Tuberculosis (TB) remains one of the deadliest infectious diseases worldwide, posing great social and economic burden to affected countries. Novel vaccine approaches are needed to increase protective immunity against the causative agent Mycobacterium tuberculosis (Mtb) and to reduce the development of active TB disease in latently infected individuals. Donor-unrestricted T cell responses represent such novel potential vaccine targets. HLA-E-restricted T cell responses have been shown to play an important role in protection against TB and other infections, and recent studies have demonstrated that these cells can be primed in vitro. However, the identification of novel pathogen-derived HLA-E binding peptides presented by infected target cells has been limited by the lack of accurate prediction algorithms for HLA-E binding. In this study, we developed an improved HLA-E binding peptide prediction algorithm and implemented it to identify (to our knowledge) novel Mtb-derived peptides with capacity to induce CD8+ T cell activation and that were recognized by specific HLA-E-restricted T cells in Mycobacterium-exposed humans. Altogether, we present a novel algorithm for the identification of pathogen- or self-derived HLA-E-presented peptides.

Characterization of immune cells is essential to advance our understanding of immunology and flow cytometry is an important tool in this context. Addressing both cellular phenotype and antigen‐specific functional responses of the same cells is valuable to achieve a more integrated understanding of immune cell behavior and maximizes information obtained from precious samples. Until recently, panel size was limiting, resulting in panels generally focused on either deep immunophenotyping or functional readouts. Ongoing developments in the field of (spectral) flow cytometry have made panels of 30⁺ markers more accessible, opening up possibilities for advanced integrated analyses. Here, we optimized immune phenotyping by co‐detection of markers covering chemokine receptors, cytokines and specific T cell/peptide tetramer interaction using a 32‐color panel. Such panels enable integrated analysis of cellular phenotypes and markers assessing the quality of immune responses and will contribute to our understanding of the immune system.

The virtually monomorphic antigen presentation molecule HLA-E can present self- and non-self peptides to the NKG2A/CD94 co-receptor inhibitory complex expressed on natural killer (NK) cells and to T cell receptors (TCRs) expressed on T cells. HLA-E presents self-peptides to NKG2A/CD94 to regulate tissue homeostasis, whereas HLA-E restricted T cells mediate regulatory and cytotoxic responses toward pathogen-infected cells. In this study, we directly compared HLA-E/peptide recognition and signaling between NKG2A/CD94 and 2 HLA-E restricted TCRs that can recognize self-peptides or identical peptide mimics from the viral UL40 protein of cytomegalovirus using position substituted peptide variants. We show that position 7 is critical for interaction with NKG2A/CD94, whereas position 8 is important for interaction with the TCRs. The Arginine at position 5 of these peptides is an essential residue for recognition by both receptors. Thus, NKG2A/CD94 and TCRs have different requirements for recognition of peptides presented in HLA-E.

There is growing interest in HLA‐E‐restricted T‐cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide‐specific T cells has allowed the in‐depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T‐cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA‐E monomers and multimers allowing the high‐throughput production of HLA‐E multimers. We optimized the thermal‐mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high‐throughput identification and analysis of HLA‐E‐binding peptides which could be involved in T‐cell and NK cell‐mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA‐E with this receptor. In particular, our results reveal that interactions of HLA‐E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA‐E multimer technology based on thermal‐mediated peptide exchange allowing us to investigate the molecular requirements for HLA‐E/peptide interaction with its receptors.

Background: Novel vaccines targeting the world’s deadliest pathogen Mycobacterium tuberculosis (Mtb) are urgently needed as the efficacy of the Bacillus Calmette–Guérin (BCG) vaccine in its current use is limited. HLA-E is a virtually monomorphic unconventional antigen presentation molecule, and HLA-E-restricted Mtb-specific CD8⁺ T cells can control intracellular Mtb growth, making HLA-E a promising vaccine target for Mtb. Methods: In this study, we evaluated the frequency and phenotype of HLA-E-restricted Mtb-specific CD4⁺/CD8⁺ T cells in the circulation and bronchoalveolar lavage fluid of two independent non-human primate (NHP) studies and from humans receiving BCG either intradermally or mucosally. Results: BCG vaccination followed by Mtb challenge in NHPs did not affect the frequency of circulating and local HLA-E–Mtb CD4⁺ and CD8⁺ T cells, and we saw the same in humans receiving BCG. HLA-E–Mtb T cell frequencies were significantly increased after Mtb challenge in unvaccinated NHPs, which was correlated with higher TB pathology. Conclusions: Together, HLA-E–Mtb-restricted T cells are minimally induced by BCG in humans and rhesus macaques (RMs) but can be elicited after Mtb infection in unvaccinated RMs. These results give new insights into targeting HLA-E as a potential immune mechanism against TB.