title: Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA.
abstract: Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000818:Animals; D000921:Antibody-Producing Cells / Q000378:metabolism*; D000954:Antigens, Surface / Q000032:analysis*; D000955:Antigens, T-Independent / Q000276:immunology; D001402:B-Lymphocytes / Q000145:classification* / Q000166:cytology / Q000276:immunology; D002003:Brucella abortus / Q000276:immunology*; D002453:Cell Cycle; D002469:Cell Separation; D005362:Ficoll / Q000031:analogs & derivatives / Q000276:immunology*; D006462:Hemolytic Plaque Technique; D007376:Interleukin-2 / Q000502:physiology; D008213:Lymphocyte Activation; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008808:Mice, Inbred CBA; D009578:Nitrobenzenes / Q000276:immunology*; D010641:Phenotype; D011134:Polysaccharides / Q000276:immunology*; D014301:Trinitrobenzenes / Q000276:immunology*; D000511:alpha-Macroglobulins / Q000494:pharmacology
chemical_list: D000954:Antigens, Surface; D000955:Antigens, T-Independent; D007376:Interleukin-2; D009578:Nitrobenzenes; D011134:Polysaccharides; C020529:TNP-ficoll; D014301:Trinitrobenzenes; D000511:alpha-Macroglobulins; C032306:polyclonal B cell activator; D005362:Ficoll