IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

Abstract

IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans.

Figure 1

IRAK-4 and MyD88 deficiencies impair T-independent IgM responses. Quantity of total and bacterial array carbohydrates bound by serum (A) IgM and (B) IgG. Control (n = 8), IRAK-4 deficient (n = 8), MyD88 deficient (n = 3). (C) Example CFG array results with dotted line illustrating RFU cutoff for bound carbohydrates. Bar represents mean; whiskers represent standard error of the mean. ***P < .001.

Figure 2

IRAK-4 and MyD88 deficiencies impair antibacterial IgM responses. Levels of (A) IgM and (B) IgG specific for SP capsules, PC, SP TA, and SA TA. MyD88-deficient subjects (▲). (C) Amount of C3a released from serum after incubation with designated pneumococcal antigens. Control (n = 8), IRAK-4 deficient (n = 8), MyD88 deficient (n = 3). Data are results from 1 of 2 replicate experiments. (D) Levels of C3a detected in serum after incubation with PC correlate with levels of PC-specific IgM. P value represents the Spearman rank correlation test. *P < .05, **P < .01, ***P < .001.

Figure 3

Specific IgM responses correlate with IgM⁺IgD⁺CD27⁺ B cells. (A) Serum IgM recognizing SP capsules, PC, SP TA, and SA TA have positive correlation with percentage of IgM⁺IgD⁺CD27⁺ B cells. (B) Only serum IgG for SP capsules positively correlates with IgM⁺IgD⁺CD27⁺ B cells. P value represents the Spearman rank correlation test. (C) IgM⁺IgD⁺CD27⁺ B cells as a percentage of total B cells. **P < .01.

Figure 4

Splenectomy impairs specific IgM in correlation with levels of IgM⁺IgD⁺CD27⁺ B cells. (A) IgM⁺IgD⁺CD27⁺ percentage of total B cells. (B) Serum IgM binding SP capsules and (C) PC. (D) S. pneumoniae cell wall, but not capsules, blocks binding of PC-specific IgM. (E) Serum IgM recognizing SP capsules and PC correlate with percentage of IgM⁺IgD⁺CD27⁺ B cells. Controls (□); splenectomized subjects (▪). P value represents the Spearman rank correlation test. Dotted line indicates 95% confidence interval. Control (n = 7); splenectomy (n = 21). Results derived from 1 of 2 replicate experiments. Bar represents mean; whiskers represent standard error of the mean. *P < .05, **P < .01.

Figure 5

PC-specific IgM is produced by human CD27⁺ B cells upon IRAK-4– and MyD88-dependent TLR stimulation. (A) CD27⁺ B cells produce higher levels of total IgM and IgG when stimulated with TLR7 and TLR9 agonists. (B) PC-specific antibodies are produced by CD27⁺IgM⁺ B cells. Data from sorted PBMC samples derived from 6 unique individuals. (C) IRAK-4 and MyD88 deficiencies impair production of total and PC-specific IgM in response to TLR7 and TLR9 stimulation of PBMC cultures. Control (n = 4), IRAK-4/MyD88 deficient (n = 4). *P < .05, **P < .01, ***P < .001.

Figure 6

IRAK-4 and MyD88 deficiencies impair IgD⁺IgM⁺CD27⁺ B-cell proliferation. (A) TLR agonists enrich IgM⁺IgD⁺CD27⁺ B cells in culture of control PBMCs, but not IRAK-4– or MyD88-deficient PBMCs. Gated on CD19⁺, CD27⁺. Representative of 4 experiments. (B) Quantification of total lymphocytes, IgM⁺IgD⁺ and IgM⁻IgD⁻ CD27⁺ B cells with and without TLR stimulation. Data from 4 individuals per group. Box represents mean; whiskers represent standard error of mean. (C) Dilution of CFSE after TLR stimulation. Representative of 3 experiments. *P < .05.