The Antigen Presenting Potential of CD21low B Cells

Abstract

Human CD21^low B cells are expanded in autoimmune (AI) diseases and display a unique phenotype with high expression of co-stimulatory molecules, compatible with a potential role as antigen-presenting cells (APCs). Thus, we addressed the co-stimulatory capacity of naïve-like, IgM-memory, switched memory and CD27^negIgD^neg memory CD21^low B cells in allogenic co-cultures with CD4 T cells. CD21^low B cells of patients with AI disorders expressed high levels of not only CD86, CD80, and HLA-DR (memory B cells) but also PD-L1 ex vivo and efficiently co-stimulated CD4 T cells of healthy donors (HD), as measured by upregulation of CD25, CD69, inducible co-stimulator (ICOS), and programmed cell death protein 1 (PD-1) and induction of cytokines. While the co-stimulatory capacity of the different CD21^low B-cell populations was over all comparable to CD21^pos counterparts of patients and HD, especially switched memory CD21^low B cells lacked the increased capacity of CD21^pos switched memory B-cells to induce high expression of ICOS, IL-2, IL-10, and IFN-γ. Acknowledging the limitation of the in vitro setting, CD21^low B cells do not seem to preferentially support a specific T_h effector response. In summary, our data implies that CD21^low B cells of patients with AI diseases can become competent APCs and may, when enriched for autoreactive B-cell receptors (BCR), potentially contribute to AI reactions as cognate interaction partners of autoreactive T cells at sites of inflammation.

Keywords: CD21low B cells; T cell; antigen-presenting cells; autoimmunity; co-stimulation.

Figure 1

(A) General gating strategy for different CD21^low and CD21^pos B-cell subsets. CD19^pos B cells delineated by CD19 and CD21 were separated into naïve (a), IgM memory (IgM mem) (b), switched memory (sw mem) (c), and CD27^negIgD^neg memory (CD27^negIgD^neg mem) B cells (d) by expression of IgD and CD27. Subsequent gating on CD21^posCD38^{low-intermediate} and CD21^lowCD38^low B cells allowed the differentiation of CD21^pos and CD21^low B cells, respectively. (B) FACS plots showing CD21 and CD11c, FCRL5, CD95, ICAM-1, CD40 and CXCR5 in naive, IgM mem, sw mem and CD27^negIgD^neg mem B cells in one representative AI patient. (C) Graphs show the MFI of CD80, CD86 and HLA-DR in CD21^pos B-cell subpopulations of healthy donors (HD) and CD21^pos and CD21^low B-cell subsets of patients with autoimmune (AI) diseases in naïve, IgM mem, sw mem and CD27^negIgD^neg mem B cells. Statistical differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RA, SLE, sarcoidosis, psoriatic arthritis, and spondyloarthropathy were marked by different symbols as indicated in the legend. ns, not significant.

Figure 2

(A) Representative histogram overlays for the expression of CD80 and CD86 on naïve B cells of HD unstimulated, stimulated with SEB, CpG and in T-B co-cultures without or in the presence of SEB. (B) Histogram overlays for CD80 (upper line) and CD86 (bottom line) of naïve, IgM mem, switched mem and CD27^negIgD^neg mem CD21^pos B cells of one representative HD (black solid line) and CD21^pos (blue solid line) and CD21^low B cells (red solid line) of one representative AI patient in Staphylococcus enterotoxin B (SEB-) stimulated co-cultures with allogenic T cells and statistical analysis thereof. Differences were considered significant at *p < 0.05 and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Figure 3

(A) Representative histogram overlay displaying CD69 expression in unstimulated T cells (T only) or T-B co-cultures with naïve CD21^pos B cells of a HD or naïve CD21^low B cells of an AI patients in the absence (T + (B) or presence of SEB (T + B + SEB) gated on naïve T cells. (B–D) Histogram overlays for CD69, CD25 and Inducible Co-stimulator (ICOS) on naïve (CD45RA^pos) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27^negIgD^neg mem B-cell populations of CD21^pos B cells of one representative HD and CD21^pos and CD21^low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. Subsets marked in green (*) were significantly different to the SEB-stimulated T-cell control (p < 0.05). All subsets were statistically significant to unstimulated T cells. Differences were considered significant at *p < 0.05 * and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Figure 4

Histogram overlays for CD69 (A), CD25 (B), and ICOS (C) expression on memory (CD45RA^neg) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27^negIgD^neg mem B-cell populations of CD21^pos B cells of one representative HD and CD21^pos and CD21^low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. (D) Representative FACS plot for CD4 and PD-1 or the FMO in unstimulated T cells, T cells stimulated with SEB and T cells co-cultured with naïve CD21^pos B cells of HD or naive CD21^low B cells of a patient in the absence or presence of SEB. Graph shows the proportion of PD-1 positive memory T cells in T-cell cultures stimulated as indicated to unstimulated T cells. Subsets marked with green stars were significantly different to the SEB-stimulated T-cell controls. Differences were considered significant at *p < 0.05. The different diseases were marked by different symbols as indicated in the legend. **p < 0.01.

Figure 5

(A) Cytokines measured in supernatants of T-B co-cultures. Production of TNF-α, IL-2, IFN-γ, IL-4, and IL-10 was measured in T-B co-cultures with different B-cell subsets of AI patients and HD. Subsets marked in green were significantly different to the SEB-stimulated T-cell control. (B) Intracellular cytokine staining of TNF-α and IL-10 versus CD19 in T-B co-cultures with and without SEB and T-cell SEB control. Representative FACS Plots of co-cultures with naïve B cells of HD are displayed. Data are representative for 1–3 times for each CD21^low B-cell subpopulation of patients and 2–4 times of each CD21^pos B-cell subpopulations of HD in 4 independent experiments. Differences were considered significant at *p < 0.05 and **p < 0.01.