NetMHCstab – predicting stability of peptide–MHC‐I complexes; impacts for cytotoxic T lymphocyte epitope discovery

Major histocompatibility complex class I (MHC‐I) molecules play an essential role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTLs) allowing the immune system to scrutinize ongoing intracellular production of proteins. In the early 1990s, immunogenicity and stability of the peptide–MHC‐I (pMHC‐I) complex were shown to be correlated. At that time, measuring stability was cumbersome and time consuming and only small data sets were analysed. Here, we investigate this fairly unexplored area on a large scale compared with earlier studies. A recent small‐scale study demonstrated that pMHC‐I complex stability was a better correlate of CTL immunogenicity than peptide–MHC‐I affinity. We here extended this study and analysed a total of 5509 distinct peptide stability measurements covering 10 different HLA class I molecules. Artificial neural networks were used to construct stability predictors capable of predicting the half‐life of the pMHC‐I complex. These predictors were shown to predict T‐cell epitopes and MHC ligands from SYFPEITHI and IEDB to form significantly more stable MHC‐I complexes compared with affinity‐matched non‐epitopes. Combining the stability predictions with a state‐of‐the‐art affinity predictions NetMHCcons significantly improved the performance for identification of T‐cell epitopes and ligands. For the HLA alleles included in the study, we could identify distinct sub‐motifs that differentiate between stable and unstable peptide binders and demonstrate that anchor positions in the N‐terminal of the binding motif (primarily P2 and P3) play a critical role for the formation of stable pMHC‐I complexes. A webserver implementing the method is available at www.cbs.dtu.dk/services/NetMHCstab .     

Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen‐specific T cells

Fluorochrome‐conjugated peptide–major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen‐specific T cells. The most common multimers, streptavidin–biotin‐based ‘tetramers’, can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen‐specific T cells within a sample, an issue that is particularly problematic when staining tumour‐specific, autoimmune or MHC class II‐restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran‐based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR–pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co‐receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state‐of‐the‐art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.     

Going Pro to enhance T‐cell immunogenicity: Easy as π?

MHC class I molecules bind intracellular oligopeptides and present them on the cell surface for CD8⁺ T‐cell activation and recognition. Strong peptide/MHC class I (pMHC) interactions typically induce the best CD8⁺ T‐cell responses; however, many immunotherapeutic tumor‐specific peptides bind MHC with low affinity. To overcome this, immunologists can carefully alter peptides for enhanced MHC affinity but often at the cost of decreased T‐cell recognition. A new report published in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43:3051–3060] shows that the substitution of proline at the third residue (p3P) of a common tumor peptide increases pMHC affinity and complex stability while enhancing T‐cell receptor recognition. X‐ray crystallography indicates that stability is generated through newly introduced CH‐π bonding between p3P and a conserved residue (Y159) in the MHC heavy chain. This finding highlights a previously unappreciated role for CH‐π bonding in MHC peptide binding, and importantly, arms immunologists with a novel and possibly general approach for increasing pMHC stability without compromising T‐cell recognition.     

On integrity in immunity during ontogeny or how thymic regulatory T cells work

The Standard model of T cell recognition asserts that T cell receptor (TCR) specificities are positively and negatively selected during ontogeny in the thymus and that peripheral T cell repertoire has mild self‐major histocompatibility complex (MHC) reactivity, known as MHC restriction of foreign antigen. Thus, the TCR must bind both a restrictive molecule (MHC allele) and a peptide reclining in its groove (pMHC ligand) in order to transmit signal into a T cell. The Standard and Cohn's Tritope models suggest contradictory roles for complementarity‐determining regions (CDRs) of the TCRs. Here, I discuss both concepts and propose a different solution to ontogenetic mechanism for TCR‐MHC–conserved interaction. I suggest that double (CD4⁺CD8⁺)‐positive (DP) developing thymocytes compete with their αβTCRs for binding to self‐pMHC on cortical thymic epithelial cells (cTECs) that present a selected set of tissue‐restricted antigens. The competition between DPs involves TCR editing and secondary rearrangements, similar to germinal‐centre B cell somatic hypermutation. These processes would generate cells with higher TCR affinity for self‐pMHC, facilitating sufficiently long binding to cTECs to become thymic T regulatory cells (tTregs). Furthermore, CD4⁺ Foxp3⁺ tTregs can be generated by mTECs via Aire‐dependent and Aire‐independent pathways, and additionally on thymic bone marrow–derived APCs including thymic Aire‐expressing B cells. Thymic Tregs differ from the induced peripheral Tregs, which comprise the negative feedback loop to restrain immune responses. The implication of thymocytes’ competition for the highest binding to self‐pMHC is the co‐evolution of species‐specific αβTCR V regions with MHC alleles.     

A study of CDR3 loop dynamics reveals distinct mechanisms of peptide recognition by T‐cell receptors exhibiting different levels of cross‐reactivity

T‐cell receptors (TCRs) can productively interact with many different peptides bound within the MHC binding groove. This property varies with the level of cross‐reactivity of TCRs; some TCRs are particularly hyper cross‐reactive while others exhibit greater specificity. To elucidate the mechanism behind these differences, we studied five TCRs in complex with the same class II MHC (1A^b)‐peptide (3K), that are known to exhibit different levels of cross‐reactivity. Although these complexes have similar binding affinities, the interface areas between the TCR and the peptide–MHC (pMHC) differ significantly. We investigated static and dynamic structural features of the TCR–pMHC complexes and of TCRs in a free state, as well as the relationship between binding affinity and interface area. It was found that the TCRs known to exhibit lower levels of cross‐reactivity bound to pMHC using an induced‐fitting mechanism, forming large and tight interfaces rich in specific hydrogen bonds. In contrast, TCRs known to exhibit high levels of cross‐reactivity used a more rigid binding mechanism where non‐specific π‐interactions involving the bulky Trp residue in CDR3β dominated. As entropy loss upon binding in these highly degenerate and rigid TCRs is smaller than that in less degenerate TCRs, they can better tolerate changes in residues distal from the major contacts with MHC‐bound peptide. Hence, our dynamics study revealed that differences in the peptide recognition mechanisms by TCRs appear to correlate with the levels of T‐cell cross‐reactivity.     

Antigen‐loaded ER microsomes from APC induce potent immune responses against viral infection

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER‐enriched microsomal membranes (microsomes) isolated from DC contain more peptide‐receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide–MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide‐loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide‐pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide–MHC complexes armed microsomes from DC can be an important alternative to DC‐based vaccines for protection from viral infection.     

Tricks with tetramers: how to get the most from multimeric peptide–MHC

The development of fluorochrome‐conjugated peptide–major histocompatibility complex (pMHC) multimers in conjunction with continuing advances in flow cytometry has transformed the study of antigen‐specific T cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we bring together and discuss some of the ‘tricks’ that can be used to get the most out of pMHC multimers. These include: (1) simple procedures that can substantially enhance the staining intensity of cognate T cells with pMHC multimers; (2) the use of pMHC multimers to stain T cells with very‐low‐affinity T‐cell receptor (TCR)/pMHC interactions, such as those that typically predominate in tumour‐specific responses; and (3) the physical grading and clonotypic dissection of antigen‐specific T cells based on the affinity of their cognate TCR using mutant pMHC multimers in conjunction with new approaches to the molecular analysis of TCR gene expression. We also examine how soluble pMHC can be used to examine T‐cell activation, manipulate T‐cell responses and study allogeneic and superantigen interactions with TCRs. Finally, we discuss the problems that arise with pMHC class II (pMHCII) multimers because of the low affinity of TCR/pMHCII interactions and lack of ‘coreceptor help’.     

Weakly self‐reactive T‐cell clones can homeostatically expand when present at low numbers

T‐cell division is central to maintaining a stable T‐cell pool in adults. It also enables T‐cell expansion in neonates, and after depletion by chemotherapy, bone marrow transplantation, or infection. The same signals required for T‐cell survival in lymphoreplete settings, IL‐7 and T‐cell receptor (TCR) interactions with self‐peptide MHC (pMHC), induce division when T‐cell numbers are low. The strength of reactivity for self‐pMHC has been shown to correlate with the capacity of T cells to undergo lymphopenia‐induced proliferation (LIP), in that weakly self‐reactive T cells are unable to divide, implying that T‐cell reconstitution would significantly skew the TCR repertoire toward TCRs with greater self‐reactivity and thus compromise T‐cell diversity. Here, we show that while CD4⁺ T cells with low self‐pMHC reactivity experience more intense competition, they are able to divide when present at low enough cell numbers. Thus, at physiological precursor frequencies CD4⁺ T cells with low self‐pMHC reactivity are able to contribute to the reconstitution of the T‐cell pool.     

Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude

Cell-mediated immunity stems from the proliferation of naive T lymphocytes expressing T cell antigen receptors (TCRs) specific for foreign peptides bound to host major histocompatibility complex (MHC) molecules. Because of the tremendous diversity of the T cell repertoire, naive T cells specific for any one peptide:MHC complex (pMHC) are extremely rare. Thus, it is not known how many naive T cells of any given pMHC specificity exist in the body or how that number influences the immune response. By using soluble pMHC class II (pMHCII) tetramers and magnetic bead enrichment, we found that three different pMHCII-specific naive CD4(+) T cell populations vary in frequency from 20 to 200 cells per mouse. Moreover, naive population size predicted the size and TCR diversity of the primary CD4(+) T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides are stronger immunogens than others.     

The cellular environment regulates in situ kinetics of T‐cell receptor interaction with peptide major histocompatibility complex

T cells recognize antigens at the two‐dimensional (2D) interface with antigen‐presenting cells (APCs), which trigger T‐cell effector functions. T‐cell functional outcomes correlate with 2D kinetics of membrane‐embedded T‐cell receptors (TCRs) binding to surface‐tethered peptide‐major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR–pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T‐cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR–pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on‐rates but had no effect on 2D off‐rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on‐rate was insensitive to cellular perturbations and the force‐dependent off‐rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR–pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T‐cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.     

Is there a place and role for endocytic TCR signaling?

T‐lymphocyte activation relies on the cognate recognition by the TCR of the MHC‐associated peptide ligand (pMHC) presented at the surface of an antigen‐presenting cell (APC). This leads to the dynamic formation of a cognate contact between the T lymphocyte and the APC: the immune synapse (IS). Engagement of the TCR by the pMHC in the synaptic zone induces a cascade of signaling events leading to phosphorylation and dephosphorylation of proteins and lipids, which ultimately shapes the response of T lymphocytes. Although the engagement of the T‐cell receptor (TCR) takes place at the plasma membrane, the TCR/CD3 complexes and the signaling molecules involved in transduction of the TCR signal are also present in intracellular membrane pools. These pools, which are both endocytic and exocytic, have tentatively been characterized by several groups including ours. We will herein summarize what is known on the intracellular pools of TCR signaling components. We will discuss their origin and the mechanisms involved in their mobility at the IS. Finally, we will propose several hypotheses concerning the functional role(s) that these intracellular pools might play in T‐cell activation. We will also discuss the tools that could be used to test these hypotheses.     

T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR‐binding parameters

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self‐peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide–MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN‐γ production by T cells, and 1G4‐pMHC dissociation rates measured in soluble phase or on surface‐bound molecules, displayed six‐ to sevenfold variation among pMHCs. When T cells were dropped onto pMHC‐coated surfaces, rapid spreading occurred after a 2‐min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all‐or‐none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.     

All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigen-processing pathway

Summary: The end result of the antigen‐processing pathway is the display of peptide‐bound major histocompatibility complex I (pMHC I) molecules. The pMHC I molecules are expressed on the cell surface where they can be surveyed by CD8⁺ T cells for abnormal proteins. MHC I molecules present a large repertoire of peptides that fit perfectly in their binding grooves and represent the otherwise hidden intracellular contents. Many peptides originate as defective ribosomal products in the cytoplasm. In a stepwise manner, the antigen‐processing pathway generates and protects the proteolytic intermediates until they yield the final peptides that can fit the MHC I in the endoplasmic reticulum.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

A Public T cell Receptor Recognized by a Monoclonal Antibody Specific for the D‐J Junction of the β‐chain

It is becoming increasingly clear that T cell responses against many antigens are dominated by public α/β T cell receptors (TCRs) with restricted heterogeneity. Because expression of public TCRs may be related to resistance, or predisposition to diseases, it is relevant to measure their frequencies. Although staining with tetrameric peptide/major histocompatibility complex (pMHC) molecules gives information about specificity, it does not give information about the TCR composition of the individual T cells that stain. Moreover, next‐generation sequencing of TCR does not yield information on pairing of α‐ and β‐chains in single T cells. In an effort to overcome these limitations, we have here investigated the possibility of raising a monoclonal antibody (moAb) that recognizes a public TCR. As a model system, we have used T cells responding to the 91–101 CDR3 peptide of an Ig L‐chain (λ2³¹⁵), presented by the MHC class II molecule I‐E^d. The CD4⁺ T cell responses against this pMHC are dominated by a receptor composed of Vα3Jα1;Vβ6DβJβ1.1. Even the V(D)J junctions are to a large extent shared between T cell clones derived from different BALB/c mice. We here describe a murine moAb (AB10) of B10.D2 origin that recognizes this public TCR, while binding to peripheral T cells is negligible. Binding of the moAb is abrogated by introduction of two Gly residues in the D‐J junction of the CDR3 of the β‐chain. A model for the public TCR determinant is presented.     

pMHC 多聚体技术研究进展

抗原肽结合的主要组织相容性复合物(pMHC)通过形成多聚体可有效提高结合到位于T 细胞表面上的T 细胞受体(TCR)的亲合力。自从20 年前pMHC 四聚体首次被用于抗原特异性T 细胞检测以来,pMHC 四聚体已成为免疫分析中最重要的检测工具之一。近年来pMHC 多聚体在四聚体的基础上又取得了较大进展,更高价的pMHC 多聚体被研制出来以提高免疫检测的灵敏度;可逆化的pMHC 多聚体由于可以从T 细胞表面解离而避免了对T 细胞的功能损伤,也被发展用于抗原特异的T 细胞分离。pMHC 多聚体作为一类分子工具在抗原特异的T 细胞分析和免疫治疗中具有重要作用,对它的充分了解和有效运用能帮助我们更好地进行科学和临床应用。     

Degenerate recognition of T cell epitopes: impact of T cell receptor reserve and stability of peptide:MHC complexes

The concept of molecular mimicry suggests that microbial pathogens might activate antigen-specific T cells that then cross-react with endogenous antigens and result in the generation of autoimmunity. Here we discuss several under-represented factors impacting the ability of TCRs to recognize a wide spectrum of related peptide:MHC (pMHC) ligands. Two of these factors include the affinity of the peptide for MHC and the level of TCR expression. Thymocytes that recognize peptides with low affinity for MHC avoid negative selection, but mature T cells, by virtue of increased TCR expression, will proliferate in response to these same unstable pMHC complexes. While the expression of a reserve of antigen receptors expands the potential number of epitopes for which a T cell can cross-react, phosphatase activity provides a tuning mechanism to increase the threshold level of activation. Thus, degenerate recognition of T cell epitopes involves the stability of the peptide:MHC complex, the number of TCR expressed, and the level of phosphatase activity.     

Nature and nurture: T-cell receptor-dependent and T-cell receptor-independent differentiation cues in the selection of the memory T-cell pool

The initiation of a T‐cell response begins with the interaction of an individual T‐cell clone with its cognate antigen presented by MHC. Although the strength of the T‐cell receptor (TCR) –antigen–MHC (TCR‐pMHC) interaction plays an important and obvious role in the recruitment of T cells into the immune response, evidence in recent years has suggested that the strength of this initial interaction can influence various other aspects of the fate of an individual T‐cell clone and its daughter cells. In this review, we will describe differences in the way CD4⁺ and CD8⁺ T cells incorporate antigen‐driven differentiation and survival signals during the response to acute infection. Furthermore, we will discuss increasing evidence that the quality and/or quantity of the initial TCR‐pMHC interaction can drive the differentiation and long‐term survival of T helper type 1 memory populations.     

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.     

Improved methods for predicting peptide binding affinity to MHC class II molecules

Major histocompatibility complex class II (MHC‐II) molecules are expressed on the surface of professional antigen‐presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC‐II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T‐cell epitopes. We here present updated versions of two MHC–II–peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC–peptide binding affinity data obtained from the Immune Epitope Database covering HLA‐DR, HLA‐DQ, HLA‐DP and H‐2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2 .