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. Author manuscript; available in PMC: 2022 Apr 25.
Published in final edited form as: J Immunol Methods. 2021 Dec 2;501:113198. doi: 10.1016/j.jim.2021.113198

Multiparameter immunohistochemistry analysis of HIV DNA, RNA and immune checkpoints in lymph node tissue

Zuwena A Richardson a, Claire Deleage b, Candani SA Tutuka c,d, Marzena Walkiewicz c,e, Perla M Del Río-Estrada f, Rachel D Pascoe a, Vanessa A Evans a, Gustavo Reyesteran f, Michael Gonzales g, Samuel Roberts-Thomson g, Mauricio González-Navarro f, Fernanda Torres-Ruiz f, Jacob D Estes h, Sharon R Lewin a,i,j, Paul U Cameron a,d,k,*
PMCID: PMC9036546  NIHMSID: NIHMS1796507  PMID: 34863818

Abstract

The main barrier to a cure for HIV is the persistence of long-lived and proliferating latently infected CD4+ T-cells despite antiretroviral therapy (ART). Latency is well characterized in multiple CD4+ T-cell subsets, however, the contribution of regulatory T-cells (Tregs) expressing FoxP3 as well as immune checkpoints (ICs) PD-1 and CTLA-4 as targets for productive and latent HIV infection in people living with HIV on suppressive ART is less well defined. We used multiplex detection of HIV DNA and RNA with immunohistochemistry (mIHC) on formalin-fixed paraffin embedded (FFPE) cells to simultaneously detect HIV RNA and DNA and cellular markers. HIV DNA and RNA were detected by in situ hybridization (ISH) (RNA/DNAscope) and IHC was used to detect cellular markers (CD4, PD-1, FoxP3, and CTLA-4) by incorporating the tyramide system amplification (TSA) system. We evaluated latently infected cell lines, a primary cell model of HIV latency and excisional lymph node (LN) biopsies collected from people living with HIV (PLWH) on and off ART. We clearly detected infected cells that coexpressed HIV RNA and DNA (active replication) and DNA only (latently infected cells) in combination with IHC markers in the in vitro infection model as well as LN tissue from PLWH both on and off ART. Combining ISH targeting HIV RNA and DNA with IHC provides a platform to detect and quantify HIV persistence within cells identified by multiple markers in tissue samples from PLWH on ART or to study HIV latency.

Keywords: HIV latency, Immune checkpoints, Immunohistochemistry, In situ hybridization, RNAscope, Microscopy, Lymph nodes

1. Introduction

Antiretroviral therapy (ART) has led to a significant decline in mortality and morbidity in people living with HIV (PLWH), however treatment is life long and there is no cure. Despite rapid decline of HIV RNA in plasma to undetectable levels following ART, virus persists in long lived and proliferating latently infected CD4+ T-cells (Chun et al., 1995; Hosmane et al., 2017). Latency is defined as the integration of intact virus into the host genome but failure to complete the virus life cycle in the absence of T-cell stimulation (Brooks et al., 2003; Hermankova et al., 2003) and can be distinguished by detection of HIV DNA but not HIV RNA (Chun et al., 1998; Finzi et al., 1999). Latently infected cells are rare in blood and are found at far higher frequency in tissue such as lymph node (LN) and the gastrointestinal (GI) tract (Yukl et al., 2010; Chun et al., 2008; Yukl et al., 2013; Khoury et al., 2017). Identification of specific subpopulations of latently infected cells by multiple markers, specifically in tissue from PLWH on ART, is of high interest but better tools are needed for such studies.

New strategies are needed to understand the complexity of HIV persistence in tissue sites to allow analysis of the cell phenotype in situ and provide spatial information about the exact location of the infected cell in tissue structures (Aguzzi and Krautler, 2010; Josefsson et al., 2013; Rato et al., 2017). The lack of sensitivity of traditional in situ hybridisation (ISH) approaches has led to development of a new and more sensitive ISH method of RNAscope and DNAscope. These use fluorescent labelled probes in either formalin-fixed, paraffin embedded (FFPE) tissues samples or fresh cryopreserved tissue. This method identifies RNA and DNA in individual cells and uses bright-field microscope (for chromogenic detection), or a fluorescent microscope (for fluorescent labels) (Wang et al., 2012).

The combination of both DNA and RNAscope in the same tissue section collected from either simian immunodeficiency virus (SIV) infected nonhuman primates (NHP) or PLWH allows for the discrimination of viral DNA (vDNA+) cells that are transcriptionally silent, from those that are actively transcribing viral RNA (vRNA+) cells. These methods demonstrate the importance of the B cell follicles (BCFs) as an important site for persistence of vRNA+ cells in LN from PLWH on ART (Deleage et al., 2016) and in all lymphoid tissue (i.e. LN, gut, lungs and spleen) in NHP even after years of ART (Estes et al., 2017).

There is currently no surface marker that can distinguish latently infected from productively infected cells in PLWH on ART. There are many cellular markers that are enriched for HIV persistence and these include immune checkpoint (IC) markers which can be found on certain CD4+ T cell subsets, including regulatory T-cells (Tregs) (Takahashi et al., 2000; Amarnath et al., 2010). We and others have shown that on ART, virus can persist in cells that express PD-1 (Evans et al., 2018; Fromentin et al., 2019) and these PD-1 high cells are frequently found in the BCFs (Banga et al., 2016; Fukazawa et al., 2015) and have features consistent with T follicular helper (Tfh) cells. However, HIV can persist in cells other than PD-1 hi cells, including cells that express other IC markers such as TIM-3 (Rallón et al., 2018), TIGIT (Fromentin et al., 2016; Yin et al., 2018; Vendrame et al., 2020), and CTLA-4 (McGary et al., 2017), which are largely found in the extrafollicular areas. Therefore characterization of HIV and multiple markers in tissue is of great importance and has been studied in FFPE tissue (McGary et al., 2017), however, further progress to stain more antigens on the same tissue has been limited.

Advances in immunohistochemistry (IHC) now allows simultaneous in situ detection of RNA and multiple surface proteins in FFPE tissues (Wee et al., 2018), using the Tyramide signal amplification (TSA) Opal system and the Vectra multispectral IHC (mIHC) imaging system from Perkin-Elmer and could potentially be applied to fully understand where HIV persists in LN tissue in PLWH on ART. In this study, we report the development of a seven-color mIHC panel, combining both HIV DNA and RNAscope with IHC, allowing us to simultaneously characterize HIV RNA, HIV DNA, CD4, FoxP3, PD-1, CTLA-4 and DAPI in FFPE tissue using the TSA Opal system. Sections where then scanned and analysed using the Vectra 3 quantitative imaging system from Perkin-Elmer. We could simultaneously detect HIV RNA and DNA, in combination with up to seven cellular markers. In agreement with previous studies both vRNA and DNA could be detected in LN tissue both on and off ART and we found co-localisation of vRNA with FoxP3 and CTLA-4 consistent with Tregs being a potential reservoir for HIV on ART.

2. Materials & methods

2.1. Cell lines

ACH-2 cells (subclone A3.01) obtained from NIH AIDS reagent program contain an integrated copy of HIV and although commonly used as a model for HIV latency, have evidence of multiple diverse integration sites consistent with some low level virus replication (Symons et al., 2017). ACH-2 cells were cultured in Roswell Park Memorial (RPMI) 1640 supplemented with 10 mM N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2 mM l-glutamine, 10%; heat inactivated fetal bovine serum (FBS). The parental cell line, Jurkat cells (clone E6–1) were cultured in RPMI 1640 (RF10) supplemented with 2 mM l-glutamine, 1% Penicillin-Streptomycin and 10% FBS.

The J-Lat isoclone 6.3 contains a single copy of integrated HIV which lacks multiple HIV genes including envelope but includes both Tat and green fluorescent protein (GFP) open reading frames both under the control of the HIV promoter in the 5′ long terminal repeat (LTR) (Jordan et al., 2003); NIH AIDS reagent program]. The cell line was cultured in RPMI 1640, 10% FBS, supplemented with penicillin (100 Ul/ml) and streptomycin (100 μg/ml). All cells were maintained in a humidified incubator at 37 °C with 5% CO2.

ACH-2 cells were mixed with uninfected Jurkat cells to obtain serial dilutions of 10–20 × 106 total cells at different ratios of infected to uninfected cells of 1:2, 1:10 and 0:1. J-Lat cells were mixed with uninfected Jurkat cells at a ratio of 1:2. For some experiments, ACH-2 and J-Lat 6.3 cells were first stimulated with phorbol myristyl acetate (PMA; 10 nM) for 24 h and then diluted with Jurkats at a ratio of 1:2.

2.2. An in vitro HIV latency model using resting CD4+ T-cells and monocytes

Resting CD4+ T-cells and monocytes from peripheral blood mononuclear cells (PBMC) collected from healthy donors (Australian Red Cross, Melbourne, Australia), were cultured in 10% RPMI 1640 medium with antibiotics (penicillin–streptomycin–glutamine; RF10). These cells were infected with NL4.3 which contained the CCR5–using envelope protein from AD8 and expressed GFP (GFP) in place of the nef protein or under the control of an internal ribosome entry site (IRES) element (NL (AD8)Δnef-GFP and NL(AD8)IRES–GFP respectively) were produced by transfection of 293 T-cells at a multiplicity of infection (MOI) of 0.5 as determined by limiting dilution on TZM-bl (Reed and Muench, 1938). Resting CD4+ T-cells were isolated by negative selection using a panel of monoclonal antibodies and magnetic bead sorting (autoMACS; Miltenyi Biotec, San Diego, CA) as previously described (Evans et al., 2013). Monocytes (CD14+) were isolated from syngeneic donors using positive selection for CD14 on by magnetic bead sorting (autoMACS; Miltenyi Biotec). Monocytes with a purity ≥95% and resting CD4+ T-cells with a purity of >95% were used. Resting CD4+ T-cells were cultured alone or with syngeneic monocytes at a ratio of 10:1 for 24 h in the presence of IL-2 (2 U/ml; Roche Diagnostics, Mannheim, Germany) and Staphylococcus enterotoxin B (SEB) (10 ng/ml) before infection with either reporter virus for 2 h. After washing, cells were cultured for five days in IL-2 (2 U/ml) supplemented media without additional SEB. Productive infection was measured by flow cytometry at day five post infection. Monocytes were excluded by gating for HLA-DRlo CD3+ T cells. CD4+ T-cells that were non-productively infected (EGFP−) were sorted by flow cytometry using a FACSAria (BD Biosciences, San Jose, CA). Latent infection was determined following activation of 200,000 sorted CD4+ T-cells (EGFP−) with immobilized anti-CD3 (7 μg/ml; Beckman Coulter, Brea, CA) supplemented with soluble CD28 (7 μg/ml; BD Biosciences). Sorted cells were harvested 72 h after stimulation in RF10 media supplemented with IL-2 (10 U/ml) and IL-7 (1 ng/ml) with anti-CD3/CD28 and raltegravir (1 μM; National Institutes of Health AIDS Reagent Program) was added to media to prevent spreading infection. The expression of inducible virus was quantified by GFP expression using flow cytometry (FACSCanto; BD Biosciences).

2.3. Fixation and embedding of cell pellets in paraffin

Cells were washed in PBS and fixed in 4% paraformaldehyde (PFA) at room temperature for 24 h with mild aggregation on a slow speed rocker. Fixed cells were washed to remove PFA, which was replaced with 80% ethanol, on a slow speed rocker for mixing. Cells were then washed and in a drop wise manner, suspended in liquefied Histogel biopsy gel (Fisher Scientific, Hampton, New Hampshire) pre-warmed to 50 °C in a water bath and centrifuged for five minutes at 2000 ×g. After centrifugation, cells were cooled for 5–10 min at 4 °C in covered by 80% ethanol and the cell pellet was paraffin embedded.

2.4. Human subjects

Four participants underwent elective surgical resection of LNs. All participants were PLWH and were either naïve to ART (n = 2; with HIV RNA >150,000 copies/ml) or on suppressive ART (n = 2; defined as HIV RNA <90 copies/ml for at least 2 years). LNs were collected at the Centro de Investigación en Enfermedades Infecciosas (CIENI-IN ER), Mexico City, Mexico and the study was approved by the local human and research ethics committee (B03–16). Clinical details are provided in Supplementary Table 1.

2.5. Histology and immunohistochemistry

Immunohistochemistry was performed on FFPE LN tissue using a peroxidase-based method on 5 μm sections on Superfrost® plus microscope slides (Thermo Scientific). Specimen slides were incubated at 60 °C for 45–60 min for the melting and fixing of specimens onto microscope slides. In order to deparaffinise, slides were washed in xylene and rehydrated in ethanol of 100% to 70%. Slides were then treated with hydrogen peroxidase (Chem-Supply, Gillman, Australia) in 0.3% H2O2 (v/v) in double-distilled H2O (ddH2O) for 15 min at room temperature. Heat induced epitope retrieval was then performed until boiling was achieved, followed with 90 °C for 15 min by microwave treatment (MWT) in the appropriate retrieval buffer optimized for each target epitope. Specimens were then blocked using Background Sniper (Biocare Medical, Concord, CA) for 15 min prior to primary antibody application.

2.6. HIV-1 RNA and DNA target probes

The HIV RNA probe used was designed to hybridize to viral RNA in gag, pol, vif, vpr, tat, rev, env, nef, and vpx genes (vRNA anti-sense probe, ACD catalog: ADV416111) as well as HIV DNA probe targeting the Gag-Pol coding region (vDNA sense probe, ACD catalog: ADV425531). All probes were purchased from Advances Cell Diagnostics (ACD Newark, CA) and a complete list of the sequence of each probe used has been previously published (Deleage et al., 2016).

2.7. HIV- RNA and DNA in situ hybridization

For the detection of vRNA and vDNA, we used the RNAscope 2.5 brown kit (Deleage et al., 2016), with some modifications (Deleage et al., 2016). In brief, probes were visualized by hybridizing with pre-amplifiers, amplifiers in a humidified HybEZ oven, and finally, fluorescent label with TSA amplification system (Perkin Elmer, Waltham, Massachusetts). Pre-amplifier 1 was hybridized at 40 °C for 30 min. Following washing of samples twice, then hybridized with Amplifier two in a humidified at 40 °C for 15 min. Again following two washes, amplifier three was hybridized at 40 °C for 30 min. After a further two washes, Amplifier four was hybridized at 40 °C for 15 min, washed twice, following hybridization of Amplifier five for 30 min at 40 °C, again with two washes and lastly incubation of Amplifier 6 for 15 min at 40 °C for 15 min with a final two washes. For vRNA detection, slides were first washed once in Tris Buffered saline (TBS) with Tween (VWR International, Radnor, Pennsylvania) and incubated for 4 min with either 3,3′-diaminobenzidine (DAB) for 5–10 min or TSA dyes. TSA dye 520 (1:700) was used for 4 min for labelling of vRNA, TSA dye 570 (1:700) for two minutes for labelling of vDNA and washed twice in TBS-Tween for five minutes. Lastly, slides were incubated with either DAPI (Perkin Elmer) or counterstained with hematoxylin and eosin (H&E) for one-two minutes. Slides were incubated with DAPI (1:2) for either four (human LN tissue) or six minutes (cell lines or primary model of latency). For the removal of DAPI, slides were washed twice with TBS-Tween for five minutes.

2.8. Simultaneous detection of vDNA and vRNA

In order to visualize vDNA and vRNA+ cell simultaneously, we combined both DNA and RNAscope (DNA/RNAscope). Following fixation and pretreatment as described for RNA and DNAscope (Deleage et al., 2016), slides were incubated for two hours at 40 °C with RNA probes as described previously, following amplification, washes, labelling with TSA 520 (1:700) for four minutes and MWT retrieval. For vDNA, slides were hybridized with HIV DNA sense probes overnight at 40 °C, followed by amplification, washes, labelling with TSA 570 (1:700) for two minutes and counterstained with DAPI.

2.9. Multiplex immunohistochemistry (OPAL™)

Rabbit monoclonal antibodies to CD4 (Cell Marque, Rocklin, CA) clone 104R-1, 1/100, high pH retrieval), mouse monoclonal antibody to FOXP3 (Abcam, Cambridge, UK), clone IgG1, 1/1000, high pH retrieval), mouse polyclonal antibody to PD-1 (Abcam, IgG1 Nat105, 1/500, low pH retrieval), mouse monoclonal antibody to CTLA-4 (MyBiosource, San Diego, CA) IgG2a/k, 1/100, high pH retrieval) were used. Antibodies were diluted using a background-reducing antibody diluent buffer S3022 (Agilent, Santa Clara, CA). A horseradish hydrogen peroxidase (HRP) linked anti-mouse and anti-rabbit secondary antibody, EnvisionTM HRP (Agilent), was used for each primary antibody species according to the manufacturer’s recommendation. Immunofluorescent signal was visualized using the TSA amplification system, OPAL™ 7-color fluorescent IHC kit (Perkin Elmer), TSA dyes 540, 620, 650, and 690 (1:50) for ten minutes, counterstained with Spectral DAPI. All slides were imaged on the Vectra® 3 Quantitative Pathology Imaging System (Perkin Elmer). Images were then examined using color separation and inForm® Software v2.1 (Perkin Elmer). All slides were scanned on the Vectra at 10× magnification in order to select for high-powered imaging at 20× (resolution of 0.5 μm per pixel) using Phenochart (Perkin Elmer).

2.10. Quantitative image analysis

HALO® image analysis software from Indica Labs (versions 2.0, Albuquerque, New Mexico) was used for quantitative IHC assessment of the number of cells positive for each stain as described above, including vDNA+ and vRNA+ cells in each compartment and the number of cells with different co-localization. For the evaluation of probe signals for both vDNA and vRNA+ cells in cell lines, primary T-cells and LN tissue samples, HALO’S Fluorescence In Situ Hybridization (FISH) probing module was used to quantify co-expression of fluorescent HIV DNA and RNA probes on a per cell basis and cell classification of each probe (0, 1+, 2+, 3+ and 4+) using ACD guidelines for scoring. HALO cell-based segmentation algorithm was used to accurately segment nuclear DAPI counterstained cells. Once segmentation was verified, the HALO’S Highplex Fluorescence algorithm was designed for each fluorescent probe using adjustable thresholding for positive signals (Horai et al., 2019). Signals were determined by comparing the Opal single stains with the multiplex signal (Stack et al., 2014a). Negative control slides consisted of primary antibodies (excluding Opal), LN tissue from HIV-1 negative individuals and LN tissue from PLWH incubated with ACD negative control probes. Only cells above the positive threshold were scored. This approach allowed for the evaluation of cell phenotypes (DAPI, DNA, RNA, CD4, FoxP3, PD-1 and CTLA-4) and subsets (Tregs, nonTregs and nonCD4 expressing PD-1+/− and CTLA-4+/−). Regions of interest (ROI) containing TCZs and BCFs were manually selected by outlining regions and any tissue or staining artefacts were excluded. The same algorithm design was used for each ROI per slide based on staining intensity and adjusted for each patient to account for staining variability.

2.11. Statistical analysis

Statistical analysis was performed and graphs plotted using Graphpad Prism (version 8.2.1, GraphPad Software, La Jolla, CA). Given the small sample numbers, we assumed normality and used a Two-tailed paired t-test to determine difference in vDNA− and vRNA+ cells between unstimulated and stimulated ACH-2 and J-Lat cells. Details of significant differences between data groups have been described in each figure legend and P values less than 0.05 was considered significant.

3. Results

3.1. Identification of latent and productively infected cells using HIV DNA and RNA probes in cell lines

We first optimized our method for tissue analyses using the T-cell lines ACH-2 and J-Lats. We used unstimulated ACH-2 cells which contains 2 integrated copies of HIV-1 DNA (Symons et al., 2017) and have evidence of low level virus replication (Symons et al., 2017). These cells were diluted in the uninfected parental Jurkat cell line. Latently infected cells were defined as vDNA+, vRNA− cells and productively infected cells as vDNA+ vRNA+ or vDNA− vRNA+ cells. We assessed the specificity for each probe for HIV RNA and HIV DNA using the in situ hybridization (ISH) assays (RNAscope and DNAscope).

We detected no signals using the no probe control (Supplemental Fig. S1A, i) and uninfected Jurkat cells alone (Supplemental Fig. S1A, ii). When unstimulated ACH-2 cells were diluted at a ratio of 1:10 in uninfected Jurkat cells, we were able to detect vRNA+ cells with chromogen diaminobenzidine (DAB), as a brown signal in 10% of cells, indicating highly specific detection of productively infected cells. The brown cells were densely stained, encompassing the entire cell body (Supplemental Fig. S1A, iii). Similar results were obtained with a higher ratio of infected cells and a corresponding increase in DAB positive or vRNA+ cells (Supplemental Fig. S1A, iv).

We then evaluated HIV DNA probes using the same samples. With the DNAscope probes, we demonstrated no DAB positive signals in the no probe control (Supplemental Fig. 1B, i) and in Jurkat cells (Supplemental Fig. S1B, ii). When we used the HIV DNA probes in mixtures of PMA-activated ACH-2 and Jurkat cells, there were punctuate signals of one or two dots (vDNA+ cells) within the nucleus. The frequency of cells visually scored as vDNA+ decreased accordingly with the proportion of diluted PMA-activated ACH-2 with uninfected Jurkat cells (Supplemental Fig. S1B, iii and iv, black arrows). Collectively, this data indicate that the HIV RNA probes could detect vRNA+ cells and vDNA in PMA-stimulated ACH-2 cells as previously reported (Deleage et al., 2016).

After successfully identifying vDNA and vRNA+ cells by bright field microscopy using DAB, we next sought to identify both HIV DNA and/or RNA using dual ISH and IHC and imaged on the multispectral imaging platform Vectra (Perkin-Elmer). This approach was further investigated in combination with the TSA system, which has previously demonstrated to be more sensitive than conventional DAB or fluorescence IHC (Stack et al., 2014b), but has been difficult to simultaneously examine HIV RNA and DNA with cellular markers (mIHC >5-plex) (Millar, 2020; Vasquez et al., 2018).

We normalized the background to a no probe control (Fig. 1A, i), and detected green vRNA+ cells, when ACH-2 were at a frequency of 1 in 10 uninfected Jurkat cells (Fig. 1A, ii). We used a similar approach for DNA probes using a no probe control (Fig. 1B, i) and detected red vDNA+ cells as one or multiple distinct punctuate red dots within the nuclei (Fig. 1B, ii). To determine whether both RNA and DNA probes could be detected simultaneously, we used a dilution of ACH-2 at a frequency of 1 ACH-2 cell to 1 uninfected Jurkat cells, and detected vRNA+ cells (green) within the cytoplasm (Fig. 1C, i, iii) and punctuate dots in red, indicative of vDNA+ cells (Fig. 1C, ii, iii). In these cultures, we could detect both productive (vRNA+ vDNA+, white arrow 1.) and latent infection (vRNA-vDNA+ cells, white arrow 2.).

Fig. 1.

Fig. 1.

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Assessment of HIV RNA and DNA probes in ACH-2 cells using immunofluorescence detection. Unstimulated ACH-2 cells were diluted 1:10 with uninfected Jurkat cells and hybridized with either (A) RNA (green) or (B) DNA (red) probes. The left panels contain no target probe controls. (C) (i) Unmixed composite image of both RNA and DNA probes showing simultaneous detection of vRNA and vDNA+ in a cell, consistent with a productively infected cell (white arrow 1) and a vDNA+ cell, consistent with a latently infected cell (white arrow 2) (ii) vRNA probe only indicated in green and (iii) vDNA probe only indicated in red. All specimens were counterstained with DAPI nuclear staining in blue and all micrographs were taken at 40× magnification on the Vectra ®. Scale bars are 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. Quantification of probes in cell lines

We next quantified the number of vDNA and/or vRNA+ cells using Halo imaging analysis software in the infected cell lines, ACH-2 and J-Lat which were either unstimulated or stimulated with PMA. Simultaneous visualization of vDNA+ (red) and/or vRNA+ cells (green) was possible in ACH-2 (Fig. 2A) and J-Lat cell lines (Fig. 2B). Both fluorescence signals for HIV DNA and RNA were visible in unstimulated and stimulated ACH-2 cells (Fig. 2A) and there was a significant increase in the number of vDNA and vRNA+ cells in both latently and productively infected cells after stimulation (Fig. 2C). In unstimulated J-Lat cells, the majority of cells were vDNA+ cells (red) (Fig. 2B) with the number of vRNA+ cells significantly increasing after stimulation (Fig. 2D). These data are consistent with ACH-2 cells being infected with replication competent virus (Symons et al., 2017; Emiliani et al., 1996) while J-Lat cells are infected with replication defective virus, however, once these cells are stimulated the activation of the LTR leads to expression of viral RNA (Zhang et al., 2018). After stimulation with PMA, the ratio of vRNA to vDNA+ cells increased in both ACH-2 and J-Lat cells (Fig. 2E). We used the Halo analysis software to quantify the number of HIV DNA and RNA positive probe signals in each cell. HIV DNA and/or RNA probe signals were quantified using ACD recommended RNAscope scoring guideline which consist of cells that are 0+ (no probe signal), 1+ (1–3 probes), 2+ (4–9 probes), 3+ (10–13 probes) and 4+ (>14). In the ACH-2 cells, we observed multiple copies with both DNA and RNA probes within each cell, mainly in the 1+ and 2+ categories (Fig. 2F) with the frequency of DNA and RNA increasing in both categories after stimulation (Fig. 2G). In the J-Lat cells, HIV DNA+ cells were in the 1+ expression category with a few HIV RNA+ cells (Fig. 2 H). Following stimulation, the number of HIV DNA and RNA cells increased and were mostly in the 1+ expression category (Fig. 2I). One interesting observation while utilizing image analysis on the Halo platform, we were able to enumerate multiple copies of vDNA in J-Lat 6.3 cells pre and post-activation, despite there being no evidence that suggests that these cells contain multiple copies (Symons et al., 2017).

Fig. 2.

Fig. 2.

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Sensitivity of HIV DNA and RNA probes in unstimulated and stimulated ACH-2 cells and J-Lat cells. Representative image of (A) ACH2 and (B) J-Lat cells that were (i) unstimulated and stained with no probe control (ii) unstimulated and hybridized with HIV DNA and RNA probes (iii) stimulated with PMA and stained with no probe control and (iv) stimulated with PMA and hybridized with HIV DNA and RNA probes. DAPI was used for nuclear counterstain and all micrographs were taken at 20× magnification on the Vectra®. Scale bars are 20 μm. Proportion of vDNA+ and vRNA+ cells in unstimulated and stimulated (C) ACH-2 and (D) J-Lat cells. (E) Ratio of vDNA+ and vRNA+ cells in unstimulated and stimulated ACH-2 and J-Lat cells. (C-E). Each color represents a region of interest. Columns represent the mean of four regions. Proportion of cells that express different frequencies of vRNA and vDNA based on the ACD scoring system of grade 0 to 2 defined as the number of RNA and DNA copies for (F) unstimulated ACH-2; (G) stimulated ACH2; (H) unstimulated J-Lats; and (I) stimulated J-Lats. Columns represent the mean of three regions of interest and the numbers above each column represents the mean value. Comparisons between conditions were performed with a t-test. * P < 0.05; ** P < 0.01.

3.3. HIV RNA and DNA detection in an in vitro model of inducible latency

We next investigated expression of virus using the DNA and RNA probes and the TSA system in an in vitro infection model for HIV latency (Evans et al., 2013). Resting CD4+T-cells were cultured in the presence and absence or syngeneic monocytes, with all culture media being supplemented with IL-2 (2 U/ml). Following 24 h of culture, CD4+ T-cells alone and CD4+T-cells-monocytes co-cultures were infected with NL (AD8)-nef/eGFP at an MOI of 0.5 for 2 h (Fig. 3A). These cells were then collected and processed to generate FFPE blocks. We did not observe vDNA+ nor vRNA+ cells in CD4+T-cells alone or CD4+T-cells-monocytes co-cultures 2 h following infection (Fig. 3B i and ii). At day 5 post-infection, the GFP+ cells contained both vRNA (green) and/or vDNA (red) + cells consistent with productively infected cells (Fig. 4B, iii). In contrast, GFP- cells contained few vRNA+ cells and the majority of cells contained integrated vDNA+, suggesting that these cells were latently infected (Fig. 3B, iv). Finally, 3 days following activation of GFP− cells, i.e. at day 8, we observed an increase in vRNA+ (green) and/or vDNA + cells, (red) (Fig. 3B, v), consistent with inducible latent infection.

Fig. 3.

Fig. 3.

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HIV DNA and RNA probes to detect latent and productive infection in an in vitro latency model. (A) Schematic diagram of experimental approach. Resting CD4+ T cells (orange) were cultured with monocytes (grey) for 24 h and infected with NL(AD8)-nef/GFP virus (green) at an MOI of 0.5 for 2 h. Non-productively infected (GFP−) cells were sorted at day 5 post infection and the number of productively infected (GFP+) cells was determined by flow cytometry. Sorted GFP− cells were stimulated with anti-CD3/CD28+IL-7+IL-2+raltegravir for 3 days to induce expression of virus from latently infected cells. Cells were harvested at 2 h, day 5 and 8 and formalin-fixed and paraffin-embedded (FFPE), sectioned and stained for vRNA and vDNA as indicated by black arrows. (B) Representative image of vDNA (red) and vRNA (green) showing (i) T-cells alone; (ii) T-cells and monocytes; (iii) eGFP+ T-cells and monocytes; (iv) eGFP− T-cells; and (v) activated eGFP− T-cells. (C) Mean number of vDNA+ (open column) and vRNA+ (grey column) cells when analyzing GFP+ (productive), GFP− (latent) and inducible GFP+ cells. (D) Mean ratio of vRNA+ and DNA+ cells in the same cell populations. DAPI was used for nuclear counterstain and all micrographs were taken at 20× magnification on the Vectra. The lower limit of detection of each assay is represented by a dotted line. Columns represent the mean of t sections per slide. Each coloured symbol in panels C and D represents a region of interest. Scale bars are 20 μm. t-test; mean; * P < 0.05; ** P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Fig. 4.

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HIV RNA and DNA probes to detect HIV persistence in LNs from PLWH. Representative image of LN tissue from a person living with HIV off ART using (i) low and (ii) high magnification with (A) HIV RNA probes demonstrating vRNA (green) in the BCF region (white arrows); (B) HIV DNA probes demonstrating vDNA (red) as a punctate mark (white arrows); and (C) both HIV RNA and DNA probes showing vRNA (green), vDNA (red) and DAPI (blue) with the magnified image indicating simultaneous detection of both vDNA and vRNA+ cells (arrow 1) and single detection of vDNA+ cells (arrow 2). Additional panels show an unmixed composite image for (iii) vDNA+ cells and (iv) vRNA+ cells. (D) The number of vRNA+ and vDNA+ cells in LN tissue from 4 HIV-infected participants (2 on ART and 2 off ART), with the mean number from four sections shown as a column. The ratio of vRNA and vDNA+ cells is shown as a number. Each symbol represents a region of interest. All specimens were subjected to DAPI nuclear staining, shown in blue and all micrographs were taken at 20× magnification on Vectra® multispectral IHC imaging platform. Scale bars are 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To address the efficiency of viral detection using our mIHC approach, we quantified the proportion of productive, latent and inducible infection in our in vitro model of latency (Fig. 3C). In productive infection, we found similar levels of vDNA+ and vRNA+ cells. In latent infection, we detected a higher frequency of vDNA+ cells compared to vRNA+ cells. After activating these latent cells (inducible infection), we observed an increase in vRNA+ cells, with roughly equivalent levels of vDNA+ cells when we compared cultures pre and post stimulation (Fig. 3C). Finally we examined the ratio of vRNA+ to vDNA+ cells and observed a significant decrease in the ratio of vRNA+ to vDNA+ cells in latent cells compared to productive and inducible infection (Fig. 3D). Together these findings are consistent with the establishment of latency in this in vitro cell model, and that following stimulation, expression of viral RNA was induced using DNAscope and RNAscope.

3.4. Visualization of vDNA and vRNA in LN tissue from PLWH on ART

We next evaluated FFPE LN tissue from PLWH on ART using RNAscope and DNAscope and the TSA detection system. We observed vRNA+ cells in LN tissue (Fig. 4A, white arrows) located in the B cell follicles, as previously described (Deleage et al., 2016; Tacchetti et al., 1997; Fletcher et al., 2014). Using DNAscope in combination with the TSA system, we detected punctuate signals within the nuclei (Fig. 4B, white arrows) indicative of HIV provirus. We detected both productively infected cells, ie vDNA+ and vRNA+ cells (Fig. 5C, ii, arrow 1) as well as latently infected cells i.e. vDNA+ cells only (Fig. 4C, ii, second arrow) . Finally we quantified the frequency of productive and latent cells in LN tissue from PLWH on and off ART. Both vDNA+ and vRNA+ cells were detected in all samples, with less frequent vDNA+ and vRNA+ cells on ART compared to those off ART (Fig. 4D). In addition, the vRNA+ to vDNA+ ratio was lower on ART compared to off ART. Together, this suggests that the frequency of HIV RNA is less than the frequency of HIV DNA in PLWH on ART.

Fig. 5.

Fig. 5.

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Multiplex detection with HIV RNA and DNA probes and surface markers in LN tissue from PLWH. Representative images of LN tissue sections from a person living with HIV off ART depicting simultaneous detection of HIV DNA (red) and RNAscope (green) in combination with antibodies to CD4 (cyan), FoxP3 (white), CTLA-4 (magenta) and PD-1 (orange). (i) Whole slide scan and (ii) composite image (enlarged section of whole slide scan, white box) depicting colocalization of vDNA+ cell (arrow 1) with CD4+ T cells, PD-1 and CTLA-4 and vDNA+ and vRNA+ cell (arrow 2) with CD4+ T cells, PD-1 and CTLA-4. Representative unmixed composite images of (iii) HIV DNA and RNA and (iv) HIV DNA, RNA and CD4+ T-cells. DAPI was used for nuclear counterstain and micrographs were taken at 10× (whole slide scan) and 20 X magnifications using the Vectra™ multispectral imaging software. Scale bars are 500 μm (whole slide scan) and 20 μm. B) Images were analysed with HALO to quantify the total events (number) of HIV-1 RNA+ and/or DNA+ positive cells and denoted as total cells (DAPI+), CD4neg (CD4− FoxP3−), CD4pos (CD4+), nonTregs (CD4+FoxP3−) and Tregs (CD4+ FoxP3+) in the BCF (grey symbols) or TCZ (blue symbols) from participants off ART (closed symbol) and on ART (open symbol). C) The absolute number of HIV-1 RNA+ and/or DNA+ positive cells on total cells prior-to ART (i) and on ART (ii) and (D) the frequency of infection compared with the total amount of HTV-1 RNA+ and/or DNA+ cells is shown as log 10 for PLWH off ART (i) and on ART (ii) in TCZ and the BCF of LN. Each participant is shown by a different colour (n=7 subjects). Grey shaded area highlights people living with HIV-1 on ART. TCZ: T-cell zone; BCF: B-cell follicle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5. Co-expression of cell markers in productive and latently infected cells

To identify if IC markers such as PD-1 and CTLA-4 were co-located with latent or productively infected cells, we performed a multiplex assay with TSA, combining ISH and IHC to examine FFPE LN tissue from PLWH on and off ART. Slides were stained first for RNA and DNA, followed by antibodies to CD4, FoxP-3, PD-1 and CTLA-4 with imaging on the Vectra multispectral IHC imaging platform (Fig. 5). We were able to demonstrate latent infection (vDNA+ and vRNA− cells) co-expressing the cellular markers CD4, CTLA-4 and PD-1 (Fig. 5, arrow 1) and a productively infected cell (vDNA+ and vRNA+ cells) co-expressing CD4, PD-1 and CTLA-4 (Fig. 5, arrow 2).

We then looked at the distribution of productively and latently infected cells using RNA/DNAscope. Given the small numbers of participants, we are only able to describe trends and not apply any statistical tests. We found a similar frequency of HIV-1 RNA+ and/or DNA+ cells in the BCF and TCZ (Fig. 5B) in participants both prior to and following ART. In addition, cells that were positive for HIV-1 were evenly distributed in both PLWH on and off ART (Fig. 5C). Lastly, when we looked at the frequency of HIV-1+ cells per million of total cells, there was a higher frequency of HIV-1 RNA+ and/or DNA+ cells in the BCFs compared to the TCZ in four out of four PLWH prior-to ART and in two out of three in PLWH during ART (Fig. 5D). Overall, in agreement with the literature (Estes et al., 2017; McGary et al., 2017), our results show that HIV-1-infected cells can be found distributed between the BCF and the TCZ, with the BCFs containing a higher frequency of infected cells per million total cells.

4. Discussion

Despite the extensive use of flow cytometry (FACS) for characterizing immune subsets, FACS does not reveal the spatial location of these cells or the cellular immune neighborhood, which are potentially essential indicators for understanding where and how HIV can persist on ART. Advances in multiparameter imaging for nucleic acid and proteins are rapidly changing the field of pathology, enabling a more detailed description of the anatomic and cellular sites of HIV persistence on ART. The development of RNAscope and DNAscope has allowed the detection of vDNA and vRNA+ cells in FFPE tissue, discriminating between vDNA+ cells that are transcriptionally silent (vRNA−) and cells that are actively transcribing viral RNA (vRNA+) (Deleage et al., 2016). In combination with IHC, co-expression of vDNA is seen in immune subsets in histological sites (Deleage et al., 2016; McGary et al., 2017).

Despite the advances in combining ISH with IHC, to our knowledge this is the first demonstration of >5-plex mIHC with detection of HIV DNA and RNA in FFPE tissue. To achieve this we have modified the already established RNAscope and DNAscope approaches by incorporating the TSA system from Perkin Elmer and extended with TSA based mIHC. The previous studies of HIV combining ISH and IHC have used TSA with fluorescence antibodies and chromogenic staining (Deleage et al., 2016; McGary et al., 2017; Vasquez et al., 2018). The combination of high sensitivity of TSA based fluorescence detection and dilute protease step allows the detection of the attenuated immunohistochemical signal seen with combination of ISH and IHC (Millar, 2020). The additional intracellular marker FoxP3 and surface markers has been used to identify Tregs with greater specificity than that used in the previous studies where the number of phenotyping markers was limited (McGary et al., 2017).

We first demonstrated that these technologies can be applied to in vitro cell models of HIV latency. We first optimized this approach using latently infected cell lines and primary cells. Using this approach, we detected multiple copies of HIV DNA in each cell. One potential explanation for this is that the probes are targeting a small portion of the HIV genome, and as a result, a few double Z-probe pairs bound to the same integrated HIV genome can be detected. This was recently also observed in a study in which two copies of vDNA+ signal were found in a J-Lat 10.6 cell (Ukah et al., 2018). The presence of vRNA+ cells does not distinguish between cells with intact and defective proviruses, as a result, the size of the reservoir of intact virus is overestimated in LNs in PLWH on ART.

In LN tissue from PLWH, both vRNA and vDNA+ cells were detected, however, there was a lower ratio of vRNA: vDNA in individuals on ART compared to off ART. This is consistent with studies showing that RNA+ cells are substantially reduced by ART (Symons et al., 2017; Jordan et al., 2003; Reed and Muench, 1938).Using the TSA fluorescent 7-plex platform consisting of probes for HIV DNA and HIV RNA and antibodies to CD4, FoxP3, CTLA-4, PD-1 and DAPI in one single tissue, we developed a technique to further characterize cells that are either latently or productively infected. Combining ISH and IHC can offer new insights into HIV persistence on ART in tissue. Furthermore, this technique can be applied to a wide range of viruses and pathological studies.

Supplementary Material

Supp Table S1 & Figure S1

Acknowledgements

We acknowledge the participants who donated their LNs for this study, Dr. Mauricio González-Navarro and Dr. Fernanda Torres-Ruiz for performing the LN biopsies (Centro de Investigación en Enfermdades Infecciosas, Mexico city, Mexico). We thank Caroline Tumpach and Michael Roche for providing us with cells (RMIT, Melbourne Australia and Peter Doherty Institute, Melbourne Australia), Dr. Andreas Behren (Olivia Newton John Cancer Institute) for using his lab and imaging facility, Metta Jana, Rejhan Idriza and Cameron Skinner for the analysis and imaging facility (Petermac, Melbourne, Australia), Dr. Byron Martina for proof-reading (Erasmus MC, Rotterdam, The Netherlands), T. Luka, C. Li for flow cytometric cell sorting (University of Melbourne Flow Cytometry Facility, Melbourne, Australia). This work was funded by grants to Sharon Lewin from National Institutes of Health Delaney AIDS Research Enterprise to Find a Cure Collaboratory (Grant UM1AI126611-01). JD Este was funded from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases and National Institute of Allergy and Infectious Diseases grants R01 DK119945 (JDE) and R01 AI143411-01A1 (JDE). SRL is an NHMRC Practitioner Fellow

Footnotes

Declaration of Competing Interest

No conflict of interest to declare.

Appendix A.: Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jim.2021.113198.

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Supplementary Materials

Supp Table S1 & Figure S1
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