Human spleen organoid single-cell trancsriptomics, enriched for γδ T cells

Vaccine effectiveness against malaria is dramatically reduced in malaria-exposed compared to malaria-naïve populations, potentially due to altered immune responses in secondary lymphoid organs following repeated infection. Newly developed human tonsil and spleen organoids, which replicate key features of B and T cell immunity, provide an exciting opportunity to overcome challenges of other models and to improve our understanding of innate-adaptive interactions in lymphoid tissue. The objectives of this study were to use these organoids to investigate the impact of malaria parasites on 1) cells within lymphoid tissues and 2) responses to a heterologous antigen.
To better characterize the impact of iRBC stimulation on human lymphoid reponses, we cocultured three human spleen organoids with iRBC and uninfected red blood cells for 10 days. We then enriched for Vd2+ cells using fluorescence-activated cell sorting, and used the BD Rhapsody platform to generate multiomic single-cell data, comb..., Spleens organoids were prepared as per Wager et al. 2021 (PMC7891554). Briefly, cryopreserved splenic cell suspensions were thawed and counted, 6 x 106 cells in 100 µl immune organoid media were added to each transwell (Millipore PICM01250) in 12-well plates. Immune organoid media containing 0.5 µl g/mL recombinant human B cell activation factor (BAFF, Biolegend 559608) was added to the outside of the well. Spleen tissue from 3 donors was used and an aliquot of cells from d0 was frozen for each. Organoids were set up and stimulated with uRBC or 3D7 iRBC (12 x 106 iRBC or uRBC/well) for 10 days with media changes every 3-4 days. Stimulated cells, as well as d0 cells, were then stained with an anti-Vδ2+ antibody (PerCP-conjugated, 1:50) and a cell viability dye (Live/Dead Aqua, 1:400) for 30 minutes, washed, and then sorted on a Sony cell sorter. For each sample, 8,000-40,000 Vδ2+ γδ T cells were sorted and depending on the number collected, 10,000-30,000 Vδ2- cells were sorted in order t..., # Human spleen organoid single-cell trancsriptomics, enriched for γδ T cells

Dataset DOI: [10.5061/dryad.3r2280gw3](https://doi.org/10.5061/dryad.3r2280gw3)

## Description of the data and file structure

BD Rhapsody v2 beads were used to capture transcriptomes, TCR sequences and oligo tagged antibodies bound to surface proteins of γδ-enriched spleen organoids. Two Seurat objects were generated from the alignments of these sequencing libaries for, one object for each lane on the sequencer that was used. The SampleMetadata.xslx file contains metadata on the samples that were used in this experiment.

### Files and variables

#### File: \_1_p258b_CKDL240012182-1A_225CC7LT4_L2_1_Seurat.rds

**Description:** Seurat object made from libraries sequences in lane 2

#### File: \_1_p258a_CKDL240012182-1A_225CC7LT4_L2_1_Seurat.rds

**Description:** Seurat object made from libraries sequences in lane 1

Both RDS files contain Seurat objects containing transcriptomic, antigen receptor sequence, an..., No personally identifiable data is contained in the data published in this repository.

Written informed consent was obtained from all participants or legal representatives. Ethics approval for malaria-naïve samples was obtained from Stanford University’s Human and Animal Research Subjects Compliance Office (Stanford IRB-60741 and IRB-30387). ,