Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

Abstract

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.

Fig. 1

Distribution of PAB⁺ cells. B cells were isolated from the PerC, SPL and BM of C57BL, FVB and BALB/c mice using anti-B220 magnetic beads. The positively selected cells then were incubated with β-gal, Tg or TT, stained with antibody to B-220, and analysed by FACS. The percentage of double-positive cells (Ag-binding B cells) is indicated.

Fig. 2

PAB⁺ and PAB^– cells in the PerC. PAB⁺ and PAB^– cells from the PerC of BALB/c mice were positively selected as described in Materials and methods. The binding of Ags (β-gal, Tg, actin and insulin) and the expression of cell surface markers was determined by FACS analysis. Rat IgG served as a negative control. The percentage of positively stained cells is indicated.

Fig. 3

PAB⁺ and PAB^– cells in the SPL. PAB⁺ and PAB^– cells from the SPL of BALB/c mice were positively selected as described in Materials and Methods and analysed as indicated in Fig. 2 for Ag-binding and expression of cell surface markers.

Fig. 4

Relationship of PAB⁺ cells from the PerC to B-1 cell surface markers. B cells from the PerC of BALB/c mice were positively selected with anti-B220 magnetic beads. (a–f) Cells then were incubated with antibodies to cell surface markers (anti-IgM, anti-IgD, anti-Mac-1, anti-CD23 and anti-CD5) and different antigens. The percentage of gated cells binding antigens in each region (R) is shown in the histograms. (e, f) Based on high antigen-binding and high FSC, gated cells were analysed to determine their relationship to B-1 cell surface markers.

Fig. 5

Relationship of PAB⁺ cells from the SPL to B-1 cells surface markers. (a–c) B cells from the SPL of BALB/c mice were positively selected with anti-B220 magnetic beads. Cells then were incubated with antibodies to cell surface markers (anti-IgM, anti-IgD, anti-Mac-1, and anti-CD23) and different antigens. Percentage of gated cells binding antigens in each region (R) is shown in the histograms. (d) Binding of antigens to cells from the MZ (CD21^hiCD23^int) and FO (CD21^intCD23^hi) of the SPL. (e) B cells from the SPL of C57BL/6 IgM^+/+ and IgM^–/– mice were isolated with anti-B220 magnetic beads and the expression of cell surface markers and the binding of β-gal determined.

Fig. 6

PAB⁺ cells in PP and LP. (a) B cells from the PP and LP were positively selected by anti-B220 magnetic beads, and two-colour stained with various combination of anti-B220, β-gal, Tg, or TT. The percentage of B220 cells that bound antigens was determined. (b) B cells from the SPL, PerC, PP and LP of BALB/c mice were positively selected with anti-B220 magnetic beads and two-colour stained with various combinations of anti-B220, anti-IgM, anti-IgD or anti-IgA. (c, d) B cells from PP and LP were positively isolated with anti-B220 magnetic beads, incubated with β-gal or Tg and three-colour stained with various combinations of anti-B220, anti-IgM or anti-IgA to determine the percentage of IgM and IgA cells that bound β-gal or Tg. (e) PAB⁺ and PAB^– cells from the PerC and PP of C57BL/6 mice were positively selected as described in Materials and Methods. The cells then were incubated with LPS, E. coli or Staph. aureus (SA) and percent binding determined.

Fig. 7

PAB cells in the thymus. (a) B cells from the thymus were positively and negatively selected by 2 passages with anti-220 magnetic beads. The B220⁺ and B220^– populations then were incubated with antibody to B220 and one of several different antigens (i.e. β-gal, Tg, TT, Ins, Actin) and analysed by FACS. (b) B220^– cells stained with antibodies to the T cell markers CD5 and CD3 and the B cell marker IgM. (c) To determine their phenotypes, high β-gal binding-cells with high FSC were gated and incubated with antibodies to IgM, IgD, Mac-1, CD5 and CD23. (d) PAB⁺ and PAB^– cells from the thymus of BALB/c mice were positively selected as described in Materials and Methods and analysed as indicated in Fig. 2 for Ag-binding and expression of cell surface markers.