Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival

Abstract

Chronic engagement of the B cell receptor by soluble autoantigen leads to reduced B cell survival. Using the Ig and hen egg lysozyme double transgenic mouse model, we demonstrate that the survival of soluble autoantigen-engaged B cells is further reduced in mice lacking CD4 T cells or deficient in CD40. Mixed bone marrow chimera experiments reveal that, under homeostatic conditions, the CD40L-CD40 pathway can augment autoreactive B cell survival in a non-cell-autonomous manner. Naive CD4 T cells are shown to constitutively express CD40L mRNA and protein, although cell surface CD40L abundance is low because of engagement with CD40 on other cells. These observations indicate that the CD40L-CD40 pathway can augment the survival of autoantigen-engaged B cells in the absence of T cell activation. We propose that constitutive CD40L expression by naive CD4 T cells influences the composition of the B cell repertoire and may also affect the homeostasis of other cell types such as regulatory T cells in lymphoid organs.

Fig. 1.

Reduced survival of HEL autoantigen-binding B cells in T cell-deficient mice and in mice lacking CD40. (A) Adult mice of the indicated types were given water containing BrdU for 5 days and analyzed. Spleen and lymph node (LN) cells were stained for CD19, HEL, AA4, and CD23 to distinguish immature (AA4+CD23−), transitional (T2- and T3-inclusive and AA4+CD23+), and mature (AA4−CD23+) HEL-binding B cells (8). Bone marrow (BM) cells were stained for B220, IgM, and IgD, and immature B cells were identified as B220+IgM+IgD−. Spleen cells were also stained for B220, HEL binding, and BrdU. Data show three to six mice per group from two separate experiments. (B) Bone marrow from CD40^+/+ Ig Tg or CD40^−/− Ig Tg mice was used to reconstitute lethally irradiated non-Tg and HEL Tg recipient mice. After 6–8 weeks, mice were given water containing BrdU for 5 days and then analyzed as in A. Data show six mice per group from one set of bone marrow chimeras and are representative of two experiments. (C) Examples of AA4 and CD23 profiles of splenic B cells of mice in B. Cells were stained for B220, HEL binding, CD23, and AA4 and pregated on size, B220, and HEL binding (B220⁺HEL⁺). (D) Bone marrow from CD45.1-positive CD40^+/+ Ig Tg mice and CD45.2-positive CD40^+/+ Ig Tg or CD45.2-positive CD40^−/− Ig Tg mice was mixed at a 1:1 ratio and used to reconstitute lethally irradiated HEL Tg recipient mice. After 6–8 weeks, spleen cells were stained for CD45.1, CD19, AA4, CD23, and HEL-binding. Data show three to four mice per group from one set of bone marrow chimeras and are representative of two experiments. ∗, P < 0.002 by Student’s t test.

Fig. 2.

CD40L blocking and depletion of CD4 T cells lead to similar reductions in autoantigen-binding B cell numbers. (A) Non-Tg, Ig Tg, and Ig/HEL Tg mice were treated with 250 μg of anti-mouse CD40L (clone MR1) or control IgG on days 0, 2, and 5. After 7 days, HEL binding (in Ig Tg and Ig/HEL Tg mice) or total (in non-Tg mice) B cells in spleen and lymph nodes were enumerated. Ig/HEL Tg data show six mice from two experiments; non-Tg and Ig Tg data show three mice from one experiment. (B) CD45.1-positive Ig/HEL Tg cells were transferred into CD45.2-positive Ig/HEL Tg recipients 1 h after treatment of recipient mice with anti-CD40L or control antibody. Mice were treated and analyzed as in A by using anti-CD45.1 to identify the transferred cells. Data show five to six mice from two experiments. (C) Depletion of CD4⁺ T cells leads to decreased survival of autoreactive B cells. Ig/HEL Tg mice were treated with 1 mg of anti-mouse CD4 (clone GK1.5) (d0, d4) to deplete CD4 T cells. After 9 days, HEL-binding B cells were enumerated as in A and B. Data show nine mice from three separate experiments. Tx, antibody-treated. ∗, P < 0.05 by Student’s t test.

Fig. 3.

CD40L is constitutively expressed by naive CD4 T cells. (A) CD40L transcript abundance in spleen tissue harvested from WT (n = 5), CD40^−/− (n = 5), or TCR-β^−/−δ^−/− (n = 3) mice. CD40L and hypoxanthine phosphoribosyltransferase (HPRT) transcripts were quantitated by real-time PCR. (B) CD40L transcript abundance in T cell subsets sorted from WT splenocytes by using the following criteria: total CD4⁺, total CD8⁺, naive CD4⁺ (CD4⁺CD25⁻CD62L^hiCD44^lo), naive CD8⁺ (CD8⁺ CD62L^hi), and CD4⁺CD25⁺. Data show results from two separate experiments. Fresh and PMA and ionomycin (P/I)-treated CD4⁺ T cells from spleens were prepared as in Materials and Methods. (C and D) Flow cytometric analysis of CD40L expression by CD4 and CD8 T cells. Spleen and lymph node (LN) cells from WT, CD40^−/−, and CD40L^−/− mice were stained for CD4, CD8, CD62L, CD25, and CD40L. Data are representative of more than five experiments. SA-PE, streptavidin phycoerythrin. (E and F) Flow cytometric analysis of CD40L (E) and CD69 (F) expression on in vitro incubated WT CD4 cells. Cells in the absence or presence of puromycin (puro) were incubated on ice (fresh) or at 37°C without (rest) or with P/I for 2 or 4 h. Cells were harvested and stained for CD4, CD40L, l-selectin, and CD69. The background staining of CD40L^−/− cells is included for comparison.

Fig. 4.

CD40 on B cells, as well as non-B cells, causes the down-modulation of CD40L on naive T cells. (A) CD40L expression in WT, CD40^−/−, and μMT spleen, lymph nodes (LN), and blood, with the corresponding CD40L^−/− cell populations used as staining controls. CD40L expression on CD4+CD62L^hi cells is shown, and the data are representative of five mice of each type from four experiments. (B) CD40L expression on CD4⁺CD62L^hi T cells in CD40^−/− hosts 2.5 days after transfer of 5 × 10⁶ CD40^+/+ B cells compared with T cells from WT or CD40^−/− mice not receiving transferred cells. (C) CD40L expression on CD4⁺CD62L^hi T cells in CD40^−/− hosts 3 h or 2.5 days after transfer of WT or CD40^−/− splenocytes containing 10⁷ B cells. Data in B and C are representative of three experiments. SA-PE, streptavidin phycoerythrin; SA-APC, streptavidin allophycocyanin.