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Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors.

Duty JA, Szodoray P, Zheng NY, Koelsch KA, Zhang Q, Swiatkowski M, Mathias M, Garman L, Helms C, Nakken B, Smith K, Farris AD, Wilson PC - J. Exp. Med. (2008)

Bottom Line: Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA.Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic.This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology and Cancer, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

ABSTRACT
Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

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Related in: MedlinePlus

BND cells have reduced tyrosine phosphorylation, suggesting that they are natural anergic B cells. (A) Representative histogram of treated BND and naive cells sorted according to a least-touch strategy (see Materials and methods). 105 cells were incubated with anti-IgD Fab'2-FITC + IgM Fab'2 for 45 s at various concentrations, fixed, and permeabilized, followed by staining by anti-pTyr and intracellular anti-CD20. Histograms were built by FACS analysis and treated populations were compared with untreated controls. Cells were also stimulated with pervanadate as a positive control (right histogram). Green lines are used as qualitative references to mark the median fluorescence of the untreated controls, allowing a visual comparison of fluorescence shift with treated samples. (B) Cells were stimulated with anti-IgD Fab'2 FITC + IgM Fab'2 at concentrations of 10, 50, and 100 μg/ml to determine concentration dependency and were plotted as mean fluorescence intensity (MFI) ± SD of pTyr-PE (for each concentration point, n = 3 groups of 1–3 donors; *, P < 0.05 and **, P < 0.01 for 10 and 50 μg/ml, respectively, by Student's t test). Class switched B cells could also be gated (IgD−, IgM−, and CD20+; black line) from the peripheral set and are included as an internal negative control. Isotype controls (gray line) were established for the stimulated fractions. (C) Incubation of BND and naive cells with 50 μg/ml of anti-IgD Fab'2 alone or in combination with anti-IgM. Results of three donors are plotted as MFI of pTyr. Error bars represent the mean ± SD. Color lines represent individual donor variation between naive and BND population. (D) Cells were sorted as in A but stained with antiphosphorylated Src (pTyr 416; pSrc). Representative histograms are shown for naive and BND cells, untreated and treated with anti-IgD Fab'2 FITC + anti-IgM Fab'2. Bar graphs represent the mean absolute number of units/molecules ±SD of phosphorylated Src as determined by Sphero Rainbow Calibration beads (Invitrogen) from three healthy donors and compared between BND (red) and naive (blue) populations for both treated and untreated fractions. Significance for treated fractions (P < 0.05) was established by a Student's t test.
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fig6: BND cells have reduced tyrosine phosphorylation, suggesting that they are natural anergic B cells. (A) Representative histogram of treated BND and naive cells sorted according to a least-touch strategy (see Materials and methods). 105 cells were incubated with anti-IgD Fab'2-FITC + IgM Fab'2 for 45 s at various concentrations, fixed, and permeabilized, followed by staining by anti-pTyr and intracellular anti-CD20. Histograms were built by FACS analysis and treated populations were compared with untreated controls. Cells were also stimulated with pervanadate as a positive control (right histogram). Green lines are used as qualitative references to mark the median fluorescence of the untreated controls, allowing a visual comparison of fluorescence shift with treated samples. (B) Cells were stimulated with anti-IgD Fab'2 FITC + IgM Fab'2 at concentrations of 10, 50, and 100 μg/ml to determine concentration dependency and were plotted as mean fluorescence intensity (MFI) ± SD of pTyr-PE (for each concentration point, n = 3 groups of 1–3 donors; *, P < 0.05 and **, P < 0.01 for 10 and 50 μg/ml, respectively, by Student's t test). Class switched B cells could also be gated (IgD−, IgM−, and CD20+; black line) from the peripheral set and are included as an internal negative control. Isotype controls (gray line) were established for the stimulated fractions. (C) Incubation of BND and naive cells with 50 μg/ml of anti-IgD Fab'2 alone or in combination with anti-IgM. Results of three donors are plotted as MFI of pTyr. Error bars represent the mean ± SD. Color lines represent individual donor variation between naive and BND population. (D) Cells were sorted as in A but stained with antiphosphorylated Src (pTyr 416; pSrc). Representative histograms are shown for naive and BND cells, untreated and treated with anti-IgD Fab'2 FITC + anti-IgM Fab'2. Bar graphs represent the mean absolute number of units/molecules ±SD of phosphorylated Src as determined by Sphero Rainbow Calibration beads (Invitrogen) from three healthy donors and compared between BND (red) and naive (blue) populations for both treated and untreated fractions. Significance for treated fractions (P < 0.05) was established by a Student's t test.

Mentions: In addition to calcium flux, the signaling cascade seconds and minutes after BCR cross-linkage results in overall increases of cellular pTyr levels. Because relatively few BND cells can be isolated at any one time from human peripheral blood, we chose to analyze pTyr levels by using flow cytometry or “PhosFlow.” This powerful technique allows the quantification of both the pTyr magnitude for each individual cell and the frequency of cells affected. With this assay, total activated B cells are rapidly fixed seconds after receptor cross-linkage and before any manipulation that might reduce detectable pTyr levels. The phenotypes and pTyr levels of individual cells can then be resolved retrospectively by using immunostaining and detection on a flow cytometer (Fig. 6 A). Each B cell population was fractionated and depleted of other cell types. Naive and BND cells were first enriched through sorting with anti-CD27 and anti-IgM antibodies. Naive cells are predominantly in the total CD27− fraction, BND cells are predominantly in the IgM−CD27− fraction, and untreated cells that will not be activated by anti-IgM/D treatment are predominantly in the CD27−IgD−IgM− fraction (determined separately to be mostly IgG, IgA, or IgE+ B cells). By spiking the anti-IgM/IgD Fab′2 cross-linking mix with a fluorescently labeled (FITC) anti-IgD and then staining with intracellular anti-CD20 and anti-pTyr after stimulation, fixation, and permeabilization, we could gate and resolve each B cell population to high purity (Fig. 6 A, left). As indicated in the middle of Fig. 6 A, poststimulation pTyr levels were much greater in naive B cells than in BND cells after treatment with a combination of polyclonal anti-IgD/IgM for an optimal 45 s (determined separately to be the peak of activation; Fig. S2 A). The reduced pTyr induction is not caused by increased cell death or lack of viability in the BND population, as both they and naive cells are stimulated to the same level with the phosphatase inhibitor pervanadate (orthovandate H2O2; Fig. 6 A, right histogram).


Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors.

Duty JA, Szodoray P, Zheng NY, Koelsch KA, Zhang Q, Swiatkowski M, Mathias M, Garman L, Helms C, Nakken B, Smith K, Farris AD, Wilson PC - J. Exp. Med. (2008)

BND cells have reduced tyrosine phosphorylation, suggesting that they are natural anergic B cells. (A) Representative histogram of treated BND and naive cells sorted according to a least-touch strategy (see Materials and methods). 105 cells were incubated with anti-IgD Fab'2-FITC + IgM Fab'2 for 45 s at various concentrations, fixed, and permeabilized, followed by staining by anti-pTyr and intracellular anti-CD20. Histograms were built by FACS analysis and treated populations were compared with untreated controls. Cells were also stimulated with pervanadate as a positive control (right histogram). Green lines are used as qualitative references to mark the median fluorescence of the untreated controls, allowing a visual comparison of fluorescence shift with treated samples. (B) Cells were stimulated with anti-IgD Fab'2 FITC + IgM Fab'2 at concentrations of 10, 50, and 100 μg/ml to determine concentration dependency and were plotted as mean fluorescence intensity (MFI) ± SD of pTyr-PE (for each concentration point, n = 3 groups of 1–3 donors; *, P < 0.05 and **, P < 0.01 for 10 and 50 μg/ml, respectively, by Student's t test). Class switched B cells could also be gated (IgD−, IgM−, and CD20+; black line) from the peripheral set and are included as an internal negative control. Isotype controls (gray line) were established for the stimulated fractions. (C) Incubation of BND and naive cells with 50 μg/ml of anti-IgD Fab'2 alone or in combination with anti-IgM. Results of three donors are plotted as MFI of pTyr. Error bars represent the mean ± SD. Color lines represent individual donor variation between naive and BND population. (D) Cells were sorted as in A but stained with antiphosphorylated Src (pTyr 416; pSrc). Representative histograms are shown for naive and BND cells, untreated and treated with anti-IgD Fab'2 FITC + anti-IgM Fab'2. Bar graphs represent the mean absolute number of units/molecules ±SD of phosphorylated Src as determined by Sphero Rainbow Calibration beads (Invitrogen) from three healthy donors and compared between BND (red) and naive (blue) populations for both treated and untreated fractions. Significance for treated fractions (P < 0.05) was established by a Student's t test.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626668&req=5

fig6: BND cells have reduced tyrosine phosphorylation, suggesting that they are natural anergic B cells. (A) Representative histogram of treated BND and naive cells sorted according to a least-touch strategy (see Materials and methods). 105 cells were incubated with anti-IgD Fab'2-FITC + IgM Fab'2 for 45 s at various concentrations, fixed, and permeabilized, followed by staining by anti-pTyr and intracellular anti-CD20. Histograms were built by FACS analysis and treated populations were compared with untreated controls. Cells were also stimulated with pervanadate as a positive control (right histogram). Green lines are used as qualitative references to mark the median fluorescence of the untreated controls, allowing a visual comparison of fluorescence shift with treated samples. (B) Cells were stimulated with anti-IgD Fab'2 FITC + IgM Fab'2 at concentrations of 10, 50, and 100 μg/ml to determine concentration dependency and were plotted as mean fluorescence intensity (MFI) ± SD of pTyr-PE (for each concentration point, n = 3 groups of 1–3 donors; *, P < 0.05 and **, P < 0.01 for 10 and 50 μg/ml, respectively, by Student's t test). Class switched B cells could also be gated (IgD−, IgM−, and CD20+; black line) from the peripheral set and are included as an internal negative control. Isotype controls (gray line) were established for the stimulated fractions. (C) Incubation of BND and naive cells with 50 μg/ml of anti-IgD Fab'2 alone or in combination with anti-IgM. Results of three donors are plotted as MFI of pTyr. Error bars represent the mean ± SD. Color lines represent individual donor variation between naive and BND population. (D) Cells were sorted as in A but stained with antiphosphorylated Src (pTyr 416; pSrc). Representative histograms are shown for naive and BND cells, untreated and treated with anti-IgD Fab'2 FITC + anti-IgM Fab'2. Bar graphs represent the mean absolute number of units/molecules ±SD of phosphorylated Src as determined by Sphero Rainbow Calibration beads (Invitrogen) from three healthy donors and compared between BND (red) and naive (blue) populations for both treated and untreated fractions. Significance for treated fractions (P < 0.05) was established by a Student's t test.
Mentions: In addition to calcium flux, the signaling cascade seconds and minutes after BCR cross-linkage results in overall increases of cellular pTyr levels. Because relatively few BND cells can be isolated at any one time from human peripheral blood, we chose to analyze pTyr levels by using flow cytometry or “PhosFlow.” This powerful technique allows the quantification of both the pTyr magnitude for each individual cell and the frequency of cells affected. With this assay, total activated B cells are rapidly fixed seconds after receptor cross-linkage and before any manipulation that might reduce detectable pTyr levels. The phenotypes and pTyr levels of individual cells can then be resolved retrospectively by using immunostaining and detection on a flow cytometer (Fig. 6 A). Each B cell population was fractionated and depleted of other cell types. Naive and BND cells were first enriched through sorting with anti-CD27 and anti-IgM antibodies. Naive cells are predominantly in the total CD27− fraction, BND cells are predominantly in the IgM−CD27− fraction, and untreated cells that will not be activated by anti-IgM/D treatment are predominantly in the CD27−IgD−IgM− fraction (determined separately to be mostly IgG, IgA, or IgE+ B cells). By spiking the anti-IgM/IgD Fab′2 cross-linking mix with a fluorescently labeled (FITC) anti-IgD and then staining with intracellular anti-CD20 and anti-pTyr after stimulation, fixation, and permeabilization, we could gate and resolve each B cell population to high purity (Fig. 6 A, left). As indicated in the middle of Fig. 6 A, poststimulation pTyr levels were much greater in naive B cells than in BND cells after treatment with a combination of polyclonal anti-IgD/IgM for an optimal 45 s (determined separately to be the peak of activation; Fig. S2 A). The reduced pTyr induction is not caused by increased cell death or lack of viability in the BND population, as both they and naive cells are stimulated to the same level with the phosphatase inhibitor pervanadate (orthovandate H2O2; Fig. 6 A, right histogram).

Bottom Line: Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA.Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic.This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology and Cancer, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

ABSTRACT
Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

Show MeSH
Related in: MedlinePlus