HLA-E Ligand Research Articles

All-Atom Simulations Reveal the Intricacies of Signal Transduction upon Binding of the HLA-E Ligand to the Transmembrane Inhibitory CD94/NKG2A Receptor.

Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.

Do variations in the HLA-E ligand encoded by UL40 distinguish individuals susceptible to HCMV disease?

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15–23 encoded by the HCMV gene UL40 match positions 3–11 of HLA-A and HLA-C, and constitute a “signal peptide” able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL – a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort.

What a Difference an Amino Acid Makes: An All-Atom Simulation Study of Nonameric Peptides in Inhibitory HLA-E/NKG2A/CD94 Immune Complexes.

MHC class I antigen E (HLA-E), a ligand for the inhibitory NKG2A/CD94 receptor of the immune system, is responsible for evading the immune surveillance in several settings, including senescent cell accumulation and tumor persistence. The formation of this ligand-receptor interaction promotes the inhibition of the cytolytic action of immune system natural killer (NK) cells and CD8+ T-cells expressing this receptor. The final outcome of the HLA-E/NKG2A/CD94 interaction on target cells is also highly dependent on the identity of the nonameric peptide incorporated into the HLA-E ligand. To better understand the role played by a nonameric peptide in these immune complexes, we performed a series of multi-microsecond all-atom molecular dynamics simulations. We generated natural and alternative variants of the nonameric peptide bound to the HLA-E ligand alone or in the HLA-E/NKG2A/CD94 complexes. A systematic study of molecular recognition between HLA-E and peptides led to the development of new variants that differ at the strategic 6th position (P6) of the peptide and have favorable in silico properties comparable to those of natural binding peptides. Further examination of a selected subset of peptides in full complexes revealed a new variant that, according to our previously derived atomistic model, can interfere with the signal transduction via HLA-E/NKG2A/CD94 and thus prevent the target cell from evading immune clearance by NK and CD8+ T-cells. These simulations provide an atomistic picture of how a small change in amino acid sequence can lead to a profound effect on binding and molecular recognition. Furthermore, our study also provides new data on the peptide interaction motifs as well as the energetic and conformational properties of the binding interface, laying the structure-based foundation for future development of potential therapeutic peptides, peptidomimetics, or even small molecules that would bind to the HLA-E ligand and abrogate NKG2A/CD94 recognition. Such external intervention would be useful in the emerging field of targeting senescent cells in a variety of age-related diseases, as well as in novel cancer immunotherapies.

Abstract 5653: Discovery of Anti-NKG2A antibody from immunized phage display approach

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c and SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no detectable binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. The final lead is a humanized antibody which behaves well in stability test. Lab scale processes are successfully developed and confirmed. The safety and efficacy will be further validated. Citation Format: Teddy Yang, Jing Gao, Jingyun Yao, Dongxu Wang, Ken Dai. Discovery of Anti-NKG2A antibody from immunized phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5653.

Subordinate Effect of -21M HLA-B Dimorphism on NK Cell Repertoire Diversity and Function in HIV-1 Infected Individuals of African Origin.

Natural Killer (NK) cells play an important role in antiviral defense and their potent effector function identifies them as key candidates for immunotherapeutic interventions in chronic viral infections. Their remarkable functional agility is achieved by virtue of a wide array of germline-encoded inhibitory and activating receptors ensuring a self-tolerant and tunable repertoire. NK cell diversity is generated by a combination of factors including genetic determinants and infections/environmental factors, which together shape the NK cell pool and functional potential. Recently a genetic polymorphism at position -21 of HLA-B, which influences the supply of HLA-E binding peptides and availability of HLA-E for recognition by the inhibitory NK cell receptor NKG2A, was shown to have a marked influence on NK cell functionality in healthy human cytomegalovirus (HCMV) seronegative Caucasian individuals. In this study, -21 methionine (M)-expressing alleles supplying HLA-E binding peptides were largely poor ligands for inhibitory killer immunoglobulin-like receptors (KIRs), and a bias to NKG2A-mediated education of functionally-potent NK cells was observed. Here, we investigated the effect of this polymorphism on the phenotype and functional capacity of peripheral blood NK cells in a cohort of 36 African individuals with human immunodeficiency virus type 1 (HIV-1)/HCMV co-infection. A similarly profound influence of dimorphism at position -21 of HLA-B on NK cells was not evident in these subjects. They predominantly expressed African specific HLA-B and -C alleles that contribute a distinct supply of NKG2A and KIR ligands, and these genetic differences were compounded by the marked effect of HIV-1/HCMV co-infection on NK cell differentiation. Together, these factors resulted in a lack of correlation of the HLA-B -21 polymorphism with surface abundance of HLA-E and loss of the NK cell functional advantage in subjects with -21M HLA-B alleles. Instead, our data suggest that during HIV/HCMV co-infection exposure of NK cells to an environment that displays altered HLA-E ligands drives adaptive NKG2C+ NK cell expansions influencing effector responses. Increased efforts to understand how NK cells are functionally calibrated to self-HLA during chronic viral infections will pave the way to developing targeted therapeutic interventions to overcome the current barriers to enhancing immune-based antiviral control.

Is there a role played by HLA-E, if any, in HPV immune evasion?

Cervical cancer incidence worldwide exceeds half a million new cases per year. The human papillomavirus (HPV) being the major causative agent of CC uses a variety of strategies to evade immune surveillance, where the immune status varies amongst individuals. This immune evasion altered by HPV is reflected in persistent infections, causing the evolution of cervical neoplasia. The role of the immune system in viral recognition and elimination is of extreme relevance in the development of CC. The interactions of the HLA-E ligand in the target cell along with CD94/NKG2 receptors, which are expressed predominantly, but not exclusively, on NK cells' surface, are responsible for activating or inhibiting cytotoxic activity according to their function. The engagement between HLA-E and CD94/NKG2 molecules is one of the fundamental surveillance mechanisms in patients with CIN I, II and III, where HLA-E expression increases significantly, especially in HPV 16 and 18 infections. Higher HLA-E expression was observed in most histopathological types of CC, and at the same time was correlated to best survival of the patient. This review aims to summarize and discuss the immunological role of HLA-E in the context of HPV infection and immune system evasion, and the oncogenic process of cervical cancer.

Abstract 2387: NKG2A-antagonizing monoclonal antibodies identified through phage display

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c or SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. In summary, isolation and characterization of a panel of NKG2A-specific antagonizing antibodies enable us to further determine the feasibility of NKG2A-targeted immunotherapy of cancer. Citation Format: Jing Gao, Lili Hu, Ruirui Sui, Cuicui Guo, Lina Wang, Teddy Yang, Jack Han, Qing Duan, Louis Liu. NKG2A-antagonizing monoclonal antibodies identified through phage display [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2387.

Expression analysis of HLA-E and NKG2A and NKG2C receptors points at a role for natural killer function in ankylosing spondylitis

BackgroundAnkylosing spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of human leucocyte antigen (HLA)-B27 alleles. HLA-E molecules are non-classical major histocompatibility complex (MHC) class I...

T cell recognition of Mycobacterium tuberculosis peptides presented by HLA-E derived from infected human cells.

HLA-E is a non-conventional MHC Class I molecule that has been recently demonstrated to present pathogen-derived ligands, resulting in the TCR-dependent activation of αβ CD8+ T cells. The goal of this study was to characterize the ligandome displayed by HLA-E following infection with Mycobacterium tuberculosis (Mtb) using an in-depth mass spectrometry approach. Here we identified 28 Mtb ligands derived from 13 different source proteins, including the Esx family of proteins. When tested for activity with CD8+ T cells isolated from sixteen donors, nine of the ligands elicited an IFN-γ response from at least one donor, with fourteen of 16 donors responding to the Rv0634A19-29 peptide. Further evaluation of this immunodominant peptide response confirmed HLA-E restriction and the presence of Rv0634A19-29-reactive CD8+ T cells in the peripheral blood of human donors. The identification of an Mtb HLA-E ligand that is commonly recognized may provide a target for a non-traditional vaccine strategy.

SAT0253 HLA-E as ligand for NKG2A/NKG2C in ankylosing spondylitis: Increased expression of HLA-E and prevalence of the inhibitory receptor

BackgroundAnkylosing Spondylitis (AS) is a complex chronic inflammatory disease strongly associated with the majority of HLA-B27 alleles. HLA-E are non classical MHC class I molecules that specifically interact with the...

A Viral, Transporter Associated with Antigen Processing (TAP)-independent, High Affinity Ligand with Alternative Interactions Endogenously Presented by the Nonclassical Human Leukocyte Antigen E Class I Molecule

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Structural basis for NKG2A/CD94 recognition of HLA-E

The NKG2x/CD94 (x = A, C, E) natural killer-cell receptors perform an important role in immunosurveillance by binding to HLA-E complexes that exclusively present peptides derived from MHC class I leader sequences, thereby monitoring MHC class I expression. We have determined the crystal structure of the NKG2A/CD94/HLA-E complex at 4.4-A resolution, revealing two critical aspects of this interaction. First, the C-terminal region of the peptide, which displays the most variability among class I leader sequences, interacts entirely with CD94, the invariant component of these receptors. Second, residues 167-170 of NKG2A/C account for the approximately 6-fold-higher affinity of the inhibitory NKG2A/CD94 receptor compared to its activating NKG2C/CD94 counterpart. These residues do not contact HLA-E or peptide directly but instead form part of the heterodimer interface with CD94. An evolutionary analysis across primates reveals that whereas CD94 is evolving under purifying selection, both NKG2A and NKG2C are evolving under positive selection. Specifically, residues at the CD94 interface have evolved under positive selection, suggesting that the evolution of these genes is driven by an interaction with pathogen-derived ligands. Consistent with this possibility, we show that NKG2C/CD94, but not NKG2A/CD94, weakly but specifically binds to the CMV MHC-homologue UL18. Thus, the evolution of the NKG2x/CD94 family of receptors has likely been shaped both by the need to bind the invariant HLA-E ligand and the need to avoid subversion by pathogen-derived decoys.

Uncommon Endocytic and Trafficking Pathway of the Natural Killer Cell CD94/NKG2A Inhibitory Receptor

The CD94/NKG2A inhibitory receptor, expressed by natural killer and T cells, is constantly exposed to its HLA-E ligand expressed by surrounding cells. Ligand exposure often induces receptor downregulation. For CD94/NKG2A, this could potentiate activation receptor(s) induced responses to normal bystander cells. We investigated CD94/NKG2A endocytosis and found that it occurs by an amiloride-sensitive, Rac1-dependent macropinocytic-like process; however, it does not require clathrin, dynamin, ADP ribosylation factor-6, phosphoinositide-3 kinase or the actin cytoskeleton. Once endocytosed, CD94/NKG2A traffics to early endosomal antigen 1(+), Rab5(+) early endosomes. It does appear in Rab4(+) early/sorting endosome, but, in the time period examined, fails to reach Rab11(+) recycling or Rab7(+) late endosomes or lysosome-associated membrane protein-1(+) lysosomes. These results indicate that CD94/NKG2A utilizes a previously undescribed endocytic mechanism coupled with an abbreviated trafficking pattern, perhaps to insure surface expression.

The Heterodimeric Assembly of the CD94-NKG2 Receptor Family and Implications for Human Leukocyte Antigen-E Recognition

The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

Interactions between NKG2x Immunoreceptors and HLA-E Ligands Display Overlapping Affinities and Thermodynamics

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.

Regulation of FasL expression in natural killer cells

Fas ligand (FasL)-mediated cytotoxicity is initiated in natural killer (NK) cells through ligation of their activating receptors. The CD16 receptor has been shown to induce FasL expression and cytotoxicity in NK cells. In this study, we made the novel observation that FasL expression was upregulated in NKL cells stimulated through 2B4 and LFA-1 activating receptors, implying a role for FasL-mediated cytotoxicity early in the immune response. Coligation with CD94/NKG2A human leukocyte antigen (HLA) class I inhibitory receptor did not block the induced FasL expression; therefore, these opposing pathways appear to function independently. We also showed, however, that FasL-mediated cytotoxicity was downregulated in CD94/NKG2A-expressing LAK cells in response to the HLA-E ligand, suggesting a mechanism by which aberrant cells expressing class I may evade FasL-mediated cytotoxicity. Thus we show for the first time that 2B4, LFA-1, and CD94/NKG2A receptors are involved in modulating FasL expression and, therefore, cytotoxicity mediated by NK cells.

Truncated HLA-G isoforms are retained in the endoplasmic reticulum and insufficiently provide HLA-E ligands

The preferential expression of the non-polymorphic human leukocyte antigen G (HLA-G) on invading extravillous cytotrophoblast cells that are, with the exception of HLA-C and -E, HLA class I negative led to the hypothesis that HLA-G plays a major role in controlling the effector functions of the large granular leukocytes (LGL), a specialized natural killer (NK) cell population present in large numbers in the decidua. Transcription of the HLA-G gene is characterized by extensive alternative splicing producing at least seven potentially membrane bound or secreted isoforms. Except for HLA-G1 and its soluble variant (HLA-G1s), there is still dispute as to whether any of the other isoforms displays a major immunological function. Here we describe that the membrane-bound isoforms HLA-G2, -G3, and G4 as well as the soluble variant of HLA-G2 (HLA-G2s) do not egress the endoplasmic reticulum as determined by Endo H sensitivity assays. Moreover these isoforms seem not to have a major immunological function with respect to NK cell inhibition by providing a ligand for HLA-E, which would allow the interaction of this molecule with the inhibitory CD94/NKG2A NK cell receptor.

Truncated HLA-G isoforms are retained in the endoplasmic reticulum and insufficiently provide HLA-E ligands

The preferential expression of the non-polymorphic human leukocyte antigen G (HLA-G) on invading extravillous cytotrophoblast cells that are, with the exception of HLA-C and -E, HLA class I negative led to the hypothesis that HLA-G plays a major role in controlling the effector functions of the large granular leukocytes (LGL), a specialized natural killer (NK) cell population present in large numbers in the decidua. Transcription of the HLA-G gene is characterized by extensive alternative splicing producing at least seven potentially membrane bound or secreted isoforms. Except for HLA-G1 and its soluble variant (HLA-G1s), there is still dispute as to whether any of the other isoforms displays a major immunological function. Here we describe that the membrane-bound isoforms HLA-G2, -G3, and G4 as well as the soluble variant of HLA-G2 (HLA-G2s) do not egress the endoplasmic reticulum as determined by Endo H sensitivity assays. Moreover these isoforms seem not to have a major immunological function with respect to NK cell inhibition by providing a ligand for HLA-E, which would allow the interaction of this molecule with the inhibitory CD94/NKG2A NK cell receptor.

Implications of HLA-E allele expression and different HLA-E ligand diversity for the regulation of NK cells

The interaction of HLA-E with CD94/NKG2A is dependant on the binding of HLA class I signal sequence derived peptides to HLA-E. In the caucasoid population two HLA-E alleles are observed at equal frequencies. Here we study the functional differences between the two HLA-E molecules with regard to cell surface expression, peptide binding, and potential to inhibit lytic activity of a CD94/NKG2A + NK cell line. In contrast to the HLA-E R allele, the HLA-E G allele shows considerable cell surface expression even in the absence of endogenous HLA class I signal sequence derived HLA-E ligands. Eighteen HLA-E allele/HLA-E ligand combinations were analyzed. No correlation between cell surface expression of HLA-E and NK cell inhibition was observed. The peptides present in the signal sequences of HLA-B15, -Cw0402, and -Cw7 bound to both HLA-E alleles but did not lead to an inhibition of NK cell lysis. In our experimental system the peptides A2 and G were not effective with regard to NK cell inhibition when bound to the HLA-E R allele. These results may be of functional significance particularly in the placenta where the only HLA-E ligands are derived from HLA-G and -C.

Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis.

Human CMV has evolved multiple strategies to interfere with immune recognition of the host. A variety of mechanisms target Ag presentation by MHC class I molecules resulting in a reduced class I cell-surface expression. This down-regulation of class I molecules is expected to trigger NK cytotoxicity, which would have to be counteracted by the virus to establish long-term infection. Here we describe that the human CMV open reading frame UL40 encodes a canonical ligand for HLA-E, identical with the HLA-Cw03 signal sequence-derived peptide. Expression of UL40 in HLA-E-positive target cells conferred resistance to NK cell lysis via the CD94/NKG2A receptor. Generation of the UL40-derived HLA-E ligand was also observed in TAP-deficient cells. The presence of a functional TAP-independent HLA-E ligand in the UL40 signal sequence implicates this viral gene as an important negative regulator of NK activity.