Identification of a novel conserved signaling motif in CD200 receptor required for its inhibitory function.

The inhibitory signaling of CD200 receptor 1 (CD200R) has been attributed to its NPxY signaling motif. However, NPxY-motifs are present in multiple protein families and are mostly known to mediate protein trafficking between subcellular locations rather than signaling. Therefore, we investigated whether additional motifs specify the inhibitory function of CD200R. We performed phylogenetic analysis of the intracellular domain of CD200R in mammals, birds, bony fish, amphibians and reptiles. Indeed, the tyrosine of the NPxY-motif is fully conserved across species, in line with its central role in CD200R signaling. In contrast, P295 of the NPxY-motif is not conserved. Instead, a conserved stretch of negatively charged amino acids, EEDE279, and two conserved residues P285 and K292 in the flanking region prior to the NPxY-motif are required for CD200R mediated inhibition of p-Erk, p-Akt308, p-Akt473, p-rpS6 and LPS-induced IL-8 secretion. Altogether, we show that instead of the more common NPxY-motif, CD200R signaling can be assigned to a unique signaling motif in mammals defined by: EEDExxPYxxYxxKxNxxY.

Revealing factors determining immunodominant responses against dominant epitopes.

Upon recognition of peptide-MHC complexes by T cell receptors (TCR), the cognate T cells expand and differentiate into effector T cells to generate protective immunity. Despite the fact that any immune response generates a diverse set of TCR clones against a particular epitope, only a few clones are highly expanded in any immune response. Previous studies observed that the highest frequency clones usually control viral infections better than subdominant clones, but the reasons for this dominance among T cell clones are still unclear. Here, we used publicly available TCR amino acid sequences to study which factors determine whether a response becomes immunodominance (ID) per donor; we classified the largest T cell clone as the epitope-specific dominant clone and all the other clones as subdominant responses (SD). We observed a distinctively hydrophobic CDR3 in ID responses against a dominant epitope from influenza A virus, compared to the SD responses. The common V-J combinations were shared between ID and SD responses, suggesting that the biased V-J recombination events are restricted by epitope specificity; thus, the immunodominance is not directly determined by a bias combination of V and J genetic segments. Our findings reveal a close similarity of global sequence properties between dominant and subdominant clones of epitope-specific responses but detectable distinctive amino acid enrichments in ID. Taken together, we believe this first comparative study of immunodominant and subdominant TCR sequences can guide further studies to resolve factors determining the immunodominance of antiviral as well as tumor-specific T cell responses.

Exploratory Study of Predicted Indirectly ReCognizable HLA Epitopes in Mismatched Hematopoietic Cell Transplantations.

HLA-mismatches in hematopoietic stem-cell transplantation are associated with an impaired overall survival (OS). The aim of this study is to explore whether the Predicted Indirectly ReCognizable HLA-Epitopes (PIRCHE) algorithm can be used to identify HLA-mismatches that are related to an impaired transplant outcome. PIRCHE are computationally predicted peptides derived from the patient's mismatched-HLA molecules that can be presented by donor-patient shared HLA. We retrospectively scored PIRCHE numbers either presented on HLA class-I (PIRCHE-I) or class-II (PIRCHE-II) for a Dutch multicenter cohort of 103 patients who received a single HLA-mismatched (9/10) unrelated donor transplant in an early phase of their disease. These patients were divided into low and high PIRCHE-I and PIRCHE-II groups, based on their PIRCHE scores, and compared using multivariate statistical analysis methods. The high PIRCHE-II group had a significantly impaired OS compared to the low PIRCHE-II group and the 10/10 reference group (HR: 1.86, 95%-CI: 1.02-3.40; and HR: 2.65, 95%-CI: 1.53-4.60, respectively). Overall, PIRCHE-II seem to have a more prominent effect on OS than PIRCHE-I. This impaired OS is probably due to an increased risk for severe acute graft-vs.-host disease. These data suggest that high PIRCHE-II scores may be used to identify non-permissible HLA mismatches within single HLA-mismatched hematopoietic stem-cell transplantations.

Immune biomarkers for predicting response to adoptive cell transfer as cancer treatment.

Adoptive cell transfer (ACT) is a form of personalised immunotherapy which has shown promising results in metastasised cancer. For this treatment, autologous T lymphocytes are selected and stimulated in vitro before re-administration in large numbers. However, only a fraction of patients benefit from ACT, and it is not yet known what biomarkers can predict treatment outcome. In this review, we describe what tumour characteristics are associated with response to ACT. Based on the current knowledge, the best candidate biomarker for a good anti-tumour response seems to be a large number of neoantigens with a homogeneous distribution across the tumour in combination with sufficient MHC-I expression level. Additionally, it is necessary to be able to isolate a diverse population of T cells reactive to these neoantigens from tumour tissue or peripheral blood. Additional promising candidate biomarkers shared with other cancer immunotherapies are a large number of tumour-infiltrating cytotoxic and memory T cells, normal levels of glycolysis, and a pro-inflammatory cytokine profile within the tumour. Intense research in this field will hopefully result in identification of more biomarkers for cancers with low mutational load.

Specificity of inhibitory KIRs enables NK cells to detect changes in an altered peptide environment.

The activity of natural killer (NK) cells is tightly regulated by inhibitory and activating receptors. Inhibitory killer immunoglobulin-like receptors (iKIRs) survey the surface of target cells by monitoring the expression of human leukocyte antigen (HLA) class I. The binding of iKIRs has been shown to be sensitive to the peptides presented by HLA class I, implying that iKIRs have the ability to detect the changes in the repertoire of peptide-HLA class I complexes (pHLA), a process occurring during viral infection and in tumor cells. To study how the pHLA repertoire changes upon infection, and whether an iKIR is able to detect these changes, we study peptides eluted from cells prior and after infection with measles virus (MV). Remarkably, most changes in the repertoire of potential iKIR ligands are predicted to be caused by the altered expression of self-peptides. We show that an iKIR can detect these changes in the presented peptides only if it is sufficiently specific, e.g., if iKIRs can distinguish between different amino acids in the contact residues (e.g., position 7 and 8). Our analysis further indicates that one single iKIR per host is not sufficient to detect changes in the peptide repertoire, suggesting that a multigene family encoding for different iKIRs is required for successful peptide recognition.

The evolution of natural killer cell receptors.

Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

Can Selective MHC Downregulation Explain the Specificity and Genetic Diversity of NK Cell Receptors?

Natural killer (NK) cells express inhibiting receptors (iNKRs), which specifically bind MHC-I molecules on the surface of healthy cells. When the expression of MHC-I on the cell surface decreases, which might occur during certain viral infections and cancer, iNKRs lose inhibiting signals and the infected cells become target for NK cell activation (missing-self detection). Although the detection of MHC-I deficient cells can be achieved by conserved receptor-ligand interactions, several iNKRs are encoded by gene families with a remarkable genetic diversity, containing many haplotypes varying in gene content and allelic polymorphism. So far, the biological function of this expansion within the NKR cluster has remained poorly understood. Here, we investigate whether the evolution of diverse iNKRs genes can be driven by a specific viral immunoevasive mechanism: selective MHC downregulation. Several viruses, including EBV, CMV, and HIV, decrease the expression of MHC-I to escape from T cell responses. This downregulation does not always affect all MHC loci in the same way, as viruses target particular MHC molecules. To study the selection pressure of selective MHC downregulation on iNKRs, we have developed an agent-based model simulating an evolutionary scenario of hosts infected with herpes-like viruses, which are able to selectively downregulate the expression of MHC-I molecules on the cell surface. We show that iNKRs evolve specificity and, depending on the similarity of MHC alleles within each locus and the differences between the loci, they can specialize to a particular MHC-I locus. The easier it is to classify an MHC allele to its locus, the lower the required diversity of the NKRs. Thus, the diversification of the iNKR cluster depends on the locus specific MHC structure.

A Coevolutionary Arms Race between Hosts and Viruses Drives Polymorphism and Polygenicity of NK Cell Receptors.

Natural killer cell receptors (NKRs) monitor the expression of major histocompatibility class I (MHC-I) and stress molecules to detect unhealthy tissue, such as infected or tumor cells. The NKR gene family shows a remarkable genetic diversity, containing several genes encoding receptors with activating and inhibiting signaling, and varying in gene content and allelic polymorphism. The expansion of the NKR genes is species-specific, with different species evolving alternative expanded NKR genes, which encode structurally different proteins, yet perform comparable functions. So far, the biological function of this expansion within the NKR cluster has remained poorly understood. To study the evolution of NKRs, we have developed an agent-based model implementing a coevolutionary scenario between hosts and herpes-like viruses that are able to evade the immune response by downregulating the expression of MHC-I on the cell surface. We show that hosts evolve specific inhibitory NKRs, specialized to particular MHC-I alleles in the population. Viruses in our simulations readily evolve proteins mimicking the MHC molecules of their host, even in the absence of MHC-I downregulation. As a result, the NKR locus becomes polygenic and polymorphic, encoding both specific inhibiting and activating receptors to optimally protect the hosts from coevolving viruses.

Biomarkers in cancer immunotherapy.

Antibodies against T cell checkpoint molecules have started to revolutionize cancer treatment. Nevertheless, less than half of all patients respond to these immunotherapies. Recent work supports the potential value of biomarkers that predict therapy outcome and inspires the development of assay systems that interrogate other aspects of the cancer-immunity cycle.

In vitro expansion of antigen-specific CD8(+) T cells distorts the T-cell repertoire.

Short-term in vitro expansion of antigen-specific T cells is an appreciated assay for the analysis of small memory T-cell populations. However, how well short-term expanded T cells represent the direct ex vivo situation remains to be elucidated. In this study we compared the clonality of Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific CD8(+) T cells directly ex vivo and after in vitro stimulation with antigen. Our data show that the antigen-specific T cell repertoire significantly alters after in vitro culture. Clear shifts in clonotype hierarchy were observed, with the most dominant ex vivo clonotype decreasing after stimulation at the expense of several previously subdominant clonotypes. Notably, these alterations were more pronounced in polyclonal T-cell populations compared to mono- or oligoclonal repertoires. Furthermore, TCR diversity significantly increased after culture with antigen. These results suggest that the T-cell repertoire is highly subjective to variation after in vitro stimulation with antigen. Hence, although short-term expansion of T cells provides a simple and efficient tool to examine antigen-specific immune responses, caution is required if T-cell populations are expanded prior to detailed, clonotypic analyses or other repertoire-based investigations.

Properties of MHC class I presented peptides that enhance immunogenicity.

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4-6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.

Virus encoded MHC-like decoys diversify the inhibitory KIR repertoire.

Natural killer (NK) cells are circulating lymphocytes that play an important role in the control of viral infections and tumors. Their functions are regulated by several activating and inhibitory receptors. A subset of these receptors in human NK cells are the killer immunoglobulin-like receptors (KIRs), which interact with the highly polymorphic MHC class I molecules. One important function of NK cells is to detect cells that have down-regulated MHC expression (missing-self). Because MHC molecules have non polymorphic regions, their expression could have been monitored with a limited set of monomorphic receptors. Surprisingly, the KIR family has a remarkable genetic diversity, the function of which remains poorly understood. The mouse cytomegalovirus (MCMV) is able to evade NK cell responses by coding "decoy" molecules that mimic MHC class I. This interaction was suggested to have driven the evolution of novel NK cell receptors. Inspired by the MCMV system, we develop an agent-based model of a host population infected with viruses that are able to evolve MHC down-regulation and decoy molecules. Our simulations show that specific recognition of MHC class I molecules by inhibitory KIRs provides excellent protection against viruses evolving decoys, and that the diversity of inhibitory KIRs will subsequently evolve as a result of the required discrimination between host MHC molecules and decoy molecules.

CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αß TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

A comparative analysis of viral peptides presented by contemporary human and chimpanzee MHC class I molecules.

Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less polymorphic than human MHC (HLA) genes, which is surprising because chimpanzee is the older species of the two and is therefore expected to display more variation. To quantify the effect of the reduced polymorphism, we compared the peptide binding repertoire of human and chimpanzee MHC molecules. Using a peptide-MHC binding predictor and proteomes of >900 mammalian viruses, we show that, at the population level, the total peptide binding repertoire of Patr-A molecules is ~36% lower than that of their human counterparts, whereas the reduction of the peptide binding repertoire of the Patr-B locus is only 15%. In line with these results, different Patr-A molecules turn out to have largely overlapping peptide binding repertoires, whereas the Patr-B molecules are more distinct from each other. This difference is somewhat less apparent at the individual level, where we found that only 25% of the viruses are significantly better presented by "simulated" humans with heterozygous HLA-A and -B loci. Taken together, our results indicate that the Patr-B molecules recovered more after the selective sweep, whereas the Patr-A locus shows the most signs of the selective sweep with regard to its peptide binding repertoire.

HLA class I allele promiscuity revisited.

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (> 50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that "functional" HLA polymorphism on individual and population level is probably much lower than previously anticipated.

A comparative study of HLA binding affinity and ligand diversity: implications for generating immunodominant CD8+ T cell responses.

Conventional CD8(+) T cell responses against intracellular infectious agents are initiated upon recognition of pathogen-derived peptides presented at the cell surface of infected cells in the context of MHC class I molecules. Among the major MHC class I loci, HLA-B is the swiftest evolving and the most polymorphic locus. Additionally, responses restricted by HLA-B molecules tend to be dominant, and most associations with susceptibility or protection against infectious diseases have been assigned to HLA-B alleles. To assess whether the differences in responses mediated via two major HLA class I loci, HLA-B and HLA-A, may already begin at the Ag presentation level, we have analyzed the diversity and binding affinity of their peptide repertoire by making use of curated pathogen-derived epitope data retrieved from the Immune Epitope Database and Analysis Resource, as well as in silico predicted epitopes. In contrast to our expectations, HLA-B alleles were found to have a less diverse peptide repertoire, which points toward a more restricted binding motif, and the respective average peptide binding affinity was shown to be lower than that of HLA-A-restricted epitopes. This unexpected observation gives rise to new hypotheses concerning the mechanisms underlying immunodominance of CD8(+) T cell responses.

Amino acid similarity accounts for T cell cross-reactivity and for "holes" in the T cell repertoire.

BACKGROUND: Cytotoxic T cell (CTL) cross-reactivity is believed to play a pivotal role in generating immune responses but the extent and mechanisms of CTL cross-reactivity remain largely unknown. Several studies suggest that CTL clones can recognize highly diverse peptides, some sharing no obvious sequence identity. The emerging realization in the field is that T cell receptors (TcR) recognize multiple distinct ligands. PRINCIPAL FINDINGS: First, we analyzed peptide scans of the HIV epitope SLFNTVATL (SFL9) and found that TCR specificity is position dependent and that biochemically similar amino acid substitutions do not drastically affect recognition. Inspired by this, we developed a general model of TCR peptide recognition using amino acid similarity matrices and found that such a model was able to predict the cross-reactivity of a diverse set of CTL epitopes. With this model, we were able to demonstrate that seemingly distinct T cell epitopes, i.e., ones with low sequence identity, are in fact more biochemically similar than expected. Additionally, an analysis of HIV immunogenicity data with our model showed that CTLs have the tendency to respond mostly to peptides that do not resemble self-antigens. CONCLUSIONS: T cell cross-reactivity can thus, to an extent greater than earlier appreciated, be explained by amino acid similarity. The results presented in this paper will help resolving some of the long-lasting discussions in the field of T cell cross-reactivity.

An unanticipated lack of consensus cytotoxic T lymphocyte epitopes in HIV-1 databases: the contribution of prediction programs.

BACKGROUND: Most consensus HIV-1-specific cytotoxic T lymphocytes epitopes presented via intensively studied HLA molecules are thought to be known OBJECTIVE: To identify possible novel HIV-1 epitopes for HLA-B27 and HLA-B57; two HLA types which are abundantly studied because of their correlation with slow HIV disease progression. METHODS: HIV-1 consensus subtype B sequences were analysed using peptide prediction programs based on major histocompatibility complex binding, proteasomal cleavage and TAP (transporter associated with antigen processing) transport. Recognition of the novel identified epitopes by cytotoxic T lymphocytes was tested using interferon-gamma ELISpot assay. RESULTS: In total, 22 novel epitopes predicted to be presented by either HLA-B27 or HLA-B57 were selected. Of these, 86% elicited significant immune responses in HIV-1-infected individuals. CONCLUSIONS: These data show that numerous HIV-1 epitopes remain to be identified, and that prediction programs are powerful tools for this purpose.

Increased sequence diversity coverage improves detection of HIV-specific T cell responses.

The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss responses to regions where test set and infecting virus differ. In this study, we report the design of a peptide test set with significantly increased coverage of HIV sequence diversity by including alternative amino acids at variable positions during the peptide synthesis step. In an IFN-gamma ELISpot assay, these "toggled" peptides detected HIV-specific CD4(+) and CD8(+) T cell responses of significantly higher breadth and magnitude than matched consensus peptides. The observed increases were explained by a closer match of the toggled peptides to the autologous viral sequence. Toggled peptides therefore afford a cost-effective and significantly more complete view of the host immune response to HIV and are directly applicable to other variable pathogens.