3BP2 is a pleckstrin homology (PH) domain- and Src homology 2 (SH2) domain-containing adapter protein that has been linked through genetic evidence to a rare human disease called cherubism 146. 3BP2 was originally cloned in a screen to identify c-Abl SH3 binding proteins 23,24. In overexpression studies, 3BP2 has been implicated as a positive regulatory adapter molecule coupled to immunoreceptor on T cells 67,69,70, B cells 68, NK cells 71-73 and mast cells 74,75. It was also evident that 3BP2 forms complexes with a number of signaling molecules, such as Zap-70, LAT, phospholipase C-γ1 (PLC-γ1), Grb2, Cbl, and Fyn in Jurkat cells 67 and Vav1, Vav2, PLC-γ, and Syk in Daudi B cells 68. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of the biological function of 3BP2 has yet to emerge. To elucidate the in vivo function of 3BP2, our laboratory has generated 3BP2 gene-deficient mice through homologous recombination 452. The 3BP2-deficient (3BP2-/-) mice were born at the expected Mendelian frequency and were fertile and viable. 3BP2-/- mice accumulate splenic marginal-zone (MZ) B cells, possess a reduced frequency of peritoneal B-1 B cells, and have a diminished thymus-independent type 2 (TI-2) antigen response. 3BP2-/- B cells demonstrate diminished proliferation and cell survival following cross-linking of the B-cell receptor (BCR). Following BCR ligation, 3BP2 might be recruited to BCR complex through its inducible interaction with BCR costimulatory molecule CD19. In the absence of 3BP2, the activation of BCR downstream effectors such as MAPK Erk1/2, JNK, and c-Abl is normal; however, 3BP2 deficiency leads to defects in Syk phosphorylation and calcium flux. In addition to defects in peripheral B cell activities, 3BP2 deficiency contributes to defects in neutrophil activities. In response to the chemotactic peptide, fMLF, 3BP2-/- neutrophils fail to establish directional migration in vitro. There is a defect in the accumulation of filamentous actin at the leading edge of migrating 3BP2-/- neutrophils which might be responsible for the random movement of these cells under shallow gradient of fMLF. In vivo, there is a delay in the recruitment of circulating neutrophils to the site of chemically induced inflammation in 3BP2-/- mice. Compared to wildtype neutrophils, 3BP2-/- neutrophils fail to properly produce superoxide anion (O2-) following fMLF stimulation. Defects in both directional migration and superoxide production of 3BP2-/- neutrophils might contribute to the reduction in bacteria clearance and the increased mortality in 3BP2-/- mice post Listeria Monocytogenes infection. In Chapter 1 of this thesis, I have reviewed basic structures and functions of the domain modules found in adapter proteins. In addition, I have reviewed the findings from numerous reports on the function of 3BP2 in different cell types. A discussion of the physical appearance and some of the initial characterization of 3BP2-deficient mice (3BP2-/-) we have generated in our laboratory are included in Chapter 1. The second part of Chapter 1 consists of an introduction on B cell receptor signaling pathway and B-cell development and activation. A discussion of G protein-coupled receptor-mediated neutrophil functions can also be found in Chapter 1. Chapter 2 contains all the methods and materials used in my study. Chapter 3 includes the characterization of peripheral B cell compartment of 3BP2-/- mice as well as the role of 3BP2 downstream of B-cell antigen receptor and in T-independent immune response. In chapter 4, I present data from experiments designed to examine the role of 3BP2 downstream of a G protein-coupled receptor, fMLF receptor, of neutrophils. I also show the requirement of 3BP2 in the clearance of Listeria Monocytogenes. In chapter 5, I propose two models for 3BP2 action based on the findings in B cells and neutrophils and discuss future areas for investigation.

B cells and in particular IL-10-secreting B cells emerge as important players in immune balance during pregnancy. We have recently revealed that CD19-deficient (CD19−/−), B cell-specific IL-10-deficient (BIL-10−/−) and B cell-deficient µMT pregnant mice are highly susceptible to LPSinduced preterm birth (PTB). We aimed to analyze the ability of IL-10-secreting cells to protect from PTB and the underlying mechanisms. Wild type (WT), CD19−/−, BIL-10−/− and µMT mice were treated with LPS at gd16 and the cellular immune response was investigated 24 h later. LPS-treated BIL-10−/− dams showed a more pronounced PTB phenotype compared to WT, CD19−/− and µMT females, and increased inflammatory and reduced anti-inflammatory mediator concentrations in the peritoneal cavity and serum. CD19−/−, BIL-10−/− and µMT mice displayed altered immune cell population frequencies in the blood and uterus with lower numbers of IL-10-secreting B cells and T cells. BIL-10−/− mothers presented decreased frequencies of uterine CD4+CD25+Foxp3+ Treg cells. Co-stimulatory molecules are critical for feto-maternal tolerance and IL-10 secretion. We found dysregulated PD-1 expression in peripheral blood and ICOS expression in the uterus of CD19−/−, BIL-10−/− and µMT dams. Our data show that B cell-specific IL-10-signaling is essential for a balanced maternal immune response to an inflammatory stimulant that cannot be hampered without IL-10-secreting B cells.

B cells and in particular IL-10-secreting B cells emerge as important players in immune balance during pregnancy. We have recently revealed that CD19-deficient (CD19−/−), B cell-specific IL-10-deficient (BIL-10−/−) and B cell-deficient μMT pregnant mice are highly susceptible to LPS- induced preterm birth (PTB). We aimed to analyze the ability of IL-10-secreting cells to protect from PTB and the underlying mechanisms. Wild type (WT), CD19−/−, BIL-10−/− and μMT mice were treated with LPS at gd16 and the cellular immune response was investigated 24 h later. LPS-treated BIL-10−/− dams showed a more pronounced PTB phenotype compared to WT, CD19−/− and μMT females, and increased inflammatory and reduced anti-inflammatory mediator concentrations in the peritoneal cavity and serum. CD19−/−, BIL-10−/− and μMT mice displayed altered immune cell population frequencies in the blood and uterus with lower numbers of IL-10-secreting B cells and T cells. BIL-10−/− mothers presented decreased frequencies of uterine CD4+CD25+Foxp3+ Treg cells. Co-stimulatory molecules are critical for feto-maternal tolerance and IL-10 secretion. We found dysregulated PD-1 expression in peripheral blood and ICOS expression in the uterus of CD19−/−, BIL-10−/− and μMT dams. Our data show that B cell-specific IL-10-signaling is essential for a balanced maternal immune response to an inflammatory stimulant that cannot be hampered without IL-10-secreting B cells

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: BACKGROUND Lymphomas arise in cells of the immune system. They vary widely in cytomorphology, immunophenotype and clinical course as well as response to treatment - all of which are factors that determine prognosis. The substantial geographical differences that exist for Hodgkin and other lymphoproliferative disorders have been previously reported from the Lymphoma Reclassification Project. OBJECTIVE This investigation was in two parts. In the first we tested the hypothesis that, using comparable treatment regimens, outcome from a private academic centre in the Western Cape region of South Africa would be similar to that from the first world. To this end a series of 512 individuals were analysed. In the second tissue samples from these patients where the most common subtype is diffuse large B-cell lymphoma (n=93) randomly receiving either standard combination chemotherapy in the form of the CHOP regimen or the identical program with rituximab which is an anti-CD20 monoclonal antibody were further investigated in cooperation with the University of Nebraska Medical Centre using an immunohistochemistry based method, also known as tissue microarray. PATIENTS AND METHODS In the first cohort consecutive and comprehensive records of patients diagnosed with lymphoma aged 14 years and older seen between October 1998 in July 2006, were scrutinized. After exclusion for a variety of reasons 398 cases suitable for further definitive study remained. In the second group tissue samples from a total of 149 biopsy proven de novo diffuse large Bcell variants could be further evaluated. After additional refinement a total of 93 remained that met all the entry criteria for the study in which 48 received chemotherapy alone and the remaining 45 chemo-immunotherapy. Demographic features were well matched. The initial stratification of these 93 cases, based on phenotyping with immunohistochemistry employing a tissue microarray method, yielded two populations depending on the criteria used. Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predict response. Finally, for each variable, clinical characteristics and survival outcome were compared between chemotherapy as a single modality or the same regimen combined with rituximab. STATISTICAL ANALYSIS Overall and event-free survival were calculated as the years from diagnosis to death, loss to follow-up, first progression or relapse respectively. The Kaplan Meier method was used to estimate distribution and the log rank test to compare survival between groups. Patient characteristics for each cohort specified were tested with Chi-squared or Fisher's exact test for small samples. RESULTS In the first part of this study all 398 cases were retrospectively analysed and showed a similar treatment outcome with regards to overall and event-free survival at 36 months when compared to first world reference centers. Adverse factors identified were similar to published experience comprising constitutional symptoms, prior treatment with chemotherapy, intermediate or high-risk scores as defined by the International Prognostic Index, histologic grading and certain anatomical sites of primary tumour. In contrast gender, staging by Rye or Rai classification, retroviral infection and prior treatment with radiotherapy were without effect. In the second part tissue samples from 93 de novo DLBCL, subtyped using an investigational immunohistochemical based Tissue Micro Array (TMA) were contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre or activated B-cells. Not surprisingly when compared to DNA-based gene expression profiling, as a reference point, concordance was different being 86 as opposed to 93% for the respective algorithms. The rationale for this exercise was to determine relative cost-effectiveness in an affordable but accurate technology that may be applicable to under resourced areas of the globe. Additionally the prognostic marker expression for BCL2 and LMO2 was investigated with regards to treatment outcome. Using the investigational tissue microarray approach the addition of rituximab to standard chemotherapy group did not show any significant improvement on 5 years overall (63% vs 59%, p 0.68) or event-free survival (42% vs 39%, p 0.94). Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% vs 55%, p 0.32) as well as event-free survival (44% vs 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of rituximab. According to Choi, both regimens (chemotherapy with or without the addition of rituximab) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. However, in contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. These results must be interpreted with care due to the very low sample size and the follow up time of only 9 months. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS – regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. Again the reservation about small numbers and the 14 months observation period apply. DISCUSSION AND CONCLUSION Most of the studies examining the proportions of subtypes in large series of DLBCL patients have been predominantly carried out on Western populations. While 50% of patients in North America or Europe express GCB phenotypes, this is only 31% in their Asian counterparts. Thus far, no study has been published on lymphoma subtypes in South African populations using a Tissue Micro Array (TMA) method. Despite certain limitations of this study, due to a variety of reasons such as loss of analysable data, variability of representation of the South African population as a whole or low sample size leading to a potential source of error, these results confirm that lymphoproliferative disorders are heterogeneous. Neverless this group can be treated successfully if exact staging, classification and risk assessment defines the holistic management plan – no matter if in a developed or under resourced setting. With the introduction of sophisticated methods such as gene expression profiling (GEP), diffuse large B-cell lymphoma (DLBCL) can be classified into the prognostically favourable germinal center B-cell–like (GCB) and the more aggressive activated B-cell–like (ABC) subtypes. Against the background of resource restriction we therefore used an immunohistochemical (IHC) stain method to analyse formalin-fixed, paraffin-embedded tissue samples. Here the algorithm by Choi et al., originally described by Hans et al., showing a high concordance rate, was comparable to the GEP classification. The use of the IHC based TMA methodology was shown to be a simple, cost effective and a robust alternative to GEP which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome when using these regimens is needed and would provide convincing evidence for the use of this monoclonal antibody in a resource constrained setting. / AFRIKAANSE OPSOMMING: AGTERGROND Limfome ontstaan vanuit selle van die immuunsisteem. Hulle toon ‘n groot verskil met betrekking tot sitomorfologie, immunofenotipe, kliniese verloop, asook respons op behandeling – almal faktore wat prognose bepaal. Die Limfoom Herklassifikasieprojek het getoon dat daar substansiële geografiese verskille bestaan vir Hodgkin se limfoom en ander limfoproliferatiewe toestande. Huidige navorsing oor hierdie maligniteite skep die indruk dat hulle skaars is in Afrika, met die inligting wat wel beskikbaar is hoofsaaklik gebaseer op gevallestudies deur klinici en patoloë. DOEL Hierdie studie bestaan uit twee dele. In die eerste toets ons die hipotese dat die uitkomste vir die behandeling van limfoom in ‘n privaat akademiese instansie geleë in die Wes-Kaap, Suid- Afrika, dieselfde is as wat in die res van die wêreld gesien word indien soortgelyke behandeling toedgedien word.`n Reeks van 512 pasiënte is in die analise gebruik. Vir die tweede hipotese het`n kohort van die mees algemene subtipe, naamlik diffuse groot B-sellimfoom (DLBCL) (n = 93), lukraak òf die standaardkombinasie-chemoterapie in die vorm van CHOP òf `n identiese program met rituximab, `n anti-CD20 monoklonale teenliggaam, gekry. Die teenliggaam is in samewerking met die Universiteit van Nebraska se Mediese Sentrum getoets met behulp van `n immunohistochemies-gebaseerde metode, ook bekend as weefsel mikro-skikking (tissue microarray or TMA). PASIËNTE EN METODE Vir die eerste kohort is die opeenvolgende en volledige rekords van pasiënte wat ouer as 14 was en tussen Oktober 1998 en Julie 2006 in `n privaat-gebaseerde akademiese instansie gediagnoseer is, noukeurig ondersoek. Na sekere uitsluitingskriteria toegepas is, was daar 398 gevalle wat geskik was vir verdere analise. In die tweede deel van die navorsing kon 149 pasiënte wat nuut met diffuse groot B-sel-limfoom (DLBCL) gediagnoseer is, verder geëvalueer word. Drie-en-neëntig van hierdie 149 pasiënte het aan die kriteria vir insluiting voldoen; 48 het slegs die standaard chemoterapie (CHOP) ontvang en die oorblywende 45 het chemo-immunoterapie (chemoterapie plus rituximab (R-CHOP) ontvang. Die demografiese eienskappe van beide groepe het goed vergelyk. Vanweë die kriteria wat gebruik is, het die aanvanklike stratifikasie van 93 gevalle, wat deur middel van die TMA-metode op fenotipering met immunohistochemie gebaseer was, twee populasies gelewer. Hierdie primêre subdivisies is verder geëvalueer vir uitdrukking van BCL2 en LMO2, beide erkende voorspellers van respons. Laastens is kliniese eienskappe en oorlewingsuitkoms ná behandeling met chemoterapie as `n enkel modaliteit of dieselfde chemoterapie met rituximab, vir elke veranderlike met mekaar vergelyk. STATISTIESE ANALISE Algehele en insident-vrye oorlewing is bereken as die jare vanaf diagnose tot sterfte, verdwyning van pasiënte tydens opvolg, eerste progressie of terugval van die toestand. Die Kaplan Meier-metode is gebruik om distribusie te bepaal en die log rank-toets om oorlewing tussen die groepe te vergelyk. Pasiëntkenmerke vir elke gespesifiseerde kohort is met die Chikwadraattoets of Fisher se eksakte toets in die geval van kleiner groepe getoets. RESULTATE In die eerste gedeelte van die studie is al 398 gevalle terugwerkend geanaliseer en daar is gevind dat die behandelingsuitkoms met betrekking tot totale oorlewing en insident-vrye oorlewing teen 36 maande vergelykbaar was met uitkomste in eerstewêreldsentrums. Nadelige faktore met betrekking tot oorlewing was soortgelyk aan reeds gepubliseerde data en het die volgende ingesluit: gestelsimptome, voorafgaande behandeling met chemoterapie, intermediêre of hoë-risiko tellings soos deur die Internasionale Prognostiese Indeks bepaal, histologiese gradering en sekere anatomiese setels van primêre tumor. In teenstelling met internasionale ondervinding het geslag, steiering volgens Rye- of Rai-klassifikasie, retrovirale status en vorige behandeling met radioterapie geen invloed gehad nie. In die tweede gedeelte is weefsel van 93 nuut-gediagnoseerde DLBCL-pasiënte wat deur middel van die TMA-metode subtipeer is, vergelyk met naastenby ooreenstemmende kategorieë soos deur Hans et al. met `n drie-merkerpaneel in kiem- en nie-kiemsentrumsubtipes, of deur Choi et al. met twee bykomende teenliggame in kiemsentrum of geaktiveerde B-selle gedefinieer. Met die DNA-gebaseerde geen-uitdrukkingsprofiel as `n verwysingspunt, was die ooreenstemming tussen die onderskeie algoritmes na verwagting verskillend, met 86% teenoor 93%. Die onderliggende rasionaal was om die relatiewe lonendheid te bepaal van `n bekostigbare maar akkurate tegnologie, wat moontlik in gebiede met onvoldoende hulpbronne toegepas kon word Verder is die prognostiese merkeruitdrukking vir BCL2 en LMO2 ook met die oog op die uitkomste van behandeling ondersoek. In vergelyking met standaard chemoterapie, het die gebruik van die TMA-tegniek met toevoeging van rituximab geen noemenswaardige verbetering in die algehele 5-jaar oorlewing (63% vs 59%, p = 0.68) óf insident-vrye oorlewing (42% vs 39%, p = 0.94) getoon nie. Insgelyks was daar geen duidelike verskille in subtipe-analise nie. Dit is egter interessant dat, met die toepassing van die Choi-kriteria, sitotoksiese middels op hul eie, in teenstelling met die geaktiveerde B-sel-subtipe, nie ‘n statisties belangrike neiging tot beter oorlewing (74% vs 55%, p = 0.32) of insident-vrye oorlewing (44% vs 40%, p = 0.42) vir die kiemsentrumsubtipe (GCB) getoon het nie. Dit was selfs nie eers moontlik om ‘n klein verskil in die teenwoordigheid van rituximab te demonstreer nie. Volgens Choi, het chemoterapie (met óf sonder die toevoeging van rituximab) by vergelyking van kiemsentrumsubtipes met nie-kiemsentrumsubtipes soortgelyke resultate gelewer as die Hans algoritme na 5-jaar oorlewing, sowel as 3-jaar insident-vrye oorlewing. Ten spyte van ’n klein steekproef het verdere subtipe-analise van BCL2- en LMO2- merkeruitdrukking van die onderskeie immunohistochemiese (IHC) subtipes die volgende aan die lig gebring: Analise met gebruik van die Choi-kriteria vir subtipe-analise vir BCL2- uitdrukking het, ongeag die subtipe (GCB of ABC) en die behandelingsmodaliteit (chemoterapie met of sonder toevoeging van rituximab), geen verskil getoon in 5-jaar oorlewing en insident-vrye oorlewing nie. Daar was egter `n noemenswaardige verskil ten opsigte van beter insident-vrye oorlewing (p = 0.0015) in die BCL2-positiewe groep van die ABC-subtipes wat met rituximab-bevattende chemoterapie behandel is. Hierdie resultate moet egter met sorg geïnterpreteer word weens die klein steekproef en kort opvolgperiode van slegs nege maande. Soortgelyke resultate met betrekking tot die uitkoms vir oorlewing is vir LMO2 gelewer, naamlik geen verskil ten opsigte van 5-jaar oorlewing of insident-vrye oorlewing ongeag die subtipe of behandelingsmodaliteit. Hier ook was daar egter beter insident-vrye oorlewing (p = 0.039) in die LMO2-positiewe groep van die ABC-subtipe wanneer rituximab-bevattende chemoterapie toegedien is. Weereens moet die resultate met sorg interpreteer word weens die klein steekproef en die kort opvolgperiode van 14 maande. BESPREKING EN GEVOLGTREKKING Die meeste studies wat reeds die verhoudings van subtipes in groot getalle DLBCL-pasiënte ondersoek het, is onder westerse populasies uitgevoer en daar bestaan onsekerheid oor of dieselfde verhoudings in Afrika van toepassing is. Terwyl 50% van alle DLBCL-pasiënte in Noord-Amerika of Europa die GCB-fenotipe uitdruk, kom dit in slegs 31% van hierdie pasiënte in Asië tot uitdrukking. Geen studie is tot dusver met behulp van die TMA-metode onder limfoomsubtipes in populasies in Afrika onderneem nie, veral nie onder pasiënte met verswakte immuunstelsels nie. Ten spyte van sekere beperkings van hierdie studie, waarvoor daar verskeie redes is, soos die verlies van analiseerbare data, die wisselende verteenwoordiging van die Suid-Afrikaanse bevolking as ‘n geheel en die klein steekproef, wat vergissing in die hand kan werk, bevestig die resultate dat limfoproliferatiewe toestande heterogeen is. Desnieteenstaande kan hierdie groep met sukses behandel word indien ‘n holistiese bestuurplan presies volgens stadiums, klassifikasie en risiko-assessering uitgevoer word – ongeag of dit in `n ontwikkelde gebied of een met min hulpbronne plaasvind. Met die beskikbaarheid van ingewikkelde metodes soos geen-ekspressie profielskepping (GEP), kan DLBCL in prognosties-voordelige kiemsentrum B-sel-agtige (GCB) en die meer aggressiewe geaktiveerde B-sel-subtipes geklassifiseer word. Teen die agtergrond van beperkte hulpbronne, is immunohistochemiese (IHC) kleuringsmetodes gebruik om - weefselmonsters wat in formalien gefikseer en in paraffien ingebed was, te analiseer. Hier was die algoritme wat oorspronklik deur Hans et al. beskryf is en deur Choi et al. verder ontwikkel is, en hoë ooreenstemming toon, vergelykbaar met die GEP-klassifikasie. Die gebruik van die IHC-gebaseerde metodologie is as ‘n eenvoudige, effektief lonende en kragtige alternatief tot GEP, wat tans as die goudstandaard vir klassifikasie van limfoom beskou word, bewys. Dit verskaf ‘n nuttige prognose-hulpmiddel vir die stratifisering van DLBCL of ander entiteite in die toekoms, selfs wanneer gevriesde weefselmonsters nie vir GEP-analise beskikbaar is nie. Onder die huidige begrotingsbeperkings in staatshospitale is middels soos rituximab by die protokolle vir die behandeling van limfoproliferatiewe toestande uitgesluit. Plaaslike terapeutiese middel-komitees beskou die nagenoeg 15% algehele oorlewingsvoordeel teen vyf jaar, wat vir DLBCL moontlik is met die toevoeging van rituximab by kombinasiechemoterapie, as te randstandig om die bykomende onkoste te regverdig. Daarvolgens is dit noodsaaklik om te demonstreer dat `n spesifieke molekulêre subtipe tot ’n beter uitkoms lei wanneer hierdie metode gebruik word en dat dit oortuigende bewyse sal lewer vir die gebruik van hierdie monoklonale teenliggaam in `n omgewing met beperkte hulpbronne.

The primary approach to the control of meningococcal disease remains effective vaccination programmes in susceptible populations. Vaccines against serogroups A, C, W and Y offer broad protection against meningococci and both polysaccharide and conjugate quadrivalent vaccines are licensed for use in the UK. Previous studies have assessed the antibody response to meningococcal polysaccharide and conjugate vaccines, but there is limited information on the nature of the B cell response to these antigens. As part of a clinical trial using both polysaccharide (MenACWY-PS) and conjugate (MenACWY-CRM) vaccines in adult volunteers, this DPhil reports the analysis of subsets of antigen specific B-cells produced in response to either vaccine. Prior MenACWY-PS impaired the response to a subsequent dose of MenACWY-CRM. This may be due to MenACWY-PS driving terminal differentiation of antigen specific cells into plasma cells, without replenishment of the memory B cell pool. In addition, despite prior data indicating that it may act as a thymus dependent antigen, the serogroup A polysaccharide component of MenACWY-PS appears to behave in the same way as serogroup C, W & Y polysaccharide components. Antibody molecules recognise and bind to a multitude of conformational epitopes. This variability is enabled by the complexities of immunoglobulin variable domain gene recombination which can generate a vast potential repertoire of unique antibody molecules. However, the diversity of the antibody repertoire is more restricted against specific antigens and within defined B cell subsets. In this DPhil, ‘next generation’ sequencing technologies were used to investigate the diversity of the B cell variable domain before and after vaccination of adult volunteers. Individuals at baseline were found to have distinct antibody repertoires. Vaccination with a Haemophilus influenzae type b (Hib) conjugate vaccine resulted in an oligoclonal antibody response, with enrichment for Hib specific canonical antibody sequences.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

La réponse immunitaires est régulée par des molécules de co-signalisation, qui, en apportant des signaux inhibiteurs ou activateurs, modulent les fonctions lymphocytaires. Nous avons focalisé notre étude sur deux co-effecteurs et membres de la famille CD28/B7 : PD-1 (Programmed Death 1) et BTLA (B and T Lymphocyte Attenuator), tous deux décrits, à l’image de CTLA-4, comme inhibitrices de l’activation lymphocytaire T. Au cours de ma thèse, nous avons pu évaluer l’expression et le rôle de PD-1 et BTLA sur les lymphocytes B normaux et pathologiques. Dans une première partie, nos travaux ont montré que ces molécules sont exprimées à la surface des sous populations B du sang périphérique et des tissus et qu’elles sont également finement régulées au cours de l’activation B. Par la suite nous avons démontré pour la première fois que BTLA et PD-1, à l’image des cellules T, sont recrutées, après activation, au niveau du récepteur de la cellule B et qu’elles exercent un rôle inhibiteur sur l’activation de ces mêmes cellules. Dans une seconde partie, grâce au développement d’un outil en cytométrie multicouleurs, nous avons analysé, dans une cohorte de 72 lymphomes B, l’infiltrat immunitaire ainsi que l’expression de PD-1, BTLA et leurs ligands. Ce travail, par son analyse de la physiologie B d’une part et de pathologies tumorales B d’autre part, donne un nouvel éclairage sur les rôles complexes des co-récepteurs BTLA et PD-1. / The immune response is regulated by co-signaling molecules, which, by providing signals inhibitors or activators, modulate lymphocyte functions. We focused our study on two co-effectors and CD28/B7 family members: PD-1 (Programmed Death 1) and BTLA (B and T Lymphocyte Attenuator), both described, like CTLA-4, as inhibitors of T lymphocyte activation. This work allowed us to evaluate the expression and the role of BTLA and PD-1 on normal and tumoral B lymphocytes. In the first part, our work has shown that these molecules are expressed on B cell subsets of peripheral blood and tissues and are also finely regulated during the B cell activation. Subsequently, we have demonstrated for the first time that BTLA and PD-1, like T cells, are recruited after activation at the B cell receptor, and they exert an inhibitory role on the activation of these cells. In the second part, through the development of a multicolor cytometry tool, we analyzed the immune infiltrate and the expression of PD-1, BTLA and their ligands in a cohort of 72 B-cell lymphomas. Taken together, these results, by his analysis of the physiology of B cell from B cell malignancies, give a new insight into the complex roles of BTLA and PD-1 co-receptors.

As células produtoras de anticorpos desempenham um papel chave na resposta efetora a microrganismos, sendo o foco principal da maioria das vacinas existentes. Entretanto, elas podem ter efeitos deletérios em doenças autoimunes e na rejeição a transplantes. Apesar de grandes avanços no controle da resposta humoral, alguns desafios permanecem e neste contexto, novos alvos terapêuticos têm sido explorados. Sabe-se que alterações metabólicas decorrentes da ativação de células B estão intimamente relacionadas com a função efetora destas células, o que anteriormente se imaginava ser apenas um reflexo de crescimento e proliferação celular. Estas alterações são controladas por sensores metabólicos ativados logo após a ativação de células B, como o mTOR, o qual é componente central de dois complexos: mTORC1 e mTORC2. Estudos anteriores já reportaram o papel positivo exercido por mTOR na via glicolítica em células T, bem como na função efetora destas células. Aqui, nós formulamos a hipótese de que a via do mTOR favorece a via glicolítica em detrimento de OXPHOS em células B, e que estas alterações metabólicas impactam as funções efetoras das mesmas. Desta maneira, para investigarmos alterações nestas vias em decorrência de mTOR, células B foram isoladas de animais controle (CT), ou de animais com células B deficientes de mTORC1 (RaptorΔB) ou mTORC2 (RictorΔB) e então estimuladas com LPS (lipopolissacarídeo) in vitro. Nossos dados indicam que a deficiência de mTORC2 beneficia OXPHOS em detrimento da via gicolítica, bem como a ativação de células B e a formação de plasmablastos. Na sequência, confirmamos que a redução nas taxas de glicólise, assim como a elevação da oxidação lipídica e de OXPHOS são cruciais para manter a elevada ativação de células B e formação de plasmablastos a partir de células B RaptorΔB e RictorΔB. Constatou-se ainda que a produção total de IgM é elevada em células RictorΔB após estímulo com LPS. Entretanto, identificamos que isso é decorrente do aumento de plasmablastos formados e não da capacidade individual de secreção dos mesmos. Diferentemente das células B deficientes, observamos que plasmablastos RaptorΔB e RictorΔB reduzem a atividade mitocondrial. Na sequência, confirmamos que a atividade mitocondrial via oxidação lipídica é fundamental para a produção de anticorpos. Além disso, demonstramos que a deficiência de mTORC2 eleva a troca de isotipo, enquanto a de mTORC1 a diminui após estimulo com LPS e IL4. Posteriormente, o impacto da deficiência de mTORC2 em células B foi avaliado in vivo em modelo de transplante de pele. Neste caso, não observamos diferenças significativas na sobrevida do enxerto entre CT e RictorΔB, mas foi constatado que apesar de ambos apresentarem formação de plasmócitos similares, animais deficientes apresentaram um número significativamente menor de células B na periferia. Assim, concluímos que a deficiência de mTORC1 ou mTORC2 em células B implica em uma maior diferenciação de plasmablastos e em uma maior produção total de anticorpos em RictorΔB in vitro, enquanto um papel funcional para essas moléculas no contexto das células B in vivo ainda precise ser determinado. / Antibodies are produced by Antibody Secreting Cells (ASCs), which are essential to fight infections. They are also the basis of most successful vaccines available, however they can present deleterious effects in autoimmune diseases and in graft rejection. Even though there have been great improvements in controlling the humoral response, its proper manipulation still remains a challenge, thus new targets need to be explored. It is known that metabolic shifts that occur upon B cell activation are not only essential for cell growth and proliferation, but are also interconnected with these cells effector function. Metabolic shifts are controlled by metabolic sensors, as the mTOR, which is a core component of two complexes, mTORC1 and mTORC2. Previous studies with T cells have already reported that mTOR exerts a positive role on glycolysis, which in turn impacts the effector function of T cells. We then hypothesized that mTOR favors glycolysis over Oxidative Phosphorylation (OXPHOS) in B cells, and that these metabolic changes impact the effector function of B cells. Thus, to investigate the impact of the mTOR pathway on B cells, we isolated B cells from mice with mTORC1 deficient B cells (RaptorΔB) or mTORC2 deficient B cells (RictorΔB) or Control mice (CT), and stimulated them in vitro with lipopolysaccharide (LPS). Our results indicate that the deficiency of mTORC2 favors OXPHOS over glycolysis, as well as B cell activation markers expression and plasmablast formation. Next, we confirmed that the reduced glycolysis levels, improved lipid oxidation and OXPHOS are in fact crucial for the enhanced activation and plasmablast formation observed in RaptorΔB and RictorΔB B cells. We also described that IgM secretion was elevated in B cells from RictorΔB after stimuli with LPS, however we found that this increase was due to the overall increase in plasmablasts in this group and not to their individual antibody secretion capacity. Interestingly, RaptorΔB and RictorΔB plasmablasts differently from B cells, reduce their mitochondrial activity. Subsequently, we confirmed that the mitochondria via lipid oxidation is actually essential for antibody secretion. In addition, we showed that mTORC2 deficiency increases isotype switching, while mTORC1 deficiency diminishes it when IL4 was added to LPS. We then sought to determine if mTORC2 deficiency in B cells would present an impact in vivo in a skin graft model. However, we did not observe a significant difference in graft survival between the CT and RictorΔB mice and in plasmacyte numbers, but we did observe a significant reduction in B cells in the periphery. Thus, we conclude that mTORC1 and mTORC2 deficiency leads to an improved plasmablast differentiation and an overall increase in antibody secretion in the last one in vitro, whereas the role of these sensors remains to be determined in vivo.