Biased immunoglobulin heavy variable (IGHV) gene usage and distinctive B-cell receptor (BCR) features have been reported in chronic lymphocytic leukaemia (CLL), which may reflect clonal selection by antigens during disease development. Furthermore, the IGHV gene mutation status distinguishes two clinical entities of CLL, where patients with unmutated IGHV genes have an inferior prognosis compared to those with mutated IGHV genes. Recently, one subgroup of CLL patients expressing the IGHV3-21 gene was found to display highly similar immunoglobulin (IG) gene features, even within the heavy chain complementarity-determining region 3 (HCDR3). Patients in this subgroup typically had a poor prognosis. In paper I, we aimed to identify further subgroups with restricted BCR features among 346 CLL cases. Six subsets were defined which carried virtually identical BCRs in terms of rearranged heavy and light chain (LC) IG genes and CDR3 length and composition. In paper II, we investigated 90 IGHV3-21 cases from diverse geographical locations. We confirmed the highly restricted HCDR3 characteristics in 56% of patients and a biased usage of the IGLV3-21 gene in 72% of cases. Survival analysis also confirmed the poor outcome of this group, irrespective of IGHV gene mutation status and geographical origin. Papers III and IV involved a large-scale analysis of IGH and IG kappa and lambda (IGK/L) gene rearrangements, to define subsets with ‘stereotyped’ BCRs and also to systematically examine the somatic hypermutation (SHM) features of the IG genes in CLL. We studied a cohort of 1967 IGH and 891 IGK/L gene sequences from 1939 patients from 6 European institutions. Over 5300 IGH and ~4700 IGK/L sequences from non-CLL B cells were used as a control data set. In total, 110 CLL stereotyped subsets were defined according to HCDR3 homology. Striking IGK/L gene biases were also evident within subsets, along with distinctive K/LCDR3 features, such as length and amino acid composition. At cohort level, the patterns of mutation appeared to be consistent with that of a canonical SHM mechanism. However, at a subgroup level, certain stereotyped subsets, e.g. IGHV3-21/IGLV3-21 and IGHV4-34/IGKV2-30 CLL, deviated from this pattern. Furthermore, recurrent ‘stereotyped’ mutations occurred in cases belonging to subsets with restricted HCDR3s, in both IGHV and IGK/LV genes, which were subset- and CLL-biased when compared to non-CLL B cells. In conclusion, our findings implicate antigen selection as a significant factor in the pathogenesis of CLL, particularly in cases carrying stereotyped BCRs. The presence of stereotyped mutations throughout the VH and VL domain also indicates involvement of IG regions other than the CDR3 in antigen recognition. Finally, biased IGK/L gene usage and specific K/LCDR3 features are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains.

Η αναιμία σχετιζόμενη με τη νόσο στη χρόνια λεμφογενή λευχαιμία (ΧΛΛ) είναι διάγνωση εξ΄ αποκλεισμού και διαπιστώνεται στις περιπτώσεις που δεν ανευρίσκεται άλλο εμφανές αίτιο αναιμίας. Δεδομένου ότι η παθογένεια της αναιμίας αυτής δεν είναι διευκρινισμένη, μελετήσαμε διάφορες παραμέτρους της ερυθροποίησης σε πρωτοδιαγνωσθέντες ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και τις συγκρίναμε με τα αντίστοιχα ευρήματα ασθενών με ΧΛΛ χωρίς αναιμία και χωρίς να έχουν λάβει θεραπεία και με αυτά των υγιών μαρτύρων. Η διήθηση του μυελού των οστών από τα κακοήθη Β-λεμφοκύτταρα δεν ήταν αποκλειστικά υπεύθυνη για την πρόκληση αναιμίας. Τα CD34+ κύτταρα του μυελού των οστών στη ΧΛΛ δεν εμφάνισαν ενδογενή διαταραχή στη στροφή και περαιτέρω διαφοροποίησή τους προς την ερυθρά σειρά. Επιπλέον, δε διαπιστώθηκαν ανεπαρκή επίπεδα ερυθροποιητίνης ορού ούτε ελαττωματική απάντηση των ερυθροποιητικών προδρομικών κυττάρων στην ΕΡΟ στον άξονα ΕΡΟ-ΕΡΟ υποδοχέα κατά τη διέγερσή τους με ΕΡΟ ± TNF-α. Από την άλλη μεριά, τα επίπεδα του TNF-α βρέθηκαν αυξημένα στους ασθενείς με ΧΛΛ και αναιμία σχετιζόμενη με τη νόσο και φάνηκε ο TNF-α να ασκεί άμεση κατασταλτική δράση στη διαφοροποίηση των CD34+ κυττάρων προς κύτταρα της ερυθράς σειράς in vitro. Η μελέτη μας έδειξε ότι η αναιμία σχετιζόμενη με τη νόσο στη ΧΛΛ δεν οφείλεται σε ενδογενείς διαταραχές των ερυθροποιητικών προδρομικών κυττάρων, αλλά πιθανότατα να αποδίδεται στην απευθείας κατασταλτική επίδραση του TNF-α . / Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. To investigate its underlying mechanisms we studied parameters of erythropoiesis at cellular and molecular level in newly diagnosed CLL patients with disease-related anemia in comparison with those of non-anemic CLL patients and normal controls. Bone marrow (BM) infiltration by leukemic B cells was not exclusively responsible for the presence and the severity of disease-related anemia and BM CD34+ cells were intrinsically capable of generating erythroid precursors. No deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO ± Tumor Necrosis Factor-α (TNF-α) stimulation was also observed. On the other hand, serum TNF-α levels were found increased in patients with CLL and disease-related anemia and TNF-α appeared to exert a direct inhibitory effect on the differentiation of CD34+ cells towards the erythroid lineage in vitro. Our study showed that disease-related anemia in CLL was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-α on the erythroid production.

Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy.

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-08-29 14:53:18.766

To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

Monoclonal B lymphocytosis (MBL) has been recognized as a novel diagnostic condition. This study aims at the identification of clonal lymphocytosis in the patients with asymptomatic lymphocytosis. A total of 203 patients were evaluated for clonal B and T lymphocytosis by using flow cytometry and multiplex-PCR. Among them clonal B- or T-cells were detected in 54.2% of the cases, of which 38.4% were clonal B-cells and 15.8% were clonal T-cells cases. By immunophenotype, MBL was classified into the chronic lymphocytic leukemia (CLL) type (21.7%) and non-CLL-type (7.4%). Flow cytometry analysis and cell counts were used to determine the size of clonal population, and the data indicate that MBL and CLL are present in a continuous spectrum of clonal expansion. The findings may contribute to the current understanding of MBL and evaluation of incidental lymphocytosis. Further studies are required to evaluate clonal progression as a precursor stage of lymphoid malignancy.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.