Single-cell profiling identifies a CD8bright CD244bright Natural Killer cell subset that reflects disease activity in HLA-A29-positive birdshot chorioretinopathy.

Birdshot chorioretinopathy is an inflammatory eye condition strongly associated with MHC-I allele HLA-A29. The striking association with MHC-I suggests involvement of T cells, whereas natural killer (NK) cell involvement remains largely unstudied. Here we show that HLA-A29-positive birdshot chorioretinopathy patients have a skewed NK cell pool containing expanded CD16 positive NK cells which produce more proinflammatory cytokines. These NK cells contain populations that express CD8A which is involved in MHC-I recognition on target cells, display gene signatures indicative of high cytotoxic activity (GZMB, PRF1 and ISG15), and signaling through NK cell receptor CD244 (SH2D1B). Long-term monitoring of a cohort of birdshot chorioretinopathy patients with active disease identifies a population of CD8bright CD244bright NK cells, which rapidly declines to normal levels upon clinical remission following successful treatment. Collectively, these studies implicate CD8bright CD244bright NK cells in birdshot chorioretinopathy.

Extracellular vesicle-packaged lncRNA from cancer-associated fibroblasts promotes immune evasion by downregulating HLA-A in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by immune evasion that contribute to poor prognosis. Cancer-associated fibroblasts (CAFs) play a pivotal role in orchestrating the PDAC tumour microenvironment. We investigated the role of CAF-derived extracellular vesicle (EV)-packaged long non-coding RNAs (lncRNAs) in immune evasion and explored gene therapy using engineered EVs loading small interfering RNAs (siRNAs) as a potential therapeutic strategy. Our findings highlight the significance of EV-packaged lncRNA RP11-161H23.5 from CAF in promoting PDAC immune evasion by downregulating HLA-A expression, a key component of antigen presentation. Mechanistically, RP11-161H23.5 forms a complex with CNOT4, a subunit of the mRNA deadenylase CCR4-NOT complex, enhancing the degradation of HLA-A mRNA by shortening its poly(A) tail. This immune evasion mechanism compromises the anti-tumour immune response. To combat this, we propose an innovative approach utilising engineered EVs as natural and biocompatible nanocarriers for siRNA-based gene therapy and this strategy holds promise for enhancing the effectiveness of immunotherapy in PDAC. Overall, our study sheds light on the critical role of CAF-derived EV-packaged lncRNA RP11-161H23.5/CNOT4/HLA-A axis in PDAC immune evasion and presents a novel avenue for therapeutic intervention.

Uncommon P1 Anchor-featured Viral T Cell Epitope Preference within HLA-A*2601 and HLA-A*0101 Individuals.

The individual HLA-related susceptibility to emerging viral diseases such as COVID-19 underscores the importance of understanding how HLA polymorphism influences peptide presentation and T cell recognition. Similar to HLA-A*0101, which is one of the earliest identified HLA alleles among the human population, HLA-A*2601 possesses a similar characteristic for the binding peptide and acts as a prevalent allomorph in HLA-I. In this study, we found that, compared with HLA-A*0101, HLA-A*2601 individuals exhibit distinctive features for the T cell responses to SARS-CoV-2 and influenza virus after infection and/or vaccination. The heterogeneous T cell responses can be attributed to the distinct preference of HLA-A*2601 and HLA-A*0101 to T cell epitope motifs with negative-charged residues at the P1 and P3 positions, respectively. Furthermore, we determined the crystal structures of the HLA-A*2601 complexed to four peptides derived from SARS-CoV-2 and human papillomavirus, with one structure of HLA-A*0101 for comparison. The shallow pocket C of HLA-A*2601 results in the promiscuous presentation of peptides with "switchable" bulged conformations because of the secondary anchor in the median portion. Notably, the hydrogen bond network formed between the negative-charged P1 anchors and the HLA-A*2601-specific residues lead to a "closed" conformation and solid placement for the P1 secondary anchor accommodation in pocket A. This insight sheds light on the intricate relationship between HLA I allelic allomorphs, peptide binding, and the immune response and provides valuable implications for understanding disease susceptibility and potential vaccine design.

HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma.

BACKGROUND: Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance. METHODS: We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients. RESULTS: Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC. CONCLUSIONS: HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.

A bioinformatic analysis of T-cell epitope diversity in SARS-CoV-2 variants: association with COVID-19 clinical severity in the United States population.

Long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the identification of T-cell epitopes affecting host immunogenicity. In this computational study, we explored the CD8+ epitope diversity estimated in 27 of the most common HLA-A and HLA-B alleles, representing most of the United States population. Analysis of 16 SARS-CoV-2 variants [B.1, Alpha (B.1.1.7), five Delta (AY.100, AY.25, AY.3, AY.3.1, AY.44), and nine Omicron (BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ.1, BQ.1.1, XBB.1, XBB.1.5)] in analyzed MHC class I alleles revealed that SARS-CoV-2 CD8+ epitope conservation was estimated at 87.6%-96.5% in spike (S), 92.5%-99.6% in membrane (M), and 94.6%-99% in nucleocapsid (N). As the virus mutated, an increasing proportion of S epitopes experienced reduced predicted binding affinity: 70% of Omicron BQ.1-XBB.1.5 S epitopes experienced decreased predicted binding, as compared with ~3% and ~15% in the earlier strains Delta AY.100-AY.44 and Omicron BA.1-BA.5, respectively. Additionally, we identified several novel candidate HLA alleles that may be more susceptible to severe disease, notably HLA-A*32:01, HLA-A*26:01, and HLA-B*53:01, and relatively protected from disease, such as HLA-A*31:01, HLA-B*40:01, HLA-B*44:03, and HLA-B*57:01. Our findings support the hypothesis that viral genetic variation affecting CD8 T-cell epitope immunogenicity contributes to determining the clinical severity of acute COVID-19. Achieving long-term COVID-19 immunity will require an understanding of the relationship between T cells, SARS-CoV-2 variants, and host MHC class I genetics. This project is one of the first to explore the SARS-CoV-2 CD8+ epitope diversity that putatively impacts much of the United States population.

Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Distribution of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 allele frequencies in patients with COVID-19 bilateral pneumonia in Russians, living in the Chelyabinsk region (Russia).

In this population-based case-control study conducted in the Chelyabinsk region of Russia, we examined the distribution of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, in a group of 100 patients with confirmed COVID-19 bilateral pneumonia. Typing was performed by NGS and statistical calculations were carried out with the Arlequin program. HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were compared between patients with COVID-19 and 99 healthy controls. We identified that COVID-19 susceptibility is associated with alleles and genotypes rs9277534A (disequilibrium with HLA-DPB1*02:01, -02:02, -04:01, -04:02, -17:01 alleles) with low expression of protein products HLA-DPB1 (pc < 0.028) and homozygosity at HLA-C*04 (p = 0.024, pc = 0.312). Allele HLA-A*01:01 was decreased in a group of patients with severe forms of bilateral pneumonia, and therefore it may be considered as a protective factor for the development of severe symptoms of COVID-19 (p = 0.009, pc = 0.225). Our studies provide further evidence for the functional association between HLA genes and COVID-19.

Cutting Edge: Unconventional CD8+ T Cell Recognition of a Naturally Occurring HLA-A*02:01-Restricted 20mer Epitope.

Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.

Minimal Information about MHC Multimers (MIAMM).

With the goal of improving the reproducibility and annotatability of MHC multimer reagent data, we present the establishment of a new data standard: Minimal Information about MHC Multimers (https://miamm.lji.org/). Multimers are engineered reagents composed of a ligand and a MHC, which can be represented in a standardized format using ontology terminology. We provide an online Web site to host the details of the standard, as well as a validation tool to assist with the adoption of the standard. We hope that this publication will bring increased awareness of Minimal Information about MHC Multimers and drive acceptance, ultimately improving the quality and documentation of multimer data in the scientific literature.

HLA-A∗02:01 restricted T cell receptors against the highly conserved SARS-CoV-2 polymerase cross-react with human coronaviruses.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

HLA associations, somatic loss of HLA expression, and clinical outcomes in immune aplastic anemia.

Immune aplastic anemia (AA) features somatic loss of HLA class I allele expression on bone marrow cells, consistent with a mechanism of escape from T-cell-mediated destruction of hematopoietic stem and progenitor cells. The clinical significance of HLA abnormalities has not been well characterized. We examined the somatic loss of HLA class I alleles and correlated HLA loss and mutation-associated HLA genotypes with clinical presentation and outcomes after immunosuppressive therapy in 544 AA patients. HLA class I allele loss was detected in 92 (22%) of the 412 patients tested, in whom there were 393 somatic HLA gene mutations and 40 instances of loss of heterozygosity. Most frequently affected was HLA-B*14:02, followed by HLA-A*02:01, HLA-B*40:02, HLA-B*08:01, and HLA-B*07:02. HLA-B*14:02, HLA-B*40:02, and HLA-B*07:02 were also overrepresented in AA. High-risk clonal evolution was correlated with HLA loss, HLA-B*14:02 genotype, and older age, which yielded a valid prediction model. In 2 patients, we traced monosomy 7 clonal evolution from preexisting clones harboring somatic mutations in HLA-A*02:01 and HLA-B*40:02. Loss of HLA-B*40:02 correlated with higher blood counts. HLA-B*07:02 and HLA-B*40:01 genotypes and their loss correlated with late-onset of AA. Our results suggest the presence of specific immune mechanisms of molecular pathogenesis with clinical implications. HLA genotyping and screening for HLA loss may be of value in the management of immune AA. This study was registered at clinicaltrials.gov as NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167.

Combined presentation and immunogenicity analysis reveals a recurrent RAS.Q61K neoantigen in melanoma.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote "off-the-shelf" precision immunotherapies, alleviating limitations of personalized treatments.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody-antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to "shared" antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Novel mumps virus epitopes reveal robust cytotoxic T cell responses after natural infection but not after vaccination.

Mumps is nowadays re-emerging despite vaccination. The contribution of T cell immunity to protection against mumps has not been clearly defined. Previously, we described a set of 41 peptides that were eluted from human leukocyte antigen (HLA) class I molecules of mumps virus (MuV)-infected cells. Here, we confirmed immunogenicity of five novel HLA-B*07:02- and HLA-A*01:01-restricted MuV T cell epitopes from this set of peptides. High frequencies of T cells against these five MuV epitopes could be detected ex vivo in all tested mumps patients. Moreover, these epitope-specific T cells derived from mumps patients displayed strong cytotoxic activity. In contrast, only marginal T cell responses against these novel MuV epitopes could be detected in recently vaccinated persons, corroborating earlier findings. Identifying which MuV epitopes are dominantly targeted in the mumps-specific CD8+ T- response is an important step towards better understanding in the discrepancies between natural infection or vaccination-induced cell-mediated immune protection.

Establishment of a novel cell-based assay using HLA-transfected cells to detect HLA antibodies.

The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA). Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined. All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided. By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

ERAP2 Increases the Abundance of a Peptide Submotif Highly Selective for the Birdshot Uveitis-Associated HLA-A29.

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.

Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01.

Human cytomegalovirus (CMV) infection is widespread among adults (60-90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in immunocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide-MHC complex, pMHC) composed of MHC molecules with embedded short peptides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (495NLVPMVATV503; hereafter, CMVpp65495-503) in complex with MHC-I molecule human leukocyte antigen (HLA)-A*02:01 (CMVpp65495-503/HLA-A*02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions using yeast surface display technology. Compared with the parental Ab, the final generated Ab (C1-17) showed ~67-fold enhanced binding affinity (KD ≈ 5.2 nM) for the soluble pMHC, thereby detecting the cell surface-displayed CMVpp65495-503/HLA-A*02:01 complex with high sensitivity and exquisite specificity. Thus, the new high-affinity TCR-like Ab may be used for the detection and treatment of CMV infection.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.