NFAT5 contributes to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and decrease of T regulatory cells in female mice.

Multiple sclerosis disproportionally affects women. The present study was undertaken to determine whether NFAT5 contributed to the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and if it did, whether the impact was sex associated. NFAT5 haplodeficiency reduced the disease severity only in female mice. This effect was associated with significant increases in frequency of T regulatory (Treg) cells in the CNS (from 1.45 ± 0.39% to 3.73 ± 0.94%) and spleen from (0.31 ± 0.06% to 0.94 ± 0.29%) without significantly affecting the CNS CD4+ subsets frequency. NFAT5 haploinsufficiency also significantly reduced the frequency of CD11c+CD8α+ dendritic cells in the female CNS. However, increase of their frequency in the CNS via intraperitoneal Flt3L injection at peak EAE had no significant effect on the disease courses. We conclude that NFAT5 contributes to pathogenesis of EAE in female mice, possibly through decreasing tissue specific frequency of Treg cells.

The role of pirfenidone in the treatment of interstitial pneumonia with autoimmune features.

OBJECTIVES: No approved pharmacotherapies are available for patients with interstitial pneumonia with autoimmune features (IPAF). In the present work, we aimed to evaluate the efficacy and safety of pirfenidone for the treatment of IPAF. METHODS: A retrospective cohort study consisting of patients who met diagnostic criteria for IPAF was performed after a multidisciplinary review, and the patients receiving pirfenidone were compared with those in the non-pirfenidone group. The baseline data and diagnostic characteristics of patients were assessed. Pulmonary function and prednisone dose were analysed by a mix-effects model. RESULTS: A total of 184 patients, who met the diagnostic criteria of IPAF, were divided into two groups: pirfenidone group (n=81) and non-pirfenidone group (n=103). Patients in the pirfenidone group had a lower forced vital capacity (FVC%, p<0.001) and a lower diffusion capacity for carbon monoxide (DLCO%, p=0.003). The pirfenidone group exhibited a greater increase of FVC% at 6 (p=0.003), 12 (p=0.013), and 24 (p=0.003) months. After adjustment for sex, age, UIP pattern, baseline FVC% and DLCO%, patients in the pirfenidone group continued to show a greater improvement in FVC% (χ2(1)=4.59, p=0.032). Subgroup analysis identified superior therapeutic effects of pirfenidone in patients with dosage >600 mg/day (p=0.010) and medication course >12 months (p=0.007). Besides, the pirfenidone group had a lower prednisone dose than the non-pirfenidone group after 12 months of treatment (p=0.002). Moreover, 17 patients (19.32%) experienced side effects after taking pirfenidone, including one case of anaphylactic shock. CONCLUSIONS: Pirfenidone (600-1,800 mg/day) might help improve FVC, with an acceptable safety and tolerability profile in IPAF patients.

Engineered regulatory T cells expressing myelin-specific chimeric antigen receptors suppress EAE progression.

The expansion of polyclonal T regulatory cells (Tregs) offers great promise for the treatment of immune-mediated diseases, such as multiple sclerosis (MS). However, polyclonal Tregs can be non-specifically immunosuppressive. Based on the advancements with chimeric antigen receptor (CAR) therapy in leukemia, we previously engineered Tregs to express a T-cell receptor (TCR) specific for a myelin basic protein (MBP) peptide. These TCR-engineered specific Tregs suppressed the proliferation of MBP-reactive T effector cells and ameliorated myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Herein, we extend this approach by creating human regulatory T cells expressing functional single-chain chimeric antigen receptors (scFv CAR), targeting either MBP or MOG. These scFv CAR-transduced Tregs retained FoxP3 and Helios, characteristic of Treg cells, after long-term expansion in vitro. Importantly, these engineered CNS targeting CAR-Tregs were able to suppress autoimmune pathology in EAE, demonstrating that these Tregs have the potential to be used as a cellular therapy for MS patients.

Targeting Antigen-Specific B Cells Using Antigen-Expressing Transduced Regulatory T Cells.

Controlling immune responses in autoimmunity and to biotherapeutics is an unmet need. In hemophilia, for example, up to one third of patients receiving therapeutic factor VIII (FVIII) infusions develop neutralizing Abs termed "inhibitors." To address this problem in a mouse model of hemophilia A, we used an Ag-specific regulatory T cell (Treg) approach in which we created a novel B cell-targeting chimeric receptor composed of an FVIII Ag domain linked with the CD28-CD3ζ transmembrane and signaling domains. We termed these "BAR" for B cell-targeting Ab receptors. CD4+CD25hiCD127low human Tregs were retrovirally transduced to express a BAR containing the immunodominant FVIII C2 or A2 domains (C2- and A2-BAR). Such BAR-Tregs specifically suppressed the recall Ab response of spleen cultures from FVIII-immunized mice in vitro and completely prevented anti-FVIII Ab development in response to FVIII immunization. Mechanistic studies with purified B cells and T cells from tolerized or control recipients demonstrated that the FVIII-specific B cells were directly suppressed or anergized, whereas the T cell response remained intact. Taken together, we report in this study a successful proof-of-principle strategy using Ag-expressing Tregs to directly target specific B cells, an approach which could be adapted to address other adverse immune responses as well.

Engineered ovalbumin-expressing regulatory T cells protect against anaphylaxis in ovalbumin-sensitized mice.

Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells, and specifically block effector cell responses. Our lab recently developed antigen-specific regulatory T-cell (Treg) therapies as a treatment for adverse responses. Recently, we created a chimeric antigen receptor (CAR) approach in which we engineered a target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells and sensitized mast cells in an allergy model. We named this receptor "BAR" for B-cell Antibody Receptor. Murine or human Tregs, transduced with a BAR containing OVA or control Tregs expressing an unrelated antigen, were successfully expanded in vitro and tested in the murine OVA-alum allergy model with measurable titers of anti-OVA IgE. Because BAR Tregs express the target antigen and could interact with specific IgE on sensitized mast cells, we first demonstrated that intravenously injected OVA-BAR Tregs did not directly lead to a drop in temperature or release of mediators in plasma indicative of anaphylaxis. Forty-eight hours later, mice were challenged intraperitoneally with 200 µg OVA to induce an anaphylactic reaction, and temperature immediately measured for 30 min. We found that OVA-BAR Tregs protected mice from hypothermia, whereas mice given control BARs (expressing an unrelated antigen) or PBS showed substantial temperature drops indicative of anaphylaxis when systemically challenged with OVA. Importantly, this effect was also demonstrated in a passive anaphylaxis model in which mice that received anti-OVA IgE antibody were protected from hypothermia when treated with OVA-BAR Tregs prior to systemic OVA challenge. These results provide proof of principle that engineered allergen-specific T-regulatory cells can provide clinical protection against severe allergic reactions in individuals already IgE-sensitized to an allergen.

FVIII-specific human chimeric antigen receptor T-regulatory cells suppress T- and B-cell responses to FVIII.

Replacement therapy with factor VIII (FVIII) is used in patients with hemophilia A for treatment of bleeding episodes or for prophylaxis. A common and serious problem with this therapy is the patient's immune response to FVIII, because of a lack of tolerance, leading to the formation of inhibitory antibodies. Development of tolerogenic therapies, other than standard immune tolerance induction (ITI), is an unmet goal. We previously generated engineered antigen-specific regulatory T cells (Tregs), created by transduction of a recombinant T-cell receptor (TCR) isolated from a hemophilia A subject's T-cell clone. The resulting engineered T cells suppressed both T- and B-cell effector responses to FVIII. In this study, we have engineered an FVIII-specific chimeric antigen receptor (ANS8 CAR) using a FVIII-specific scFv derived from a synthetic phage display library. Transduced ANS8 CAR T cells specific for the A2 domain proliferated in response to FVIII and ANS8 CAR Tregs were able to suppress the proliferation of FVIII-specific T-effector cells with specificity for a different FVIII domain in vitro. These data suggest that engineered cells are able to promote bystander suppression. Importantly, ANS8 CAR-transduced Tregs also were able to suppress the recall antibody response of murine splenocytes from FVIII knockout mice to FVIII in vitro and in vivo. In conclusion, CAR-transduced Tregs are a promising approach for future tolerogenic treatment of hemophilia A patients with inhibitors.

Engineered MBP-specific human Tregs ameliorate MOG-induced EAE through IL-2-triggered inhibition of effector T cells.

Expanded polyclonal T regulatory cells (Tregs) offer great promise for the treatment of immune-mediated diseases. Inhibition by Tregs is under the control of the T-cell receptor (TCR). Therefore, we created Tregs with defined antigen specificity, using a recombinant T-cell receptor isolated from a myelin-basic protein specific T-cell clone of a multiple sclerosis (MS) patient (Ob2F3). We expressed this TCR using a retroviral expression vector in human Tregs from peripheral blood. We observed that transduced Tregs were activated in vitro in response to myelin basic protein (MBP) peptide on DR15 antigen-presenting cells (APC) and upregulated Treg markers, Foxp3, LAP and Helios. These engineered MBP-specific Tregs could suppress MBP-specific T effector cells, and were also able to suppress T cells with other specificities after Tregs had been activated through the TCR. Importantly, we showed that these engineered Tregs were able to function effectively in the presence of strong TLR-induced inflammatory signals, and that MBP-specific Tregs ameliorated EAE in myelin oligodendrocyte glycoprotein (MOG)-immunized DR15 transgenic mice. We further demonstrated in vitro that IL-2 produced by neighboring effector T cells activated MBP-specific Tregs, initiating contact-independent suppression to T effectors in local milieu. Mechanistic studies demonstrated that bystander suppression in vivo may involve transfer of soluble mediators, enhanced by cell contact between Tregs and effectors. Taken together, we show that engineered clonal MBP-specific Tregs are able to suppress autoimmune pathology in EAE. This approach may serve as a cellular therapy for MS patients with the common DR15 haplotype that is associated with disease susceptibility.

Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance.

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses.

Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject's T-cell clone, into expanded human FoxP3(+) Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients.

Tolerogenic nanoparticles to induce immunologic tolerance: Prevention and reversal of FVIII inhibitor formation.

The immune response of hemophilia A patients to administered FVIII is a major complication that obviates this very therapy. We have recently described the use of synthetic, biodegradable nanoparticles carrying rapamycin and FVIII peptide antigens, to induce antigen-specific tolerance. Herein we test the tolerogenicity of nanoparticles that contains full length FVIII protein in hemophilia A mice, focusing on anti-FVIII humoral immune response. As expected, recipients of tolerogenic nanoparticles remained unresponsive to FVIII despite multiple challenges for up to 6 months. Furthermore, therapeutic treatments in FVIII-immunized mice with pre-existing anti-FVIII antibodies resulted in diminished antibody titers, albeit efficacy required longer therapy with the tolerogenic nanoparticles. Interestingly, durable FVIII-specific tolerance was also achieved in animals co-administered with FVIII admixed with nanoparticles encapsulating rapamycin alone. These results suggest that nanoparticles carrying rapamycin and FVIII can be employed to induce specific tolerance to prevent and even reverse inhibitor formation.

Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor.

Introduction: Solid cancers Myeloid cells are prevalent in solid cancers, but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME), which hinders the effectiveness of cancer immunotherapies. Myeloid cells' natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers, including tumor infiltration, tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1, which is often upregulated by solid cancers to evade immune responses. Method: Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1, while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells, we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally, we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy's effectiveness in vivo. Result: Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1ß. Moreover, CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors, infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Conclusion: Taken together, these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers.

The highly selective and sensitive fluorescence probe for detection of copper (II) ions and its bioimaging in vitro and vivo.

We designed and developed the probe W-3 for detection of Cu2+. The results showed probe can selectively detect Cu2+, accompanied by noticeable color change. The probe can detect the Cu2+ in water samples and drinks based on absorption detection. In addition, the combination of portable test paper and the smartphone platform obtained great convenience for on-site and visual detection of Cu2+, with satisfactory sensitivity and reliability. More importantly, the fluorescence probe W-3 can be used for the detection of Cu2+ in cells and mice. Therefore, the W-3 provided potential chemical tools for detecting Cu2+ in vitro and vivo.

A fluorescence probe with targeted mitochondria for detecting hydrogen peroxide in vitro and in diabetic mice.

We designed and prepared probe W-1 for the detection of H2O2. W-1 showed excellent selectivity for H2O2 and was accompanied by colorimetric signal changes. The excellent linear relationship between fluorescence intensity and H2O2 concentration (0-100 µM) provided favorable conditions for its quantitative detection. In addition, the combination of portable test strips with a smartphone platform provided great convenience for on-site visual detection of H2O2. Moreover, W-1 possessed targeting mitochondria property and could be applied to image the exogenous and endogenous H2O2 in cells to distinguish normal cells and cancer cells. Lastly, W-1 was used for monitoring the H2O2 fluctuation of the diabetic process in mice, and the results showed an increase in H2O2 levels in diabetes. Therefore, the probe provided a tool for understanding the pathological and physiological mechanisms of diabetes by imaging H2O2.

Development of the near-infrared fluorescence probe for detection of ONOO- and its application in vitro and vivo.

Diabetes has become one of the most common endocrine and metabolic diseases threatening human health, which can induce mitochondrial dysfunction and exacerbate the excessive production of reactive oxygen species (ROS). Among them, ONOO- level fluctuation was closely related to diabetes. Hence, it is of great significance to develop a near-infrared fluorescence probe for visualizing ONOO- level fluctuations in diabetes. In this paper, we constructed a fluorescence probe YBL with dicyano-isophorone derivative as fluorophore and diphenyl phosphate as ONOO- response site, which can detect ONOO- with the low detection limit (39.8 nM) and exhibit excellent selectivity and sensitivity. The probe YBL has been applied to monitor intracellular ONOO- level fluctuations. Meanwhile, the image results showed that high sugar promoted the increase of ONOO- level in cells. More important, the probe YBL can be used for imaging in mice, and the results showed that content of ONOO- was increased in diabetic mice. Therefore, the probe YBL provided a tool for understanding diabetes progression by imaging ONOO-.

Multifunctional fluorescence probe for simultaneous detection of viscosity, polarity, and ONOO- and its bioimaging in vitro and vivo.

Intracellular microenvironment (viscosity and polarity) and peroxynitrite ions (ONOO-) are involved in maintaining cell morphology, cell function, and signaling so that it is crucial to explore their level changes in vitro and vivo. In this work, we designed and synthesized a mitochondria-targeted fluorescence probe XBL for monitoring the dynamic changes of viscosity, polarity, and ONOO- based on TICT and ICT mechanism. The fluorescence spectra showed obvious changes for polarity at 500 nm as well as ONOO- and viscosity at 660 nm, respectively. The XBL can image simultaneously viscosity, polarity, and ONOO- in cells, and the results showed excess ONOO- leaded to the increase of viscosity in mitochondrial. The ferroptosis process was accompanied by increase of intracellular viscosity and ONOO- levels (or decrease of polarity), which allowed us to better understand the relevant physiological and pathological processes. The XBL can distinguish normal cells and cancerous cells by the fluorescence intensity changes in green and red channels, and image viscosity in inflamed mice. Thus, XBL can provided the chemical tool to understand the physiological and pathological mechanisms of disease by simultaneous detection of viscosity, polarity and ONOO-.

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice.

We herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.

Role of autophagy in acute myeloid leukemia therapy.

Despite its dual role in determining cell fate in a wide array of solid cancer cell lines, autophagy has been robustly shown to suppress or kill acute myeloid leukemia cells via degradation of the oncogenic fusion protein that drives leukemogenesis. However, autophagy also induces the demise of acute leukemia cells that do not express the known fusion protein, though the molecular mechanism remains elusive. Nevertheless, since it can induce cooperation with apoptosis and differentiation in response to autophagic signals, autophagy can be manipulated for a better therapy on acute myeloid leukemia.

Survival of 48866 cancer patients: results from Nantong area, China.

Objective: This study aimed to provide a realistic observation of survival by major site for 48,866 cancer patients treated at a tertiary cancer hospital in a rural area of China. Methods: Patients with cancer registered between 2007 and 2017 in the Nantong rural area were followed up. The starting date for survival calculation was the date of the first diagnosis of cancer at the Nantong Tumor Hospital, and the closing date was December 31, 2020. Observed survival (OS) was analyzed according to ICD-10 site, sex, age, region, and hospitalization period using the life table method and compared using the Wilcoxon (Gehan) statistic. Results: The overall 5-year OS rate was 40.48% for all 48,866 patients, 30.19% for males, and 51.90% for females. The top five cancer sites, accounting for 60.51% of the total cases, were the esophagus, lung, stomach, liver, and cervix, with 5-year OS rates of 33.72%, 18.64%, 32.10%, 19.04%, and 71.51%, respectively. The highest 5-year OS was observed in the thyroid (87.52%) and the lowest was in the pancreas (6.37%). Survival was significantly higher in younger patients than in older patients, with 5-year OSs of 69.26% and 19.84% in those aged 20-29 and 90-99 years, respectively. Five-year OSs improved significantly from 39.35% in 2007-2011 to 41.26% in 2012-2017. Conclusion: Overall survival improved over the years, although the improvement at some sites was not significant. The observed survival varies from region to region, reflecting differences in the patterns of major sites, disparities in proportions of hospitalization, and demographic characteristics.

Preclinical In Vitro and In Vivo Models for Adoptive Cell Therapy of Cancer.

ABSTRACT: Adoptive cellular therapies are making major strides in the treatment of cancer, both for hematologic and solid tumors. These cellular products include chimeric antigen receptor T cells and T-cell receptor-modified T cells, tumor-infiltrating lymphocytes, marrow-infiltrating T cells, natural killer cells as well as macrophage-based therapeutics. Advancement in genomics, computational biology, immunology, and cell therapy manufacturing has facilitated advancement of adoptive T cell therapies into the clinic, whereas clinical efficacy has driven Food and Drug Administration approvals. The growth of adoptive cellular therapy has, in turn, led to innovation in the preclinical models available, from ex vivo cell-based models to in vivo xenograft models of treatment. This review focuses on the development and application of in vitro models and in vivo models (cell line xenograft, humanized mice, and patient-derived xenograft models) that directly evaluate these human cellular products.

B cells "transduced" with TAT-fusion proteins can induce tolerance and protect mice from diabetes and EAE.

Antigen-immunoglobulin fusion protein expressing B cells have been shown as excellent tolerogenic antigen-presenting cells in multiple disease models. Using efficient protein transduction by fusion with a HIV TAT protein transduction domain, we herein tested the TAT-fusion protein transduced B cells for their effects in antigen-specific tolerance induction in two animal models, experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes. We demonstrated that transfer of TAT-MOG35-55 (myelin oligodendrocyte glycoprotein)-Ig 'transduced B cells' 10 days after EAE induction significantly protected mice from disease. Similarly, the onset of disease was delayed when NOD mice received insulin specific TAT-B9-23-B cells. Surprisingly, no protection against EAE was observed in a prophylactic protocol when transduced B cells were given before disease induction. Moreover, TAT-ovalbumin transduced cells were tolerogenic in primed but not naïve mice. Our results suggest that TAT-fusion protein transduced B cells were tolerogenic in antigen primed recipients, a condition clinically relevant to autoimmune diseases.