Major Histocompatibility Complex-peptide Complexes Research Articles

Recent advances in cellular immunotherapy for lymphoid malignancies.

Cellular immunotherapy with chimeric antigen receptor (CAR) T-cells has revolutionized the treatment of lymphoid malignancies. This review addresses the need for CAR expression in our endogenous T-cells to kill tumor cells with a focus on the basic principles of T-cell receptor recognition of major histocompatibility complex-peptide complexes. We review the factors associated with CAR T-cell outcomes and recent efforts to employ CAR T-cells in earlier lines of therapy. We also discuss the value of bispecific T-cell engagers as off-the-shelf products with better toxicity profiles. Finally, natural killer cells are discussed as an important cellular immunotherapy platform with the potential to broaden immunotherapeutic applications beyond lymphoid malignancies.

Assays to Detect Cross-Dressing by Dendritic Cells In Vivo and In Vitro.

The presentation of peptides derived from exogenous antigens on major histocompatibility complex (MHC) class I molecules of antigen-presenting cells (APCs), termed cross-presentation, is crucial for the activation of cytotoxic T-lymphocytes during cell-mediated immune response. Typically, the APCs acquire exogenous antigens by (i) endocytosis of soluble antigens present in their external milieu, or (ii) through phagocytosis of dying/dead cancer cells or infected cells, followed by intracellular processing, before presentation by MHC I on the surface, or (iii) uptake of heat shock protein-peptide complexes generated in the antigen donor cells (3). In a fourth new mechanism, preformed peptide-MHC complexes can be directly transferred from the surface of antigen donor cells (i.e., cancer cells or infected cells) to that of APCs, without the need of further processing, in a process referred to as cross-dressing. Recently, the importance of cross-dressing in dendritic cell-mediated antitumor immunity and antiviral immunity has been demonstrated. Here, we describe a protocol to study cross-dressing of dendritic cells with tumor antigens.

Opening opportunities for Kd determination and screening of MHC peptide complexes

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength. As a complement to computational prediction tools, our method estimates binding strength of even low-affinity peptides to MHC class I complexes quickly and efficiently. It has huge potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

Evolutionary divergence of HLA class I genotype impacts efficacy of cancer immunotherapy.

Functional diversity of the highly polymorphic human leukocyte antigen class I (HLA-I) genes underlies successful immunologic control of both infectious disease and cancer. The divergent allele advantage hypothesis dictates that an HLA-I genotype with two alleles with sequences that are more divergent enables presentation of more diverse immunopeptidomes1-3. However, the effect of sequence divergence between HLA-I alleles-a quantifiable measure of HLA-I evolution-on the efficacy of immune checkpoint inhibitor (ICI) treatment for cancer remains unknown. In the present study the germline HLA-I evolutionary divergence (HED) of patients with cancer treated with ICIs was determined by quantifying the physiochemical sequence divergence between HLA-I alleles of each patient's genotype. HED was a strong determinant of survival after treatment with ICIs. Even among patients fully heterozygous at HLA-I, patients with an HED in the upper quartile respond better to ICIs than patients with a low HED. Furthermore, HED strongly impacts the diversity of tumor, viral and self-immunopeptidomes and intratumoral T cell receptor clonality. Similar to tumor mutation burden, HED is a fundamental metric of diversity at the major histocompatibility complex-peptide complex, which dictates ICI efficacy. The data link divergent HLA allele advantage to immunotherapy efficacy and unveil how ICI response relies on the evolved efficiency of HLA-mediated immunity.

Unbiased Identification of T-Cell Receptors Targeting Immunodominant Peptide-MHC Complexes for T-Cell Receptor Immunotherapy.

T-cell receptor (TCR) immunotherapy uses T cells engineered with new TCRs to enable detection and killing of cancer cells. Efficacy of TCR immunotherapy depends on targeting antigenic peptides that are efficiently presented by the best-suited major histocompatibility complex (MHC) molecules of cancer cells. However, efficient strategies are lacking to easily identify TCRs recognizing immunodominant peptide-MHC (pMHC) combinations utilizing any of the six possible MHC class I alleles of a cancer cell. We generated an MHC cell library and developed a platform approach to detect, isolate, and re-express TCRs specific for immunodominant pMHCs. The platform approach was applied to identify a human papillomavirus (HPV16) oncogene E5-specific TCR, recognizing a novel, naturally processed pMHC (HLA-B*15:01) and a cytomegalovirus-specific TCR targeting an immunodominant pMHC (HLA-B*07:02). The platform provides a useful tool to isolate in an unbiased manner TCRs specific for novel and immunodominant pMHC targets for use in TCR immunotherapy.

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry

Major histocompatibility complex linked databases and prediction tools for designing vaccines

Presently, the major histocompatibility complex (MHC) is receiving considerable interest owing to its remarkable role in antigen presentation and vaccine design. The specific databases and prediction approaches related to MHC sequences, structures and binding/nonbinding peptides have been aggressively developed in the past two decades with their own benchmarks and standards. Before using these databases and prediction tools, it is important to analyze why and how the tools are constructed along with their strengths and limitations. The current review presents insights into web-based immunological bioinformatics resources that include searchable databases of MHC sequences, epitopes and prediction tools that are linked to MHC based vaccine design, including population coverage analysis. In T cell epitope forecasts, MHC class I binding predictions are very accurate for most of the identified MHC alleles. However, these predictions could be further improved by integrating proteasome cleavage (in conjugation with transporter associated with antigen processing (TAP) binding) prediction, as well as T cell receptor binding prediction. On the other hand, MHC class II restricted epitope predictions display relatively low accuracy compared to MHC class I. To date, pan-specific tools have been developed, which not only deliver significantly improved predictions in terms of accuracy, but also in terms of the coverage of MHC alleles and supertypes. In addition, structural modeling and simulation systems for peptide–MHC complexes enable the molecular-level investigation of immune processes. Finally, epitope prediction tools, and their assessments and guidelines, have been presented to immunologist for the design of novel vaccine and diagnostics.

B cell modulation in transplantation.

B cells are cells of the lymphoid lineage characterized by the surface expression of antibody/immunoglobulin (Ig) as the B cell receptor (BCR). B cells are the precursors of antibody-forming plasma cells. It has recently been appreciated that subpopulations of B cells may also act as immunoregulatory cells. These ‘regulatory’ B cells were first described in the mouse and subsequently in humans 1,2, and mediate immunoregulation via the production of interleukin (IL)-10 and IL-35 3. B cells bind antigen via their BCR, and the outcome of this interaction depends upon the affinity of the BCR for the antigen and the environmental context. For example, B cell activation can be facilitated by the presence of complement components (C3b and C3d binding to CD21/CR2). B cells may also receive proactivation and survival signals via cytokines, including BAFF (B cell-activating factor belonging to the tumour necrosis factor family, also known as BLyS). Over-expression of BAFF is sufficient to drive abnormal B cell survival, hypergammaglobulinaemia and a lupus-like autoimmune disease in mice independent of T cell help 4. If the BCR aggregation is sufficient to induce signalling, the B cell internalizes bound antigen, processes it and presents in the context of major histocompatibility complex (MHC) class II. At the border between the B cell follicle and T cell zone of secondary lymphoid organs, the B cell MHC–peptide complex may be recognized by a cognate T cell. Following this T–B interaction, B cells return to the follicle to form the germinal centre, where they undergo somatic hypermutation and class-switch recombination. Here, B cells with a higher affinity for antigen are positively selected and differentiate into either memory B cells or plasma cells. A subset of CD4 T cells located within B cell follicles and characterized by expression of the transcription repressor Bcl-6 [known as T follicular helper (Tfh) cells] are essential for the development of germinal centre B cells 5. A small proportion of plasma cells arising from the germinal centre become established as long-lived plasma cells in the bone marrow. Given the potent and widespread effects of B cell activation, it is critical that there are also stringent control measures to prevent inappropriate B cell responses. To this end, the B cell expresses a number of inhibitory receptors, for example FcγRIIB, CD22, CD72 and PIR-B 6. In recent years, there has been increasing interest in how B cells, plasma cells and their associated antibody respond to allografts 7. Sensitized patients with preformed human leucocyte antigen (HLA) antibodies have an increased risk of acute and chronic antibody-mediated rejection (AMR), which significantly impacts allograft longevity 8. Furthermore, it is now well established that the appearance of de-novo donor-specific antibodies (DSAs) is associated with AMR and chronic allograft attrition 9. Outside their remit of antibody production, there is an appreciation that B cells may play a role in acute cellular rejection, also known as T cell-mediated rejection (TCMR). In contrast to these negative effects of B cells and antibody on the allograft, there is a growing body of evidence that B cells may be beneficial for long-term graft survival; a number of studies have shown a B cell transcriptomic signature in tolerant transplant recipients 10,11 and an up-regulation of B cell biomarkers in rejection-free transplant recipients 12 that may be due to the effects of ‘regulatory’ B cells. Possible strategies to target B cells in transplantation include: (i) B cell depletion; (ii) modulation of B cell activation; (iii) increase B cell inhibition; and (iv) enhancing the generation of regulatory B cells.

Cell:cell interactions in the immune system

Video podcast available Go to www.immunologicalreviews.com to watch an interview with Guest Editor Facundo Batista. The cells of the immune system are continually on the move! Naive T cells in a human collectively crawl over 10 000 miles a day in search of antigens (1). Each dendritic cell (DC) touches 5000 T cells an hour to scan repertoires over a million deep (2, 3). The adaptive immune system functions through a vast network of provisional cell–cell interactions that are continuously changing. When this frenetic scanning finds a fit between T-cell antigen receptor (TCR) and DC antigen, the interaction can be prolonged with a pronounced deceleration of the T cell and finally a long-lived interaction that has been described as an immunological synapse (2, 4). In vitro studies on the immunological synapse suggest a profound supramolecular organization that serves focused trans-synaptic and juxtracrine information transfer between cells (5–8). Immunological synapses are not restricted to T cells but are also formed by natural killer (NK) cells, B cells, granulocytes, and phagocytes using related immunotyrosine-based activation motif-containing receptors (9, 10). These interactions are profoundly influenced by the three-dimensional (3D) tissue environment (11). This volume of Immunological Reviews takes a broader look at the cell–cell interactions in the immune system from the molecular organization of antigen receptors during surveillance to the organization of stromal cell networks in lymph nodes, bone marrow, and the lung (Fig. 1). The organization of the plasma membrane is a fundamental issue in biology that sets the stage for cell–cell interaction and communication. The classical fluid mosaic model suggested membrane proteins diffusing in a lipid sea with potential for interactions with cytoplasmic and extra-cytoplasmic binding partners that account for regulation and processes like adhesion and receptor capping. Observations on lipid phases in model systems with disordered, liquidordered, and gel phases in order of increasing viscosity suggested that such phases could also exist in biological membranes on some scale (12). Biophysical observations of liquid-ordered domains enriched in various lipid-modified proteins have led to models in which lipids have a role in providing a higher level of organization (13). Most recently, evidence for protein-driven domains, referred to as Immunological Reviews 2013 Vol. 251: 7–12 Printed in Singapore. All rights reserved

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein-peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein-peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody-peptide interaction characteristics, by combining large-scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide-binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low-affinity antibody-peptide interactions. The molecular mechanism for the degenerate peptide-binding specificity appeared to be executed through the use of 2-3 semi-conserved anchor residues in the C-terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex-peptide complexes. In the long-term, this knowledge will be instrumental for advancing our fundamental understanding of protein-peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide-binding proteins in general, in various biotechnical and medical applications.

Constitutive and Inflammatory Immunopeptidome of Pancreatic β-Cells

Type 1 diabetes is characterized by the autoimmune destruction of pancreatic β-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 β-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography–tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant Kd-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by β-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206–214 revealed low-level presentation by Kd (∼25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed Kd-restricted peptide Janus kinase-1355–363 (∼15,000 copies/cell). We have successfully sequenced peptides from NIT-1 β-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.

Autoantibodies against the signal peptide domain of MUC1 in patients with multiple myeloma: Implications for disease diagnosis and prognosis

Naturally generated autoantibodies to tumor-associated antigens such as MUC1 can assist in cancer diagnosis and prognosis. While previous studies have concentrated on the tandem repeat array domain of MUC1, here we focused on MUC1's signal peptide domain. We used ELISA assays with MUC1-specific epitopes and antibodies to quantify soluble MUC1 antigen and anti-MUC1 autoantibodies against the tandem repeat array and signal peptide domains in 15 naïve donors and 27 multiple myeloma cancer patients. We showed a significant increase in up to 24-fold (P<0.004) only in the levels of anti-MUC1 signal peptide autoantibodies in the sera of multiple myeloma patients vs. naïve donors. This increase stemmed chiefly from the preferred immunogenicity of the signal peptide. Moreover, a significant positive correlation (R(2)=0.5361, P<0.048, Pearson correlation) was shown between the levels of soluble MUC1 and anti-MUC1 signal peptide autoantibodies in multiple myeloma patients with progressive disease while under therapy. This is an initial report on the existence of autoantibodies to a signal peptide domain in general and to the MUC1 signal peptide domain in particular in cancer patients. The autoantibodies had MUC1 rather than signal peptide specificity. The specific nature of the antigen leading to generation of these autoantibodies is still unclear because it is unlikely that the target antigen is a major histo-compatibility complex-peptide complex and we could not trace soluble MUC1 signal peptide fragments in naïve donors and multiple myeloma patients. Further validation of these findings may improve diagnostic and prognostic capabilities for MUC1-positive multiple myeloma patients and potentially, patients with other MUC1-positive cancers, as well.

T-cell-receptor-like antibodies – generation, function and applications

Tumour and virus-infected cells are recognised by CD8+ cytotoxic T cells that, in response, are activated to eliminate these cells. In order to be activated, the clonotypic T-cell receptor (TCR) needs to encounter a specific peptide antigen presented by the membrane surface major histocompatibility complex (MHC) molecule. Cells that have undergone malignant transformation or viral infection present peptides derived from tumour-associated antigens or viral proteins on their MHC class I molecules. Therefore, disease-specific MHC-peptide complexes are desirable targets for immunotherapeutic approaches. One such approach transforms the unique fine specificity but low intrinsic affinity of TCRs to MHC-peptide complexes into high-affinity soluble antibody molecules endowed with a TCR-like specificity towards tumour or viral epitopes. These antibodies, termed TCR-like antibodies, are being developed as a new class of immunotherapeutics that can target tumour and virus-infected cells and mediate their specific killing. In addition to their therapeutic capabilities, TCR-like antibodies are being developed as diagnostic reagents for cancer and infectious diseases, and serve as valuable research tools for studying MHC class I antigen presentation.

Dynamical Characterization of Two Differentially Disease Associated MHC Class I Proteins in Complex with Viral and Self-Peptides

Major histocompatibility complex (MHC) class I proteins are expressed on the cell surface where they present foreign and self-peptides to effector cells of the immune system. While an understanding of the structural prerequisites for antigen presentation has already been achieved, insight into subtype- or peptide-dependent dynamical characteristics of a peptide–MHC antigen is so far largely obscure. We approached this problem by employing 400-ns molecular dynamics simulations with two human MHC class I subtypes as model systems: the ankylosing spondylitis-associated HLA-B∗27:05 and the non-ankylosing spondylitis-associated HLA-B∗27:09. Both proteins differ only by a micropolymorphism at the floor of the peptide binding groove (Asp116His). A viral (pLMP2) and three self-peptides (pVIPR, pGR, and TIS) were evaluated. The stability of the binding grooves was found to be both subtype dependent and peptide dependent. A detachment from the C- and/or N-terminal pockets was observed for all peptides except TIS, resulting in a stabilization of the α1-helix in both TIS-displaying subtypes. Estimates of the entropy associated with the bound peptides showed an increased entropy for pLMP2 presented by B∗27:05 as compared to B∗27:09, in contrast to the self-peptides. Additionally, the flexibility of the α1-helix that is probably important for receptor binding to the B27:peptide epitope is significantly enhanced for B∗27:05. These in silico results show that the dynamic properties of peptide–MHC complexes are affected both by the bound peptide and by micropolymorphisms of the heavy chain. Our findings suggest a role for the conformational flexibility of MHC class I molecules in the context of recognition by receptors on effector cells.

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.

Naturally occurring CD4 + T-cell epitope variants act as altered peptide ligands leading to impaired helper T-cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3 358-375 of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3 358-375 peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3 358-375 stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3 358-375. In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide–MHC complexes were able to specifically bind to NS3 358-375 T-cell clones and that both the variant and NS3 358-375 tetramers were able to bind to the same CD4 + T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope.

Human leukocyte antigens A*3001 and A*3002 show distinct peptide-binding patterns of the Mycobacterium tuberculosis protein TB10.4: consequences for immune recognition.

High-tuberculosis (TB)-burden countries are located in sub-Saharan Africa. We examined the frequency of human leukocyte antigen (HLA) alleles, followed by recombinant expression of the most frequent HLA-A alleles, i.e., HLA-A*3001 and HLA-A*3002, to study differences in mycobacterial peptide presentation and CD8(+) T-cell recognition. We screened a peptide library (9-mer peptides with an 8-amino-acid overlap) for binding, affinity, and off-rate of the Mycobacterium tuberculosis-associated antigen TB10.4 and identified only three TB10.4 peptides with considerable binding to HLA-A*3001. In contrast, 22 peptides bound to HLA-A*3002. This reflects a marked difference in the binding preference between the two alleles, with A*3002 tolerating a more promiscuous peptide-binding pattern and A*3001 accommodating only a very selective peptide repertoire. Subsequent analysis of the affinity and off-rate of the binding peptides revealed a strong affinity (8 nM to 7 μM) and moderate off-rate (20 min to 3 h) for both alleles. Construction of HLA-A*3001 and HLA-A*3002 tetramers containing selected binding peptides from TB10.4, including a peptide which was shared among both alleles, QIMYNYPAM (TB10.4(3-11)), allowed us to enumerate epitope-specific T cells in HLA-A*3001- and HLA-A*3002-typed patients with active TB. HLA-A*3001 and HLA-A*3002 major histocompatibility complex-peptide complexes were recognized in individuals with active TB, irrespective of their homozygous HLA-A*3001 or HLA-A*3002 genetic background. The antigen-specific T cells exhibited the CD45RA(+) CCR7(+) precursor phenotype and the interleukin-7 receptor (CD127), which were different from the phenotype and receptor exhibited by the parental CD8(+) T-cell population.

Costly major histocompatibility complex signals produced only by reproductively active males, but not females, must be validated by a ‘maleness signal’ in three-spined sticklebacks

Olfactory information about individual major histocompatibility complex (MHC) immune genotypes is important for mate choice in several species. For example, during the mate choice decisions of three-spined sticklebacks, females assess males on the basis of odour cues that convey information about their MHC diversity. Here, we show that an additional 'maleness' signal is needed to validate the MHC signal. Furthermore, using interaction between natural odour of sticklebacks and synthetic MHC-ligand peptides, we show that MHC signals are conditional on the reproductive state in males. By contrast, we find that gravid females do not produce such signals. Since MHC olfactory signals relevant to mate choice decisions are conditional upon gender and reproductive state, we suggest that their manufacture is likely to be costly to senders, and therefore, potentially conditional on the health/parasitization status of the sender. We hypothesize that shedding of peptide-MHC complexes compromises immune function, selecting against unconditional use of these signals.

Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner

TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide-MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax(11-19)] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a 'polar hydrophobicity' mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

Cloning and expression of three peptide-linked β2-microglobulin molecules in Escherichia coli with an isocaudamer technique

The application of the peptide-linked beta2-microglobulin (beta2m) strategy is limited in some cases due to the incompatibility between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted to overcome this restriction. Three peptide-linked beta2m genes, HBc(18-27)-hbeta2m gene, OVA(257-264)-mbeta2m gene and HER2/neu(369-377)-mbeta2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked beta2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes.