Paul J. Travers

The Impact of Duration versus Extent of TCR Occupancy on T Cell Activation: A Revision of the Kinetic Proofreading Model

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands.

The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37 degrees C compared to 25 degrees C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37 degrees C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus, there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

Artificial exosomes as tools for basic and clinical immunology.

Dendritic cell derived exosomes are able to mediate and modulate immune responses in vivo by semi-direct T cell activation. T cells can eradicate primary, metastatic, relapsed tumours and ameliorate otherwise fatal viral infections. Not surprisingly activation and expansion of T cells has become one of the main focuses for immunotherapy. Using nanotechnology, we have developed targeted and traceable in vivo artificial exosomes by coating liposomes (FDA approved) with an optimized number of MHC Class I/peptide complexes and a selected specific range of ligands for adhesion, early activation, late activation and survival T cell receptors. These targeted artificial exosomes are traceable both in vitro and in vivo via fluorescent and Magnetic Resonance Imaging and facilitate imaging of specific areas by applying localised nuclear magnetic interactions of hydrogens via super paramagnetic labels. Here we show that artificial exosomes activate and expand functional antigen specific T cells at sufficient levels. This novel system has potential basic and clinical applications in immunology where the study of membrane interactions is desired.

The role of Vδ2-negative γδ T cells during cytomegalovirus reactivation in recipients of allogeneic stem cell transplantation

Reactivation of cytomegalovirus (CMV) remains a serious complication after allogeneic stem cell transplantation, but the role of γδ T cells is undefined. We have studied the immune reconstitution of Vδ2negative (Vδ2neg) γδ T cells, including Vδ1 and Vδ3 subsets and Vδ2positive (Vδ2pos) γδ T cells in 40 patients during the first 24 months after stem cell transplantation. Significant long-term expansions of Vδ2neg but not Vδ2pos γδ T cells were observed during CMV reactivation early after transplantation, suggesting direct involvement of γδ T cells in anti-CMV immune responses. Similarly, significantly higher numbers of Vδ2neg γδ T cells were detected in CMV-seropositive healthy persons compared with seronegative donors; the absolute numbers of Vδ2pos cells were not significantly different. The expansion of Vδ2neg γδ T cells appeared to be CMV-related because it was absent in CMV-negative/Epstein-Barr virus-positive patients. T-cell receptor-δ chain determining region 3 spectratyping of Vδ2neg γδ T cells in healthy subjects and patients showed restricted clonality. Polyclonal Vδ2neg cell lines generated from CMV-seropositive healthy donors and from a recipient of a graft from a CMV-positive donor lysed CMV-infected targets in all cases. Our study shows new evidence for role of γδ T cells in the immune response to CMV reactivation in transplantation recipients.

Novel mechanisms of fibroblast growth factor receptor 1 regulation by extracellular matrix protein anosmin-1.

Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoform-specific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2–D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2·heparin complex, thus preventing FGFR1·FGF2·heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2·FGFR1 complex, generating an anosmin-1·FGFR1·FGF2·heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1·FGF2·heparin complex.

Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as β-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

Cytomegalovirus-specific CD8(+) T cells targeting different HLA/peptide combinations correlate with protection but at different threshold frequencies.

Cytomegalovirus (CMV) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (HSCT). Due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor‐derived CMV‐specific CD8+ T cells, have been considered. Levels of such cells correlating with protection against CMV infection and disease have only been reported in patients expressing HLA‐A*0201 and HLA‐B*0702. This is despite an increasing number of reports describing cells targeting CMV peptides presented by other human leucocyte antigens (HLAs). Considering several frequent HLA alleles, our findings suggest that HLA‐A*2402/pp65 (341‐349)‐ and HLA‐B*3501/pp65 (123‐131)‐specific CD8+ T cells correlate with protection from CMV reactivation at significantly lower cell levels than HLA‐A*0101/pp50 (245‐253)‐ and HLA‐A*0201/pp65 (495‐503)‐specific CD8+ T cells, both in HSCT recipients post‐transplant and in healthy CMV seropositive volunteers. This may result from a differing efficiency of the responses restricted by the two sets of HLA alleles. These findings add to the knowledge of immunodominance and differences in antigen processing that are coordinated in individuals with different HLA alleles and have direct implications for therapy and monitoring in patients.

SiPep: a system for the prediction of tissue-specific minor histocompatibility antigens

Approximately 50 years ago it was found that inbred strains of mice were able to reject tumours and skin grafts from major histocompatibility complex (MHC) identical donors. They proposed that additional transplantation antigens must exist outside the MHC. These were described as minor histocompatibility antigens (mHAgs). Since then, related studies in humans have identified 16 human mHAgs. The aim of this work is to increase the number of known mHAgs by prediction of candidate minor histocompatibility loci by identifying coding single nucleotide polymorphisms (SNPs) where the amino acid variation lies within an MHC‐binding peptide and alters the ability of that peptide to bind.

Understanding Hematopoietic Stem Cell Development through Functional Correlation of Their Proliferative Status with the Intra-aortic Cluster Architecture

Summary During development, hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region through a process of multi-step maturation and expansion. While proliferation of adult HSCs is implicated in the balance between self-renewal and differentiation, very little is known about the proliferation status of nascent HSCs in the AGM region. Using Fucci reporter mice that enable in vivo visualization of cell-cycle status, we detect increased proliferation during pre-HSC expansion followed by a slowing down of cycling once cells start to acquire a definitive HSC state, similar to fetal liver HSCs. We observe time-specific changes in intra-aortic hematopoietic clusters corresponding to HSC maturation stages. The proliferative architecture of the clusters is maintained in an orderly anatomical manner with slowly cycling cells at the base and more actively proliferating cells at the more apical part of the cluster, which correlates with c-KIT expression levels, thus providing an anatomical basis for the role of SCF in HSC maturation.

Cytomegalovirus-specific CD8+ T cells targeting different peptide/HLA combinations demonstrate varying T-cell receptor diversity

Cytomegalovirus (CMV) infection and reactivation pose a serious threat for patients after haematopoietic stem cell transplantation. We have previously shown that CD8+ T cells targeting different CMV epitopes correlate with protection at different threshold frequencies in those patients. To investigate if this may relate to a different quality of these cells here we analyse the T‐cell receptor diversity of pp50 (245–253)/HLA‐A*0101 specific CD8+ T cells with that of CD8+ T cells targeting various pp65 peptides. The results from this pilot study show differences in the breadth of the T‐cell receptor usage of the different cell populations. We observe for the first time that the T‐cell receptor Vβ CDR3 spectratypes used by CMV pp50 (245–253)/HLA‐A*0101‐specific CD8+ T cells can reach higher numbers than those used by CD8+ T cells targeting various pp65 peptides in our patient cohort. This merits further investigation into the effectiveness of the different CMV‐specific T cells and their impact on immunosenescence, which is important to eventually define the most useful source of adoptive therapy and monitoring protocols for cytomegalovirus‐specific immune responses.