T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.

As new therapeutic targets and drugs are discovered for B-cell lymphoma and other cancers, companion diagnostics are also needed to determine target engagement, therapeutic efficacy, and patient segmentation for clinical trials. We first employed synthetic chemistry to build a platform for modifying small molecule drugs into imaging probes, using the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor AZD2281 (Olaparib) as a model for technology development. Our results showed that small-molecule companion imaging drugs can be used for fluorescence imaging in cells, as well as for pharmacokinetic studies and positron emission tomography (PET) imaging in vivo, without significantly perturbing their target binding properties or cellular uptake. To apply this approach to B-cell lymphoma drugs currently in clinical trials, we modified an irreversible inhibitor of Bruton's Tyrosine Kinase (BTK), PCI-32765 (Ibrutinib), with the fluorophore Bodipy FL (BFL), and used it for imaging in cells and in a mouse window-chamber xenograft model. The excellent co-localization of our probe (Ibrutinib-BFL) with BTK demonstrated its utility for studying additional BTK inhibitors and as a companion imaging probe. In parallel, we hypothesized that central nervous system (CNS) lymphoma diagnosis from paucicellular cerebrospinal fluid (CSF) samples could be improved with molecular profiling of putative lymphoma cells trapped in a customized microfluidic chip. Following fabrication and characterization of a polydimethylsiloxane (PDMS) diagnostic device containing an array of affinity-free single-cell capture sites, we were able to efficiently recover >90% of lymphocytes, perform immunostaining on chip, and apply an image-processing algorithm to group cells based on their molecular marker expression, such as kappa/lambda light chain restriction. Additionally, in combination with Ibrutinib-BFL or other imaging drugs, we demonstrated the potential for on-chip drug imaging for use in conjunction with drug development. Finally, we applied bioorthogonal conjugation chemistries on cellulose paper for potential applications in lowering the cost of drug screening. We anticipate that these approaches will enable direct, molecular information for personalized treatment decisions in B-cell lymphomas, as well as provide a roadmap for the development of companion diagnostic probes and devices for additional indications.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 6 uppsatser.

No description available.

Les polynucléaires neutrophiles ont longtemps été considérés comme des cellules n’intervenant que dans la réponse immune innée. Cependant, au cours de ces dernières années, de nombreuses publications suggèrent que ces cellules, retrouvées au sein du microenvironnement de nombreux cancers, pourraient également jouer un rôle dans la tumorigénèse et la progression tumorale. Ces études mettent en évidence leur fréquence comme marqueur pronostique dans différents cancers solides, mais peu de travaux se sont intéressés à la caractérisation fonctionnelle de ces cellules dans la progression tumorale. Dans de nombreux cancers dont les lymphomes B issus du centre germinatif, les cellules tumorales, qui sont incapables de proliférer et de survivre seules, sont dépendantes de leur microenvironnement de soutien. Dans cette étude, nous avons évalué la fonctionnalité des polynucléaires neutrophiles dans la croissance des lymphomes B. Ainsi, nous avons démontré pour la première fois que les polynucléaires neutrophiles soutiennent directement la croissance et la survie des cellules tumorales de lymphomes B. De plus, un dialogue bidirectionnel existe entre les polynucléaires neutrophiles et les cellules stromales. D’une part, les cellules stromales soutiennent la survie des polynucléaires neutrophiles, qui en retour induisent les caractéristiques d’un stroma lymphoïde. L’induction de ce phénotype permet aux cellules stromales d’acquérir de meilleures capacités de soutien envers les cellules tumorales. Cette étude confirme donc que les polynucléaires neutrophiles sont une composante importante du microenvironnement tumoral, et pourraient devenir une nouvelle cible thérapeutique pour le traitement des lymphomes B issus du centre germinatif. / For long time, neutrophils have only been considered as cells involved in the innate immune response. More recently, in descriptive publications, neutrophils were found in the microenvironment of many solid cancers, hypothesizing that they could also play a role in tumorigenesis and cancer progression. These studies highlighted the prognostic value of their frequency, but few of them focused on the functional characterization of these cells in tumor growth. In many cancers, including germinal centre-derived B-cell lymphomas, tumor cells are dependent on their microenvironment to proliferate and survive. In this study, we focused on the role of neutrophils in the progression of B-cell lymphomas, and for the first time we demonstrated that neutrophils directly support the growth and survival of tumor Bcells. In addition, we highlighted the existence of bidirectional cooperation between neutrophils and stromal cells. In one hand stromal cells support the survival of neutrophils. On the other hand, neutrophils induce a lymphoid stroma phenotype which is well known to enhance their supportive effect on tumor cells. This study demonstrates that neutrophils are a significant component of the tumor microenvironment and may be considered as a potential therapeutic target for the treatment of B-cell lymphomas.

No description available.

Thesis (M.A.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / High dose chemotherapy followed by autologous stem cell transplantation (ASCT) remains the standard of care for patients with relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL) who are chemosensitive to salvage therapy. There is now evidence that the achievement of complete remission by PET scan (PET-CR) after salvage therapy is a favorable determinant of ASCT outcome, implying that PET response should be part of the prognostic assessment for patients considering ASCT. However, it is unclear whether other prognostic factors are still relevant in patients getting post-salvage PET scanning. Moreover, while ASCT is often also used for patients with R/R transformed indolent lymphoma (TIL), there are no data on whether prognostic factors that are important for DLBCL patients, especially PET response to salvage, are similarly prognostic in this population. We conducted a retrospective study of 143 patients with R/R DLBCL and TIL who were transplanted in the last decade and had a post-salvage PET scan prior to ASCT. We examined prognostic factors in both groups, and constructed a prognostic score for DLBCL patients. For patients with DLBCL, post-salvage PET response was an important prognostic factor. Advanced age and symptomatic relapse were also significantly associated with inferior outcome. A simple score could stratify patients into 3 risk groups with 4-year post-ASCT overall survival of 84%, 59%, and 10%, and 4-year progression-free survival of 67%, 41% and 0% (p<0.0001 for both). However, none of those factors (including PET response to salvage) could be demonstrated for TIL, likely because of the limited sample size. Our novel prognostic score for DLBCL patients undergoing ASCT may be useful for prognostication, for stratification in clinical trials, and to motivate the design of new strategies for patients in the highrisk group, who may not derive benefit from standard ASCT. Those factors, however, do not apply to patients with TIL, which has important implications for their treatment and inclusion in ASCT clinical trials with larger sample sizes. / 2031-01-01

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22727号 / 医博第4645号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 濵﨑 洋子, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les lymphomes cutanés primitifs B comprennent 2 formes indolentes (lymphomes des centres folliculaires et de la zone marginale) et une forme clinique agressive, le lymphome B diffus à grandes cellules de type jambe. Si le lymphome des centres folliculaires ne présente le plus souvent pas la translocation t(14;18) à l'origine d'une dérégulation de BCL2 caractéristique des lymphomes folliculaires ganglionnaires, elle peut être identifiée en FISH dans 8,7% des cas et représenter un risque d'extension extra-cutanée. En revanche, l'étude de l'oncogenèse des lymphomes B de type jambe révèle des mécanismes communs d'oncogenèse avec les lymphomes B diffus à grandes cellules ganglionnaires, avec une répartition différente des altérations. Notamment, la mutation du gène MYD88L265P qui encode une protéine adaptatrice de la voie des Toll-like récepteurs responsable de l'activation constitutive de la voie NFκB, est très fréquemment observée (69% des cas) et est associée à une survie spécifique plus courte. De plus, contrairement aux autres lymphomes cutanés B, les cellules tumorales sont porteuses de multiples anomalies comme des translocations ou des délétions. D'autres arguments issus de l'analyse des séquences des gènes des immunoglobulines nous permettent de présumer que la cellule d'origine est un lymphocyte B mature, post-centre germinatif. Le fort taux de mutations identifiées reflète l'hypermutation somatique acquise à l'occasion du passage par le centre germinatif, mais l'expression d'un isotype primaire d'anticorps (IgM) suggère un blocage de la différentiation plasmocytaire terminale notamment pour la commutation isotypique. / Cutaneous B-cell lymphomas are represented by indolent B-cell lymphomas (follicle center and marginal zone), and primary cutaneous diffuse large B-cell lymphoma, leg-type which is characterized by an aggressive behavior. Primary cutaneous follicle center lymphoma usually do not harbor the t(14;18) translocation, which is characteristic of nodal follicular lymphoma and conduct to BCL2 overexpression. However, it can be observed by FISH in 8.7% of cutaneous cases and seems to be associated with extra-cutaneous disease. In contrast, primary cutaneous diffuse large B-cell lymphoma, leg-type shows common genetic alterations with its nodal counterpart diffuse large B-cell lymphoma, suggestive of common oncogenesis pathways, with distinct frequencies and repartition ofmutations. Especially, the MYD88L265P mutation that encodes an important adaptator protein of the Toll-like receptor pathway, activating NFκB, is very frequent (69% of cases) and associated with a shorter specific survival. Moreover, contrary to indolent primary cutaneous B-cell lymphoma, tumour cells often harbor multiple genetic alterations such as translocations and deletions. The analysis of the immunoglobulin genes sequences led us to suppose that the cell of origin could be a post germinal-center mature B-cell. Highly mutated sequences are the reflection of the germinal center passage, but IgM expression suggests a terminal differentiation blockage, notably with a class switch recombination defect.

The human positive regulatory domain I binding factor 1 (PRDI-BF1/PRDM1) promotes differentiation of mature B cells into antibody secreting plasma cells. In contrast ectopic expression of PRDM1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1. The first study presented demonstrates that in lymphoma cells stimulation through the B cell receptor rapidly induces endogenous PRDM1 at the level of transcription. This study provides evidence that the PRDM1 promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for B cell receptor induced expression of PRDM1 in lymphoma cells and in PU.1 positive myeloma cells. Furthermore, activation is associated with loss of the co-repressor TLE4 from the PU.1 complex. The second study establishes the requirement for PRDM1 in Mantle cell lymphoma (MCL) response to Bortezomib. MCL, an aggressive form of B cell lymphoma, has poor disease- free survival rate. The proteasome inhibitor, Bortezomib, is approved for treatment of relapsed and refractory MCL. However, the precise mechanism of action of Bortezomib is not well understood. Bortezomib rapidly induces transcription of PRDM1 along with apoptosis in MCL cell lines and primary MCL tumor samples. Knockdown of PRDM1 inhibits Bortezomib-induced apoptosis, while ectopic expression of PRDM1 alone leads to apoptosis in MCL. MKI67 and PCNA, which are required for proliferation and survival, were identified as novel direct targets of PRDM1 in MCL. Chromatin immunoprecipitation and knockdown studies reveal specific repression of MKI67 and PCNA is mediated by PRDM1 in response to Bortezomib. Furthermore promoter studies demonstrate that PRDM1 functions through a specific site in the proximal promoter region of PCNA and through a distal upstream repression domain on the MKI67 promoter. Together these findings establish PRDM1 as a key mediator of Bortezomib activity in MCL through suppression of proliferation and survival genes. The third study presented demonstrates use of Tandem affinity purification technique followed by mass spectrometry to identify PRDM1 and Reptin52 protein interactions. The observations in this study provide preliminary evidence of novel mechanism of regulation of PRDM1 protein function.