Dendritic Cell Maturation Regulates TSPAN7 Function in HIV-1 Transfer to CD4+ T Lymphocytes

Abstract

Dendritic cells (DCs) serve a key function in host defense, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. DCs express cell surface receptors for HIV-1 entry, but are relatively resistant to productive viral replication. They do, however, facilitate infection of co-cultured T-helper cells through a process referred to as trans-infection. We previously showed that tetraspanin 7 (TSPAN7), a transmembrane protein, is involved, through positive regulation of actin nucleation, in the transfer of HIV-1 from the dendrites of immature monocyte-derived DCs (iMDDCs) to activated CD4⁺ T lymphocytes. Various molecular mechanisms have been described regarding HIV-1 trans-infection and seem to depend on DC maturation status. We sought to investigate the crosstalk between DC maturation status, TSPAN7 expression and trans-infection. We followed trans-infection through co-culture of iMDDCs with CD4⁺ T lymphocytes, in the presence of CXCR4-tropic replicative-competent HIV-1 expressing GFP. T cell infection, DC maturation status and dendrite morphogenesis were assessed through time both by flow cytometry and confocal microscopy. Our previously described TSPAN7/actin nucleation-dependent mechanism of HIV-1 transfer appeared to be mostly observed during the first 20 h of co-culture experiments and to be independent of HIV replication. In the course of co-culture experiments, we observed a progressive maturation of MDDCs, correlated with a decrease in TSPAN7 expression, a drastic loss of dendrites and a change in the shape of DCs. A TSPAN7 and actin nucleation-independent mechanism of trans-infection, relying on HIV-1 replication, was then at play. We discovered that TSPAN7 expression is downregulated in response to different innate immune stimuli driving DC maturation, explaining the requirement for a TSPAN7/actin nucleation-independent mechanism of HIV transfer from mature MDDCs (mMDDCs) to T lymphocytes. As previously described, this mechanism relies on the capture of HIV-1 by the I-type lectin CD169/Siglec-1 on mMDDCs and the formation of a "big invaginated pocket" at the surface of DCs, both events being tightly regulated by DC maturation. Interestingly, in iMDDCs, although CD169/Siglec-1 can capture HIV-1, this capture does not lead to HIV-1 transfer to T lymphocytes.

Keywords: HIV-1; TSPAN7; actin nucleation; dendritic cell maturation; kinetic of transfer; trans-infection.

Figure 1

Kinetics of TSPAN7/actin nucleation-dependent mechanism of HIV-1 transfer from immature MDDCs to CD4⁺ T lymphocytes. (A) Scheme depicting the experimental layout used throughout this manuscript to assess MDDC-mediated HIV-1 trans-infection of CD4⁺ T cells. MDDC, monocyte-derived dendritic cell; VLP, virus-like particles; IL-2, interleukin-2; PHA-L, phytohaemagglutinin/leucoagglutinin; IL-4, interleukin-4; GM-CSF, granulocytes-macrophage colony-stimulating factor. (B) Flow cytometry plots showing GFP and P24 expression levels in cells pre-gated on SSC FSC, living CD3⁺ singlets, 20 or 40 h after the start of co-culture; percentage of CD4⁺ T cells infected by X4–HIV-1–GFP (GFP⁺ and P24⁺) is shown above the gates. Top panel: T cells were cultured with X4–HIV-1–GFP for 20 or 40 h in the absence or presence of MDDCs at a 1:1 ratio and with (bottom panel) or without (top panel) 1 μM Nelfinavir (NFV; an HIV protease inhibitor). (C) Bar graph showing the fold increase in T cell infection by X4–HIV-1–GFP following co-culture with MDDCs as compared to CD4⁺ T cells alone [i.e., % infected T cells (GFP⁺ P24⁺) cultured with MDDCs/% infected T cells without MDDCs] as measured by flow cytometry (see B). For example, the first bar graph on the left means that, for donor A, 10 times more CD4⁺ T cells were infected when MDDCs were present than when T cells were cultured alone with X4–HIV-1–GFP. Mean ± Standard Deviation (SD) of technical triplicates are presented for three healthy blood donors (D) Percentage of variation of X4–HIV-1–GFP transfer with MDDCs transduced with two different shRNAs against TSPAN7 (shRNA 1 and 3) previously validated (Ménager and Littman, 2016) vs. a non-specific shRNA (scramble shRNA), observed by flow cytometry at 20 or 40 h of co-culture. Dot plots on the right of each time point represent variation of transfer when, in addition to knockdown, 1 μM of NFV was added to the co-culture. Mean ± Standard Deviation (SD) of seven healthy blood donors in the context of 4 independent experiments. (E) Bar graph showing the percent variation of X4–HIV-1–GFP transfer (based on fold increase in CD4⁺ T cell infection) between condition where DMSO (drug carrier) was used [0% variation ± Standard Deviation (SD)] vs. 100 μM CK-666 to inhibit the ARP2/3 complex and actin nucleation. CK-666 was added during the first or the last 20 h of co-culture or during the entire experiment. The experiment was performed in three unrelated healthy blood donors, with the mean ± SD of technical triplicates in each condition and for each donor. This experiment is representative of three independent experiments. (C–E) NS, not significant. ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Figure 2

Change of shape and loss of dendrites for MDDCs during HIV-1 transfer experiments. (A) Four hundred nanometers of Z-stacks showing by confocal microscopy, iMDDCs and CD4⁺T cells co-cultured with X4–HIV-1–iGag-GFP for 4, 20, and 40 h. Actin was detected in red following phalloidin staining and nuclei stained in blue by DAPI. Using ImageJ software, perimeter of each MDDC was manually traced in white (middle panel) alongside dendrites (bottom panel). (B) Same as in (A) with iMDDCs transduced with TSPAN7 shRNA. (C) Same as in (A) with MDDCs stimulated with 1 μg/ml LPS for 24 h before analysis by confocal microscopy. (A–C) Data are from a donor representative of five unrelated donors studied in three independent experiments.

Figure 3

Impact of MDDC maturation on TSPAN7 expression and function. (A) Flow cytometry plots showing CD4⁺ T cells infected with X4–HIV-1–GFP, in the absence (top panel) or presence (middle panel) of iMDDCs. In the bottom panel, the maturation state of MDDCs was evaluated by gating on CD86 expression, upon co-culture with CD4⁺ T cells and X4–HIV-1–GFP measured at 4, 20, and 40 h. Cells were pre-gated as follows: SSC FSC, singlets, living cells, CD3⁺ T cells (top and middle panel), or DC-SIGN⁺ MDDCs (bottom panel) (B) Maturation state of MDDCs at 2 and 40 h following treatment with Poly(I:C) (1 μg/ml), LPS (1 μg/ml) or infection with a VSV-G-pseudotyped single-round HIV-1–GFP (VSV–G-HIV-1-GFP), represented as (G)–HIV-1 on the figure, in the presence of the viral protein Vpx to allow infection and innate sensing. CD86 monitoring by flow cytometry was used to assess maturation status and GFP expression for HIV replication. MDDCs were pre-gated following the same gating strategy as mentioned in (A). (C,D) Quantitative PCR (qPCR) measurement of the Log2 fold change of TSPAN7 mRNA normalized by the housekeeping gene GAPDH, 40 h after MDDCs stimulation by LPS (1 μg/ml)/Poly(I:C) (1 μg/ml), infection with VSV-G–HIV-1–GFP + Vpx or in the presence of X4–HIV-1–GFP (represented as X4–HIV-1) or left unstimulated. Fold change expression was normalized to the level of TSPAN7 detected in iMDDCs. Experiments were performed 3 times in 6 independent blood donors. Donor N in (C) and donor O in (D) are representative of an experiment performed in six unrelated blood donors in the context of six independent experiments, using two different sets of qPCR primers to detect specific expression of TSPAN7. NS, not significant. ^****p < 0.0001.

Figure 4

Role of TSPAN7 and actin nucleation in HIV transfer from mature MDDCs to T cells. (A) Percentage change of HIV-1 transfer following inhibition of actin nucleation by CK-666 (100 μM), compared to DMSO-treated cells; in immature MDDCs (iMDDCs) and MDDCs matured by 100 ng/ml LPS, 48 h before transfer experiment (mMDDCs). Mean ± SD of triplicates for four different healthy blood donors are represented. (B) Percentage change of X4–HIV-1–GFP transfer following TSPAN7 knockdown compared to scramble shRNA-expressing cells, in iMDDCs and MDDCs matured by 100 ng/ml LPS, 48 h before transfer experiments. Mean ± SD of six different healthy blood donors. (C) Percentage change of X4–HIV-1–GFP transfer according to kinetic of maturation of MDDCs [LPS added 48 or 24 h before co-culture or at the time of co-culture (time 0)], compared to iMDDCs. Mean ± SD of six different healthy blood donors. (A–C) NS, not significant. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001. (D) Confocal microscopy images of MDDCs at different stages of maturation: iMDDCs (left panel); mMDDCs stimulated by LPS (100 ng/ml) 48 h before (middle panel) or at the same time of start of co-culture (right panel). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue), respectively and Siglec-1/CD169 was detected using an anti-CD169 antibody conjugated to APC (magenta). Incoming X4–HIV-1–Gag-iGFP particles are seen in green based on (GFP presence). Four hundred nanometers of Z-stacks were taken 20 h after the start of the co-culture with CD4⁺ T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Figure 5

CD169, as an HIV-1 receptor, mostly impacts transfer from mature MDDCs rather than immature MDDCs. (A) Flow cytometry plots showing CD86, DC-SIGN, and CD169 expression levels on MDDCs (pre-gated on SSC FSC, living cells, CD3⁻ cells and singlets). Panels show the expression of these proteins in iMDDCs (left panel) and MDDCs with LPS pretreatment at 100 ng/ml for 48 or 24 h before co-culture (middle and right panels, respectively). (B) Percentage of variation of HIV-1 transfer when using iMDDCs or LPS-treated MDDCs (100 ng/ml LPS for different lengths of time) incubated with a blocking antibody against CD169 as compared to an isotype control for each condition. Results are displayed for 4 different blood donors with the mean ± SD of technical triplicates. (C) Percent of variation in HIV-1 transfer to assess the impact of blocking CD169 and TSPAN7 knockdown as compared to scramble shRNA on MDDCs matured with LPS for 48 h treated by an isotype control. Mean ± SD of seven different blood donors in 4 experiments. (B,C) NS, not significant. ^**p < 0.01; ^***p < 0.001. (D) Confocal microscopy images of iMDDCs (left panel) and mature MDDCs (mMDDCs) right panel, to assess the degree of colocalization between CD169 (magenta) and incoming X4–HIV-1–Gag-iGFP (green). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue). Four hundred nanometers of Z-stacks were taken 40 h after the start of the co-culture with CD4⁺ T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Figure 6

Model comparing the contribution of TSPAN7 and actin nucleation during the different identified phases of HIV transfer when performing co-culture experiments with iMDDCs vs. LPS-matured MDDCs. On the left: mechanism showing the transfer of HIV-1 from LPS-matured MDDCs to CD4⁺ T lymphocytes; as described in the literature, the transfer relies mostly on the capture of HIV-1 by Siglec-1/CD169 and the formation of a big invaginated pocket. TSPAN7 and actin nucleation roles seem rather limited. On the right, a first phase of transfer, dependent on TSPAN7, actin nucleation and dendrite formation is observed during the first 20 h of co-culture with iMDDCs. Seventy-five percent of the transfer is taking place during this first phase, independently of HIV-1 replication and capture by CD169/Siglec-1. A second phase of transfer, less dependent on TSPAN7 and actin nucleation, but dependent on HIV-1 replication is observed during the last 20 h of co-culture. Of note, a decrease of TSPAN7 expression correlated with a maturation of MDDCs was detected after 20 h of co-culture and leads to a decrease in actin nucleation, inducing a shortening of dendrite and a change in shape of the cells. Previously reported scientific data are shown in green and the new results presented in this manuscript are in blue. Points that will require further investigation in this model are stated in red.