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Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x(L), Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-x(L) and Mcl-1. Expression of Bcl-x(L), Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-x(L) and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatemnt with specific small molecule inhibitors of Bcl-x(L) (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-x(L) induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-x(L) and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-x(L) in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-x(L) as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-x

Cet article propose un temoignage du travail du psychologue en psychiatrie, de nos jours (periode precedant l’apparition de la covid-19). Le contexte de transformation des structures hospitalieres et d’evolution des pratiques soignantes est decrit de facon concrete. L’auteure illustre son travail de positionnement aupres des patients et de l’equipe, au fil du deploiement de deux dispositifs d’une unite d’entree : la reunion soignants-soignes et la reunion clinique. L’article cherche a mettre en evidence la fonction intermediaire du psychologue, qui joue un role d’articulation sur de nombreux plans, entre patients et soignants, entre dimension personnelle et dimension groupale et collective, entre reperes theoriques et pratiques effectives, etc. La capacite des professionnels a s’engager dans les dispositifs est soulignee, pour peu que le travail (re)trouve sens. En conclusion, l’auteure suggere des conditions pour un travail d’equipe de qualite, dans lequel le psychologue trouvera sa place.

Memory T follicular helper (TFH) cells play an essential role to induce secondary antibody response by providing help to memory and naïve B cells. Here, we show that the transcription factor Tox2 is vital for the maintenance of TFH cells in germinal centers (GCs) and the generation of memory TFH cells. High Tox2 expression was almost exclusive to GC TFH cells among human tonsillar and blood CD4+ T cell subsets. Tox2 overexpression maintained the expression of TFH-associated genes in T cell receptor–stimulated human GC TFH cells and inhibited their spontaneous conversion into TH1-like cells. Tox2-deficient mice displayed impaired secondary TFH cell expansion upon reimmunization with an antigen and upon secondary infection with a heterologous influenza virus. Collectively, our study shows that Tox2 is highly integrated into establishment of durable GC TFH cell responses and development of memory TFH cells in mice and humans., 効率よい抗体反応の形成に必要なTリンパ球因子の発見. 京都大学プレスリリース. 2021-10-15., Molecules for building stronger antibodies. 京都大学プレスリリース. 2021-10-15.

Since metastatic colorectal cancer (CRC) is a leading cause of cancer-related death, therapeutic approaches overcoming primary and acquired therapy resistance are an urgent medical need. In this study, the efficacy and toxicity of high-affinity inhibitors targeting antiapoptotic BCL-2 proteins (BCL-2, BCL-XL, and MCL-1) were evaluated. By RNA sequencing analysis of a pan-cancer cohort comprising >1500 patients and subsequent prediction of protein activity, BCL-XL was identified as the only antiapoptotic BCL-2 protein that is overactivated in CRC. Consistently, pharmacologic and genetic inhibition of BCL-XL induced apoptosis in human CRC cell lines. In a combined treatment approach, targeting BCL-XL augmented the efficacy of chemotherapy in vitro, in a murine CRC model, and in human ex vivo derived CRC tissue cultures. Collectively, these data show that targeting of BCL-XL is efficient and safe in preclinical CRC models, observations that pave the way for clinical translation.

Autism and speech and language deficits are predominantly found in boys, however the causative mechanisms for this sex bias are unknown. Human FOXP1 is associated with autism, intellectual disability and speech and language deficits. Its closely related family member FOXP2 is involved in speech and language disorder and Foxp2 deficient mice have demonstrated an absence of ultrasonic vocalizations (USVs). Since Foxp1 and Foxp2 form heterodimers for transcriptional regulation, we investigated USV in neonatal brain-specific Foxp1 KO mice. Foxp1 KO pups had strongly reduced USV and lacked the sex-specific call rate from WT pups, indicating that Foxp1 is essential for normal USV. As expression differences of Foxp1 or Foxp2 could explain the sex-dimorphic vocalization in WT animals, we quantified both proteins in the striatum and cortex at P7.5 and detected a sex-specific expression of Foxp2 in the striatum. We further analyzed Foxp1 and Foxp2 expression in the striatum and cortex of CD1 mice at different embryonic and postnatal stages and observed sex differences in both genes at E17.5 and P7.5. Sex hormones, especially androgens are known to play a crucial role in the sexual differentiation of vocalizations in many vertebrates. We show that Foxp1 and the androgen receptor are co-expressed in striatal medium spiny neurons and that brain-specific androgen receptor KO (ArNesCre) mice exhibit reduced Foxp1 expression in the striatum at E17.5 and P7.5 and an increased Foxp2 level in the cortex at P7.5. Thus, androgens may contribute to sex-specific differences in Foxp1 and Foxp2 expression and USV.

LCAs of electric cars and electrolytic hydrogen production are governed by the consumption of electricity. Therefore, LCA benchmarking is prone to choices on electricity data. There are four issues: (1) leading Life Cycle Impact (LCI) databases suffer from inconvenient uncertainties and inaccuracies, (2) electricity mix in countries is rapidly changing, year after year, (3) the electricity mix is strongly fluctuating on an hourly and daily basis, which requires time-based allocation approaches, and (4) how to deal with nuclear power in benchmarking. This analysis shows that: (a) the differences of the GHG emissions of the country production mix in leading databases are rather high (30%), (b) in LCA, a distinction must be made between bundled and unbundled registered electricity certificates (RECs) and guarantees of origin (GOs), the residual mix should not be applied in LCA because of its huge inaccuracy, (c) time-based allocation rules for renewables are required to cope with periods of overproduction, (d) benchmarking of electricity is highly affected by the choice of midpoints and/or endpoint systems, and (e) there is an urgent need for a new LCI database, based on measured emission data, continuously kept up-to-date, transparent, and open access.

Summary IL-12 is important for the differentiation of T follicular helper (Tfh) cells, as well as Th1 cells in humans. Still, how IL-12 signals regulate Tfh versus Th1 cell differentiation remains poorly characterized. Here we aimed to determine the molecular mechanisms that regulate the differentiation and the function of IL-12-stimulated human naive CD4 + T cells. We found that T-bet promoted the expression of CXCR5 in human CD4 + T cells. We provide evidence that T-bet does not strongly inhibit the Tfh cell differentiation program per se but diminishes the functions to provide help to B cells. This study also suggests that IRF4 plays an important role in driving the early differentiation of IL-12-stimulated CD4 + T cells toward Tfh and away from Th1 by inhibiting the expression of Eomesodermin. Thus, the fate of IL-12-stimulated CD4 + T cells is determined through interplay of multiple transcription factors at early stages.

Our understanding of the origin and functions of human blood CXCR5(+) CD4(+) T cells found in human blood has changed dramatically in the past years. These cells are currently considered to represent a circulating memory compartment of T follicular helper (Tfh) lineage cells. Recent studies have shown that blood memory Tfh cells are composed of phenotypically and functionally distinct subsets. Here, we review the current understanding of human blood memory Tfh cells and the subsets within this compartment. We present a strategy to define these subsets based on cell surface profiles. Finally, we discuss how increased understanding of the biology of blood memory Tfh cells may contribute insight into the pathogenesis of autoimmune diseases and the mode of action of vaccines.

Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4+ helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-β (TGF-β) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.

Antibody responses represent a key immune protection mechanism. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset that provides help to B cells to generate an antibody response. Tfh cells together with B cells form germinal centers (GCs), the site where high-affinity B cells are selected and differentiate into either memory B cells or long-lived plasma cells. We show here that interleukin-12 receptor β1 (IL-12Rβ1)-mediated signaling is important for in vivo Tfh response in humans. Although not prone to B cell-deficient-associated infections, subjects lacking functional IL-12Rβ1, a receptor for IL-12 and IL-23, displayed substantially less circulating memory Tfh and memory B cells than control subjects. GC formation in lymph nodes was also impaired in IL-12Rβ1-deficient subjects. Consistently, the avidity of tetanus toxoid-specific serum antibodies was substantially lower in these subjects than in age-matched controls. Tfh cells in tonsils from control individuals displayed the active form of signal transducer and activator of transcription 4 (STAT4), demonstrating that IL-12 is also acting on Tfh cells in GCs. Thus, our study shows that the IL-12-STAT4 axis is associated with the development and the functions of Tfh cells in vivo in humans.

Here, we define the landscape and dynamics of active regulatory DNA in cutaneous T cell lymphoma (CTCL) by ATAC-seq. Analysis of 111 human CTCL and control samples revealed extensive chromatin signatures that distinguished leukemic, host, and normal CD4+ T cells. We identify three dominant patterns of transcription factor (TF) activation that drive leukemia regulomes, as well as TF deactivations that alter host T cells in CTCL patients. Clinical response to histone deacetylase inhibitors (HDACi) is strongly associated with a concurrent gain in chromatin accessibility. HDACi causes distinct chromatin responses in leukemic and host CD4+ T cells, reprogramming host T cells toward normalcy. These results provide a foundational framework to study personal regulomes in human cancer and epigenetic therapy.

The immune mechanism leading to the generation of protective antibody responses following influenza trivalent inactivated vaccine (TIV) vaccinations remains largely uncharacterized. We recently reported that TIV vaccination induced a transient increase of circulating ICOS+PD-1+CXCR3+ T follicular helper (cTfh) cells in blood, which positively correlated with the induction of protective antibody responses measured at day 28. However, whether and how these T cells directly contribute to antibody response remains unclear. In this study, we analyzed the changes after TIV vaccination in the amount and the avidity of the polyclonal antibodies specific for the HA1 subunit of the pandemic H1N1 virus, and analyzed the correlation with the increase of ICOS+PD-1+CXCR3+ cTfh cells. We found that both the amount and the avidity of specific antibodies rapidly increased during the first 7 days after TIV. Importantly, the increase of ICOS+PD-1+CXCR3+ cTfh cells strongly correlated with the increase in the avidity of antibodies, particularly in subjects who did not have high affinity antibodies at baseline. We propose that ICOS+PD-1+CXCR3+ Tfh cells directly contribute to the generation of high-avidity antibodies after TIV vaccinations by selectively interacting with high affinity B cells at extrafollicular sites.

Dendritic cells (DCs) were discovered in 1973 by Ralph Steinman as a previously undefined cell type in the mouse spleen and are now recognized as a group of related cell populations that induce and regulate adaptive immune responses. Studies of the past decade show that, both in mice and humans, DCs are composed of subsets that differ in their localization, phenotype, and functions. These progresses in our understanding of DC biology provide a new framework for improving human health. In this review, we discuss human DC subsets in the context of their medical applications, with a particular focus on DC targeting.

T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.

Systemic lupus erythematosus (SLE) is a chronic systemic inflammatory autoimmune disease characterized by a breakdown of tolerance to self. The autoantibodies generated in SLE are directed against nuclear components, with which they form immune complexes (ICs). ICs play key roles in organ and tissue damage, as well as in the activation of the innate and adaptive immune system during the disease course. Therefore, it is of prime importance to understand the mechanisms responsible for the development of B cells producing these pathogenic autoantibodies. There is compelling evidence that T follicular helper (Tfh) cells play a fundamental role in this process. In this review, we will summarize the current knowledge regarding the involvement of Tfh cells in SLE pathogenesis, and discuss potential strategies to target Tfh cells and/or molecules as a therapeutic modality of SLE.

Multiple Sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease which results from the destruction of myelin and associated collateral tissue damage within the central nervous system (CNS). MS is a highly diverse disease with different clinical profiles. During the past decade, several new treatment options have been introduced, but no treatment completely stops the disease progression. Therefore deeper understanding of the disease mechanism is necessary to develop novel therapeutic strategies. While yet to be proven, there is evidence suggesting the involvement of T follicular helper (Tfh) cells, a CD4 T cell subset specialized for the provision of help to B cells, in the pathogenesis of MS. In this review, I will discuss the potential pathogenic roles of Tfh cells in the course of MS.

Since the discovery of Th1 and Th2 cells in the late 1980s, the family of effector CD4(+) helper T (Th) cell subsets has expanded. The differentiation of naive CD4(+) T cells is largely determined when they interact with dendritic cells (DCs) in lymphoid organs, and cytokines play a major role in the regulation of Th differentiation in the early stages. Recent studies show that the developmental mechanism of certain Th subsets is not fully shared between mice and humans. Here we will review recent discoveries on the roles of cytokines in the regulation of Th differentiation in humans, and discuss the differences between mice and humans in the developmental mechanisms of several Th subsets, including Th17 cells and T follicular helper (Tfh) cells. We propose that the differentiation of human Th subsets is largely regulated by the three cytokines, IL-12, IL-23, and TGF-β.

Human blood contains a memory counterpart of T follicular helper (T(FH)) cells. Blood T(FH) cells are composed of subsets that differ in phenotype and function. Recent studies show that analysis of blood circulating memory T(FH) cells can provide clues to understand the mode of actions of vaccines and the pathogenesis of human autoimmune diseases. We will describe here a detailed protocol to analyze the memory T(FH) subsets in human whole blood samples. We will also describe a protocol to assess the helper capacity of blood memory T(FH) subsets.

Human blood contains a memory counterpart of T follicular helper (T(FH)) cells. Blood T(FH) cells are composed of subsets that differ in phenotype and function. Recent studies show that analysis of blood circulating memory T(FH) cells can provide clues to understand the mode of actions of vaccines and the pathogenesis of human autoimmune diseases. We will describe here a detailed protocol to analyze the memory T(FH) subsets in human whole blood samples. We will also describe a protocol to assess the helper capacity of blood memory T(FH) subsets.

Objectives: Human thymus can be infected by HIV-1 with potential consequences on immune regeneration and homeostasis. We previously showed that CD4 thymocytes preferentially replicate CXCR4 tropic (X4) HIV-1 dependently on interleukin (IL)-7. Here we addressed the susceptibility of thymic dendritic cells (DC) to HIV-1 infection. Methods: We investigated the replication ability of CXCR4 or CCR5 (R5) tropic HIV-1 in thymic micro-explants as well as in isolated thymic CD11c low CD14 - DC, CDT1c high CD14 + DC and plasmacytoid DC subsets. Results: Thymic tissue was productively infected by both X4 and R5 viruses. However, X4 but not R5 HIV-1 replication was enhanced by IL-7 in thymic micro-explants, suggesting that R5 virus replication occurred in cells other than thymocytes. Indeed, we found that R5 HIV-1 replicated efficiently in DC isolated from thymic tissue. The replicative capacity of X4 and R5 viruses differed according to the different DC subsets. R5 but not X4 HIV-1 efficiently replicated in CD1 1c high CD14 + DC. In contrast, no HIV-1 replication was detected in CDT1c low CD14 - DC. Both X4 and R5 viruses efficiently replicated in plasmacytoid DC, which secreted interferon-a upon HIV-1 exposure. Productive HIV -1 infection also caused DC loss, consistent with different permissivity of each DC subset. Conclusions: Thymic DC sustain high levels of HIV-1 replication. DC might thus be the first target for R5 HIV-1 infection of thymus, acting as a Trojan horse for HIV-1 spread to thymocytes. Furthermore, DC death induced by HIV-1 infection may affect thymopoiesis.

Background Men are more susceptible to neurodevelopmental diseases such as autism and language deficits than women however, the underlying factors remain unclear. FOXP1 is associated with autism, intellectual disability and speech and language deficits, while FOXP2 is exclusively involved in speech and language disorder. FOXP1 and FOXP2 together form heterodimers for transcriptional regulation. Methods Generation of mouse models, Quantitative real-time PCR, Protein, antibodies and western blotting, Pup ultrasonic vocalization recordings, Immunofluorescence. Results In this study, we analyzed Foxp1 and Foxp2 expression in the developing mouse brain and observed sex-biased expression of Foxp1 and Foxp2 in the striatum at embryonic day (E) 17.5 and postnatal day (P) 7.5. Since the striatum is implicated in social and vocal communication, we investigated isolation-induced ultrasonic vocalization in brain-specific Foxp1NesCre mice. Foxp1NesCre pups had strongly reduced ultrasonic vocalization and lacked the sex-specific call rate from wild type pups, indicating that Foxp1 is important for normal ultrasonic vocalization. In addition, Foxp1 expression was reduced in the striatum of brain-specific androgen receptor KO mice at E17.5 and P7.5, suggesting that androgens significantly contribute to the sex-specific differences in Foxp1 expression and sex-dimorphic ultrasonic vocalization. Discussion Our findings demonstrate a spatio-temporal pattern of Foxp1 and Foxp2 expression at distinct stages in the developing mouse brain, directly or indirectly regulated by androgens. This regulation seems to mediate sex-specific differences in USV, commonly used as a readout for social behavior. The present study contributes to the understanding how testosterone may influence gene expression during human brain development, and helps to assess its impact on the etiology of social and communicative behavior in autism spectrum disorder.

Purpose. To understand the molecular mechanisms involved in protein–methylidene malonate 2.1.2 polymer interactions.

Seasonal influenza vaccine protects 60 to 90% of healthy young adults from influenza infection. The immunological events that lead to the induction of protective antibody responses remain poorly understood in humans. We identified the type of CD4+ T cells associated with protective antibody responses after seasonal influenza vaccinations. The administration of trivalent split-virus influenza vaccines induced a temporary increase of CD4+ T cells expressing ICOS, which peaked at day 7, as did plasmablasts. The induction of ICOS was largely restricted to CD4+ T cells co-expressing the chemokine receptors CXCR3 and CXCR5, a subpopulation of circulating memory T follicular helper cells. Up to 60% of these ICOS+CXCR3+CXCR5+CD4+ T cells were specific for influenza antigens and expressed interleukin-2 (IL-2), IL-10, IL-21, and interferon-γ upon antigen stimulation. The increase of ICOS+CXCR3+CXCR5+CD4+ T cells in blood correlated with the increase of preexisting antibody titers, but not with the induction of primary antibody responses. Consistently, purified ICOS+CXCR3+CXCR5+CD4+ T cells efficiently induced memory B cells, but not naïve B cells, to differentiate into plasma cells that produce influenza-specific antibodies ex vivo. Thus, the emergence of blood ICOS+CXCR3+CXCR5+CD4+ T cells correlates with the development of protective antibody responses generated by memory B cells upon seasonal influenza vaccination.

Antibody response constitutes one of the key immune protection mechanisms. T follicular helper (Tfh) cells represent the major CD4⁺ T cell subset that provides help to B cells to induce antibody response. How Tfh cells develop and how Tfh cells or their associated subsets regulate antibody response in humans remains largely unknown. In this review, we will summarize the recent discoveries on the biology of Tfh cells, with a particular focus on human Tfh cells.

Antibody response constitutes one of the key immune protection mechanisms. T follicular helper (Tfh) cells represent the major CD4+ T cell subset that provides help to B cells to induce antibody response. How Tfh cells develop and how Tfh cells or their associated subsets regulate antibody response in humans remains largely unknown. In this review, we will summarize the recent discoveries on the biology of Tfh cells, with a particular focus on human Tfh cells.

SLE is a chronic systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and generation of high-affinity pathogenic autoantibodies. These autoantibodies form, with autoantigens, immune complexes that are involved in organ and tissue damages. Understanding how the production of these pathogenic autoantibodies arises is of prime importance. T follicular helper cells (Tfh) and IL-21 have emerged as central players in this process. This article reviews the pathogenic role of Tfh cells and IL-21 in SLE.

T follicular helper (Tfh) cells represent a Th subset engaged in the help of B-cell responses in germinal centers (GCs). Tfh cells abundantly express the transcription repressor B-cell lymphoma 6 (Bcl6), a factor that is necessary and sufficient for their development in vivo. Whether Tfh or Tfh-committed cells are involved in the help of B cells outside GCs remains unclear, particularly in humans. In this study, we identified a previously undefined BCL6 -expressing CD4 + T-cell subset in human tonsils. This subset expressed IL-7 receptor and chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS) at low levels (CXCR5 lo ICOS lo ), and it was found exclusively outside GCs. CXCR5 lo ICOS lo CD4 + T cells secreted larger amounts of IL-21 and IL-10 than CXCR5 hi ICOS hi GC-Tfh cells upon activation, and they induced proliferation and differentiation of naïve B cells into Ig-producing cells more efficiently than GC-Tfh cells. However, this subset lacked the capacity to help GC-B cells because of the induction of apoptosis of GC-B cells through the FAS/FAS–ligand interaction. CXCR5 lo ICOS lo CD4 + T cells expressed equivalent amounts of BCL6 transcript with CXCR5 hi ICOS hi GC-Tfh cells, but they expressed less Bcl6 protein. Collectively, our study indicates that CXCR5 lo ICOS lo CD4 + T cells in human tonsils represent Tfh lineage-committed cells that provide help to naïve and memory B cells outside GCs.

International audience; Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

Although a fraction of human blood memory CD4(+) T cells expresses chemokine (C-X-C motif) receptor 5 (CXCR5), their relationship to T follicular helper (Tfh) cells is not well established. Here we show that human blood CXCR5(+)CD4(+) T cells share functional properties with Tfh cells and appear to represent their circulating memory compartment. Blood CXCR5(+)CD4(+) T cells comprised three subsets: T helper 1 (Th1), Th2, and Th17 cells. Th2 and Th17 cells within CXCR5(+), but not within CXCR5(-), compartment efficiently induced naive B cells to produce immunoglobulins via interleukin-21 (IL-21). In contrast, Th1 cells from both CXCR5(+) and CXCR5(-) compartments lacked the capacity to help B cells. Patients with juvenile dermatomyositis, a systemic autoimmune disease, displayed a profound skewing of blood CXCR5(+) Th cell subsets toward Th2 and Th17 cells. Importantly, the skewing of subsets correlated with disease activity and frequency of blood plasmablasts. Collectively, our study suggests that an altered balance of Tfh cell subsets contributes to human autoimmunity.

Dendritic cells (DCs) orchestrate a repertoire of immune responses that endow resistance to infection and tolerance to self. DC plasticity and subsets are prominent determinants of the quality of elicited immune responses. Different DC subsets display different receptors and surface molecules, and express different sets of cytokines/chemokines, all of which lead to distinct immunological outcomes. Recent findings on human DC subsets and their functional specialization have provided insights for the design of novel human vaccines.

Background Systemic lupus erythematosus (SLE) is a chronic systemic inflammatory autoimmune disease characterized by a breakdown of tolerance to nuclear antigens. Antigen-presenting cells including dendritic cells (DCs) are aberrantly activated in SLE patients, and promote the activation of autoreactive T and B cells. Increased activity of T follicular helper (Tfh) cells plays a major pathogenic role in systemic lupus erythematosus (SLE). However, the mechanisms that cause aberrant Tfh responses in SLE remain elusive. Objectives To identify the mechanisms responsible for Tfh activation in human SLE. Methods Adult SLE patients (total 61: 53 females and 8 males) and pediatric SLE patients (total 38: 34 females and 4 males) who met the American College of Rheumatology revised criteria for SLE (Hochberg, 1997) were enrolled. All clinical and biologically relevant information were collected. For the analysis of OX40L expression, whole blood samples were stained with anti- CD14-PC5, CD16-FITC, CD11c-APC, HLA-DR-PC7, and OX40L-PE mAbs, and red blood cells were lysed with Versalyse. For the analysis of blood Tfh cells, whole blood samples were stained with anti-CXCR5-AF488, CCR6-PE, CXCR3- PC5, CCR4-PC7, CD3-AF700, CD8-APCH7, CD4-Pacific Blue CD45RA-ECD, ICOS-APC and CD45-Pacific Orange. Naive and memory Th cells were sorted by flow cytometry, stimulated overnight in the presence or absence of sOX40L. Extensive phenotype and transcriptomic analysis was done. Results We show that the OX40 ligand (OX40L)-OX40 axis contributes to the aberrant Tfh response in SLE. OX40L was expressed by myeloid APCs, but not by B cells, in blood as well as in inflamed tissues in adult and pediatric active SLE patients. OX40L stimulation induced human CD4 + helper T (Th) cells to express Tfh-associated molecules, and was sufficient to induce them to become functional B cell helpers. We found that OX40 signals promote the expression of Tfh molecules by enhancing TCR signals, in addition to the modulation of NF-kB pathway. Finally, we show that immune complexes (ICs) containing ribonucleoprotein (RNP) present in lupus sera induce OX40L expression by myeloid APCs through activation of TLR7. Thus, our study shows that the RNP IC-OX40L axis likely provides an amplification loop of the generation of autoantibodies in SLE. Conclusions Our study provides a rationale that therapeutic modalities targeting the RNP-containing IC-OX40L-OX40 axis and TLR7 could impact the development of autoantibodies and therefore be beneficial for human SLE. Acknowledgements This work was supported by grants from Arthritis fundation and Societe Francaise de Rhumatologie. Disclosure of Interest None declared

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c + DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c + DC could be separated into two main subsets based on differential expression of CD14: CD11c + CD14 − and CD11c + CD14 + cells. Spontaneous production of IL-10 and IFN γ without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA + cells was noticed within the first 48 h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c + cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c + subset of thymic DC.

Despite efficient antiretroviral therapy (ART), CD4+ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIVmac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4+ and CD8+ memory T cells expressing activation (HLA-DR+, CD25+) and proliferation (Ki-67+) markers. It also increased naive (CD45RAbrightCD62L+) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.

The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4⁺ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1⁺CXCR5⁺CD4⁺ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV⁺ individuals.

Macrophages play a major role in HIV-1 persistence. In the present paper, we demonstrate that the absence of apoptosis in HIV-1-infected primary human monocyte-differentiated macrophages (MDM) correlates with an increase in anti-apoptotic (Bcl-2 and Bcl-x(L)) and a decrease in pro-apoptotic (Bax and Bad) proteins. This is associated with macrophage activation as shown by tumor necrosis factor (TNF) production and NF-kappaB activation upon infection. TNF production was shown to be involved in the upregulation of Bcl-2 and Bcl-x(L) because this increase was abolished by an anti-TNF anti-serum or an inhibitor of TNF synthesis. In parallel, inhibition of TNF production induced an increase in the number of apoptotic cells. Furthermore, using an inhibitor of NF-kappaB activation, we demonstrated that TNF-induced upregulation of Bcl-x(L) and Bcl-2 occurs, respectively, through a NF-kappaB-dependent and an NF-kappaB-independent pathway.

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) variants in infected individuals is associated with poor prognosis. One of the possible causes of this emergence might be the selection of X4 variants in some specific tissue compartment. We demonstrate that the thymic microenvironment favors the replication of X4 variants by positively modulating the expression and signaling of CXCR4 in mature CD4+CD8−CD3+thymocytes. Here, we show that the interaction of thymic epithelial cells (TEC) with these thymocytes in culture induces an upregulation of CXCR4 expression. The cytokine secreted by TEC, interleukin-7 (IL-7), increases cell surface expression of CXCR4 and efficiently overcomes the downregulation induced by SDF-1α, also produced by TEC. IL-7 also potentiates CXCR4 signaling, leading to actin polymerization, a process necessary for virus entry. In contrast, in intermediate CD4+CD8−CD3−thymocytes, the other subpopulation known to allow virus replication, TEC or IL-7 has little or no effect on CXCR4 expression and signaling. CCR5 is expressed at similarly low levels in the two thymocyte subpopulations, and neither its expression nor its signaling was modified by the cytokines tested. This positive regulation of CXCR4 by IL-7 in mature CD4+thymocytes correlates with their high capacity to favor X4 virus replication compared with intermediate thymocytes or peripheral blood mononuclear cells. Indeed, we observed an enrichment of X4 viruses after replication in thymocytes initially infected with a mixture of X4 (NL4-3) and R5 (NLAD8) HIV strains and after the emergence of X4 variants from an R5 primary isolate during culture in mature thymocytes.

The sequence of events and the mechanisms leading to the destruction of the thymus during human immunodeficiency virus (HIV) infection are still poorly characterized. Investigated here are the survival capacity on HIV-1 infection of the mature single-positive CD4(+)CD8(-)CD3(+) (SP CD4(+)) and the intermediate CD4(+) CD8(-)CD3(-) thymocytes previously shown to be able to replicate the virus in the thymic microenvironment. It is demonstrated that the mature SP CD4(+) thymocytes exhibit a high survival capacity despite the production of a high yield of viruses. Interleukin-7, reported to be a crucial cofactor of tumor necrosis factor (TNF) to promote HIV replication, is shown here to counteract the apoptotic activity of TNF. Resistance to apoptosis of SP CD4(+) cells is conferred by a high expression of the IL-7 receptor (IL-7R) associated with the capacity of IL-7 to permanently up-regulate Bcl-2. In addition, this high Bcl-2 level is further enhanced by infection itself. In contrast, intermediate thymocytes, which replicate the virus at a lower level, are more sensitive to apoptosis, and their differentiation into double-positive CD4(+)CD8(+)CD3(-) (DP CD3(-)) cells strongly increases their death rate on infection. This sensitivity is related to a lower expression of IL-7R and Bcl-2 in intermediate thymocytes, which further decreases at the DP CD3(-) stage. In addition, a decreased level of Bcl-2 is observed in this subset during infection. Altogether these data suggest that in vivo, HIV infection might create a persistent virus reservoir within the SP CD4(+) thymocytes, whereas the later infection of intermediate cells might lead to thymopoiesis failure.

In barley grains, lipoxygenase has been well characterized and two isoenzymes have been purified [1–3] but the characterization of hydroperoxide-metabolizing enzymes has received less attention. Hydroperoxide-consuming enzymes can be divided into two types of enzymes. Hydroperoxide lyase cleaves fatty acid hydroperoxides into aldehydes and oxoacids, and hydroperoxide dehydrase (also named hydroperoxide isomerase) catalyzes the formation of α- and γ-ketols.

Countercurrent chromatography (CCC) is a separation technique in which the stationary phase is a liquid. The liquid stationary phase retention is a critical problem in CCC. The retention of 18 organic solvents in a hydrodynamic CCC apparatus was measured with an aqueous mobile phase, the centrifuge spin rate and the mobile phase flow rate being constant, 800 rpm and 2 ml/min, respectively. Conversely, water retention was measured when the 18 solvents were the mobile phases. A direct relationship between the liquid stationary phase retention and the phase density difference was found. The liquid phase density difference is the most important parameter for stationary phase retention in a hydrodynamic CCC apparatus with coiled tubes. The chromatographic retention of formanilide was measured in biphasic systems and expressed as the formanilide partition coefficient. It is shown that the partition coefficient correlates with the Reichardt polarity index of the organic solvent when the liquid stationary phase retention volume does not.

The ability of a Quartz Crystal Microbalance to perform blood grouping in the ABO system is demonstrated. Having regard for the experimental conditions, we consider the theoretical case of an AT-cut quartz crystal simultaneously loaded with a surface mass layer and a contacting viscous liquid. Admittance spectra, measured by an impedance analyzer, have been fitted to a simple Lorentz function leading to the resonant frequency and the quality factor. Also, it has been found that immobilized blood cells behave more like a boundary liquid rather than a surface mass layer.