B cell–intrinsic TLR signals amplify but are not required for humoral immunity

Although innate signals driven by Toll-like receptors (TLRs) play a crucial role in T-dependent immune responses and serological memory, the precise cellular and time-dependent requirements for such signals remain poorly defined. To directly address the role for B cell–intrinsic TLR signals in these events, we compared the TLR response profile of germinal center (GC) versus naive mature B cell subsets. TLR responsiveness was markedly up-regulated during the GC reaction, and this change correlated with altered expression of the key adaptors MyD88, Mal, and IRAK-M. To assess the role for B cell–intrinsic signals in vivo, we transferred MyD88 wild-type or knockout B cells into B cell–deficient μMT mice and immunized recipient animals with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma globulin. All recipients exhibited similar increases in NP-specific antibody titers during primary, secondary, and long-term memory responses. The addition of lipopolysaccharide to the immunogen enhanced B cell-intrinsic, MyD88-dependent NP-specific immunoglobulin (Ig)M production, whereas NP-specific IgG increased independently of TLR signaling in B cells. Our data demonstrate that B cell–intrinsic TLR responses are up-regulated during the GC reaction, and that this change significantly promotes antigen-specific IgM production in association with TLR ligands. However, B cell–intrinsic TLR signals are not required for antibody production or maintenance

Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation

We have characterized a distinct, late transitional B cell subset, CD21int transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21int T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21int T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21int T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21int T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development

WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity

In a manner dependent on CD4 T cell help and Toll-like receptor signals, B cells lacking WASp induce autoantibody production and autoimmune disease in mice

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF 20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1β and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care

Involvement of proposed B-1 cells.

<p>(A) Absolute number of CD19⁺CD27⁺CD20⁺CD70^-CD69^-CD43⁺ B cells including CD27⁺⁺ CD38⁺⁺PC/PB after Pneumovax^®23 vaccination evaluated by flow cytometry (n = 21). (<b>B</b>) Absolute number of CD19⁺CD20⁺CD27⁺CD38^lo/intCD43⁺ B cells after exclusion of CD38⁺⁺CD27⁺⁺ PC/PB. Mean values ± SEM are indicated for each time point; (*p<0.05, ***p<0.001).</p

Immunization with Pneumovax^®23 leads to transiently decreased numbers of transitional and naïve B cells.

<p>(<b>A</b>) Absolute numbers of transitional (T1+T2) and naïve B cells after immunization. Thirteen independently performed experiments with n = 21. (<b>B</b>) Expression levels of surface IgM on CD27^-CD38^hiCD24^hi T1, CD27^-CD38^intCD24^hi T2 and CD27^-CD38^loCD24^lo naïve B cells compared between day 0 and 28. (<b>C</b>) Cell cycle analysis of transitional 1 and 2 and naive B cells (n = 9). Mean values ± SEM are indicated for each time point (*p<0.05 **p<0.01 ***p<0.001).</p

Kinetics of PnPS-specific IgA, IgM and IgG antibody secreting cells and antibodies.

<p>(<b>A</b>) Upper panel: PnPS-specific IgM, IgA and IgG as determined by ELISpot assay (n = 21). Lower panel: Representative ELISpot wells of one donor shown for IgA-producing PBMC on day 0, 7, and 14 after vaccination with Pneumovax^®23. (<b>B</b>) PnPS-specific IgM, IgA and IgG ASC in sort-purified CD19⁺CD27⁺⁺ plasma blasts/plasma cells, CD19⁺CD27⁺ memory B cells and CD19⁺CD27^- naïve B cells at day 7 post-vaccination. (<b>C</b>) Dynamics of PnPS-specific antibody responses in serum from day 0 to day 90 determined by ELISA (n = 30). (<b>D</b>) Positive correlation between PnPS-specific serum IgA on day 7 and IgA-producing cells. (<b>E</b>) Sputum/serum ratio of PnPs-specific Ig on day 28. (<b>F</b>) Positive correlation between PnPS-specific serum IgA on day 14 and IgA in sputum on day 28. Mean values ± SEM are indicated for each time point; (*p<0.05, **p<0.01, ***p<0.001).</p