The antibodies 3D12 and 4D12 recognise distinct epitopes and conformations of HLA-E.

The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.

The MHC-E peptide ligands for checkpoint CD94/NKG2A are governed by inflammatory signals, whereas LILRB1/2 receptors are peptide indifferent.

The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.

Commensal bacteria maintain a Qa-1b-restricted unconventional CD8+ T population in gut epithelium.

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8+ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1b-restricted CD8+ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1b-dependent unconventional phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigens, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Moreover, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1b-restricted IEL landscape.

The promiscuous development of an unconventional Qa1b-restricted T cell population.

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αß+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αß intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αß T cells. QFL T cells require the MHC I subunit ß-2 microglobulin (ß2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αß+CD4- pathway for development of CD8αα IELs.

Discovery of HLA-E-Presented Epitopes: MHC-E/Peptide Binding and T-Cell Recognition.

Understanding the interactions involved during the immunological synapse between peptide, HLA-E molecules, and TCR is crucial to effectively target protective HLA-E-restricted T-cell responses in humans. Here we describe three techniques based on the generation of MHC-E/peptide complexes (MHC-E generically includes HLA-E-like molecules in human and nonhuman species, while HLA-E specifically refers to human molecules), which allow to investigate MHC-E/peptide binding at the molecular level through binding assays and by using peptide loaded HLA-E tetramers, to detect, isolate, and study peptide-specific HLA-E-restricted human T-cells.

Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes.

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.

Antigen presentation by MHC-E: a putative target for vaccination?

The essentially monomorphic human antigen presentation molecule HLA-E is an interesting candidate target to enable vaccination irrespective of genetic diversity. Predictive HLA-E peptide-binding motifs have been refined to facilitate HLA-E peptide discovery. HLA-E can accommodate structurally divergent peptides of both self and microbial origin. Intracellular processing and presentation pathways for peptides by HLA-E for T cell receptor (TCR) recognition remain to be elucidated. Recent studies show that, unlike canonical peptides, inhibition of the transporter associated with antigen presentation (TAP) is essential to allow HLA-E antigen presentation in cytomegalovirus (CMV) infection and possibly also of other non-canonical peptides. We propose three alternative and TAP-independent MHC-E antigen-presentation pathways, including for Mycobacterium tuberculosis infections. These insights may help in designing potential HLA-E targeting vaccines against tumors and pathogens.

Vaccine-Mediated Inhibition of the Transporter Associated with Antigen Processing Is Insufficient To Induce Major Histocompatibility Complex E-Restricted CD8+ T Cells in Nonhuman Primates.

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.