (Ms anti-human CD22). (PPT 666 kb

Differentiation potential of TN populations. A

<p>. Individual thymocytes were cultured with the OP9-DL4 cell line and sampled at different time points after plating. The depicted results are from 14-day cultures, since the T cell phenotype did not change after that time point. Plating efficiency: the percentage of plated wells in which progeny were detected; % T cells: the percentage of positive wells containing T cells. Bipotent, TCRαβ⁺-restricted and TCRγδ⁺-restricted cells were identified according to the dot plots shown below. <b>Please note</b>: in positive wells in which T cells were not detected, the average clone size was 100 cells/well; in both bipotent and TCR-αβ−only cultures, clone sizes averaged 2×10⁵ cells/well; in γδ−only cultures, average clone sizes were 5×10⁴ cells/well. Results are pooled from three independent experiments, each of which gave equivalent results. <b>B</b> Expression of different components of the pre-TCR in TN populations. <i>TRB</i> locus DJ rearrangements. <b>C</b>. <i>Rag1</i>, <i>CD3ε</i> and <i>pT</i>α expression in individual cells. Upper graphs show expression frequencies determined in 80 individual cells. Lower graphs show the number of mRNA molecules expressed by each individual cell studied (n = 40 cells). Each positive cell is represented by a dot, negative cells are not shown and bars represent mean expression values. Statistical analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073098#pone-0073098-g002" target="_blank">Fig 2</a>. <b>D</b>. Intracellular TCRβ and CD3ε expression in different TN subsets in one representative experiment of four independent experiments with equivalent results.</p

Evaluation of committed progenitors: all values were obtained from figures within thymic (TN) or committed (Com) progenitors.

<p>Evaluation of committed progenitors: all values were obtained from figures within thymic (TN) or committed (Com) progenitors.</p

Gene expression in TN3a and TN3b populations. A

<p>. TN3a and TN3b populations were sorted as single cells and tested for the expression of each gene. Upper graphs show expression frequencies (determined in 80 individual cells). Lower graphs show the number of mRNA molecules expressed by each individual cell studied (n = 40 cells), depicted as described in Fig. 1. <b>B</b>. Gene co-expression patterns in individual TN4 cells. Each horizontal row corresponds to the same (numbered) cell. Each column shows the expression of a different gene, with the number of mRNA molecules/cell represented according to the adjacent color log scale. Empty symbols represent cells not expressing that particular mRNA (i.e. fewer than two mRNA molecules per cell). The black symbol corresponds to a positive cell in which quantification was not performed.</p

Single-cell quantitative gene expression profiling.

<p>(<b>A</b>) Overall strategy. (<b>B</b>) Validation of the primer pairs used to quantify gene expression. Graphs show triplicates of independent qRT-PCRs for each gene. Upper graphs: amplifications in which all genes were reverse-transcribed and amplified together in the first RT-PCR. Lower graphs: amplifications in which each gene was reverse-transcribed and amplified separately in the first RT-PCR. The histograms compare PCR efficiency in the two conditions. (<b>C</b>) Examples of differences between population-based readouts and single-cell readouts. A mature monoclonal CD8 T cell population was sorted and tested on the same day for expression of two different genes (genes a and b). Upper graphs: cells were sorted at 100 cells/well, in order to mimic population studies in which only average gene expression can be evaluated. The results demonstrate that amplifications of genes a and b have the same efficiency and that both genes are expressed to the same extent. On the basis of this data, one would conclude that the two genes are similarly expressed in this cell population. Lower graphs: each well received a single cell that was tested for the expression of genes a and b. In contrast to the population studies, these single-cell studies reveal that the respective expression levels of genes a and b genes are very different: gene b is expressed at low levels by all cells and gene a is expressed at high levels by only 10% of the cells.</p

Transcription factor interactions during T cell commitment

<p>. <b>(A, B).</b> CD45.2⁺ 15-day fetal liver cells from <i>Gata3⁺/+</i> (left) <i>Gata3⁻/−</i> (middle) and <i>Gata3⁺/−</i> (right) mice were injected into sublethally irradiated CD45.1 mice. The CD45.2⁺ cells present in the thymus one month later were studied. (<b>A</b>) the phenotype of TN thymocytes. Upper graphs show CD44/CD25 profiles and lower graphs show c-Kit expression in gated CD44⁺ cells. (<b>B</b>) <i>Notch1</i> expression in TN1 cells from these chimeras. (<b>C</b>–<b>E</b>) Expression of different TFs during T cell commitment. B–E: Upper graphs depict expression frequencies (the proportion of positive cells) and lower grafts show the mRNA expression level in each individual cell (represented by a dot) from three independent experiments. Expression-negative cells and cells expressing fewer than 10 mRNAs/cell are not shown. Bars represent mean expression levels. Statistical analysis was performed using Fisher's exact test for expression frequencies and a Mann-Whitney rank sum test for expression levels. Asterisks correspond to a comparison of the population of interest with the population in the previous transition stage: * p<0.05, ** p<0.01 and *** p<0.001. <b>F.</b> The co-expression of the different genes was studied in forty individual cells. Each dot represents an individual cell, plotted simultaneously for the number of <i>Pu1</i> mRNA molecules on the X axis and the number of mRNA molecules coded as either Notch1 (upper graph) or Bc11B on the Y axis (lower graph). The correlation between the respective expressions of each pair of genes was studied in a Goodman-Kruskal gamma test, which assesses the correlation's significance (via a p-value) and nature (via the gamma coefficient, which is negative for a negative correlation and positive for a positive correlation). A polynomial trend curve is shown for each correlation.</p