HTLV-1 HBZ cooperates with JunD to enhance transcription of the human telomerase reverse transcriptase gene (hTERT)

<p>Abstract</p> <p>Background</p> <p>Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription.</p> <p>Results</p> <p>Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter.</p> <p>Conclusion</p> <p>These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.</p

Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria

SummaryHuman group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127+ ILC1, and intraepithelial CD103+ ILC1. In inflamed intestinal tissues from Crohn’s disease patients, numbers of CD127+ ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127+ ILC1 is reversible in vitro and in vivo. CD127+ ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn’s disease patients a higher proportion of CD14+ dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127+ ILC1. In contrast, CD14− DCs promoted differentiation from CD127+ ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127+ ILC1 and ILC3 in the gut

HTLV-1 propels thymic human T cell development in “human immune system” Rag2-/- gamma c-/- Mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that TaxHTLV-1 interferes with ß- selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a “Human Immune System” (HIS) Rag2-/-γc-/- mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2-/-γc-/- mice, mature single-positive (SP) CD4+ and CD8+ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2-/-γc-/- animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice

Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-Associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γsignature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB

HTLV-1 propels thymic human T cell development in “human immune system” Rag2-/- IL-2R γc-/- Mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that TaxHTLV-1 interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a “Human Immune System” (HIS) Rag2-/-γc-/- mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2-/-γc-/- mice, mature single-positive (SP) CD4+ and CD8+ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2-/-γc-/- animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1

Co-display of diverse spike proteins on nanoparticles broadens sarbecovirus neutralizing antibody responses

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection

Brouwer et al. present preclinical evidence in support of a COVID-19 vaccine candidate, designed as a self-assembling two-component protein nanoparticle displaying multiple copies of the SARS-CoV-2 spike protein, which induces strong neutralizing antibody responses and protects from high-dose SARS-CoV-2 challenge.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication i

Développement d’un modèle de souris Rag2-/-γc-/- humanisée pour l’étude de l’infection et de la leucémogénèse associée à HTLV-1

Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.Le virus HTLV-1 (Human T-cell Leukemia Virus Type 1) est l’agent étiologique de la Leucémie T de l’adulte (ATL) qui est caractérisée par la prolifération de cellules T CD4+ activées. L’absence de modèle animal fiable reproduisant la leucémogénèse associée à l’infection a ralenti la compréhension des étapes précoces du processus leucémogène et le développement de stratégies thérapeutiques efficaces. Récemment l’amélioration des modèles de souris humanisées a permis la reconstitution d’un système immunitaire humain dans des souris. L’injection de cellules souches hématopoïétiques purifiées à partir de sang de cordon humain dans des souris nouveau-nées de la lignée Rag2-/-γc-/- conduit à la formation de novo de cellules dendritiques, B et T humaines. Ces dernières étant la cible de l’infection par HTLV-1, nous avons infecté des souris humanisées avec des cellules productrices de HTLV-1. Cette inoculation conduit à l’infection stable des cellules humaines dans la souris humanisée et la formation de lymphome ou de leucémie à cellules T humaines activées. Cette infection altère le développement des cellules T dans le thymus conduisant à un phénotype plus mature des thymocytes. Ce modèle animal reproduisant l’infection et la pathogénèse associée nous a permis de suivre l’évolution de la clonalité du virus au sein des différents organes lymphoïdes. Basées sur ces observations, des tests préliminaires ont permis d’étudier une nouvelle approche thérapeutique potentiellement applicable en clinique humaine. Ce travail nous a également permis d’affiner le protocole conduisant à l’humanisation des souris afin d’obtenir une meilleure reconstitution humaine dans ce modèle

Développement d un modèle de souris Rag2-/-gc-/- humanisée pour l étude de l infection et de la leucémogénèse associée à HTLV-1

Le virus HTLV-1 (Human T-cell Leukemia Virus Type 1) est l agent étiologique de la Leucémie T de l adulte (ATL) qui est caractérisée par la prolifération de cellules T CD4+ activées. L absence de modèle animal fiable reproduisant la leucémogénèse associée à l infection a ralenti la compréhension des étapes précoces du processus leucémogène et le développement de stratégies thérapeutiques efficaces. Récemment l amélioration des modèles de souris humanisées a permis la reconstitution d un système immunitaire humain dans des souris. L injection de cellules souches hématopoïétiques purifiées à partir de sang de cordon humain dans des souris nouveau-nées de la lignée Rag2-/-gc-/- conduit à la formation de novo de cellules dendritiques, B et T humaines. Ces dernières étant la cible de l infection par HTLV-1, nous avons infecté des souris humanisées avec des cellules productrices de HTLV-1. Cette inoculation conduit à l infection stable des cellules humaines dans la souris humanisée et la formation de lymphome ou de leucémie à cellules T humaines activées. Cette infection altère le développement des cellules T dans le thymus conduisant à un phénotype plus mature des thymocytes. Ce modèle animal reproduisant l infection et la pathogénèse associée nous a permis de suivre l évolution de la clonalité du virus au sein des différents organes lymphoïdes. Basées sur ces observations, des tests préliminaires ont permis d étudier une nouvelle approche thérapeutique potentiellement applicable en clinique humaine. Ce travail nous a également permis d affiner le protocole conduisant à l humanisation des souris afin d obtenir une meilleure reconstitution humaine dans ce modèle.Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-gc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.LYON-ENS Sciences (693872304) / SudocSudocFranceF