RNA-seq of HLA-A2 restricted human CD8+ T cells against SARS-CoV-2 from 11 COVID-19 patients
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PROVIDER: PRJEB83834 | ENA |
REPOSITORIES: ENA
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Project description:A cryovial that contains frozen PBMCs (10^6 cells/vial) from each of 11 COVID-19 patients was thawed in 37oC water bath. Immediately after, thawed cells were transferred to 15-mL Falcon tube that contained 10 mL of cold culture medium. After centrifugation at 150g for 5 min, the cell pellets were resuspended in complete RPMI + 10% heat-inactivated FCS medium and plated onto 12-well plate at a concentration of 5 x 10^6 cells/3 mL/well. Then a peptide cocktail was added at a concentration of 5 μg/mL for 16hr in 5% CO2 37oC incubator. After 16-hour incubation, cells were washed 3 times and incubated for 10 minutes with Fc receptor block, following each sample was stained with a total of 6 HLA-A2+/peptide fluorescent tetramer (Tet) and pentamer (Pent) for 10 min. After cell washing at twice, each sample was stained with cell hashing antibodies for 20 min, respectively and then washed at three times and finally pooled. The combined cells were stained with CITE-seq and anti-human CD8 FACS antibody for 30 minutes. After cell washing at twice, viable HLA-A2+/peptide Tet/Pent positive CD8+ T cells were sorted by FACS Aria II. The scRNA-Seq libraries including GEX and CITE-seq libraries were prepared using the 10x Chromium single-cell 3’ reagent kits (v3.1 Chemistry), per manufacturer’s instructions. As a result, we have 2 separate groups of the single cell datasets. MKH datasets (MKH_GEX_... and MKH_FB...) contain RNA-seq outcomes of HLA-A2 restricted human CD8+ T cells against SARS-CoV-2 from 5 COVID-19 patients. MKK datasets contain RNA-seq outcomes of HLA-A2 restricted human CD8+ T cells against SARS-CoV-2 from 6 COVID-19 patients. Cell hashing and CITE-seq antibody information is available in the csv files.
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Project description:Few non-classical HLA-E restricted HIV-specific epitopes have been described, and even less is known about the functional profile of responding CD8 T cells (CD8s). This study evaluates the functional characteristics of CD8s targeting the Gag epitope (KAFSPEVIPMF or KF11) based on their restriction by either HLA-E (E-CD8s) or HLA-B57 (B57-CD8s). CD8s from 8 people with HIV (PWH) were cocultured with KF11 peptide presented by cell lines expressing HLA-B*57:01, HLA-E*01:01 or E*01:03. CD8s were analyzed through single-cell (sc) RNA and TCR sequencing. Additionally, supernatants were analyzed for soluble proteomics using a Luminex assay. B57-CD8s secreted higher levels of cytotoxic cytokines such as IFNγ, while E-CD8s produced more chemotactic cytokines, including RANTES, CXCL10 (IP-10), and IL27confirmed through scRNAseq. Despite distinct cytokine profiles, TCR clonotypes stimulated by KF11 were cross-restricted by HLA-B*57 and HLA-E*01/03. In vitro T cell reporter assays clearly demonstrated this cross-restriction. A TRAV5-containing metaclonotype cluster was seen in PWH with lower viral loads. These findings demonstrate that HIV-specific CD8s in PWH exhibit cross HLA-B*57 and HLA-E*01/03 restriction, resulting in functionally distinct immune responses that may contribute to HIV control.
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