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Diss. (sammanfattning) Göteborg : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

<p>E and Id proteins are members of the basic helix-loop-helix (bHLH) transcription regulator family. These proteins control a broad range of lymphocyte biology, from the development of multiple lineages to execution of their effector functions. With the development of new experiment models, novel functions of E and Id proteins continued to be discovered. In this thesis, I focused my study on the role of Id2 in gamma delta T cells and CD4<super>+</super> alpha beta T cells, as well as the role of Id3 in B cells.</p><p> Id proteins have been shown to control gamma delta T cell development. Id3 knockout mice demonstrate a dramatic expansion of innate-like Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells in the neonatal stage, suggesting that Id3 is an inhibitor of their development. Interestingly, Id3 knockout mice with a B6/129 mix background have much less expansion of the Vgamma1.1<super>+</super> Vdelta6.3<super>+</super> T cells compared to mice with pure B6 background. Genetic studies showed that this difference is strongly influences by a chromosome region very close to the Id2 locus. Using the Id2<super>f/f</super> CD4Cre<super>+</super> mice, I found that Id2 is also an inhibitor of gamma delta T cell development. Deletion of Id2 alone is sufficient to enhance the maturation of these cells in the thymus and induce a moderate expansion of gamma delta T cells in the periphery. This study demonstrated the delicate balance of transcription control in cells of the immune system.</p><p> The Id2<super>f/f</super> CD4Cre<super>+</super> mice also enabled me to study the role of Id2 in peripheral CD4<super>+</super> alpha beta T cell functions, which was difficult in the past because Id2 knockout mice lack lymph node development. I found that CD4 T cells in these mice have a profound defect in mounting immune responses, demonstrated by a complete resistance to induction of experimental autoimmune encephalomyelitis (EAE). I found that Id2-deficient CD4 T cells fail to infiltrate the central nervous system, and the effector CD4 T cell population is smaller compared to that in control mice. Id2 is important for the survival and proliferation of effector CD4 T cells, and this phenotype was correlated with an increased expression of <italic>Bim</italic> and <italic>SOCS3</italic>. This study revealed a novel role of Id2 in the functioning of CD4<super>+ </super>alpha beta T cells.</p><p> Switching my focus to B cells, recent next generation sequencing of human Burkitt lymphoma samples revealed that a significant proportion of them have mutations of Id3. This finding suggests that Id3 may be a tumor suppressor gene in the lymphoid system. Utilizing various Id3 knockout and conditional knockout mouse models, I showed that Id3 deficiency can accelerate lymphoid tumor genesis driven by the over-expression of oncogene c-Myc. This work may lead to development of a more realistic mouse model of human Burkitt lymphoma, allowing more mechanistic studies and perhaps preclinical tests of new therapies.</p> / Dissertation

Burkitt lymphoma (BL) was the first tumor found to be strongly associated with Epstein-Barr virus (EBV). Almost 100% of the lymphoma cells are cycling, necessitating dose- and time-intense multi-agent chemotherapy regimens to achieve a cure of the disease. Whilst standard risk BL can be cured with this approach, high risk BL with leukaemic and CNS disease has significantly inferior survival. The intensive chemotherapy regimen causes considerable toxicity to the patients and relapse of BL is largely incurable. Thus, novel therapeutic approaches for high risk and relapsed BL are needed. Histone deacetylase inhibitors (HDACis) represent a novel class of drugs with potent anti-cancer effect in a wide range of malignancies. In the first part of this study, we tested HDACis of different classes for their ability to inhibit cell proliferation and activate the lytic cycle of EBV in a panel of EBV-positive BL cells of different latent viral gene expression patterns (type I, Wp-restricted and type III latency with highly restrictive, partial and full spectrum of EBV latent gene expression, respectively). Different HDACis could inhibit proliferation of EBV-positive BL cells in a time- and dose-dependent manner but only weakly activate the viral lytic cycle indicating that the drugs’ cytotoxic effect is independent of the EBV lytic cycle. Of note, BL cells of Wp-restricted or type III latency were more resistant to killing by HDACis than those of latency I, suggesting a possible link between relative resistance to the drug and expression of the latent viral genes. Bortezomib, a proteasome inhibitor, may have synergistic action with HDACis on lymphoid malignancies. We hypothesized that Bortezomib could potentiate the killing of EBV-positive BL cells by HDACis. In the second part, we tested the effect of combination of a FDA-approved HDACi, suberoylanilide hydroxamic acid (SAHA) and Bortezomib in the same panel of BL cells and also EBV-transformed lymphoblastoid cell lines (LCLs) which represent an in-vitro model of EBV-associated post-transplant lymphoproliferative disorder (PTLD). Interestingly, combination of SAHA and Bortezomib significantly enhanced the killing of BL cells of Wp-restricted or type III latency. Furthermore, the resistance to either SAHA or Bortezomib alone in contrast to synergistic killing by the combination of the two drugs could be observed in LCLs which also have the type III latency pattern. Compared with either drug alone, combination of SAHA and Bortezomib induced enhanced apoptosis in Wp-restricetd BL cells and LCLs as shown by the increase in the percentage of annexin V-positive cell, sub-G1 population and the proteolytic cleavage of apoptotic markers including PARP, caspase-3 and -9. The drug combination hyper-acetylated histone and induced cell cycle arrest. Combination of SAHA and Bortezomib was further shown to suppress the growth of BL xenograft in nude mice. In conclusion, our data indicated that expression of partial or full spectrum of viral latent genes in EBV-positive BL cells of Wp-restricted or type III latency confers resistance of the tumor cells to cytotoxic effect of HDACis. Bortezomib could potentiate SAHA-induced apoptosis of both BL cells and LCLs and might overcome mechanism of drug resistance. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Thesis (doctor of medicine)--Rijksuniversiteit te Groningen, 1979.

Endemic Burkitt lymphoma (eBL) is the leading pediatric cancer in sub-Saharan Africa and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Current treatment options in Africa are combination chemotherapy with a survival rate hovering around 50%. Relapsed or refractory eBL patients have failed to receive any targeted treatments in the clinic. Our focus was to delineate immune responses in eBL, interrogate the tumor variation in responses to targeted treatments and develop mouse models that can be used to target essential mediators of tumor pathogenesis. Immune-based treatments including immune checkpoint inhibition have recently become an effective therapeutic modality in oncology. However, some B cell lymphomas such as Hodgkin Lymphoma (HL), are more receptive to checkpoint inhibition than others suggesting a need to understand the efficacy of checkpoint inhibition on different lymphoma subtypes. Checkpoint inhibitors act by blocking inhibitory receptors on T cells and improving anti-tumor responses. One of the goals of this thesis was to characterize checkpoint inhibitors on Tumor-infiltrating lymphocytes (TILs) in eBL tumors and to identify T cell subsets that exhibit increased expression of inhibitory receptors, poor cytokine production, poor proliferation and express transcription factors associated with exhaustion. Using scRNA seq, we identified T cell clusters that co-expressed inhibitory receptors, poor proliferative markers but also sustained costimulatory signals, as well as cytokine expression suggesting a pre dysfunctional state and not terminally exhausted state. Furthermore, we quantified the dominant co-inhibitory receptors PD1 and TIGIT that are upregulated in the tumor microenvironment via immunohistochemistry (IHC) and in peripheral blood of eBL patients via flow cytometry. We compared eBL patients with healthy pediatric cohorts with a history of persistent malaria exposure to those who had little to no malaria infections, to understand uniquely T cell mediated responses in BL children. Tumors had high co-expression of PD1 and TIGIT but fewer PD1 only populations, suggesting that both ligands may play a role in restraining immune activation via IHC. Next, we investigated if PD1 ligands or TIGIT ligands were overexpressed in eBL tumors. Nectin-2, TIGIT ligand was highly expressed in eBL tumors but was not highly correlated with TIGIT expression. These studies provide insights for PD1/ TIGIT blockade in Burkitt lymphoma patients. Additionally, we established new patient-derived cell lines from eBL tumors to study tumor variation and to study targeted treatments. We established five new patient-derived eBL lines BL717, BL 719, BL720, BL725, and BL740 that were interrogated for their inter-patient variation by studying their gene expression profiles. Further, we developed a patient cell-line derived xenograft (CDX) mouse model by injecting newly patient-derived BL cell lines in immunodeficient mice (NSG BL) and studying BL tumorigenesis. Having successfully established NSG BL tumors, we observed differences in tumor growth sensitivity and survival. We tested rituximab efficacy, one of the most established treatments for B cell lymphomas in our mouse model. We also identified pathways associated with unfolded protein response (UPR) and the mammalian target of rapamycin (mTOR) signaling, as well as apoptosis in one of the cell line xenografts, BL740, in response to rituximab. BL717, BL720 cell line xenograft failed to control tumor growth and was enriched in IFN-ɑ signature genes. This mouse model will prove to be useful to study combination therapy against eBL tumors as well as mechanisms of resistance to drug targets. Collectively, these studies provide insights into intratumoral variation including subtypes during tumor progression and expression profiles of TILs in eBL tumors. This will be important in designing new therapeutic strategies as well as help pose novel therapeutic targets.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.