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T cell immune evasion by SARS-CoV-2 JN.1 escapees targeting two cytotoxic T cell epitope hotspots

Nature Immunology volume 26, pages 265–278 (2025) Cite this article

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Abstract

Although antibody escape is observed in emerging severe acute respiratory syndrome coronavirus 2 variants, T cell escape, especially after the global circulation of BA.2.86/JN.1, is unexplored. Here we demonstrate that T cell evasion exists in epitope hotspots spanning BA.2.86/JN.1 mutations. The newly emerging Q229K at this conserved nucleocapsid protein site impairs HLA-A2 epitope hotspot recognition. The association between HLA-A24 convalescents and T cell immune escape points to the spike (S) protein epitope S448–456NYNYLYRLF, with multiple mutations from Delta to JN.1, including L452Q, L452R, F456L, N450D and L452W, and N450D, L452W and L455S. A cliff drop of immune responses was observed for S448–456NYNYRYRLF (Delta/BA.5.2) and S448–456NYDYWYRSF (JN.1), but with immune preservation of S448–456NYDYWYRLF (BA.2.86). Structural analyses showed that hydrophobicity exposure determines the pronounced escape of L452R and L455S mutants, which was further confirmed by T cell receptor binding. This study highlights the characteristics and molecular mechanisms of the T cell immune escape for JN.1 and provides new insights into understanding the dominant circulation of variants, from the viewpoint of cytotoxic T cell evasion.

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Fig. 1: Cellular immunity to peptides with BA.2.86 and JN.1 mutations.
The alternative text for this image may have been generated using AI.
Fig. 2: Evasion of T cell responses of BA.2.86 and JN.1 in individuals previously infected with SARS-CoV-2.
The alternative text for this image may have been generated using AI.
Fig. 3: Cross-immunity and evading-immunity responses to natural mutations at S448–456 in different variants.
The alternative text for this image may have been generated using AI.
Fig. 4: The mutations at S448–456 across natural variants have diverse impacts on T cell responses and HLA-A*2402 binding.
The alternative text for this image may have been generated using AI.
Fig. 5: Characterization of TCR repertoire specific to S448–456 and functional validation.
The alternative text for this image may have been generated using AI.
Fig. 6: Structure of HLA-A*2402/S448–456NYNYLYRLF and mutants.
The alternative text for this image may have been generated using AI.
Fig. 7: The BA.2.86/JN.1 N protein Q229K mutation leads to T cell immune escape of a highly conserved HLA-A2 epitope hotspot region.
The alternative text for this image may have been generated using AI.

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Data availability

The GenBank accession numbers used are listed in the ‘Sequence analysis’ section. Source data are provided with this paper.

Code availability

The crystal structures reported in this study have been deposited in the Protein Data Bank under the accession nos. 8YZZ, 8Z08, 8Z07, 8YZR, 8YZW, 8Z06 and 8Z05.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (grant no. 2022YFC2604100 to J.L. and grant no. 2023YFC3041500 to J.L.), and the National Natural Science Foundation of China (grant no. 92269203 to J.L.). This work was also supported by the Center for Biosafety Mega-Science, Chinese Academy of Sciences and the User Experiment Assist System of Shanghai Synchrotron Radiation Facility. We thank Y. Chen, B. Zhou and Z. Yang (Institute of Biophysics, Chinese Academy of Sciences) for technical help with the Biacore experiments.

Author information

Author notes

  1. These authors contributed equally: Jinmin Tian, Bingli Shang, Jianing Zhang, Yuanyuan Guo, Min Li, Yuechao Hu.

Authors and Affiliations

  1. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

    Jinmin Tian, Bingli Shang, Jianing Zhang, Yuanyuan Guo, Min Li, Yuechao Hu, Dan Bai, Junying She, Yang Han, Peipei Guo, Mengkun Huang, Yalan Wang, Maoshun Liu, Beiwei Ye, Yaxin Guo, Mengjie Yang, Ying Lin, Ting Zhang, Xin Sun, Xiaoju Yuan, Danni Zhang, Ziqian Xu, Yingze Zhao & Jun Liu

  2. NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

    Jinmin Tian, Bingli Shang, Jianing Zhang, Yuanyuan Guo, Min Li, Yuechao Hu, Junying She, Yang Han, Peipei Guo, Mengkun Huang, Yalan Wang, Maoshun Liu, Beiwei Ye, Yaxin Guo, Mengjie Yang, Ting Zhang, Xin Sun, Xiaoju Yuan, Danni Zhang, Ziqian Xu, Yingze Zhao, George F. Gao & Jun Liu

  3. School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China

    Bingli Shang, Junying She, Mengjie Yang & Jun Liu

  4. Department of Epidemiology, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, China

    Yuanyuan Guo, Peipei Guo, Ying Lin & Xin Sun

  5. School of Ophthalmology & Optometry, Wenzhou Medical University, Wenzhou, China

    Dan Bai, Yang Han & George F. Gao

  6. Collaborative Innovation Centre of Regenerative Medicine and Medical BioResource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning, China

    Mengkun Huang & Ting Zhang

  7. Beijing Ditan Hospital, Capital Medical University, Beijing, China

    Jie Zhang & Rui Song

  8. Beijing Institute of Infectious Diseases, Beijing, China

    Jie Zhang

  9. Research Unit of Adaptive Evolution and Control of Emerging Viruses, Chinese Academy of Medical Sciences, Beijing, China

    Ziqian Xu, Yingze Zhao, George F. Gao & Jun Liu

  10. CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, China

    Yan Chai, Jianxun Qi, Kefang Liu, Shuguang Tan & George F. Gao

  11. The Fifth Hospital of Shijiazhuang, Shijiazhuang, China

    Jikun Zhou

  12. The D. H. Chen School of Universal Health, Zhejiang University, Hangzhou, China

    George F. Gao

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  1. Jinmin Tian
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Contributions

J.L. and G.F.G. conceived and designed the study. R.S., J. Zhou, J.T., Y.Z., Yuanyuan Guo, Jie Zhang, B.Y. and M. Liu collected the samples. J.T., B.S., Yuanyuan Guo, Y. Hu, J.S., M.Y., P.G., M.H. and D.B. conducted the experiments. Y.Z., B.Y., Yaxin Guo, Y.W., Y. Han, Jianing Zhang, T.Z., X.S., X.Y., Z.X., Y.L., J.Q., Y.C., D.Z., K.L., S.T., R.S. and J. Zhou provided technical support and experimental assistance. J.T., B.S., Jianing Zhang, M. Li, Yuanyuan Guo, Y. Hu and J.L. analyzed and interpreted the data. J.T., B.S., J.Z., M. Li and J.L. wrote the initial draft of the paper. All authors contributed intellectually and approved the paper.

Corresponding authors

Correspondence to Yingze Zhao, Jikun Zhou, Rui Song, George F. Gao or Jun Liu.

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Nature Immunology thanks Tao Dong, Katherine Kedzierska and Leo Swadling for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Identifying MHC I epitopes eliciting positive T cell responses in convalescents, related to Fig. 1.

Specific T cell responses were detected after in vitro culture of the PBMCs for 9 days under the stimulation of the prototype-derived MHC-I epitope pool. T cell responses against the 12 positive peptides including two HLA-A24/A2 epitopes hotspots were tested in donors infected with SARS-CoV-2 (n = 19) (Supplementary Table 1, 6 and 9). The results for donors whose PBMCs did not respond to the 12 peptides are not shown. The white represents the negative control, with the gray for the stimulated peptide. The x-axis displays donor IDs, which correspond to the individual samples analyzed in this study.

Source data

Extended Data Fig. 2 S1 peptide pool-specific T cell responses in the prototype group and XBB group, related to Fig. 2.

a, Cellular immunity to S1 protein of prototype and BA.2.12.1/BA.5.2 strains in 48 convalescents infected with the prototype strain (prototype group). Specific T cell responses were detected after in vitro culture of the PBMCs for 9 days under the stimulation of the prototype-derived S1 peptide pool. The prototype-derived S1 peptide pool-specific T cell responses and also the cross-reactivity to other different Omicron strains-derived S1 peptide pools were tested by using ELISpot assays. b. Comparison of T cell responses in populations with three HLA I supertypes (HLA-A24, HLA-A2, and HLA-A3) in the prototype group (n = 47, one sample excluded due to the HLA-A information unavailable). Escape value indicated the T cell responses of the prototype minus the T cell responses of BA.5.2 (Escape value = T cell response of prototype - T cell response of BA.5.2). c, Comparison of the cellular immunity to S1 protein responses between the prototype and BA.5.2 strain. Red (HLA-A24) and blue (non-HLA-A24) lines indicate T cell responses decreased by more than 20%. d. Comparison of T cell responses in populations with three HLA I supertypes (HLA-A24, HLA-A2, and HLA-A3) in 30 convalescents infected with the XBB strain (XBB group). Escape value indicated a percentage decline of the T cell responses, which is calculated as (T cell response of XBB - T cell response of BA.2.86) / T cell response of XBB. e. Comparison of T cell responses in populations with three HLA I supertypes (HLA-A24, HLA-A2, and HLA-A3) in the prototype group (n = 47). Escape value indicated a percentage decline of the T cell responses, which is calculated as (T cell response of prototype - T cell response of BA.5.2) / T cell response of prototype. The top and bottom of each rectangular box denote the interquartile range (IQR) of each group, with the median shown inside the box for panel a. Data are shown as mean ± s.e.m. for b, d, and e. A two-tailed Wilcoxon matched-pairs signed-rank test was used for a and c. A two-tailed Mann-Whitney U-test was used for b, d, and e. Abbreviation: SFCs, spot-forming cells.

Source data

Extended Data Fig. 3 Tetramer staining of S448-456, related to Fig. 4.

a, Gating strategy for tetramer staining. b, The tetramer staining of S448-456NYNYLYRLF (prototype), S448-456NYNYQYRLF (BA.2.12.1), and S448-456NYNYRYRLF (Delta/BA.5.2) from a BA.2.75-infected (the same as prototype epitope here) individual. And the tetramer+ T cells of S448-456 were further sorted for TCR screening.

Extended Data Fig. 4 Characterization of TCR repertoire specific to S448-456NYNYLYRLF and S448-456NYNYQYRLF.

PBMCs from an individual infected with BA.2.75 (conserved in the sequence of S448-456NYNYLYRLF as prototype) were in vitro cultured for 9-12 days. CD8+ T cell lines were stained with S448-456NYNYLYRLF- and S448-456NYNYQYRLF-tetramers, and tetramer+ cells were further single-cell sorted. The TCR repertoire was determined using multiplex PCR. a, Pie chart displays the combined TRAV (left) and TRBV (middle) usage of the S448-456NYNYLYRLF (prototype)-specific TCRs. b, Pie chart displays the combined TRAV (left) and TRBV (middle) usage of the S448-456NYNYQYRLF (BA.2.12.1) cross-recognized TCRs. Bubble plots showing the paired TCRs (right). c, Summary of CDR3α (left) and CDR3β (right) lengths for S448-456NYNYLYRLF (prototype)-specific TCR clonotypes. d, Summary of CDR3α (left) and CDR3β (right) lengths for S448-456NYNYQYRLF (BA.2.12.1)-cross-recognized TCR clonotypes. e. A Venn diagram displaying the TRAV/TRAJ/CDR3α and TRBV/TRBJ/CDR3β use of S448-456NYNYLYRLF- and S448-456NYNYQYRLF-specific TCRs in an individual recovered from BA.2.75. The numbers without parentheses represent the quantities of TRAV/TRAJ/CDR3α or TRBV/TRBJ/CDR3β types. The number ‘n’ inside parentheses represents the number of TCR clones, where the overlapping portion of the two circles on the left side indicates the number of shared clones for S448-456NYNYLYRLF, and on the right side, it indicates the number of shared clones for S448-456NYNYQYRLF. f. CDR3α and CDR3β analysis of the S448-456-specific CD8+ T cells. Analysis of the motifs of CDR3α (upper) and CDR3β (lower) sequences from distinct S448-456NYNYLYRLF- and S448-456NYNYQYRLF-specific CD8+ T cell clonotypes. CDR3α motifs for the most common lengths of 15, 16 and 17 amino acids long. CDR3β motifs for the most common lengths of 13 and 14 amino acids long.

Extended Data Fig. 5 The electronic density map of S448-456NYNYLYRLF (prototype)with its mutants and N222-230LLLDRLNKL (BA.2.86/JN.1).

The electronic density map of S448-456NYNYLYRLF (prototype) (a), S448-456NYNYQYRLF (BA.2.12.1) (b), S448-456NYNYRYRLF (Delta/BA.5.2) (c), S448-456NYNYLYRLL (EG.5.1) (d), S448-456NYDYWYRLF (BA.2.86) (e), S448-456NYDYWYRSF (JN.1) (f) and N222-230LLLDRLNKL (BA.2.86/JN.1) (g).

Extended Data Table 1 Summary of the current infected status of SARS-CoV-2 donors, as represented in Fig. 1
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Extended Data Table 2 Characteristics of participants described in Figs. 2 and 3, and in the supplementary materials
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Extended Data Table 3 Data collection and refinement statistics (molecular replacement)
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Supplementary information

Supplementary Information (download PDF )

Supplementary Tables 5–9.

Reporting Summary (download PDF )

Supplementary Tables 1–4 (download XLSX )

S/M/N/ORF1ab protein-derived potential HLA-I epitopes containing mutation sites in BA.2.86 and JN.1.

Supplementary Code 1 (download PDF )

8YZR_val-report-full-annotate_P1.

Supplementary Code 2 (download PDF )

8YZW_val-report-full-annotate_P1.

Supplementary Code 3 (download PDF )

8YZZ_val-report-full-annotate_P1.

Supplementary Code 4 (download PDF )

8Y05_val-report-full-annotate_P1.

Supplementary Code 5 (download PDF )

8Y06_val-report-full-annotate_P1.

Supplementary Code 6 (download PDF )

8Y07_val-report-full-annotate_P1.

Supplementary Code 7 (download PDF )

8Y08_val-report-full-annotate_P1.

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Tian, J., Shang, B., Zhang, J. et al. T cell immune evasion by SARS-CoV-2 JN.1 escapees targeting two cytotoxic T cell epitope hotspots. Nat Immunol 26, 265–278 (2025). https://doi.org/10.1038/s41590-024-02051-0

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