Jehan J. El-Jawhari

Human tumour immune evasion via TGF-β blocks NK cell activation but not survival allowing therapeutic restoration of anti-tumour activity.

Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the interleukin (IL)-15 induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. Interleukin-15 also promotes NK cell survival and IL-15 excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-15 mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity.

Mesenchymal stem cells, autoimmunity and rheumatoid arthritis

The vast majority of literature pertaining to mesenchymal stem cells (MSC) immunomodulation has focussed on bone marrow-derived MSC that are systemically infused to alleviate inflammatory conditions. Rheumatoid arthritis (RA) is the commonest autoimmune joint disease that has witnessed significant therapeutic advances in the past decade, but remains stubbornly difficult to treat in a subset of cases. Pre-clinical research has demonstrated that bone marrow, adipose, synovial and umbilical cord-derived MSC all suppress the functions of different immune cells thus raising the possibility of new therapies for autoimmune diseases including RA. Indeed, preliminary evidence for MSC efficacy has been reported in some cases of RA and systemic lupus erythromatosis. The potential use of bone marrow-MSC (BM-MSC) for RA therapy is emerging but the use of synovial MSC (S-MSC) to suppress the exaggerated immune response within the inflamed joints remains rudimentary. Synovial fibroblasts that are likely derived from S-MSCs, also give rise to a cell-cultured progeny termed fibroblast-like synoviocytes (FLS), which are key players in the perpetuation of joint inflammation and destruction. A better understanding of the link between these cells and their biology could be a key to developing novel MSC-based strategies for therapy. The review briefly focuses on BM-MSC and gives particular attention to joint niche synovial MSC and FLS with respect to immunoregulatory potential therapy roles.

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies

Objectives. The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis. Methods. The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-&ggr; and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used. Results. Combined treatment of MSC with IL-22, IFN-&ggr; and TNF resulted in increased MSC proliferation (P = 0.008) and migration (P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone (P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure (P = 0.03, measured by calcium production). The combination of IFN-&ggr; and TNF with or without IL-22 suppressed MSC osteogenesis (P = 0.03). Conclusion. This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-&ggr;/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.

Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss®. Another collagen‐containing synthetic scaffold, Vitoss® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss® Collagen and Vitoss® was similar but greater than Orthoss® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss® Collagen and Vitoss® after 2‐week culture was also higher compared to Orthoss® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss® collagen and Vitoss® was greater compared to Orthoss® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.

The roles of immune cells in bone healing; what we know, do not know and future perspectives

Key events occurring during the bone healing include well-orchestrated and complex interactions between immune cells, multipotential stromal cells (MSCs), osteoblasts and osteoclasts. Through three overlapping phases of this physiological process, innate and adaptive immune cells, cytokines and chemokines have a significant role to play. The aim of the escalating immune response is to achieve an osseous healing in the shortest time and with the least complications facilitating the restoration of function. The uninterrupted progression of these biological events in conjunction with a favourable mechanical environment (stable fracture fixation) remains the hallmark of successful fracture healing. When failure occurs, either the biological environment or the mechanical one could have been disrupted. Not infrequently both may be compromised. Consequently, regenerative treatments involving the use of bone autograft, allograft or synthetic matrices supplemented with MSCs are increasingly used. A better understanding of the bone biology and osteoimmunology can help to improve these evolving cell-therapy based strategies. Herein, an up to date status of the role of immune cells during the different phases of bone healing is presented. Additionally, the known and yet to know events about immune cell interactions with MSCs and osteoblasts and osteoclasts and the therapeutic implications are being discussed.

Age-related Changes in Bone Marrow Mesenchymal Stromal Cells: A Potential Impact on Osteoporosis and Osteoarthritis Development

Aging at the cellular level is a complex process resulting from accumulation of various damages leading to functional impairment and a reduced quality of life at the level of the organism. With a rise in the elderly population, the worldwide incidence of osteoporosis (OP) and osteoarthritis (OA) has increased in the past few decades. A decline in the number and “fitness” of mesenchymal stromal cells (MSCs) in the bone marrow (BM) niche has been suggested as one of the factors contributing to bone abnormalities in OP and OA. It is well recognized that MSCs in vitro acquire culture-induced aging features such as gradual telomere shortening, increased numbers of senescent cells, and reduced resistance to oxidative stress as a result of serial population doublings. In contrast, there is only limited evidence that human BM-MSCs “age” similarly in vivo. This review compares the various aspects of in vitro and in vivo MSC aging and suggests how our current knowledge on rejuvenating cultured MSCs could be applied to develop future strategies to target altered bone formation processes in OP and OA.

Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.

T cell immunomodulation by clinically used allogeneic human cancellous bone fragments: a potential novel immunotherapy tool

Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.

The regenerative therapies of the ankle degeneration: a focus on multipotential mesenchymal stromal cells

The ankle degeneration ranging from focal osteochondral lesions to osteoarthritis can cause a total joint function loss. With rising life expectancy and activity of the patients, various regenerative therapies were introduced aiming to preserve the joint function via the induction of cartilage and bone repair. Here, biological events and mechanical changes of the ankle degeneration were discussed. The regenerative therapies were reviewed versus the standard surgical treatment. We especially focused on the use of mesenchymal (multipotential) stromal cells (MSCs) highlighting their dual functions of regeneration and cell modulation with an emphasis on the emerging MSC-based clinical studies. Being at an early step, more basic and clinical research is needed to optimize the applications of all ankle regenerative therapies including MSC-based methods.

Vertebral body versus iliac crest bone marrow as a source of multipotential stromal cells: Comparison of processing techniques, tri-lineage differentiation and application on a scaffold for spine fusion

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion.