Increased Apoptosis of Peripheral Blood and Bone Marrow B and T Cells Correlates with Advanced Stages and Poor Risk Factors in Patients with B-CLL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4162-4162
Author(s):  
Malgorzata Sieklucka ◽  
Agnieszka Bojarska-Junak ◽  
Agata Surdacka ◽  
Iwona Hus ◽  
Ewa Wasik-Szczepanek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Disease Progression ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
High Rate ◽  
Advanced Stage

Abstract B-cell chronic lymphocytic leukemia (B-CLL), is characterized by the accumulation of long-lived, neoplastic B-lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. Apoptotic processes have been shown to be altered in leukemic B cells, however, the role of apoptosis in the mechanisms of disease progression remains unclear. Recent studies suggest that the clonal excess of B-cells is caused not only by a decrease in cell death but also by increased cell proliferation. We have recently reported on a high rate of apoptosis leukemic B cells in peripheral blood (PB) of advanced stage patients and that apoptosis of PB lymphocytes from advanced-stage (III–IV acc. Rai) patients is higher than that in early-stage (0–II acc. Rai) patients. However the spontaneous apoptosis in B-CLL patients was significantly lower compared to the healthy controls that confirmed the defective apoptosis as one of the mechanisms of leukemic lymphocytes accumulation in B-CLL. Continuing our research, in the presented study we measured apoptosis of B and T cells in peripheral blood and bone marrow in correlation with the stage of B-CLL and prognostic factors. Materials and methods: Peripheral blood and bone marrow (BM) samples were obtained from 120 previously untreated B-CLL patients. An analysis of apoptosis within the B and T cells population was performed using flow cytometer and chloromethyl-X-rosamine staining (Mito Tracker Red CMXRos). CMXRos was used to detect disruptions in the mitochondrial membrane potential (ΔΨm), which is one of the earliest events in the apoptotic pathway and allow finding apoptotic cells when there are still in PB and BM. We found that ex vivo lymphocyte apoptosis was higher in BM compared to PB (p<0.05). Moreover, both B-cell and T-cell apoptosis in BM was higher than in PB (p<0.0001 and p<0.001, respectively). When compared, ex vivo apoptosis of T cells was found higher than that of B cells, both in BM (p<0.0001) and PB (p<0.0001). The percentage of apoptotic leukemic B cells correlated negatively with Bcl-2/Bax ratio in CD19+ B cells (p<0.05). Similarly, the percentage of apoptotic CD3+ cells correlated negatively with Bcl-2/Bax ratio in CD3+ cells (p<0.01). We also found that the percentage of apoptotic leukemic B cells correlated positively with the expression of proapoptotic protein Par-4 (prostate apoptosis response-4) in CD19+ B cells (p<0.01). The expression of Par-4 protein in CD19+ B cells correlated positively with the percentage of CD38+ cells (p<0.05), and it was higher in patients with CD38+ and ZAP-70+/CD38+ phenotypes (p<0.05 and p<0.01, respectively). There was a positive correlation between the expression of Par-4 protein and the lactate dehydrogenase (LDH) and β2-microglobulin serum concentrations (p<0.01 and p<0.05, respectively). Furthermore, the percentage of apoptotic CD19+ cells correlated positively with the LDH serum level (p<0.05). These data indicate that high amount of apoptotic leukemic cells in PB and BM might be considered as poor prognosis factor. Higher rate of B and T cells apoptosis in BM than in PB suggest the influence of bone marrow microenviroment on this process. Our results indicate also that high rate of T cells apoptosis might be responsible for immune dysfunction including both impaired anti-infection immunity as well as impaired anti-cancer response resulting in disease progression.

B Cell Activating Factor and c-Myc Regulate the Progression of Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 359-359
Author(s):  
Weizhou Zhang ◽  
Arnon P. Kater ◽  
Han-Yu Chuang ◽  
Thomas Enzler ◽  
George F. Widhopf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Disease Progression ◽  
Cell Population ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Low Grade ◽  
B Cell Activating Factor

Abstract Abstract 359 Chromosomal translocations involving c-Myc are frequently found in high grade lymphoma and multiple myeloma. In contrast, c-Myc translocations rarely occur in low-grade lymphomas/leukemias like chronic lymphocytic leukemia (CLL), but when present they are associated with rapid disease progression and bad prognosis. Overexpression of c-myc may also be the result of increased transcription by several proto-oncogene transcription factors, including NF-kB. Mice with c-Myc de-regulation at different stages of B cell development develop either aggressive B cells lymphomas or plasma cell neoplasm. So far, no c-Myc mouse model developed low-grade lymphoma/leukemia. iMycCa mice develop an expansion of CD5+ peritoneal B1 cells, as compared with WT littermates mice. These mice have a normal life-span and very rarely develop B cell lymphoma at older age. Interestingly, in iMycCa mice mature B cells, but not plasma cells,could be rescued from apoptosis by administration of B cell-activating factor belonging to the TNF family (BAFF). To our surprise, double transgenic iMycCa/Baff-Tg (Myc/Baff) mice developed a disease resembling human CLL, with dramatically shorter mean survival than parental strains, due to early onset and rapid clonal expansion of a mature CD5+B220low B cell population. Those cells transferred the disease into Baff-Tg (Baff) mice with marked infiltration in lymphoid organs and bone marrow. Gene-expression analyses revealed that among the genes altered in Myc/Baff CD5+B220lowleukemia cells were those with known relevance to human CLL disease, including elevated anti-apoptotic Bcl2 family members. Apart from studies on individual genes, sub-network analysis was performed which showed enrichment of apoptosis-related and stress-induced gene sets in Myc/Baff CD5+CD3- leukemia cells. The NF-kB gene set, a major target downstream of BAFF signaling, was also enriched in Myc/Baff CD5+CD3- leukemia cells. We observed a continuum in levels of c-MYC mRNA in 166 samples using Affymetrix array analyses. Changes in c-Myc protein expression were confirmed by immunoblot analyses and correlated with disease progression. In accordance with the functions of c-Myc as a promoter of cell cycle progression, as well as apoptosis, we found enhanced spontaneous cell death in vitro in CLL cells expressing high levels of c-Myc, which could be abrogated by co culture with BAFF expressing nurse-like cells (NLC) or recombinant BAFF. In addition to its anti-apoptotic role, BAFF treatment of primary human CLL cells led to dramatically enhanced expression of c-Myc through the IKK/NF-kB pathway. Inhibition of the NF-kB pathway significantly reduced viability of both Myc/Baff CD5+CD3- leukemia cells and human CLL cells co-cultured with NLC. Also it significantly lowered CD5+B220low leukemia cell population in blood and spleen, and prevented the infiltration of leukemia cells into lymph nodes and bone marrow of transplanted mice. This study demonstrates a potential pathologic role for c-Myc, in the pathogenesis and progression of CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 767-774 ◽  
Author(s):  
LA Fernandez ◽  
JM MacSween ◽  
GR Langley
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Suppressor Activity ◽  
Normal Peripheral Blood

Abstract The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T- cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.

scholarly journals Abnormal B-Cell and Tfh-Cell Profiles in Patients With Parkinson Disease

2021 ◽  
Vol 9 (2) ◽  
pp. e1125
Author(s):  
Rui Li ◽  
Thomas Francis Tropea ◽  
Laura Rosa Baratta ◽  
Leah Zuroff ◽  
Maria E. Diaz-Ortiz ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Parkinson Disease ◽  
B Cell ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
Cell Counts

Background and ObjectivesThere has been growing interest in potential roles of the immune system in the pathogenesis of Parkinson disease (PD). The aim of the current study was to comprehensively characterize phenotypic and functional profiles of circulating immune cells in patients with PD vs controls.MethodsPeripheral blood was collected from patients with PD and age- and sex-matched neurologically normal controls (NCs) in 2 independent cohorts (discovery and validation). Comprehensive multicolor flow cytometry was performed on whole blood leukocytes and peripheral blood mononuclear cells to characterize different immune subsets and their ex vivo responses.ResultsThe discovery cohort included 17 NCs and 12 participants with PD, and the validation cohort included 18 NCs and 18 participants with PD. Among major immune cell types, B cells appeared to be preferentially affected in PD. Proliferating B cell counts were decreased in patients with PD compared with controls. Proportions of B-cell subsets with regulatory capacity such as transitional B cells were preferentially reduced in the patients with PD, whereas proportions of proinflammatory cytokine-producing B cells increased, resulting in a proinflammatory shift of their B-cell functional cytokine responses. Unsupervised principal component analysis revealed increased expression of TNFα and GM-CSF by both B cells and T cells of patients with PD. In addition, levels of follicular T cells, an important B-cell helper T-cell population, decreased in the patients with PD, correlating with their B-cell abnormality.DiscussionOur findings define a novel signature of peripheral immune cells and implicate aberrant Tfh:B-cell interactions in patients with PD.

VCAM-1 Expression in B-Lymphopoiesis and Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3247-3247
Author(s):  
Faith M. Young ◽  
Raymond E. Felgar ◽  
Antonia P. Eyssallenne ◽  
Andrea Bottaro ◽  
Timothy P. Bushnell
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Surface Expression ◽  
Lymphoid Cells ◽  
B Lymphoid

Abstract Vascular Cell Adhesion Molecule-1 (VCAM; CD106), a member of the Ig Superfamily of molecules, binds to the β-1 integrin, Very Late Antigen-4 (VLA-4; CD49d); this interaction plays an integral role in leukocyte trafficking as well as lymphocyte-stromal cell interactions. VCAM can be shed from the surface of cells, and, in humans, serum levels of soluble VCAM (sVCAM) parallel activity and remission states in acute lymphocytic leukemia (ALL) and inflammation. Although widely investigated as a stromal-cell associated molecule, our lab and others have recently identified VCAM expression on normal bone-marrow derived B-lymphoid cells. Using FACS technology, we found that surface expression of VCAM is closely modulated at specific stages of B cell development, with relatively high levels on the pro-B cell population, down-modulation in pre-B cells at the onset of immunoglobulin (Ig) gene rearrangement, and subsequent re-expression at variable levels in immature and mature peripheral B cell subsets. We have verified VCAM transcripts by cDNA PCR in highly purified populations of murine precursor B cells. Normal human bone marrow precursor B-lymphoid populations (hematogones) also demonstrate VCAM surface protein expression. Finally, in an animal model of BCR/ABL+ ALL, we found that VCAM expression is dramatically increased on lymphoblasts when compared to normal reference populations in bone marrow and spleen. VCAM expression in human lymphoid malignancies is currently under investigation. Antibody-mediated VCAM cross-linking on primary B-cell precursors ex-vivo generates intracellular reactive oxygen species, demonstrating that signaling through this molecule has functional consequences. Intriguingly, in-vivo, VCAM expression is limited to B-lymphoid cells harvested from tissues such as bone marrow, spleen and lymph node; since, in the same animal, peripheral blood lymphocytes and peritoneal cells do not express readily detectable levels of the surface antigen. VCAM-expressing B-lymphoid cells cultured ex-vivo gradually lose surface expression over 24 hours. The tissue-associated modulation of VCAM expression is preserved in the murine Ph+ lymphoblasts; leukemia cells isolated from the peripheral blood express very low levels of surface VCAM compared to those harvested from bone marrow or spleen. Our data suggests that VCAM expression is dependent on tissue-specific microenvironmental signals in-vivo. B-lymphoid expression of both VCAM and its ligand VLA-4 is a surprising finding that has broad implications regarding leukemic cell interaction with endothelial cells, the bone marrow retention and trafficking of precursor- and leukemic-B cell populations, and the interpretation of an extensive experimental database predicated on the stromal-cell specificity of VCAM expression and function.

Effects Of Bendamustine Plus Rituximab On The Distribution Of Normal Peripheral Blood Leucocyte Populations In Advanced-Stage Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5289-5289 ◽  
Author(s):  
Georgiana Grigore ◽  
Martin Perez-Andres ◽  
Susana Barrena ◽  
Rosa Ana Rivas ◽  
Marcos González ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cd4 T Cell ◽  
Advanced Stage ◽  
Time Point

Abstract Introduction Management of B-cell chronic lymphocytic leukemia (CLL) is currently undergoing profound changes. Accordingly, new treatment options with an expected less toxicity than standard regimens are been explored. Recent results show that chemoimmunotherapy may improve the life expectancy of CLLpatients and has proven to be more efficient than chemotherapy alone in depleting malignant cells. Despite its efficacy, little is known about its precise immunomodulatory effects. Aim To evaluate the effects of chemoimmunotherapy with bendamustine plusrituximab (BR) on the distribution of normal residual leucocyte populations in peripheral blood (PB) from advanced-stage CLL patients, with special emphasis on maturation-associated B-cell subsets (immature, naïve, memory IgM/IgG/IgA and plasma cells). Material and Methods Distribution of PB neoplastic cells and residual normal immune cell subpopulations were analyzed in 72 CLL patients with advanced disease (Binet B/C), before therapy (M0) and after 1 course of BR (M1). The same analysis was repeated 3 months after completing treatment (M3) in 31/72 patients. PB leucocyte cell subsets were identified at each time-point by 8-color flow cytometry with monoclonal antibody reagents against CD3, CD4, CD5, CD8, TCRgd, CD19, CD20, CD27, CD38, CD45, CD56, sIgM, sIgA, sIgG, sIgLambda and sIgKappa. Results After the first BR course, absolute counts of all PB myeloid subsets were significantly decreased as compared to time M0, including neutrophils (2,744±1,830 vs 4,764±2,906 cells/uL, p<0.001), eosinophils (132±185 vs 215±245 cells/uL; p<0.001), basophils (37±28 vs 59±47 cells/uL, p<0.001), monocytes (334±280 vs 504±424 cells/uL, p=0.001) and dendritic cells (DCs, 41±40 vs 89±168 cells/uL, p=0.02), as well as NK cells (120±147 vs 550±599 cells/uL, p<0.001). At M3, all these populations remained decreased when compared to M0, but at similar levels to M1 (except for the absolute number of DCs, found to be increased vs. M1 -74±46 vs 41±40 cells/uL, p=0.008- and closer to M0). In turn, total T cells were reduced in M1 as compared to M0 values (818±655 vs 3,905±2,375 cells/uL, p<0.001), due to decreased numbers of CD4+ (424±376 vs 1,573±1,204 cells/uL, p<0.001), CD8+ (342±330 vs 1,334±1,218 cells/uL, p<0.001) and TCRgd (21±28 vs 141±289 cells/uL, p=0.001) T cells, leading to an increased CD4/CD8 ratio (1.8±1.3 vs 1.4±0.8, p=0.004). Also, decreased levels of CD4 (222±156 cells/uL), CD8 (501±544 cells/uL) and TCRgd (21±40 cells/uL) T cells were observed at time M3 vs. baseline values. No changes (p>0.05) were observed for CD8 and TCRgd for M3 vs. M1, while CD4+ T-cell numbers were significantly reduced (p=0.006), resulting in an inverted CD4/CD8 ratio (0.9±1.0 vs. 1.8±1.3, p=0.005) at the M3 time-point. As regards B cells, the absolute count of both neoplastic and normal B lymphocytes were significantly decreased at time M1 vs. M0 (3,363±9,353 vs 53,521±56,602 CLL cells/uL and 2±6 vs 58±107 normal B-cells/uL, p=0.006 and p<0.001, respectively). Within the normal residual B-cell compartment, we found significantly decreased numbers of immature (0.07±0.22 vs 6.55±21.64 cells/uL, p=0.01) and memory (1.3±14.7 vs 35.1±43.6 cells/uL, p<0.001) B cells -including sIgM (0.5±2.3 vs 14.5±24.8 cells/uL, p<0.001), sIgG (0.2±1.0 vs 11.5±17.2 cells/uL; p<0.001) and sIgA (0.6±3.1 vs 9.5±12.5 cells/uL, p<0.001) memory B cells-. At time M3, decreased (p<0.01) naïve (0.46±2.58 cells/uL) and memory B-cells (1.34±6.75 cells/uL), including IgM (0.46±2.58 cells/uL), IgG (0.34±1.69 cells/uL) and IgA (0.09±0.31 cells/uL), but not immature cells (2.28±8.84 cells/uL, p=0.9), were observed as compared to time M0. Differences did not reach statistical significance when comparing M3 vs. M1. The number of circulating plasma cells did not significantly vary during treatment. Conclusions All PB leucocyte subsets are affected by BR treatment in advanced-stage CLL. Interestingly, at time M3 the CD4+ T-cell subset continues to be decreased, while the other T-cell compartments seem to remain stable. Also, normal B cells are affected by BR treatment, and the depletion induced after one course therapy is maintained even three months after finishing BR therapy, except for immature B cells, that seem to be the first to recover in PB. Further studies will offer a more accurate insight into the biology of cell recovery during and after BR therapy in CLL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Tissue Counterpart to Monoclonal B-Cell Lymphocytosis

2021 ◽  
Author(s):  
Gabriel K. Habermehl ◽  
Lisa Durkin ◽  
Eric D. Hsi
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
High Count

Context.— B-cell clones discovered in tissue biopsies, without overt lymphoma, may represent a tissue counterpart to peripheral blood monoclonal B-cell lymphocytosis (MBL), herein termed tMBL. Objective.— To characterize the clinicopathologic features of tMBL. Design.— During a 10-year period, we retrospectively identified non–bone marrow/peripheral blood cases with monotypic B cells detected by tissue-based flow cytometry, but without an identifiable lymphomatous infiltrate on routine histopathology. We excluded cases with prior diagnosis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma or MBL. Results.— Fifty-four cases were identified (35 lymph node, 3 splenic, and 16 soft tissue/viscera). Forty-six cases were CLL-type, 2 were atypical CLL, and 6 were non-CLL. tMBL was detectable by immunohistochemistry in 14 cases (26%, all CLL-type). Concurrent blood flow cytometry, available in 10 cases, showed 4 with low-count MBL (3 CLL-type, 1 with non-CLL–type), 5 with high-count MBL (all CLL-type), and 1 case negative for clonal population. With median follow-up of 51 months, 2 patients had progression of disease (CLL, 68.7 months; and diffuse large B-cell lymphoma, 5.9 months). Patients with IHC-detectable tMBL had increased monoclonal B cells per total lymphocyte events (P = .01), morphologic evidence of bone marrow involvement (P = .04), higher white blood cell count (P = .02), and increased absolute lymphocyte count (P = .02). Conclusions.— tMBL spans an immunophenotypic spectrum similar to MBL, is detectable by immunohistochemistry in a minority of cases (often CLL immunophenotype), and is likely systemic in most cases. Development of overt lymphoma is uncommon but may occur, warranting clinical follow-up.

Analysis of ex vivo Apoptosis of B and T cells from Peripheral Blood and Bone Marrow of Patients with Chronic Lymphocytic Leukemia

2012 ◽  
Vol 43 (4) ◽  
pp. 336-341 ◽  
Author(s):  
Agnieszka Bojarska-Junak ◽  
Iwona Hus ◽  
Karolina Olszewska-Bożek ◽  
Sylwia Chocholska ◽  
Ewa Wąsik-Szczepanek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Lymphocytic Leukemia

scholarly journals Telomere Dynamics in Monoclonal B-Cell Lymphocytosis and Binet a Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5266-5266
Author(s):  
Felipe M Furtado ◽  
Daniel M Matos ◽  
Natalia Ferreira Scatena ◽  
Barbara Santana ◽  
Priscila Santos Sheucher ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Disease Progression ◽  
Telomere Length ◽  
Peripheral Blood ◽  
Population Screening ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes

Abstract Monoclonal B-Cell lymphocytosis (MBL) is an asymptomatic clinical entity characterized by monoclonal B-cell proliferation not meeting criteria for chronic lymphocytic leukemia. It is hypothesized that MBL may precede all CLL cases, but the molecular mechanisms responsible for disease progression and evolution are not known. Telomeres are the ends of linear chromosomes composed of tandem hexameric nucleotide repeats (5'-TTAGGG-3') coated by specific protective proteins (shelterin complex). Telomeres are usually short in CLL and telomere length is an independent prognostic factor for disease progression. Additionally, telomere attrition has been shown to increase chromosomal instability and acquisition of karyotypic abnormalities in CLL (Brugat, Blood 116:239-249, 2010). However, the telomere length of MBL clonal cells is not known. The purpose of this study was to evaluate the telomere length of clonal B-cells in Binet A CLL patients and subjects with clinic or population-screening MBL (Rawstron, Curr Hematol Malig Rep 8:52-59,2013).Twenty Binet A CLL patients, four with population-screening MBL detected in a previous study (Matos, Br J Haematol 147:339-46, 2009), and nine subjects with clinic MBL were studied. Mononuclear cells from the peripheral blood were separated by density gradient centrifugation and CD19+CD5+ cells were sorted in a FACSAria (Becton Dickinson) or a JSAN (Bay Bioscience) flow cytometers. Telomere length (TL) was determined in sorted CD19+CD5+ cells by quantitative polymerase chain reaction (qPCR) and expressed as telomere/single gene (T/S) ratio in comparison to a standard; TL also was adjusted for age. TL was measured in peripheral blood leukocytes from 52 healthy volunteers aged 50 to 86 years and used as controls. The CLL group had a medium age of 70 years (range, 58 to 81), the clinic MBL, 79 years (range, 57 to 97), and the population-screening MBL, 67 years (range, 53 to 82). In the CLL group, half of the patients was female; and all individuals were male in the clinic MBL group; in the population-screening MBL, 3/4 were males. The medium total lymphocytes count for the CLL patients was 36650/μL (range, 7500 to 147000), 3833 (range 2900 to 5900) in the clinic MBL, and 1625 (range 900 to 2100) in the population-screening MBL. The median percentage of CD19+CD5+ cells in total B-lymphocytes was 78% (range 59 to 91), 38% (range, 6 to 61), and 1.89% (range, 0.97 to 3.4) in CLL, clinic MBL, and population-screening MBL, respectively. Median TL was 0.32 (range, 0.13 to 0.78), 0.21 (range, 0.13 to 0.48) and 0.42 (range, 0.36 to 0.45) for CLL, clinic MBL and population-screening MBL, respectively. Age-adjusted TL was shorter in CLL and clinic MBL as compared to healthy controls (P<0.05; Figure). TL also tended to be shorter in population-screening MBL in comparison to healthy subjects, although not reaching statistic significance probably due to the low number of individuals in this group (Figure). In healthy individuals, TL of peripheral blood leukocytes shortened with aging, but in the three patient groups anlayzed, clonal B-cells TLs were equally short regardless of patient's age. These findings indicate that telomere erosion is an early phenomenon of leukemogenesis in B-clonal disorders and that MBL and CLL represent different stages of the same disease with different tumor burden. Disclosures No relevant conflicts of interest to declare.

Quantification of Recent Bone Marrow B-Cell and Thymic T-Cell Emigrants in Chemotherapy Naïve Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2353-2353
Author(s):  
Marina Motta ◽  
Claudia Ghidini ◽  
Cinzia Zanotti ◽  
Marco Chiarini ◽  
Luigi Caimi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mutational Status ◽  
Cd4 Lymphocytes

Abstract Abstract 2353 Poster Board II-330 Background Chronic lymphocytic leukemia (CLL) results in accumulation of mature, malignant, monoclonal B cells in blood, lymph nodes, spleen, liver, and bone marrow. Patients with CLL have fundamental defects in both humoral and cell-mediated immunity that significantly impact on its clinical course. It is still not known if these immune abnormalities include a decrease of new B- and T-cell mobilization from their production sites. During early lymphocyte development and differentiation, occurring in bone marrow and in thymus, B- and T-cells undergo rearrangements of B-cell and T-cell receptor genes whereby specific chromosomal sequences are excised to produce episomal DNA products identified respectively as T-cell receptor excisional circles (TRECs) and Kappa-deleting recombination excision circles (KRECs). Being stable circular fragments of DNA, TRECs and KRECs do not replicate during the mitotic process required for cell proliferation and, therefore, they are diluted out by cell divisions and are lost when the cells die. Since T-cells leaving the thymus are 70% TRECs+ and KRECs are present randomly in about 50% of neo-produced normal B-cells, the quantification of TRECs and KRECs allows a good estimation of the thymic and bone marrow output. Aim To investigate the extent of neo-synthesis of normal B and T cells in untreated patients with CLL. Patients and Methods Twelve previously untreated CLL patients were enrolled in this study. M:F ratio was 5:1; median age was 66; 7 patients had mutated IgVH genes, while 5 patients had unmutated IgVH genes; all patients were stage A Binet; FISH analysis including del(13q), del(11q), del(17p) and trisomy 12 detected 13q deletion in 6 patients while the other 6 patients presented no abnormalities. TRECs and KRECs were measured in mononuclear cells isolated from peripheral blood by duplex quantitative Real-Time PCR. This new assay, based on the use of a standard curve prepared with a plasmid containing fragments of TRECs, KRECs and of a reference gene, allows to quantify neo-produced B and T lymphocytes. T-cell subpopulations were determined by flow cytometry as follows: recent thymic emigrants (RTE) as cells with CD4+CD45RA+CCR7+CD31+ phenotype, regulatory T cells (Treg), as CD4+CD25int/highCD127low, RTE-Treg as Treg expressing CCR7+CD31+CD25int/high markers, effector memory (TEM) and central memory (TCM) T cells as lymphocytes displaying CD4+CD45RA-CCR7- and CD4+CD45RA-CCR7+ phenotype. Results The overall number of CD4+ lymphocytes was increased in chemotherapy-naïve patients with CLL (1585/μl vs 953/μl; p=0.02). TRECs were significantly higher in CLL patients compared to age-matched healthy controls (2.9/ml vs 1.4/ml; p=0.04), while the proportion of RTE was lower (24.9% vs 32.4%; p=0.02). No significant differences were observed in the percentage of Treg and RTE-Treg as well as of TCM. On the contrary, the percentage of TEM was higher (22.4% vs 10.4%; p=0.01) in CLL patients. The number of KRECs was lower in CLL patients than in controls (3.8/ml vs 7.4/ml; p=0.004). No correlation between IgVH gene mutational status, Ig levels and KRECs was found. Conclusions The neo-synthesis of normal T, with the exception of Treg, and B cells is reduced in patients with CLL compared to controls, even in a good prognosis, chemotherapy-naïve subset. The increased number of TRECs+ cells and TEM together with low RTE could be ascribed to the increased number of circulating CD4+ lymphocytes which do not appear to be recently mobilized from the thymus (CD31- cells) or to undergo peripheral expansion, but to accumulate in the peripheral blood. The low number of KRECs compared to normal controls could be interpreted as a consequence of the abnormal leukemic B-cell expansion which may impair normal B cell neo-synthesis. The level of KRECs did not seem to correlate to the mutational status of IgVH genes or the Ig level but a larger number of patients is needed to confirm these preliminary data and to establish whether the analysis of TRECs and KRECs may help clarify the complex immune abnormalities in CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 767-774 ◽  
Author(s):  
LA Fernandez ◽  
JM MacSween ◽  
GR Langley
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Suppressor Activity ◽  
Normal Peripheral Blood

The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T- cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.

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