Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

University of Edinburgh

Longitudinal studies suggest that a set of immune parameters including high percentages of peripheral CD8+, CD28-, CD57+ T lymphocytes, low CD4 and B cell counts, and poor T cell proliferative responses to mitogens is associated with decreased remaining longevity in the free-living very elderly (> 85 years). This combination of immune parameters was also significantly associated with an inverted CD4/CD8 ratio and cytomegalovirus seropositivity. Here, using tetramer technology, we show markedly increased numbers of CD8+ T cells bearing receptors for one single CMV epitope in the very elderly. Moreover, the fraction of these tetramer-reactive cells secreting interferon-gamma after specific antigenic stimulation was significantly lower in the old than in the young, as was the percentage of CD28-positive cells in this population. Therefore, we conclude that marked expansions of CMV-specific CD8+ T cells have occurred and that the obsession of a large fraction of the entire CD8+ T cell subset with one single viral epitope may contribute to the increased incidence of infectious disease in the elderly by shrinking the T cell repertoire available for responses to other antigens.

Large numbers of dysfunctional CD8+ T lymphocytes bearing receptors for a single dominant CMV epitope in the very old.

A "gold standard" method for the diagnosis of bacterial vaginosis (BV) is lacking. The clinical criteria described by the Amsel technique are subjective and difficult to quantify. Alternatively, the reading of Gram-stained vaginal smears by scoring techniques such as those that use the Nugent or Hay-Ison scoring systems is again subjective, requires expert personnel to perform the reading, and is infrequently used clinically. Recently, a new diagnostic device, the Osmetech Microbial Analyzer--Bacterial Vaginosis (OMA-BV), which determines a patient's BV status on the basis of measurement of the amount of acetic acid present in a vaginal swab specimen, was approved by the Food and Drug Administration. The present study uses the conducting polymer gas-sensing technology of OMA-BV to measure the concentration of acetic acid in the headspace above vaginal swab specimens from patients undergoing treatment for BV with metronidazole. In 97.8% of the cases the level of acetic acid detected fell sharply during the treatment period, crossing from above to below the diagnostic threshold of 900 ppm. The diagnosis obtained on the basis of the level of vaginal acetic acid was compared with the diagnoses obtained by use of the Amsel criteria and the Nugent scoring system both at the time of initial entry into the study and at the repeat samplings on days 7 and 14. The results obtained with OMA-BV showed overall agreements compared with the results of the Amsel and Nugent tests of 98 and 94%, respectively, for the 34 patients monitored through the treatment process. This provides further evidence that the measurement of vaginal acetic acid by headspace analysis with conducting polymer sensors is a valid alternative to present tests for the diagnosis of BV.

Analysis of vaginal acetic acid in patients undergoing treatment for bacterial vaginosis.

Immunotherapeutic aspects of stem cell transplantation.

Human leukocyte antigen (HLA)-DM is a critical participant in antigen presentation that catalyzes the dissociation of the Class II-associated Invariant chain-derived Peptide (CLIP) from the major histocompatibility complex (MHC) Class II molecules. There is competition amongst peptides for access to an MHC Class II groove and it has been hypothesised that DM functions as a 'peptide editor' that catalyzes the replacement of one peptide for another within the groove. It is established that the DM catalyst interacts directly with the MHC Class II but the precise location of the interface is unknown. Here, we combine previously described mutational data with molecular docking and energy minimisation simulations to identify a putative interaction site of >4000A2 which agrees with known point mutational data for both the DR and DM molecule. The docked structure is validated by comparison with experimental data and previously determined properties of protein-protein interfaces. A possible dissociation mechanism is suggested by the presence of an acidic cluster near the N terminus of the bound peptide.

Identification of the HLA-DM/HLA-DR interface.

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.

Artificial exosomes as tools for basic and clinical immunology.

The BCR/ABL p210 fusion protein has long been considered an ideal target antigen for the development of immunotherapeutic strategies in chronic myeloid leukaemia (CML) due to its central role in malignant transformation and to its unique novel amino acid sequence solely expressed in leukaemia cells. However, the feasibility to expand BCR-ABL-specific T cells remains still controversial. Using BCR/ABL peptide/MHC tetramers, significantly higher frequencies of tetramer positive cells were detected in the peripheral blood of HLA-A*0301 (mean 0.38%) and HLA-B*0801 (mean 0.28%) CML patients than in healthy donors (P = 0.0025 and 0.0026, respectively). However, following stimulation with autologous peptide-pulsed DCs, BCR/ABL-specific T cells were only expanded from some healthy donors, suggesting that CML patients may have a specific immune deficit with respect to the BCR/ABL antigen.

BCR/ABL-specific CD8+ T cells can be detected from CML patients, but are only expanded from healthy donors.

Soluble MHC-peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8(+) T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens.

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses.

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.

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

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.

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 HLAA* 0201/pp65 (495-503)-specific CD8+ T cells, both in HSCT recipients posttransplant 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.

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.

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

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.

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

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.

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

Athletes undergoing intensive training schedules have chronic exposure to stress-induced hormones such as cortisol that can depress immune function. We compared the circulating levels of T cell receptor excision circles (TREC), a marker of recent thymic emigrants, as well as the levels of naïve and memory subsets in a group of elite endurance athletes and in controls. The athletes showed a reduction in absolute numbers of naïve T cells, particularly in CD4 T cells. In contrast, memory cells were increased. TREC levels in the athletes were significantly reduced compared to age-matched controls. Such changes resemble premature ageing of the T cell component of the immune system. Since thymic production of T cells naturally decline with age, these results raise the concern that prolonging high intensity exercise into the 4th decade of life may have deleterious consequences for athletes' health.

Paul Travers

MRC Centre for Regenerative Medicine

University of Edinburgh

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Paul Travers

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University of Edinburgh

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

MRC Centre for Regenerative Medicine