CD4 T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1

Abstract

This report investigates the role of OX40 ligand (OX40L) and its receptor, OX40, expressed on activated B and T cells, respectively, in promoting the differentiation of T helper type 2 (Th2) CD4 T cells. These molecules are expressed in vivo by day 2 after priming with T cell- dependent antigens. Their expression coincides with the appearance of immunoglobulin (Ig)G switch transcripts and mRNA for interleukin (IL)-4 and interferon (IFN)-gamma, suggesting that this molecular interaction plays a role in early cognate interactions between B and T cells. In vitro, we report that costimulation of naive, CD62Lhigh CD4 T cells through OX40 promotes IL-4 expression and upregulates mRNA for the chemokine receptor, blr-1, whose ligand is expressed in B follicles and attracts lymphocytes to this location. Furthermore, T cell stimulation through OX40 inhibits IFN-gamma expression in both CD8 T cells and IL-12-stimulated CD4 T cells. Although this signal initiates IL-4 expression, IL-4 itself is strongly synergistic. Our data suggest that OX40L on antigen-activated B cells instructs naive T cells to differentiate into Th2 cells and migrate into B follicles, where T cell-dependent germinal centers develop.

Figure 1

Induction of OX40 and OX40L after immunization. Time course of induction of mRNA for OX40 and OX40L in popliteal LNs in mice after immunization with NP-CγG (plus killed Bordetella pertussis 5 × 10⁸) or MMTV. Symbols show the amount of mRNA for OX40L (A and B) and OX40 mRNA (C and D) per LN section by semiquantitative PCR. Symbols, Results for individual mice; solid lines, median values.

Figure 2

Intracellular staining for IL-4 and IFN-γ on activated subpopulations of T cells. Purified subpopulations of CD4 and CD8 T cells, which had been activated for 3 d with immobilized mAb to CD3 and soluble mAb to CD28, were restimulated through CD3 for 4 h in the presence of GolgiStop™ to cause accumulation of intracellular cytokines before staining on the FACs^®. Histograms show the proportion of cells positive compared with a negative control mAb: CD4⁺CD62L^low T cells enriched for activated and memory T cells (A) IL-4 and (B) IFN-γ; CD8⁺ CD62L^high naive T cells (C) IL-4 and (D) IFN-γ; and CD4⁺CD62L^hi naive T cells (E) IL-4 and (F) IFN-γ. lo, Low; hi, high.

Figure 3

Induction of IL-4 expression by OX40L. CD62L^high CD8 and CD4 T cells were activated through CD3 and CD28 for 3 d in the presence of a fixed OX40L transfectant or the control parental cell line. Cells were restimulated through CD3 for 4 h in the presence of GolgiStop™ and stained for intracellular expression. (A) Staining with a control mAb (striped bars), anti-CD40L (black bars), anti–IL-4 (white bars), and anti–IFN-γ (stippled bars), on purified CD4 or CD8 CD62L^high T cells. The above data are representative of at least 10 different experiments. (B) Timing of induction of IL-4 expression in naive T cells by OX40L. Cells were stimulated as above. Data are representative of two experiments. hi, High.

Figure 3

Induction of IL-4 expression by OX40L. CD62L^high CD8 and CD4 T cells were activated through CD3 and CD28 for 3 d in the presence of a fixed OX40L transfectant or the control parental cell line. Cells were restimulated through CD3 for 4 h in the presence of GolgiStop™ and stained for intracellular expression. (A) Staining with a control mAb (striped bars), anti-CD40L (black bars), anti–IL-4 (white bars), and anti–IFN-γ (stippled bars), on purified CD4 or CD8 CD62L^high T cells. The above data are representative of at least 10 different experiments. (B) Timing of induction of IL-4 expression in naive T cells by OX40L. Cells were stimulated as above. Data are representative of two experiments. hi, High.

Figure 4

(A) Dependence of OX40L effect on IL-4 and IFN-γ secretion. Purified CD4⁺CD62L^high T cells were stimulated as described in the legend to Fig. 3 in the presence or absence of neutralizing mAbs to IL-4 and IFN-γ at a final concentration of 50 μg/ml. Before restimulation, cells were washed three times in medium to remove excess cytokine antibody. This was done to prevent interference with subsequent staining for intracellular cytokines. A shows the percentage of IL-4– or IFN-γ–positive CD4⁺ T cells when T cells were stimulated alone, with J558L, or with OX40L-fixed transfectants. Data shown are representative of six experiments in Balb/c mice. C57Bl/6 mice gave qualitatively similar results, but the degree of IL-4 expression was less (three experiments). Striped bars, Control; white bars, IL-4; stippled bars, IFN-γ. (B) Effect of OX40L on intracellular expression of IFN-γ in CD4 and CD8 T cells. CD4 and CD8 T cells were activated for 3 d as described in the legend to Fig. 2. To induce IFN-γ expression in CD4 T cells, IL-12 was added to cultures at a final concentration of 50 ng/ml. a, c, e, and g, Effects of OX40L and blocking cytokine mAbs on IFN-γ expression in CD4 T cells. b, d, f, and h, Effects on CD8 T cells. Results are representative of five separate experiments. hi, High.

Figure 4

(A) Dependence of OX40L effect on IL-4 and IFN-γ secretion. Purified CD4⁺CD62L^high T cells were stimulated as described in the legend to Fig. 3 in the presence or absence of neutralizing mAbs to IL-4 and IFN-γ at a final concentration of 50 μg/ml. Before restimulation, cells were washed three times in medium to remove excess cytokine antibody. This was done to prevent interference with subsequent staining for intracellular cytokines. A shows the percentage of IL-4– or IFN-γ–positive CD4⁺ T cells when T cells were stimulated alone, with J558L, or with OX40L-fixed transfectants. Data shown are representative of six experiments in Balb/c mice. C57Bl/6 mice gave qualitatively similar results, but the degree of IL-4 expression was less (three experiments). Striped bars, Control; white bars, IL-4; stippled bars, IFN-γ. (B) Effect of OX40L on intracellular expression of IFN-γ in CD4 and CD8 T cells. CD4 and CD8 T cells were activated for 3 d as described in the legend to Fig. 2. To induce IFN-γ expression in CD4 T cells, IL-12 was added to cultures at a final concentration of 50 ng/ml. a, c, e, and g, Effects of OX40L and blocking cytokine mAbs on IFN-γ expression in CD4 T cells. b, d, f, and h, Effects on CD8 T cells. Results are representative of five separate experiments. hi, High.

Figure 5

OX40L induces IL-4 and blr-1 mRNA in naive CD4 T cells. The ethidium bromide gel shows semiquantitative RT-PCR for IL-4, IFN-γ, and blr-1 in cell samples of activated CD4 CD62L^high T cells at the times indicated after culture. Amounts of mRNA were adjusted to give comparable β-actin signals. T cells had been stimulated in the presence of fixed parental cell line J558L or OX40L transfectant with and without mAb to IL-4 and IFN-γ. RT-PCR of the fixed cell lines did not yield cDNA that could be amplified. Results are representative of three experiments. Nil, no blocking mAb added.