Allogenic bone marrow-derived mesenchymal stromal cell-based therapy for patients with chronic low back pain: a prospective, multicentre, randomised placebo controlled trial (RESPINE study).

OBJECTIVES: To assess the efficacy of a single intradiscal injection of allogeneic bone marrow mesenchymal stromal cells (BM-MSCs) versus a sham placebo in patients with chronic low back pain (LBP). METHODS: Participants were randomised in a prospective, double-blind, controlled study to receive either sham injection or intradiscal injection of 20 million allogeneic BM-MSC, between April 2018 and December 2022. The first co-primary endpoint was the rate of responders defined by improvement of the Visual Analogue Scale (VAS) for pain of at least 20% and 20 mm, or improvement of the Oswestry Disability Index (ODI) of 20% between baseline and month 12. The secondary structural co-primary endpoint was assessed by the disc fluid content measured by quantitative MRI T2, between baseline and month 12. Secondary endpoints included pain VAS, ODI, the Short Form (SF)-36 and the minimal clinically important difference in all timepoints (1, 3, 6, 12 and 24 months). We determined the immune response associated with allogeneic cell injection between baseline and 6 months. Serious adverse events (SAEs) were recorded. RESULTS: 114 patients were randomised (n=58, BM-MSC group; n=56, sham placebo group). At 12 months, the primary outcome was not reached (74% in the BM-MSC group vs 69% in the placebo group; p=0.77). The groups did not differ in all secondary outcomes. No SAE related to the intervention occurred. CONCLUSIONS: While our study did not conclusively demonstrate the efficacy of allogeneic BM-MSCs for LBP, the procedure was safe. Long-term outcomes of MSC therapy for LBP are still being studied. TRIAL REGISTRATION NUMBER: EudraCT 2017-002092-25/ClinicalTrials.gov: NCT03737461.

Nonclassical CD4+CD49b+ Regulatory T Cells as a Better Alternative to Conventional CD4+CD25+ T Cells To Dampen Arthritis Severity.

Promising immunotherapeutic strategies are emerging to restore tolerance in autoimmune diseases by triggering an increase in the number and/or the function of endogenous regulatory T (Treg) cells, which actively control pathological immune responses. Evidence suggests a remarkable heterogeneity in peripheral Treg cells that warrants their better characterization in terms of phenotype and suppressive function, to determine which subset may be optimally suitable for a given clinical situation. We found that repetitive injections of immature dendritic cells expanded Foxp3-negative CD49b(+) Treg cells that displayed an effector memory phenotype. These expanded Treg cells were isolated ex vivo for transcriptome analysis and found to contain multiple transcripts of the canonical Treg signature shared mainly by CD25(+) but also by other subphenotypes. We characterized the CD49b(+) Treg cell phenotype, underscoring its similarities with the CD25(+) Treg cell phenotype and highlighting some differential expression patterns for several markers, including lymphocyte activation gene 3, KLRG1, CD103, ICOS, CTLA-4, and granzyme B. Comparison of the CD25(+) and CD49b(+) Treg cells' suppressive mechanisms, in vitro and in vivo, revealed the latter's potent suppressive activity, which was partly dependent on IL-10 secretion. Altogether, our results strongly suggest that expression of several canonical Treg cell markers and suppressive function could be Foxp3 independent, and underscore the therapeutic potential of IL-10-secreting CD49b(+) Treg cells in arthritis.

A new autoinflammatory and autoimmune syndrome associated with NLRP1 mutations: NAIAD (NLRP1-associated autoinflammation with arthritis and dyskeratosis).

OBJECTIVES: Inflammasomes are multiprotein complexes that sense pathogens and trigger biological mechanisms to control infection. Nucleotide-binding oligomerisation domain-like receptor (NLR) containing a PYRIN domain 1 (NLRP1), NLRP3 and NLRC4 plays a key role in this innate immune system by directly assembling in inflammasomes and regulating inflammation. Mutations in NLRP3 and NLRC4 are linked to hereditary autoinflammatory diseases, whereas polymorphisms in NLRP1 are associated with autoimmune disorders such as vitiligo and rheumatoid arthritis. Whether human NLRP1 mutation is associated with autoinflammation remains to be determined. METHODS: To search for novel genes involved in systemic juvenile idiopathic arthritis, we performed homozygosity mapping and exome sequencing to identify causative genes. Immunoassays were performed with blood samples from patients. RESULTS: We identified a novel disease in three patients from two unrelated families presenting diffuse skin dyskeratosis, autoinflammation, autoimmunity, arthritis and high transitional B-cell level. Molecular screening revealed a non-synonymous homozygous mutation in NLRP1 (c.2176C>T; p.Arg726Trp) in two cousins born of related parents originating from Algeria and a de novo heterozygous mutation (c.3641C>G, p.Pro1214Arg) in a girl of Dutch origin. The three patients showed elevated systemic levels of caspase-1 and interleukin 18, which suggested involvement of NLRP1 inflammasome. CONCLUSIONS: We demonstrate the responsibility of human NLRP1 in a novel autoinflammatory disorder that we propose to call NAIAD for NLRP1-associated autoinflammation with arthritis and dyskeratosis. This disease could be a novel autoimmuno-inflammatory disease combining autoinflammatory and autoimmune features. Our data, combined with that in the literature, highlight the pleomorphic role of NLRP1 in inflammation and immunity. TRIAL REGISTRATION NUMBER: NCT02067962; Results.

DX5+ CD4+ T cells modulate CD4+ T-cell response via inhibition of IL-12 production by DCs.

DX5(+) CD4(+) T cells have been shown to dampen collagen-induced arthritis and delayed-type hypersensitivity reactions in mice. These cells are also potent modulators of T-helper cell responses through direct effects on CD4(+) T cells in an IL-4 dependent manner. To further characterize this T-cell population, we studied their effect on DCs and the potential consequences on T-cell activation. Here, we show that mouse DX5(+) CD4(+) T cells modulate DCs by robustly inhibiting IL-12 production. This modulation is IL-10 dependent and does not require cell contact. Furthermore, DX5(+) CD4(+) T cells modulate the surface phenotype of LPS-matured DCs. DCs modulated by DX5(+) CD4(+) T-cell supernatant express high levels of the co-inhibitor molecules PDL-1 and PDL-2. OVA-specific CD4(+) T cells primed with DCs exposed to DX5(+) CD4(+) T-cell supernatant produce less IFN-γ than CD4(+) T cells primed by DCs exposed to either medium or DX5(-) CD4(+) T-cell supernatant. The addition of IL-12 to the co-culture with DX5(+) DCs restores IFN-γ production. When IL-10 present in the DX5(+) CD4(+) T-cell supernatant is blocked, DCs re-establish their ability to produce IL-12 and to efficiently prime CD4(+) T cells. These data show that DX5(+) CD4(+) T cells can indirectly affect the outcome of the T-cell response by inducing DCs that have poor Th1 stimulatory function.

Nicotinamide phosphoribosyltransferase/visfatin expression by inflammatory monocytes mediates arthritis pathogenesis.

OBJECTIVES: Nicotinamide phosphoribosyltransferase (NAMPT)/pre-B-cell colony-enhancing factor/visfatin exerts multiple functions and has been implicated in the pathogenesis of rheumatoid arthritis. To gain insight into its role in arthritis and given that NAMPT is identified as a novel mediator of innate immunity, we addressed the function of monocyte-derived NAMPT in experimental arthritis by selective gene knockdown in inflammatory monocytes. METHODS: siRNA uptake and NAMPT expression were determined in Ly6Chigh and Ly6Clow monocyte subsets following intravenous injection of siRNA against NAMPT (siNAMPT) or non-targeting siRNA (siCT) formulated with the DMAPAP cationic liposome into mice. Mice with established collagen-induced arthritis (CIA) were treated weekly after disease onset with siNAMPT or siCT and clinical features were assessed. T-helper cell frequencies, cytokine production and percentage of IL-6-producing Ly6Chigh monocytes were analysed. Using a co-culture system consisting of purified CD14 monocytes and autologous CD4 T cells, NAMPT and cytokine production, and the percentage of IL-17-producing CD4 T cells, were determined following transfection of CD14 monocytes with siCT or siNAMPT. RESULTS: On intravenous injection, siRNA was preferentially engulfed by Ly6Chigh monocytes, and siRNA-mediated silencing of NAMPT expression in Ly6Chigh monocytes inhibited CIA progression. This effect was associated with reduced IL-6 production by Ly6Chigh monocytes, reduced proportion of Th17 cells and autoantibody titers, and decreased activation and infiltration of monocytes/macrophages and neutrophils in arthritic joints. Moreover, NAMPT-RNAi-silenced CD14 monocytes were found to reduce the percentage of IL-17-producing CD4 T cells in vitro. CONCLUSIONS: Our results show that the expression of NAMPT in Ly6Chigh monocytes promotes many downstream effects involved in inflammatory arthritis and demonstrate the utility of targeting disease-causing genes, such as NAMPT, in Ly6Chigh monocytes for therapeutic intervention in arthritis.

Intra-articular delivery of full-length antibodies through the use of an in situ forming depot.

Monoclonal antibodies (mAbs) are large size molecules that have demonstrated high therapeutic potential for the treatment of cancer or autoimmune diseases. Despite some excellent results, their intravenous administration results in high plasma concentration. This triggers off-target effects and sometimes poor targeted tissue distribution. To circumvent this issue, we investigated a local controlled-delivery approach using an in situ forming depot technology. Two clinically relevant mAbs, rituximab (RTX) and daratumumab (DARA), were formulated using an injectable technology based on biodegradable PEG-PLA copolymers. The stability and controlled release features of the formulations were investigated. HPLC and mass spectrometry revealed the preservation of the protein structure. In vitro binding of formulated antibodies to their target antigens and to their cellular FcγRIIIa natural killer cell receptor was fully maintained. Furthermore, encapsulated RTX was as efficient as classical intravenous RTX treatment to inhibit the in vivo tumor growth of malignant human B cells in immunodeficient NSG mice. Finally, the intra-articular administration of the formulated mAbs yielded a sustained local release associated with a lower plasma concentration compared to the intra-articular delivery of non-encapsulated mAbs. Our results demonstrate that the utilization of this polymeric technology is a reliable alternative for the local delivery of fully functional clinically relevant mAbs.

DX5(+)CD4(+) T cells modulate cytokine production by CD4(+) T cells towards IL-10 via the production of IL-4.

CD4(+) Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune-modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5(+)CD4(+) T cells, which upon adoptive transfer show potent regulatory properties in murine collagen-induced arthritis as well as in delayed-hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5(+)CD4(+) T cells on other CD4(+) T cells in vitro. Although proliferation of naïve CD4(+) T cells upon antigenic triggering was not altered in the presence of DX5(+)CD4(+) T cells, there was a striking difference in cytokine production. In the presence of DX5(+)CD4(+) T cells, an IL-10-producing CD4(+) T-cell response was induced instead of a predominant IFN-γ-producing Th1 response. This modulation did not require cell-cell contact. Instead, IL-4 produced by DX5(+)CD4(+) T cells was primarily involved in the inhibition of IFN-γ and promotion of IL-10 production by CD4(+) T cells. Together, our data indicate that DX5(+)CD4(+) T cells modulate the outcome of Th-responses by diverting Th1-induction into Th responses characterized by the production of IL-10.

DC-induced CD8(+) T-cell response is inhibited by MHC class II-dependent DX5(+)CD4(+) Treg.

CD4(+) T cells are important for CD8(+) T-cell priming by providing cognate signals for DC maturation. We analyzed the capacity of CD4(+) T cells to influence CD8(+) T-cell responses induced by activated DC. Surprisingly, mice depleted for CD4(+) cells were able to generate stronger antigen-specific CD8(+) T-cell responses after DC vaccination than non-depleted mice. The same observation was made when mice were vaccinated with MHC class II(-/-) DC, indicating the presence of a MHC class II-dependent CD4(+) T-cell population inhibiting CD8(+) T-cell responses. Recently we described the expansion of DX5(+)CD4(+) T cells, a T-cell population displaying immune regulatory properties, upon vaccination with DC. Intriguingly, we now observe an inverse correlation between CD8(+) T-cell induction and expansion of DX5(+)CD4(+) T cells as the latter cells did not expand after vaccination with MHC class II(-/-) DC. In vitro, DX5(+)CD4(+) T cells were able to limit proliferation, modulate cytokine production and induce Foxp3(+) expression in OVA-specific CD8(+) T cells. Together, our data show an inhibitory role of CD4(+) T cells on the induction of CD8(+) T-cell responses by activated DC and indicate the involvement of DX5(+)CD4(+), but not CD4(+)CD25(+), T cells in this process.

Adoptive transfer of IL-10-secreting CD4+CD49b+ regulatory T cells suppresses ongoing arthritis.

We have previously demonstrated, in the collagen-induced arthritis model (CIA), that repetitive injections of immature bone-marrow-derived dendritic cells (iDCs) induce the expansion of a population of CD4CD49b-expressing cells, and that their adoptive transfer results in protection against CIA in a prophylactic setting. However, the in vivo mechanism responsible for their expansion, as well as their therapeutic potential in established disease remains to be defined. In the present study, we show that expression of the MHC class II molecules on iDCs is required for their expansion thus identifying these cells as MHC class II-restricted T cells. Using adoptive transfer of Thy1.1 positive cells, it is shown that iDC-induced CD4(+)CD49b(+) T cells home to the lymph nodes draining the inflamed tissue. The high immunomodulatory potential of these cells was underscored following their adoptive transfer in a model of contact hypersensitivity. Finally, we assessed and compared the therapeutic potential of iDC-inducible CD4(+)CD49b(+) T cells with that of iDCs in established CIA. Repetitive injections of iDCs in arthritic mice failed to decrease the severity of established disease. In contrast however, a single injection of iDC-induced CD4(+)CD49b(+) T cells reversed clinical symptoms of arthritis and provided long-lasting protection. Together, our data indicate that iDC-induced CD4(+)CD49b(+) T cells are bona fide T regulatory cells with strong immunomodulatory properties that are not only able to prevent disease onset, but also to interfere with an ongoing inflammatory immune response.

Differential Accumulation and Activation of Monocyte and Dendritic Cell Subsets in Inflamed Synovial Fluid Discriminates Between Juvenile Idiopathic Arthritis and Septic Arthritis.

Despite their distinct etiology, several lines of evidence suggest that innate immunity plays a pivotal role in both juvenile idiopathic arthritis (JIA) and septic arthritis (SA) pathophysiology. Indeed, monocytes and dendritic cells (DC) are involved in the first line of defense against pathogens and play a critical role in initiating and orchestrating the immune response. The aim of this study was to compare the number and phenotype of monocytes and DCs in peripheral blood (PB) and synovial fluid (SF) from patients with JIA and SA to identify specific cell subsets and activation markers associated with pathophysiological mechanisms and that could be used as biomarkers to discriminate both diseases. The proportion of intermediate and non-classical monocytes in the SF and PB, respectively, were significantly higher in JIA than in SA patients. In contrast the proportion of classical monocytes and their absolute numbers were higher in the SF from SA compared with JIA patients. Higher expression of CD64 on non-classical monocyte was observed in PB from SA compared with JIA patients. In SF, higher expression of CD64 on classical and intermediate monocyte as well as higher CD163 expression on intermediate monocytes was observed in SA compared with JIA patients. Moreover, whereas the number of conventional (cDC), plasmacytoid (pDC) and inflammatory (infDC) DCs was comparable between groups in PB, the number of CD141+ cDCs and CD123+ pDCs in the SF was significantly higher in JIA than in SA patients. CD14+ infDCs represented the major DC subset in the SF of both groups with potent activation assessed by high expression of HLA-DR and CD86 and significant up-regulation of HLA-DR expression in SA compared with JIA patients. Finally, higher activation of SF DC subsets was monitored in SA compared with JIA with significant up-regulation of CD86 and PDL2 expression on several DC subsets. Our results show the differential accumulation and activation of innate immune cells between septic and inflammatory arthritis. They strongly indicate that the relative high numbers of CD141+ cDC and CD123+ pDCs in SF are specific for JIA while the over-activation of DC and monocyte subsets is specific for SA.

Antitumoral activity and osteogenic potential of mesenchymal stem cells expressing the urokinase-type plasminogen antagonist amino-terminal fragment in a murine model of osteolytic tumor.

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

Tripartite siRNA micelles as controlled delivery systems for primary dendritic cells.

Dendritic cells (DCs) are key cells in immunology that are able to stimulate or inhibit the immune response. RNA interference has appeared of great interest to modulate the expression of immunogenic or tolerogenic molecules. In our study, pH-sensitive polyion complex micelles based on a double-hydrophilic block copolymer and poly-L-lysine were formulated to entrap a small interfering RNA (siRNA). We show that siRNA-loaded micelles were cytotolerant and efficiently endocytosed by DCs. siRNA targeting eGFP, used as model siRNA, was released into the cytosol following endocytosis of the micelles and the silencing of eGFP expression was observed in DC isolated from transgenic mice. Our results underscore the potential of pH-sensitive polyion complex micelles to formulate therapeutic siRNA for DC engineering in order to maintain the homeostasis of the immune response.

Arthritis sensory and motor scale: predicting functional deficits from the clinical score in collagen-induced arthritis.

BACKGROUND: In the collagen-induced arthritis (CIA) mouse model, inflammation readouts are usually quantified using operator-dependent clinical scoring systems, and no systematic relationship with functional deficits has been detected. In this study, we extensively quantified sensory and motor deficits in CIA mice during natural disease progression and therapeutic treatment. Then, we used these data to build a scale to predict functional deficits on the basis of the classical clinical score. METHODS: Using the CIA mouse model, we longitudinally screened multiple approaches to assess locomotion (open field test, Catwalk™), sensitivity (Von Frey, Hargreaves, static weight-bearing tests), and inflammation (skin temperature), and identified the most accurate tests to correlate sensory and motor deficits with disease severity, measured by clinical score. We then used these tests to characterize functional deficits in control (naïve and mice injected with complete Freund's adjuvant) and CIA mice, either untreated or treated with methotrexate to prevent functional deficits. By mathematical approaches, we finally investigated the relationship between functional deficits and clinical score. RESULTS: We found that the functional disability scores obtained with the open field, Catwalk™, Hargreaves, and skin temperature tests significantly correlated with the clinical score in CIA mice, either untreated or treated with methotrexate. Mathematical correlation showed that motor deficits, robustly characterized by two different tests, were twice more responsive than thermal sensitivity deficits. CONCLUSION: We propose the arthritis sensory and motor (ArthriSM) scale as a new theranostic tool to predict motor and sensory deficit based on the clinical score, in the experimental mouse model of CIA. This ArthriSM scale may facilitate the transfer of knowledge between preclinical and clinical studies.

Polyoxidonium® Activates Cytotoxic Lymphocyte Responses Through Dendritic Cell Maturation: Clinical Effects in Breast Cancer.

Immunotherapy, which is seen as a major tool for cancer treatment, requires, in some cases, the presence of several agents to maximize its effects. Adjuvants can enhance the effect of other agents. However, despite their long-time use, only a few adjuvants are licensed today, and their use in cancer treatment is rare. Azoximer bromide, marketed under the trade name Polyoxidonium® (PO), is a copolymer of N-oxidized 1,4-ethylenepiperazine and (N-carboxyethyl)-1,4-ethylene piperazinium bromide. It has been described as an immune adjuvant and immunomodulator that is clinically used with excellent tolerance. PO is used in the treatment and prophylaxis of diseases connected with damage to the immune system, and there is interest in testing it in antitumor therapy. We show here that PO treatment for 1 week induced positive pathological changes in 6 out of 20 patients with breast cancer, including complete response in a triple-negative patient. This correlated with an increased tumor CD4+ T-lymphocyte infiltration. The immune effects of PO are associated with myeloid cell activation, and little is known about the action of PO on lymphocyte lineages, such as natural killer (NK) and T cells. We reveal that PO increases T-cell proliferation in vitro without negative effects on any activation marker. PO does not affect dendritic cell (DC) viability and increases the expansion of immature DC (iDC) and mature DC (mDC) at 100 µg/ml, and it stimulates expression of several DC co-stimulatory molecules, inducing the proliferation of allogeneic T cells. In contrast, PO decreases DC viability when added at day 5 post-expansion. PO is not toxic for NK cells at doses up to 100 µM and does not affect their activation, maturation, and cytotoxicity but tends to increase degranulation. This could be beneficial against target cells that show low sensitivity to NK cells, e.g., solid tumor cells. Finally, we have found great variability in PO response between donors. In summary, our in vitro results show that PO increases the number of costimulatory molecules on DC that prime T cells, favoring the production of effector T cells. This may support the future clinical development of PO in cancer treatment.

Injection of Adipose-Derived Stromal Cells in the Knee of Patients with Severe Osteoarthritis has a Systemic Effect and Promotes an Anti-Inflammatory Phenotype of Circulating Immune Cells.

Rationale: Recent studies confirmed that osteoarthritis (OA) is associated with systemic inflammation. Adipose-derived stromal cells (ASCs) could become the most promising cell-based therapy in OA, based not only on their differentiation capacities and trophic and paracrine effects on the existing cartilage, but also on their immunomodulatory properties. Here, we wanted to determine the biological effect of autologous ASC intra-articular (IA) injection. Method: To this aim, we monitored the profile of immune cells in fresh peripheral blood after IA injection of autologous ASCs in the knee of 18 patients with severe OA (ADIPOA phase I study). Specifically, we used 8-color flow cytometry antibody panels to characterize the frequencies of innate and adaptive immune cell subsets (monocytes, dendritic cells, regulatory T cells and B cells) in blood samples at baseline (before injection) and one week, one month and three months after ASC injection. Results: We found that the percentage of CD4+CD25highCD127lowFOXP3+ regulatory T cells was significantly increased at 1 month after ASC injection, and this effect persisted for at least 3 months. Moreover, CD24highCD38high transitional B cells also were increased, whereas the percentage of classical CD14+ monocytes was decreased, at 3 months after ASC injection. These results suggest a global switch toward regulatory immune cells following IA injection of ASCs, underscoring the safety of ASC-based therapy. We did not find any correlation between the scores for the Visual Analogic Scale for pain, the Western Ontario and McMaster Universities Osteoarthritis Index (pain subscale and total score) at baseline and the immune cell profile changes, but this could be due to the small number of analyzed patients. Conclusion: ASCs may drive an immediate local response by releasing paracrine factors and cytokines, and our results suggest that ASCs could also initiate a cascade resulting in a long-lasting systemic immune modulation.

Micro-CT combined with bioluminescence imaging: a dynamic approach to detect early tumor-bone interaction in a tumor osteolysis murine model.

Prostate cancer (CaP) cells possess high affinity for bone marrow and predilection to induce bone metastasis. Although the end result of metastasis is predominantly osteoblastic, most patients present mixed lesions with osteolytic component which could initiate and precede bone formation. A precise characterization of tumor-induced bone resorption is thus necessary for early evaluation of therapeutic efficiency. Herein, we investigate the advantage of combining micro-computed tomography (microCT) and in vivo bioluminescence imaging (BLI) to determine the kinetics of the intraosseous CaP growth and bone lesions appearance in an experimental murine model. To mimic established osteolytic bone metastasis, the left tibiae of SCID mice were injected with the human CaP cell line PC-3 expressing luciferase (PC-3 Luc). Noninvasive monitoring of tumor progression was followed weekly by BLI during 4 weeks and bone morphometric parameters were quantified by microCT. Data were compared with conventional radiological and histological analyses. While BLI monitoring in vivo revealed an exponential growth of PC-3 Luc after 2 weeks, a decrease of bone density and bone mineral content was evidenced by microCT as early as 7 days post-injection, reaching significant values at day 21 (30% and 25% loss, respectively), compared with mock-injected controls. Enhanced osteoclast TRAP activity was observed during the first two weeks, highlighting an active interaction between low proliferative PC-3 cells and osteoclasts at the early stage of tumor establishment in bone. Tumor growth detected by BLI was tightly correlated to the osteolysis assessed by microCT (p<0.05). Our results show that the combination of microCT and BLI applied to this tumor osteolysis murine model allows early measurement of intraosseous tumor growth and bone destruction, as well as correlation between both processes kinetics. This model will help to assess new therapeutic approaches targeting intraosseous tumor growth or tumor/osteoclast crosstalk.

Earlier onset of syngeneic tumors in the presence of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are widely investigated for cell therapy purposes as support of hematopoietic cell transplantation, skeletal tissue regeneration, or as a cell delivery system of therapeutic agents in cancer. However, because of their immunosuppressive capacities, we investigated the effect of MSC on the development of syngeneic tumors. METHODS: The murine MSC line C3H10T1/2 was coinjected with the Renca adenocarcinoma or the B16 melanoma cell lines in BALB/c mice. RESULTS: The injection of MSC permitted the growth of the allogeneic B16 tumor cells and reduced the delay of tumor appearance when Renca cells were implanted, without modifying the kinetics of tumor growth. This effect was observed even with a low ratio of cancer cells, mimicking minimal residual disease. In this last case, no MSC were detected in the tumor mass, suggesting that cell contact was not necessary. The presence of MSC did not enhance the development of lung metastasis after systemic injection of Renca cells. Because the proliferative rate of Renca cells was not affected by in vitro coculture with MSC, this observation is likely due to a systemic suppressive effect on the host immune system. CONCLUSIONS: Altogether, these data suggest that MSC did not interfere with the kinetics of tumor development but may reduce the delay for tumor occurrence. An important finding of this study is that a low but relevant amount of MSC may induce tumor rejection.

Cellular senescence impact on immune cell fate and function.

Cellular senescence occurs not only in cultured fibroblasts, but also in undifferentiated and specialized cells from various tissues of all ages, in vitro and in vivo. Here, we review recent findings on the role of cellular senescence in immune cell fate decisions in macrophage polarization, natural killer cell phenotype, and following T-lymphocyte activation. We also introduce the involvement of the onset of cellular senescence in some immune responses including T-helper lymphocyte-dependent tissue homeostatic functions and T-regulatory cell-dependent suppressive mechanisms. Altogether, these data propose that cellular senescence plays a wide-reaching role as a homeostatic orchestrator.

Systemic LPS Translocation Activates Cross-Presenting Dendritic Cells but Is Dispensable for the Breakdown of CD8+ T Cell Peripheral Tolerance in Irradiated Mice.

Lymphodepletion is currently used to enhance the efficacy of cytotoxic T lymphocyte adoptive transfer immunotherapy against cancer. This beneficial effect of conditioning regimens is due, at least in part, to promoting the breakdown of peripheral CD8+ T cell tolerance. Lymphodepletion by total body irradiation induces systemic translocation of commensal bacteria LPS from the gastrointestinal tract. Since LPS is a potent activator of the innate immune system, including antigen presenting dendritic cells, we hypothesized that LPS translocation could be required for the breakdown of peripheral tolerance observed in irradiated mice. To address this issue, we have treated irradiated mice with antibiotics in order to prevent LPS translocation and utilized them in T cell adoptive transfer experiments. Surprisingly, we found that despite of completely blocking LPS translocation into the bloodstream, antibiotic treatment did not prevent the breakdown of peripheral tolerance. Although irradiation induced the activation of cross-presenting CD8+ dendritic cells in the lymphoid tissue, LPS could not solely account for this effect. Activation of dendritic cells by mechanisms other than LPS translocation is sufficient to promote the differentiation of potentially autoreactive CD8+ T cells into effectors in irradiated mice. Our data indicate that LPS translocation is dispensable for the breakdown of CD8+ T cell tolerance in irradiated mice.

Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases.