BACKGROUND: Regulatory T cells (Tregs) are an essential subset of T cells that despite over 10 years of research have yet to be fully characterized. These suppressive immune cells, derived from the thymus, express high levels of interleukin-2 receptor alpha-chain (CD25). Tregs are needed to maintain self-tolerance and to control responses to non-self-antigen. The mechanism of Treg repression is unknown. The direct cell-to-cell contact through binding of cell surface molecules as well as secretion of suppressive cytokines has been shown to suppress the proliferation of Thl and Th2 cells against auto, allo and foreign antigens. The role of Tregs in regulating B cell response is also uncertain. The objective of this study is to determine how the removal of T regulatory cells can increase B cell responses in vitro. METHOD: This study focuses on the effect Tregs have on the generation of an antigen specific immune response in vitro. T cells with and without Tregs were co-cultured with monocyte derived dendrtic cells. The antigen specific activation was determined by analyzing cytokine production using intracellular cytokine staining, a flow based assay. Next, the effects of Tregs on both recall and naive B cell responses was analyzed using a co-culture of B cells, CD4 T cells with and without Tregs and monocyte-derived dendritic cells. Analysis of lymphoproliferation, activation, and antibody production was analyzed by using flow cytometry, Elispot and ELISA assays. RESULTS: An antigen specific response against gp120 was generated in naive T cell culture. Tregs were shown to inhibit antigen specific cytokine production in CD4 T cell culture to de novo antigens. The activation in the absence of Tregs was superior to the addition of exogenous factors of IL-2 and IL-7 with a third less non-specific background activation. When analyzed in a TT recall B cell assay, however, the removal of Tregs proved to have an inhibitory effect on antigen secreting cells detected by Elispot. This inhibition appeared at both a 1: 1 and 1 :4 T to B cell ratios though was slightly decreased at the 1 :4 ratio. The same was true for na1ve B cell assay showing a decrease in the generation ofMSPl-42 IgM antigen secreting cells. ELISA assays also confirmed the results showing a nearly 2.5 fold decrease in the amount of MSP 1-42 specific IgM Ab in the L TE cell culture supernatant. Conclusion: While the removal of T regulatory cells is beneficial for the activation of na1ve T cells, the removal of Tregs seems to be inhibitory to B cell activation in the LTE. This inhibitory effect maybe due to T cells becoming too stimulatory before culture with B cells. Studies involving a wider range of T to B cell ratios and culture times may be beneficial to determine if the depletion of Tregs would benefit this culture method.

Streptococcus pneumoniae is a significant cause of mortality and morbidity in both children and older adults, with infection resulting in invasive disease, pneumonia and otitis media. The inclusion of pneumococcal conjugate vaccines in routine infant immunisation programmes has had a major impact on disease rates. Vaccine-induced protection against pneumococcal infection is thought to be mediated by the generation of persistent serotype-specific functional antibodies and antigen-specific memory B cells, the latter capable of generating a rapid secondary antibody response on re-exposure to antigen. Although many studies have investigated the immunogenicity of pneumococcal vaccines in different age groups by measuring serotype-specific antibodies, there is more limited information about the B cells underlying such an immune response. Important areas to investigate include the identity of the B cell subsets involved in antibody production and the potential link between memory B cells (B_MEM) and persistent antibody production by long-lived plasma cells. In this thesis I have investigated in detail the immune response to pneumococcal vaccines given to children and adults by a variety of different methods. By examining the variability of a B_MEM ELISpot method, it was shown that this assay is robust and reproducible and can be performed on fresh or frozen samples and in different laboratories. Using this technique, in a study of pre-school children, it was demonstrated for the first time that the level of pre-existing serotype 3-specific antibody is negatively correlated with, and may directly impair the B_MEM response to a booster dose of 13-valent pneumococcal conjugate vaccine (PCV-13) containing serotype 3 glycoconjugate. In the same study, it was shown that antibody persistence against most vaccine serotypes can be expected until the age of 3.5 years. A novel antigen-labelling technique was used in a detailed kinetics study of antigen-specific B cell subsets in response to either PCV-13 or 23-valent pneumococcal polysaccharide vaccine in adults. The results of this study revealed distinct B cell subset response patterns that were observed in all study participants indicating that IgM B_MEM seem to play a major role in the immune response to pneumococcal vaccines. In addition, in the same study, genome wide analysis of gene expression was performed and it was shown that vaccination with either a pneumococcal conjugate or polysaccharide vaccine results in a marked difference in numbers of differentially expressed genes 8 days following vaccination. A further tool likely to be of use in investigating B cell responses is the analysis of the antibody repertoire using next-generation sequencing techniques. In order to test the ability of these methods to detect vaccine responses, a large dataset of high-throughput B cell receptor sequences was analysed and revealed convergence of antigen-specific complementary-determining region (CDR)₃ amino acid (AA) sequences following vaccination and identified antigen-specific sequences. It was further demonstrated that for sequences directed against the H. influenzae type b (Hib) polysaccharide, diversity of immunoglobulin gene rearrangements is much greater than previously recognised. Frequencies of Hib-specific CDR₃ AA sequences were linked with anti-Hib avidity indices highlighting the potential of this method as an alternative (functional) measure of vaccine immunogenicity. These data suggest that studying the B cells and antibody repertoire post-vaccination can give novel insights into the biology that underlies the immune responses.

Le potentiel régulateur des lymphocytes B (LB), largement associé avec la production d’interleukine-10 (IL-10), a été mis en évidence dans des modèles murins de pathologies spécifiques d’Ag. Les cellules B transitionnelles (Tr.) CD24fortes CD38fortes ont été décrites comme régulatrices, au travers de la production d’IL-10, de l’inhibition de la prolifération T, ainsi que de la suppression de la réponse inflammatoire des cellules T. Les LB transitionnels représentent un stade de développement central dans la maturation des cellules B, en faisant le lien entre les cellules immatures de la moelle osseuse et celles matures situées dans les organes lymphoïdes secondaires. Dans une première étude, nous montrons que cette population est hétérogène, et composée de LB Tr. de type 1 (T1), T2, T3 et Tr. CD27+. Les LB T3 anergiques semblent jouer un rôle dans la tolérance périphérique en limitant la prolifération des lymphocytes T (LT) CD4+, tandis que les LB Tr. CD27+ IL-10+ nouvellement décrits inhibent la différenciation des LT CD4+ en cellules productrices d’IFN-γ et de TNF-α. Notons que les LB T1 et Tr. CD27+ se différencient rapidement en cellules productrices d’Ac suite à la reconnaissance de signaux de l’immunité innée. La production d’IL-10 est en partie dépendante des signaux perçus, provenant du microenvironnement. Nous avons décrit dans un second travail que les LB s’adaptent aux cellules avec lesquelles ils sont cultivés. En effet, les cellules B régulent spécifiquement les LT CD4+ mémoires (et non naïfs), en limitant leur prolifération avant d’induire une mort cellulaire. Ces caractéristiques fonctionnelles pourraient être associées avec une modification du programme transcriptionnel, permise par la plasticité des cellules B, qui se polarisent en LB régulateurs (Breg) de façon ciblée. L’expression des gènes PRDM1 et IL10 serait associée avec une signature Breg spécifique en culture mixte autologue, en opposition avec celle des gènes NFκB1 et BCL6. La transplantation rénale est un excellent modèle physiopathologique, pour étudier l’importance de certaines populations de LB dans la tolérance immunologique. L’étude BHL (B lymphocytes in humoral rejection and alloimmunisation) nous a permis de confirmer que les LB Tr. ont probablement un rôle important dans cette tolérance du greffon. La présence d’anticorps spécifiques du donneur (DSA) semble limiter l’émergence des LB Tr., même si le pourcentage de cellules B CD24fortes CD38fortes n’est a priori pas associé avec la capacité du compartiment lymphocytaire B à réguler la prolifération des cellules T des patients alloimmunisés. / Regulatory B cells (Breg) were first reported to be interleukine-10 (IL-10) producing B cells in mice. The almost concurrent discovery of Breg cells drew interest toward potential links with transitional B cells because of phenotypic and functional similarities. In addition with IL-10 production, CD24high CD38high transitional B cells limit the proliferation of T cells and the polarization of CD4+ T cells into Th1 cells. Transitional B cells represent a central developmental stage in B-cell maturation, linking generation in the bone marrow with differentiation in periphery. In a first study, we reveal for the first time that human transitional B cells encompass not only transitional type 1 and type 2 B cells, but also distinct anergic type 3 B cells, as well as IL-10-producing CD27+ transitional B cells. Interestingly, the latter two subsets differentially regulate CD4+ T-cell proliferation and polarization toward Th1 effector cells. Additional experiments showed that type 1 and CD27+ transitional B cells are capable to differentiate into antibody secreting cells after toll-like receptor 9 engagement. In a second work, we wanted to explore the ability of B cells to target T-cell populations. We demonstrate that B cells can be suppressive cells. B cells are capable to target CD4+ memory T-cell, limiting the proliferation and inducing the death of this T-cell population. At the opposite, B cells seem to be effector of CD4+ naïve T-cell functions. These properties are probably associated with a specific transcriptional program. Thus, we observed that suppressive B cells overexpress PRDM1 and IL10, whereas effector B cells preferentially express BCL6 and NFκB1 in in vitro mixed culture. In the last part, we worked on B-cell phenotype and functions in transplanted patients. BHL (B lymphocytes in humoral rejection and alloimmunisation) is a clinical study that aims to better understand the role of B cells in the alloimmunisation and the chronic rejection occurring after renal transplantation. Donor specific antibodies (DSA) seem to limit the expansion of transitional B cells, which are probably not associated with the ability of B cells to regulate T-cell proliferation in DSA+ patients.

Les alloanticorps anti-HLA sont dirigés vis-à-vis de différents épitopes des molécules du système HLA. Cette immunisation survient lors d'une transplantation d'organe, de transfusions sanguines ou d'une grossesse. On retrouve aussi ces anticorps, lorsque les techniques de détection sont sensibles, en l'absence de tout évènement immunisant. En transplantation d'organe, rénale en particulier, la présence d’anticorps anti-HLA, du fait des lésions de rejet humoral qu'ils induisent, constitue une des premières causes de perte de fonction des greffons à moyen et long terme. Néanmoins, les cellules lymphocytaires qui sont la source de ces anticorps anti-HLA demeurent mal identifiées.Dans la première partie de ce travail, nous avons étudié, dans une cohorte de patients en attente de transplantation rénale, la distribution des différentes sous-populations lymphocytaires B circulantes par cytométrie de flux en relation avec la nature des évènements immunisants vis-à-vis du système HLA, la présence et la diversité des anticorps anti-HLA. Nous avons étudié en parallèle les concentrations sériques de BAFF ("B cell activating factor belonging to the TNF family"), principal facteur impliqué dans la survie et la différenciation des lymphocytes B matures. Nous avons retrouvé une association entre la présence et la diversité des anticorps anti-HLA, et l'augmentation de la proportion de lymphocytes B naïfs activés Bm2, par rapport aux autres sous-populations lymphocytaires B, et indépendamment de l'existence d'évènements immunisants. Les concentrations sériques de BAFF étaient également associées positivement à la présence et à la diversité des anticorps anti-HLA. Ces données suggèrent que l'augmentation des lymphocytes B naïfs activés et des concentrations sériques de BAFF favorise le développement des anticorps anti-HLA à la suite d'un événement immunisant. A l'instar du mécanisme évoqué en auto-immunité, BAFF pourrait intervenir en présence de l'alloantigène en favorisant la survie de clones B alloréactifs.Dans la deuxième partie de notre travail, nous nous sommes intéressés plus particulièrement à l'implication des lymphocytes B mémoire alloréactifs dans la réponse humorale anti-HLA. Pour détecter les lymphocytes B mémoire circulants, nous avons utilisé un test de stimulation polyclonale permettant leur différenciation en plasmablastes puis nous avons recherché et étudié la spécificité des anticorps anti-HLA produits dans les surnageants de culture. Un premier résultat important a été la possibilité de détecter, chez les patients présentant des anticorps anti-HLA, des lymphocytes B mémoire alloréactifs circulants plusieurs années après un événement immunisant. En deuxième lieu, la présence de ces lymphocytes B mémoire était associée au nombre d'évènements immunisants. En effet, les patients ayant développé, en l'absence d'événement immunisant des anticorps anti-HLA - dont nous montrons par ailleurs le caractère potentiellement pathogène - n'ont pas présenté de lymphocytes B mémoire alloréactifs circulants. Enfin, à l'aide du logiciel HLAMatchmaker, nous avons montré que les anticorps produits par les lymphocytes B mémoire étaient dirigés contre un nombre restreint d'épitopes partagés par plusieurs antigènes HLA, ce qui suggère une oligoclonalité du contingent B mémoire alloréactif. Chez les mêmes patients, les anticorps anti-HLA circulants présentaient une diversité de spécificité plus large, étant dirigés contre de multiples épitopes HLA. Ces résultats suggèrent l'existence d'au moins deux types de réponse humorale vis-à-vis des alloantigènes HLA : l'une aboutissant à la production de lymphocytes B mémoire et de plasmocytes à la suite d'une réaction de centre germinatif T-dépendante, l'autre impliquant seulement des plasmocytes, possiblement issus de réponses extra-folliculaires. Les facteurs orientant vers l’un ou l’autre type de réponse sont encore mal définis mais pourraient impliquer la dose et la voie d'exposition aux alloantigènes. / Anti-HLA antibodies are directed against various epitopes of HLA molecules. They develop during organ transplantations, red cell transfusions or pregnancies. But anti-HLA antibodies are also detected with sensitive assays in the absence of any sensitizing event. In renal transplantation, anti-HLA antibodies, through the development of antibody-mediated rejection, represent the first cause of late allograft loss. Nevertheless, the mechanisms and the exact nature of B cells involved in anti-HLA antibodies synthesis are poorly understood.In a first part, we studied by flow cytometry in patients awaiting kidney transplantation the distribution of the different peripheral B cell subsets in relation with immunizing events, titer and diversity of anti-HLA antibodies. We also studied the serum levels of BAFF ("B cell activating factor belonging to the TNF family"), the main factor involved in survival and differentiation of mature B cells. We found an association between the presence and the diversity of anti-HLA antibodies, and the proportion of activated naive Bm2 B cells, at the expense of other subsets, independently of immunizing events. BAFF serum levels were also positively associated with the presence and the diversity of anti-HLA antibodies. These data suggest that the increase in activated naive B cells and in BAFF levels facilitate the development of anti-HLA antibodies, following an immunizing event. Similarly to what is observed in autoimmunity, BAFF could help to the positive selection of alloreactive B cell clones, in the presence of alloantigen.In a second part, we focused on the role of circulating alloreactive memory B cells in anti-HLA humoral response. To detect those alloreactive memory B cells, we used a polyclonal stimulation assay allowing the differentiation of memory B cells into plasmablasts and we studied the specificity of anti-HLA antibodies recovered from culture supernatant. A first important result was the detection, decades after an imunizing event, of specific alloreactive memory B cells, even in the absence of the antigen. The detection of those circulating alloreactive memory B cells was related to the strength of immunizing events, i.e. the number of different immunizing events in the history of patients. Indeed, patients with anti-HLA antibodies with no history of immunizing event had no circulating alloreactive memory B cells. Eventually, with HLAMatchmaker software, we showed that antibodies produced by memory B cells were directed against a limited number of epitopes shared by HLA antigens, which suggests an oligoclonality of the alloreactive memory B cell population. By comparison, serum antibodies displayed a greater diversity, with multiple epitopic specificities. These results suggest two distinct cellular arms of humoral response towards HLA epitopes: medullar plasma cells, involved in long term HLA antibodies synthesis, and memory B cells waiting for a recall response in the presence of the antigen. The factors involved in the choice of those two cellular fates are poorly understood but may involve dose and route of exposition to the alloantigen.

No description available.

No description available.

L’infection au VIH a plusieurs effets délétères, dont la dérégulation du compartiment des lymphocytes B. Cette dérégulation s’installe rapidement après l’infection et perdure au-delà de la thérapie antirétrovirale, pouvant mener à diverses maladies auto-immunes ainsi qu’à des lymphomes. Chez les individus atteints du VIH, cette dérégulation mène à l’augmentation de la fréquence des cellules B précurseurs de la zone marginale (MZ) dans le sang ainsi qu’à leur production d’IL-10, à l’augmentation du B-cell activating factor (BAFF), et à l’hyperglobulinémie. De plus, Nef a été détecté dans le sérum et dans les cellules dendritiques des patients infectés, les niveaux de Nef des patients corrélant avec leurs niveaux de BAFF. L’analyse d’un RNAseq effectué sur des cellules B MZ précurseurs démontre la diminution hautement significative d’expression des NR4As et de CD83 chez les patients VIH+ progresseurs comparativement aux contrôles VIH- et aux élites contrôleurs. Notre équipe a d’ailleurs démontré le potentiel et la fonction Breg associés à l’expression des NR4As et CD83 chez les cellules B MZ précurseurs du sang et d’amygdales. Aussi, la majorité de cette population co-exprime CD73 et CD39, molécules impliquées dans la synthèse de l’adénosine, cette dernière ayant un contrôle sur l’expression des NR4As. Nous voulions donc étudier l’effet de niveaux élevés de BAFF et de Nef sur la modulation de l’expression des NR4As, de CD83, CD73 et CD39 chez les cellules B MZ d'amygdales et l’effet de la déhydroergotamine (DHE) sur ce modèle, des études ayant déjà illustré sa modulation positive des NR4As. Nous étions aussi intéressés par la production d’IL-10 par ces cellules B MZ ainsi que l’effet de Nef sur son expression. Nous avons trouvé qu’après incubation avec BAFF et Nef, l’expression de NR4A1 et de CD83 était souvent plus basse qu’après une incubation avec BAFF seul qui semblait augmenter l’expression de ces dernières, ce qui concorde avec les résultats du RNAseq mentionné plus haut. Après une incubation avec Nef seul, l’expression des NR4As et de CD83 est similaire à l’expression mesurée après une incubation sans traitement, mais certains patients ont démontré une diminution légère de l’expression de ces molécules, chose normale puisque les échantillons contenaient tous des niveaux basaux de BAFF. De plus, nos résultats démontrent que le DHE augmente l’expression de NR4A1 et 3 après que leur expression ait été diminuée par Nef et son effet semble être plus important au niveau des cellules B MZ et MZ précurseurs. Ces résultats suggèrent l’utilité du DHE pour la diminution des niveaux inflammatoires chez les individus atteints du VIH, les NR4As ayant un rôle anti-inflammatoire par la diminution des fréquences de NFkB, molécule modulée positivement par Nef. Nous avons remarqué que les populations exprimant IL-10 co-expriment principalement CD10. L’effet de Nef n’a malheureusement pas été remarqué sur l’expression d’IL-10, d’autres études étant nécessaires avec nos populations d’amygdales. Bref, nos résultats suggèrent l’utilité d’agents thérapeutiques ciblant les NR4As en addition à la thérapie antirétrovirale, leur modulation chez les lymphocytes B MZ et MZ précurseurs pouvant être un aspect clé du contrôle des niveaux inflammatoires chez les individus atteints du VIH. / HIV infection is accompanied by many deleterious effects, including B cell dysfunction. This dysfunction begins rapidly after infection and persists throughout the course of infection, without being fully restored by antiretroviral therapy. These alterations can lead to lymphomas and a multitude of auto-immune diseases. In people living with HIV, B-cell deregulation leads to an increase in “precursor-like” marginal zone (MZ) B cell frequency and their secretion of IL-10, to an increase in B-cell activating factor (BAFF), and to hyperglobulinemia. In addition, Nef has been detected in the serum and dendritic cells of infected individuals, its levels correlating with BAFF levels in affected patients. The analysis of an RNA-seq performed on precursor-like MZ B cells indicated a highly significant drop in NR4A1-3 and CD83 levels in HIV+ progressors compared to HIV- controls and HIV+ elite controllers. In fact, our team has previously demonstrated regulatory “Breg” potential associated to NR4A and CD83 expression in blood and tonsil precursor-like MZ B cells. The majority of this population also co-expresses CD73 and CD39, molecules involved in adenosine synthesis, adenosine being a regulator of NR4A expression. Therefore, we wanted to study the effects of BAFF and Nef on the modulation of NR4A, CD83, CD73 and CD39 expression in tonsil MZ B cells as well as the effect of dihydroergotamine (DHE) on this model, studies having already shown its positive modulation on the NR4As. We were also interested in the effects of Nef on IL-10 production by MZ B cells. We found that after incubation with BAFF and Nef, NR4A1 and CD83 expression was often lower than after an incubation with BAFF only, which seemed to increase their expression. This corresponds with the results of the RNA-seq mentioned above. After incubation with Nef alone, NR4A and CD83 expression is similar to the expression levels found after incubation without treatment. Some patients did demonstrate a slight decrease in the expression of these molecules, a normal observation considering all samples contain a basal level of BAFF. In addition, our results demonstrate that DHE increases NR4A1 and NR4A3 levels after their expression is initially decreased iv by Nef, and its effect seems more significant in MZ and precursor-like MZ B cells. These results suggest the usefulness of DHE for the reduction of inflammatory levels in individuals living with HIV, the NR4As having an anti-inflammatory role by decreasing the frequency of NFkB, a molecule positively modulated by Nef. Furthermore, we noted the populations expressing IL-10 mainly co-express CD10. Unfortunately, Nef’s previously reported effect on IL-10 expression was not noticed, indicating the need for further studies using our tonsil samples. In conclusion, our results suggest the value of therapeutic agents targeting NR4As in addition to antiretroviral therapy, their modulation of MZ and precursor-like MZ B cells possibly being the key to controlling inflammatory levels in individuals living with HIV.

The Human immunodeficiency virus (HIV) pandemic has resulted in a heavy global disease burden, and clinically causes Acquired Immuno-Deficiency Syndrome (AIDS). The development of highly active antiretroviral therapy (HAART) has achieved remarkable control of the rapidly evolving HIV. However, HIV remains neither curable nor preventable by vaccine, and in the developing regions worst affected by HIV, HAART remains inaccessible to most patients. Furthermore, the change in both immunology and viral evolution during chronic HIV infection and its relation to AIDS pathogenesis remains unknown. Following the failure of recent HIV vaccines, it is believed that a better understanding of host-pathogen interaction is vital to advance therapeutic (vaccine and drug) design. In this thesis, I have performed an investigation of viral adaptation in response to different selection forces during advanced HIV infection and AIDS. The thesis first examined a case study that reveals the potential role of B cell-mediated neutralising antibody (NAb) in chronic HIV infection through the unexpected effect of B cell depletion agent, anti-CD20 (Rituximab). Here, longitudinal results have shown that viral load (VL), env gene diversity, and NAb sensitive strains increased during B cell and NAb depletion as a result of Rituximab administration, and reversed as B cells recovered. The study provides preliminary evidence to support the idea that NAb may be effective at suppressing HIV. The rest of the thesis focused on the cross-sectional cohort at Bloemfontein, South Africa (n=1491), a resource-limited region affected by the pandemic. Here, we used methods that include molecular and pretherapy drug resistance epidemiology, mathematical modelling, phylogenetically adjusted bioinformatics analysis and in vitro viral replication capacity (VRC) assay to study materials including cohort demography, plasma samples, CD4 cell count, VL, viral genetic sequences and host human leukocyte antigen (HLA) tissue types. Our analysis was further augmented by the additional data kindly contributed by our neighbouring Durban cohort collaborators (n=775), which also includes an IFN! ELISPOT assay that measures cytotoxic T lymphocyte (CTL) responses. Using the HIV pol sequencing data and phylogenetic analysis we confirmed that the local molecular epidemiology is similar to the circulating strains documented in the regional database. However, the pretherapy drug resistance mutation screening results have revealed an unexpected high incidence of drug-induced viral mutants in the AIDS patients with CD4 counts <100 cells/μl. According to mathematical modelling, this finding is attributable to additional sources of antiretroviral therapy exposure, which warrants public health caution. The investigation then focused on studying the changes in HLA class I mediated CTL selection and viral evolution as CD4 counts are reduced in AIDS. Interestingly we have noted evidence that suggest weakening CTL immune selection against gag during AIDS is associated with increased viral fitness (measured by VRC) and reversion of previous immune-escape mutations which conferred high fitness costs. In conclusion, this thesis compared different sources of host and drug mediated HIV selection and its implication for viral evolution. The identification of more bottleneck sites conferring high fitness costs to the selection of escape mutants is expected to be helpful in the design of future therapeutics (via vaccine, drug, immune therapy, or public health strategy). As we have learnt from the principle of combinational ARV, it would be desirable for a vaccine to select HIV at multiple sites of high escape-mutation fitness cost, hence offering protective effect.

Student Number : 9708297R - MSc (Med) dissertation - School of Medicine - Faculty of Health Sciences / B chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins. B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α. The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis. It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro. IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.

Alguns fatores de virulência em bactérias de microbiota normal, tais como sideróforos moléculas captadoras de ferro e determinadas fímbrias, possibilitam que esses microorganismos causem infecção quando a colonização ocorre fora de seu habitat normal. Dentre as diferentes espécies bacterianas da microbiota normal com potencial para causar doenças, como as infecções do trato urinário (ITUs), destaca-se Escherichia coli. Certas cepas dessa espécie bacteriana apresentam um plasmídeo (pColV) que contém um gene que codifica IutA, o receptor para a aerobactina férrica, que é um sideróforo frequentemente associado às ITUs. Recentemente, nosso grupo estabeleceu que IutA apresenta a capacidade de induzir proliferação de linfócitos B. Neste trabalho, objetivamos identificar as moléculas e os mecanismos que modulam a proliferação de linfócitos B induzida por IutA recombinante (rIutA) de E. coli. Para avaliar se a proliferação era dependente de outras células, foram realizados ensaios de proliferação de células B marcadas com CFSE utilizando o sobrenadante de macrófagos ou células dendríticas estimulados com rIutA, por 24 horas, ou coculturas em placas de transwell. As análises desses ensaios revelaram que a proliferação das células B induzida por rIutA é dependente de moléculas liberadas por células acessórias, ou seja, ocorre de forma indireta. Os resultados dos ensaios utilizando células deficientes da molécula adaptadora MyD88 mostraram dependência da sinalização por essa molécula nos linfócitos B, mas não nas células acessórias, para que ocorresse a proliferação. Posteriormente, os ensaios in vitro utilizando células de animais deficientes para TLR4, TLR2 e IL-33R mostraram que a sinalização por esses receptores é dispensável. Contrariamente, a utilização do antagonista do receptor de IL-1 reduziu significativamente a proliferação de células B tratadas com esse antagonista. Além disso, identificamos que rIutA leva à expressão de IL-1 em macrófagos e células dendríticas estimuladas com essa proteína. Assim, nossos resultados sugerem que rIutA de E. coli induz a proliferação policlonal de linfócitos B de maneira independente de células T, por um mecanismo mediado por células acessórias, como macrófagos e células dendríticas. Embora não identifiquemos o receptor macrofágico ou das células dendríticas a qual rIutA se liga, sugerimos que a ação de rIutA sobre essas células induz a produção de IL-1 que age sobre seu receptor em células B, induzindo-as a proliferação. Esses resultados abrem perspectivas de estudo de IutA como molécula estimuladora do tecido linfóide associado a mucosa, assim como evasina de E. coli patogênicas. / Indigenous bacteria may contain some virulence factors, such as siderophores iron chelator molecules and fimbriae, that allow these microorganisms to become pathogens in sites others than their normal habitat. Among the different indigenous bacterial species in the gut, Escherichia coli is one with potential to cause infections, mainly urinary tract infection (UTI). Certain strains of E. coli have a plasmid (pColV), which encode ferric aerobactin outer membrane receptor, IutA, often associated with UTI. Our group has recently described IutA as an inducer of B cell proliferation. Here, we identify the molecules and mechanisms that modulate the proliferation of B lymphocytes induced by recombinant IutA (rIutA) from E. coli. To determine whether the B cell proliferation induced by rIutA is dependent on other cell, we carried out assays with CFSE-labeled B cells cocultured separately with macrophages or dendritic cells stimulated with rIutA using transwell membranes or incubated with conditioned medium from these cells. The analysis of the results showed that rIutA indirectly induced the proliferation of B cells in a manner dependent on molecules released by accessory cells. When we analyzed the ability of rIutA in inducing proliferation of cells from mice deficient in adapter molecule MyD88, we found that this signaling molecule is crucial for signaling induced by rIutA in B cells, but not in accessory cells. A similar analysis with cells from mice deficient in Toll like receptor (TLR) 4, TLR2 or inteukin (IL-) 33 receptor revealed that these receptors were not required for rIutA signaling of any tested cells. Conversely, the pretreatment of the B cells with IL-1 receptor antagonist significantly decreased the proliferation of these cells in response to conditioned medium from cultures of IutAstimulated macrophages. Moreover, we determined that rIutA induced the expression of IL-1 in macrophages and dendritic cells stimulated with IutA. Altogether, our results suggest that IutA from E. coli induces polyclonal B-cell proliferation independently of T cells in a mechanism mediated by accessory cells such as macrophages and dendritic cells. Although the IutA-binding receptors from macrophages and dendritic cells have not been identified, we suggested that rIutA induces these cells to produce IL-1, which in turns acts on its receptor on B cells, triggering proliferation. These results open perspectives for studying IutA as a molecule that stimulates the mucosa-associated lymphoid tissue and as an immune-evasion molecule from pathogenic E. coli.