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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I1 - Cell Barcoding for Pooled Screening in Multiplexed CD8+ T Cell Phenotyping

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  CD8+ cytotoxic T cells have immense therapeutic potential due to their ability to target and kill infected or cancerous cells, and many novel cancer immunotherapies focus on the engineering of these cells toward specified targets. However, the dynamism of these cells and their regulatory networks in vivo gives rise to significant heterogeneity in both cell subtype and anti-tumor efficacy among activated T cell populations. Characterizing the dynamic regulatory networks of T cell activation and stimulation is essential for developing more precise cell therapies, and novel spatial-omics tools bring multiplexed stimulation studies within reach. A critical and under-investigated part of these studies is the barcoding of cells from distinct conditions so that they can be stained and screened together. Here, we investigated a double-oligonucleotide barcoding strategy for pooled screening of T cells cultured in multiplexed stimulation conditions.

Materials and

Methods:  In a CODEX experiment, cells are stained with primary antibodies that have been conjugated to unique oligonucleotides using a DBCO click-chemistry reaction. The CODEX machine cyclically binds complementary fluorescent oligonucleotides 3 channels at a time (Cy3, ATTO550, and A488), takes an image, then strips away the fluorescent oligonucleotides with 80% DMSO. With this, many markers can be probed in the same fluorescent channel as long as the markers are imaged in separate cycles; a single multicycle run can probe 50+ markers on the same cell population while also allowing for computational de-barcoding of distinct subpopulations.

For this experiment, PMEL mouse splenocytes were thawed and activated against GP100 melanoma antigen, then stimulated via co-culture with artificial antigen-presenting cells (aAPCs). The aAPCs were made via conjugation of costimulatory signals anti-CD3e and anti-CD28 to magnetic microparticles. Some cells were instead stimulated with plate-adhered anti-CD3e alongside anti-CD28 in the media, which allowed downstream comparison between plate stimulation and aAPC stimulation conditions. Cells were harvested and barcoded using a novel double-oligo method that stains cells with unique combinations of oligo-conjugated CD45 and CD90 antibodies. Samples were pooled, stained with 22 distinct markers used to characterize CD8+ T cells, and imaged using the Akoya PhenoCycler. Cell segmentation of the raw CODEX data was performed using SPACEc, and computational barcoding was performed using manual gating.

Results, Conclusions, and Discussions: Barcoded and pooled cells from three stimulation conditions appeared as unique subpopulations in CODEX multiplexed imaging. Barcodes 1 and 2 appear on two different plate-stimulated populations, and Barcode 3 appear on the aAPC-stimulated cells [Fig. A]. Plots of paired barcoding oligonucleotides show distinct clustering for two of the three paired barcodes [Fig B]. Plots of unpaired barcoding oligonucleotides also illustrate cell populations with distinct barcodes, with 15.32% of cells in the A17+ region and 12.64% of cells in the A17+ region. Only 0.69% of cells were “noisy,” showing high staining of both barcodes [Fig. C]. This establishes a proof-of-concept for CD45/CD90 double-oligo barcoding. Future steps will involve conjugation of CD45 and CD90 antibodies to additional oligonucleotides to increase the number of possible barcodes. Likewise, barcodes will be permuted with each other to drastically increase our ability to study unique cell stimulation conditions in pooled multiplex cycles.

Acknowledgements (Optional):  This work was supported by the Duke Cancer Institute and the Pratt School of Engineering.