Structured Illumination Microscopy Improves Visualization of Lytic Granules in HIV-1 Specific Cytotoxic T-Lymphocyte Immunological Synapses

Some HIV-1-infected people, known as “elite controllers (EC),” demonstrate superior virus control maintaining undetectable (<50 RNA copies/ml) viral loads even in the absence of treatment. ¹ While it is well known that HIV-1-specific cytotoxic T lymphocytes (CTLs) are critical for viral control and that human leukocyte antigen (HLA) haplotype is associated with progression status, ^2,3 the exact mechanisms underlying EC superior virus control remain unknown. Optimal cytotoxicity depends on the quality of cytotoxic immunological synapses (IS). ⁴ Conventional approaches such as confocal microscopy cannot reveal the detail of cytotoxic IS.

Many critical questions about CTLs and HIV-1-infected target cell's interface can be addressed only by using superresolution imaging. Here we examined cytotoxic IS and the loading and delivery of perforin at IS by superresolution structured illumination microscopy (SIM). To compare the perforin loading and delivery at IS between TCR clonotypes, we first imaged the IS under conventional widefield fluorescence microscopy (Fig. 1A). The polarized lytic granule stained by perforin (a marker for lytic granules) was released at the cytotoxic immunological synapses and delivered into HIV-1-infected GXR [a green fluorescent protein (GFP) reporter T cell line, derived from the human lymphoblastoid CD4⁺ T cell line CEM, that fluoresces green after infection with HIV-1] cells after the mixture of HIV-1-specific CTLs with HIV-1-infected GXR for 30 min. However, conventional widefield fluorescence microscopy cannot distinguish the accumulated lytic granules at IS, as shown in the lower panel of Fig. 1A.

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FIG. 1.

Comparison of perforin imaging between widefield fluorescence and structured illumination microscopy (SIM). HIV-1 KK10-specific cytotoxic T-lymphocyte (CTL) clone cells were mixed with HIV-1-infected HLA-B*27-expressing GFP reporter GXR cells for 30 min. Cells were fixed and stained with monoclonal antibody against perforin and followed by Alexa Fluor dye-conjugated secondary antibodies. F-actin was stained with phalloidin-Alexa Fluor 647. Three-dimensional images were collected on a Zeiss ELYRA superresolution microscope using a plan apochromat 63×1.4 oil immersion objective. The middle panels are the same region in the target cells under widefield fluorescence microscopy (A) and superresolution SIM (B). Scale bars, 50 nm.

To distinguish the accumulated lytic granules, we used superresolution SIM (Zeiss ELYRA) to visualize the individual lytic granules. SIM clearly dissects the number and size of lytic granules at IS (lower panel of Fig. 1B). This is the first superresolution image of HIV-1 cytotoxic IS, which allows us to determine the structure and function of HIV-1-specific CTL IS in greater detail and yields critical insights into how these findings relate to viral control.