Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF-κB Ligand, and TNF-α in the Activation of Dendritic Cells and the Expansion of Viral Specific CD8+ T Cell Memory Responses in HIV-1-Infected and HIV-1-Uninfected Individuals

Abstract

Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-κB ligand (RANKL), and TNF-α, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-α alone to mature DCs and to expand CTL. In healthy volunteers, TNF-α or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF-α or RANKL in combination with CD40L were variable. TNF-α and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-α enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4+ T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4+ T cells can provide help to CD8+ T cells independently of CD40L, RANKL, or TNF-α.

CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

“…The method for generation of MDDCs has been described previously [26]. Briefly, purified monocytes obtained using Monocyte Negative Isolation Kit (Dynal, Oslo, Norway) were cultured in RPMI-1640 medium supplemented with 10% FBS, 2mM L-glutamine, 25mM HEPES, 100 U/ml of penicillin and 100μg/ml streptomycin in the presence of 50ng/ml human rGM-CSF and 100ng/ml human rIL-4 (PeproTech, Rocky Hill, NJ).…”

“…Peptide-specific CTL were expanded by a modification of a method previously described [26]. Briefly, CD4 + T cell-containing or -depleted PBMCs were cocultured for 10 d with autologous MDDCs that were either pulsed or not pulsed with HLA-restricted epitopes of HIV-1 proteins or EBV proteins.…”

“…A recombinant canarypox vector expressing a membrane form of human CD40L, vA3131-2, was constructed and expression of human CD40L in infected cells was verified by flow cytometry and Western blot (data not shown). Previous studies show that CD40L could activate and mature human monocyte derived dendritic cells, which then can expand immune responses [26,37,38]. Therefore, we first studied the effect of CD40L expressing from the canarypox vector on human immature monocyte derived dendritic cells (iMDDCs).…”

“…To determine whether CD40L expressed from canarypox vector can expand virus-specific memory CTL responses, we used an in vitro co-culture method in which peptide-pulsed MDDCs stimulate CD8 + T cells in the absence of exogenous cytokines, as previously described [26]. In this co-culture system, expansion of CD8 + T cells is highly dependent on the presence of CD4 + T cells providing help.…”

“…We previously reported that soluble recombinant human CD40L protein could enhance ex vivo viral specific CD8 + T cell memory responses in HIV-1-infected and healthy individuals [25][26][27]. To test whether CD40L can be used as an adjuvant for HIV-1 ALVAC vaccines, we constructed recombinant canarypox viruses expressing CD40L and evaluated their effect on immune responses elicited by HIV-1 ALVAC vaccine in mice.…”