Background: Defects in thyroid hormone synthesis at birth lead to congenital hypothyroidism (CH). Recently, pathogenic variants in the SLC26A7 gene have been linked to dyshormonogenetic goitrous CH. This anion transporter is highly expressed in the thyroid and is involved in thyroid hormone synthesis; however, its exact function and cellular localization remain unclear. In this study, we investigated SLC26A7 variants in Finnish patients with CH, characterized the phenotypes, and analyzed thyroid-specific gene expression.Methods: SLC26A7 variants were identified from a clinical CH cohort (n = 139) using exome sequencing, and the FinnGen database (R12 release) was screened for disease associations. Thyroid histology and thyroid-specific gene expression were analyzed in six human samples (including two homozygous SLC26A7 pathogenic variant carriers, patients with goitrous and hyperactive thyroids, and normal controls) and in thyroids from different mouse models (including hypo- and hyperthyroid mice, thyroid-specific G-protein deficient, and Slc26a7-knockout mice).Results: Four CH patients from four novel families carried the homozygous SLC26A7 (c.1893delT, p.F631Lfs*8) pathogenic variant. Two had large trachea-compressing goiters, requiring thyroidectomy already at birth. In addition, one homozygous participant with normal CH screening results developed hypothyroidism at age 16, and one patient with heterozygous SLC26A7 pathogenic variant had permanent CH at birth. Dentofacial abnormalities were frequently noted, including enamel hypoplasia (in four carriers), pro- or retrognathia, and malocclusion requiring orthodontic treatment (in 8/24 carriers). Thyrocyte hypertrophy with large colloid aggregates was a hallmark of homozygous patients. FinnGen screening revealed a 75-fold enrichment of the variant in the Finnish population, identifying a few other homozygous and seven heterozygous cases with early-onset hypothyroidism and dentofacial abnormalities. In human thyrocytes, SLC26A7 was localized to the basolateral membrane, with intense staining in hyperthyroid samples, while in mouse thyroid models, its expression pattern depended on dietary iodide levels, thyrotropin signaling, and GNAS activity.Conclusions: We describe variable phenotypes associated with the SLC26A7 pathogenic variant, ranging from severe CH with large congenital goiters to delayed onset hypothyroidism and dentofacial abnormalities. SLC26A7 shows thyrotropin-, GNAS-, and dietary iodine-dependent basolateral localization, suggesting their role in phenotypic variations.

Foxp3-expressing regulatory T (Treg) cells protect against systemic autoimmunity. However, little is known about the significance of Treg cells in inflammation-experienced tissues. Here, we use an experimental autoimmune encephalomyelitis model and show that Treg cells accumulate and persist in the central nervous system (CNS) long after the resolution of the bulk of the inflammatory infiltrate. CNS-specific depletion of postinflammatory Treg cells, but not systemic depletion of Treg cells, results in autoimmune inflammatory flares in the CNS by residual local effector T cells. Expression of the NAD-consuming ectoenzyme CD38 is crucial for the functional adaptation of postinflammatory CNS Treg cells to a stressful microenvironment, in which access to interleukin-2 (IL-2) is limited. CD38 counteracts ADP-ribosylation of the IL-2 receptor and thus maintains its high sensitivity to IL-2. This fully functional high-affinity IL-2 receptor prevents the loss of tissue-resident antigen-specific Treg cells. These 'stress-tolerant' CNS Treg cells impede the collapse of immune homeostasis in the CNS once acute inflammation is controlled.

Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is a recently defined neurological autoimmune condition. The pathogenesis of the disease remains poorly understood, and no specific therapies are currently approved. Here, a comprehensive single-cell immunophenotyping of peripheral blood mononuclear cells from two independent cohorts of patients with MOGAD revealed pronounced immune perturbations in MOGAD when compared with healthy controls and patients with multiple sclerosis. Patients with MOGAD displayed an expansion of CXCR5-CD21- activated naïve and double-negative B cell subsets, a feature shared with patients with systemic lupus erythematosus. In addition, we observed altered Fc gamma receptor expression in natural killer cells, monocytes, and dendritic cells. Within the T cell compartment, CXCR3+CD4+ memory T cells were reduced in the circulation of patients with MOGAD compared with healthy controls, and this result was mirrored in a transgenic mouse model that showed retention of these cells in the inflamed central nervous system. Together, these results demonstrate profound systemic immune cell alterations in MOGAD and contribute to a better understanding of this distinct disease entity.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen 1 . The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP4 2 . However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Autoreactive encephalitogenic T cells exist in the healthy immune repertoire but need a trigger to induce CNS inflammation. The underlying mechanisms remain elusive, whereby microbiota were shown to be involved in the manifestation of CNS autoimmunity. Here, we used intravital imaging to explore how microbiota affect the T cells as trigger of CNS inflammation. Encephalitogenic CD4+ T cells transduced with the calcium-sensing protein Twitch-2B showed calcium signaling with higher frequency than polyclonal T cells in the small intestinal lamina propria (LP) but not in Peyer's patches. Interestingly, nonencephalitogenic T cells specific for OVA and LCMV also showed calcium signaling in the LP, indicating a general stimulating effect of microbiota. The observed calcium signaling was microbiota and MHC class II dependent as it was significantly reduced in germfree animals and after administration of anti-MHC class II antibody, respectively. As a consequence of T cell stimulation in the small intestine, the encephalitogenic T cells start expressing Th17-axis genes. Finally, we show the migration of CD4+ T cells from the small intestine into the CNS. In summary, our direct in vivo visualization revealed that microbiota induced T cell activation in the LP, which directed T cells to adopt a Th17-like phenotype as a trigger of CNS inflammation.

Background: The human adrenal cortex undergoes several rapid remodeling steps during its lifetime. In rodents, similar remodeling occurs postnatally in the "X-zone" layer through unknown mechanisms. Furthermore, little is known regarding the impact of thyroid hormone (TH) on adrenal glands in humans.Methods: To investigate the impact of TH on adrenal pathophysiology, we created two genetic murine models mimicking human nonautoimmune hypothyroidism and hyperthyroidism. Moreover, we analyzed serum thyrotropin (TSH) and steroid hormone concentrations in patients diagnosed with congenital hypothyroidism and premature adrenarche (PA).Results: We found that TH receptor beta-mediated hypertrophy of the X-zone significantly elevated the adrenal weights of hyperthyroid women. In the hypothyroid model, the X-zone was poorly developed in both sexes. Moreover, large reciprocal changes in the expression levels of genes that regulate adrenal cortical function were observed with both models. Unexpectedly, up- and downregulation of several genes involved in catecholamine synthesis were detected in the adrenal glands of the hypothyroid and hyperthyroid models, respectively. Furthermore, TSH and adrenal steroid concentrations correlated positively in pediatric patients with congenital hypothyroidism and PA.Conclusions: Our results revealed that congenital hypothyroidism and hyperthyroidism functionally affect adrenal gland development and related steroidogenic activity, as well as the adrenal medulla.

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or ‘mixed’ priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell–induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease. Korn and colleagues demonstrate that the site of T cell priming (gut versus skin draining lymph nodes) dictates their effector phenotypes and homing to distal sites of immunopathology.

Macrophages, which are highly diverse in different tissues, play a complex and vital role in tissue development, homeostasis, and inflammation. The origin and heterogeneity of tissue‐resident monocytes and macrophages in ovaries remains unknown. Here we identify three tissue‐resident monocyte populations and five macrophage populations in the adult ovaries using high‐dimensional single cell mass cytometry. Ontogenic analyses using cell fate mapping models and cell depletion experiments revealed the infiltration of ovaries by both yolk sac and fetal liver‐derived macrophages already during the embryonic development. Moreover, we found that both embryonic and bone marrow‐derived macrophages contribute to the distinct ovarian macrophage subpopulations in the adults. These assays also showed that fetal‐derived MHC II‐negative macrophages differentiate postnatally in the maturing ovary to MHC II‐positive cells. Our analyses further unraveled that the developmentally distinct macrophage types share overlapping distribution and scavenging function in the ovaries under homeostatic conditions. In conclusion, we report here the first comprehensive analyses of ovarian monocytes and macrophages. In addition, we show that the mechanisms controlling monocyte immigration, the phenotype of different pools of interstitial macrophages, and the interconversion capacity of fetal‐derived macrophages in ovaries are remarkably different from those seen in other tissue niches.

In the testis, interstitial macrophages are thought to be derived from the yolk sac during fetal development, and later replaced by bone marrow-derived macrophages. By contrast, the peritubular macrophages have been reported to emerge first in the postnatal testis and solely represent descendants of bone marrow-derived monocytes. Here, we define new monocyte and macrophage types in the fetal and postnatal testis using high-dimensional single-cell analyses. Our results show that interstitial macrophages have a dominant contribution from fetal liver-derived precursors, while peritubular macrophages are generated already at birth from embryonic precursors. We find that bone marrow-derived monocytes do not substantially contribute to the replenishment of the testicular macrophage pool even after systemic macrophage depletion. The presence of macrophages prenatally, but not postnatally, is necessary for normal spermatogenesis. Our multifaceted data thus challenge the current paradigms in testicular macrophage biology by delineating their differentiation, homeostasis and functions.

Abstract Background: Tumor-associated macrophages (TAM) play an important role in tumor-promoting inflammation and have been associated with poor prognosis in many human cancers. Recently, we showed that class A scavenger receptor MARCO is a specific marker for the immune-suppressive tumor-promoting TAM subtype in different tumor models. Targeting MARCO by monoclonal antibodies (Ab) could reprogram the protumorigenic TAM towards a proinflammatory phenotype with antitumor activity, leading to decreased tumor growth and metastases formation in three different mouse tumor models. Aim(s): The current immunotherapies are estimated to treat less than 50% of all cancers; therefore, it is of high importance to develop new targets and combinatorial therapies. In this study, we aim to define the role of scavenger receptor MARCO on TAM in more detail and the mechanisms of how targeting MARCO by monoclonal Ab modulates macrophage phenotype and tumor microenvironment. Moreover, we are investigating how it can be used as a possible anticancer therapeutic approach used as single or combinatorial treatment. Results: Our recent data demonstrate that using anti-MARCO Ab in combination with other immune checkpoint inhibitors, such as anti-CTLA-4 Ab, further decreases the tumor volume by 40-50% in the B16 melanoma model. For example, different checkpoint Ab combined with anti-MARCO Ab shows a reduction from average tumor volume 500mm3 in single treatments to 250mm3 (p=0.0101) in the combinatorial treatment. Currently, we are investigating what happens in the tumor microenvironment on a cellular level after using the different combinatorial treatments. Moreover, we see increased ATP release of MARCO+ macrophages in vitro after engagement of MARCO as well as enhanced glycolysis in anti-MARCO Ab treated macrophages compared to the control group. Conclusion: Checkpoint inhibitors targeting T cells are already used in the clinics and the anti-MARCO Ab targeting TAM is a promising candidate to use in combination with checkpoint inhibitors to even further increase the survival rate of patients. The understanding of MARCO signaling and how this leads to a shift towards proinflammatory macrophages would not just benefit the field of basic macrophage biology but will also be an important step in translating the anti-MARCO Ab therapy from mice to humans as a new approach for cancer immunotherapy. Citation Format: Vanessa F. Boura, Sofia Tyystjärvi, Ganna Oliynyk, Marie Arsenian-Henriksson, Mikael C.I. Karlsson, Silke Sohn. Targeting scavenger receptors for immunotherapy of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B09.

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast.