Cryopreserved PBMC samples collected from 30 healthy volunteers (HV) and 10 antiretroviral therapy (ART)-suppressed PLWH were used in this study. PBMCs from HV were obtained under the Gilead blood collection program, while PBMCs collected from ART suppressed PLWH were procured by Cell Quest under the protocol “Leukapheresis on Adult Participants with HIV-1 to Obtain Leukocytes for Testing of Virus Eradication Strategies Protocol”. PLWH were selected based on the following inclusion criteria: Either male or female, at least 18 years of age, and diagnosed with HIV-1 infection. All participants had to be on an ART regimen for at least 12 months prior to enrollment in the study and had undetectable plasma viral loads below 50 copies per mL based on Cobas (Roche) Amplicor (or similar) assay. Additionally, all participants had well preserved CD4 cell counts above 350 cells/ml. Individuals who tested positive for hepatitis B or C viruses or had a history of ART drug-resistance, switched regimens, or women who were or became pregnant or breastfeeding, were excluded from the study. All study participants provided informed consent prior to enrollment into the study.

Cryopreserved PBMCs were thawed in a 37°C water bath, washed in standard media and resuspended in PBMC plating media (RPMI with 10% FBS and supplements) to bring the final cell count per well to 35,000, as calculated from the pre-thaw PBMC count. A Microlab STAR liquid handler (Hamilton) was used to add 20 μL of the cell’s suspension into each well of 384-well plates (Greiner), pre-filled with 10 μL PBMC plating media at room temperature. The numbers of PBMCs in each well were very consistent ( Supplementary Figure S1A ). The plate was incubated at room temperature for 10 minutes, then centrifuged for one minute at 140 RCF, followed by an incubation at 37°C and 5% CO₂ for 30 minutes to promote attachment to the plate. FBS was then added to bring the final concentration to 10% per well prior to addition of the different TLR stimulants. Two concentrations of each TLR agonists were evaluated, either high or low, except TLR4 ( Table 1 ). The appropriate concentration of TLR agonists was then added into each well of a 384 well plate, using a Mantis liquid dispenser (Formulatrix), and incubated for 6, 24, or 48 hours at 37°C, 5% CO₂.

At the end of each incubation period, cell culture supernatants were collected from each well and stored at −80°C. These were later thawed, treated with 1% Triton X-100 for 2 hours at room temperature, and refrozen at −80°C before shipping on dry ice to Nomic Bio (Montreal) for the quantification of cytokine and chemokine responses.

Frozen cell-culture supernatants were shipped on dry ice to Nomic Bio (Montreal) for analysis of a custom panel of 51 cytokines and chemokines using standard protocols (22). Briefly, the frozen supernatants were thawed and diluted 1:1 using R10 media (RPMI 1640 supplemented with 10% heat inactivated FBS, 2% Pen/Strep), and nELISA beads were added to each well of the 384 well plates and incubated for 3 hours at room temperature. The target-bound beads were washed with Wash Buffer and resuspended in Assay Buffer, prior to the addition of Displacement Oligo, all reagents included in the assay kit. The target-bound-oligo-bead mixture was then incubated at room temperature for 30 minutes, then washed with Wash Buffer and resuspended in Assay Buffer. The target-bound-oligo beads were then read by flow cytometry on a ZE5 Cell Analyzer (Bio-Rad).

Secreted protein data was normalized as fold-changes above the reference DMSO/basal control per plate, donor, and protein grouping. The median concentrations for the controls and analytes (serum protein-cytokine, chemokine or factor), were calculated. The fold-change per analyte per donor was averaged to generate per-donor measurements, and per donor measurements compared between groups.

PBMCs were stained with 0.5 µM MitoTracker Deep Red FM (ThermoFisher) and 0.5 µg/mL Annexin V CF750 (Biotium) for 30 minutes at 37°C and 5% CO². The stained cells were then fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked, and permeabilized using 1% bovine growth serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 15 minutes at room temperature. Following fixation and permeabilization, the cells were stained with Concanavalin A Alexa Fluor 488 (ThermoFisher) at 25 µg/mL and Phalloidin Phenovue Fluor 568 (Perkin Elmer) at 66 nM in staining buffer (1% bovine growth serum in PBS) overnight at 4°C. Finally, the cells were washed 3 times with PBS, stained with Hoechst 33342 (ThermoFisher) at 0.2 µg/mL in PBS for 15 min, and finally washed 3× times with PBS, prior to imaging. The fixation, washing, and staining steps were done on a Biotek EL406 washer dispenser (Agilent). Imaging was performed on an ImageXpress^® Micro Confocal high content imager (Molecular Devices).

Extracting image features using artificial intelligence embeddings was performed as previously described (23). High content images were captured for the entire contents of each well, and each image was scored for an ‘on basis’ and ‘off basis’ effect (24). ‘On basis’ referring to the distance along the projected vector in an embedded space from the centroid of the negative control cluster to the centroid of the positive control cluster. ‘Off basis’ was calculated as the orthogonal distance from projection to corresponding data points per embedded space. While assay performance metrics for phenotypic assays rarely achieved levels of significance seen in target- or molecular-based assays, for this study we achieved a beta value (strictly standardized mean difference) of 2.68, which is interpreted as excellent or good strengths of differences for moderate or strong positive controls, respectively (25) ( Supplementary Figure S1B ).

TLR messenger RNA transcript expression calculated as nTPM (normalized transcripts per million), from whole blood of 6 healthy donors sorted by flow cytometry into 18 immune cell types, was retrieved from Human Protein Atlas database (35). Single-cell RNA sequencing (scRNAseq) datasets generated from PBMCs of healthy donors were retrieved from 10x Genomics database for PLWH and NCBI SRA BioProject PRJNA783363 (36) (132,247 cells in total). Analysis of transcriptomic data was performed using Seurat R package (version 5.1) (37, 38). The gene counts were normalized and scaled using the following quality control filters: Cells with less than 200 unique genes detected or with greater than 5% mitochondrial genes were excluded from the analysis. 2,000 variable genes were identified, and 30 principal components (PC) used for dimensionality reduction. Shared Nearest Neighbor (SNN) graph (K = 20) and Louvain method were used for cell type clustering with the resolution parameter of 0.5. Uniform Manifold Approximation and Projection (UMAP) embeddings were calculated for data visualization using the first 30 PC dimensions. The datasets were integrated using Harmony R package (version 1.2) (39).

Statistical analysis used are defined in the figure legends.

A selection of TLR agonists were profiled in vitro using human PBMCs from healthy donors and ART-suppressed PLWH ( Figure 1A , Table 1 ). TLR agonist concentrations used and summarized in Table 1 , were previously validated to be sufficient to stimulate healthy human PBMCs over 48 hours in vitro (data not shown). Using PBMCs from 30 healthy donors across 3 timepoints (6 hours, 24 hours, and 48 hours), quality control experiments were conducted to build robust models of TLR activation for both fluorescence imaging and cytokine profiling ( Supplementary Figure S1 ).

To understand TLR mRNA expression patterns in PBMCs, we retrieved publicly available RNAseq datasets and analyzed mRNA gene expression levels of TLR4, TLR7, TLR8 and TLR9 (35, 36). The single cell RNA sequencing (scRNA seq) data from healthy volunteers and PLWH were clustered into nine immune cell types: T cells, cycling T cells, B cells, plasma cells, natural killer (NK) cells, monocytes, conventional dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs) and red blood cells (RBCs) ( Figure 2A ). The immune cell types were assigned based on phenotypic marker gene expression levels and blood transcription module (BTM) scores ( Figure 2B , Supplementary Table S1 ) (40). Pseudobulk expression levels of TLRs across these immune cells were consistent with the literature (35): TLR4 and TLR8 were primarily expressed on monocytes and cDCs, whereas TLR7 and TLR9 were predominantly expressed on pDCs and B cells ( Figure 2C ). TLR7 was also expressed in monocytes and cDCs, unlike TLR9. These transcriptomic expression patterns were consistent with the bulk RNAseq data from blood cells sorted by flow cytometry ( Supplementary Figure S2 ). Within the data from healthy individuals and ART-suppressed PLWH, no significant differences in TLR mRNA distribution over the different immune cell types was observed ( Figure 2C ).

To characterize the specific cytokine, chemokine and soluble analyte profiles elicited by each TLR pathway in healthy volunteers (HV), supernatants were collected 6- and 48-hours post-stimulation with TLR4, TLR7, TLR7/8, TLR8 and TLR9 agonists. Supernatants were evaluated using a cytokine and chemokine multiplex assay, and the magnitude of fold change in cytokine responses upon TLR stimulation was normalized to those responses upon DMSO stimulation for each donor. At 6 hours post-stimulation, cytokine and chemokine shifts were observed in wells stimulated by TLR4, TLR7/8, and TLR8 agonists. Specifically, there were greater fold increases in CCL4, IL-1β, IL-6, and TNF-α and fold decreases in CCL2, IL-1Rα, MMP-1, OPN, and IL-6Rα ( Figure 3B ). TLR7-associated stimuli resulted in similar increases as TLR4 and TLR8 ( Figure 3A ). However, increases in CXCL1 and CXCL5 were also observed while PBMCs stimulated with TLR9 agonists had minimal fluctuations in cytokine profiles at 6 hours.

At 48 hours post-stimulation, increases in CCL1, CCL2, CCL4, CXCL1 and IL-6 were observed in all wells with all TLR stimuli to varying degrees of magnitude fold change. Like the 6-hour timepoint post-stimulation, the magnitude of cytokine responses was greater in the TLR4, TLR7 and TLR8 pathways relative to TLR9.

Based on shape and size, we investigated if the TLR agonists influenced the proportions of specific PBMC sub-populations in culture. Two subsets were expected, “small round cells” (which range from 6 to 15 µm, to be lymphocytes) and “large round cells” (with an approximate size of 20 µm, to be monocytes or monocyte-derived macrophages). Small round cells had minimal cytoplasm and prominently featured nuclei, as indicated by Hoechst staining. Large round cells, however, were presented with an array of definable features using other cell biological stains (e.g., F-actin for cytoskeleton) in addition to the nucleus given their pronounced cytoplasmic areas. Examples of the described small and large round cells are shown in Figure 1B . These populations were quantified longitudinally across healthy PBMC cultures. We limited our identification and quantification to round cells, which excluded the quantification of dendritic cells, as they are expected to have a prevalence of only 1⁠–2% in PBMCs.

The fraction of small round cells with TLR4 and TLR8 agonist stimulations was higher than the DMSO control across all three timepoints ( Figure 4A ). This effect was most pronounced with TLR8 agonists and was significantly higher than TLR4 pathway stimulation at all three timepoints. The small round cell frequency was higher with TLR7/8 agonists stimulations than DMSO control, but not those with TLR7 agonists, suggesting the compositional shift towards small round cells was driven primarily by their TLR8 activity. TLR9 agonists did not have a significant effect on small round cell populations across the time course.

In contrast, the fraction of large round cells decreased relative to DMSO in response to TLR4 and TLR8 agonists across all time points ( Figure 4B ). The effect was more noticeable for TLR8 agonists than for TLR4 agonists. Once again, the effects of TLR7/8 agonists paralleled TLR8 pathway activation but remained distinct from TLR7 agonists. We also observed a higher frequency of large round cells in those stimulated with TLR9 pathway agonists than those stimulated with DMSO, but only after 24 hours of culture. We did not detect a difference in small or large round cell populations for those stimulated with TLR7 agonist at any time point. Overall, individual TLR agonists targeting the same receptor showed similar trends to one another ( Supplementary Figures S3A, B ). Correlation analysis for the different TLR mechanisms confirmed that TLR4 and TLR8 pathway stimulations had similar effects on large and small round cell populations, while TLR7 and TLR9 pathway stimulation were more analogous to each other ( Figure 4C ).

To determine if the TLR-induced changes in proportions of small and large round cell populations may be due to cell death, cell surface expression of annexin V was quantified. Phosphatidylserine is a phospholipid usually found on the inner surface of the plasma membrane, but its expression inverts externally to the outer surface of the plasma membrane during early apoptosis. Phosphatidylserine binds preferentially to annexin V, which can be used as a marker of apoptosis (41). In our PBMC cultures, annexin V staining was highest at the 6-hour timepoint and decreased progressively over 48 hours ( Figure 5A ). At 6 hours, an increase in annexin V relative to the DMSO control was detected with TLR4, TLR7/8 and TLR8 agonist stimulations ( Figure 5A , Supplementary Figure S4 ). In comparison to DMSO, TLR4, TLR7/8, TLR8 and TLR9 agonist stimulations were all significantly increased for the annexin V signal at 24 hours, with those elevations being maintained at 48 hours. Individual TLR agonists sharing the same pathway showed similar trends to one another ( Supplementary Figure S4 ). When annexin V changes were compared to small and large round cell population shifts at the 24-hour timepoint, unique patterns were identified for each of the TLR agonists ( Figure 5B ). As described previously, TLR4 stimulation showed increases in small round cells and decreases in large round cells at this timepoint. However, when a correlational analysis was used to compare the levels of annexin V positive cells, we found the frequencies of small and large round cells negatively and positively correlated with annexin V expression, respectively. TLR7 and TLR9 agonists did not correlate with the changes in annexin V positivity across wells as indicated by low slope values. Interestingly, the pronounced increases in small round cells for TLR8 agonists were accompanied by a cluster of annexin V positivity in those wells that had the highest numbers of small round cells. Those same wells had the largest decrease in large round cells, suggesting that the compositional shift favoring small round cells with TLR8 pathway stimulation could be due to the death of large round cells.

Another conventional hallmark of cell death is nuclear fragmentation, an event that occurs much later in apoptosis than phosphatidylserine expression inversion to the exterior plasma membrane, and which is also associated with other forms of cell death (42, 43). Therefore, we quantified PBMCs with fragmented nuclei in response to different TLR agonist stimulations over time from our high content images. As opposed to annexin V staining, which decreased over 48 hours, the number of cells with fragmented nuclei grew in number during the time course ( Figure 5C ). Significant increases in this cell death marker relative to DMSO were found at the 48-hour timepoint for TLR4, TLR7/8 and TLR8 agonists, a result that corresponded with annexin V expression at the 6-hour timepoint. We also detected a marked decrease in fragmented nuclei at 48 hours for TLR9 agonists compared to the DMSO control. When fragmented nuclei changes were compared to the fraction of small round cells at the 48-hour timepoint, we found that all TLRs trended upward with positive slopes (range 0.02266 to 0.03142) ( Figure 5D ). We observed the opposite effect for fragmented nuclei correlations relative to the proportion of large round cells (negative slopes ranging from –0.06132 to –0.08836).

Antigen-presenting cells protect the body by engulfing foreign particles, bacteria, and dead or dying cells in response to external signals, including those that activate TLRs (4), through a process called phagocytosis. Within PBMCs, larger cells (including macrophages and monocytes) perform this function. Using high-content imaging, we quantified cells with phagocytic morphology in vitro in healthy PBMC cultures treated with or without TLR agonists ( Figure 1B ). Phagocytic cell fractions were normalized to those of large round cells per well and reported as ‘phagocytosis/large round cells’. The proportions of phagocytosis/large round cells were highest at the 6-hour timepoint and declined over time ( Figure 6A , Supplementary Figure S5 ). The pattern of phagocytic activity was consistent with those from previous reports (44, 45). As early as 6 hours, an increase in phagocytosis on large round cells was detected for all TLR agonists relative to DMSO, with the largest changes resulting from TLR7 and TLR8 stimulation. Elevations were maintained throughout the time course, except for TLR9 agonists at the 48-hour timepoint. Shifts in the fraction of phagocytic (y-axis) or large round (x-axis) cells relative to DMSO were plotted for each TLR agonist at each timepoint to compare MOA response patterns ( Figure 6B ). Similar to what was observed in our cell composition and death analyses, we found that TLR7 and TLR9 agonists clustered together and TLR4 and TLR8 agonists clustered together, and this effect was most pronounced at the 6-hour timepoint. When phagocytosis changes were compared to small and large round cell changes at the 24-hour timepoint for each well, a positive association was detected between increased phagocytosis and increased large round cells ( Figure 6C ). Overall, as the proportion of small round cells increased, the amount of phagocytosis decreased, and the opposite was true as populations shifted to favor large round cells. This is evident for TLR9 agonists which previously had increased the relative proportion of large round cells ( Figure 4A ). However, all TLR agonist pathways exhibited a negative slope for phagocytosis when compared to small round cells and a positive slope when compared to large round cells, this is consistent with cellular functions of the immune cell types in PBMC cultures that are known to perform phagocytosis.

To determine if TLR stimulation influenced actin dynamics in stimulated cells, we first trained single cell models to recognize cells with contracted or distributed cytoskeletons as markers of movement or immobility, respectively. We then applied the trained models to quantify small round cells that were positive for motility features in healthy PBMCs ( Figure 7 ). Of note, not all small round cells stained positive for actin. Detection of actin contraction indicated that TLR4 and TLR8 stimulations had a significantly higher proportion of small round cells with the contracted actin feature (Figure 7A). These changes were maintained at 24- and 48-hours with the greatest amplitude observed for TLR8 agonists (median difference vs DMSO of 1.55- and 1.91-fold higher, respectively). There was no observed effect of TLR7 or TLR9 agonists on actin contraction. The disassembly of actin filaments is associated with cells that are not moving. However, the pool of actin filaments is maintained if a stimulus for cell movement is received (46, 47). We found that small round cells with dispersed actin decreased over time and most TLR stimulations did not change this feature when compared to the DMSO control ( Figure 7B ). The only exception was with TLR9 agonists at the 48-hour timepoint, which did retain more actin dispersed small round cells than the DMSO control, although still at lower levels relative to TLR9 at earlier timepoints. Overall, individual TLR agonists that utilized the same signaling pathway exhibited remarkably similar effects on actin contraction and dispersion ( Supplementary Figure S6A, B ).

Podosomes are another example of actin-rich structures that are present at the periphery of the plasma membrane on a multitude of migrating cell types, including certain immune cells, such as macrophages and monocytes (48). Podosomes commonly display a polarized pattern on migrating cells, usually located on the leading edge of a migrating cell. In our study we observed higher levels of podosome positive large round cells treated with TLR4 and TLR9 agonists at the 6-hour timepoint ( Figure 7C , Supplementary Figure S6C ). Additionally, TLR7 agonists increased the proportion of large round cells with podosomes at both the 24- and 48-hour time points. We also detected lower levels of podosome positive large round cells following TLR8 stimulation, but only at 24-hours.

Next, we quantified and compared the cytokine and chemokine responses by PBMCs from healthy volunteers (HV) and PLWH on ART, following 6- and 48-hour stimulations with TLR4, TLR7, TLR7/8, TLR8 or TLR9 agonists. Cell culture supernatants from stimulated or unstimulated (DMSO) PBMCs were collected for use in quantifying 51 secreted proteins. All TLR-stimulated secreted protein responses were normalized to DMSO control.

Overall, secreted protein responses in the supernatants of PBMCs of PLWH on ART were frequently lower following TLR stimulations when compared to those of HV, except for soluble CD14 (sCD14), CCL5 and CXCL4. Notably, CCL5 and CXCL4 are ligands for HIV co-receptor CCR5 ( Figure 8 , Supplementary Figures S7 , S8 ). Similar increases in levels of HIV co-receptor ligand in blood acts as surrogate markers for HIV infection (49). PBMCs of PLWH at 6 hours, resulted in lower MMP3 responses with TLR4 stimulation and lower CCL2 levels with either TLR7 or TLR7/8 stimulations. However, higher CRP levels were found with only TLR7/8 stimulation when compared to HV ( Figure 8 , Supplementary Figure S7 ). Similarly, 6 hours of TLR8 stimulation led to lower Activin A, CCL1, CCL2, IL-1β, IL-6, IL-8, and MMP-9 responses, but higher sCD14 and CRP responses in PBMCs of PLWH in comparison to HV. CCL1 and MMP-9 responses were lower, while CCL5 and CXCL4 responses were increased in PBMCs of PLWH after 6 hours of TLR9 stimulation.

Longer TLR stimulation of PBMCs (48 hours) resulted in more striking differences in secreted protein responses between HV and PLWH ( Figure 8 ). Specifically, PBMCs from PLWH at 48 hours of TLR4 stimulation had lower CCL2, IL-16, IL-6Rα, and TNF-RII responses, but higher CXCL4 production than HV. Moreover, PBMCs of PLWH produced lower amphiregulin, CCL2, IL-1RA/RN, IL-6Rα, osteopontin and TNF-RII, but higher CCL5 and CXCL4 responses to TLR7 stimulation than HV, while 48 hours of TLR7/8 stimulation resulted in lower CCL1, CCL2, CCL22, CXCL1, CXCL5, CXCL16, IL-6Rα, MMP-9, sCD14 and TNF-RII responses ( Figure 8 ). A dichotomy was observed in TLR8 responses after 48 hours stimulation, with lower activin A, CCL19, CXCL16, IL-1RA/RN IL-16Rα, MMP-9 and TNFRII responses, but higher CCL2 responses in PBMCs of PLWH compared to controls. Opposing response patterns were also observed after 48 hours of TLR9 stimulation, with lower CCL1, CCL2, CXCL16, IL-6Rα, IL-6, and MMP-1 responses, but higher amphiregulin, CCL5 and CXCL4 responses by PBMCs of PLWH compared to HV ( Figure 8 , Supplementary Figure S8 ).

Overall, secreted protein responses induced by the different TLR pathways were lower in PBMCs from PLWH, except for sCD14, CCL2, CCL5 and CXCL4, which were higher in response to TLR4, TLR7 and TLR9 stimulations, and CRP, which was higher in PLWH only after early TLR7 and TLR8 stimulation. As CCR5 is the cognate ligands from HIV co-receptor, binding CCL5 these increased responses could be relevant to overall pathogenesis.

Using the same high content imaging, features defining cellular size, cell motility, and nuclear shape, were analyzed from PBMCs of HV and PLWH with TLR4, TLR7, TLR7/8, TLR8, or TLR9 stimulations after 6, 24, and 48 hours of stimulation. The resulting high-content images were analyzed using the same machine-learning trained algorithms described above ( Figure 1B ). The results were plotted as color-coded heatmaps representing the fraction of cells per well that were positive for each listed feature or normalized using previously described criteria ( Table 2 ), with significant changes relative to DMSO indicated ( Supplementary Figure S9 ).

Changes in the cell features were heterogeneous in the two groups ( Figure 9 , Supplementary Figure S9 ). Six hours of TLR7 stimulation resulted in a lower fraction of actin dispersed cells, but a higher fraction of actin contracted cells in PBMCs of PLWH compared to HV. A smaller fraction of cells was positive for annexin V, but a more substantial fraction of large round cells was undergoing phagocytosis in PBMCs of PLWH following TLR7/8 stimulation than HV. Following overnight stimulation with TLR7, a greater increase in the fraction of cells with fragmented nuclei in PBMCs of PLWH was observed compared to those of healthy controls. In contrast, 48 hours of TLR7/8 stimulation resulted in a reduced fraction of annexin V positive cells in PBMCs of PLWH than HV. After 24 hours, there was a significantly lower number of large round cells but a higher number of large round cells undergoing phagocytosis in PBMCs of PLWH with TLR9 stimulation. Moreover, 48 hours of TLR4 stimulation led to larger fractions of fragmented cells and phagocytosis, while TLR7 stimulation resulted in higher frequencies of small round cells, higher fractions of PBMCs with dispersed actin and large round cells with no podosomes, in PBMCs of PLWH compared to healthy controls. 48 hours of TLR7 stimulation also led to higher numbers of large round cells lacking podosomes, while 6 and 48 hours of TLR9 stimulation resulted in a higher fraction of large round cells with and without podosomes in PBMCs from PLWH compared to controls ( Figure 9 ). Interestingly, unstimulated PBMCs from PLWH also had higher numbers of cells with fragmented nuclei and large round cells lacking podosomes, but fewer cells were positive for annexin V and actin contraction between 6-24 hours of culture ( Figure 9 , Supplementary Figure S9 ).

Overall, the image analysis of cell features revealed a heterogeneity in responses in the two study groups, with significant differences being observed in the proportions of cells undergoing phagocytosis, nuclear fragmentation and actin contraction or dispersal.