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Patients with kidney failure are at increased risk during the COVID-19 pandemic and effective vaccinations are needed. It is not known how efficient mRNA vaccines mount 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 25 HC, 44 DP and 40 KTR. Markedly impaired anti-BNT162b2 responses were identified among KTR and DP compared to 100% seroconversion in HC. In DP, the response was delayed (3-4 weeks after boost) and reduced with anti-S1 IgG positivity in 31 (70.5%) and anti-S1 IgA in 30 (68.2%) of 44, respectively. In contrast, KTR did not develop IgG response except one patient who had 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 these RBD+ B cells were enriched among pre-switch and naïve B cells from DP and KTR. Single cell transcriptome and CITE-seq analyses found reduced frequencies of plasmablasts, TCF7+CD27+GZMK+ T cells and proliferating MKI67-expressing lymphocytes among KTR non-responders. Importantly, the frequency and absolute number of antigen-specific circulating plasmablasts in the whole cohort correlated with the Ig response, a characteristic not reported for other vaccinations. In conclusion, this data indicate that lack of T cell help related to immunosuppression results in impaired germinal center differentiation of B and plasma cell memory. There is an urgent need to improve vaccination protocols in patients after kidney transplantation or on chronic dialysis.One Sentence SummaryKidney transplant recipients and dialysis patients show a markedly diminished humoral response and impaired molecular B cell memory formation upon vaccination with BNT162b2.

Why do we still have no cure for chronic inflammatory diseases? One reason could be that current therapies are based on the assumption that chronic inflammation is driven by persistent 'acute' immune reactions. Here we discuss a paradigm shift by suggesting that beyond these reactions, chronic inflammation is driven by imprinted, pathogenic 'memory' cells of the immune system. This rationale is based on the observation that in patients with chronic inflammatory rheumatic diseases refractory to conventional immunosuppressive therapies, therapy-free remission can be achieved by resetting the immune system; that is, by ablating immune cells and regenerating the immune system from stem cells. The success of this approach identifies antigen-experienced and imprinted immune cells as essential and sufficient drivers of inflammation. The 'dark side' of immunological memory primarily involves memory plasma cells secreting pathogenic antibodies and memory T lymphocytes secreting pathogenic cytokines and chemokines, but can also involve cells of innate immunity. New therapeutic strategies should address the persistence of these memory cells. Selective targeting of pathogenic immune memory cells could be based on their specificity, which is challenging, or on their lifestyle, which differs from that of protective immune memory cells, in particular for pathogenic T lymphocytes. The adaptations of such pathogenic memory cells to chronic inflammation offers entirely new therapeutic options for their selective ablation and the regeneration of immunological tolerance.

Bone growth requires a specialised, highly angiogenic blood vessel subtype, so-called type H vessels1,2, which pave the way for osteoblasts surrounding these vessels3. At the end of adolescence, type H endothelial cells differentiate into quiescent type L endothelium lacking the capacity to promote bone growth. Until now, the signals that switch off type H vessel identity and thus arrest adolescent bone growth have remained ill defined. Here we show that mechanical forces, associated with increased body weight at the end of adolescence, trigger the mechanoreceptor PIEZO1 and thereby mediate enhanced production of the kinase FAM20C in osteoblasts. FAM20C phosphorylates dentin matrix protein 1 (DMP1)4, previously identified as a key factor in bone mineralization5. This phosphorylation elicits a burst in DMP1 secretion from osteoblasts. Extracellular DMP1 inhibits vascular endothelial growth factor (VEGF) signalling by preventing VEGFR2 phosphorylation and VEGFR3 expression on the tip cells of type H endothelium. DMP1-mediated VEGF inhibition transforms bone growth-promoting type H vessels into quiescent type L vasculature to arrest bone growth and enhance bone mineralization. This molecular mechanism links mechanical forces and the termination of bone growth via accumulation of an extracellular matrix protein and its regulation of vascular subtypes. It suggests new options for the treatment of diseases characterised by inappropriate turnover or invasion of bone such as osteoarthritis, osteoporosis and osteosarcoma.

© 2021 The Authors., The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., Hyun-Dong Chang is supported by the Dr. Rolf M. Schwiete Foundation. Susanne Melzer and Attila Tarnok thank De Novo Software for providing FCS Express. Enrico Lugli is supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC IG20676). Gabriele De Simone and Giovanni Galletti were supported by Fellowships from the Fondazione Italiana per la Ricerca sul Cancro-Associazione Italiana per la Ricerca sul Cancro (FIRC-AIRC). Jun Dong is supported by Deutsche Forschungsgemeinschft (DFG, German Research Foundation) Projektnummer 389687267 and Chinesisch-Deutsches Zentrum für Wissenschaftsförderung [Sino-German Center for Research Promotion (SGC)] grant C-0072. Nicola Gagliani, Samuel Huber and Franziska Muscate are supported by DFG fundings: SFB841,GA 2441/3-1, HU 1714/10-1. The tetramer APC-conjugated H-2K (d) HIV-1 gag197-205 AMQMLKETI used in TDS assay for mouse blood T cells was obtained through the NIH Tetramer Facility. Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14-1. Supported by the following grants: AIRC 5X1000 2018 id. 21147 (Lorenzo Moretta); AIRC IG 2017 id. 19920 (Lorenzo Moretta); RC-2020 OPBG (Lorenzo Moretta); AIRC and European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 800924 (Linda Quatrini). Dirk Baumjohann was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Emmy Noether Programme BA 5132/1-2 (252623821) and Germany's Excellence Strategy EXC2151 (390873048).

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 PLZF

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

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.

RORγt

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.

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.

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

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.

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.

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.

MicroRNAs (miRNAs) are endogenously encoded ∼22 nt small non-coding RNAs. They function as key players of many cellular processes by base pairing with target mRNAs and thereby impairing gene expression at the post-transcriptional level. Recent findings demonstrate a critical role of many miRNAs in immune cell differentiation and immune responses, which is also associated with the development and progression of many tumor and non-tumor diseases. Here we review the multifaceted miRNA-148/-152 family members consisting of miR-148a, miR-148b and miR-152. Next to regulation mechanisms that control the expression of this miRNA family, we will focus on (i) the role of miR-148a in regulating B and T lymphocyte function and its role in associated diseases and (ii) the importance of miR-148/-152 family members for cancer initiation, tumor growth and metastasis formation. In addition, this review aims to highlight some selected targets of the miRNA-148/-152 family members, which are involved in the biology of cancer and maintenance of epigenetic patterns. In conclusion, members of the miR-148/-152 family might represent prognostic markers and/or potential therapeutic targets for treatment of autoimmune disorders, chronic inflammatory diseases and multiple types of cancer.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Cellular and antibody-mediated rejection processes and also interstitial fibrosis/tubular atrophy (IFTA) lead to allograft dysfunction and loss. The search for accurate, specific and non-invasive diagnostic tools is still ongoing and essential for successful treatment of renal transplanted patients. Molecular markers in blood cells and serum may serve as diagnostic tools but studies with high patient numbers and differential groups are rare. We validated the potential value of several markers on mRNA level in blood cells and serum protein level in 166 samples from kidney transplanted patients under standard immunosuppressive therapy (steroids ± mycophenolic acid ± calcineurin inhibitor) with stable graft function, urinary tract infection (UTI), IFTA, antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR) applying RT-PCR and ELISA. The mRNA expression of RANTES, granulysin, granzyme-B, IP-10, Mic-A and Interferon-γ in blood cells did not distinguish specifically between the different pathologies. We furthermore discovered that the mRNA expression of the chemokine IL-8 is significantly lower in samples from IFTA patients than in samples from patients with stable graft function (p

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.

CD4 T cell memory is fundamental for long-lasting immunity and effective secondary responses following infection or vaccination. We have previously found that memoryCD4 T cells specific for systemic antigens preferentially reside in the bone marrow (BM)and arise from splenic CD49b+T-bet+ CD4 T cells. However, how BM-homing memory precursors are generated during an immune reaction is unknown. We show here that BM memory precursors are generated via augmented rates of cell division throughout a primary immune response. Treatment with the cytostatic drug cyclophosphamide or blockade of the CD28/B7 co-stimulatory pathway at the beginning of the contraction phase abrogates the generation of BM memory precursors. We determine that,following a critical number of cell divisions, memory precursors down regulate CCR7 and upregulate IL-2Rb, indicating that loss of CCR7 and gain of IL-2 signal are required for the migration of memory precursors toward the BM. Refereed/Peer-reviewed

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.

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.

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.

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.

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.

ICOS signaling is required for inhibition of the transcription factor Klf2, which controls expression of genes expressed by follicular T helper (Tfh) cells. When ICOS signaling is blocked, Tfh cells lose expression of characteristic Tfh genes and revert to an effector phenotype, resulting in disruption of the germinal center response., The co-stimulators ICOS (inducible T cell co-stimulator) and CD28 are both important for T follicular helper (TFH) cells, yet their individual contributions are unclear. Here, we show that each molecule plays an exclusive role at different stages of TFH cell development. While CD28 regulated early expression of the master transcription factor Bcl-6, ICOS co-stimulation was essential to maintain the phenotype by regulating the novel TFH transcription factor Klf2 via Foxo1. Klf2 directly binds to Cxcr5, Ccr7, Psgl-1, and S1pr1, and low levels of Klf2 were essential to maintain this typical TFH homing receptor pattern. Blocking ICOS resulted in relocation of fully developed TFH cells back to the T cell zone and reversion of their phenotype to non-TFH effector cells, which ultimately resulted in breakdown of the germinal center response. Our study describes for the first time the exclusive role of ICOS and its downstream signaling in the maintenance of TFH cells by controlling their anatomical localization in the B cell follicle.

Foxp3

It is current belief that numbers of CD8+ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8+ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8+ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8+ memory T lymphocytes are maintained by proliferation. The numbers of CD8+ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8+ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.

Background Recent reports have shown dysregulated microRNAs (miRNAs) in murine models of lupus, among them increased expression of microRNA-182 (miRNA-182), which has been demonstrated to target the transcription factor FOXO1 in activated murine CD4 + T cells, leading to spontaneous T cell activation and clonal expansion. Here we aimed to investigate the expression of miR-182 and FOXO1 in T cells from human SLE patients. Methods Expression levels of miR-182 were analysed with RT-PCR in purified peripheral blood CD4 + T cells from 9 patients with SLE and age/sex-matched healthy controls (HC). Multicolor flow cytometry was performed to analyse CD4 + T cell expression for FOXO1 , Ki-67, Foxp3, the interleukin-7 receptor-α (CD127) and phosphorylated STAT-5a (pSTAT5). Analysis of serum IL-7 levels was performed with ELISA in 27 SLE patients and HC. Induction of miR-182 was assessed in vitro after polyclonal T cell stimulation in the presence of IL-7, and inhibition of T cell proliferation investigated using mir-182 antagomirs. Results MiRNA-182 was significantly upregulated in CD4 + T cells from SLE patients compared to HC, while the FOXO1 expression was significantly decreased. The percentage of proliferating Ki-67 + conventional Foxp3 - CD4 + T cells T CON was significantly higher in SLE compared to HC (3.85% vs. 1.58%, p CON displayed decreased expression levels for the FOXO1 target gene CD127 (MFI 2021 vs. 2553, p=0.049) and serum IL-7 levels were significantly higher in SLE compared to HC (17.0 pg/ml vs. 10.2 pg/ml, p=0.001). In vitro , miR-182 could be induced by IL-7, and specific inhibition of miR-182 inhibited T cell proliferation and survival. Conclusion Our data suggest that enhanced IL7R/STAT5 signalling mediates the induction of miR182 expression, which promotes the proliferation of conventional Foxp3 - T cells SLE. Collectively, our data provide new insights in the pathophysiology of T cell hyperactivity in SLE and identifies miR-182 as a candidate target for future therapeutic approaches.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory., Highlights • Th1 cells expressing miR-148a mediate colitis in a murine model of IBD. • Antagomir-148a inhibits colitis by selectively depleting Th1 cells from the colon. • Antagomir-148a does not affect the protective immunological memory.

Background: Not every T helper type 2 (Th2) lymphocyte imprinted to express interleukin-4 (IL-4) does so when activated. Results: Preventing nuclear translocation of the nuclear factor of activated T cells (NFAT) reduces the number of Th2 lymphocytes reexpressing IL-4. Conclusion: NFAT is the limiting factor determining digital IL-4 expression in Th2 lymphocytes. Significance: This might help us to understand the regulation of immunopathology in allergy and asthma., Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.

The transcriptional regulator Blimp-1 is absolutely required for IL-10 production in Th1 cells and limits inflammatory effector T cell responses downstream of IL-12 and IL-27., Secretion of the immunosuppressive cytokine interleukin (IL) 10 by effector T cells is an essential mechanism of self-limitation during infection. However, the transcriptional regulation of IL-10 expression in proinflammatory T helper (Th) 1 cells is insufficiently understood. We report a crucial role for the transcriptional regulator Blimp-1, induced by IL-12 in a STAT4-dependent manner, in controlling IL-10 expression in Th1 cells. Blimp-1 deficiency led to excessive inflammation during Toxoplasma gondii infection with increased mortality. IL-10 production from Th1 cells was strictly dependent on Blimp-1 but was further enhanced by the synergistic function of c-Maf, a transcriptional regulator of IL-10 induced by multiple factors, such as the Notch pathway. We found Blimp-1 expression, which was also broadly induced by IL-27 in effector T cells, to be antagonized by transforming growth factor (TGF) β. While effectively blocking IL-10 production from Th1 cells, TGF-β shifted IL-10 regulation from a Blimp-1–dependent to a Blimp-1–independent pathway in IL-27–induced Tr1 (T regulatory 1) cells. Our findings further illustrate how IL-10 regulation in Th cells relies on several transcriptional programs that integrate various signals from the environment to fine-tune expression of this critical immunosuppressive cytokine.

Context Antibody-mediated rejection (ABMR) after kidney transplantation (KTx) remains the crucial obstacle to successful long-term graft function. The identification of gene signatures involved in ABMR could grant the basis for better prevention and treatment strategies. Objective The identification of gene signatures in whole blood cells specific for ABMR after KTx. Materials and methods Total RNA from blood cells of 16 kidney-transplanted patients with ABMR, stable graft function (SGF), and with T-cell-mediated rejection (TCMR) was isolated. Gene expression was determined by high-throughput sequencing followed by validation and analyses of differentially expressed candidates on mRNA level and on protein level in a large patient cohort (n = 185) in patients with SGF, urinary tract infection (UTI), borderline rejection (BL), TCMR, ABMR, and interstitial fibrosis and tubular atrophy. Results From the 570 genes detected, 111 discriminated ABMR from SGF and TCMR. A distinct enrichment of interferon (IFN) type I and type II signature gene set was observed. The expression of candidate genes IFIT1, ETV7, and RSAD2 distinguished ABMR patients from patients with SGF and also TCMR, whereas ETV7 and RSAD2 differentiated ABMR also from BL. Conclusion The IFN-inducible genes ETV7 and RSAD2 represent specific biomarkers for ABMR episodes after KTx.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Summary Humoral rejection processes may lead to allograft injury and subsequent dysfunction. Today, only one B-cell-specific agent is in clinical use and the effects of standard and new immunosuppressant substances on B-cell activation and function are not fully clarified. The impact of sotrastaurin, mycophenolic acid and everolimus on human B-lymphocyte function was assessed by analysing proliferation, apoptosis, CD80/CD86 expression and immunoglobulin and IL-10 production in primary stimulated B cells. In addition, B-cell co-cultures with pre-activated T cells were performed to evaluate the effect of the different immunosuppressive agents on T-cell-dependent immunoglobulin production. Sotrastaurin did not inhibit B-cell proliferation, CD80/CD86 expression, and IgG production and had only minor effects on IgM levels at the highest concentration administered. In contrast, mycophenolic acid and everolimus had strong effects on all B-cell functions in a dose-dependent manner. All immunosuppressive agents caused decreased immunoglobulin levels in T-cell-dependent B-cell cultures. The data provided here suggest that mycophenolic acid and everolimus, but not sotrastaurin, are potent inhibitors of human B-lymphocyte function and activation.

The Forkhead box O (FoxO) family of transcription factors is important for the maintenance of immunological homeostasis and tolerance by controlling the development and function of B and T lymphocytes. Because dysregulation in FoxO activity can result in chronic inflammation and autoimmunity, the transcriptional activity of FoxO proteins is tightly controlled and generally dependent on complex posttranslational modifications that lead either to their nuclear entry and subsequent activation or, alternatively, to their nuclear export. The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) axis represents the major pathway phosphorylating and thereby inactivating FoxO proteins. However, recent results have revealed an additional posttranscriptional mechanism of FoxO inactivation by microRNAs. The discovery of this molecular pathway may provide a new therapeutic avenue for the modulation of FoxO activity in immune-mediated diseases using either microRNA targeting antagomirs or synthetic microRNA mimics, a topic that is addressed in this review.

The prevalence of acute renal allograft rejection has decreased substantially in past decades due to new and more specific immunosuppressive compounds but improvements in long-term graft function have not been achieved. There is a large need for new immunosuppressive agents that lack toxicity of current agents such as calcineurin inhibitors but show high synergistic efficiency in preventing rejection processes.This review summarizes data concerning the pharmacokinetics, pharmacodynamics and clinical efficacy of the new PKC inhibitor sotrastaurin with a focus on renal transplantation. The article contains information that has been presented at international transplant meetings and congresses and that has been published between 2006 and 2010. Additionally, current ongoing trials are described in detail.Immunosuppressive regimens after kidney transplantation consist of a combination of several agents in order to minimize drug toxicity. Therefore, the reader is presented with the most up-to-date/current developments in sotrastaurin applications in Phase I and II trials with emphasis on data maintained from studies that combined sotrastaurin with established agents such as mycophenolic acid and tacrolimus.Several trials are ongoing and planned to determine the optimal immunosuppressive regimen to benefit from sotrastaurin's distinct mechanism of action.

The induction of heme oxygenase 1 (HO-1) by a single treatment with cobalt protoporphyrin (CoPPIX) protects against inflammatory liver failure and ischemia reperfusion injury after allotransplantation. In this context, the HO-1-mediated inhibition of donor-derived dendritic cell maturation and migration is discussed as one of the key events of graft protection. To investigate the poorly understood mechanism of CoPPIX-induced HO-1 activity in more detail, we performed gene expression analysis in murine liver, revealing the up-regulation of STAT3 after CoPPIX treatment. By using wild-type and HO-1-deficient dendritic cells we demonstrated that LPS-induced maturation is dependent on STAT3 phosphorylation and independent of HO-1 activity. In summary, our observations revise our understanding of the anti-inflammatory properties of HO-1 and highlight the immunomodulatory capacity of STAT3, which might be of further interest for targeting undesired immune responses, including ischemia reperfusion injury.

Regulatory T-cells induced via IL-2 and TGFβ in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFβ counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFβ. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation., Antigen stimulation in vivo can reprogram T regulatory cells to lose the expression of Foxp3 and become effector cells. Here the authors show that the mechanism involves dephosphorylation of STAT5 by PTPN2 and downregulation of Foxo1 by miR-182.

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.

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.