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Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

The COVID-19 pandemic has shown the potentially devastating impact of novel respiratory infections worldwide. Insightful data obtained in the last years have shed light on the pathophysiology of SARS-CoV-2 infection and the role of the inflammatory response in driving both the resolution of the disease and uncontrolled deleterious inflammatory status in severe cases. In this mini-review, we cover some important aspects of the role of T cells in COVID-19 with a special focus on the local response in the lung. We focus on the reported T cell phenotypes in mild, moderate, and severe COVID-19, focusing on lung inflammation and on both the protective and damaging roles of the T cell response, also highlighting the open questions in the field.

Background and aims The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. Methods Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. Results Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. Conclusions This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology., Graphical abstract

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies. Mutations in the RNA-binding proteins Roquin-1 or Regnase-1 cause systemic autoimmunity. Heissmeyer and colleagues show that Roquin-1 and Regnase-1 physically interact and thereby regulate CD4+ and CD8+ T cell metabolism and functionality.

IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood pressure and end-organ damage is unknown. We examined the role of IL-18 in the development of renal inflammation and injury in a mouse model of low-renin hypertension. Hypertension was induced in male C57BL6/J (WT) and IL-18−/−mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% drinking saline (1K/DOCA/salt). Normotensive controls received uninephrectomy and placebo (1K/placebo). Blood pressure was measured via tail cuff or radiotelemetry. After 21 days, kidneys were harvested for (immuno)histochemical, quantitative-PCR and flow cytometric analyses of fibrosis, inflammation, and immune cell infiltration. 1K/DOCA/salt-treated WT mice developed hypertension, renal fibrosis, upregulation of proinflammatory genes, and accumulation of CD3+T cells in the kidneys. They also displayed increased expression of IL-18 on tubular epithelial cells. IL-18−/−mice were profoundly protected from hypertension, renal fibrosis, and inflammation. Bone marrow transplantation between WT and IL-18−/−mice revealed that IL-18-deficiency in non-bone marrow-derived cells alone afforded equivalent protection against hypertension and renal injury as global IL-18 deficiency. IL-18 receptor subunits—interleukin-18 receptor 1 and IL-18R accessory protein—were upregulated in kidneys of 1K/DOCA/salt-treated WT mice and localized to T cells and tubular epithelial cells. T cells from kidneys of 1K/DOCA/salt-treated mice produced interferon-γ upon ex vivo stimulation with IL-18, whereas those from 1K/placebo mice did not. In conclusion, IL-18 production by tubular epithelial cells contributes to elevated blood pressure, renal inflammation, and fibrosis in 1K/DOCA/salt-treated mice, highlighting it as a promising therapeutic target for hypertension and kidney disease.

Heindl et al. describe the local proliferation and clustering of T cells in the brain of mice and humans after stroke. This previously unrecognized phenomenon could explain why blocking cerebral leukocyte invasion might fail to improve long-term stroke recovery., Neuroinflammation is an emerging focus of translational stroke research. Preclinical studies have demonstrated a critical role for brain-invading lymphocytes in post-stroke pathophysiology. Reducing cerebral lymphocyte invasion by anti-CD49d antibodies consistently improves outcome in the acute phase after experimental stroke models. However, clinical trials testing this approach failed to show efficacy in stroke patients for the chronic outcome 3 mo after stroke. Here, we identify a potential mechanistic reason for this phenomenon by detecting chronic T cell accumulation—evading the systemic therapy—in the post-ischemic brain. We observed a persistent accumulation of T cells in mice and human autopsy samples for more than 1 mo after stroke. Cerebral T cell accumulation in the post-ischemic brain was driven by increased local T cell proliferation rather than by T cell invasion. This observation urges re-evaluation of current immunotherapeutic approaches, which target circulating lymphocytes for promoting recovery after stroke., Graphical Abstract

The proximal DNA damage response kinase ATM is frequently inactivated in human malignancies. Germline mutations in the ATM gene cause Ataxia-telangiectasia (A-T), characterized by cerebellar ataxia and cancer predisposition. Whether ATM deficiency impacts on tumor initiation or also on the maintenance of the malignant state is unclear. Here, we show that Atm reactivation in initially Atm-deficient B- and T cell lymphomas induces tumor regression. We further find a reduced T cell abundance in B cell lymphomas from Atm-defective mice and A-T patients. Using T cell-specific Atm-knockout models, as well as allogeneic transplantation experiments, we pinpoint impaired immune surveillance as a contributor to cancer predisposition and development. Moreover, we demonstrate that Atm-deficient T cells display impaired proliferation capacity upon stimulation, due to replication stress. Altogether, our data indicate that T cell-specific restoration of ATM activity or allogeneic hematopoietic stem cell transplantation may prevent lymphomagenesis in A-T patients.

Autoimmune disorders of the central nervous system (CNS) comprise a broad spectrum of clinical entities. The stratification of patients based on the recognized autoantigen is of great importance for therapy optimization and for concepts of pathogenicity, but for most of these patients, the actual target of their autoimmune response is unknown. Here we investigated oligodendrocyte myelin glycoprotein (OMGP) as autoimmune target, because OMGP is expressed specifically in the CNS and there on oligodendrocytes and neurons. Using a stringent cell-based assay, we detected autoantibodies to OMGP in serum of 8/352 patients with multiple sclerosis, 1/28 children with acute disseminated encephalomyelitis and unexpectedly, also in one patient with psychosis, but in none of 114 healthy controls. Since OMGP is GPI-anchored, we validated its recognition also in GPI-anchored form. The autoantibodies to OMGP were largely IgG1 with a contribution of IgG4, indicating cognate T cell help. We found high levels of soluble OMGP in human spinal fluid, presumably due to shedding of the GPI-linked OMGP. Analyzing the pathogenic relevance of autoimmunity to OMGP in an animal model, we found that OMGP-specific T cells induce a novel type of experimental autoimmune encephalomyelitis dominated by meningitis above the cortical convexities. This unusual localization may be directed by intrathecal uptake and presentation of OMGP by meningeal phagocytes. Together, OMGP-directed autoimmunity provides a new element of heterogeneity, helping to improve the stratification of patients for diagnostic and therapeutic purposes. Electronic supplementary material The online version of this article (10.1186/s40478-020-01086-2) contains supplementary material, which is available to authorized users.

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases., Graphical Abstract, The chemokine CCL17 promotes T cell attraction to peripheral tissues and thereby exacerbates inflammatory reactions. Using SELEX, Fülle et al. selected high-affinity aptamers, which neutralize CCL17 in murine contact hypersensitivity. Because of their small size and structural properties, such aptamers may represent new versatile drugs for allergic skin diseases.

The CNS is protected by the immune system, including cells that reside directly within the CNS and help to ensure proper neural function, as well as cells that traffic into the CNS with disease. The CNS-resident immune system is comprised mainly of innate immune cells and operates under homeostatic conditions. These myeloid cells in the CNS parenchyma and at CNS-periphery interfaces are highly specialized but also extremely plastic cells that immediately react to any changes in CNS homeostasis and become reactive in the context of neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease. However, when the blood-brain barrier is impaired during CNS diseases such as multiple sclerosis or altered with cerebral ischemia, peripheral adaptive and innate immune cells, including monocytes, neutrophils, T cells and B cells, can enter the CNS, where they execute distinct cell-mediated effects. On the basis of these observations, we assess strategies for targeting peripheral immune cells to reduce CNS disease burden.

CRELD1 is a pivotal factor for heart development, the function of which is unknown in adult life. We here provide evidence that CRELD1 is an important gatekeeper of immune system homeostasis. Exploiting expression variance in large human cohorts contrasting individuals with the lowest and highest CRELD1 expression levels revealed strong phenotypic, functional and transcriptional differences, including reduced CD4+ T cell numbers. These findings were validated in T cell–specific Creld1-deficient mice. Loss of Creld1 was associated with simultaneous overactivation and increased apoptosis, resulting in a net loss of T cells with age. Creld1 was transcriptionally and functionally linked to Wnt signaling. Collectively, gene expression variance in large human cohorts combined with murine genetic models, transcriptomics and functional testing defines CRELD1 as an important modulator of immune homeostasis. Within a human cohort, wide variation can occur with constitutively expressed proteins. Aschenbrenner and colleagues found that individuals with lower CRELD1 expression have decreased frequencies of naive CD4+ T cells. Mice with conditional Creld1 deficiency also exhibit a phenotype associated with immunological aging.

B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (μMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.

Cross-talk between the peripheral immune system and the central nervous system is important for physiological brain health. T cells are required to maintain normal baseline levels of neural precursor proliferation in the hippocampus of adult mice. We show here that neither T cells, B cells, natural killer cells nor natural killer T cells are required for the increase in hippocampal precursor proliferation that occurs in response to physical exercise. In addition, we demonstrate that a subpopulation of T cells, regulatory T cells, is not involved in maintaining baseline levels of neural precursor proliferation. Even when applied at supraphysiological numbers, populations of both naive and stimulated lymphocytes had no effect on hippocampal precursor proliferation in vitro. In addition, physical activity had no effect on peripheral immune cells in terms of distribution in the bone marrow, lymph nodes or spleen, activation state or chemokine receptor (CXCR4 and CCR9) expression. Together these results suggest that lymphocytes are not involved in translating the peripheral effects of exercise to the neurogenic niche in the hippocampus and further support the idea that the exercise-induced regulation of adult neurogenesis is mechanistically distinct from its baseline control., Frontiers in Immunology, 9, ISSN:1664-3224

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

T cells critically depend on reprogramming of metabolic signatures to meet the bioenergetic demands during activation and clonal expansion. Here we identify the transcription factor Nur77 as a cell-intrinsic modulator of T cell activation. Nur77-deficient T cells are highly proliferative, and lack of Nur77 is associated with enhanced T cell activation and increased susceptibility for T cell-mediated inflammatory diseases, such as CNS autoimmunity, allergic contact dermatitis and collagen-induced arthritis. Importantly, Nur77 serves as key regulator of energy metabolism in T cells, restricting mitochondrial respiration and glycolysis and controlling switching between different energy pathways. Transcriptional network analysis revealed that Nur77 modulates the expression of metabolic genes, most likely in close interaction with other transcription factors, especially estrogen-related receptor α. In summary, we identify Nur77 as a transcriptional regulator of T cell metabolism, which elevates the threshold for T cell activation and confers protection in different T cell-mediated inflammatory diseases.

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn−/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn−/− T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn−/− T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn−/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

Despite their importance for signalling events, protein-protein interactions cannot easily be analyzed on a single cell level. We developed a robust automated FRET measurement system implemented on a commercial flow cytometer allowing for rapid profiling of molecular associations in living cells. We used this method to measure the most proximal signaling events on human T lymphocyte activation, which preceded calcium influx, and could automatically detect T cell receptor/CD3 complex clustering defects in immunocompromised patients. © 2016 International Society for Advancement of Cytometry.

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody-Fc-fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP-ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2-specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc-fusion protein of s + 16a (s + 16-mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near-infrared fluorescence imaging. Nanobody s + 16a(680) and s + 16mFc(680) labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102(680) required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2-positive lymph nodes but not of ART2-negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc(680) showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a(680) , since renal elimination of unbound s + 16a(680) significantly reduced background signals. Our results indicate that small single-domain nanobodies are best suited for short-term uses, such as noninvasive imaging, whereas larger nanobody-Fc-fusion proteins are better suited for long-term uses, such as therapy of inflammation and tumors.

Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.

Development of type-II collagen (CII)-induced arthritis (CIA) is dependent on a T-cell mediated activation of autoreactive B cells. However, it is still unclear if B cells can present CII to T cells. To investigate the role of B cells as antigen-presenting cells (APCs) for CII, we purified B cells from lymph nodes of immunized and nonimmunized mice. These B cells were used as APC for antigen-specific T-cell hybridomas. B cells from naïve mice did present native, triple-helical, CII (nCII) but also ovalbumin (OVA) and denatured CII (dCII) to antigen-specific T-cell hybridomas. In addition, B cells primed with nCII or OVA, but not dCII, activated the antigen-specific T-cell hybridomas two to three times better than naïve B cells. We conclude that antigen-primed B cells have the capacity to process and present CII to primed T cells, and antigen-primed antigen-specific B cells are more efficient as APC than naïve B cells. We further conclude that B cells have the potential to play an important role as APC in the development of CIA.