[Development of allogeneic CAR T-cells]. / Développement des CAR-T allogéniques.

Chimeric antigen receptor (CAR) T-cell therapy represents a major breakthrough in the field of hematology. "Off-the-shelf" allogeneic CAR T-cells from donors have many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches, possible standardization of the cell product, time for multiple cell modifications, redosing and decreased cost. However, allogeneic T-cells possess foreign immunological identities that can lead to graft-versus-host disease (GvHD) and their rejection by the host immune system. In this review, we describe the different approaches to produce allogeneic CAR T-cells with limited potential for GvHD and that can persist in the recipient. The preliminary clinical results obtained with the first generation of allogeneic CAR T-cells are presented as well as the perspectives in hematological malignancies and solid tumors.

[Allogeneic CAR-NK cells: A promising alternative to autologous CAR-T cells - State of the art, sources of NK cells, limits and perspectives]. / Les CAR-NK allogéniques : une alternative prometteuse aux CAR-T autologues ­ État de la question, sources de cellules NK, limites et perspectives.

Immunotherapy with chimeric antigen receptor engineered-T cells (CAR-T) has revolutionized the landscape of treatment of relapsed or refractory B-cell. However, the use of autologous T cells has limitations: variable quality of collected effector T cells, duration of the process sometimes incompatible with uncontrolled hemopathy, limited number of available CAR cells, sometimes fatal toxicities, extremely high cost. Natural Killer (NK) cells are an interesting alternative to T cells. NK cells are very powerful cytotoxic effectors that have demonstrated an anti-tumor effect after haploidentical hematopoietic stem cells transplantation or in adoptive cell therapy against a number of solid or hematological tumors. Mainly, they can be used in allogeneic situations without causing major toxic side effects. The sources of NK cells are multiple: cell line, cord blood, peripheral blood, induced pluripotent stem cells. Recent advances in manufacturing engineered CAR-NK cells make it possible to promote antibody-dependent cell-mediated cytotoxicity (ADCC), as well as the activation and persistence of these cells, notably via the cytokine Il-15. The majority of the reports on CAR-NK cells concern pre-clinical or early clinical trials. However, the many advantages of "off-the-shelf" allogeneic CAR-NK cells provide great potential in cancer treatments.

Development of Stem Cell-Derived Immune Cells for Off-the-Shelf Cancer Immunotherapies.

Cell-based cancer immunotherapy has revolutionized the treatment of hematological malignancies. Specifically, autologous chimeric antigen receptor-engineered T (CAR-T) cell therapies have received approvals for treating leukemias, lymphomas, and multiple myeloma following unprecedented clinical response rates. A critical barrier to the widespread usage of current CAR-T cell products is their autologous nature, which renders these cellular products patient-selective, costly, and challenging to manufacture. Allogeneic cell products can be scalable and readily administrable but face critical concerns of graft-versus-host disease (GvHD), a life-threatening adverse event in which therapeutic cells attack host tissues, and allorejection, in which host immune cells eliminate therapeutic cells, thereby limiting their antitumor efficacy. In this review, we discuss recent advances in developing stem cell-engineered allogeneic cell therapies that aim to overcome the limitations of current autologous and allogeneic cell therapies, with a special focus on stem cell-engineered conventional αß T cells, unconventional T (iNKT, MAIT, and γδ T) cells, and natural killer (NK) cells.

The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Large-Scale Generation of Human Allospecific Induced Tregs With Functional Stability for Use in Immunotherapy in Transplantation.

Regulatory T cells play an important role in the control of autoimmune diseases and maintenance of tolerance. In the context of transplantation, regulatory T cells (Tregs) have been proposed as new therapeutic tools that may induce allospecific tolerance toward the graft, avoiding the side effects induced by generalized immunosuppressors. Although most clinical trials are based on the use of thymic Tregs in adoptive therapy, some reports suggest the potential use of in vitro induced Tregs (iTregs), based on their functional stability under inflammatory conditions, indicating an advantage in a setting of allograft rejection. The aim of this work was to generate and expand large numbers of allospecific Tregs that maintain stable suppressive function in the presence of pro-inflammatory cytokines. Dendritic cells were derived from monocytes isolated from healthy donors and were co-cultured with CTV-labeled naïve T cells from unrelated individuals, in the presence of TGF-ß1, IL-2, and retinoic acid. After 7 days of co-culture, proliferating CD4+CD25++CTV- cells (allospecific iTregs) were sorted and polyclonally expanded for 6 weeks in the presence of TGF-ß1, IL-2, and rapamycin. After 6 weeks of polyclonal activation, iTregs were expanded 230,000 times, giving rise to 4,600 million allospecific iTregs. Allospecific iTregs were able to specifically suppress the proliferation of autologous CD4+ and CD8+ T cells in response to the allo-MoDCs used for iTreg generation, but not to third-party allo-MoDCs. Importantly, 88.5% of the expanded cells were CD4+CD25+FOXP3+, expressed high levels of CCR4 and CXCR3, and maintained their phenotype and suppressive function in the presence of TNF-α and IL-6. Finally, analysis of the methylation status of the FOXP3 TSDR locus demonstrated a 40% demethylation in the purified allospecific iTreg, prior to the polyclonal expansion. Interestingly, the phenotype and suppressive activity of expanded allospecific iTregs were maintained after 6 weeks of expansion, despite an increase in the methylation status of the FOXP3 TSDR. In conclusion, this is the first report that demonstrates a large-scale generation of allospecific iTregs that preserve a stable phenotype and suppressor function in the presence of pro-inflammatory cytokines and pave the way for adoptive cell therapy with iTregs in transplanted patients.

Regenerative potential of allogeneic adipose tissue-derived mesenchymal cells in canine cutaneous wounds.

BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest over the past decade as a novel therapeutic treatment for a variety of diseases. Emerging studies have indicated that MSCs could enhance the repair of injured skin in canine cutaneous wounds. CASE PRESENTATION: A healthy 2 years old Bodeguero Andaluz dog was presented with multiple skin bite wounds. Antibiotic and anti-inflammatory therapy was administered for 8 days. On day three, 107 allogeneic adipose-derived mesenchymal stem cells (ASCs) were intradermally injected approximately equidistant to the ASCs treated wounds. Control wounds underwent conventional treatment with a topical antibacterial ointment until wound healing and closure. Wounds, skin morphology and healing progress were monitored via serial photographs and histopathology of biopsies obtained at day seven after ASC treatment. Histopathology revealed absence of inflammatory infiltrates and presence of multiple hair follicles in contrast to the non-ASCs treated control wounds indicating that ASC treatment promoted epidermal and dermal regeneration. ASCs were identified by flow cytometry and RT-PCR. The immunomodulatory role of ASCs was evidenced by coculturing peripheral blood mononuclear cells with allogeneic ASCs. Phytohemagglutinin was administered to stimulate lymphocyte proliferation. Cells were harvested and stained with an anticanine CD3-FITC antibody. The ASCs inhibited proliferation of T lymphocytes, which was quantified by reduction of carboxyfluorescein succinimidyl ester intensity using flow cytometry. CONCLUSIONS: Compared with conventional treatment, wounds treated with ASCs showed a higher regenerative capacity with earlier and faster closure in this dog.

Low immunogenic endothelial cells endothelialize the Left Ventricular Assist Device.

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.

Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers.

BACKGROUND: Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. METHODS: To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. RESULTS: Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. CONCLUSIONS: Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.

A fibrinous and allogeneic fibroblast-enriched membrane as a biocompatible material can improve diabetic wound healing.

The application of conventional approaches to diabetic wound regeneration has some limitations. Thus, skin substitutes could be a new therapeutic possibility. In this regard, fibrin scaffolds are promising materials due to their desirable characteristics. Since defective fibroblasts caused by diabetes can disrupt regeneration, it seems that the use of living cells can improve the healing process. Thus, based on this fact, a cellular fibrin membrane was used to evaluate the diabetic wound healing in rats. The fibrin membrane was fabricated using fresh frozen plasma on which isolated fibroblasts were cultured. The wound model was created on 36 diabetic rats that were randomly divided into three groups: control, membrane, and cellular fibrin membrane (CM). Wound photogramography and immuno-histopathological staining were performed during consecutive days after treatment. Macroscopic evaluation of the wounds indicated a noteworthy enhancement of wound closure in the CM group. In the CM group, the re-epithelialization rate on day 7, 10 (p < 0.001), and 14 (p < 0.05), the fibroblast percentage on day 3 (p < 0.01) and 7 (p < 0.05) and the collagenization in all days were significantly higher than those of other groups (p < 0.001). The fibroblast number in the CM group on day 10 was significantly (p < 0.01) lower than that in the other groups. Contrary to the neutrophil and angiogenesis percentages that had no significant difference among the groups at different points of time (p > 0.05), the macrophage percentage on day 7 (P < 0.01), 10, and 14 (p < 0.05) was significantly lower in the CM group as compared to that in other groups. Overall, it seems that the use of a fibroblast-loaded fibrin membrane is an attractive strategy to promote diabetic wound healing.

Whole-Tooth Regeneration by Allogeneic Cell Reassociation in Pig Jawbone.

IMPACT STATEMENT: The methods developed in this study to manipulate pig tooth germ cells in vitro and in vivo provide a reference for studying whole-tooth regeneration and tooth development in large animals. Of importance, compared with conventional ectopic tooth regeneration, conducted in the omentum, subcutaneous tissues, or kidney capsule (among other locations) with low with immune reactivity in rodent models, this study achieved orthotopic regeneration and development of whole teeth in a large mammal, representing a large stride toward the realization of tooth regenerative therapy for humans with missing teeth.

Update regarding xenotransplantation in Japan.

To overcome the donor shortage, a promising solution could be xenotransplantation. The pig is generally considered the most suitable donor species for xenotransplantation. A clinical xenotransplantation has not been conducted in Japan. However, many progresses have recently been made in this field. Japan has regulations for conducting cell xenotransplantation and guidelines to prevent zoonosis. Most Japanese patients and their family members have a positive opinion about islet xenotransplantation. A grant for clinical islet xenotransplantation research and development has been approved, and germ-free pigs have been developed. Further research may bring the successful application of xenotransplantation closer to reality.

Repair of bone defects in rat radii with a composite of allogeneic adipose-derived stem cells and heterogeneous deproteinized bone.

BACKGROUND: In the bone tissue engineering domain, seed cells, scaffold and cell-scaffold composites are three focuses. In this study, the feasibility of using allogeneic adipose-derived stem cells(ADSCs) combined with heterogeneous deproteinized bone (HDB) to repair segmental radial defects was investigated by observing the repair of the defect area. METHODS: ADSCs were cultured in vitro, purified, antigen-detected and osteogenic differentiation potency-measured; then, the ADSCs of the third generation were seeded into HDB to prepare an ADSCs-HDB composite partly with osteogenesis induced cells. Sixty Wistar rats were randomly divided into four groups with 15 in each group. A bone defect (4 mm in length) was created at the left radius in each rat. Two kinds of ADSCs-HDB composites were implanted in the ADSCs osteogenesis group or ADSCs group; HDB was implanted in the negative control group; nothing was filled in the blank control group. The bone defect repair was evaluated by gross observation, molybdenum target X-ray examination and histological analyses after surgery. RESULTS: Gross observation: the bone defect area was completely filled and difficult to recognize in the ADSCs osteogenesis group. The connection of the ADSCs group was strong, but the implants were clearly identifiable. The joints of the negative control group were slightly thick but the connection was unstable. In the blank control group, kermesinus tissue was between the two ends and bones were not connected after 8 weeks. Molybdenum target X-ray examinations: In the ADSCs osteogenesis group, evident bridges in the graft were observed in the defects in the fourth week; the defects were filled with new bone completely and a marrow cavity appeared at 8 weeks. In the ADSCs group, there were some callus formations, but the radial defect was still obvious at 8 weeks. In the negative control group, fracture lines were clear. In the blank control group, no osseous bridges were observed, which resulted in bone nonunion eventually in 8 weeks. There were significant differences in the callus density between experimental groups and the blank control group at 4 and 8 weeks (P < 0.01). Histological measures showed that the rate and quality of the new bone formation and remodelling was significantly different between the experimental and control groups. CONCLUSIONS: A composite of ADSCs-HDB has a strong osteogenic ability. It can repair segmental bone defects well and is promising to serve as grafting material in bone tissue engineering.