Inadequate T follicular cell help impairs B cell immunity during HIV infection.

The majority of HIV-infected individuals fail to produce protective antibodies and have diminished responses to new immunizations. We report here that even though there is an expansion of follicular helper T (TFH) cells in HIV-infected individuals, the cells are unable to provide adequate B cell help. We found a higher frequency of programmed cell death ligand 1 (PD-L1)(+) germinal center B cells from lymph nodes of HIV-infected individuals suggesting a potential role for PD-1-PD-L1 interaction in regulating TFH cell function. In fact, we show that engagement of PD-1 on TFH cells leads to a reduction in cell proliferation, activation, inducible T-cell co-stimulator (ICOS) expression and interleukin-21 (IL-21) cytokine secretion. Blocking PD-1 signaling enhances HIV-specific immunoglobulin production in vitro. We further show that at least part of this defect involves IL-21, as addition of this cytokine rescues antibody responses and plasma cell generation in vitro. Our results suggest that deregulation of TFH cell-mediated B cell help diminishes B cell responses during HIV infection and may be related to PD-1 triggering on TFH cells. These results demonstrate a role for TFH cell impairment in HIV pathogenesis and suggest that enhancing their function could have a major impact on the outcome and control of HIV infection, preventing future infections and improving immune responses to vaccinations.

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

TSLP promotes influenza-specific CD8+ T-cell responses by augmenting local inflammatory dendritic cell function.

Thymic stromal lymphopoietin (TSLP) is a mucosal tissue-associated cytokine that has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Although TSLP is also produced upon viral infection in vitro, the role of TSLP in antiviral immunity is unknown. In this study we report a novel role for TSLP in promoting viral clearance and virus-specific CD8+ T-cell responses during influenza A infection. Comparing the immune responses of wild-type and TSLP receptor (TSLPR)-deficient mice, we show that TSLP was required for the expansion and activation of virus-specific effector CD8+ T cells in the lung, but not the lymph node. The mechanism involved TSLPR signaling on newly recruited CD11b+ inflammatory dendritic cells (DCs) that acted to enhance interleukin-15 production and expression of the costimulatory molecule CD70. Taken together, these data highlight the pleiotropic activities of TSLP and provide evidence for its beneficial role in antiviral immunity.

Autocrine Action of IGF2 Regulates Adult β-Cell Mass and Function.

Insulin-like growth factor 2 (IGF2), produced and secreted by adult β-cells, functions as an autocrine activator of the β-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in β-cell and whole-body physiology is not known. Here, we studied mice with β-cell-specific inactivation of Igf2 (βIGF2KO mice) and assessed β-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female βIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female βIGF2KO mice displayed lower GSIS compared with control mice, but reduced β-cell mass was observed only in female βIGF2KO mice. During pregnancy, there was no increase in β-cell proliferation and mass in βIGF2KO mice. Finally, β-cell mass expansion in response to acute induction of insulin resistance was lower in βIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult β-cell mass and function to preserve in vivo GSIS in aging and to adapt β-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

MicroRNAs and the functional β cell mass: For better or worse.

Insulin secretion from pancreatic β cells plays a central role in the control of blood glucose levels. The amount of insulin released by β cells is precisely adjusted to match organism requirements. A number of conditions that arise during life, including pregnancy and obesity, can result in a decreased sensitivity of insulin target tissues and a consequent rise in insulin needs. To preserve glucose homoeostasis, the augmented insulin demand requires a compensatory expansion of the pancreatic β cell mass and an increase in its secretory activity. This compensatory process is accompanied by modifications in β cell gene expression, although the molecular mechanisms underlying the phenomenon are still poorly understood. Emerging evidence indicates that at least part of these compensatory events may be orchestrated by changes in the level of a novel class of gene regulators, the microRNAs. Indeed, several of these small, non-coding RNAs have either positive or negative impacts on β cell proliferation and survival. The studies reviewed here suggest that the balance between the actions of these two groups of microRNAs, which have opposing functional effects, can determine whether β cells expand sufficiently to maintain blood glucose levels in the normal range or fail to meet insulin demand and thus lead, as a consequence, towards diabetes manifestation. A better understanding of the mechanisms governing changes in the microRNA profile will open the way for the development of new strategies to prevent and/or treat both type 2 and gestational diabetes.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Follicular helper T cell in immunity and autoimmunity

The traditional concept that effector T helper (Th) responses are mediated by Th1/Th2 cell subtypes has been broadened by the recent demonstration of two new effector T helper cells, the IL-17 producing cells (Th17) and the follicular helper T cells (Tfh). These new subsets have many features in common, such as the ability to produce IL-21 and to express the IL-23 receptor (IL23R), the inducible co-stimulatory molecule ICOS, and the transcription factor c-Maf, all of them essential for expansion and establishment of the final pool of both subsets. Tfh cells differ from Th17 by their ability to home to B cell areas in secondary lymphoid tissue through interactions mediated by the chemokine receptor CXCR5 and its ligand CXCL13. These CXCR5+ CD4+ T cells are considered an effector T cell type specialized in B cell help, with a transcriptional profile distinct from Th1 and Th2 cells. The role of Tfh cells and its primary product, IL-21, on B-cell activation and differentiation is essential for humoral immunity against infectious agents. However, when deregulated, Tfh cells could represent an important mechanism contributing to exacerbated humoral response and autoantibody production in autoimmune diseases. This review highlights the importance of Tfh cells by focusing on their biology and differentiation processes in the context of normal immune response to infectious microorganisms and their role in the pathogenesis of autoimmune diseases.

L’implication du Stromal Cell-derived Factor-1 alpha dans le remodelage cardiaque une semaine après un infarctus du myocarde

L’injection de cellules souches provenant de la moelle osseuse est reconnue pour améliorer la fonction ventriculaire ainsi que le remodelage cicatriciel après un infarctus du myocarde (IM). Le Stromal Cell-derived factor-1 alpha (SDF-1 alpha), une chimiokine induite par l’ischémie cardiaque, représente une grande importance en raison de son rôle dans le recrutement de cellules inflammatoires et de cellules souches de la moelle osseuse vers les sites endommagés. Quoique les recherches sur le rôle de la chimiokine SDF-1 alpha dans le remodelage ventriculaire se multiplient, son implication dans la phase aiguë du remodelage reste inexplorée. Le but de la présente étude est de déterminer l’effet du SDF-1 alpha sur la taille de la cicatrice, l’hypertrophie cardiaque ainsi que la fonction ventriculaire chez des rats et des souris une semaine après un IM. La stratégie utilisée implique l’administration de l’AMD3100 (1 mg/kg, 24 heures après l’IM, pendant 6 jours), l’antagoniste sélectif du récepteur du SDF-1 alpha, le CXCR4. Ce récepteur est couplé à une protéine G alpha i et induit la migration et la prolifération cellulaire. Chez les rats du groupe IM, l’expression de la chimiokine a été détectée surtout dans les cellules musculaires lisses et les cellules endothéliales des vaisseaux cicatriciels. Le profil d’expression de la chimiokine dans le cœur infarci indique un gradient de concentration vers la cicatrice. Une semaine après l’IM, le traitement avec l’AMD3100 a diminué la taille de la cicatrice, résultant en une amélioration de la fonction ventriculaire et une diminution de l’élévation de l’expression de l’ARNm de l’ANP dans le ventricule gauche non infarci (VGNI). Chez les souris, le traitement avec l’AMD3100 a engendré les mêmes effets, soit une diminution de la taille de la cicatrice ainsi qu’une amélioration de la fonction ventriculaire. La réduction de la taille de la région infarcie chez les souris traitées avec l’AMD3100 est associée avec une atténuation de l’infiltration des neutrophiles dans la région ischémique. Ces résultats suggèrent que le blocage pharmacologique de l’axe SDF-1 alpha/CXCR4 lors de la phase aiguë du remodelage ventriculaire après un IM diminue la taille de la cicatrice et améliore la fonction ventriculaire, en partie, par la diminution de la réaction inflammatoire.

Étude du mécanisme par lequel la thérapie à l'IL7 induit l'expansion homéostatique des lymphocytes T CD4+

Dans les cas de lymphopénie, les lymphocytes T résiduels prolifèrent exagérément dans un phénomène appelé «expansion homéostatique périphérique» (HPE), qui est efficace pour la régénération des T CD8+, mais inefficace pour les T CD4+. L’interleukine-7 (IL7) est une cytokine homéostatique utilisée afin d’augmenter les comptes lymphocytaires T des patients lymphopéniques. Toutefois, la raison de l’expansion préférentielle des lymphocytes T CD8+ par l’IL7 demeure toujours inconnue. Nous montrons que cette expansion est due au fait que l’IL7 induit une prolifération efficace des T CD8+ périphériques (CD8+PERI) ainsi que des émigrants thymiques CD8+ (CD8+RTEs). Par contre, l’effet prolifératif de l’IL7 est restreint presqu’uniquement aux CD4+RTEs même si les CD4+PERI survivent mieux que les CD4+RTEs. De plus faibles doses d’IL7 sont nécessaires aux CD4+RTEs afin de phosphoryler STAT5 ou de proliférer comparativement aux CD4+PERI et nous démontrons que les contacts TCR/CMHII sont nécessaires à la prolifération induite par l’IL7 des CD4+RTEs en périphérie. De fait, augmenter au Flt3 ligand le nombre de cellules dendritiques périphériques d’une souris donneuse, avant de transférer ses TPERI dans des souris receveuses traitées à l’IL7 induit une prolifération significative des CD4+PERI. Nos résultats indiquent donc que l’abondance des contacts TCR/CMHII reçus dans le thymus semble contrôler la sensibilité à l’IL7 des CD4+RTEs. Finalement, l’observation que les CD8+PERI et CD8+RTEs prolifèrent pareillement pendant la thérapie à l’IL7, alors que la prolifération des T CD4+ est largement restreinte aux RTEs expliquerait pourquoi, dans les cas de lymphopénie, la régénération des T CD4+ est aussi dépendante de la thymopoïèse.

Expansion des mégacaryocytes par HoxB4 pour accélérer la reconstitution plaquettaire

La greffe de cellules souches hématopoïétiques est parfois le seul traitement efficace contre les cancers hématologiques ainsi que plusieurs autres désordres reliés au système hématopoïétique. La greffe autologue est souvent le traitement de choix pour les patients atteints de lymphome ou de myélome. Dans ce cas, les cellules souches hématopoïétiques (CSH) du patient sont récoltées et congelées. Le patient subit ensuite des traitements de chimiothérapie et/ou radiothérapie qui éliminent les cellules malignes, mais détruisent aussi son système hématopoïétique. Ce dernier sera ensuite reconstitué par la greffe de CSH. Ces traitements ont pour conséquence de plonger le patient en état d’aplasie pour une période variant de 2 à 4 semaines. La thrombocytopénie (faible taux de plaquettes) est une complication majeure nécessitant des transfusions plaquettaires répétées et associée à une augmentation de la mortalité hémorragique post-transplantation. Il serait particulièrement intéressant de développer une thérapie accélérant la reconstitution des mégacaryocytes (MK), ce qui aurait pour effet de raccourcir la période de thrombopénie et donc de diminuer les besoins transfusionnels en plaquettes et potentiellement augmenter la survie. HOXB4 est un facteur de transcription qui a déjà démontré sa capacité à expandre les CSH et les progéniteurs multipotents (CFU-GEMM) donnant naissance aux MK. Il est donc un bon candidat pour l’expansion des progéniteurs MK. Comme la protéine HoxB4 a par contre une courte demi-vie (~1.1h), des protéines HoxB4 de deuxième génération avec une plus grande stabilité intracellulaire ont été créées (1423 (HoxB4L7A), 1426 (HoxB4Y23A) et 1427 (HoxB4Y28A)). Nous avons donc étudié la capacité d’HoxB4 sauvage et de deuxième génération à expandre les CSH, ainsi que les MK donnant naissance aux plaquettes. La surexpression rétrovirale de ces protéines HoxB4Y23A et HoxB4Y28A conduit à une expansion des progéniteurs MK murins in vitro supérieure à HoxB4-wt, 1423 et au contrôle GFP. La reconstitution plaquettaire in vivo dans un modèle murin a ensuite été évaluée par des transplantations primaires et secondaires. Les résultats révèlent que la surexpression rétrovirale des différents HoxB4 n’apporte pas de bénéfice significatif à la reconstitution plaquettaire des souris. Lorsque cultivées dans un milieu favorisant la différenciation mégacaryocytaire, le traitement de cellules CD34+ dérivées du sang de cordon ombilical avec les protéines recombinantes TATHoxB4WT ou de seconde génération n’a pas augmenté la production plaquettaire. Par contre, de manière intéressante, les cellules CD34+ provenant de sang mobilisé de patients atteints de myélome et mises en culture dans un milieu favorisant l’expansion des CSH ont montré des différences significatives dans la différenciation des progéniteurs MK en présence de la protéine recombinante TATHoxB4. La protéine HOXB4 possède donc un avenir prometteur quant à une amélioration de l’état thrombocytopénique chez les patients.