Traditional immunohistochemistry techniques are limited in the number of fluorescent detection channels, markers, and staining resolution, which restricts the ability to fully evaluate bulk tissue samples and biopsies. Recent technical advancements, such as co-detection by indexing and spatial proteomics, have enabled in situ visualization of tissues by combining multiplex assays, repetitive staining, and quantitative image analysis. These technologies can identify cellular co-expression, cellular spatial relationships, tissue heterogeneity, and detect low-abundance molecules, which are critical for basic immunology, disease evaluation, and therapeutic evaluation studies. However, these methods require specially conjugated antibodies and protocols to analyze highly multiplexed tissue imaging (HMTI) readouts. Here, we utilize imaging cytometry as a viable alternative that also enables individual cellular cytometry analyses in two-dimensional formats. This technique has been used to investigate human and mouse tissues but is underexplored in the translationally relevant rhesus macaque (RM) model. Here, we demonstrate the use of this platform to image RM placenta, jejunum, kidney, and liver stored in OCT, using commercially available fluorophore-conjugated antibodies to identify structural markers (cytokeratin and vimentin), pan-immune cells (CD45), T cells (CD3 and CD8), natural killer cells (NKG2A/C), monocytes (CD16) and macrophages (CD68 and CD163), without any customization. We compared these preclinical samples to human samples to emphasize the potential for cross-species translational analyses using this platform. The flexibility to perform multiple rounds of photobleaching and fluorophore-based staining, combined with the ability to compensate for autofluorescence, makes this technology extremely valuable for deciphering tissue architecture and the spatial distribution of immune cells. Furthermore, we leveraged the platform’s ability to export data in HMTI format and flow cytometry standard format, compatible with other quantitative downstream analysis pipelines, to simultaneously visualize the spatial distribution of various cell populations.
Hari Balachandran, C. Manickam, Rhianna A. Jones et al.· JIM - Journal of Immunologic...· 0 citations
Natural killer (NK) cells are critical effector cells of the innate immune system. Classically, NK cells mediate antibody-dependent responses via CD16, the IgG Fc receptor. However, IgG is the minority of Ig expressed at mucosal sites, whereas IgA is > 90% of Ig in mucosal secretions. Thus, harnessing NK cells for mucosal responses via IgA is an attractive therapeutic target. In this study, we demonstrate highly efficient CD89 knock-in that enables potent IgA- and IgG-mediated responses in NK cells.
Primary human NK cells were expanded and activated with irradiated K562-mbIL15 cells for 7 days before nucleofection with a transposon vector including the CD89 open reading frame. Knock-in efficiency was determined 4 days post-nucleofection and CD89+ NK cells were FACS purified and expanded with feeder cells. Functional assays were carried out using CD89+ NK cells and monoclonal rituximab antibodies expressing IgG, IgA1, or IgA2 Fc regions. CD89+ NK cells were co-cultured with Raji cells (10:1 E:T) and combinations of rituximab antibodies for 6-hours before staining and data acquisition via flow cytometry.
CD89+ NK cells retained baseline phenotypes compared to non-nucleofected NK cells, including natural cytotoxicity and co-stimulatory receptors, intracellular signaling molecules, and markers of activation. Most importantly, CD89+ NK cells also retained CD16 expression, allowing them to respond to both IgG and IgA antibody activation. CD89+ NK cells display similar activation profiles as measured by CD107a, interferon-γ, and MIP-1β when activated with IgG, IgA1, or IgA2. As expected, CD89- NK cell controls only responded to IgG and not to IgA, confirming CD89-specific activation.
NK cells represent a promising avenue for cell-based therapeutics due to various benefits compared to T cell-based therapies. Through our current study we have shown consistent and robust engineering of NK cells to express CD89 to give them the ability to simultaneously respond to IgA and IgG.
NIAID grants R21AI184422 and P01AI162242
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Kyle W. Kroll, Andrew Hudson, R. Reeves· Journal of Immunology· 0 citations
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