In vitro characterization of cellular responses elicited by endosomal TLR agonists encapsulated in Qβ virus-like particles

Abstract

Background: Despite promising clinical data for Toll-like receptor-9 agonists encapsulated in virus-like particles (TLR9a VLPs), the relative potency and mechanisms of TLR7a and dual TLR7/8a VLPs remain undefined. TLR9a VLPs, also known as Vidutolimod or CMP-001 is a novel TLR9a encapsulated in Qβ VLPs, which can activate plasmacytoid DCs (pDCs) and promote T cell activation. Other endosomal TLRs such as TLR7 and TLR8 expressed in DCs have been studied in several preclinical and clinical studies; however, their immune-activating properties when encapsulated in VLPs have not been tested before. Here, we utilized a series of in vitro experiments to test and compare immune cell activation stimulated by agonists to TLR7 (TLR7a), TLR7/8 (TLR7/8a), and TLR9 (TLR9a) when encapsulated in Qβ VLPs.

Methods: Activation of immune cells (monocytes, natural killer (NK) cells, T cells, pDCs, and monocytic DCs (mDCs)) in response to TLR7a, TLR7/8a and TLR9a VLPs, was evaluated using flow cytometry, intracellular cytokine staining (ICS) and ELISA. Neutralizing cytokine antibodies, immune cell depletion kits and transwell models were used to determine the contribution of select cytokines and antigen presenting cells (APCs) in VLP-mediated immune cell activation.

Results: Results showed that all three VLPs activated pDCs and monocytes. However, TLR7/8a VLPs were most effective at NK and T cell activation compared to the other VLPs. NK cells were a major source of IFNγ, whereas pDCs were the main source of IFNα and TNFα production in response to the VLPs. Neutralizing antibodies against TNFα (but not IFNα) showed significant suppressive effects on TLR7/8a VLP-mediated activation of CD4 + and CD8 + T cells. Depletion of APCs completely abrogated TLR7/8 VLP-mediated activation of CD4 + and CD8 + T cells. Lastly, TLR7/8a VLP-mediated activation of T cells was highly dependent on direct contact with pDCs (and not DC1 and DC2 subsets).

Conclusions: In summary, endosomal TLRa VLPs all have the ability to activate pDCs, however, combined TLR7/8 activation using TLR7/8a VLPs was significantly more effective than the other VLPs at activating T cells and was dependent on direct contact with pDCs. Therefore, TLR7/8a VLPs may potentially induce a robust anti-tumor immune response and warrant further investigation for cancer therapy.

Keywords: CD4; CD8; DCs; T cells; TLR; Vidutolimod.

Fig. 1

Endosomal TLRa VLPs trigger the release of both IFNα and TNFα from human PBMCs. Peripheral blood mononuclear cells (PBMCs) were treated with escalating doses (0.1–50 µg/mL) of virus-like particles (VLPs) encapsulating TLR7, TLR9 and TLR7/8 agonists (in addition to identical doses of anti-Qβ) for 24 h then analyzed for interferon alpha (IFNα) A, tumor necrosis factor alpha (TNFα) B, and interferon gamma (IFNγ) C in cell culture media by ELISA. Bars represent the mean of n = 3 biological replicates. Error bars represent SE from the mean. *p <.05; **p <.01; ***p <.001

Fig. 2

Endosomal TLRa VLPs activate immune cells. Peripheral blood mononuclear cells (PBMCs) were treated with escalating doses (1–50 µg/mL) of virus-like particles (VLPs) encapsulating TLR7, TLR9 and TLR7/8 agonists (in addition to identical doses of anti-Qβ) for 24 h then analyzed by flow cytometry for the indicated activated immune cells. A Flow cytometry gating strategy to identify CD40 + monocytes (antibody-dependent, cell-mediated cytotoxicity), and plasmacytoid dendritic cells (pDCs) B and CD69 + CD4 + T cells (D), CD8 + T cells E and natural killer (NK) cells F from human PBMCs. Average values were normalized to succinate control and plotted as fold change. Bars represent the mean of n = 3 biological replicates. Error bars represent SE from the mean. *p <.05; **p <.01; ***p <.001; ****p <.0001

Fig. 3

Endosomal TLRa VLPs induce the production of cytokines in immune cells. Freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated by the indicated VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) for 2 h then treated with brefeldin A for 16–18 h. Surface and intracellular staining was performed to identify IFNα, TNFα and IFNγ positive monocytes A plasmacytoid dendritic cells (pDCs) B monocytic DCs C CD4 + T cells D CD8 + T cells E, ad natural killer (NK) cells F by flow cytometry analyses. Succinate buffer was used as a control. Error bars represent SE from the mean. Bars represent the mean of n = 3 biological replicates. *p <.05; **p <.01; ***p <.001; ****p <.0001

Fig. 4

TNFα partially mediates TLR7/8a VLP-induced activation of T cells. Freshly isolated peripheral blood mononuclear cells were treated with TLR7/8a VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) with and without neutralizing antibodies (20 µg/mL) to IFNα (nIFNα Ab), TNFα (nTNFα Ab), and nIFNα Ab + nTNFα Ab for 24 h. IgG and succinate buffer were used as controls. Cells were then harvested and analyzed by flow cytometry for CD4 + T cell A CD8 + T cell B and NK cell C activation. Error bars represent SE from the mean. Bars represent the mean of n = 3 biological replicates. **, ***, and **** indicates p <.01, p <.001, and p <.0001 versus Succinate. ¥ indicates p <.05 versus TLR7/8 VLP

Fig. 5

TLR7/8a VLP-mediated T cell activation is dependent on antigen presenting cells. T cells were isolated from unfractionated PBMCs (A) and treated with TLR7/8a VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) for 24 h then analyzed for CD69 surface expression on CD4+ B and CD8+ C T cells. Subsets of immune cells (NK cells, monocytes and APCs) were depleted from PBMCs A treated with TLR7/8a VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) for 24 h then analyzed for CD69 surface expression on CD4+ D and CD8+ E and NK cells F. Succinate buffer (Succ) was used as a control. Average values were normalized to succinate control and plotted as fold change. Error bars represent SE from the mean. Bars represent the mean of n = 3 biological replicates. ***p <.001; ns: non-significant

Fig. 6

TLR7/8a VLP-mediated activation of T cells is dependent on pDC help. Subsets of dendritic cells (pDC, cDC1 and cDC2) were isolated from fresh PBMCs and co-cultured with freshly isolated T cells from the same donor. The DC: T cell co-cultures were treated with TLR7/8a VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) for 24 h and analyzed by flow cytometry for CD69 surface expression on CD4+ B and CD8 + T C cells. Succinate, PBS and EMPTY VLPs were used as controls. Average values were normalized to PBS control and plotted as fold change. Bars represent the mean of n = 3 biological replicates. *p <.05; **p <.01; ***p <.001; ****p <.0001

Fig. 7

TLR7/8a VLP-mediated T cell activation through pDCs is contact dependent. Human T cells and pDCs were isolated from PBMCs then cultured in transwell plates where pDC and T cells were separated by a 0.4 μm pore polycarbonate membrane insert. pDCs were treated with TLR7/8a VLPs (10 µg/mL) plus anti-Qβ (10 µg/mL) for 24 h. Succinate was used as a control. Cells from the bottom chamber were stained for CD304 expression to check for any pDCs that migrated across the membrane A. T cells from the bottom chamber were collected and analyzed through flow cytometry for changes in CD69 surface expression on CD4+ B and CD8+ C T cells. Average values were normalized to succinate control and plotted as fold change. Error bars represent SE from the mean. *p <.01 versus succinate control; ns: non-significant

Fig. 8

Graphical illustration of TLRa VLP-mediated immune cell activation and potential anti-tumor immune response