1,721,015 research outputs found
CAR-iNKT cells as a novel immunotherapy for B cells malignancies
Get PDFAnti-CD19 chimeric antigen receptor T cell (CAR19-T) immunotherapy has shown curative potential in B cell malignancies. However, clinical remissions in relapsed/refractory CD19+ lymphomas and lymphoproliferative disorders are often short-lived, with therapeutic benefit for less than half of patients, highlighting the need for more effective CAR-based strategies.
iNKT cells are rare but powerful immunoregulatory and cytotoxic T lymphocytes, playing a pivotal anti-tumour role. They are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA I-like molecule, expressed on malignant CD19+ B cells in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) cells, while in up to 50% of patients with chronic lymphocytic leukaemia (CLL) CD1d expression is very low or negative.
I tested the hypotheses that a) bi-specific CAR19-iNKT cells, targeting simultaneously CD19 and CD1d, via the CD19-specific CAR and their natural invariant TCR respectively, would be more effective than CAR19-T cells against CD19+CD1d+ B cell malignancies and b) transcriptional enhancement of CD1d expression would increase the CAR19-iNKT cytotoxic effect, including against CLL cells.
I established and optimized a novel, highly efficient protocol for manufacturing clinical scale CAR19-iNKT cells. In vitro validation demonstrated that CAR19-iNKT cells are CD19-specific, retain their natural CD1d-restricted reactivity and exert additive dual-specific cytotoxicity against CD1d+CD19+ targets. Compared to same-donor CAR19-T, CAR19-iNKT cells display a significantly higher expandability and proliferative potential, they are equally or more effective in killing CD19+CD1d+ lymphoid cell lines and consistently more effective against primary MCL, MZL and CLL cells.
Notably, I found that in CD1dlow/– primary CLL cells, surface CD1d expression can be restored by clinically relevant concentrations of all-trans retinoic acid (ATRA) and, in line with my hypothesis, CAR19-iNKT but not CAR19T cells displayed higher cytotoxic activity against ATRA-treated CLL cells.
Finally, in an NSG xenograft model of lymphoma, CAR19-iNKT cell immunotherapy led to a significantly improved overall survival, with earlier, more profound and sustained complete responses, which resulted in a significantly improved tumour-free survival as well as eradication of CNS lymphoma.
I conclude that CAR19-iNKT are more effective than CAR19-T cells against CD1d+CD19+ B cell malignancies in vitro and in vivo. This, together with the previously demonstrated ability of third donor-derived iNKT cells to protect from acute Graft-versus-Host Disease (aGVHD), raise the prospect of developing a more effective ‘off-the-shelf’ CAR19-iNKT immunotherapy for lymphomas. Furthermore, the finding that ATRA-mediated restoration of CD1d expression enhances the anti-lymphoma effect of CAR19-iNKT immunotherapy against CD1d low/negative tumour cells in vitro suggests that CAR-iNKT cells, in combination with transcriptional/epigenetic modulation of CD1d, may represent a highly efficient platform for CAR-based immunotherapy also for other CD1d-negative disorders.Open Acces
Novel modification of Human Myeloma proteasomes and development of non-active site directed inhibitors
Get PDFMultiple Myeloma (MM) is a plasma cell malignancy that is characterised by bone lesions and production of excessive amounts of monoclonal protein. Treatment involves the use of chemotherapy agents, immunomodulatory agents and proteasome inhibitors (PI). The 26S proteasome is a 2.5 MDa molecular machine that is integral to the viability of all eukaryote cell. Its main function is to hydrolyse proteins that are marked for degradation by a poly-ubiquitin chain. Although MM is treatable, it is as yet incurable with mean survival of ~6 years.
In this thesis, I show that human proteasomes contain a charged polymeric posttranslational modification (PTM), one which has some similarity to poly-ADP-ribose. This modification is not normally resolvable via normal SDS-PAGE electrophoresis, but can be resolved by the lesser used CTAB-PAGE or after separation of the proteasome from other cellular components. This modification appears to be present predominantly in the nucleus of the cell, and may provide a mechanism for how nuclear proteasomes interact with chromatin, DNA and other nuclear components.
The use of proteasome inhibitors as a valid therapy for MM has been hypothesised to be due to a high proteasome load in MM; therefore, a small amount of inhibition would suffice to perturb proteostasis. However, I show that myeloma cells experience severe proteasome inhibition upon treatment with compounds such as Bortezomib, to a degree that far exceeds the levels of inhibition observed with purified proteasomes. This suggests that, when PIs engage with proteasomal active-sites, they trigger a cellular mechanism which exacerbates this inhibition to a far greater degree than would otherwise be achieved. I excluded trivial explanations including additional binding of the inhibitor, caspase mediated proteasome inhibition or cell death initiation. Intriguingly, I found that early changes to CTAB-PAGE detectable PTMs coincided with PIs’ ability to achieve an excessive degree of cellular proteasome inhibition.
In addition to this work I continue the development of an allosteric proteasome inhibitor identified by a phage display technique. Through a number of rounds of chemical optimisation, I show the ability of these compounds to inhibit proteasome degradation of an ubiquinated substrate and a lethality in myeloma cells at 25 nM.Open Acces
Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency
Get PDFRESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas
de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de
superfície celular ancoradas a GPI apresentam várias funções essenciais para a
manutenção celular. A deficiência na síntese de GPI é o que caracteriza
principalmente a deficiência hereditária em GPI, um grupo de doenças
autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV,
PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a
ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento,
impondo a compactação da cromatina, associada à hipoacetilação de histonas, e
consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da
primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as
proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência
Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência
de hemólise intravascular e anemia, sendo que estas duas últimas características
definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada
por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os
tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre
células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a
deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas
e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular.
Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI
nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA
variam entre células primárias hematopoiéticas normais. Adicionalmente, a
configuração dos nucleossomas no promotor de PIGM é mais compacta em células
B do que em células eritróides e tal está correlacionado com os níveis de expressão
de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação
para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor
de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição.
Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel
repressor em vez de activador da expressão de PIGM. Resultados preliminares
sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a
transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia
directamente a transcrição de PIGM em ambas as células B e eritróide.
Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de
PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides
Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC.
Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição
de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com
IGD associada a PIGM, uma das principais características que define a HPN.
Por último, resultados preliminares mostraram que a repressão da transcrição de
PIGM devida à mutação patogénica -270C>G está associada com a diminuição da
frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”,
sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada.
No seu conjunto, os resultados apresentados nesta tese contribuem para o
conhecimento do controlo transcricional de um gene housekeeping, específico-detecido,
por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of
proteins for cell surface anchoring. GPI-anchored proteins have various functions
that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases
caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis.
A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts
binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction
associated with histone hypoacetylation, thus abrogating transcription of PIGM. As
a consequence of PIGM transcriptional repression, addition of the first mannose
residue onto the GPI core and thus GPI production are impaired; and expression of
GPI-anchored proteins on the surface of cells is severely impaired. Patients with
PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not
intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA.
Although the disease-causing mutation in IGD is constitutional and present in all
tissues, the degree of GPI deficiency is variable and differs between cells of the
same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes
and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the
haematopoietic cells of PIG-M-associated IGD are not known and constitute the
general aim of this thesis.
Firstly, I found that PIGM mRNA levels are variable amongst normal primary
haematopoietic cells. In addition, the nucleosome configuration in the promoter of
PIGM is more compacted in B cells than in erythroid cells and this correlated with
the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of
several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a
repressor rather than an activator of PIGM expression. Preliminary data suggested
that KLF1, an erythroid-specific TF, regulates PIGM transcription but
independently of the -270GC motif.
Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates
PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B
cells in which active PIGM transcription requires binding of the generic TF Sp1 to
the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by
binding upstream of but not to the -270GC-rich motif. Additionally, I showed that
Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells.
These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a
defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a
shared regulatory link between these genes and PIGM.
Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and
generic TFs
The role and regulation of CD1d in normal and pathological B cells
Get PDFThis work provides novel insights into the intersection between two critical areas of immunology, the CD1d-invariant NKT (iNKT) axis and B cells. CD1d is a non-polymorphic, MHC class I-like molecule, which presents phospho- and glycosphingo-lipid antigens to a subset of CD1d-restricted T cells called iNKT cells. CD1d is expressed on a variety of antigen presenting cells and the CD1d-iNKT cell axis regulates nearly all aspects of the innate and adaptive immune response. Expression of CD1d on B cells allows these cells to form cognate interactions with iNKT cells. Emerging evidence suggests, however, that expression of CD1d on B cells is variable, both on “normal” B cells during humoral immune responses, and also on “pathological” B cells in certain B cell disorders.
In this work, I investigate in detail the expression of CD1d on B cells across a range of conditions. Using both human and murine germinal centre (GC) B cells as a model for normal B cells, I show for the first time that CD1d expression changes dynamically, both at the surface protein and transcriptional level. CD1d falls to a nadir as a naïve B cell enters the GC, and subsequently rises again in post GC B cells. I then provide evidence that the loss of CD1d expression is paralleled in pathological B cells, specifically in Epstein-Barr Virus infection of B cells and the plasma cell disorder Multiple Myeloma. In these conditions, CD1d is again downregulated at both the surface and transcriptional level.
Having established that CD1d expression is lost in certain scenarios, I attempt to elucidate the biological significance of this downregulation. I approach this aim by attempting to constitutively express CD1d in murine GC B cells, and subsequently assessing the GC reaction. Constitutive expression of CD1d is achieved by two distinct methods, either by using adoptive transfer of Cd1d1 transduced haematopoietic stem cells or by using a CD1d transgenic mouse model. I show that both of these approaches represent a feasible way to constitutively express CD1d in murine GC B cells, but cannot establish a definitive biological role for CD1d downregulation in murine GC B cells.
Finally, I investigate the transcriptional mechanisms governing the downregulation of CD1d, in either normal or pathological B cells. By analysing GC and MM B cells, I provide the first evidence that both retinoic acid signalling and bivalent chromatin domains act as a dual regulatory mechanism of CD1d. These findings demonstrate a new concept in the field of MM biology, specifically that MM B cells (like lymphomas) are able to “hijack” physiological pathways for disease propagation. Although previous work has highlighted the ability of all-trans retinoic acid (ATRA) alone to increase CD1d expression, this uncovering of a dual mechanism provides the rationale to use both ATRA and the polycomb-repressive complex inhibitor GSK343 to restore CD1d expression. I show that this is a more effective strategy than ATRA alone in increasing CD1d expression. Although this will need to be validated in further in vivo models, this ability to further increase CD1d expression may be of great therapeutic importance in the emerging field of tumour immunotherapy.Open Acces
Investigation of the use of histone deacetylase inhibitors for the treatment of inherited disorders of the glycolytic pathway
Get PDFHistone acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) regulate gene expression by activating or repressing transcription, respectively. HDAC inhibitors (HDACIs) are a diverse class of drugs used to treat haemoglobinopathies, urea cycle disorders and several types of malignancies. Recent evidence from genome-wide as well as gene-specific epigenetic studies suggest a model whereby active genes are more likely than silent genes to be hyperacetylated and increase their transcription levels in response to HDACIs, a process underpinned by the dynamic recruitment and antagonistic activities of HATs and HDACs. Based on this model and from a therapeutic perspective, I hypothesised that the ability of HDACIs to increase expression of active genes might be relevant for diseases caused by genes that encode proteins with enzymatic function. HDACI-mediated increase in gene transcription, even in the presence of missense, disease-causing mutations, might lead to increased enzymatic activity and amelioration of the cellular and clinical phenotype. I tested this hypothesis on a group of genes involved in the glycolytic and pentose phosphate pathway (GPPP) which, when mutated, cause chronic or episodic haemolytic anaemia.
Using RT-qPCR (B cell lines) and gene expression profiling (primary, in vitro generated human erythroid precursors and CD4+ T cells) I found that of the 17 GPPP genes, only Glucose-6-Phosphate Dehydrogenase (G6PD) mRNA levels increased in response to HDACIs in a time-dependent manner. Epigenetic analysis in B cells by ChIP-qPCR showed that histone hyper-acetylation and increased recruitment of HATs and HDACs underpin the selective G6PD transcriptional activation in response to HDACIs. Pharmacological and genetic assays showed that increase in G6PD transcription was also dependent on Sp1, a generic transcription factor known to recruit both HDACs and HATs.
Finally, I directly tested the hypothesis that HDACIs may increase enzymatic activity in G6PD deficient cells. Using B cell lines and primary erythroid cells from patients with G6PD deficiency, I found that HDACIs induce the same epigenetic changes in the mutant as in the wild type G6PD gene; more importantly, they lead to increased levels of the mutant mRNA and protein, associated with an up to 3-fold increase in enzymatic activity. These findings are potentially of great therapeutic significance for correction of G6PD deficiency in up to 300 million individuals worldwide with the polymorphic variants of G6PD deficiency (e.g., G6PDMed and G6PDA-).Open Acces
Investigation of the cellular pathogenesis of paroxysmal nocturnal haemoglobinuria
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Molecular and cellular pathogenesis of the erythroid defect and the therapeutic effect of glucocorticoids in Diamond-Blackfan anaemia
Get PDFDiamond-Blackfan Anaemia (DBA) is a rare inherited bone marrow (BM) failure syndrome characterised by selective anaemia, congenital anomalies and predisposition to malignancy. DBA is caused by monoallelic, loss-of-function mutations in ribosomal protein (RP) genes. Delineation of the precise erythroid defect underpinning anaemia in DBA has been hampered by a lack of markers that define cells giving rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies, i.e., the clonogenic assays that quantify early and late erythroid progenitor (EP) potential respectively. By combining flow-cytometry, cell-sorting and single cell clonogenic assays, I identify Lin-CD34+CD38+CD45RA-CD123-CD71+CD41a-CD105-CD36- BM cells as early EP (EEP) and Lin-CD34+/-CD38+CD45RA-CD123-CD71+CD41a-CD105+CD36+ cells as late EP (LEP) giving rise to BFU-E and CFU-E respectively. By applying these definitions to DBA, I reveal, for the first time, that both quantitative and qualitative defects in EEP and LEP contribute to defective erythropoiesis in DBA and its restoration by corticosteroids (CS). I also demonstrate that, while an EP defect associated with profound loss of erythroblasts (EB) is present in transfusion-dependent (TD) patients with RPS gene-associated DBA, EP/EB are relatively preserved in TD patients with RPL gene variants. Transcriptome profiling by RNA-sequencing of FACS-purified EB from RPL genotype patients compared with age-matched controls, provides a unique dataset showing a distinct DBA molecular signature, characterised by p53 and inflammatory signalling pathways. Remarkably, the cellular differences between RPL and RPS gene-associated DBA correlate with divergent clinical phenotypes: patients with RPL rather than RPS gene pathogenic variants are more likely to present with anaemia at an older age, and to respond to CS. Finally, although DBA is rare, I demonstrate its wider utility as a paradigm of disordered erythroid commitment, providing insight into normal haematopoiesis. The strategies described for prospective isolation of EP/EB constitute an original contribution to knowledge that will facilitate more incisive study of normal and aberrant erythropoiesis.Open Acces
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Cellular and Molecular Characterisation of Familial Haemophagocytic Lymphohistiocytosis Type 1
Get PDFHaemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition in
which absent or markedly reduced T cell and natural killer (NK) cell cytotoxicity results
in uncontrolled proliferation of T cells, activation of macrophages, hypercytokinaemia,
pancytopaenia, and hepatosplenomegaly. Familial HLH (FHL), an autosomal recessive
disorder affecting young infants and children, is fatal unless treated with chemotherapy
and allogeneic haemopoietic stem cell transplantation. Mutations in the gene encoding
perforin, a lytic protein involved in cell cytotoxicity, account for 30% of cases of FHL
(FHL II). Mutations in genes encoding hMunc 13-4, syntaxin 11, and syntaxin binding
protein 2, all essential for perforin release and T/NK cell cytotoxicity, have also been
identified in FHL (FHL III IV and V respectively). A previous report form our group
showed that in the UK, in consanguineous families of Pakistani descent, FHL (designated
FHL I) maps to 9q21.3-22 in an area spanning 5Mb and containing 14 genes. The aim of
my project was to identify the gene responsible for FHL I and to study the function of the
corresponding protein. Based on the premise that similar to other types of FHL, the
protein product of FHL I candidate gene would function in the perforin-dependent
cytotoxic pathway, 4 out of the 14 genes emerged as strong functional candidates: KIF27,
RASEF, UBQLN1 and FRMD3.
My mutation screening strategy aimed at amplifying and sequencing all exons and intronexon
boundaries of the candidate genes, followed by screening the remaining genes of the
FHL I locus. The material used was either genomic DNA extracted from 4 parental and 1
patient B cell line, or cDNA from the latter. In 2 newly identified patients, genomic DNA
from granulocytes was used. PCR amplification of genomic DNA and direct sequencing
or cloning and sequencing of individual clones or in some cases direct sequencing of the
full length cDNA from the patient B cell line did not reveal any mutations in any of the
known genes in the FHL I locus. For KIF27, mutations were excluded in gene regulatory
areas by amplification and direct sequencing of the whole promoter region, up to 1kb
upstream of the transcription start site. KIF27 protein expression was also studied by
immunoblotting and immunostaining followed by confocal microscopy but no
abnormality was identified at protein level. Promoter area mutation was also excluded in UBQLN1, the other strong candidate. The gene responsible for FHL I remains
unidentified. Further refinement of the locus with high-density SNP arrays in newly
diagnosed patients demonstrating linkage to the FHL I locus might help identify the gene
responsible for FHL I
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