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To investigate their importance in non-Hodgkin’s lymphoma (NHL), I enumerated Treg cells in peripheral blood mononuclear cells (PBMC) and involved tissues from 30 newly diagnosed patients CD25+FoxP3+CD127^lowCD4+ Treg cells were increased markedly in PBMC (median=20.4% CD4 T cells, n=20) versus healthy controls (median=3.2%, n=13:<i> p</i>&lt;0.001, rank sum test) and correlated with serum lactate dehydrogenase (n=14; R_s=0.79, <i>p</i>&lt;0.001) and disease stage. I documented poor proliferation of T cells with mitogen ConA and almost none with recall antigens PPD and DPT in both PBMC and involved tissue samples (n=9). T cell hyporesponsiveness was reversed by depleting CD25+ cells (n=4), or by adding anti-CDLA-4 (n=3), supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. As a high percentage of Treg cells were also present in involved tissues (patients’ involved tissues median=38.8% of CD4 T cells (n=15) vs. reactive nodes median=11.6% of CD4 T cells (n=2); <i>p</i>=0.02, rank sum test), I determined if tumour cells could induce a T regulatory phenotype. I incubated CD25+ depleted PBMC with tumour cells <i>in vitro</i> for five days. A dose and time dependent T regulatory phenotype induction from CD25+ depleted PBMC fractions were seen (n=6, maximum induction of 86.7%). Partial induction was seen when these fractions were separated with transwells. These ‘induced Treg cells’ were FACS sorted and suppressed effector T cells proliferation. I conclude that NHL cells are powerful inducers of Treg cells. These cells circulate systemically and induce active immune tolerance both systematically and within tumour microenvironment, thus representing a new therapeutic target in NHL.

NK cell lymphoma is one of the cellular malignancies that arise from lymphocytes. Due to its rarity and aggressiveness the detailed molecular pathogenesis of NK cell lymphoma remains to be discovered. There are recent studies showing that the master regulator of B-cell differentiation into plasma cells, the Positive Regulatory Domain containing 1, with ZNF domain(PRDM1) has tumor suppressive function not only in diffuse large B-cell lymphoma (DLBCL), but also in NK cell lymphoma. The PRDM1 has two isoforms, αand β, where the former one is a functional isoform and the latter is a defective isoform with shortened and disrupted positive regulatory domain formed from transcription of internal promoter. By semi -quantitative RT-PCR, PRDM1-αexpression was found to be absent in 80% (4/5) NK cell lines while present in the normal NK cells. Loss of PRDM1 expression suggests its role as tumor suppressor. In order to study the tumor suppressive role of the αisoform of PRDM1, short-hairpin RNA (shRNA) with isoform specific sequence is used to knockdown the expression of PRDM1-αin NK cell lines. Western blot result showed about 40% decrease of PRDM1-αprotein after knockdown. Retroviral infection of the NK cell lines, NKYS and YT which have endogenous α-isoforms of expression, for the delivery of the shRNA was done and were subsequently subjected to in vitro functional analyses including MTS assay, colony formation assay, cell viability test and cell cycle analysis to determine potential effect of the loss of PRDM1-αon the NK cell lines. The PRDM1-αprotein isoform is expected to be able to repress excessive growth of NK cell line. When this isoform is inactivated, the NK cell lines are expected to proliferate significantly than the negative control counterpart in functional analyses. However in this study only YTcell line showed significant proliferation advantage in MTS and colony formation assay after the knockdown of PRDM1-α by shRNA. Cell viability assays and cell cycle analyses failed to show significant changes in both NK cell lines and yet even showed inhibitory effect after the knockdown of the gene. Ectopic expression of PRDM1-αby retroviral infection was done in KHYG cell line to further evaluate its tumor suppressive function. Apoptotic assay on the KHYG cells with ectopic expression of PRDM1-αwas performed and percentage of cells with late apoptosis was found to be significantly higher in this cell line. This suggests that one of the mechanisms for PRDM1-αto act as tumor suppressor is via the apoptosis pathway which in turn promotes the cell death. Future studies will be made to further investigate the effects of knockdown of PRDM-1αby designing another shRNA sequence which knockdown the expression of gene by at least 50% and to further investigate the role of PRDM1-αinthe pathogenesis NK cell lymphomaby proliferation assays, colony formation assay and cell cycle analysis. / published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Doctor of Medicine

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 140-160).

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 152-179).

Thesis (Ph. D.)--Washington State University, December 2008. / Title from PDF title page (viewed on Oct. 22, 2009). "Pharmacology/Toxicology Graduate Program." Includes bibliographical references (p. 90-97).

Thesis (M.D.)--Aberdeen University, 2009. / Title from web page (viewed on July 28, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 297-327).

La translocation t(1;9)(q24 ;q34), qui engendre la protéine de fusion RCSD1-ABL1, a été identifiés dans des cas de LAL de mauvais pronostic. Les propriétés oncogéniques du partenaire RCSD1, sa structure et son rôle dans l’activité et la signalisation de RCSD1-ABL1 restent inconnus. Nous avons récemment rapporté le cas d’une LAL-B t(1;9)(q24 ;q34) montrant une résistance à un grand nombre d’ITK, mais une sensibilité inattendue au ponatinib, en l’absence de mutation d’ABL1 susceptible d’expliquer ces réponses.Dans le but de caractériser la protéine RCSD1-ABL1, de comprendre ces profils de réponse aux ITK et de rechercher un traitement optimal de ces leucémies, nous avons cloné le produit de fusion à partir des blastes leucémiques de la patiente. Les constructions obtenues ont été transfectées dans le modèle cellulaire BaF3, ce qui nous a permis : 1) de démontrer et 2) de disséquer pour la première fois l’oncogénicité et la signalisation de la protéine de fusion, au moins partiellement distincte de celle de BCR-ABL1 et notamment concernant l’activation de la voie JAK/STAT; 3) de purifier RCSD1-ABL1 et de révéler l’impact inattendu du bras N-terminal RCSD1 sur l’activité catalytique de l’enzyme et sa sensibilité aux ITK ; 4) d’intégrer ces données et de démontrer l’effet potentialisateur d’inhibiteurs de la voie JAK/STAT sur l’activité des ITK dans les cellules transduites par RCSD1-ABL1 mais pas celles exprimant BCR-ABL1. Enfin, le profilage chémo-génomique de prélèvements issus de 3 patients nous a permis de conforter nos résultats, et de proposer des bases précliniques de traitements personnalisés ciblant ces mécanismes. / The t(1;9)(q24;q34) translocation, generating the RCSD1-ABL1 fusion protein, is found in bad prognosis LAL cases. The oncogenic properties of RCSD1-ABL1 are unknown and the structure of the RCSD1 portion as well as its impact on RCSD1-ABL1 activity and signaling is yet to be determined. We recently reported the case of a patient with ALL associated with a RCSD1-ABL1 rearrangement that was resistant or poorly responsive to a large number of TKIs but was sensitive to Ponatinib, with no mutation that could explain this.In order to characterize this fusion protein, understand its response profile to TKI and optimize therapeutic approaches for these patients, we cloned the RCSD1-ABL1 gene from the patient sample and expressed it in the cellular model BaF3. This allowed us to 1) Demonstrate and 2) Study for the first time the oncogenic properties and signaling of the fusion protein, which is partially distinct from that of BCR-ABL1, especially regarding the JAK/STAT pathway; 3) Purify RCSD1-ABL1 and reveal the impact of the RCSD1 N-terminal portion on the enzyme activity and its TKI sensitivity; 4) Integrate this data and demonstrate the potentiating effect of JAT/STAT pathway inhibitors on TIK activity in cells expressing RCSD1-ABL1 but not in cells expressing BCR-ABL1.Finally, the chemo-genomic profiling of samples from three B-ALL t(1;9)(q24 ;q34) allowed us to consolidate our results and to propose preclinical bases for personalized treatments targeting the identified mechanisms.