J C Cambier

Sorting of B lymphoblasts based upon cell diameter provides cell populations enriched in different stages of cell cycle

Here we report analysis and correlation of changes in cell size and cycle state resulting from exposure of murine B lymphocytes to the mitogens lipopolysaccharide (LPS) and dextran sulfate (DxSO4). Cell cycle changes are assessed by flow cytofluorometric analysis of acridine orange stained cells. Cell diameters are determined by flow cytometric analysis of the pulse-width (time of flight) of the axial light extinction signal. Results indicate that within 12 h of exposure of B cell populations to these mitogens, cells displaying increased diameter and containing increased RNA can be detected. Under these conditions, increased RNA content is considered indicative of G0 to G1 transition or entry into cell cycle (Darzynkiewicz et al., 1976). Progressive increases in cell size and transition through G1, S, G2, and M occur in parallel during 48 h of culture with mitogens. Sorting of cells based upon size followed by cell cycle analysis reveals a direct correlation between cell size and cycle phase. Specifically, cells 4.5-5.5 microns in diameter are in primarily G0. Cells 5.5-7.0 microns in diameter are in early G1. Populations of cells 7.0-10 microns in diameter are comprised of late G1 and S phase cells. Populations of cells 10-12 microns in diameter consist of S, G2, and M phase cells. The importance of this correlation is discussed in view of needs to more rigorously define B cell populations for investigations of biochemical events of and accessory cell requirements for activation of B lymphocytes.

Hyper-Ia antigen expression on B cells from B6-lpr/lpr mice correlates with manifestations of the autoimmune state.

To investigate the state of activation of B cells from mice with the lpr gene defect, membrane Ia antigen (mIa) expression was analyzed on B cells from B6-lpr/lpr (lpr) and control B6- +/-/+/- mice. B cells from lpr mice exhibited marked increases in levels of mIa as determined by flow cytometry using a monoclonal anti-I-Ab,d reagent. This increase, which was progressive with age, suggests that phenotypic alteration of B-cell mIa expression is a consequence of lpr gene action. Since B-cell activation manifest by elevated mIa expression may promote productive interactions with helper T cells, these observations suggest an important role for B-cell abnormalities in the etiology of lpr-induced autoimmune disease.

Isolated phosphorylcholine binding lymphocytes. I. Use of a cleavable crosslinking reagent for solid-phase adsorbent isolation of functional antigen binding cells

Isolation and characterization of BALB/c lymphocytes specific for phosphorylcholine is described. The isolation protocol utilizes phosphorylcholine coupled to gelatin coated plates via the cleavable crosslinking reagent N-succinimidyl 3-(2-pyridyldiothio)propionate (SPDP). The procedure is rapid, requiring only 1-2 h and conducted entirely at 0-4 degrees C. Hapten binding cells are eluted by vigorous pipetting at 4 degrees C with medium containing 20% fetal calf serum. Approximately 70% of isolated cells rebind antigen as assessed using a PC Brucella abortus rosette assay while 50% express the TEPC15 idiotype. Approximately 85% of isolated and idiotype positive cells are B cells while the remainder are T cells. Limiting dilution analysis revealed that approximately 1/5 of PC binding cells isolated from the spleens of normal mice respond to lipopolysaccharide plus dextran sulfate by production of anti-PC antibody. Approximately 1/11 respond to PC Brucella abortus and 1/250 respond to PC sheep erythrocytes plus primed T cells by anti-PC antibody production. The results clearly demonstrate the effectiveness of this technique for isolation of highly enriched, functional, antigen specific lymphocytes.