Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic pattern recognition receptor that senses short double-stranded RNA with uncapped 5'-triphosphate (3p-RNA). Upon activation, RIG-I induces type I interferons and proinflammatory cytokines, thereby promoting adaptive immunity. Thus, RIG-I activation is a promising approach for creating a proinflammatory tumor microenvironment. In this study, we investigated its therapeutic potential in hepatocellular carcinoma (HCC). We explored and confirmed RIG-I expression and signaling in human HCC samples and cell lines. The therapeutic potential of RIG-I activation by 3p-RNA for the treatment of HCC was investigated in vitro and in syngeneic murine orthotopic tumor models. In vivo, 3p-RNA treatment significantly reduced the tumor burden, delayed disease progression, and achieved partial complete remission of RIL-175 tumors with durable immune memory. However, no therapeutic effects were observed in the Hep-55.1C model. Tumor clearance depended on CD4⁺ and CD8⁺ T cells, but not NK cells. Additionally, 3p-RNA induced PD-L1 expression on HCC cells, enhancing their sensitivity to anti-PD-1 immune checkpoint therapy in vivo. RIG-I activation via 3p-RNA therapy shows promise as an immunotherapeutic strategy for hepatocellular carcinoma (HCC). Future investigations need to focus on tumor-intrinsic factors to understand heterogeneity between tumors and to overcome resistance mechanisms.

Metabolic activity shapes cell fate but remains challenging to capture in vivo with high resolution. Here we performed longitudinal metabolic and phenotypic profiling of human antigen-specific CD8+ T cells after yellow fever vaccination using flow cytometry and single-cell RNA sequencing. As assessed by protein translation rates, CD8+ T cells upregulated glycolysis to fuel anabolic needs for proliferation but predominantly used oxidative phosphorylation for energy production during the acute phase (days 7-28) after vaccination. Simultaneously, CD8+CD62L+CD45RA- central memory T cells were the most metabolically active subset, whereas CD8+CD62L-CD45RA+ effector T cells underwent metabolic shutdown. Weakly differentiated CD8+CD62L+CD45RA+CD95- naive-like memory T cells showed minimal activity, relied solely on oxidative phosphorylation and were preferentially maintained 26 years postvaccination, reinforcing the link between cellular quiescence and longevity. Our study highlights quiescence as a key feature for long-term immunological memory formation in humans.

The live-attenuated yellow fever 17D (YF17D) vaccine is a model of acute viral infection that induces long-lasting protective immunity. Among immunocompetent adults, responses to YF17D vary significantly. To understand the sources of this variability, we investigate the influence of sex, age, human leukocyte antigen (HLA) type, and 20 prior infections on basal immune parameters and the cellular and antibody response to YF17D in 250 healthy young individuals. Multivariate regression found that sex and cytomegalovirus (CMV) infection significantly contribute to baseline immune variation but do not affect vaccine responses except for reduced YF17D-specific CD8+ frequencies in CMV-infected males. However, the abundance at baseline of non-specific cytokine-expressing T helper cells in circulation is associated with stronger vaccine responses, a state that smoking favors. Additionally, an elevated baseline level of interferon-stimulated CXCL10 is linked to poorer vaccination outcomes. Altogether, YF17D reactivity is conditioned by the baseline immune status independent of sex and CMV-related variations.

STAT3-hyper-IgE syndrome (STAT3-HIES) is an inborn error of immunity caused by heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3). In this study, we evaluate the functional relevance of a previously undescribed heterozygous STAT3 variant in a patient with clinical findings of STAT3-HIES. Flow cytometry, quantitative real-time PCR, pull-down assays, native PAGE, DNA-binding ELISA, and 3D-structural data analysis were performed. Genetic analysis identified the heterozygous STAT3 variant NM_139276.2:c.2127G>C (NP_644805.1:p.(K709N); short: p.K709N) in a patient with a clinical and laboratory phenotype characteristic of STAT3-HIES, including early onset severe eczema, chronic lung disease, eosinophilia, and elevated serum IgE levels. While STAT3 p.K709N did not significantly affect STAT3 phosphorylation, STAT3 target gene expression was impaired in patient cells. Expression of STAT3 p.K709N and wild-type STAT3 in STAT3-deficient cells indicated a dominant-negative effect by the mutation. Analysis of 3D-structural data and modeling suggested a central role of the affected amino acid K709 in stabilizing a C-terminal loop in STAT3 essential for dimer formation. Consequently, p.K709N resulted in diminished STAT3 dimerization and reduced DNA binding in patient cells. Functional analyses verified STAT3 p.K709N to cause STAT3-HIES and suggest that STAT3 p.K709N impairs STAT3 dimer formation.

Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) requires lifelong glucocorticoid replacement to manage cortisol deficiency and excessive androgen production. Conventional circadian treatment (CT) tries to mimic natural cortisol rhythms, whereas reverse-circadian treatment (RC) prioritizes the suppression of adrenal androgen excess overnight through evening dosing. Limited data exist on the immunological impact of these regimens. A bi-centric study was conducted, including 41 pediatric and adolescent CAH patients. Peripheral blood samples were collected from patients on conventional treatment (n = 38) or RC (n = 16), with 11 RC patients switching to conventional treatment. Immune cell phenotypes, cytokine profiles, and natural killer (NK) cell cytotoxicity were assessed. Patients receiving RC showed lower percentages of CD4+CD25+ T cells (p = 0.0139). After the switch, patients with RC presented with a higher percentage of non-classical monocytes (p = 0.0255) and a lower percentage of Th17 cells (p = 0.0195). A lower expression of CD107 was observed with RC (p < 0.0001), as well as a higher percentage of NKp30 (p = 0.0189). Comparing patients after the switch from RC to HC, patients with RC presented with a lower NKG2D expression (p = 0.0420). Both conventional treatment and RC exhibited distinct immunological impacts, with CT showing modest advantages in normalizing immune phenotypes. These findings suggest that CT may offer immunological benefits for managing young patients with congenital adrenal hyperplasia.

Vaccines represent a major public health intervention against infectious diseases and potentially cancer. Surrogate markers of vaccine efficacy usually rely on neutralizing antibody titers afflicted by high interindividual variabilities. Automated multiplexed T cell assays currently allow to test the clinical relevance of T lymphocyte responses during vaccine rollout. We examined cellular and/or humoral immune responses in five independent cohorts of health care workers, young healthy individuals and patients with cancer (melanoma or lung cancer) receiving various immunizing formulations (non-replicating, viral/tumoral, mRNA/peptides/cellular/viral particles). Here we show that about 20% of vaccinees to non-replicating formulations fail to mount protective antibody and Th1/Tc1 responses while 9% receiving a live vaccine were hyperresponders. Vaccine outliers could at least in part be attributed to gut dysbiosis at baseline, evaluated by shotgun metagenomics-based machine learning or the TOPOSCORE. These findings highlight the requirement of diagnostic tools to identify intestinal dysbiosis, as well as microbiota-centered interventions to optimize the efficiency of mass vaccinations.

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14, and 28 d after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on days 3 and 7 after vaccination as well as cell type-specific responses and response kinetics. Single-cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines, and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses.

The live-attenuated yellow fever vaccine 17D-204 (YF17D) activates robust innate immune responses followed by rapid induction of adaptive immunity resulting in long-lasting protection. YF17D triggers the production of type I interferons (IFNs) which have a dual role in antigen presenting cells regulating their infection and contributing to their activation. Infection with YF17D was detected in primary human blood monocytes and conventional dendritic cells (DCs) and in monocyte-derived DCs but was highly restricted by type I IFN. Blocking IFNAR signaling in YF17D-infected PBMC from vaccinated donors resulted in increased activation of YF17D-specific CD8+ T cells. Consistently, peak IFN-alpha plasma levels correlated inversely with the CD8+ T cells response in YF17D vaccinees. Loss of function experiments demonstrated a dominant role of retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and mitochondrial antiviral signaling protein (MAVS) for type I IFN induction and restriction of YF17D. The type I IFN response was mediated by 5’ tri- or diphosphate dsRNA intermediates that are formed during YF17D infection. In vivo proximity labelling (IPL) of RIG-I and next-generation sequencing confirmed interaction of RIG-I with YF17D-dsRNA in infected cells. Thus, YF17D-triggered RLR-signaling restricts viral replication through type I IFN and thus limits the production of viral antigens that can be presented to T cells. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 391217598, 369799452 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-17-CE15-0031-01 European Union Horizon Europe, 101137459 - Yellow4FLAVI Helmholtz Association of German Research Centres, https://ror.org/0281dp749, iMed consortium Evangelisches Studienwerk Villigst, https://ror.org/05az3m671 Friedrich Baur Stiftung, https://ror.org/032a9fh96

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.

Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14 and 28 days after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on day 3 and 7 after vaccination as well as cell type specific responses and temporal dynamics. Single cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses. ### Competing Interest Statement The authors have declared no competing interest.

The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

Abstract Disclosure: H.F. Nowotny: None. T. Marchant Seiter: None. J. Ju: None. A. Gottschlich: None. H. Schneider: None. S. Zopp: None. F. Vogel: None. L. Tschaidse: None. M.K. Auer: None. C. Lottspeich: None. S. Kobold: None. S. Rothenfusser: None. F. Beuschlein: None. M. Reincke: None. L. Braun: None. N. Reisch: None. Background: Increased risk of infection, adrenal crises and a higher mortality rate have been reported for patients with primary adrenal insufficiency (PAI). Such dismal outcomes have been inferred to immune cell dysregulation as a consequence of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid deficiency. Methods: The present cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 patients after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 patients with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable glucocorticoid replacement regimen using a median dose of 25 mg hydrocortisone (IQR 5 mg for BADx and AD and IQR 10 mg for CAH) per day. Heparinized blood samples were processed to isolate peripheral blood mononuclear cells. All samples were processed for analysis of immune cell subsets using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate (PMA)/Ionomycin, as well as analysis of natural killer (NK-) cell cytotoxicity and clock gene expression. Results: Analysis of immune cell profiles revealed reduced percentages of IFNγ-secreting T helper (Th1) in AD (p = 0.0024) and cytotoxic (Tc1) cells in CAH (p = 0.0055) and AD patients (p = 0.0075) compared to controls. Moreover, we observed a downregulation of the percentage of IL-4+ Th2 cells in AD patients (p = 0.0157) and Tc2 cells in AD (p = 0.0154) and CAH patients (p = 0.0012) compared to controls. Th17 and Tc17 cells were also downregulated in patients with AD compared to the other patient groups and controls (p < 0.0001). NK-cell cytotoxicity (NKCC) using bioluminescence measurements was reduced in all subsets of PAI patients compared to controls with the lowest change in patients with CAH (mean specific lysis 57.5% in controls, 21.7% in CAH, -0.5% in AD and -28.7% in BADx). The percentage of activated NK-cells was upregulated in BADx (p = 0.0008) and AD patients (p = 0.0348) compared to controls, also expressed as an increase in CD107a expression as a marker of NK-cell degranulation (BADx p < 0.0001; AD p = 0.0002). While the percentage of NK-cell activating receptor expressing NK-cells did not differ from controls, expression percentage of NK-cell inhibiting receptor CD94 was upregulated in both BADx and AD patients (p < 0.0001). Conclusion: In patients with three different etiologies of PAI, distinct differences in T- and NK-cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight so far unsuspected differences in immune cell composition and NK-cell function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment. Presentation: Saturday, June 17, 2023

Supplementary Figure 1-Treatment with CpG causes a reduction of Treg within the CD4 T cell population and an increase of CD8+ tumor-infiltrating cells.

Supplementary Figure S1 shows that RIG-I, MDA and IRF7 mRNA expression are correlated to the Type I IFN signature in melanoma, while MAVS, TBK1 and IRF3 are not. Supplementary Figure S2 shows a list of gene products correlated with RIG-I expression based on TCGA RNAseq data from 384 melanomas. Ranking is done based on Person correlation coefficients. Supplementary Figure S3 shows a graphical illustration of RIG-I and CXCL-10 expression correlation based on TCGA RNAseq data of melanoma samples. Supplementary Figure S4 shows the results of the analysis of mRNA expression of IFN-β, RIG-I and IP-10 following RNA treatment of C8161 parental cells and CRISPR/Cas generated KO clones. Cells were treated with RIG-I agonist ppp-RNA and respective controls at 100nM. Relative mRNA levels were determined by RT-qPCR 48 hours post treatment by reverse transfection with synthetic ppp-RNA and respective OH-RNA controls as indicated. RIG-I knockout variants are characterized by absence of RIG-I agonist triggered mRNA upregulation secretion. Error bars represent standard deviation. Supplementary Figure S5 shows the flow cytometry analysis of surface antigen expression following ppp-RNA treatment of parental and KO variants of C8161 (a-c) and B16-F10 (d-f). Cells were treated with ppp-RNA and respective controls as indicated at 100nM and analyzed 48 hours post treatment. Supplementary Figure S6 shows IP 10 quantification results of C57BL/6 female mice (n=4 per group), carrying Hepa1-6 liver tumors treated with 50ug RNA complexed with In vivo Jet-PEI (i.v.). Serum was collected for IP-10 quantification 4h post RNA delivery. Error bars indicate standard deviation. Supplementary Figure S7 shows the serum cytokine response profiles 6 hours post first intratumoral treatment with RNA as indicated. Error bars reflect standard deviation. Supplementary Figure S8 shows flow cytometry based analysis of immune cell populations isolated from spleens of B16-F10 tumor bearing mice treated intratumorally with ppp-RNA or respective controls. Spleens were harvested 48h after last treatment. Error bars indicate standard deviation. Supplementary Figure S9 shows the serum cytokine response profile 6 hours post first intratumoral treatment with RNA as indicated. Error bars represent standard deviation. Supplementary Table S1 shows a list of genes comprising the type I IFN signature Supplementary Table S2 shows the results of a Pearson Correlation analysis of OVA-specific CTL frequency versus cytokine expression in the respective animals

Supplementary Figure 6-Type I interferon is a key mediator in the process of CCL22 suppression and tumor regression.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

Dysregulation of the myeloid cell compartment is a feature of severe disease in hospitalized COVID-19 patients. Here, we investigated the response of circulating dendritic cell (DC) and monocyte subpopulations in SARS-CoV-2 infected outpatients with mild disease and compared it to the response of healthy individuals to yellow fever vaccine virus YF17D as a model of a well-coordinated response to viral infection. In SARS-CoV-2-infected outpatients circulating DCs were persistently reduced for several weeks whereas after YF17D vaccination DC numbers were decreased temporarily and rapidly replenished by increased proliferation until 14 days after vaccination. The majority of COVID-19 outpatients showed high expression of CD86 and PD-L1 in monocytes and DCs early on, resembling the dynamic after YF17D vaccination. In a subgroup of patients, low CD86 and high PD-L1 expression were detected in monocytes and DCs coinciding with symptoms, higher age, and lower lymphocyte counts. This phenotype was similar to that observed in severely ill COVID-19 patients, but less pronounced. Thus, prolonged reduction and dysregulated activation of blood DCs and monocytes were seen in a subgroup of symptomatic non-hospitalized COVID-19 patients while a transient coordinated activation was characteristic for the majority of patients with mild COVID-19 and the response to YF17D vaccination.

Supplementary Figure 5-CCL22 secretion of human tumor cell lines upon IFN-γ stimulation.

Background Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens.Methods Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo.Results MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model.Conclusion The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.