IFN-Gamma and TNF-Alpha as a Priming Strategy to Enhance the Immunomodulatory Capacity of Secretomes from Menstrual Blood-Derived Stromal Cells

Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.

Enhancement of Neuroblastoma NK-Cell-Mediated Lysis through NF-kB p65 Subunit-Induced Expression of FAS and PVR, the Loss of Which Is Associated with Poor Patient Outcome

High-risk neuroblastoma (NB) is a rare childhood cancer whose aggressiveness is due to a variety of chromosomal genetic aberrations, including those conferring immune evasion. Indeed, NB cells adopt several molecular strategies to evade recognition by the immune system, including the downregulation of ligands for NK-cell-activating receptors. To date, while molecular strategies aimed at enhancing the expression of ligands for NKG2D- and DNAM-1-activating receptors have been explored, no evidence has been reported on the immunomodulatory mechanisms acting on the expression of death receptors such as Fas in NB cells. Here, we demonstrated that transient overexpression of the NF-kB p65 subunit upregulates the surface expression of Fas and PVR, the ligand of DNAM-1, thus making NB cell lines significantly more susceptible to NK-cell-mediated apoptosis, recognition, and killing. In contrast, IFNγ and TNFα treatment, although it induced the upregulation of FAS in NB cells and consequently enhanced NK-cell-mediated apoptosis, triggered immune evasion processes, including the strong upregulation of MHC class I and IDO1, both of which are involved in mechanisms leading to the impairment of a proper NK-cell-mediated killing of NB. In addition, high-resolution array CGH analysis performed in our cohort of NB patients revealed that the loss of FAS and/or PVR genes correlated with low survival independently of the disease stage. Our data identify the status of the FAS and PVR genes as prognostic biomarkers of NB that may predict the efficacy of NK-cell-based immunotherapy of NB. Overall, restoration of surface expression of Fas and PVR, through transient upregulation of NF-kB, may be a clue to a novel NK-cell-based immunotherapy of NB.

The Synergistic Effect of Corticosteroids and Mycophenolic Acid on Chemokines in Orbital Cells From Patients With Graves’ Ophthalmopathy

In recent studies, an improvement of the response rate to therapy has been reported with corticosteroids and mycophenolic acid in patients with Graves’ ophthalmopathy (GO). In GO, retro-orbital cells (fibroblasts, preadipocytes, and extraocular muscle cells) secrete Th1 and Th2 chemokines stimulated by cytokines. Until now, no studies are present in literature regarding the effect of corticosteroids and mycophenolic acid on the secretion of chemokines in GO orbital cells. For this reason, the effect of increasing concentrations of mycophenolic acid or corticosteroids on the secretion of either the Th1 (CXCL10) and Th2 (CCL2) chemokines was tested in primary cultures of myoblasts, preadipocytes and fibroblasts obtained from GO patients. CXCL10 was undetectable in the supernatants of the retro-orbital cells in primary cultures; its release was induced dose-dependently by IFNγ, while TNFα alone had no effect. On the contrary CCL2 release (that was produced in low amounts basally) was dose-dependently induced by TNFα, while IFNγ alone had no effect. In both cases the combination of TNFα and IFNγ had a significant synergistic effect on CXCL10 and CCL2 secretion. The release of these chemokines was dose-dependently inhibited by increasing concentrations of mycophenolic acid, or corticosteroids (in a pharmacological range), in presence of IFNγ and TNFα stimulation. Moreover, the association of corticosteroids and mycophenolic acid (in presence of IFNγ and TNFα) had a stronger inhibitory effect on the chemokines release. In conclusion, in GO orbital cells, mycophenolic acid and/or corticosteroids (in a pharmacological range) have an inhibitory role on the secretion of both Th1 (CXCL10) and Th2 (CCL2) chemokines. This suggests a possible therapeutic role of these drugs.

EV PD-L1 Contributes to Immunosuppressive CD8+ T Cells in Peripheral Blood of Pediatric Wilms Tumor

Wilms tumor (WT) is the most common renal cancer and the most prevalent abdominal cancer in children. Children with recurrent or progressive forms of WT could benefit from novel immune-targeted approaches. While the immune status of these patients, especially the immunosuppression of peripheral T cells, was rarely reported. The present study enrolled a consecutive series of 14 Chinese WT children and 14 age- and gender-matched healthy controls. We demonstrated that plasma extracellular vesicular (EV) PD-L1 levels significantly increased in WT patients than in healthy controls. EV PD-L1 significantly inhibited the activation of human CD8+ T cells by down-regulating the cell surface CD69 expression and the intracellular IFNγ and TNFα production in vitro. In peripheral CD8+ T cells of WT patients, the intracellular IFNγ and TNFα production significantly decreased than healthy controls. The level of plasma EV PD-L1 significantly correlated with the intracellular TNFα production in peripheral CD8+ T cells of WT patients. In conclusion, the significantly increased plasma EV PD-L1 in WT patients contributed to the immunosuppression of peripheral CD8+ T cells. Monitoring the level of plasma EV PD-L1 will be helpful for the selection of immune-targeted therapies for WT patients.

Defective CD19+CD24hiCD38hi transitional B-cell function in patients with relapsing-remitting MS

Background: Multiple sclerosis (MS) is characterized by central nervous system (CNS) infiltration of T and B cells, excess inflammatory cytokine and chemokine production and failure of immune regulation. CD19+CD24hiCD38hi transitional B cells producing interleukin (IL)-10 have been shown to suppress interferon-γ (IFNγ) and tumour necrosis factor-α (TNFα) production by CD4+ T cells and to be dysfunctional in autoimmune arthritis and systemic lupus erythematosus. Objective: We hypothesized that transitional B-cell-dependent immune regulation could be defective in MS and examined their function in healthy subjects and patients with relapsing-remitting multiple sclerosis (RRMS). Methods: A total of 62 healthy donors and 21 RRMS subjects donated peripheral blood for the study. IL-10-producing B cells, IFNγ and TNFα-producing T cells and proliferating T cells were quantified by flow cytometry. Results: In healthy individuals, CD19+CD24hiCD38hi transitional B cells produce more IL-10 than CD19+CD24+CD38+ naive and CD19+CD24hiCD38− memory B cells and are able to suppress CD4+ T-cell proliferation and IFNγ and TNFα-production. In subjects with RRMS, CD19+CD24hiCD38hi transitional B cells produce significantly less IL-10 and to fail to suppress effector T-cell function. Conclusion: CD19+CD24hiCD38hi transitional B cells physiologically represent the most potent regulatory B-cell subset and are functionally defective in patients with RRMS, an abnormality that may contribute to the immune pathological process.

Lymphocyte senescence in COPD is associated with decreased sirtuin 1 expression in steroid resistant pro-inflammatory lymphocytes

Background: The class III NAD-dependent histone deacetylase (HDAC) sirtuin 1 (SIRT1) is an important regulator of senescence, aging, and inflammation. SIRT1de-acetylates chromatin histones, thereby silencing inflammatory gene transcription. We have reported increased steroid-resistant senescent pro-inflammatory CD28nullCD8+ T cells in patients with chronic obstructive pulmonary disease (COPD). We hypothesized that SIRT1 is reduced in these cells in COPD, and that treatment with SIRT1 activators (resveratrol, curcumin) and agents preventing NAD depletion (theophylline) would upregulate SIRT1 and reduce pro-inflammatory cytokine expression in these steroid-resistant cells. Methods: Blood was collected from n = 10 COPD and n = 10 aged-matched controls. Expression of CD28, SIRT1, and pro-inflammatory cytokines was determined in CD8+ and CD8– T and natural killer T (NKT)-like cells cultured in the presence of ±1 µM prednisolone, ±5 mg/L theophylline, ±1 µM curcumin, ±25 µM resveratrol, using flow cytometry and immunofluorescence. Results: There was an increase in the percentage of CD28nullCD8+ T and NKT-like cells in COPD patients compared with controls. Decreased SIRT1 expression was identified in CD28nullCD8+T and NKT-like cells compared with CD28+ counterparts from both patients and controls (e.g. CD28null 11 ± 3% versus CD28+ 57 ± 9%). Loss of SIRT1 was associated with increased production of IFNγ and TNFα, steroid resistance, and disease severity. SIRT1 expression was upregulated in the presence of all drugs and was associated with a decrease in steroid resistance and IFNγ and TNFα production by CD28nullCD8+T and NKT-like cells. The presence of the SIRT1 inhibitor, EX-527 negated [by 92 ± 12% (median ± SEM)] the effect of the SIRT1 activator SRT720 on the percentage of CD8+ T cells producing IFNγ and TNFα. Conclusions: Steroid resistance in pro-inflammatory CD28nullCD8+ T and NKT-like cells is associated with decreased SIRT1 expression. Treatment with prednisolone, in combination with theophylline, curcumin or resveratrol increases SIRT1 expression, restores steroid sensitivity, and inhibits pro-inflammatory cytokine production from these cells and may reduce systemic inflammation in COPD. The reviews of this paper are available via the supplemental material section.

IL-33 Therapy Prevents Acute Lung Injury after Transplantation Via IL-9-Producing Type 2 Innate Lymphoid Cells Induction

Idiopathic pneumonia syndrome (IPS) is a noninfectious acute lung injury, often fatal, following allogeneic hematopoietic cell transplantation (HCT). Similar to graft-versus-host disease (GVHD), IPS is mediated by type 1 cytopathic T cells accompanied with high levels of proinflammatory cytokines. We previously showed that elevated plasma soluble Stimulation-2 (sST2), which acts as a decoy receptor for IL-33, is a risk factor of death by GVHD (N. Engl. J. Med, 2013) or by IPS (Biol Blood Marrow Transplant, 2018). ST2 blockade of the excess of sST2 with a neutralizing antibody or small molecules released plasmatic IL-33, increasing its availability to cytoprotective T cells expressing the transmembrane molecule form of ST2, such as regulatory T cells (Tregs) reducing the type 1 proinflammatory T cell-response (Sci Transl Med, 2015; JCI Insight, 2019). The membrane-ST2 is also expressed on type 2 innate lymphoid cells (ILC2s), mostly present in lungs. Herein, we first confirmed in a cohort of 673 HCT patients that plasma sST2 measured 14 days following HCT is increased 10 fold in IPS patients (n=22) as compared to controls with no IPS/GVHD (n=271), and is 6 fold higher as compared to GVHD patients (n=380) (Figure 1A). Patients with IPS and high sST2 levels above the median of 200 ng/ml, were significantly more likely to die than patients with lower sST2 levels (Figure 1B). In a therapeutic translational purpose, we then inquired if local administration of IL-33 via intranasal route at a dose of 500 ng/mouse daily (5 doses from day -1 to +3) will ameliorate the recipients' pulmonary function tests in a major-mismatched B6 → Balb/c HCT murine model. Allogeneic recipients that received IL-33 improved their lung compliance (C), lung resistance (R), and elastance (E) as compared to vehicle treated mice (Figure 2A). Based on our patients' data, we further explored the sST2/IL-33 ratio. Although the treatment was local, plasma IL-33 increased at day +7 post-HCT and therefore the sST2/IL-33 ratio was significantly decreased in IL-33 treated mice (Figure 2B). Parraleling this decrease, both systemic IFNγ and TNFα at day +7 post-HCT were significantly lower in mice treated with IL-33 compared to vehicle treated mice (Figure 2B). Findings in the plasma were also correlated with a local decrease of IFNγ secretion in the bronchoalveolar lavage of IL-33 treated mice (not shown). The frequencies and numbers of donor CD45.1+ IFNg+CD4+ and IFNg+CD8+ donor T cells in the lungs of IL-33 treated mice were also significantly decreased as compared to vehicle treated mice (Figure 2C). We next sought to determine if IL-33 had an impact on recipient ILC2s (CD45.2+Lin-CD90.2+GATA3+ST2+). As shown in Figure 2D, recipient mice treated with IL-33 had significant higher frequencies of lung ILC2s at day +7 post-HCT compared to mice treated with vehicle. RNA-seq analysis of sorted ILC2s from the lungs of naïve GATA3 reporter mice treated with IL-33 showed increased Il9 and PU.1 transcripts in lung ILC2s, validated at the protein level in allogeneic mice treated with IL-33 as compared to allogeneic vehicle treated mice in which ILC2s were undetectebale (Figure 2D). As antibody (Ab) injection is more clinically relevant than local cytokine instillation, and since we have shown that anti-ST2 Ab results in IL-33 increase, we tested this in a minor-mismatched B6 → C3H.SW HCT murine model, and respectively treated mice with anti-ST2 Ab 100μg/dose every other day (6 doses total) or anti-ST2 Ab 200μg/dose for 2 doses at days -1 and +1 or isotype 100μg/dose for 6 doses. Prophylactic administration of anti-ST2 Ab with 6 doses and 2 doses significantly decreases mortality as compared to isotype with six doses allowing a better survival than the peritransplant administration (Figure 3A). Plasma IL-33 increased in both anti-ST2 treated groups vs. isotype (Figure 3B). Consistently, both plasma IFNγ and TNFα were significantly decreased in anti-ST2 Ab treated groups (Figures 3C, 3D). Percentages of cytopathic lung donor CD4+IFNγ+ and CD8+IFNγ+ T cells were decreased (Figure 3E) while cytoprotective lung recipient total, IL-9+, and PU.1+ ILC2s were increased in anti-ST2 Ab treated groups vs. isotype (Figures 3F, 3G). Tregs in both anti-ST2 Ab treated groups were concomitantly increased (Figure 3H). We concluded that not only is sST2 a prognostic biomarker for IPS but it is also a promising therapeutic target that may prevent IPS via IL-33 induced IL-9 secreting ILC2s. Disclosures Paczesny: Viracor Eurofins Clinical Diagnostic: Patents & Royalties.

P12.03 Bi-specific T cell engagers targeting IL13Rá2 activate tumor-infiltrating lymphocytes and improve survival in pre-clinical models of glioblastoma

BACKGROUND The outstanding efficacy of bi-specific T-cell engagers (BiTE against hematological malignancies offers hope that they can similarly target solid tumors like GBM. In this study, we have designed a BiTE protein with specificity to the tumor-associated antigen, IL13Rα2, and investigated how BiTE protein engages a host’s T cell immune response to promote anti-glioma activity in pre-clinical models of GBM. MATERIAL AND METHODS BiTE molecule consisting of two single chain variable regions (scFv) of antibodies against either murine or human CD3ε and scFv47 against human IL13Rα2 connected through a flexible linker (BiTEIL13Rα2) were sub-cloned in a lentiviral expression cassette. The BiTE molecule (BiTEIL13Rα2off) modified to abrogate the interaction of scFv47 with IL13Rα2 served as a negative control. The BiTE proteins were isolated from the supernatants of HEK293T cells using His-Tag affinity chromatography and validated in SDS-PAGE, Western Blot, and ELISA assays. The BiTEIL13Rα2-induced T cells activation was measured in (i) cytotoxicity assay against IL13Rα2+ glioma cells, (ii) flow cytometry measuring for CD69 and CD25 T cells’ activation markers, and (iii) the production of cytokines, IFNγ and TNFα. For in vivo analysis, VmDk and C57Bl/6 mice bearing established intracranial glioma were treated systemically with BiTE proteins. The survival of the mice was recorded and analyzed using the log-rank test. RESULTS Here we show that BiTEIL13Rα2 specifically binds to IL13Rα2 but not to IL13Rα1, whereas BiTEIL13Rα2off has no binding activity to both IL13 receptors. The co-culture of naïve murine or donor’s human CD3+ T cells with IL13Rα2+ glioma cells in the presence of BiTEIL13Rα2 but not with BiTEIL13Rα2off (i) activates CD3+CD8+ T cells as judged by upregulation of CD69, CD25, and production of IFNγ and TNFα and (ii) results in concentration- and antigen-dependent cytotoxicity in glioma cells. Furthermore, a direct comparison of CD3+ T cells obtained from the peripheral blood and tumor tissue of GBM patients revealed that BiTEIL13Rα2 induces a potent cytotoxic activity of CD3+ T cells against IL13Rα2+ glioma cells. Finally, treatment of immunocompetent mice bearing IL13Rα2+ murine glioma with BiTEIL13Rα2 resulted in a higher frequency of intratumoral CD8+ T cells, and significant (p<0.05) improvement of survival over a negative control, BiTEIL13Rα2off group of mice. CONCLUSION Our data demonstrate that BiTEIL13Rα2 protein activates CD3+ T cells in an antigen-specific fashion. Furthermore, systemic treatment with BiTEIL13Rα2 protein confers a significant survival benefit in pre-clinical syngeneic glioma models, warranting investigations in other IL13Rα2-expressing cancers and translation to clinical settings.