CD4+ T cells and CD40 participate in selection and homeostasis of peripheral B cells

Abstract

Control of peripheral B cell development and homeostasis depends critically on coordinate signals received through the BAFFRs and BCRs. The extent to which other signals contribute to this process, however, remains undefined. We present data indicating that CD4(+) T cells directly influence naive B cell development via CD40 signaling. Loss of CD4(+) T cells or CD40-CD40L interaction leads to reduced B cell homeostatic proliferation and hindered B cell reconstitution posttransplantation. Furthermore, we demonstrate that in the absence of CD40 signals, these events are modulated by BCR self-reactivity. Strikingly, murine models lacking CD40 reveal a broadly altered BCR specificity and limited diversity by both single-cell cloning and high-throughput sequencing techniques. Collectively, our results imply that any setting of T cell lymphopenia or reduced CD40 function, including B cell recovery following transplantation, will impact the naive B cell repertoire.

FIGURE 1.

CD40 signaling promotes B cell HP. (A–E) B cell HP measured by transfer of splenic B cells into B cell–deficient μMT hosts. Ly5.2 congenically marked WT or CD40 KO B cells were transplanted in Ly5.1 μMT–recipient mice (A) Gating on congenically marked donor B cells (left panel) and proliferating cells by CFSE dilution (right panel). (B) B cell HP measured 5–7 d after transfer of WT B cells to μMT mice. Left panel, Mice receiving WT B cells were treated with either isotype control or CD4-depleting Ab. Right panel, Transfer of WT versus CD40 KO B cells to μMT recipients. (C) Summary of HP data from multiple experiments, data are normalized to either WT B cells or isotype control treated recipients for each individual experiment. (D) Transfer of B cells and/or CD40 sufficient or deficient CD4⁺ T cells into Rag KO recipients. (E) Surface phenotype of WT donor B cells prior to transfer into μMT recipients for HP experiment (left panel) and after HP (middle panel); CD40 KO B cells after HP (right panel). (F) HP experiment using donor B cells from M167-Tg mice, showing proliferation of Id⁻ and Id⁺ cells (left panel) and the frequency of Id⁺ cells in nonproliferating or proliferating cells (right panel). (G) KREC assay on sorted FM and MZ cells from WT, CD40 KO, and CD40L KO mice. (H) Serum BAFF levels measured by ELISA in WT, CD40 KO, and CD40L KO mice. Error bars show SEM. Data representative of at least three experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.

FIGURE 2.

CD40 signaling promotes B cell development in mixed BM chimeras. (A) WT/CD40 KO–mixed BM chimeras created by transfer of congenically marked BM to μMT recipients. (B) The percentage of ly5.1⁺ (WT) and ly5.2⁺ (CD40 KO) cells in each B cell subset at 3 mo posttransplant using a 65:35 (CD40 KO:WT) ratio of donor BM, either untreated (left panel) or depleted of CD4 T cells (right panel); n = 23 mice. (C) Analysis of BM chimeras created with a 90:10 (CD40 KO:WT) ratio of donor BM, untreated (left panel), or depleted of CD4 T cells (right panel); n = 26 mice. (D) Mean BAFFR and TACI surface expression in BM chimeras created using both ly5.1⁺ (WT) and ly5.2⁺ (CD40 KO) WT donors, either untreated (left panels) or depleted of CD4 T cells (right panels); n = 26 mice. Surface expression of BAFFR (E) and TACI (F) after in vitro stimulation of B cells with agonistic anti-CD40 and anti-IgM Abs. (G) Quantitative PCR of subsets sorted from mixed BM chimeras; mRNA levels of A1 (top panel) and BclxL (bottom panel) relative to β₂-microglobulin. (H) Cell cycle analysis by DAPI and PyroninY staining; percentage of cells in a combined S and G2 gate in WT/CD40 KO chimeras and control chimeras created using WT ly5.1⁺ and WT ly5.2⁺ BM. (I) KRECs assay on FM and MZ cell subsets sorted from untreated and CD4-depleted 65:35 CD40 KO:WT mixed BM chimeras; n = 8 mice. Error bars show SEM. Data representative of at least three experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.

FIGURE 3.

B cell development depends on CD40 in the absence of BCR signaling. (A) B cell subset gating on xid mice (left panel) and xid mice depleted of CD4 T cells (right panel). (B) Average splenic B cell subset percentages (left panel) and absolute numbers (right panel) in xid mice and xid mice depleted of CD4 T cells. (C) B cell subset gating on tec/Btk DKO mice treated with isotype (left panel) or CD4-depleting Ab (right panel). (D) Average splenic B cell subset percentages (left panel) and numbers (right panel) in tec/Btk DKO mice treated with isotype or CD4-depleting Ab. (E) B cell subset percentages (left panel) and numbers (right panel) in MD4-Tg mice and MD4 × CD40 KO mice. Error bars show SEM. Data representative of at least two experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.

FIGURE 4.

Increased Ag-mediated positive selection in the absence of CD40. (A–D) Selection of Id⁺ cells analyzed in CD40^−/− × M167-Tg mice versus CD40^+/− littermate controls. Comparison of splenic B cell subset percentages (A) and numbers (B). Relative frequency (C) and total number (D) of Id⁺ cells in splenic B cell subsets. (E and F) Analysis of mixed BM chimeras using both CD40^+/− × M167 and CD40^−/− × M167 donor BM at a ratio of 65:35, respectively. (E) Frequency of CD40^pos and CD40^neg cells in B cell subsets. (F) Percentage M167 Id⁺ cells in CD40^pos and CD40^neg B cell subsets. Error bars show SEM. Data representative of at least two experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.

FIGURE 5.

Molecular characteristics and specificity profiles of the naive BCR repertoire in WT and CD40 KO mice. (A) Relative frequency of each ANA-IFA pattern in cloned BCRs, with total number of Abs evaluated for each subset shown in center of pie charts. (B) Autoantigen array data generated using Abs derived from WT versus CD40 KO FM and MZ cells. 60 Abs at equal concentration were pooled for each subset and normalized signal intensities plotted for each Ag. (C) BCR IgH sequence characteristics, specifically N-1 addition of cloned rAbs. Total number of BCR clones sequenced are 54 FM negative, 18 WT FM positive, 23 CD40 KO FM positive, 41 MZ negative, 22 WT MZ positive, and 22 CD40 KO MZ positive. (D–F) High-throughput BCR H chain sequencing of FM and MZ subsets sorted from WT and CD40 KO mice and 90:10 CD40 KO:WT mixed bone marrow chimeric mice. Analysis includes CDRH3 length of WT and CD40 KO mice (D), average Kyte–Doolittle hydrophobicity index (E), and N-1 addition of sorted subsets in mixed BM chimeras (F). (G–I) BCR diversity analyzed by sequencing splenic B cells derived from Vk8-Tg versusVk8-CD40^−/−-Tg mice showing empirical cumulative distribution function for VDJ combination frequencies in Vk8-Tg and Vk8CD40^−/− B cells (G), individual rarefaction demonstrating the approach to saturation of VDJ combinatorial diversity as sequencing depth increases (H), and diversity index calculations using SPADE (I). Error bars show SEM. Data representative of at least two experiments. *p < 0.05, **p < 0.005, ***p < 0.0005.