Sample sparing platform for microscopy shows that CD45RB ligation increases Treg sensitivity to activation signals in vitro

Abstract Small subpopulations of cells, e.g. regulatory T cells, often play key roles in the function of the entire immune system. While their smaller number makes them difficult to work with in conventional immunocytochemistry assays or flow cytometry, microscopy can provide functional and spatial information on a single cell basis. Combined with surface modification technology such as synthetic lipid bilayers and micropatterned antibodies, we are in a unique position to be able to study the spatial mobility of intracellular signals (e.g. show that mobility of Lck differs between mouse and human T cells). Even still, conventional microscopy platforms such as 96-well plates or flow cell systems require a minimum of 10k to 1 million cells and does not allow for an interchangeable surface. Conical wells with a 1mm bottom and 5mm top diameter (microwells), laid out in a 96-well configuration were mass manufactured out of polypropylene from Protolabs, and adhered to surface modified glass coverslips. 1k cells seeded and centrifuged at 100g for 10 mins resulted in 81% of the cells landing on an image-able area. Based on the report that anti-CD45RB mAb MB23G2 enhances graft-survival, we tested the hypothesis that anti-CD45RB ligation in vitro increases Treg sensitivity to activation signals. 3 different activation signal levels (1, 5, and 20 μg/mL), 2 cell types, and 2 treatment conditions conventionally required at least 150k cells per sample, or 1.8M cells total. On the microwell, we performed the experiment with 8k cells per sample, with quadruplicates for a total of 384k cells, to show that the ratio of phosphorylation of Lck at Tyr 394 to 505 sites, and alignment to micropatterned anti-CD3 dots are increased in Tregs treated with anti-CD45RB.