Studying Autoimmune Disease

Systemic lupus erythematosus (SLE) is an autoimmune disease which affects about 1.5 million people in the USA. It results in chronic inflammation of various organs leading to permanent organ damage. When SLE infects the glomeruli of the kidney, termed lupus nephritis (LN), the small blood vessels in the kidney become inflamed resulting in potential kidney failure. Understanding the disease forming pathway in LN will be important for the development of effective therapies to treat this autoimmune disease [1,2]. It has been shown that human T lymphocytes with a characteristic defect in potassium channel behavior contributes to the hyperactivity of SLE T lymphocytes [2]. Therefore, it may be possible to target ion channels for new therapies to suppress hyperactive T lymphocytes of SLE patients with a focus on their chemotactic and cytotoxic capabilities [1,2].

For the study of kidney specimens, an imaging solution is needed for fast screening of large specimen overviews at high magnification. Widefield, camera-based fluorescence microscopy offers ease of use, speed, and detection sensitivity, however, when imaging thick 3D specimens image contrast is reduced and structural details are obscured due to the out-of-focus blur (haze) [3]. This haze is produced by detected fluorescence signals which are emitted from out-of-focus planes in the specimen [3].

Formalin-fixed paraffin-embedded (FFPE) kidney tissue biopsy specimens of LN patients were utilized for this study. Before imaging, the FFPE specimens were deparaffinized and stained with Alexa-Fluor-647-conjugated rabbit anti-human CD8 antibodies (green) and Alexa-Fluor-594-conjugated mouse anti-CD45RO antibodies (magenta) to label memory T lymphocytes, a type of white blood cell [4]. Nuclei in the specimens were stained with DAPI (blue). The specimens were imaged on the THUNDER Imager 3D Cell Culture using large volume Computational Clearing (LVCC) [3].

Kidney specimens were examined for memory T lymphocyte infiltration. The images in figure 1 show the extent of T cell infiltration where magenta is detected. The THUNDER images after LVCC processing are clearer and sharper than the raw widefield images. These results show that THUNDER Imagers can reveal biological events that are typically obscured in the “haze" when imaged using conventional widefield microscopes.

1. M. Khodoun, A.A. Chimote, F.Z. Ilyas, H.J. Duncan, H. Moncrieffe, K. Shashi Kant, L. Conforti, Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus, Science Advances (2020) vol. 6, no. 47, eabd1471, DOI: 10.1126/sciadv.abd1471.
2. S.A. Nicolaou, P. Szigligeti, L. Neumeier, S. Molleran Lee, H.J. Duncan, S.K. Kant, A.B. Mongey, A.H. Filipovich, L. Conforti, Altered dynamics of Kv1.3 channel compartmentalization in the immunological synapse in systemic lupus erythematosus, Journal Immunol. (2007) vol. 179, iss. 1, pp. 346-56, DOI: 10.4049/jimmunol.179.1.346.
3. J. Schumacher, L. Bertrand, THUNDER Technology Note: THUNDER Imagers: How Do They Really Work?, Science Lab (2019) Leica Microsystems.
4. R. Warrington, W. Watson, H.L. Kim, F. Romana Antonetti, An introduction to immunology and immunopathology, Allergy, Asthma & Clinical Immunology (2011) vol. 7, suppl. 1, DOI: 10.1186/1710-1492-7-S1-S1.