Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

Abstract

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8⁺ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N^361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N^361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N^361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N^361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8⁺ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

Keywords: CD8+T cell; HLA class I; Lung organoid; SARS-CoV-2; T cell epitope; TCR.

Figure 1

CD8⁺ T cell responses of SARS-CoV-2 peptides. IFN-γ ELISPOT assay results for the PBMCs of COVID-19 convalescent patients with (A) HLA-A2⁺, (B) HLA-A24⁺ or (C) HLA-A∗11:01⁺ allele, stimulated with mixed peptides (top) and single reactive peptide (middle and bottom). A stimulation with an equimolar amount of DMSO was performed as negative control, and Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (P/I) was performed as positive control. Results are expressed as number of peptide-specific IFN-γ spots/2 × 10⁴ PBMCs of each patient, subtracted those from the corresponding DMSO groups. N = 8. For peptide grouping: 26 HLA-A∗02:01-restricted peptides were pooled into 5 mixtures (M1-M5), 22 HLA-A∗24:02-restricted peptides were pooled into 4 mixtures (M6-M9), and 30 HLA-A∗11:01-restricted peptides were pooled into 5 mixtures (M10-M14). Each mixture contained 3–6 peptides. Positive peptide mixture and single peptide with the most dominant reactivity were marked in bold. For the single dominant peptide, P63 was from M3, P45 was from M6 and P64 was from M14. C8–C41: COVID-19 convalescent patients. Data were representative of two independent experiments.

Figure 2

The isolation and validation of the SARS-CoV-2 specific TCRs of CD8⁺ T cells. (A) After stimulated by peptide mixtures and expanded for 10 days, single IFN-γ secreting T cell from three COVID-19 convalescent PBMC samples (C40: HLA-A∗11:01⁺, C33: HLA-A24⁺ and C27: HLA-A2⁺) were individually stimulated by P64, P45 and P63, from three corresponding HLA subtypes, and isolated by flow cytometric sorter. (B) The TCRs of sorted cells were amplified by RT-PCR and the TCR repertoire was analyzed with the IMGT/V-Questtool (http://www.imgt.org/) to obtain dominant TCR clones. Unique clones were marked in white. Different colors represented relative dominant clones (copy≧2). (C,D) Specificity verification for the top 4 P64-TCR clones in CD8⁺ Jurkat cells. P64-HLA-A∗11:01 tetramer (Tetramer) and anti-mouse TCR (mTCR) antibody were applied to evaluate the specific binding of P64-TCRs (C). CD69 was used to determine the specific activation of TCR-transduced CD8⁺ Jurkat cells, after co-cultured with HLA-A∗11:01 expressingCOS-7 cells pulsed with P64 (D). Grey represented DMSO-stimulated samples and pink represented P64-stimulated samples. Data were presented as mean ± SD, n = 3, ∗∗∗P < 0.001. Representative data of two independent experiments were shown.

Figure 3

Functional characterization of the N^361-369-specific TCR 1 and TCR 4. (A) Flow cytometric analysis of the transfection efficiency of TCR 1 and TCR 4 in CD4⁺ and CD8⁺ T cells, by N^361-369-HLA-A∗11:01 tetramer. (B) Binding assay of TCR-transduced CD8^- and CD8⁺ Jurkat cells with N^361-369-HLA-A∗11:01 tetramer. (C) Flow cytometric analysis of T cell activation evaluated by CD137 expression. TCR-transduced CD8⁺ T cells and CD4⁺ T cells were individually co-cultured with HLA-A∗11⁺ K-562 cells pulsed with N^361-369, or an equimolar amount of DMSO, for 24 h. Expression of CD137 on CD8⁺ T cells (left), CD4⁺ T cells (middle) was evaluated and did further statistical analysis (right). (D) N^361-369 loaded HLA-A∗11:01⁺ K-562 cells lysis by TCR 1-CD8⁺ T cells after 8 h at different E:T ratios. DMSO was used as negative control. Reported values are the mean of triplicates, with error bars indicating. E, Effector T cells. T, Target cells. (E) Detection of IFN-γ, IL-2 and TNF-α secretion from TCR 1 and TCR 4-transduced CD4⁺ T cells by ELISA, after co-cultured with HLA-A∗11:01⁺ K-562 cells loaded with N^361-369 for 24 h. Data were presented as mean ± SD, n = 3, ∗∗P < 0.01, ∗∗∗P < 0.001. Representative data of two independent experiments were shown.

Figure 4

Evaluation of the affinity and the cross-reactivity of TCR 1 and TCR 4 to the N^361-369 mutants. (A) EC₅₀ analysis with IFN-γ secretion assessed by ELISA. HLA-A∗11⁺ K-562 cells loaded with titrating concentrations of the N^361-369 peptide were co-cultured with the TCR-CD4⁺ T cells or the TCR-CD8⁺ T cells for 24 h. Individual EC₅₀ of N^361-369 was calculated using nonlinear regression analysis. (B) Nine N^361-369 variants were aligned with the wild type peptide and the homologous MERS peptide (left). Individual mutations were marked in red. HLA-A∗11:01 binding affinity of each peptide was determined by the peptide exchange rates and shown by grey bars (n = 2). The reactivity of TCR 1- and TCR 4-CD8⁺ T cells to the N^361-369 mutant peptides were tested by IFN-γ ELISA assay and shown in connecting lines (n = 3). Data were presented as mean ± SD. Representative data of two independent experiments were shown.

Figure 5

The reactivity of TCR-transduced CD8⁺ T cells to the endogenously presented N^361-369 epitope on APCs. The reactivity assay of TCR 1- and TCR 4-transduced CD8⁺ T cells following co-culturing with (A,B) HLA-A∗11:01⁺ DCs loaded with N or (C,D) HLA-A∗11:01⁺ lung organoids endogenously expressing N protein. (A,C) CD137 expression was evaluated by flow cytometry. (B,D) IFN-γ secretion was determined by ELISA. (E) Cytotoxicity assay of TCR 1-CD8⁺ T cells against HLA-A∗11:01⁺ lung organoids exogenously pulsed with N^361-369 peptide (left). DMSO was used as negative control. T cells were stained with blue dye and lung organoids were stained in green. Target cell lysis was quantified using Calcein-AM release assay (right). (F) Cytotoxicity assay of TCR 1-CD8⁺ T cells against HLA-A∗11:01⁺ lung organoids endogenously expressing the N protein (left). Organoids expressing the S protein was applied as negative control. T cells were stained in red and lung organoids were stained in green. Target cell lysis was quantified by LDH release assay (right). Corresponding microphotographs of brightfield displayed all cells in view. White arrows show examples of organoids that are efficiently lysed. Bar: 200 μm. Data were presented as the mean ± SD, n = 3, ∗P < 0.05, ∗∗∗P < 0.001. Representative data of two independent experiments were shown.