Enhancement of the Tolerogenic Phenotype in the Liver by ImmTOR Nanoparticles

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4–CD8– (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.

POS0072 COMPREHENSIVE IMMUNE PROFILING OF 20 CHILDREN WITH MULTISYSTEM INFLAMMATORY SYNDROME

Background:Multisystem inflammatory syndrome in children (MIS-C) is a rare complication of SARS-CoV-2 infection in the pediatric population, caused by extensive activation of immune system. The understanding of the distorted immune response is still in the early stages.Objectives:To analyze comprehensively immune profile in MIS-C patients including detailed serologic response to SARS-CoV-2 in comparison with control groups.Methods:Blood samples of consecutive MIS-C patients were collected at admission. Flow cytometric analysis of all lymphocyte populations including T and B cell differentiation was performed. Immunophenotyping was performed by six-color panels for the detection of lymphocyte subpopulations. Anti-SARS-CoV-2 specific antibodies were measured in the patients serum. The IgA and IgG antibodies against S protein, the IgG S1 and S2 specific antibodies, antibodies against nucleoprotein and neutralising antibodies were measured. Patients were assessed for a wide range of auto-antibodies, namely ANA, anti-ENA (Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, Pm/Scl 100, Scl-70), myositis specific antibodies (EJ, MDA-5, TIH-Y, Ro52, SAE-1, SAE-2, NXP-2), anti-dsDNA, anti-phopholipid antibodies (aCl IgA, IgG, IgM, antiβ2GPI IgG, IgM) and ANCA. Control groups to compare specific antibody response consisted of 14 healthy children and 19 healthy adults, who had SARS-CoV-2 infection in the last 2 months.Results:Samples of 20 patients were included (14/20 boys, median age 12.4 years). Patients had higher percentage of double negative T cells and low numbers of of cytokine producing T cells Th1, Th2 and Th17. . Numbers of immune competent and CD21+ transitional B cells were also lowered. All patients had positive antibodies against SARS-CoV-2 including neutralising antibodies. Nine (9/19; 47 %) patients had high titer (≥1:160) of neutralising antibodies. Results were compared with 2 control groups; 14 healthy children (7/14 boys; median age 8 years,) and 19 healthy adults, who all experienced SARS-CoV-2 infection in the last two months. Patients with MIS-C had significantly higher levels of anti-S IgA (p<0.0001), patients with MIS-C and healthy children had significantly higher titers of anti-S1 (p=0.001) and significantly lower titers of anti-S2 (p=0.016) in comparison to adults (Figure 1). No differences were found in the titers of neutralising antibodies and anti-N antibodies. All patients were ANA negative, 19/20 patients were anti-ENA negative, whereas 1 patient had anti-Ro antibodies in low titre. Three patients had aCL IgG in medium titre and 2 patients anti-beta2GPI IgG in low titre. Patients were negative for all other autoantibodies.Conclusion:The immune response in MIS-C patients is specific with most prominent differences in elevated percentage of double negative T cells and low numbers of Th1, Th2, Th17 and CD21+ transitional B cells. MIS-C patients have distinct serologic response with high anti-S IgA, high anti-S1 and low anti-S2 titres.Figure 1.Antibody titres in patient group and control groups. Mean value with SEM s shown.Disclosure of Interests:None declared.

Examining the Relationship between Circulating CD4− CD8− Double-Negative T Cells and Outcomes of Immuno-Checkpoint Inhibitor Therapy—Looking for Biomarkers and Therapeutic Targets in Metastatic Melanoma

Background: The role of circulating CD4−/CD8− double-negative T cells (DNTs) in the immune response to melanoma is poorly understood, as are the effects of checkpoint inhibitors on T cell subpopulations. Methods: We performed a basal and longitudinal assessment of circulating immune cells, including DNTs, in metastatic melanoma patients treated with checkpoint blockade in a single-center cohort, and examined the correlations levels of immune cells with clinical features and therapy outcomes. Results: Sixty-eight patients (48 ipilimumab, 20 PD1 inhibitors) were enrolled in the study. Our analysis indicated that better outcomes were associated with normal LDH, fewer than three metastatic sites, an ECOG performance status of 0, M1a stage, lower WBC and a higher lymphocyte count. The increase in lymphocyte count and decrease of DNTs were significantly associated with the achievement of an overall response. The median value of DNT decreased while the CD4+ and NK cells increased in patients that responded to treatment compare to those who did not respond to treatment. Conclusions: DNT cells change during treatment with checkpoint inhibitors and may be adept at sensing the immune response to melanoma. The complementary variation of DNT cells with respect to CD4+ and other immune actors may improve the reliability of lymphocyte assessment. Further investigation of DNT as a potential target in checkpoint inhibitor resistant melanoma is warranted.

CD4 derived double negative T cells prevent the development and progression of nonalcoholic steatohepatitis

Hepatic inflammation is the driving force for the development and progression of NASH. Treatment targeting inflammation is believed to be beneficial. In this study, adoptive transfer of CD4+ T cells converted double negative T cells (cDNT) protects mice from diet-induced liver fat accumulation, lobular inflammation and focal necrosis. cDNT selectively suppress liver-infiltrating Th17 cells and proinflammatory M1 macrophages. IL-10 secreted by M2 macrophages decreases the survival and function of cDNT to protect M2 macrophages from cDNT-mediated lysis. NKG2A, a cell inhibitory molecule, contributes to IL-10 induced apoptosis and dampened suppressive function of cDNT. In conclusion, ex vivo-generated cDNT exert potent protection in diet induced obesity, type 2 diabetes and NASH. The improvement of outcome is due to the inhibition on liver inflammatory cells. This study supports the concept and the feasibility of potentially utilizing this autologous immune cell-based therapy for the treatment of NASH.