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The human leukocyte antigen (HLA)-Ib molecule, HLA-F, is known as a CD4+ T-cell protein and mediator of HIV progression. While HLA-Ia molecules do not have the chance to select and present viral peptides for immune recognition due to protein downregulation, HLA-F is upregulated. Post HIV infection, HLA-F loses the affinity to its activating receptor KIR3DS1 on NK cells leading to progression of the HIV infection. Several studies aimed to solve the question of the biophysical interface between HLA ligands and their cognate receptors. It became clear that even an invariant HLA molecule can be structurally modified by the variability of the bound peptide. We recently discovered the ability of HLA-F to select and present peptides and the HLA-F allele-specific peptide selection from the proteomic content using soluble HLA (sHLA) technology and a sophisticated MS method. We established recombinant K562 cells that express membrane-bound HLA-F*01:01, 01:03 or 01:04 complexes. While a recombinant soluble form of KIR3DS1 did not bind to the peptide-HLA-F complexes, acid elution of the peptides resulted in the presentation of HLA-F open conformers, and the binding of the soluble KIR3DS1 receptor increased. We used CD4+/HIV- and CD4+/HIV+ cells and performed an MS proteome analysis. We could detect hemoglobin as significantly upregulated in CD4+ T-cells post HIV infection. The expression of cellular hemoglobin in nonerythroid cells has been described, yet HLA-Ib presentation of hemoglobin-derived peptides is novel. Peptide sequence analysis from HLA-F allelic variants featured hemoglobin peptides as dominant and shared. The reciprocal experiment of binding hemoglobin peptide fractions to the HLA-F open conformers resulted in significantly diminished receptor recognition. These results underpin the molecular involvement of HLA-F and its designated peptide ligand in HIV immune escape.

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Tumor immune escape is associated with both, the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which are consequently discussed as clinical biomarker for disease status and outcome of cancer patients. HLA-G preferentially interacts with the inhibitory receptor immunoglobulin-like transcript (ILT) receptor-2 in the blood and can be secreted as free soluble molecules (sHLA-G) or via extracellular vesicles (EV). To investigate the contribution of these two forms to the expression of checkpoint molecules in peripheral blood, we primed peripheral blood mononuclear cells with purified soluble sHLA-G1 protein, or EV preparations derived from SUM149 cells transfected with membrane-bound HLA-G1 or control vector prior to anti-CD3/CD28 T cell activation. Our study demonstrated that priming of PBMC with sHLA-G1 protein prior to 48 h activation resulted in enhanced frequencies of ILT-2 expressing CD8+ T cells, and in an upregulation of immune checkpoint molecules CTLA-4, PD-1, TIM-3, and CD95 exclusively on ILT-2 positive CD8+ T cells. In contrast, when PBMC were primed with EV (containing HLA-G1 or not) upregulation of CTLA-4, PD-1, TIM-3, and CD95 occurred exclusively on ILT-2 negative CD8+ T cells. Taken together, our data suggest that priming with sHLA-G forms induces a pronounced immunosuppressive/exhausted phenotype and that priming with sHLA-G1 protein or EV derived from HLA-G1 positive or negative SUM149 cells affects CD8+ T cells complementary by targeting either the ILT-2 positive or negative subpopulation, respectively, after T cell activation.

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Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting K562 cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using K562-G*01:03 or K562-G*01:01 cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.

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Peptide-dependent engagement between human leucocyte antigens class I (HLA-I) molecules and their cognate receptors has been extensively analyzed. HLA-F belongs to the non-classical HLA-Ib molecules with marginal polymorphic nature and tissue restricted distribution. The three common allelic variants HLA-F*01:01/01:03/01:04 are distinguished by polymorphism outside the peptide binding pockets (residue 50, α1 or residue 251, α3) and are therefore not considered relevant for attention. However, peptide selection and presentation undergoes a most elaborated extraction from the whole available proteome. It is known that HLA-F confers a beneficial effect on disease outcome during HIV-1 infections. The interaction with the NK cell receptor initiates an antiviral downstream immune response and lead to delayed disease progression. During the time of HIV infection, HLA-F expression is upregulated, while its interaction with KIR3DS1 is diminished. The non-polymorphic nature of HLA-F facilitates the conclusion that understanding HLA-F peptide selection and presentation is essential to a comprehensive understanding of this dynamic immune response. Utilizing soluble HLA technology we recovered stable pHLA-F*01:01, 01:03 and 01:04 complexes from K562 cells and analyzed the peptides presented. Utilizing a sophisticated LC-MS-method, we analyzed the complete K562 proteome and matched the peptides presented by the respective HLA-F subtypes with detected proteins. All peptides featured a length of 8 to 24 amino acids and are not N-terminally anchored; the C-terminus is preferably anchored by Lys. To comprehend the alteration of the pHLA-F surface we structurally compared HLA-F variants bound to selected peptides. The peptides were selected from the same cellular content; however, no overlap between the proteomic source of F*01:01, 01:03 or 01:04 selected peptides could be observed. Recognizing the balance between HLA-F expression, HLA-F polymorphism and peptide selection will support to understand the role of HLA-F in viral pathogenesis.

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Drug hypersensitivity reactions that resemble acute immune reactions are linked to certain human leucocyte antigen (HLA) alleles. Severe and life-threatening Stevens Johnson Syndrome and Toxic Epidermal Necrolysis following treatment with the antiepileptic and psychotropic drug Carbamazepine are associated with HLA-B*15:02; whereas carriers of HLA-A*31:01 develop milder symptoms. It is not understood how these immunogenic differences emerge genotype-specific. For HLA-B*15:02 an altered peptide presentation has been described following exposure to the main metabolite of carbamazepine that is binding to certain amino acids in the F pocket of the HLA molecule. The difference in the molecular mechanism of these diseases has not been comprehensively analyzed, yet; and is addressed in this study. Soluble HLA-technology was utilized to examine peptide presentation of HLA-A*31:01 in presence and absence of carbamazepine and its main metabolite and to examine the mode of peptide loading. Proteome analysis of drug-treated and untreated cells was performed. Alterations in sA*31:01-presented peptides after treatment with carbamazepine revealed different half-life times of peptide-HLA- or peptide-drug-HLA complexes. Together with observed changes in the proteome elicited through carbamazepine or its metabolite these results illustrate the mechanistic differences in carbamazepine hypersensitivity for HLA-A*31:01 or B*15:02 patients and constitute the bridge between pharmacology and pharmacogenetics for personalized therapeutics.

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Human cytomegalovirus (HCMV) is ubiquitously prevalent. HCMV infection is typically asymptomatic and controlled by the immune system in healthy individuals, yet HCMV can be severely pathogenic for the fetus during pregnancy and in immunocompromised persons, such as transplant recipients or HIV infected patients. HCMV has co-evolved with the hosts, developed strategies to hide from immune effector cells and to successfully survive in the human organism. One strategy for evading or delaying the immune response is maintenance of the viral genome to establish the phase of latency. Furthermore, HCMV immune evasion involves the downregulation of human leukocyte antigens (HLA)-Ia molecules to hide infected cells from T-cell recognition. HCMV expresses several proteins that are described for downregulation of the HLA class I pathway via various mechanisms. Here, we review the wide range of immune evasion mechanisms of HCMV. Understanding the mechanisms of HCMV immune evasion will contribute to the development of new customized therapeutic strategies against the virus.

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HLA-F belongs to the non-classical HLA-Ib molecules with a marginal polymorphic nature and tissue-restricted distribution. HLA-F is a ligand of the NK cell receptor KIR3DS1, whose activation initiates an antiviral downstream immune response and lead to delayed disease progression of HIV-1. During the time course of HIV infection, the expression of HLA-F is upregulated while its interaction with KIR3DS1 is diminished. Understanding HLA-F peptide selection and presentation is essential to a comprehensive understanding of this dynamic immune response and the molecules function. In this study, we were able to recover stable pHLA-F*01:01 complexes and analyze the characteristics of peptides naturally presented by HLA-F. These HLA-F-restricted peptides exhibit a non-canonical length without a defined N-terminal anchor. The peptide characteristics lead to a unique presentation profile and influence the stability of the protein. Furthermore, we demonstrate that almost all source proteins of HLA-F-restricted peptides are described to interact with HIV proteins. Understanding the balance switch between HLA-Ia and HLA-F expression and peptide selection will support to understand the role of HLA-F in viral pathogenesis.

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The original version of this article contained errors. The Article Title, Figures 1 and 3, and Electronic Supplementary Materials were incorrectly shown in the wrong version. The original article has been corrected.

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T-cell receptors possess the unique ability to survey and respond to their permanently modified ligands, self HLA-I molecules bound to non-self peptides of various origin. This highly specific immune function is impaired following hematopoietic stem cell transplantation (HSCT) for a timespan of several months needed for the maturation of T-cells. Especially, the progression of HCMV disease in immunocompromised patients induces life-threatening situations. Therefore, the need for a new immune system that delivers vital and potent CD8+ T-cells carrying TCRs that recognize even one human cytomegalovirus (HCMV) peptide/HLA molecule and clear the viral infection long term becomes obvious. The transcription and translation of HCMV proteins in the lytic cycle is a precisely regulated cascade of processes, therefore, it is a highly sensitive challenge to adjust the exact time point of HCMV-peptide recruitment over self-peptides. We utilized soluble HLA technology in HCMV-infected fibroblasts and sequenced naturally sHLA-A*24:02 presented HCMV-derived peptides. One peptide of 14 AAs length derived from the IE2 antigen induced the strongest T-cell responses; this peptide can be detected with a low ranking score in general peptide prediction databanks. These results highlight the need for elaborate and HLA-allele specific peptide selection.

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On healthy cells the non-classical HLA class Ib molecule HLA-E displays the cognate ligand for the NK cell receptor NKG2A/CD94 when bound to HLA class I signal peptide sequences. In a pathogenic situation when HLA class I is absent, HLA-E is bound to a diverse set of peptides and enables the stimulatory NKG2C/CD94 receptor to bind. The activation of CD8⁺ T cells by certain p:HLA-E complexes illustrates the dual role of this low polymorphic HLA molecule in innate and adaptive immunity. Recent studies revealed a shift in the HLA-E peptide repertoire in cells with defects in the peptide loading complex machinery. We recently showed that HLA-E presents a highly diverse set of peptides in the absence of HLA class Ia and revealed a non-protective feature against NK cell cytotoxicity mediated by these peptides. In the present study we have evaluated the molecular basis for the impaired NK cell inhibition by these peptides and determined the cell surface stability of individual p:HLA-E complexes and their binding efficiency to soluble NKG2A/CD94 or NKG2C/CD94 receptors. Additionally, we analyzed the recognition of these p:HLA-E epitopes by CD8⁺ T cells. We show that non-canonical peptides provide stable cell surface expression of HLA-E, and these p:HLA-E complexes still bind to NKG2/CD94 receptors in a peptide-restricted fashion. Furthermore, individual p:HLA-E complexes elicit activation of CD8⁺ T cells with an effector memory phenotype. These novel HLA-E epitopes provide new implications for therapies targeting cells with abnormal HLA class I expression.

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Among patients treated with the anticonvulsive and psychotropic drug carbamazepine (CBZ), approximately 10% develop severe and life-threatening adverse drug reactions. These immunological conditions are resolved upon withdrawal of the medicament, suggesting that the drug does not manifest in the body in long term. The HLA allele B∗15:02 has been described to be a genomic biomarker for CBZ-mediated immune reactions. It is not well understood if the immune reactions are triggered by the original drug or by its metabolite carbamazepine-10,11-epoxide (EPX) and how the interaction between the drug and the distinct HLA molecule occurs. Genetically engineered human B-lymphoblastoid cells expressing soluble HLA-B∗15:02 molecules were treated with the drug or its metabolite. Functional pHLA complexes were purified; peptides were eluted and sequenced. Applying mass spectrometric analysis, CBZ and EPX were monitored by analyzing the heavy chain and peptide fractions separately for the presence of the drug. This method enabled the detection of the drug in a biological situation post-pHLA assembly. Both drugs were bound to the HLA-B∗15:02 heavy chain; however, solely EPX altered the peptide-binding motif of B∗15:02-restricted peptides. This observation could be explained through structural insight; EPX binds to the peptide-binding region and alters the biochemical features of the F pocket and thus the peptide motif. Understanding the nature of immunogenic interactions between CBZ and EPX with the HLA immune complex will guide towards effective and safe medications.

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