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

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; Lymph nodes; Microscopy; RNAscope.

Fig. 1.

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.)

Fig. 2.

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.

Fig. 3.

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.

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.)

Fig. 5.

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.)