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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 attenuated calcium responses compared with naive cells after BCR cross-linking. (A) A representative scheme of isolation and negative enrichment for the BND and naive populations. Negatively enriched B cells from human peripheral blood were split into two equal fractions: one stained with anti-CD27, anti-IgM, and anti-IgG (anti-BCR) to negatively gate BND cells and the other with anti-CD27 and anti-IgG (anti-BCR) to negatively gate naive cells. Cells were loaded with calcium dye Fluo-4 (Invitrogen) and transferred to flow cytometer, warmed to 37°C, and treated with different amounts and combinations of polyclonal Fab'2 BCR cross-linkers. Side stains were performed with these antibodies plus anti-CD19 and anti-IgD to test for population purity. In each case, B cell purities were >98% and BND and naive purities were >96% as indicated. Calcium curves were determined using average mean fluorescence intensity (MFI) over time. (B) Representative kinetic graphs of average Flou-4 MFI over time for naive, BND, and a B cell class-switched memory control (CS B cell) population representing IgA/E+ memory cells (CD27+, IgM−, IgG−, and IgD−; black line). Basal levels were established for 30 s and then the cells were treated with 50 μg/ml of anti-IgM Fab'2/anti-IgD Fab'2, 50 μg/ml of anti-IgD Fab'2 alone (C), or 20 μg/ml ionomycin (D). (E) Maximal peak fluorescence of Fluo-4 was averaged ±SD for B and C (*, P < 0.05; n = 3 donors; Student's t test). (F) Mean fluorescence of Fluo-4 ±SD representing the basal levels (the first 30 s) was calculated showing a slightly higher amount in the BND population.
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fig5: BND cells have attenuated calcium responses compared with naive cells after BCR cross-linking. (A) A representative scheme of isolation and negative enrichment for the BND and naive populations. Negatively enriched B cells from human peripheral blood were split into two equal fractions: one stained with anti-CD27, anti-IgM, and anti-IgG (anti-BCR) to negatively gate BND cells and the other with anti-CD27 and anti-IgG (anti-BCR) to negatively gate naive cells. Cells were loaded with calcium dye Fluo-4 (Invitrogen) and transferred to flow cytometer, warmed to 37°C, and treated with different amounts and combinations of polyclonal Fab'2 BCR cross-linkers. Side stains were performed with these antibodies plus anti-CD19 and anti-IgD to test for population purity. In each case, B cell purities were >98% and BND and naive purities were >96% as indicated. Calcium curves were determined using average mean fluorescence intensity (MFI) over time. (B) Representative kinetic graphs of average Flou-4 MFI over time for naive, BND, and a B cell class-switched memory control (CS B cell) population representing IgA/E+ memory cells (CD27+, IgM−, IgG−, and IgD−; black line). Basal levels were established for 30 s and then the cells were treated with 50 μg/ml of anti-IgM Fab'2/anti-IgD Fab'2, 50 μg/ml of anti-IgD Fab'2 alone (C), or 20 μg/ml ionomycin (D). (E) Maximal peak fluorescence of Fluo-4 was averaged ±SD for B and C (*, P < 0.05; n = 3 donors; Student's t test). (F) Mean fluorescence of Fluo-4 ±SD representing the basal levels (the first 30 s) was calculated showing a slightly higher amount in the BND population.

Mentions: In mouse models, self-reactive B cells that constitutively bind autoantigens become anergic, resulting in diminished downstream signaling events upon BCR cross-linkage (11, 48–50). We hypothesized that the BND cell population would also exhibit reduced signaling capacity upon activation because they are autoreactive. Immediately detectable measurements of BCR signaling that are reduced in anergic B cells of mice include the degree of intracellular calcium flux and an overall decreased level of pTyr. To avoid prematurely stimulating the B cells, thus depleting calcium stores and affecting pTyr levels, a negative enrichment scheme was used to isolate the BND and control B cell populations. In brief, BND and naive B cells were enriched by differentially staining peripheral blood CD27− B cells loaded with the calcium dye Fluo-4 either in the presence or absence of anti-IgM so that we could identify BND or naive cells, respectively (Fig. 5 A and Materials and methods). Baseline intracellular calcium levels were established for 30 s, followed by treatment with various anti-BCR antibodies. Physiologically, antigen should bind all surface Ig regardless of isotype (i.e., IgD versus IgM). Therefore, to most accurately mimic physiological stimulation with antigen, we first used combined anti-IgM plus anti-IgD cross-linkage to stimulate the B cells. Upon anti-IgD/IgM stimulation, the BND population showed a dramatically decreased ability to flux intracellular calcium compared with a substantial biphasic flux from the naive population (Fig. 5 B). In comparison, the BND population gave a result similar to CD27+IgM/D/G− B cell (class switched) memory population representing both IgA and IgE memory cells, which, as predicted, do not respond to anti-IgD/IgM stimulation. Representative kinetic graphs are shown for 25 μg/ml of anti-IgM/D stimulant, representing three separate donors. Similar trends were also observed when various amounts of stimulant ranging from 10–50 μg/ml were used, representing eight donors in three independent trials. On average, the peak mean fluorescence of Flou-4 was significantly higher for naive versus BND, which is indicative of a more robust flux of intracellular calcium (Fig. 5 E; P < 0.05 by Student's t test). Similarly, BCR cross-linking with anti-κ/λ resulted in reduced BND calcium flux (Fig. S2 B, available at http://www.jem.org/cgi/content/full/jem.20080611/DC1). This difference was not caused by a variegated uptake of the calcium dye Fluo-4 or reduced viability because calcium peaks between the BND and naive populations were equal when treated with the ionophore ionomycin (Fig. 5 D).


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 attenuated calcium responses compared with naive cells after BCR cross-linking. (A) A representative scheme of isolation and negative enrichment for the BND and naive populations. Negatively enriched B cells from human peripheral blood were split into two equal fractions: one stained with anti-CD27, anti-IgM, and anti-IgG (anti-BCR) to negatively gate BND cells and the other with anti-CD27 and anti-IgG (anti-BCR) to negatively gate naive cells. Cells were loaded with calcium dye Fluo-4 (Invitrogen) and transferred to flow cytometer, warmed to 37°C, and treated with different amounts and combinations of polyclonal Fab'2 BCR cross-linkers. Side stains were performed with these antibodies plus anti-CD19 and anti-IgD to test for population purity. In each case, B cell purities were >98% and BND and naive purities were >96% as indicated. Calcium curves were determined using average mean fluorescence intensity (MFI) over time. (B) Representative kinetic graphs of average Flou-4 MFI over time for naive, BND, and a B cell class-switched memory control (CS B cell) population representing IgA/E+ memory cells (CD27+, IgM−, IgG−, and IgD−; black line). Basal levels were established for 30 s and then the cells were treated with 50 μg/ml of anti-IgM Fab'2/anti-IgD Fab'2, 50 μg/ml of anti-IgD Fab'2 alone (C), or 20 μg/ml ionomycin (D). (E) Maximal peak fluorescence of Fluo-4 was averaged ±SD for B and C (*, P < 0.05; n = 3 donors; Student's t test). (F) Mean fluorescence of Fluo-4 ±SD representing the basal levels (the first 30 s) was calculated showing a slightly higher amount in the BND population.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2626668&req=5

fig5: BND cells have attenuated calcium responses compared with naive cells after BCR cross-linking. (A) A representative scheme of isolation and negative enrichment for the BND and naive populations. Negatively enriched B cells from human peripheral blood were split into two equal fractions: one stained with anti-CD27, anti-IgM, and anti-IgG (anti-BCR) to negatively gate BND cells and the other with anti-CD27 and anti-IgG (anti-BCR) to negatively gate naive cells. Cells were loaded with calcium dye Fluo-4 (Invitrogen) and transferred to flow cytometer, warmed to 37°C, and treated with different amounts and combinations of polyclonal Fab'2 BCR cross-linkers. Side stains were performed with these antibodies plus anti-CD19 and anti-IgD to test for population purity. In each case, B cell purities were >98% and BND and naive purities were >96% as indicated. Calcium curves were determined using average mean fluorescence intensity (MFI) over time. (B) Representative kinetic graphs of average Flou-4 MFI over time for naive, BND, and a B cell class-switched memory control (CS B cell) population representing IgA/E+ memory cells (CD27+, IgM−, IgG−, and IgD−; black line). Basal levels were established for 30 s and then the cells were treated with 50 μg/ml of anti-IgM Fab'2/anti-IgD Fab'2, 50 μg/ml of anti-IgD Fab'2 alone (C), or 20 μg/ml ionomycin (D). (E) Maximal peak fluorescence of Fluo-4 was averaged ±SD for B and C (*, P < 0.05; n = 3 donors; Student's t test). (F) Mean fluorescence of Fluo-4 ±SD representing the basal levels (the first 30 s) was calculated showing a slightly higher amount in the BND population.
Mentions: In mouse models, self-reactive B cells that constitutively bind autoantigens become anergic, resulting in diminished downstream signaling events upon BCR cross-linkage (11, 48–50). We hypothesized that the BND cell population would also exhibit reduced signaling capacity upon activation because they are autoreactive. Immediately detectable measurements of BCR signaling that are reduced in anergic B cells of mice include the degree of intracellular calcium flux and an overall decreased level of pTyr. To avoid prematurely stimulating the B cells, thus depleting calcium stores and affecting pTyr levels, a negative enrichment scheme was used to isolate the BND and control B cell populations. In brief, BND and naive B cells were enriched by differentially staining peripheral blood CD27− B cells loaded with the calcium dye Fluo-4 either in the presence or absence of anti-IgM so that we could identify BND or naive cells, respectively (Fig. 5 A and Materials and methods). Baseline intracellular calcium levels were established for 30 s, followed by treatment with various anti-BCR antibodies. Physiologically, antigen should bind all surface Ig regardless of isotype (i.e., IgD versus IgM). Therefore, to most accurately mimic physiological stimulation with antigen, we first used combined anti-IgM plus anti-IgD cross-linkage to stimulate the B cells. Upon anti-IgD/IgM stimulation, the BND population showed a dramatically decreased ability to flux intracellular calcium compared with a substantial biphasic flux from the naive population (Fig. 5 B). In comparison, the BND population gave a result similar to CD27+IgM/D/G− B cell (class switched) memory population representing both IgA and IgE memory cells, which, as predicted, do not respond to anti-IgD/IgM stimulation. Representative kinetic graphs are shown for 25 μg/ml of anti-IgM/D stimulant, representing three separate donors. Similar trends were also observed when various amounts of stimulant ranging from 10–50 μg/ml were used, representing eight donors in three independent trials. On average, the peak mean fluorescence of Flou-4 was significantly higher for naive versus BND, which is indicative of a more robust flux of intracellular calcium (Fig. 5 E; P < 0.05 by Student's t test). Similarly, BCR cross-linking with anti-κ/λ resulted in reduced BND calcium flux (Fig. S2 B, available at http://www.jem.org/cgi/content/full/jem.20080611/DC1). This difference was not caused by a variegated uptake of the calcium dye Fluo-4 or reduced viability because calcium peaks between the BND and naive populations were equal when treated with the ionophore ionomycin (Fig. 5 D).

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