Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells.

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19. Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19- Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19- 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells. Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

An Efficient Technique for the Long-term Preservation of SMILE Lenticules Using Desiccation.

PURPOSE: To evaluate a desiccation protocol for the long-term preservation of human small incision lenticule extraction (SMILE) lenticules and to study their integration in an in vivo rabbit model. METHODS: Lenticules were retrieved after SMILE procedures in patients, then desiccated according to a novel protocol. Histologic and electron microscopic analyses were performed. Six rabbit eyes received grafts with an inlay technique, which consisted of inserting a desiccated lenticule into a stromal pocket. Rabbits were killed at different times between 6 and 24 weeks. Rabbit corneas were analyzed using optical coherence tomography, histology, and DAPI staining. RESULTS: Microscopic analysis of desiccated lenticules showed a preserved stromal architecture after rehydration. A decellularization of the lenticules after desiccation was observed without any chemical treatment. All rabbit corneas remained clear after grafting human lenticules and no rejection occurred. Optical coherence tomography showed regular lenticular implantation and no decrease in lenticule thickness. Histologic analysis showed no inflammatory infiltration around lenticules and no nuclear material inside lenticules after 6 months. CONCLUSIONS: A favorable integration of desiccated human SMILE lenticules in rabbit corneas was observed. The refractive issue of lenticular implantation must be investigated next. Clinical trials are needed to evaluate the use of desiccated SMILE lenticules to treat hyperopia or keratoconus in humans. [J Refract Surg. 2023;39(7):491-498.].

Radiation therapy improves CAR T cell activity in acute lymphoblastic leukemia.

Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 are approved for the treatment of various CD19+ hematological malignancies. While CAR T cells induce objective responses in a majority of patients, relapse frequently occurs upon loss of CD19 expression by neoplastic cells. Radiation therapy (RT) has been successfully employed to circumvent the loss of CAR targets in preclinical models of pancreatic cancer. At least in part, this reflects the ability of RT to elicit death receptor (DR) expression by malignant cells, enabling at least some degree of CAR-independent tumor killing. In a human model of CD19+ acute lymphoblastic leukemia (ALL), we also observed DR upregulation by RT, both in vitro and in vivo. Moreover, low-dose total body irradiation (LD-TBI) delivered to ALL-bearing mice prior to CAR T cell infusion considerably extended the overall survival benefit afforded by CAR T cells alone. Such an improved therapeutic activity was accompanied by a superior expansion of CAR T cells in vivo. These data encourage the initiation of clinical trials combining LD-TBI with CAR T cells in patients with hematological malignancies.

Anti-transcription intermediary factor 1-gamma IgG2 isotype is associated with cancer in adult dermatomyositis: an ENMC multinational study.

OBJECTIVE: To assess the role of the anti-TIF1γ auto-antibody (aAb) IgG2 isotype as a biomarker of cancer in anti-TIF1γ aAb-positive adult DM. METHODS: International multicentre retrospective study with the following inclusion criteria: (i) diagnosis of DM according to ENMC criteria; (ii) presence of anti-TIF1γ IgG aAb determined using an in-house addressable laser bead immunoassay (ALBIA) from cryopreserved serums sampled at time of DM diagnosis and (iii) available baseline characteristics and follow-up data until the occurrence of cancer and/or a minimum follow-up of 1 year for patients without known cancer at diagnosis. Detection and quantification of anti-TIF1γ IgG2 aAb was done using the in-house ALBIA. In addition, a recent ELISA commercial kit was used for anti-TIF1γ IgG aAb quantification. RESULTS: A total of 132 patients (mean age 55±15 years) of whom 72 (54.5%) had an associated cancer were analysed. The association between the presence of cancer and the presence of anti-TIF1γ IgG2 aAb was statistically significant (P = 0.026), with an OR of 2.26 (95% CI: 1.10, 4.76). Patients with cancer displayed significantly higher anti-TIF1γ IgG2 aAb ALBIA values with a median value of 1.15 AU/ml (IQR: 0.14-9.76) compared with 0.50 AU/ml (IQR: 0.14-1.46) for patients without cancer (P = 0.042). In addition, patients with cancer displayed significantly higher anti-TIF1γ IgG aAb ELISA values with a median value of 127.5 AU/ml (IQR: 81.5-139.6) compared with 93.0 AU/ml (IQR: 54.0-132.9) for patients without cancer (P = 0.004). CONCLUSION: These results suggest considering anti-TIF1γ IgG2 ALBIA and IgG ELISA values as biomarkers of cancer in anti-TIF1 γ aAb-positive adult DM.

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy.

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These "off-the-shelf" AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

A Methodological Approach Using rAAV Vectors Encoding Nanobody-Based Biologics to Evaluate ARTC2.2 and P2X7 In Vivo.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Pyroptotic and Necroptotic Cell Death in the Tumor Microenvironment and Their Potential to Stimulate Anti-Tumor Immune Responses.

Cancer remains the second most common cause of death worldwide affecting around 10 million patients every year. Among the therapeutic options, chemotherapeutic drugs are widely used but often associated with side effects. In addition, toxicity against immune cells may hamper anti-tumor immune responses. Some chemotherapeutic drugs, however, preserve immune functions and some can even stimulate anti-tumor immune responses through the induction of immunogenic cell death (ICD) rather than apoptosis. ICD stimulates the immune system by several mechanisms including the release of damage-associated molecular patterns (DAMPs) from dying cells. In this review, we will discuss the consequences of inducing two recently characterized forms of ICD, i.e., pyroptosis and necroptosis, in the tumor microenvironment (TME) and the perspectives they may offer to increase the immunogenicity of the so-called cold tumors and to stimulate effective anti-tumor immune responses.

Myogenic Cell Transplantation in Genetic and Acquired Diseases of Skeletal Muscle.

This article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies.

Modifications of the BAFF/BAFF-Receptor Axis in Patients With Pemphigus Treated With Rituximab Versus Standard Corticosteroid Regimen.

The efficacy of the B-cell-depleting agent rituximab has been reported in immune diseases but relapses are frequent, suggesting the need for repeated infusions. The B-cell activating factor (BAFF) is an important factor for B cell survival, class switch recombination and selection of autoreactive B cells, as well as maintaining long-lived plasma cells. It has been hypothesized that relapses after rituximab might be due to the increase of serum BAFF levels. From the Ritux3 trial, we showed that baseline serum BAFF levels were higher in pemphigus patients than in healthy donors (308 ± 13 pg/mL versus 252 ± 28 pg/mL, p=0.037) and in patients with early relapse compared who didn't (368 ± 92 vs 297 ± 118 pg/mL, p=0.036). Rituximab and high doses of CS alone have different effects on the BAFF/BAFF-R axis. Rituximab led to an increase of BAFF levels associated to a decreased mRNA (Day 0: 12.3 ± 7.6 AU vs Month 36: 3.3 ± 4.3 AU, p=0.01) and mean fluorescence intensity of BAFF-R in non-autoreactive (Day 0: 3232 vs Month 36: 1527, mean difference: 1705, 95%CI: 624 to 2786; p=0.002) as well as on reappearing autoreactive DSG-specific B cells (Day 0: 3873 vs Month 36: 2688, mean difference: 1185, 95%CI: -380 to 2750; p=0.20). Starting high doses of corticosteroids allowed a transitory decrease of serum BAFF levels that re-increased after doses tapering whereas it did not modify BAFF-R expression in autoreactive and non-autoreactive B cells. Our results suggest that the activation of autoreactive B cells at the onset of pemphigus is likely to be related to the presence of high BAFF serum levels and that the decreased BAFF-R expression after rituximab might be responsible for the delayed generation of memory B cells, resulting in a rather long period of mild pemphigus activity after rituximab therapy. Conversely, the incomplete B cell depletion and persistent BAFF-R expression associated with high BAFF serum levels might explain the high number of relapses in patients treated with CS alone.

TRIM33 gene somatic mutations identified by next generation sequencing in neoplasms of patients with anti-TIF1γ positive cancer-associated dermatomyositis.

OBJECTIVE: To deep sequence the TRIM33 gene in tumours from patients with cancer-associated anti-TIF1γ autoantibody-positive dermatomyositis (DM) as TRIM33 somatic mutations in tumours may trigger this auto-immune disease. METHODS: Next generation sequencing of tumour DNA samples from patients with cancer-associated anti-TIF1γ autoantibody-positive DM. Fourteen tumours from 13 anti-TIF1γ autoantibody-positive DM individuals were sequenced along with two control tumours from non-DM individuals. RESULTS: Fourteen probable somatic variants from four tumours were identified in the TRIM33 gene. CONCLUSION: These results are in accordance with the previous report of Pinal-Fernandez et al. and support the hypothesis of a role of TRIM33 gene mutations in the pathophysiology of anti-TIF1γ autoantibody-positive DM.

Rituximab and Corticosteroid Effect on Desmoglein-Specific B Cells and Desmoglein-Specific T Follicular Helper Cells in Pemphigus.

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.

Syngeneic Transplantation of Rat Olfactory Stem Cells in a Vein Conduit Improves Facial Movements and Reduces Synkinesis after Facial Nerve Injury.

BACKGROUND: Posttraumatic facial paralysis is a disabling condition. Current surgical management by faciofacial nerve suture provides limited recovery. To improve the outcome, the authors evaluated an add-on strategy based on a syngeneic transplantation of nasal olfactory stem cells in a rat model of facial nerve injury. The main readouts of the study were the recording of whisking function and buccal synkinesis. METHODS: Sixty rats were allocated to three groups. Animals with a 2-mm facial nerve loss were repaired with a femoral vein, filled or not with olfactory stem cells. These two groups were compared to similarly injured rats but with a faciofacial nerve suture. Olfactory stem cells were purified from rat olfactory mucosa. Three months after surgery, facial motor performance was evaluated using video-based motion analysis and electromyography. Synkinesis was assessed by electromyography, using measure of buccal involuntary movements during blink reflex, and double retrograde labeling of regenerating motoneurons. RESULTS: The authors' study reveals that olfactory stem cell transplantation induces functional recovery in comparison to nontransplanted and faciofacial nerve suture groups. They significantly increase (1) maximal amplitude of vibrissae protraction and retraction cycles and (2) angular velocity during protraction of vibrissae. They also reduce buccal synkinesis, according to the two techniques used. However, olfactory stem cell transplantation did not improve axonal regrowth of the facial nerve, 3 months after surgery. CONCLUSIONS: The authors show here that the adjuvant strategy of syngeneic transplantation of olfactory stem cells improves functional recovery. These promising results open the way for a phase I clinical trial based on the autologous engraftment of olfactory stem cells in patients with a facial nerve paralysis.

Immune-mediated necrotizing myopathy: clinical features and pathogenesis.

Immune-mediated necrotizing myopathy (IMNM) is a group of inflammatory myopathies that was distinguished from polymyositis in 2004. Most IMNMs are associated with anti-signal recognition particle (anti-SRP) or anti-3-hydroxy-3-methylglutaryl-coA reductase (anti-HMGCR) myositis-specific autoantibodies, although ~20% of patients with IMNM remain seronegative. These associations have led to three subclasses of IMNM: anti-SRP-positive IMNM, anti-HMGCR-positive IMNM and seronegative IMNM. IMNMs are frequently rapidly progressive and severe, displaying high serum creatine kinase levels, and failure to treat IMNMs effectively may lead to severe muscle impairment. In patients with seronegative IMNM, disease can be concomitant with cancer. Research into IMNM pathogenesis has shown that anti-SRP and anti-HMGCR autoantibodies cause weakness and myofibre necrosis in mice, suggesting that, as well as being diagnostic biomarkers of IMNM, they may play a key role in disease pathogenesis. Therapeutically, treatments such as rituximab or intravenous immunoglobulins can now be discussed for IMNM, and targeted therapies, such as anticomplement therapeutics, may be a future option for patients with refractory disease.

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.

Mapping of proteomic profile and effect of the spongy layer in the human amniotic membrane.

The graft of human amniotic membrane (HAM) contributes to the healing of corneal perforating ulcers and so to save a large number of eyes suffering of severe chemical burns. This biological material is used for the treatment of ocular surface diseases because of its capacity to reduce inflammation and promote a quicker wound healing. For clinical use, the HAM is denuded from its spongy layer, but this layer can be an important source of growth factors which promote re-epithelialization. The aim of our study is to provide a general view of protein expression of the HAM and the spongy layer and therefore to determine if the spongy layer and/or a specific part of HAM have a beneficial role in the process of wound healing in patients with corneal ulcers. For this study, human placentas were obtained from healthy women after vaginal delivery or caesarean section after signing the consent form. Mapping of protein expression is done by dividing the placenta in 2 equal parts, one with spongy layer and another without (conventional HAM). Each part is also divided in 3 zones depending on the distance from the umbilical cord. The proteomic analysis was done by ELISA, targeting growth factors (EGF, HGF, KGF, NGF and TGF-beta1) and pro inflammatory cytokine TNF-α in the HAM without spongy layer and in the spongy layer. In this study we observed significant difference in the total amount of protein extract between the different donors. We do not observe a significant difference in the growth factor level between the conventional HAM and the spongy layer. No variation was observed in the expression of HGF, KGF and NGF in different zone of HAM and neither between conventional HAM and spongy layer in each zone. (*p value < 0.05, **p value<0.01,***p value < 0.001). We do detect very low dose of TNF-α and no correlation with the amount of growth factors. In our study we demonstrated that keeping the spongy layer in conventional method of handling HAM can add more GF, and so probably have a positive affect the wound healing process. Variation in some growth factors expression has been observed between the placentas and therefore this may explain the variation in clinical results. No indicator for the selection of placentas with a higher rate of growth factor was found.

Hyaluronan-based hydrogels as versatile tumor-like models: Tunable ECM and stiffness with genipin-crosslinking.

Three-dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan-based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre-functionalized our hydrogels with an adhesive polypeptide (poly-l-lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre-functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL-treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness-dependent manner. Contrary to type III collagen, type IV collagen pre-treated hydrogels supported the proliferation of glioblastoma cells. The as-desired HA-based 3D tumor-like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.

HLA-Class II Artificial Antigen Presenting Cells in CD4+ T Cell-Based Immunotherapy.

CD4+ T cells differentiate into various T helper subsets characterized by distinct cytokine secreting profiles that confer them effector functions adapted to a variety of infectious or endogenous threats. Regulatory CD4+ T cells are another specialized subset that plays a fundamental role in the maintenance of immune tolerance to self-antigens. Manipulating effector or regulatory CD4+ T cells responses is a promising immunotherapy strategy for, respectively, chronical viral infections and cancer, or severe autoimmune diseases and transplantation. Adoptive cell therapy (ACT) is an emerging approach that necessitates defining robust and efficient methods for the in vitro expansion of antigen-specific T cells then infused into patients. To address this challenge, artificial antigen presenting cells (AAPCs) have been developed. They constitute a reliable and easily usable platform to stimulate and amplify antigen-specific CD4+ T cells. Here, we review the recent advances in understanding the functions of CD4+ T cells in immunity and in immune tolerance, and their use for ACT. We also describe the characteristics of different AAPC models and the way to improve their stimulating functions. Finally, we discuss the potential interest of these AAPCs, both as fundamental tools to decipher CD4+ T cell responses and as reagents to generate clinical grade antigen-specific CD4+ T cells for immunotherapy.

The IgG2 Isotype of Anti-Transcription Intermediary Factor 1γ Autoantibodies Is a Biomarker of Cancer and Mortality in Adult Dermatomyositis.

OBJECTIVE: Anti-transcription intermediary factor 1γ (anti-TIF1γ) antibodies are the main predictors of cancer in dermatomyositis (DM). Yet, a substantial proportion of anti-TIF1γ-positive DM patients do not develop cancer. This study was undertaken to identify biomarkers to better evaluate the risk of cancer and mortality in DM. METHODS: This multicenter study was conducted in adult anti-TIF1γ-positive DM patients from August 2013 to August 2017. Anti-TIF1γ autoantibody levels and IgG subclasses were identified using a newly developed quantitative immunoassay. Age, sex, DM signs and activity, malignancy, and creatine kinase (CK) level were recorded. Risk factors were determined by univariate and multivariate analysis according to a Cox proportional hazards regression model. RESULTS: Among the 51 adult patients enrolled (mean ± SD age 61 ± 17 years; ratio of men to women 0.65), 40 (78%) had cancer and 21 (41%) died, with a mean ± SD survival time of 10 ± 6 months. Detection of anti-TIF1γ IgG2 was significantly associated with mortality (P = 0.0011) and occurrence of cancer during follow-up (P < 0.0001), with a 100% positive predictive value for cancer when the mean fluorescence intensity of anti-TIF1γ IgG2 was >385. None of the patients developed cancer after 24 months of follow-up. Univariate survival analyses showed that mortality was also associated with age >60 years (P = 0.0003), active DM (P = 0.0042), cancer (P = 0.0031), male sex (P = 0.011), and CK level >1,084 units/liter (P = 0.005). Multivariate analysis revealed that age >60 years (P = 0.015) and the presence of anti-TIF1γ IgG2 (P = 0.048) were independently associated with mortality. CONCLUSION: Our findings indicate that anti-TIF1γ IgG2 is a potential new biomarker of cancer that should be helpful in identifying the risk of mortality in anti-TIF1γ-positive DM patients.