B-cell intrinsic and extrinsic signals that regulate central tolerance of mouse and human B cells

Abstract

The random recombination of immunoglobulin V(D)J gene segments produces unique IgM antibodies that serve as the antigen receptor for each developing B cell. Hence, the newly formed B cell repertoire is comprised of a variety of specificities that display a range of reactivity with self-antigens. Newly generated IgM⁺ immature B cells that are non-autoreactive or that bind self-antigen with low avidity are licensed to leave the bone marrow with their intact antigen receptor and to travel via the blood to the peripheral lymphoid tissue for further selection and maturation. In contrast, clones with medium to high avidity for self-antigen remain within the marrow and undergo central tolerance, a process that revises their antigen receptor or eliminates the autoreactive B cell altogether. Thus, central B cell tolerance is critical for reducing the autoreactive capacity and avidity for self-antigen of our circulating B cell repertoire. Bone marrow cultures and mouse models have been instrumental for understanding the mechanisms that regulate the selection of bone marrow B cells. Here, we review recent studies that have shed new light on the contribution of the ERK, PI3K, and CXCR4 signaling pathways in the selection of mouse and human immature B cells that either bind or do not bind self-antigen.

Keywords: B cell tolerance; CXCR4; ERK; FOXO1; PI3K; autoimmunity; receptor editing.

Figure 1:. Requisite biological activities for the selection of immature B cells out of the bone marrow.

The activities listed in this box are indispensable for the selection into the periphery of newly generated B cells that retain their selected antibody and have a chance to survival and participation in immune responses.

Figure 2:. Phenotype of mouse and human immature B cells in relation to self-antigen binding.

A) Schematic representation of BCR surface expression and internalization on immature B cells responding to self-antigen with various degree of avidity. B) Markers that distinguish mouse and human immature B cells that do not engage (or minimally engage) self-antigen from those that engage self-antigen with high avidity. Markers listed in the middle are expressed by all (mouse or human) immature B cells independent of self-reactivity. Markers listed on the left or right of the scheme are those that, by their lower or higher degree of expression, distinguish non-autoreactive from high-avidity autoreactive cells.

Figure 3:. Expression of PTEN and SHIP-1 phosphatases in immature B cells.

A) Analysis of PTEN and SHIP in bone marrow immature B cells from 3–83Igi,H-2^b autoreactive (AUT) mice, expressing or not the active PI3Kα molecule P110*. Immature B cells were gated as B220⁺CD24^highCD21^– (and GFP⁺ for P110*-mb1Cre cells). Histogram plots show representative intracellular (IC) expression of PTEN and SHIP-1 in gated immature B cells. The bar graphs display mean and SD of PTEN and SHIP-1 geometric MFI form three mice per group. Symbols represent individual mice. B) Intracellular PTEN expression in 3–83 immature B cells (gated as B220⁺CD24^highIgD^–) from 3–83Igi,H-2^d (non-autoreactive, NA) and 3–83Igi,H-2^b (autoreactive, AUT) mice. Bar graph shows mean and SD from three mice per group. Symbols represent individual mice. Statistical analyses in all bar graphs were performed with a one-tailed Student’s t test. *p≤0.05; **p≤0.01.

Figure 4:. Schematic of PI3K contribution to bone marrow selection of immature B cells.

This schematic illustrates the central role PI3K plays during the selection of immature B cells, regulating molecules that enforce or relax central tolerance. Autoreactive immature B cells must diminish PI3K activity to remain in the bone marrow (via CXCR4 upregulation) and undergo receptor editing (via FOXO1). Non-autoreactive immature B cells must activate PI3K to stop VJ recombination and preserve their selected BCR (via suppressing FOXO1), and to exit the bone marrow, enter the peripheral tissue, and further their differentiation (via downmodulating CXCR4 and upregulating BAFFR).

Figure 5:. Relative S1PR and CXCR4 contribution to the bone marrow retention of autoreactive mouse and human immature B cells.

A) Schematic of the longitudinal treatment of mice and hu-mice with either FTY720 or DMSO followed by AMD3100. Blood cells were analyzed before all treatments, after FTY720 or DMSO treatment, and one hour after the last AMD3100 injection. B) Top: Representative flow cytometric analysis of 3–83Igi,H-2^b mouse immature/transitional B cells (B220⁺CD19⁺CD2⁺CD24^highIgλ^–) gated as shown in Figure S1A. The plots show the gating of IgM^lowIgD^low autoreactive immature B cells. Bottom: Frequencies (mean and SD) of IgM^lowIgD^low (left graph) and IC-Igκ⁺IgM^lowIgD^low (right graph) mouse autoreactive immature B cells within all CD24^high immature/transitional B cells in PBMCs from 3–83Igi,H-2^b treated mice (N=4–5 mice per group). Statistical analyses were performed with a one-tailed Student’s t test. C) Left: Representative flow cytometric analysis of Hcκ hu-mice human immature/transitional B cells (hCD45⁺CD20⁺CD24^highCD38^high IgM⁺ or Igκ⁺) gated as shown in Figure S1D. The plots show the gating of IC-Igκ⁺IgM^low autoreactive immature B cells. Right: Frequencies (mean and SD) of IC-Igκ⁺CD20⁺ human autoreactive immature B cells within all (IgM⁺ or Igκ⁺) CD24^highCD38^high immature/transitional B cells in PBMCs from treated hu-mice (N=5–8 hu-mice per group). Statistical analyses were performed with one-way ANOVA. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001; ns=not significant.

Figure 6:. Contribution of CXCR4 signaling to the retention and release of bone marrow immature B cells.

Expression of CXCR4 progressively decreases from pre-B cells to IgM⁺ immature B cells. Immature B cells that engage self-antigen (i.e., autoreactive cells) do not downmodulate CXCR4, and signaling by this chemokine receptor is critical for their retention in the bone marrow tissue where they undergo receptor editing. CXCR4 antagonism by the drug AMD3100 releases autoreactive immature B cells into the circulation from where they relocate into the spleen.