Soluble CD40 ligand contributes to dendritic cell-mediated T-cell dysfunction in HIV-1 infection

Abstract

Objective: Plasma soluble CD40 ligand (sCD40L) is increased during HIV-1 infection, but it is unknown whether it circulates in monomeric or multimeric forms, and whether the circulating forms have differential effects on myeloid dendritic cell function and adaptive regulation.

Design: sCD40L forms were measured in plasma samples from HIV-infected donors. The effects of sCD40L forms on dendritic cell function were measured in vitro.

Methods: To delineate which forms of sCD40L are present in plasma from HIV-infected donors, immunoblots were performed following enrichment of plasma for medium and low-abundance proteins. Dendritic cells from seronegative donors were exposed to multiple forms of sCD40L prior to Toll-like receptor stimulation and dendritic cell function and adaptive regulation was assessed in vitro.

Results: Monomeric and multimeric forms of sCD40L were identified in plasma from antiretroviral therapy-treated HIV-infected donors. Although monomeric and multimeric forms of sCD40L had differential effects on dendritic cell activation when given alone, both strongly suppressed secretion of the Th1 skewing cytokine, interleukin-12, upon subsequent Toll-like receptor stimulation. Furthermore, dendritic cells exposed to both monomeric and multimeric sCD40L induced regulatory T-cell formation and T-cell anergy.

Conclusion: Elevated sCD40L during HIV infection impairs dendritic cell function, contributing to innate and adaptive immune dysfunction. Antiretroviral adjunctive therapies that decrease sCD40L may provide immune modulatory benefits.

Figures

Figure 1. Soluble CD40L circulates in plasma as monomers and multimers, which exert differential effects on DCs

(A) sCD40L forms in plasma from 8 HIV-infected donors and 8 healthy donors were demonstrated by immunoblot analysis. (B) DC purity as measured by FACS, DCs were incubated with either sCD40L monomer (MONO), CD40L multimer (MULTI), as compared to control DC (NEG) overnight and (C) CD86 surface expression was evaluated by flow cytometry and (D) IL-6 and TNF-α production were measured in cell supernatant by cytokine bead array analysis. (E) DCs were incubated with either sCD40L monomer (MONO), CD40L multimer (MULTI) as compared to control DC (NEG) overnight and then were subsequently stimulated overnight with Poly-ICLC, and IL-12 production was measured in cell supernatants by cytokine bead array. Shown in (C), (D), and (E) are data for three donors with statistical analysis using two-tailed Student's t tests. *Represents a p-value

Figure 2. DCs incubated with sCD40L monomeric as compared to multimeric forms have differential effects on naïve CD4+ T cell skewing and proliferation

DCs were incubated with sCD40L monomer (MONO) as compared to CD40L multimer (MULTI) or control DC (NEG) overnight and were then co-cultured with CFSE-labeled allogeneic naïve CD4 T cells where (A) % CFSE dilution is represented as % proliferation and (B) culture supernatants were assayed for IFNγ using the Human CBA. (C) sCD40L-stimulated DCs were subsequently activated with Poly-ICLC and co-cultured with allogeneic naïve CD4+ T cells. Shown in (A), (B), and (C), are data for three donors with statistical analysis using two-tailed Student's t tests. *Represents a p-value

Figure 3. DCs exposed to both monomeric and multimeric sCD40L induce IDO expression and regulatory T cell phenotype

(A) DCs were incubated with sCD40L monomer (M), CD40L multimer (MT), LPS, or control (-) for 24 hours and IDO expression was measured via immunoblot, shown for 2 donors. The relative abundance of IDO is expressed as the ratio of IDO to B Actin signal intensity and is represented as a histogram on the right as the average of three donors. (B) DCs were incubated with sCD40L monomer (M), CD40L multimer (MT), LPS and interferon-γ, or control (-) for 48 hours, washed and then co-cultured with allogeneic naïve CD4 T cells at a ratio of 1:10. Phenotype of the day 7 T cell population is shown for one representative donor, gated on CD4+ T cells. Percentages of cells in each gate (Foxp3+CD127lo) are indicated, and are represented as a histogram on the right as the average of three donors. *Represents a p-value < 0.05 and was considered significant.

Figure 4. DCs exposed to both monomeric and multimeric sCD40L induce anergy, which is not reversed by IDO inhibition

(A) DCs were incubated with sCD40L monomer (MONO), CD40L multimer (MULTI), LPS, LPS and interferon-γ (LPS γ), or control (-) for 48 hours, washed and then co-cultured with allogeneic naïve CD4 T cells at a ratio of 1:10. After 6-7 days, the T cells were stained with CFSE and were mixed at various ratios with allogeneic DCs, from the original DC donor, matured overnight with R848 10uM (3M) (1:10, 1:30 and 1:300; DC: T cell ratio). After 3 days, CFSE dilution was analyzed by FACS. Data is shown for 3 donors with T cell proliferation (CFSE dilution) normalized to untreated T cells (T cells alone) stimulated with R848-DCs. *Represents a p-value