Characterization of the Plasmacytoid Dendritic Cell Response to Transmitted/Founder and Nontransmitted Variants of HIV-1

The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1.

IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.

INTRODUCTION

The high mutation rate during human immunodeficiency virus type 1 (HIV-1) replication, combined with intense selective pressures, leads to rapid viral evolution and the development of diverse viral variants, comprising the viral quasispecies, within an infected host (1). Extraordinarily, this level of diversity often arises from a single transmitted/founder (TF) virus, indicating that transmission of HIV-1 represents an extreme genetic bottleneck (2, 3). TF viruses are often nondominant variants in the blood and sexual secretions of the transmitting partner (4, 5). TF viruses are CCR5 tropic and show decreased glycosylation on the HIV-1 envelope (6–8). Genotypic studies examining TF viruses report sequence bias in the HIV-1 envelope region, including several conserved peptides near the CD4 binding site (8–10). Despite these findings, studies investigating possible mechanisms for increased viral fitness of TF viruses remain inconclusive (3, 11). Several recent studies demonstrated enhanced resistance to inactivation by alpha interferon (IFN-α) for TF viruses (12–14). However, other studies have contradicted those findings, showing no change or increased sensitivity to IFN-α among TF viruses, highlighting the need for more research (11, 14, 15).

The vast majority of IFN-α produced in response to HIV-1 infection is derived from a single relatively rare cell type, called the plasmacytoid dendritic cell (pDC) (16, 17). pDCs comprise 0.2 to 0.6% of all peripheral blood mononuclear cells (PBMCs) but have the ability to produce up to 1,000 times more IFN-α than that from any other cell in the body (18, 19). pDCs respond directly to single-stranded RNA (ssRNA), such as that comprising the HIV-1 genome, through endosomal Toll-like receptor 7 (TLR7). The downstream effects of TLR7 signaling include production of IFN-α and NF-κB-associated cytokines, such as tumor necrosis factor alpha (TNF-α) (20–23). The IFN-α produced by activated pDCs potently restricts HIV-1 replication (24). On the other hand, TNF-α drives HIV-1 replication (25).

pDCs are rapidly recruited to inflamed tissues, such as the female genital tract in the context of simian immunodeficiency virus (SIV) exposure in rhesus macaques (26, 27). It is possible that in the female genital tract, in the hours after HIV-1 exposure, pDCs encounter both TF and nontransmitted (NT) strains and contribute to a bottleneck in early infection. However, the contribution of pDCs to the earliest events in HIV-1 infection remains unexplored. We hypothesized that in order to survive a bottleneck, TF variants may suppress innate immune responses induced by pDCs as an immune evasion strategy. Alternatively, TF variants may be poised to hyperactivate pDCs, resulting in excess TNF-α fueling virus replication. To investigate these possibilities, we stimulated pDCs in vitro with a panel of confirmed TF variants of HIV-1, compared the response to that for NT viruses from the same donor pair, and assessed the secretion of IFN-α and TNF-α.

RESULTS

Donor pair-linked TF and NT viruses induce similar levels of IFN-α and TNF-α secretion from pDCs.

We hypothesized that evasion of the pDC response might be a feature of TF viruses. To investigate differences between HIV-1 isolates in early infection and those isolated from chronic infection in the ability to induce a pDC response in vitro, we acquired 20 HIV-1 subtype C full-length molecular clones that were generated as part of the Zambia-Emory HIV Research Project, as described previously (11, 28). The clones, which were derived from 5 donor pairs, included 1 confirmed TF and 3 NT variants from each donor pair (Table 1). pDCs obtained from six healthy volunteers were stimulated with each virus variant at 10 ng/ml of HIV-1 p24 for 24 to 36 h. pDC cultures without virus but with 2.5 μg/ml of imiquimod (maximal pDC activation) and with medium alone (no pDC activation) served as the positive and negative controls, respectively. Supernatants from pDC cultures were collected for determination of IFN-α and TNF-α concentrations by enzyme-linked immunosorbent assay (ELISA). A summary of all pDC donor responses to each of the viruses, broken down by virus donor pair, is shown in Fig. 1A and B. We observed considerable variability in IFN-α and TNF-α secretion between donors with the same viruses (e.g., note the variation across pDC donors in response to 331-TF). In addition, we noticed that pDCs from a given donor gave variable responses to a given group of viruses (TF/NT) in a transmission pair (e.g., note the pDC responses to 331 viruses for donors OM666 and OM914). For each of the six pDC donors, we compared the response to each TF variant to the mean of the responses to the corresponding NT viruses (Fig. 1C and D). Again, we noted considerable variability. For example, we observed increased IFN-α secretion with donor pair 331 for all pDC donors and increased TNF-α secretion with donor pair 4473 in response to TF viruses compared to that in response to NT viruses (Fig. 1C and D). For the other donor pairs, we saw insignificant trends going in both directions for both IFN-α and TNF-α. Comparing data for all NT viruses with those for the corresponding TF viruses, overall, TF viruses tended to induce more IFN-α (median [interquartile range {IQR}] of 160.4 pg/ml [48.07, 535.8 pg/ml] for TF viruses and 145.3 pg/ml [46.7, 314.7 pg/ml] for NT viruses) and TNF-α (median [IQR] of 128.2 pg/ml [32.0, 157.8 pg/ml] for TF viruses and 91.8 pg/ml [38.3, 129.8 pg/ml] for NT viruses) secretion than that by the NT viruses (Fig. 1E and F). However, the increases in production of IFN-α and TNF-α were not statistically significant (estimated difference [95% confidence interval {CI}] of 0.10 [−0.11, 0.32] [P = 0.345] for IFN-α and 0.14 [−0.03, 0.30] [P = 0.104] for TNF-α) (Table 2).

TABLE 1.

HIV-1 molecular clones used in this study

Pair ID	Sequence ID	IFN resistance (RC with IFN/RC without IFN)a	Accession no.
331	TF	3.1874	KR820323
	6	ND	KR820320
	13	ND	KR820296
	21	0.9270	KR820303
3618	TF	10.9791	KR820366
	5	8.3600	KR820354
	11	15.5614	KR820342
	15	ND	KR820345
3678	TF	11.2664	KR820393
	11	20.9117	KR820386
	14	ND	KR820371
	18	ND	KR820375
4248	TF	0.0000	KR820421
	13	ND	KR820398
	14	20.1029	KR820399
	23	ND	KR820407
4473	TF	0.0877	KR820449
	16	ND	KR820428
	17	0.0000	KR820429
	18	0.0000	KR820430

^aReplication capacity in the presence versus absence of IFN-α, as previously reported (11). ND, not done.

FIG 1.

IFN-α and TNF-α secretion from pDCs in response to TF and NT virus stimulations. pDCs derived from healthy donors (n = 6) were plated in a 96-well plate, and each well was stimulated with one of 20 viruses from the TF and NT panel of viruses at 10 ng/ml or with 2.5 μg/ml of imiquimod (positive control) or medium alone (negative control). ELISA was performed on the supernatants, with each sample measured a single time. Results for IFN-α (A) and TNF-α (B) are shown for each sample individually. (C and D) The mean of the responses to NT viruses for each donor pair was determined for each pDC donor and compared to the response to the corresponding TF virus. (E and F) To account for all NT viruses individually, the pDC response for each NT virus was compared to the corresponding TF virus response, and thus each TF virus response is reported three times, once for each corresponding NT virus. Statistical analysis for panels C and D was performed by the Wilcoxon matched-pair signed-rank test. *, P < 0.05.

TABLE 2.

Statistical analysis of changes in production of IFN-α and TNF-α

Variable	Linear regression		Robust regressiona
	Estimated difference (95% CI)	P value	Estimated difference (95% CI)	P value
IFN-α log10 change	0.10 (−0.11, 0.32)	0.345	0.10 (−0.04, 0.24)	0.168
TNF-α log10 change	0.14 (−0.03, 0.30)	0.104	0.03 (−0.06, 0.12)	0.493

^aRobust regression was performed to limit the influence of outliers on the result.

pDC maturation downstream of TLR7 signaling results in elevated expression of CD86 and TNF-apoptosis-inducing ligand (TRAIL) (20–23, 29). We thus also performed flow cytometry to assess CD86 and TRAIL on pDCs following the stimulations described for Fig. 1. We observed no differences between the responses to TF and NT viruses for either marker (data not shown).

We previously showed that strong pDC agonists, such as imiquimod, induce both TNF-α and IFN-α production from pDCs within 24 h (23, 30). Thus, we posited that a direct correlation between the secretion of TNF-α and IFN-α may provide insight into the completeness of the response to viral stimulation. When TNF-α secretion was compared to IFN-α secretion in the pDC responses to TF viruses, we found no clear relationship (r = −0.03; P = 0.87) (Fig. 2A). However, TNF-α and IFN-α secretion in response to NT viruses showed a moderate negative correlation (r = −0.38; P = 0.0002) (Fig. 2B), i.e., NT viruses tended to induce a response in which either TNF-α or IFN-α was dominant.

FIG 2.

NT and TF viruses differ in the pDC response profiles they elicit. TNF-α and IFN-α secretion levels (reported in Fig. 1) were compared for TF viruses (A) and NT viruses (B). The replication capacity (RC) of each virus was determined previously (11) by quantifying replication in the presence of IFN-α versus that when IFN-α was absent. The IFN-α secretion levels were plotted against the RC for each TF (C) and NT (D) virus. Correlation analyses were performed by the nonparametric Spearman correlation test. *, P < 0.05; ***, P < 0.001.

IFN resistance correlates with pDC activation for NT viruses.

HIV-1 is highly sensitive to inactivation by IFN-α in cell culture. The replication capacity (RC) of a subset of viruses used in this study was previously determined in the presence of IFN-α (11). Given the important role of pDCs in the production of IFN-α during HIV-1 infection, we examined the relationship between the amount of IFN-α secreted by pDCs in response to a variant and the relative IFN-α resistance of the variant. No relationship between IFN-α secretion and resistance to IFN-α was found for TF viruses (Fig. 2C). However, the amount of IFN-α produced in response to NT viruses strongly correlated with the resistance to IFN-α inactivation (r = 0.83; P = 0.03) (Fig. 2D), i.e., NT viruses that showed greater IFN-α resistance also tended to induce more IFN-α from pDCs.

DISCUSSION

It is unclear whether pDCs contribute to the bottleneck events observed in HIV-1 transmission. Within 24 h of virus exposure, pDCs accumulate within the vaginal tissues (26, 27), where they likely encounter both TF and NT viruses. Several reports have implicated IFN-α, for which pDCs are the primary producer, in the events in early HIV-1 infection (3, 11–14). However, the extent to which pDCs respond to viruses in early infection is not well understood. To investigate how pDCs respond to viruses in early infection, we compared the pDC response to TF variants of HIV-1 with that to donor pair-matched NT variants. Our findings showed some subtle differences between the pDC responses to TF and NT viruses.

We hypothesized that evasion of the pDC response might be beneficial in order for TF viruses to establish an infection. We found the contrary, in that TF viruses stimulated pDCs similarly to their stimulation by NT viruses. We did, however, observe a trend toward elevated IFN-α or TNF-α secretion in response to TF viruses. Our study was powered to detect differences of >3.55-fold for IFN-α and >1.58-fold for TNF-α with 80% confidence, and thus we could not rule out the possibility that TF viruses induce smaller differences in pDC responses than those seen with NT variants. For certain host-virus pairs, the TF virus tended to stimulate pDCs to produce more IFN-α and TNF-α, but this was less so with others, with no clear pattern. The 331 TF variant, for example, induced higher levels of IFN-α for all donors tested, but the remaining virus pairs yielded mixed results for the different donors. Since pDCs responding to TF viruses tended to produce both TNF-α and IFN-α, it is unclear whether TNF-α, a driver of HIV-1 replication (25), can override the suppressive effects of IFN-α secretion. We also examined TRAIL expression on pDCs, as this may be a mechanism for apoptosis of bystander CD4⁺ T cells (29, 31), dampening immunity in general, and saw no significant differences in induction by TF and NT variants from the different pairs (data not shown).

In order to investigate whether TF HIV-1 evades pDC activation, we used cell-free HIV-1 rather than CD4 T cell-associated HIV-1 as our pDC stimulus. While others have shown that CD4 T cell-associated virus is a stronger pDC stimulus than cell-free virus (32, 33), we chose to use cell-free virus as the pDC stimulus because one mode of mucosal transmission of HIV-1 is likely through cell-free virus found in genital secretions that are deposited along the genital mucosa. In an SIV model in which cell-free virus is used for transmission, pDCs are recruited to the female genital tract as early as 24 h after virus exposure (26, 27), and virus dissemination can be observed as early as 24 h (34). Further, during peak viremia in acute infection, cell-free virus levels reach concentrations approaching 10⁸ RNA copies per ml (35), and this is a period associated with maximum HIV transmission (36). Future studies should be done to examine cell-associated TF versus NT viruses on pDCs.

We generally found that both TF and NT HIV-1 stimulated pDCs in our study poorly. Stimulations in this study were carried out using HIV-1 that was standardized to 10 ng/ml of p24, corresponding to a range of 5.91 × 10⁷ to 2.3 × 10⁹ RNA copies/ml (5.91 × 10² to 2.3 × 10⁴ RNA copies/pDC). At this concentration of virus, we observed relatively modest pDC responses, with high heterogeneity, to both TF and NT viruses. These findings are consistent with previous work showing that HIV-1 is a poor inducer of pDC activation compared to other viruses at similar concentrations (23, 30, 32, 33). When pDCs are stimulated by potent agonists, such as imiquimod, influenza virus, or Sendai virus, activation occurs in a characteristic sequential dynamic of TNF-α followed by IFN-α production, which is not generally observed with HIV-1 as a pDC stimulus (23, 37). In this regard, TF viruses tended to behave more like these strong pDC agonists, with both TNF-α and IFN-α being produced, whereas the response to NT viruses was characterized by polarization toward either TNF-α or IFN-α production. The ability of TF viruses to induce the production of both TNF-α and IFN-α simultaneously may drive early viral expansion by producing TNF-α, which can drive virus replication (25), and might assist the virus in overcoming the negative effects of IFN-α. HIV-1 infection also drives the recruitment of pDCs to the lymph nodes (38), where the dual secretion of IFN-α and TNF-α by pDCs promotes T cell activation and creates new viral targets (17). Further work to understand the relative levels of importance of IFN-α and TNF-α induction by HIV-1 variants is needed.

The contributions of pDCs and IFN-α to the diversity of the HIV-1 quasispecies are not well understood. In nonhuman primate models in which pDCs were experimentally depleted or IFN-α was blocked, SIV disease progressed rapidly (21, 39), indicating that pDCs contribute to viral control. It is possible that HIV-1 variants that persist during chronic infection can eventually develop IFN-α resistance and still induce IFN-α from pDCs, allowing them to escape from the inhibitory effects of IFN-α. Alternatively, there also appear to be variants that are IFN-α sensitive; however, they appear to dysregulate pDC function to preferentially induce TNF-α, a potent activator of HIV replication. Interestingly, we did not observe the same relationship with TF viruses, raising the possibility that they did not undergo the same level of in vivo evolution. The viral mutations which shape the pDC response have not been explored previously. One possibility is the interaction between the viral gp120 and CD4 on pDCs. Recent studies have shown that the interaction between gp120 and CD4 can shape the ratio of IFN-α and TNF-α production (37, 40). Interestingly, one of the traits of TF viruses is conservation of residues near the CD4 binding site on gp120 (8, 9). Future studies should investigate how specific HIV-1 mutations modulate pDC responses and how the pDC responses drive the in vivo evolution of HIV-1 and its effects on transmissibility.

Despite intense interest, the unique qualities that endow TF viruses with enhanced transmissibility are still unclear. In this study, we focused on another arm of the innate immune system, the pDC. We did not find convincing evidence that TF viruses can evade the pDC responses by blocking IFN-α production. In fact, TF viruses tended to activate pDCs to a greater degree than that with their chronic coviruses. Further studies will be required to determine how the balance of IFN-α and TNF-α production from pDCs impacts early viral replication.

MATERIALS AND METHODS

Study subjects.

Infectious molecular viral clones from five transmission-linked partners enrolled in the heterosexual transmission cohort from the Zambia-Emory HIV Research Project in Lusaka, Zambia, were utilized in the study. For each transmission pair, a TF variant was identified in the recipient, along with several NT variants from the transmitting partner. A total of 5 TF viruses and 15 NT viruses, which had been sequenced and cloned into pBluescript as described previously, were used in this investigation (11, 28) (Table 1). The replication score for a subset of viruses in the presence of 5,000 IU/ml of IFN-α was previously calculated for CD8⁺ T cell-depleted PBMCs and reported as the replication score in the presence of IFN-α divided by the replication score in the absence of IFN-α (11).

Healthy donor blood used for pDC preparations was acquired in accordance with the institutional ethics board guidelines for conducting clinical research at the University of Toronto. Study approval was provided by the research ethics boards of the University of Toronto, Canada. Healthy donor samples were obtained by peripheral blood draw or leukapheresis at the University of Toronto by a registered nurse or certified phlebotomist. All participants were above 18 years of age at the time of inclusion in this study.

HIV-1 virion production.

HIV-1 viral clones in pBluescript vectors were transformed into Escherichia coli DH5α (Invitrogen) by heat shock and selected based on carbenicillin resistance per the instructions in the supplier's manual. Plasmids were harvested using a GeneJET plasmid miniprep kit (Invitrogen), and the concentration was measured by use of a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Plasmid DNAs were transfected into the HEK293T cell line (American Type Culture Collection, Manassas, VA) through use of the FuGene transfection reagent (Promega) at a FuGene/DNA ratio of 3:1, as recommended by the manufacturer. HIV-1 virions were harvested from the cell culture supernatant after 48 h and purified by use of a sucrose cushion overlay and centrifugation at 135,000 × g for 2 h. Virus concentrations were examined using p24 ELISA (NIH, Frederick, MD). Viral RNA concentrations of HIV-1 stocks were measured using the Cobas HIV-1 viral load test run on a Cobas 6800 instrument (Roche Diagnostics GmbH, Mannheim, Germany), with the assistance of the Department of Microbiology, Mount Sinai Hospital (Toronto, Canada).

pDC isolation.

Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll-Paque overlay by centrifugation at 500 × g for 30 min. Buffy coats were collected after centrifugation, washed twice with phosphate-buffered saline (PBS), and subjected to negative selection for pDC isolation (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated pDCs were suspended in R10 complete medium (RPMI 1640 medium plus 10% fetal bovine serum [FBS], penicillin, streptomycin, and l-glutamine [Thermo Fisher Scientific, Waltham, MA]) supplemented with 20 ng/ml interleukin-3 (IL-3) (R&D Systems).

In vitro pDC stimulation.

pDC stimulations were performed in 96-well flat-bottomed plates at a density of 100,000 pDCs/ml in 200 μl of R10 medium plus IL-3. Each donor's pDCs were treated with a panel of 20 HIV-1 strains, as described above, at a concentration of 10 ng/ml of p24 at 37°C. All stimulations were performed in singlet due to the technical limitations of working with pDCs. Since pDCs account for 0.2 to 0.6% of total PBMCs, in order to obtain 440,000 pDCs, the minimum cell number required to perform stimulations with all 20 viruses plus controls, we required 7.33 × 10⁷ to 2.2 × 10⁸ PBMCs, which we obtained from approximately 80 ml of blood. Culture supernatants were collected at 24 to 36 h poststimulation and stored in a −20°C freezer for cytokine secretion assay.

ELISA of cell culture supernatants.

IFN-α and TNF-α in cell culture supernatants were measured by ELISA (Mabtech, Cincinnati, OH), using the protocol described in the supplier's manual. ELISA results were read by use of Cytation imaging reader (BioTek, Winooski, VT).

Preparation of cells for flow cytometry.

Stimulated pDCs were separated during supernatant collection by centrifugation at 500 × g in a 96-well v-bottomed plate for 5 min. Cells were washed once with chilled PBS and stained with Live/Dead violet (Thermo Fisher Scientific) in the dark for 10 min at room temperature. After another wash using MACS buffer (PBS with 2% bovine serum albumin [BSA] and 2 mM EDTA), cells were stained for surface antigens at 4°C for 30 min by use of BDCA2-fluorescein isothiocyanate (BDCA2-FITC) (130-090-510; Miltenyi), CD123-phycoerythrin-Cy7 (CD123-PE-Cy7) (306010; Biolegend), CD86-peridinin chlorophyll protein-Cy5.5 (CD86-PerCP-Cy5.5) (305420; Biolegend), and TRAIL-allophycocyanin (TRAIL-APC) (308210; Biolegend) antibodies and then fixed with 2% paraformaldehyde (PFA). All samples were acquired on a BD Fortessa flow cytometer. Data were analyzed using FlowJo V10.0.7r2.

Statistical analysis.

All graphs and statistics were generated using GraphPad Prism V.6c and V.7a software (La Jolla, CA) and SAS 9.4 (SAS Institute Inc., Cary, NC). Linear regression, adjusted for donor pair and pDC donor effects, was performed to estimate the differences between TF and NT viruses. For IFN-α and TNF-α, analysis was conducted on a log₁₀ scale due to the skewness of the data. As a sensitivity analysis, robust linear regression with M estimation was also performed, as some of the data exhibited minor departures from the required statistical assumption for linear regression due to outliers.