Structural basis for ineffective T‐cell responses to MHC anchor residue‐improved “heteroclitic” peptides

MHC anchor residue‐modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild‐type peptide. The best‐studied system to date is the decamer MART‐1/Melan‐A_26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)‐A*0201 anchoring. The resulting EL AGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the EL AGIGILTV peptide can fail to recognize the natural tumor‐expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA‐A*0201‐EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA‐A*0201‐EL AGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced‐fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.     

T‐cell receptors: Tugging on the anchor for a tighter hold on the tumor‐associated peptide

Although it has been shown that human tumor‐associated, HLA anchor residue modified “heteroclitic” peptides may induce stronger immune responses than wild‐type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor‐expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584–591] highlight a novel twist on T‐cell receptor (TCR) recognition of HLA–peptide complexes. Tumor‐associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T‐cell cross‐reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan‐A/MART‐1_26‐35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8⁺ T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA‐A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide “lifts” its amino‐terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue “optimized” heteroclitic peptide, which cannot be “pulled” from the HLA groove.     

Real time detection of peptide-MHC dissociation reveals that improvement of primary MHC-binding residues can have a minimal, or no, effect on stability

The majority of known major histocompatibility complex class I (MHCI)-associated tumor-derived peptide antigens do not contain an optimal motif for MHCI binding. As a result, anchor residue-modified ‘heteroclitic’ peptides have been widely used in therapeutic cancer vaccination trials in order to enhance immune responsiveness. In general, the improved stability of these heteroclitic complexes has been inferred from their improved immunogenicity but has not been formally assessed. Here, we investigated the binding of 4 HLA A*0201-restricted tumor-derived peptides and their commonly used heteroclitic variants. We utilized a cell surface binding assay and a novel robust method for testing the durability of soluble recombinant pMHCI in real time by surface plasmon resonance. Surprisingly, we show that heteroclitic peptides designed with optimal MHC binding motifs do not always form pMHCs that are substantially more stable than their wildtype progenitors. These findings, combined with our recent discovery that TCRs can distinguish between wildtype peptides and those altered at a primary buried MHC anchor residue, suggest that altered TCR binding may account for a large part of the increased immune response that can be generated by anchor residue-modified ligands. Our results further highlight the fact that heteroclitic peptide-based immune interventions require careful evaluation to ensure that wildtype antigen specificity is maintained in vivo.     

Structural and biophysical determinants of αβ T-cell antigen recognition

The molecular rules that govern MHC restriction, and allow T-cells to differentiate between peptides derived from healthy cells and those from diseased cells, remain poorly understood. Here we provide an overview of the structural constraints that enable the T-cell receptor (TCR) to discriminate between self and non-self peptides, and summarize studies that have attempted to correlate the biophysical parameters of TCR/peptide-major histocompatibility complex (pMHC) binding with T-cell activation. We further review how the antigenic origin of peptide epitopes affects TCR binding parameters and the 'quality' of a T-cell response. Understanding the principles that govern pMHC recognition by T-cells will unlock pathways to the rational development of immunotherapeutic approaches for the treatment of infectious disease, cancer and autoimmunity.     

The molecular determinants of CD8 co-receptor function

CD8⁺ T cells respond to signals mediated through a specific interaction between the T‐cell receptor (TCR) and a composite antigen in the form of an epitopic peptide bound between the polymorphic α1 and α2 helices of an MHC class I (MHCI) molecule. The CD8 glycoprotein ‘co‐receives’ antigen by binding to an invariant region of the MHCI molecule and can enhance ligand recognition by up to 1 million‐fold. In recent years, a number of structural and biophysical investigations have shed light on the role of the CD8 co‐receptor during T‐cell antigen recognition. Here, we provide a collated resource for these data, and discuss how the structural and biophysical parameters governing CD8 co‐receptor function further our understanding of T‐cell cross‐reactivity and the productive engagement of low‐affinity antigenic ligands.     

Evolution and immunity

This report describes a meeting organized by Ken Smith and Jim Kaufman, entitled Evolution and Immunity, which took place at the University of Cambridge on 24 September 2009 to honour the anniversaries of the birth of Darwin and the first publication of The Origin of Species. Ten internationally‐known speakers described the effects of evolution on immunity, ranging in timescales from the deep‐time evolution of adaptive immune systems in vertebrates and invertebrates to the evolution of pathogens and lymphocytes within a single individual. The final talk explored the application of phylogenetic analysis to non‐biological systems.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Measuring the T‐cell down‐regulation of TCR‐zeta,ZAP‐70 and CD28 in arthritis patients: An old tool for new biomarkers

Low T‐cell receptor (TCR)/CD28 signaling lymphocytes are expanded in arthritis. We asked whether the down‐expression of TCR‐related molecules correlates with specific arthritis characteristics and if it has clinical implications. TCR‐ZETA, ZAP‐70 and CD28 expression was measured by flow cytometry in synovial fluid (SF) and peripheral blood (PB)‐derived T cells. In PB, ZETA‐downregulation in CD4⁺CD28⁺ and consequent CD4⁺CD28lowZETAlow cell expansion correlate with CRP elevation, leukocyte recruitment into SF and, primarily, disease activity (DAS). In some patients, ZETA‐downregulation extends to CD8⁺CD28null and/or CD8⁺CD28⁺ cells, and this correlates with enhanced leukocyte recruitment, multiple joint involvement, and disability index (HAQ). ZETA‐downregulation in CD4⁺CD28⁺ may also lead to CD4⁺CD28⁺ZETAnull cell expansion, which strongly correlates with HAQ. In SF, ZETA‐downregulation in CD8⁺CD28null and consequent CD8⁺CD28nullZETAlow/null cell expansion correlate with CRP elevation and neutrophilic influx into SF, whereas ZAP‐downregulation in CD8⁺CD28⁺ and consequent CD8⁺CD28lowZAPlow cell expansion strongly correlate with HAQ and DAS. ZETA‐downregulation is preponderant in SF of seronegative arthritides, with seronegative rheumatoid arthritis showing significant down‐regulation in CD8⁺CD28null, and non‐rheumatoid arthritides showing significant down‐regulation in CD4⁺CD28⁺. Altogether, we identified new molecular and cellular biomarkers of arthritis‐related T‐cell inflammation, useful for assessing arthritis activity, predicting polyarticular progression and functional impairment, characterizing seronegative arthritides, and possibly tailoring immunotherapies.     

Clonotype‐specific avidity influences the dynamics and hierarchy of virus‐specific regulatory and effector CD4+ T‐cell responses

A key component of immunity against viruses, CD4⁺ T cells expand and differentiate into functional subsets upon primary infection, where effector (Teff) cells facilitate infection control and regulatory (Treg) cells mitigate immunopathology. After secondary infection, Teff cells mount a robust response from the memory pool. Here, we show that Treg‐cell responses are diminished upon secondary infection, and Treg‐cell response dynamics are associated more with T‐cell receptors (TCRs) repertoire and avidity than with epitope specificity. In the murine model, the IA^bM₂₀₉ epitope of respiratory syncytial virus is recognized by both CD4⁺ Treg and Teff cells, while the IA^bM2₂₆ epitope is recognized almost exclusively by CD4⁺ Teff cells expressing high avidity TCR Vβ8.1/8.2 and dominating the CD4⁺ T‐cell response during primary and secondary infections. IA^bM₂₀₉‐Teff cells express relatively low avidity TCRs during early primary infection, but high avidity TCR Vβ7‐expressing IA^bM₂₀₉‐Teff cells emerge during the late phase, and become dominant after secondary infection. The emerging high avidity IA^bM₂₀₉‐Teff cells outcompete IA^bM₂₀₉‐Treg cells that share the same epitope, but have low avidity and are restricted to TCR Vβ2 and Vβ6 subpopulations. These data indicate that MHC‐peptide‐TCR interactions can produce different kinetic and functional profiles in CD4⁺ T‐cell populations even when responding to the same epitope.     

Prevention and treatment of experimental autoimmune encephalomyelitis with clonotypic CDR3 peptides: CD4+ FoxP3+ T‐regulatory cells suppress interleukin‐2‐dependent expansion of myelin basic protein‐specific T cells

T‐cell receptor (TCR)‐derived peptides are recognized by the immune system and are capable of modulating autoimmune responses. Using the myelin basic protein (MBP) TCR 1501 transgenic mouse model, we demonstrated that TCR CDR3 peptides from the transgenic TCR can provide a protective effect when therapy is initiated before the induction of experimental autoimmune encephalomyelitis (EAE). More importantly, TCR CDR3 peptide therapy can ameliorate the disease when administered after EAE onset. Concurrent with the therapeutic effects, we observed reduced T‐cell proliferation and reduced interleukin‐2 (IL‐2) levels in response to stimulation with MBP‐85‐99 peptide in splenocyte cultures from mice receiving TCR CDR3 peptides compared with that of control mice. Moreover, we found that Foxp3⁺ CD4 T cells from mice protected with TCR CDR3 peptide are preferentially expanded in the presence of IL‐2. This is supportive of a proposed mechanism where Foxp3⁺ T‐regulatory cells induced by therapy with MBP‐85‐99 TCR CDR3 peptides limit expansion and the encephalitogenic activity of MBP‐85‐99‐specific T cells by regulating the levels of secreted IL‐2.     

PD‐L1 expression on tumor or stromal cells of nodal cytotoxic T‐cell lymphoma: A clinicopathological study of 50 cases

Inhibitors of programmed cell‐death 1 (PD‐1) and programmed cell‐death ligand 1 (PD‐L1) have revolutionized cancer therapy. Nodal cytotoxic T‐cell lymphoma (CTL) is characterized by a poorer prognosis compared to nodal non‐CTLs. Here we investigated PD‐L1 expression in 50 nodal CTL patients, with and without EBV association (25 of each). We identified seven patients (14%) with neoplastic PD‐L1 (nPD‐L1) expression on tumor cells, including three males and four females, with a median age of 66 years. One of the seven cases was TCRαβ type, three were TCRγδ type and three were TCR‐silent type. Six of the seven cases exhibited a lethal clinical course despite multi‐agent chemotherapy, of whom four patients died within one year of diagnosis. Morphological findings were uniform, with six cases showing centroblastoid appearance. Among nPD‐L1⁺ cases, two of three examined had structural variations of PD‐L1 disrupting 3′‐UTR region. Notably, all of the TCRγδ‐type nodal CTL cases showed nPD‐L1 or miPD‐L1 positivity (3 and 10 cases, respectively). TCRγδ‐type cases comprised 42% of nPD‐L1⁺ cases (P = 0.043 vs. PD‐L1^?), and 35% of miPD‐L1⁺ cases (P = 0.037 vs. PD‐L1^?). The results indicate that PD‐L1⁺ nodal CTL cases, especially of the TCRγδ type, are potential candidates for anti‐PD‐1/PD‐L1 therapies.     

Synthetic Mimics of Antimicrobial Peptides (SMAMPs) in Layer‐by‐Layer Architectures: Possibilities and Limitations

Polymer‐based synthetic mimics of antimicrobial peptides (SMAMPs) show promising antimicrobial activity in solution and as surface‐attached networks. In this paper, their potential as active ingredients in layer‐by‐layer (LbL) assemblies is evaluated. These consist of the weak, anionic polyelectrolyte poly(acrylic acid), and either the hydrophobic butyl SMAMP or the hydrophilic diamine SMAMP (both of which are cationic, weak polyelectrolytes). In situ surface plasmon resonance spectroscopy is used to optimize the LbL assembly conditions. An “overshooting” is observed when depositing the SMAMP layer. Zeta potential measurements show that the layer charge inversion is reduced at each build‐up step due to layer interpenetration. Thus, the positive charge of LbL assemblies with SMAMPs as the top layer is low; a significant part is consumed to maintain layer stability. This leads to reduced antimicrobial activity. Fine‐tuning of the assembly and post‐treatment conditions leads to SMAMP‐PAA LbL systems with optimized antimicrobial activity and stability.

     

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.     

The regulatory role of interferon‐γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin‐induced pulmonary fibrosis

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild‐type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)‐γ and interleukin (IL)‐17A. Histological and biochemical analyses showed that bleomycin‐induced IP was more severe in T cell receptor (TCR‐δ‐deficient (TCRδ^–/–) mice than WT mice. In TCRδ^–/– mice, pulmonary IL‐17A⁺CD4⁺ Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ^–/– mice infused with γδT cells from WT mice, the number of pulmonary IL‐17A⁺ CD4⁺ T cells was lower than in TCRδ^–/– mice. The examination of IL‐17A^–/– TCRδ^–/– mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL‐17A⁺CD4⁺ T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4⁺ cells isolated from TCRδ^–/– mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN‐γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN‐γ‐producing γδT cells through the suppression of pulmonary IL‐17A⁺CD4⁺ T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin‐induced IP mouse model via the suppression of IL‐17A production.     

Synthesis and Hole‐Transporting Property of a Novel Poly(N‐vinylcarbazole) Copolymer with Metal‐Coordination Sites

Summary: Hole transporting poly(N‐vinylcarbazole) copolymers with phenylazomethine dendron units acting as metal ligation sites were synthesized. These polymers possess both hole‐transport and metal‐collecting units with simple σ‐bond linkages. Complexation in the phenylazomethine dendron unit within these copolymers by SnCl₂ has been successfully observed by the change in the UV‐vis spectra. The complexation changes the HOMO/LUMO energy gap that results in a spectral red‐shift. Using copolymers as a hole‐transport layer, only complexation with metal ions leads to an enhanced maximum luminescence. Such a complexation results in a high electroluminescence efficiency because the p‐type‐doped structure acts as the hole‐transport layer.

Copolymerization for the preparation of DPAGn(x)‐Cbz(y).     

Identification of leucocyte surface protein interactions by high‐throughput screening with multivalent reagents

We describe a high-throughput screening system to detect interactions between leucocyte surface proteins, taking into account that these interactions are usually of very low affinity. The method involves producing the extracellular regions of leucocyte proteins with tags so that they can be bound to nanoparticles to provide an avid reagent to screen over an array of 36 similar proteins immobilized using the Proteon™ XPR36 with detection by surface plasmon resonance. The system was tested using established interactions that could be detected without spurious binding. The ability to detect new interactions was shown by identifying a new interaction between carcinoembryonic antigen-related cell adhesion molecule 1 and carcinoembryonic antigen-related cell adhesion molecule 8.