Context:

To understand HIV persistence, it’s crucial to visualize the tissue microenvironments where the virus resides. Current methods often require removing cells from their native context. Detecting low-abundance nucleic acids and proteins in situ is essential for studying viral infections.

Method:

The authors introduce PANINI (protein and nucleic acid in situ imaging), which includes:

1. A custom branched-chain amplification method for nucleic acids using tyramide-based amplification and antibody detection.
2. An optimized antigen retrieval protocol that avoids protease treatment and allows single genomic event detection.
3. Antibody-based detection of nucleic acids and proteins using MIBI, Vectra Polaris, and CODEX platforms.

Using FFPE cell pellets and lymphoid tissues from SIV-infected and uninfected rhesus macaques, PANINI-MIBI can simultaneously detect single-integration events of SIV DNA, RNA transcripts, and protein epitopes on the same tissue section.

Results:

The researchers confirmed retroviral infection markers, including CD4+ T cell depletion, heightened NK and CD8+ T cell responses, and lack of immune infiltration in B cell follicles. Integrating CN information with markers allowed us to differentiate infection status using LDA.

They discovered a B cell response to SIV infection through IL-10 secretion, attracting and immunosuppressing macrophages via an M2-phenotypic switch. This environment, with increased IL-10, FoxO1, and HLA-DR expression around infected cells, may explain viral transcription status. Increased IL-10 protein expression and transcriptomic signatures during chronic and ART-treated infections support this.

Implication/Impact:

The PANINI platform, with a 33-marker panel for FFPE tissues, spatial analytical workflow, and multimodal analysis framework, enables reexamination of previous observations and new model development. Antibody-based multiplexed imaging offers robust readouts and molecular pathway assessment. Recent resources show high concordance between single-plex immunohistochemistry and MIBI-TOF imaging and provide a guide for selecting and validating antibodies for high-dimensional imaging.