Identify a sphingolipid analogue as a regulatory T cell enrichment agent

Andikan Akpan, Kim-Chong Yong, Li-Wen Lai

The University of Arizona, Minority Access to Research Careers Program, Marc E. Tischler, Ph. D., Director
The regulatory T cells (Treg, CD4+FoxP3+) play a central role
in the maintenance of immunological tolerance. Tregs control the intensity of immune responses by suppressing the activation of effector T cells (Teff, CD4+FoxP3-) and other inflammatory cells. This gives them the potential to treat diseases by suppressing inflammatory pathologies such as autoimmune diseases and transplant rejection. However, the inability to acquire enough CD4+ FoxP3+ Treg hinders its clinical application .

INTRODUCTION

METHODS
We isolated CD4+ cells from FoxP3+EGFP+ mice splenocytes using a magnetic negative selection kit (Miltenyi Biotec). We cultured the cells in a 96 well culture dish. Various concentrations of DMS or its vehicle control were added to each well. The nuclei of the CD4+ cells were counterstained using Hoechst 33342. We detected in real time the conversion of CD4+EGFP-(Teff) cells to CD4+EGFP+(Treg) cells using the Cellomics ArrayScan VTI HCS reader and BioApplication software (ThermoFisher) over the time course of 48 hours.
Control

Fluorescence of CD4+EGFP+ cells

T1
DMS 0.05 mM

T4

T7

T10

The transcription factor FoxP3+ is highly expressed in Tregs

and is a master regulator for Treg function. Tregs can be converted from Teffs in the periphery from various stimuli to express high levels of FoxP3.

T1

T4

T7

T10

Fig. 3 The expression of CD4+EGFP+ cells at the hour zero of the 18 hour scan (far left), hour six (middle left), hour twelve (middle right), and hour 18 (far right) acquired by the Array Scan VTI HCS reader (Cellomics).

We hypothesized that a sphingolipid analogue DMS has the

ability to convert Teffs to Tregs.

CONCLUSIONS
We have established a powerful real time in vitro assay for identifying compounds that are capable of converting Teff to Treg. We have successfully applied this assay to identify a potential Treg enrichment agent Further experiments will be conducted to examine the underlying signaling pathways for this conversion.

To test this hypothesis, we established an in vitro assay using

spleen cells isolated from Foxp3+EGFP+ transgenic mice which express EGFP only in Treg and acquiring the image in real time after drug treatment.

RESULTS
Increased expression of GFP
Well A5 Timepoint 1
2%

Well A5 Timepoint 4
5%

CD4+ T-effs: CD4+FoxP3+ T-regs:

CD4+ T-effs: CD4+FoxP3+ T-regs:

98%

95%

REFERENCES

CD4+ T-effs: CD4+FoxP3+ T-regs:

Well A5 Timepoint 7
6%

Well A5 Timepoint 10
11%

CD4+ T-effs: CD4+FoxP3+ T-regs:

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94%

89%

Fig. 1 A tumor swarmed by Teff cells and Treg cells (Shevach)

Fig. 2 Pie charts showing the amount of CD4+ FoxP3+ Tregs compared to CD4+ FoxP3- T cells for cells given 0.05mM of DMS at the zero hour of the 18 hour scan (top left), hour six (top right), hour twelve (bottom left), and hour 18 (bottom right).

**Sponsors: This research was funded by the National Institutes of Health, Grant No. T34 GM008718 (MARC) & RO1DK82718 (Lai Lab)