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.

Regulatory T cells (Tregs) play a pivotal role in modulating immune response hence supporting immune tolerance. Recently, CD45 ligation with anti-CD45RB mAb MB23G2 (anti-CD45RB) was reported to enhance graft-survival; evidence shows it was by homeostatic Treg proliferation in vivo. While many in vivo data support the notion that anti-CD45RB supports Tregs expansion, it is still unclear if the ligation ex vivo is sufficient to enhance Tregs’ avidity for activation and further promote their expansion and function. Here, we were to see if anti-CD45RB ligation can enhance Treg’s sensitivity to perceive activating signals in varying strength in vitro. To test this hypothesis, mouse CD4+ T cells (Foxp3-GFP linked B6 mouse, both Foxp3+ and Foxp3−, or Tregs and Tconvs, respectively) were seeded onto surfaces micropatterned with an antibody targeting the CD3 on T cell (anti-CD3) with and without the presence of anti-CD45RB. These surfaces were created by first microcontact printing of 2-μm diameter features of anti-CD3 at various concentrations (i.e., 1, 5, and 20 μg/mL) on glass coverslips and back-filled with ICAM-1. The effect of anti-CD45RB on Treg and Tconv activation was compared by the ratios of phosphorylation of Lck at Tyr 394 to 505 sites, cell registration on anti-CD3 dot array, and motility of each cell type on varying concentration of activation signals. Overall results show that anti-CD45RB specifically slow-down Treg motility and, therefore, enhance the probability to contact to activating signals (cell registration) and improve T cell activation indicated by ratios of phospho-Lck at active over inactive sites. This study assures the importance of further investigation on the role of anti-CD45RB in Treg activation mechanism.