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Multiparameter immunohistochemistry analysis of HIV DNA, RNA and immune checkpoints in lymph node tissue
Zuwena A Richardson1, Claire Deleage2, Candani S A Tutuka3
1The Peter Doherty Institute for Infection and Immunity, The University of Melbourne and Royal Melbourne Hospital, Melbourne, Australia.
Insights
A new method detects persistent HIV DNA and RNA in cells, even during antiretroviral therapy (ART). This technique helps study latent HIV infection in regulatory T-cells (Tregs) and immune checkpoints, crucial for finding an HIV cure.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Antiretroviral therapy (ART) suppresses HIV but cannot eliminate latent reservoirs of infected CD4+ T-cells.
- Understanding the role of regulatory T-cells (Tregs) and immune checkpoints (PD-1, CTLA-4) in persistent HIV infection is crucial for developing a cure.
Purpose of the Study:
- To develop and validate a multiplex method for detecting HIV DNA and RNA alongside cellular markers in infected cells.
- To investigate the presence and characteristics of latent HIV infection in specific CD4+ T-cell subsets, including Tregs, in people living with HIV (PLWH) on ART.
Main Methods:
- Utilized multiplex immunohistochemistry (mIHC) combining in situ hybridization (ISH) for HIV RNA/DNA detection and immunohistochemistry (IHC) for cellular markers (CD4, PD-1, FoxP3, CTLA-4).
- Employed tyramide signal amplification (TSA) for enhanced IHC detection.
- Analyzed latently infected cell lines, a primary HIV latency model, and lymph node biopsies from PLWH on and off ART.
Main Results:
- Successfully detected and differentiated between actively replicating (HIV RNA and DNA) and latently infected (HIV DNA only) cells.
- Identified HIV persistence in conjunction with cellular markers like PD-1 and FoxP3 in both in vitro models and ex vivo lymph node tissues.
- Demonstrated the co-expression of HIV DNA/RNA with immune checkpoint markers in infected cells from PLWH on ART.
Conclusions:
- The developed mIHC platform is effective for detecting and quantifying HIV persistence within specific cellular subsets in tissue samples from PLWH.
- This method provides a valuable tool for studying the mechanisms of HIV latency and persistence, particularly in relation to Tregs and immune checkpoints.
- Further research using this platform can aid in developing strategies to target and eliminate latent HIV reservoirs for a potential cure.
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.
