Removal of lymphocytes from donor RBCs

We proposed to add allogeneic human RBCs to unfractionated tonsil cell suspensions in order to optimize the use of RosetteSep for B cell isolation from tonsils. First, it was necessary to consider potential contamination of the final product with B lymphocytes from the whole blood used to obtain RBCs. In healthy individuals white blood cells make up 0.08–0.22% of cells in the blood; B lymphocytes comprise 5–25% of this fraction, depending on the age of the donor [4, 5]. Given the large excess of RBCs to be added to unfractionated tonsil cells, the use of human blood as a source of RBCs could result in significant contamination with non-tonsil B lymphocytes. In order to avoid contaminating tonsil B lymphocytes with allogeneic blood B lymphocytes during the purification, we first depleted mononuclear cells from the RBCs by centrifugation over lymphocyte separation medium. As expected, the mononuclear cells removed from whole blood were largely CD3 or CD19 positive as determined by flow cytometery (data not shown). A more sensitive measure, reverse-transcription PCR, was used to detect T and B cells in the RBC preparation before and after depletion (Figure 1). PCR products for both CD3 and CD20 were detected in samples of whole blood and the mononuclear cell fraction. The purified RBC preparation showed no CD3 or CD20 PCR product, indicating that it was free of lymphocyte contamination.

Isolation of tonsil B lymphocytes using RosetteSep

Tonsil lymphocyte suspensions include 40–80% B lymphocytes, with T lymphocytes comprising the bulk of the remaining cells. Figure 2 shows a typical purification of B lymphocytes from tonsils using RosetteSep. In the unpurified tonsil cell suspension shown, there were 58.3% B cells and 23.4% T cells (Figure 2A). RosetteSep purification of B lymphocytes without the addition of RBCs resulted in approximately 91.6% purity with 6% T cell contamination (Figure 2A). Addition of human RBCs improved the purification to 98.9%, reducing the amount of T cell contamination to 1% (Figure 2A).

Sheep RBCs have been used for tonsil B lymphocyte isolation by RosetteSep [6]. However, we found that the addition of sheep RBCs provided no improvement in B lymphocyte purity over that obtained without added RBCs (Figure 2A). Additionally, we found that tonsil B cells purified with either sheep RBCs or no added RBCs gave a reproducible PCR product for CD3 after 35 cycles, whereas there was no evidence of CD3 in tonsil B lymphocytes purified with human RBCs (Figure 2B).

To assess cell loss during this procedure, we determined the percent of CD19+ lymphocytes in unfractionated tonsil cell suspensions using flow cytometry. After purification, the total number of recovered B cells was determined and expressed as a percentage of the estimated input number. It was found that this method of B lymphocyte purification reproducibly yielded greater than 99% recovery.

To further assess the utility of this method, the B lymphocyte phenotypes were examined before and after purification, using flow cytometry to detect CD19, CD38 and IgD. Unpurified tonsil lymphocytes were gated on CD19 expression before examining the CD38/IgD profile (Figure 3, upper panels). RosetteSep with the addition of human RBCs resulted in a 99% pure population of B-lymphocytes with a CD38/IgD profile similar to that of the corresponding tonsil cells, suggesting that all B lymphocyte populations are preserved during purification (Figure 3, lower panels).

Antibodies and reagents

Phycoerythrin (PE) conjugated anti-IgD, anti-CD3 and isotype control, IgG1, were from Becton Dickinson (Mississauga, ON). Tri-color (TC) conjugated anti-CD19 was from Caltag (Burlingame, AL). FITC conjugated anti-CD38 was from Abcam (Cambridge, UK). RosetteSep human B lymphocyte enrichment cocktail was purchased from StemCell Technologies Inc. (Vancouver, BC). Sheep RBCs were purchased from Cedarlane Laboratories Ltd (Hornby, ON).

Tonsils

Tonsils were obtained from the Alberta Children's Hospital with ethical approval. Cell suspensions were prepared by cutting the tissue into fine pieces using scissors or razor blades in RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco). The tonsil tissue fragments were then forced through a nylon screen of 70 μm mesh size to disrupt the pieces into single cells. Cells were then washed twice with RPMI/FBS and counted.

Preparation of human red blood cells

Human blood was collected from healthy donors into heparin vacutainers. Mononuclear cells were removed by diluting the whole blood with an equal volume of RPMI/FBS, layering over lymphocyte separation medium (MP Biomedicals LLC, Solon, OH), and then centrifugation at 1160 × g for 20 minutes with no brake. Cells at the interphase were aspirated and discarded, while the loosely pelleted RBCs were washed and returned to their original volume using Alsever's solution. Prepared RBCs were counted and used for up to one month. All steps were performed at room temperature.

Purification of tonsil B lymphocytes

To purify B lymphocytes, 2 × 10⁸ tonsil cells were mixed with a 50-fold excess (10¹⁰) of human or sheep RBCs. The cells were centrifuged at 350 × g for 5 minutes and then resuspended in 4 mL of RPMI/FBS. 200 μl of RosetteSep was then added; the sample was gently mixed by rocking and incubated for 20 minutes. The sample was then diluted with an equal volume of phosphate buffered saline (PBS) containing 2% FBS and 1 mM EDTA. Lymphocyte separation medium was carefully layered under the preparation and centrifuged for 20 minutes at 1160 × g with no brake. Cells at the interface were collected, washed and saved for analysis. All steps were performed at room temperature.

Reverse transcription PCR

Up to 10⁷ cells were used to prepare total RNA using the RNeasy mini kit (Qiagen, Valencia, CA). 5 μg of total RNA was reverse transcribed using Superscript III (Invitrogen, Carlsbad, CA) with oligo dT primers. Polymerase chain reaction (PCR) was performed in 25 μl volumes with TAQ PCR master mix (Qiagen, Valencia, CA) using 35 cycles of 1 min at 95°C, 1 min at 58°C, and 50 sec at 72°C. Primers for CD20: forward 5' GCA GCA ACG GAG AAA AAC TC 3' and reverse 5' GAA GAA GCG TGA CAA CAC AAG 3'. Primers for CD3: forward 5' CTG GGA AGT AAT GCC AAG GA 3' and reverse 5' TAG ACC CCA ACA GCA AGG AC 3'. Primers for Beta actin: forward 5' CAC TCT TCC AGC CTT CCT TCC 3' and reverse 5' GTG TTG GCG TAC AGG TCT TTG 3'. PCR products were resolved on 1% agarose Tris-Borate-EDTA (TBE) gels with ethidium bromide and photographed to observe relative signals of each amplified transcript.

Flow cytometry

Cells were incubated with directly conjugated antibodies for 15 min at room temperature, washed and acquired on a flow cytometer (FACScan, Becton Dickinson). The data were analyzed using the FlowJo program (Tree Star Inc, San Carlo, CA).