Figure 1.
Detection of the T cell antigens CD3 and CD4 on CD20+ lymphocytes by flow cytometry.
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+ (CD3lowCD20+) lymphocytes between two independent groups of donors (fresh versus oN/4°C) was determined by the two-sided Mann-Whitney-U-Test. Outliers are depicted as circles.
Figure 2.
Co-expression of CD3 and CD20 on the cell surface is not a staining artifact.
(A) Shown is the presence of CD3lowCD20+ cells after overnight storage of blood samples at 4°C for two representative patients (#1 and #2, left). Staining of blood cells with the respective allophycocyanin (APC)-labeled mouse IgG1 isotype control excludes an artifact caused by the fluorochrome APC (#1 and #2, right). (B) Simultaneous expression of CD20 and CD3 on the surface of MACS-purified CD20+ lymphocytes is demonstrated for one representative donor by single cell analysis using confocal laser microscopy. (C) Confirmation of concomitant CD20 (FITC) and CD19 (PE) expression on MACS-purified CD20+ B cells served as positive control. Whole blood samples were stored overnight at 4°C before PBMC isolation and MACS analysis.
Figure 3.
CD3lowCD20+ cells belong to the B lymphocyte compartment.
Phenotyping of CD3lowCD20+ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3lowCD20+ 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 CD3lowCD20+ 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 CD3lowCD20+ and CD3-CD20+ B cells, respectively, excluding that the CD3lowCD20+ 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.
Figure 4.
Detection of CD3 on B cell surfaces is a result of cell number- dependent T-B cell interactions.
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 CD3lowCD20+ B cells. β-actin was used as loading control. (B) The appearance of CD3lowCD20+ 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 CD3lowCD20+ B cell population, pointing at the need for T cells. Furthermore, the quantity of CD3lowCD20+ B cells was dependent on the number of CD4+ T cells, i.e. increasing T-B cell ratios caused elevated numbers of CD3lowCD20+ B cells. Shown are the results of up to three independent experiments.
Figure 5.
Transfer of CD3 from T to B cells requires cell-cell contact and is impaired by monensin treatment.
(A) Shown are numbers of CD3lowCD20+ B cells in one patient after co-culture of MACS-purified CD4+ T cells and CD20+ B cells overnight (oN) at 4°C. While CD3lowCD20+ B cells were found to be elevated with increasing T-B cell ratios, fixation of antigens on the T cell surface using 1% PFA before co-culture with B cells oN at 4°C did prevent CD3 acquisition by CD20+ B cells. Moreover, co-culture of CD4+ T cells and CD20+ B cells using transwell (Tw) chambers (pore size of 0.4 µm) prevented the appearance of CD3lowCD20+ B cells, excluding the possibility that membrane vesicles like exosomes transfer T cell surface markers from T to B cells. (B) The occurrence of CD3lowCD20+ B cells after oN storage of blood samples at 4°C was impaired by the addition of monensin (applied by the protein transport inhibitor BD GolgiStop). Shown are the results of four independent experiments.
Figure 6.
Numbers of CD3lowCD20+ B cells are time- and temperature-dependent.
The increase in the number of CD3lowCD20+ B cells was time-dependent, but independent of storage conditions (4°C versus room temperature (RT) versus humidified atmosphere at 37°C, 5% CO2). CD3lowCD20+ 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.