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SARS-CoV-2 is a single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Given its acute and often self-limiting course, components of the innate immune system are likely central in controlling virus replication thereby determining clinical outcome. Natural killer (NK) cells are innate lymphocytes with notable activity against a broad range of viruses, including RNA viruses1,2. NK cell function may be altered during COVID-19 despite increased representation of NK cells with an activated and ‘adaptive’ phenotype3,4. Here we show that viral load decline in COVID-19 correlates with NK cell status and that NK cells can control SARS-CoV-2 replication by recognizing infected target cells. In severe COVID-19, NK cells show remarkable defects in virus control, cytokine production and cell-mediated cytotoxicity despite high expression of cytotoxic effector molecules. Single-cell RNA-sequencing (scRNA-seq) of NK cells along the time course of the entire COVID-19 disease spectrum reveals a unique gene expression signature. Transcriptional networks of interferon-driven NK cell activation are superimposed by a dominant TGFβ response signature with reduced expression of genes related to cell-cell adhesion, granule exocytosis and cell-mediated cytotoxicity. In severe COVID-19, serum levels of TGFβ peak during the first 2 weeks of infection, and serum obtained from these patients profoundly inhibits NK cell function in a TGFβ-dependent manner. Our data reveal that untimely production of TGFβ is a hallmark of severe COVID-19 and may inhibit NK cell function and early virus control.

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZFhi ILCP expressing central LTi molecules in a RORα-dependent fashion. Our data unveil an unexpected role for T-bet and RORα during embryonic ILC function and highlight that RORγt is crucial in counteracting the suppressive effects of T-bet.

ObjectiveDue to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new and pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem.DesignWe propose a new cutting strategy of the murine femur which produces three segments with a predictable mixed cell populations, where one segment contains AC and growth plate (GP) chondrocytes, another contains GP chondrocytes, and the last segment contains only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find common and distinct genes between the segments. Then, the segment containing AC chondrocytes was digested and analysed via scRNA-seq.ResultsDifferential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related- and non-related to chondrocytes, including, cartilage development (adj. p-value: 3.45E-17) and endochondral bone growth (adj. p-value 1.22E-4), respectively. ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. p-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk- and scRNA-seq contained 75 genes, where all but ten genes were previously implicated in cartilage homeostasis or osteoarthritis (OA) progression.ConclusionsOur approach has the potential to detect the scarce disease phenotypes of chondrocytes in murine OA models.

T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA). However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the transcriptional and the clonal heterogeneity of synovial T lymphocytes in JIA patients by single-cell RNA sequencing combined with T cell receptor profiling on the same cells. We identify clonally expanded subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1+ TOX+ EOMES+ population of CD4+ T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+ TOX+ BHLHE40+ population of CD4+ , and a mirror population of CD8+ T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation in situ. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.

Daratumumab, a human monoclonal antibody that targets CD38, depletes plasma cells and is approved for the treatment of multiple myeloma. Long-lived plasma cells are implicated in the pathogenesis of systemic lupus erythematosus because they secrete autoantibodies, but they are unresponsive to standard immunosuppression. We describe the use of daratumumab that induced substantial clinical responses in two patients with life-threatening lupus, with the clinical responses sustained by maintenance therapy with belimumab, an antibody to B-cell activating factor. Significant depletion of long-lived plasma cells, reduction of interferon type I activity, and down-regulation of T-cell transcripts associated with chronic inflammation were documented. (Supported by the Deutsche Forschungsgemeinschaft and others.).

Medullary thymic epithelial cells (mTECs) are key antigen-presenting cells mediating T cell tolerance to prevent harmful autoimmunity. mTECs both negatively select self-reactive T cells and promote...

The commensal microflora is a source for multiple antigens that may induce cross-reactive antibodies against host proteins and pathogens. However, whether commensal bacteria can induce cross-reactive antibodies against SARS-CoV-2 remains unknown. Here we report that several commensal bacteria contribute to the generation of cross-reactive IgA antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We identified SARS-CoV-2 unexposed individuals with RBD-binding IgA antibodies at their mucosal surfaces. Conversely, neutralising monoclonal anti-RBD antibodies recognised distinct commensal bacterial species. Some of these bacteria, such as Streptococcus salivarius, induced a cross-reactive anti-RBD antibodies upon supplementation in mice. Conversely, severely ill COVID-19 patients showed reduction of Streptococcus and Veillonella in their oropharynx and feces and a reduction of anti-RBD IgA at mucosal surfaces. Altogether, distinct microbial species of the human microbiota can induce secretory IgA antibodies cross-reactive for the RBD of SARS-CoV-2.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non‐overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long‐term maintenance of immune cells.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-β, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-β. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-β, and is distracted from itself., Our understanding on the humoral immunity induced by SARS-CoV-2 is still lacking. Here the authors analyze B cell responses at the single cell level to find that, in severe COVID-19 patients, plasmablasts shift from IFN to TGFβ instruction to produce IgA antibodies that are not specific to dominant SARS-CoV-2 antigens.

Here we have analyzed the dynamics of the adaptive immune response triggered by SARS-CoV-2 in severely affected COVID-19 patients, as reflected by activated B cells egressing into the blood, at the single cell level. Early on, before seroconversion in response to SARS-CoV-2 spike protein, activated peripheral B cells displayed a type 1 interferon-induced gene expression signature. After seroconversion, activated B cells lost this signature, expressed IL-21- and TGF-β-induced gene expression signatures, and mostly IgG1 and IgA1. In the sustained immune reaction of the COVID-19 patients, until day 59, activated peripheral B cells shifted to expression of IgA2, reflecting instruction by TGF-β. Despite the continued generation of activated B cells, those cells were not found in the lungs of deceased COVID-19 patients, nor did the IgA2 bind to dominant antigens of SARS-CoV-2. In severe COVID-19, SARS-CoV-2 thus triggers a chronic immune reaction distracted from itself and instructed by TGF-β.

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility., Nature Communications, 11 (1), ISSN:2041-1723

While it is generally accepted that tissue-resident memory T lymphocytes protect host tissues from secondary immune challenges, it is unclear whether, and if so, how they contribute to systemic secondary immune responses. Here we show that in human individuals with an established immune memory to measles, mumps and rubella viruses, when challenged with the measles-mumps-rubella (MMR) vaccine again, tissue-resident memory CD4+ T cells are mobilized into the blood within 16 to 48 hours after vaccination. These cells then leave the blood again, and apparently contribute to the systemic secondary immune reaction, as is evident from the representation of mobilized T cell receptor Vβ clonotypes among newly generated circulating memory T lymphocytes, from day 7 onwards. Mobilization of the tissue-resident memory T cells is cognate, in that memory T lymphocytes recognizing other antigens, e.g. tetanus toxin, are not mobilized, unless they cross-react with the vaccine. These data originally demonstrate the essential contribution of tissue-resident memory T cells to secondary systemic immune responses, confirming that immunological memories to systemic pathogens are maintained (also) by tissue-resident memory T cells. In practical terms, the present work defines day 1 to 2 after antigenic challenge as a time window to assess the entire immunological T cell memory for a certain pathogen, including mobilized tissue-resident memory T cells, and its correlates of effectivity.Capsule summaryThe study demonstrates the rapid and cognate mobilization of tissue-resident memory CD4+ T cells into the blood upon antigenic rechallenge, and their contribution to secondary systemic immune responses.

Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C+ NK cells has remained unclear. Here we found that adaptive NKG2C+ NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C+ NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C+ NK cell populations among HCMV-seropositive people.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the genera- tion and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer’s patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

There are currently limited insights into the progression of human primary humoral immunity despite numerous studies in experimental models. In this study, we analyzed a primary and related secondary parenteral keyhole limpet hemocyanin (KLH) immunization in five human adults. The primary challenge elicited discordant KLH-specific serum and blood effector B cell responses (i.e., dominant serum KLH-specific IgG and IgM levels versus dominant KLH-specific IgA plasmablast frequencies). Single-cell IgH sequencing revealed early appearance of highly (>15 mutations) mutated circulating KLH-specific plasmablasts 2 wk after primary KLH immunization, with simultaneous KLH-specific plasmablasts carrying non- and low-mutated IgH sequences. The data suggest that the highly mutated cells might originate from cross-reactive memory B cells (mBCs) rather than from the naive B cell repertoire, consistent with previous reported mutation rates and the presence of KLH-reactive mBCs in naive vaccinees prior to immunization. Whereas upon secondary immunization, serum Ab response kinetics and plasmablast mutation loads suggested the exclusive reactivation of KLH-specific mBCs, we, however, detected only little clonal overlap between the peripheral KLH-specific secondary plasmablast IgH repertoire and the primary plasmablast and mBC repertoire, respectively. Our data provide novel mechanistic insights into human humoral immune responses and suggest that primary KLH immunization recruits both naive B cells and cross-reactive mBCs, whereas secondary challenge exclusively recruits from a memory repertoire, with little clonal overlap with the primary response.

It is a matter of current debate whether the bone marrow is a hub for circulating memory T lymphocytes and/or the home of resident memory T lymphocytes. Here we demonstrate for CD69+ murine CD8+ , and CD69+ murine and human CD4+ memory T lymphocytes of the bone marrow, making up between 30 and 60% of bone marrow memory T lymphocytes, that they express the gene expression signature of tissue-resident memory T lymphocytes. This suggests that a substantial proportion of bone marrow memory T lymphocytes are resident. It adds to previous evidence that bone marrow memory T cells are resting in terms of mobility and proliferation, and maintain exclusive long-term memory to distinct, systemic antigens.

Summary Innate lymphoid cells (ILCs) are critical effectors of innate immunity and inflammation, whose development and activation pathways make for attractive therapeutic targets. However, human ILC generation has not been systematically explored, and previous in vitro investigations relied on the analysis of few markers or cytokines, which are suboptimal to assign lineage identity. Here, we developed a platform that reliably generated human ILC lineages from CD34+ hematopoietic progenitors derived from cord blood and bone marrow. We showed that one culture condition is insufficient to generate all ILC subsets, and instead, distinct combination of cytokines and Notch signaling are essential. The identity of natural killer (NK)/ILC1s, ILC2s, and ILC3s generated in vitro was validated by protein expression, functional assays, and both global and single-cell transcriptome analysis, recapitulating the signatures and functions of their ex vivo ILC counterparts. These data represent a resource to aid in clarifying ILC biology and differentiation.

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor α4β7 was found in CD8(+) T cells, questioning the concept. We now demonstrate that α4β7 expression in murine CD4(+) memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced α4β7. A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of α4β7 expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by α4β7 is imprinted in CD4(+) memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation. © 2017 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.

Introduction/AbstractT lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro1–29. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA)30–33. However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the heterogeneity of synovial T lymphocytes in JIA patients by single cell RNA-sequencing. We identify subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1+TOX+EOMES+ population of CD4+ T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+TOX+BHLHE40+ population of CD4+, and a mirror population of CD8+ T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.

SummaryPersistence of long-lived, memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, provided in niches organized by stromal cells. Here we describe that ex vivo we can prevent apoptosis of bone marrow plasma cells by supplying direct cell contact with stromal cells and the soluble cytokine APRIL. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the PI3K signaling pathway, leading to critical inactivation of FoxO1/3 and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Likely, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, via the NF-κB pathway, blocks activation of the endoplasmic reticulum stress-associated initiator caspase 12. Thus, stromal cell-contact induced PI3K and APRIL-induced NF-κB signaling provide necessary and complementary signals to maintain bone marrow memory plasma cells.

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-β receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.

Career situation of first and presenting author Post-doctoral fellow. Introduction The human intestine is colonized with billions of microorganisms, which form the gut microbiota, consisting of up to 1000 different bacterial species. Recent studies have implicated the intestinal microbiota in the pathogenesis of chronic inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, and Sjogren’s syndrome. Yet, we still lack the knowledge which bacteria of the gut microbiota induce, promote or inhibit chronic inflammatory inflammation. Objectives The aim of our work is to identify members of the intestinal microbiota with pro- or anti-inflammatory properties for targeted and therapeutic manipulation of the microbiota in chronic inflammatory diseases. Methods We have developed high-resolution microbiota flow cytometry which allows us to analyze the microbiota on a single cell level. This provides a non-invasive, fast and efficient diagnostic tool to visualize dramatic changes of microbiota composition in inflammatory diseases, fast and efficiently, and isolate distinct bacteria for functional and molecular analyses. Results We have identified bacteria belonging to the genus Anaeroplasma, which enhances the levels of mucosal IgA. Adoptive transfer of Anaeroplasma increases the numbers of IgA+ germinal center B cells in the Peyer’s patches and of IgA-secreting plasma cells in the lamina propria of the small intestine leading to significantly enhanced mucosal IgA levels. Anaeroplasma controls IgA expression presumably its ability to induce expression of the regulatory cytokine TGF-β in T cells, as we show here. Conclusions The anti-inflammatory properties of Anaeroplasma to induce the anti-inflammatory cytokine TGF-β, thereby also strengthening the intestinal barrier by enhancing mucosal IgA, qualify Anaeroplasma as potent probiotic for the prevention and treatment of chronic inflammation. Disclosure of Interest None declared.

Patients with kidney failure are at increased risk for SARS-CoV-2 infection making effective vaccinations a critical need. It is not known how well mRNA vaccines induce B and plasma cell responses in dialysis patients (DP) or kidney transplant recipients (KTR) compared to healthy controls (HC). We studied humoral and B cell responses of 35 HC, 44 DP and 40 KTR. Markedly impaired anti-BNT162b2 responses were identified among KTR and DP compared to HC. In DP, the response was delayed (3-4 weeks after boost) and reduced with anti-S1 IgG and IgA positivity in 70.5% and 68.2%, respectively. In contrast, KTR did not develop IgG responses except one patient who had a prior unrecognized infection and developed anti-S1 IgG. The majority of antigen-specific B cells (RBD+) were identified in the plasmablast or post-switch memory B cell compartments in HC, whereas RBD+ B cells were enriched among pre-switch and naïve B cells from DP and KTR. The frequency and absolute number of antigen-specific circulating plasmablasts in the cohort correlated with the Ig response, a characteristic not reported for other vaccinations. In conclusion, these data indicated that immunosuppression resulted in impaired protective immunity after mRNA vaccination, including Ig induction with corresponding generation of plasmablasts and memory B cells. Thus, there is an urgent need to improve vaccination protocols in patients after kidney transplantation or on chronic dialysis.

Background:We recently reported the beneficial clinical responses of the anti-CD38 monoclonal antibody daratumumab in two patients with systemic lupus erythematosus (SLE) [1]. While the primary rationale for its use was the depletion of autoantibody-producing long-lived plasma cells, daratumumab may promote additional therapeutic effects on CD38-expressing T cells, but their origin, lifestyle and role in lupus pathophysiology remains elusive.Objectives:To investigate the phenotype, transcriptional program, functional properties and clinical associations of CD4+ and CD8+CD38+ memory T cells in SLE compared to healthy controls (HC).Methods:CD38-expression on memory T cell subsets was measured by flow cytometry in 65 patients with SLE and 28 healthy controls. We investigated the functional capacity of CD38+ T cells using CFSE staining and intracellular cytokine staining after polyclonal stimulation. Additionally, we performed single-cell transcriptome and T-cell-receptor sequencing of 7 SLE patients and 7 matched healthy controls, including surface protein expression analysis using CITE-seq (RNA-barcoded) antibodies.Results:Compared to healthy controls, the frequency of CD38-expressing memory T cells in SLE was significantly increased in both CD4+ and CD8+ T cells. SLE patients with a previous or current lupus nephritis had significantly increased levels of CD8+CD38+ memory T cells compared to those without history of renal involvement. CD38+ memory T cells expressed increased levels of Ki-67 and displayed higher proliferative capacity upon polyclonal stimulation than their CD38- counterparts, both in SLE patients and HC, while they showed decreased ability to secrete IFN-γ, IL-2, GM-CSF and TNF-α. Single-cell transcriptome sequencing revealed that CD8+CD38+ memory T cells were enriched within terminally differentiated, cytotoxic CD8 T cells, and had reduced TCR repertoire diversity compared to their CD38- counterparts. CD8+CD38+ memory T cells from SLE patients had significantly higher expression of type I interferon associated genes, both compared to CD38- memory T cells from SLE patients and CD38+ cells from HCs.Conclusion:CD38+ memory T cells with increased proliferative capacity but altered effector functions are significantly expanded in peripheral blood of SLE and correlate with the lupus nephritis. Although the factors mediating their generation and their precise role in the disease pathophysiology remain to be investigated, CD38-expressing T cells may be useful as a future biomarker for lupus nephritis.References:[1]Ostendorf L, Burns M, Durek P, Heinz GA, Heinrich F, Garantziotis P, et al. Targeting CD38 with Daratumumab in Refractory Systemic Lupus Erythematosus. N Engl J Med. 2020 Sep 17;383(12):1149–55.[2]Katsuyama E, Suarez-Fueyo A, Bradley SJ, Mizui M, Marin AV, Mulki L, et al. The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections. Cell Reports. 2020 Jan;30(1):112-123.e4.Acknowledgements:We thank our patients and healthy controls for making our research possible.Disclosure of Interests:None declared.

The potential diagnostic value of circulating free miRNAs in plasma compared to miRNA expression in blood cells for rejection processes after kidney transplantation is largely unknown, but offers the potential for better and timely diagnosis of acute rejection. Free microRNA expression of specific blood cell markers was measured in 160 plasma samples from kidney transplant patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection, interstitial fibrosis and tubular atrophy, antibody-mediated rejection (ABMR), Borderline (Banff3), tubulo-interstitial (Banff4-I) and vascular rejection (Banff4-II/III) applying RT-PCR. The expression levels of specific microRNAs miR-15B, miR-103A and miR-106A discriminated patients with stable graft function significantly (p-values 0.001996, 0.0054 and 0.0019 resp.) from patients with T-cell mediated rejection (TCMR) and from patients with urinary tract infection (p-values 0.0001,0.0001 and 0.0001, resp.). A combined measurement of several microRNAs after multivariate logistic regression improved the diagnostic value supported by subsequent cross-validation. In conclusion, the measurement of circulating microRNAs in plasma from patients with renal transplants distinguishes TCMR and urinary tract infection from stable graft function. In contrast to miRNA expression measurement in blood cells it does not allow a discrimination from ABMR or interstitial fibrosis and tubular atrophy.

E- and P-selectin ligands (E- and P-ligs) guide effector memory T cells into skin and inflamed regions, mediate the inflammatory recruitment of leukocytes, and contribute to the localization of hematopoietic precursor cells. A better understanding of their molecular regulation is therefore of significant interest with regard to therapeutic approaches targeting these pathways. In this study, we examined the transcriptional regulation of fucosyltransferase 7 (FUT7), an enzyme crucial for generation of the glycosylated E- and P-ligs. We found that high expression of the coding gene fut7 in murine CD4+ T cells correlates with DNA demethylation within a minimal promoter in skin/inflammation-seeking effector memory T cells. Retinoic acid, a known inducer of the gut-homing phenotype, abrogated the activation-induced demethylation of this region, which contains a cAMP responsive element. Methylation of the promoter or mutation of the cAMP responsive element abolished promoter activity and the binding of CREB, confirming the importance of this region and of its demethylation for fut7 transcription in T cells. Furthermore, studies on human CD4+ effector memory T cells confirmed demethylation within FUT7 corresponding to high FUT7 expression. Monocytes showed an even more extensive demethylation of the FUT7 gene whereas hepatocytes, which lack selectin ligand expression, exhibited extensive methylation. In conclusion, we show that DNA demethylation within the fut7 gene controls selectin ligand expression in mice and humans, including the inducible topographic commitment of T cells for skin and inflamed sites.

The B6·Cg-Tg(Cd4-cre)1Cwi line expresses CRE recombinase under the control of the promoter and regulatory elements of the Cd4 gene. Here we show that CRE recombinase expression reduces the number and frequencies of CD4 positive subsets in a dose-dependent manner and localize the integration site of the transgenic construct to position 60335693-60341285 (qD) of chromosome 3. The insert contains at least 15 complete sequential copies of the transgenic construct. Based on this information we describe a novel PCR assay for genetic typing of transgenic mice.

Using high-resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single-cell level.

Background:Plasma Cells (PCs) are implicated in the pathogenesis of Systemic Lupus erythematosus (SLE) and their targeting proved a promising treatment modality. As there is a monoclonal therapeutic antibody targeting CD38 licensed for clinical use in multiple myeloma, plasma cell depletion via CD38 seems to represent a promising path in SLE treatment. While CD38 Is highly expressed on plasmacells, it is present on the surface of subsets of T and B lymphocytes as well as myeloid cells.Objectives:Here we aim to identify the differential expression of CD38 on peripheral blood leukocytes in SLE compared to healthy controls (HC) investigate the function of CD38+ T lymphocytesMethods:We performed flow cytometry to investigate the expression of CD38 on peripheral blood mononuclear cells of SLE patients (n=36) and HCs (n=20). We additionally analyzed the expression of T lymphocytes within the urine of patients with lupus nephritis as well as the skin of SLE patients. We investigated the inflammatory potential of CD38 positive memory T lymphocytes after stimulation and performed single-cell RNA sequencing analyses.Results:CD38 Expression is increased on certain immune cell subsets: Plasmablasts and unswitched Memory B cells, as well as plasmacytoid dendritic cells and CD16+ non-classical monocytes. We observed a drastic increase CD38 in both memory CD4 and CD8 T lymphocytes in SLE patients. These cells were mostly effector T cells (and not regulatory T cells) and expressed other markers of T cell activation and proliferation. We found an enrichment of CD38+ memory T cells in the urine of patients with lupus nephritis. After polyclonal stimulation of T cells, CD38+ produced less inflammatory cytokines. Preliminary single-cell sequencing results indicate that CD38+ CD8+ T-lymphocytes have decreased clonal diversity and that these cells express genes associated with exhaustion and type 1 interferon response.Conclusion:Increased CD38 expression on various lymphocyte subsets provides an additional rationale for investigating CD38-directed therapies in SLE. Targeting CD38 could not only deplete plasma cells but also has the potential to target interferon alpha producing plasmacytoid dendritic cells and modulate inflammatory T cell functions.Disclosure of Interests:Lennard Ostendorf: None declared, Philipp Enghard: None declared, Pawel Durek: None declared, Frederik Heinrich: None declared, Mir-Farzin Mashreghi: None declared, Gerd Rüdiger Burmester Consultant of: AbbVie Inc, Eli Lilly, Gilead, Janssen, Merck, Roche, Pfizer, and UCB Pharma, Speakers bureau: AbbVie Inc, Eli Lilly, Gilead, Janssen, Merck, Roche, Pfizer, and UCB Pharma, Andreas Radbruch: None declared, Falk Hiepe: None declared, Tobias Alexander: None declared

Summary Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor- and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)-mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells.

Item does not contain fulltext The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease. PAPERCLIP.

Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC., The contribution of intra-tumour heterogeneity is increasingly associated with resistance to therapy. Here, the authors use genomic analyses to study heterogeneity in colorectal cancer and perform in-depth reconstruction of heterogeneity in one sample.

Since the bona fide Fc receptor for IgM antibody (FcµR) was identified eight years ago, much progress has been made in defining its biochemical nature, cellular distribution, and effector function. However, there are clearly conflicting results, especially about the cellular distribution and function of murine FcµR. In this short article, we will discuss recent findings from us and other investigators along with our interpretations and comments that may help to resolve the existing puzzles and should open new avenues of investigation.

Biochemical, molecular and genetic studies emphasize the role of the potato vacuolar invertase Pain-1 in the accumulation of reducing sugars in potato tubers upon cold storage, and thereby its influence on the quality of potato chips and French fries. Previous studies showed that natural Pain-1 cDNA alleles were associated with better chip quality and higher tuber starch content. In this study, we focused on the functional characterization of these alleles. A genotype-dependent transient increase of total Pain-1 transcript levels in cold-stored tubers of six different genotypes as well as allele-specific expression patterns were detected. 3D modelling revealed putative structural differences between allelic Pain-1 proteins at the molecule's surface and at the substrate binding site. Furthermore, the yeast SUC2 mutant was complemented with Pain-1 cDNA alleles and enzymatic parameters of the heterologous expressed proteins were measured at 30 and 4 °C. Significant differences between the alleles were detected. The observed functional differences between Pain-1 alleles did not permit final conclusions on the mechanism of their association with tuber quality traits. Our results show that natural allelic variation at the functional level is present in potato, and that the heterozygous genetic background influences the manifestation of this variation.

The integrated analysis of omics datasets covering different levels of molecular organization has become a central task of systems biology. We investigated the transcriptional and metabolic response of yeast exposed to increased (37 degrees C) and lowered (10 degrees C) temperatures relative to optimal reference conditions (28 degrees C) in the context of known metabolic pathways. Pairwise metabolite correlation levels were found to carry more pathway-related information and to extend to farther distances within the metabolic pathway network than associated transcript level correlations. Metabolites were detected to correlate stronger to their cognate transcripts (metabolite is reactant of the enzyme encoded by the transcript) than to more remote or randomly chosen transcripts reflecting their close metabolic relationship. We observed a pronounced temporal hierarchy between metabolic and transcriptional molecular responses under heat and cold stress. Changes of metabolites were most significantly correlated to transcripts encoding metabolic enzymes, when metabolites were considered leading in time-lagged correlation analyses. By applying the concept of Granger causality, we detected directed relationships between metabolites and their cognate transcripts. When interpreted as substrate-to-product directions, most of these directed Granger causality pairs agreed with the KEGG-annotated preferred reaction direction. Thus, the introduced Granger causality approach may prove useful for determining the preferred direction of metabolic reactions in cellular systems. The metabolites glutamic acid and serine were identified as central causative metabolites influencing transcript levels at later time points. Selected examples are presented illustrating the intertwined relationships between metabolites and transcripts in the yeast temperature stress adaptation process.

The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows 'on-the-fly' prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.

MicroRNAs (miRNAs, miR) hold important roles in the posttranscriptional regulation of gene expression. Their function has been correlated with kidney disease, and they might represent a new class of biomarkers for frequent evaluation of renal graft status. We analyzed their potential in identifying severe T cell-mediated vascular rejection (TCMVR) (Banff 4-II/III) in kidney transplanted patients.Microarray experiments and semiquantitative real-time reverse transcription polymerase chain reaction were performed with total RNA isolated from blood cells of kidney graft recipients. Initial microarray analysis revealed 23 differentially expressed miRNAs distinguishing patients with TCMVR from patients with stable grafts. From these, we validated and further determined the expression of 6 differentially expressed miRNAs and 2 control miRNAs in 161 samples from patients with T cell-mediated rejection (Banff 3-Borderline, Banff 4-I/II/III), Banff-2 antibody-mediated rejection, Banff-5 interstitial fibrosis/tubular atrophy, in samples from stable patients and in samples from patients with urinary tract infection using real-time reverse transcription polymerase chain reaction.Expression levels of all 6 candidate miRNAs were significantly downregulated in blood of TCMVR patients compared to the other groups and displayed high sensitivities and specificities for diagnosing TCMVR. The combination of 5 miRNAs, identified by an unbiased multivariate logistic regression followed by cross-validation, enhanced the sensitivity and specificity for the diagnosis of TCMVR after renal transplantation.The combined measurement of miRNA-15B, miRNA-16, miRNA-103A, miRNA-106A, and miRNA-107 may help to better identify TCMVR after renal transplantation in a precise and clinically applicable way.

A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N-acetyl-N-hydroxy-L-ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L-N5-ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains.

Group 3 innate lymphoid cells (ILC3s) are defined by the expression of the transcription factor RORγt, which is selectively required for their development. The lineage-specified progenitors of ILC3s and their site of development after birth remain undefined. Here we identified a population of human CD34(+) hematopoietic progenitor cells (HPCs) that express RORγt and share a distinct transcriptional signature with ILC3s. RORγt(+)CD34(+) HPCs were located in tonsils and intestinal lamina propria (LP) and selectively differentiated toward ILC3s. In contrast, RORγt(-)CD34(+) HPCs could differentiate to become either ILC3s or natural killer (NK) cells, with differentiation toward ILC3 lineage determined by stem cell factor (SCF) and aryl hydrocarbon receptor (AhR) signaling. Thus, we demonstrate that in humans RORγt(+)CD34(+) cells are lineage-specified progenitors of IL-22(+) ILC3s and propose that tonsils and intestinal LP, which are enriched both in committed precursors and mature ILC3s, might represent preferential sites of ILC3 lineage differentiation.

Analysis of the signaling response of colon cancer cells to systematic perturbations reveals an EGF receptor-mediated cross-talk between the MAPK and AKT pathways. Accordingly, the predicted combinatorial treatment is shown to inhibit tumor growth in vivo., A modular response analysis model trained on perturbation data in a panel of colon cancer cells revealed a negative feedback from MAPK signaling to the EGF receptor, which leads to AKT cross-activation after MEK inhibition. The model predicted that successful inhibition of growth-factor signaling in colon cancer requires combined inhibition of MEK and EGF receptor to prevent AKT activation and tumor cell survival, which was confirmed by growth assays. A xenograft tumor model of KRAS-mutant colon cancer showed that EGFR receptor inhibition alone has no effect, but in combination with a MEK inhibitor successfully reduces tumor growth., The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small-molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model-based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR-dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF- as well as KRAS-mutated tumor cells, which we confirmed using a xenograft model.

The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27 782 predicted phosphoserine sites distributed across 17 035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link "http://phosphat.mpimp-golm.mpg.de .

Memory type 1 T helper (TH1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8+ T cells or TH1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2Chi NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells., Author Summary Upon viral infection, the innate interferon (IFN)-γ producing Natural Killer (NK) cells provide fast, but short-term protection, while adaptive T cells confer delayed, but long-lasting immunity. Once acquired, effector properties remain stably imprinted in the T cell memory progeny. Recently, it was shown that human cytomegalovirus (HCMV) infection can shape the human NK cell repertoire and drive the generation and maintenance of NK cell expansions, which express the activating receptor CD94/NKG2C and have been described as memory-like NK cells. However, the molecular mechanisms underlying NK cell adaptive properties driven by HCMV infection have not been completely defined. In this study, we identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, thus providing a molecular basis for the regulation of adaptive features in innate cells.

Summary A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 ( rds ) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 ( rpt ) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt ) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.

Summary The persistence of long-lived memory plasma cells in the bone marrow depends on survival factors available in the bone marrow, which are provided in niches organized by stromal cells. Using an ex vivo system in which we supply the known survival signals, direct cell contact to stromal cells, and the soluble cytokine a proliferation-inducing ligand (APRIL), we have elucidated the critical signaling pathways required for the survival of long-lived plasma cells. Integrin-mediated contact of bone marrow plasma cells with stromal cells activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, leading to critical inactivation of Forkhead-Box-Protein O1/3 (FoxO1/3) and preventing the activation of mitochondrial stress-associated effector caspases 3 and 7. Accordingly, inhibition of PI3K signaling in vivo ablates bone marrow plasma cells. APRIL signaling, by the nuclear factor κB (NF-κB) pathway, blocks activation of the endoplasmic-reticulum-stress-associated initiator caspase 12. Thus, stromal-cell-contact-induced PI3K and APRIL-induced NF-κB signaling provide the necessary and complementary signals to maintain bone marrow memory plasma cells., Graphical Abstract, Highlights • Description of an in vitro niche for the cultivation of primary murine plasma cells • Stromal cell contact and APRIL are essential for survival of bone marrow plasma cells • Stromal-cell-contact-induced PI3K signaling inhibits FoxO1/3 and caspase 3 activation • APRIL-induced NF-κB signaling prevents caspase 12 activation, In this study, Cornelis et al. address the molecular mechanisms underlying the survival of murine memory plasma cells in the bone marrow. The authors provide evidence that direct contact to stromal cells and exogenous APRIL provide resilience to mitochondrial and endoplasmic stress, respectively, synergistically promoting plasma cell survival.