Re-directing CD4+ T cell responses with the flanking residues of MHC class II-bound peptides: The core is not enough

Recombinant αβ T cell receptors, expressed on T cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules. There are two main subsets of αβ T cells: CD8(+) T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4(+) T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8–13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length. The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8(+) and CD4(+) T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4(+) T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4(+) T cell antigen recognition. These findings may have novel applications for the development of CD4(+) T cell peptide vaccines and diagnostics

In silico and structural analyses demonstrate that intrinsic protein motions guide T cell receptor complementarity determining region loop flexibility

T-cell immunity is controlled by T cell receptor (TCR) binding to peptide major histocompatibility complexes (pMHCs). The nature of the interaction between these two proteins has been the subject of many investigations because of its central role in immunity against pathogens, cancer, in autoimmunity, and during organ transplant rejection. Crystal structures comparing unbound and pMHC-bound TCRs have revealed flexibility at the interaction interface, particularly from the perspective of the TCR. However, crystal structures represent only a snapshot of protein conformation that could be influenced through biologically irrelevant crystal lattice contacts and other factors. Here, we solved the structures of three unbound TCRs from multiple crystals. Superposition of identical TCR structures from different crystals revealed some conformation differences of up to 5 Å in individual complementarity determining region (CDR) loops that are similar to those that have previously been attributed to antigen engagement. We then used a combination of rigidity analysis and simulations of protein motion to reveal the theoretical potential of TCR CDR loop flexibility in unbound state. These simulations of protein motion support the notion that crystal structures may only offer an artifactual indication of TCR flexibility, influenced by crystallization conditions and crystal packing that is inconsistent with the theoretical potential of intrinsic TCR motions

Seven mysteries of LAG-3: a multi-faceted immune receptor of increasing complexity

Despite three decades of research to its name and increasing interest in immunotherapies that target it, LAG-3 remains an elusive co-inhibitory receptor in comparison to the well-established PD-1 and CTLA-4. As such, LAG-3 targeting therapies have yet to achieve the clinical success of therapies targeting other checkpoints. This could, in part, be attributed to the many unanswered questions that remain regarding LAG-3 biology. Of these, we address: (i) the function of the many LAG-3-ligand interactions, (ii) the hurdles that remain to acquire a high-resolution structure of LAG-3, (iii) the under-studied LAG-3 signal transduction mechanism, (iv) the elusive soluble form of LAG-3, (v) the implications of the lack of (significant) phenotype of LAG-3 knockout mice, (vi) the reports of LAG-3 expression on the epithelium, and (vii) the conflicting reports of LAG-3 expression (and potential contributions to pathology) in the brain. These mysteries which surround LAG-3 highlight how the ever-evolving study of its biology continues to reveal ever-increasing complexity in its role as an immune receptor. Importantly, answering the questions which shroud LAG-3 in mystery will allow the maximum therapeutic benefit of LAG-3 targeting immunotherapies in cancer, autoimmunity and beyond

Molecular characterization of HLA class II binding to the LAG-3 T cell co-inhibitory receptor

Immune checkpoint inhibitors (antibodies that block the T cell co-inhibitory receptors PD-1/PD-L1 or CTLA-4) have revolutionized the treatment of some forms of cancer. Importantly, combination approaches using drugs that target both pathways have been shown to boost the efficacy of such treatments. Subsequently, several other T cell inhibitory receptors have been identified for the development of novel immune checkpoint inhibitors. Included in this list is the co-inhibitory receptor lymphocyte activation gene-3 (LAG-3), which is upregulated on T cells extracted from tumor sites that have suppressive or exhausted phenotypes. However, the molecular rules that govern the function of LAG-3 are still not understood. Using surface plasmon resonance combined with a novel bead-based assay (AlphaScreenTM), we demonstrate that LAG-3 can directly and specifically interact with intact human leukocyte antigen class II (HLA-II) heterodimers. Unlike the homologue CD4, which has an immeasurably weak affinity using these biophysical approaches, LAG3 binds with low micromolar affinity. We further validated the interaction at the cell surface by staining LAG-3+ cells with pHLA-II-multimers. These data provide new insights into the mechanism by which LAG-3 initiates T cell inhibitio

Enhanced detection of antigen-specific CD4+ T cells using altered peptide flanking residue peptide-MHC class II multimers

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions

Low-dose cyclophosphamide induces anti-tumor T-cell responses which associate with survival in metastatic colorectal cancer

Purpose: Anti-cancer T-cell responses can control tumors, but immune-suppressive mechanisms in vivo prevent their function. The role of regulatory T-cells (Tregs) in metastatic colorectal cancer (mCRC) is unclear. We have previously shown depletion of Tregs enhances CRC-specific effector T-cell responses. Low dose cyclophosphamide (CPM) targets Tregs in animal models and some human studies, however the effect of CPM in mCRC is unknown. Experimental Design: Fifty-five mCRC patients were enrolled onto a phase I/II trial and randomized to receive two week-long courses of low-dose (50mg twice-a-day) CPM or not. The absolute number, phenotype and anti-tumor function of peripheral blood-derived lymphocyte subsets were monitored throughout treatment, along with 18-month follow-up. Results: Initially CPM reduced proliferation in all lymphocyte subsets, however, a rapid mobilization of effector T-cells overcame this decrease, leading to increased absolute T-cell numbers. In contrast, a reduction in proportional and absolute Treg, B-cell and NK-cell numbers occurred. The expansion and subsequent activation of effector T-cells was focused on tumor-specific T-cells, producing both granzyme B and IFN-gamma. CPM-treated patients demonstrating the most enhanced IFN-gamma+ tumor-specific T-cell responses exhibited a significant delay in tumor progression (HR=0.29, 95% CI 0.12-0.69, P=0.0047), compared to non-responders and no-treatment controls. Conclusions: CPM-induced Treg-depletion is mirrored by a striking boost to anti-tumor immunity. This study provides the first direct evidence of the benefit of naturally primed T-cells in mCRC patients. Our results also support the concept that non-mutated self-antigens can act as useful targets for immunotherapies

Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity

Background: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. Methods: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. Results: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. Conclusions: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection

Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111–130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR–pHLA-II interactions

CD4(+)CD25(+)FOXP3(+) Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy