scholarly journals Identification of a Novel Proliferating Cell Fraction in Chronic Lymphocytic Leukaemia with High Expression of IgM and Chemokine Receptors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3711-3711
Author(s):  
Daniel Friedman ◽  
Antony Long ◽  
Piers EM Patten ◽  
Robbert Hoogeboom
Keyword(s):  
Flow Cytometry ◽  
Lymphocytic Leukaemia ◽  
High Expression ◽  
Cell Fraction ◽  
Lymphoid Tissues ◽  
Proliferating Cells ◽  
Expression Levels ◽  
Cell Fractions ◽  
Proliferating Cell

Abstract Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant CD5+ B cells in the peripheral blood (PB), secondary lymphoid tissues and bone marrow. Currently considered an incurable disease, B cell receptor (BCR) signalling plays a key role in the disease aetiology as evidenced by the therapeutic success of BCR signalling inhibitors such as ibrutinib. Previous studies using incorporation of 2H-labelling of DNA in vivo demonstrated sub-clonal heterogeneity in PB CLL cell fractions sorted based on reciprocal densities of chemokine C-X-C motif receptor 4 (CXCR4) and CD5. The CXCR4 loCD5 hi fraction was shown to be enriched in recently born proliferating cells while the CXCR4 hiCD5 lo fraction consists of resting, quiescent cells thought to reflect their migratory and BCR signalling histories in tissue. Whilst these proliferating/resting fractions have since been more closely examined, the remaining bulk PB CLL population has been left relatively unexplored leaving other therapeutically relevant cell fractions undetected. Here, we have comprehensively analysed the phenotype of subpopulations of PB cells from 11 CLL patients using flow cytometry to identify activated and proliferating cell fractions. CD19 +CD5 +cells were divided into 9 fractions based on CXCR4/CD5 densities and to permit comparisons between fractions, each cell fraction was defined as containing 1-2% of the total clonal CD19 +CD5 + population. Surprisingly, we detected enrichment for Ki67+ proliferating cells and high expression of AID in the cell fraction with highest expression levels of both CXCR4 and CD5 (CXCR4 hiCD5 hi), demonstrating that CXCR4 loCD5 hi cells are not the only proliferating fraction in the blood. Moreover, we could detect mitotic cells in the CXCR4 hiCD5 hi fraction using imaging flow cytometry of a nuclear stain. This CXCR4 hiCD5 hi fraction showed the highest surface expression levels of IgM, CD86, CCR7, CXCR3 and CXCR5 of all the fractions assessed (p<0.05), indicating they are highly activated and primed for migration to lymph nodes (LNs) for further activation and proliferation. Proliferation of CLL cells is highest in secondary lymphoid tissues, however the phenotype of proliferating cells in tissue is unknown. To examine the phenotype of proliferating CLL cells in LNs, we analysed a fine-needle aspirate obtained from an enlarged cervical node using flow cytometry and compared this to a matched PB sample. Flow cytometric gates set on the PB sample were used to define and quantify LN cell fractions. Expression levels of both Ki67 and surface IgM were highest in the CXCR4 hiCD5 hi fraction which was expanded to 20% of the CD19 +CD5 + population in the LN whilst CXCR4 loCD5 hi cells (accounting for 2% of the bulk LN population) expressed very low surface IgM and Ki67 levels, suggesting CXCR4 hiCD5 hi cells may be the most proliferative cells in CLL. The CXCR4 loCD5 hi cell fraction has been shown to be a key target of ibrutinib, however the impact of ibrutinib on the CXCR4 hiCD5 hi fraction is unknown. Administration of ibrutinib to PB CLL cells for 48hr in vitro resulted in selective targeted depletion of the CXCR4 loCD5 hi fraction, as evidenced by induction of apoptotic markers in this compartment; conversely, persistent cells after 48hr ibrutinib administration in vitro were exclusively of the CXCR4 hi phenotype. In conclusion, we have identified a potentially dangerous fraction of proliferating cells in the PB of CLL patients with high expression of CXCR4, CD5, IgM, CCR7, CXCR3 and CXCR5 open for both migration to tissue and reception of BCR signals. Furthermore, CXCR4 hiCD5 hi cells in the periphery may closely mirror tissue-resident activated cell phenotypes and may represent critical targets for therapeutic intervention, particularly in high-risk CLL patients refractory to BCR inhibitor therapies. Disclosures Patten: ROCHE: Research Funding; GILEAD SCIENCES: Honoraria, Research Funding; NOVARTIS: Honoraria; JANSSEN: Honoraria; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.

scholarly journals Loss of primitive hematopoietic progenitors in patients with human immunodeficiency virus infection

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4568-4578 ◽  
Author(s):  
A Marandin ◽  
A Katz ◽  
E Oksenhendler ◽  
M Tulliez ◽  
F Picard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Immunodeficiency Virus ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Fractions ◽  
Cd38 Antigen ◽  
Hiv 1

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, primitive cells from bone marrow aspirates of 21 patients with HIV-1 infection were quantitated by flow cytometry. The mean percentage of CD34+ cells is not significantly altered in HIV-1-infected patients in comparison with HIV-1- seronegative controls. In contrast, two- and three-color immunofluorescence analysis showed that in all HIV-1 samples, most CD34+ cells coexpressed the CD38 antigen. The proportion of HIV-1- derived CD34+ cells that did not express the CD38 antigen was significantly lower (HIV-1+: mean, 1.73%; controls: mean, 14%; P < .0005) than in controls. Moreover, of Thy-1+ cells, the proportion of CD34+ cells was twofold lower in HIV-1-infected patients (HIV-1+: mean, 12%; controls, 25%, P < .0005), which suggests that phenotypically primitive cells are depleted in HIV-1 infection. In vitro functional analysis in long-term cultures of sorted CD34+ cells from seven HIV-1 patients showed that CD34+ cells from HIV-1 patients generated much fewer colonies both in the nonadherent and adherent layers than CD34+ cells from controls after 5 weeks of culture (10-fold and four-fold less, respectively). Precise long-term culture initiating cell (LTC-IC) frequency in the CD34+ cell population was determined in three patients by limiting dilution and was markedly decreased in comparison to that of normal controls (from twofold to > sevenfold decreased). To determine if primitive cells were infected by HIV-1, both methylcellulose colonies generated from long-term culture of CD34+ cells and various CD34+ cell fractions purified by flow cytometry were evaluated for the presence of HIV-1 by polymerase chain reaction (PCR). Progeny from long-term culture was HIV-1-negative in three samples. In addition, using a sensitive PCR technique, the HIV-1 genome could not be detected in CD34+, CD34+/CD38-, and CD34+/CD4+ cells. These data show that hematologic disorders in HIV disease may be the consequence of a deficit of primitive cells. However, direct infection of these cells by HIV-1 does not seem to be responsible for this defect.

scholarly journals Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

2010 ◽  
Vol 207 (12) ◽  
pp. 2631-2645 ◽  
Author(s):  
Véronique Witko-Sarsat ◽  
Julie Mocek ◽  
Dikra Bouayad ◽  
Nicola Tamassia ◽  
Jean-Antoine Ribeil ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Nuclear Antigen ◽  
Neutrophil Apoptosis ◽  
Proliferating Cells ◽  
Rna Transfection ◽  
Neutrophil Survival ◽  
Proliferating Cell

Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand– or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

scholarly journals Proliferating cells in human eccrine and apocrine sweat glands.

1995 ◽  
Vol 43 (12) ◽  
pp. 1217-1221 ◽  
Author(s):  
Y Morimoto ◽  
K Saga
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Myoepithelial Cells ◽  
Nuclear Antigen ◽  
Sweat Glands ◽  
Secretory Cells ◽  
Proliferating Cells ◽  
Peripheral Cells ◽  
Proliferating Cell ◽  
Eccrine Sweat Glands

Morphological observations of sweat glands showed degenerated debris of secretory cells in the secretory lumen in both apocrine and eccrine sweat glands. This suggested that dead secretory cells of human eccrine and apocrine sweat glands were released into the lumen and replaced by other cells. However, we did not know which type of cells replaced lost secretory cells. Therefore, we studied the proliferating cells in human eccrine and apocrine sweat glands by labeling S-phase cells in vitro with 5-bromo-2'-deoxyuridine (BrdUrd) and by immunostaining proliferation-associated proliferating cell nuclear antigen (PCNA) with anti-PCNA monoclonal antibody. BrdUrd and anti-PCNA antibody labeled a few secretory cells in eccrine and apocrine sweat glands, but neither method labeled myoepithelial cells. Luminal and peripheral cells of the eccrine and apocrine coiled duct were labeled with both BrdUrd and PCNA. However, we could not find any highly proliferative germinative cells in coiled ducts. Our results suggest that lost secretory cells could be replaced by proliferation of secretory cells themselves rather than by proliferation of myoepithelial cells or duct cells.

scholarly journals Effect of Fluoride on Endocytosis and Surface Marker Expression Levels of Mouse B Cells In Vitro

2016 ◽  
Vol 39 (2) ◽  
pp. 596-603 ◽  
Author(s):  
Haili Ma ◽  
Zeyu Shi ◽  
Yaoze Dong ◽  
Rui Liang ◽  
Jianshan Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Immune Function ◽  
Sodium Fluoride ◽  
Surface Marker ◽  
Expression Levels ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
High Concentrations

Background/Aims: To clarify the effect of fluoride on splenic B cells, the endocytosis and surface marker expression levels of mouse splenic B cells were detected in vitro by flow cytometry. Methods: Cells were stimulated with 10 µg/mL lipopolysaccharide (LPS) and varying concentrations of Sodium Fluoride (NaF) (0, 50 µM, 100 µM, 500 µM, 1000 µM). Results: The results demonstrated that the endocytic capacity of B cells was enhanced by NaF at 50µM. NaF significantly enhanced CD80 expression at 50 µM and decreased CD86 expression at 500 µM. CD40 and CD138 expression on B cells were down-regulated at varying high concentrations of NaF. Conclusion: our results showed that the endocytic capacity, expression levels of CD40 and CD80 of B cells changed significantly at lower concentrations, whereas expression levels of CD138 and CD86 changed significantly at higher concentrations, suggesting that fluoride could inhibit immune function in animals.

Microenvironment Regulation of IL23R/IL-23 Axis Drives Chronic Lymphocytic Leukemia (CLL) Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 616-616
Author(s):  
Fortunato Morabito ◽  
Giovanna Cutrona ◽  
Anna Grazia Recchia ◽  
Marina Fabbi ◽  
Silvano Ferrini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Biological Effects ◽  
Luciferase Reporter ◽  
Side Chain ◽  
Lymphoid Tissues ◽  
Stat3 Phosphorylation

Abstract Background : CLL displays a considerable degree of clinical heterogeneity, which is in part ascribable to clone-intrinsic biological features and that are also influenced by clone-extrinsic events related to the microenvironment. Among the dynamics-taking place within the CLL microenvironment, those finalized to the induction of an overly inflammatory milieu may significantly impact on the CLL natural history by hijacking the immunological microenvironment at the same time fostering clone fitness. IL-23 acts as a prototypical pro-inflammatory mediator representing a promising therapeutic target. We analyzed the ability of CLL cells to sense IL-23 through the IL-23R complex (consisting of IL12Rß1 and IL23R subunits) expression and correlated this feature with clinical outcome. Moreover, we investigated the synthesis of IL-23 within the CLL microenvironment, and tested the biological effects of the IL-23/IL-23R axis engagement and of its interference in vitro and in vivo. Methods : IL23R complex was detected by quadruple flow cytometry staining with CD19, CD5, IL23R, and IL12Rβ1 in prospectively enrolled CLL cases (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). On human tissue specimens, lymph node and bone marrow samples from 16 CLL patients were selected for in situ immunolocalization analyses. NOD/Shi-scid/γcnull (NSG) mice were used for in vivo xenografts, in which activated autologous T cells (AAT), obtained by adding anti-CD3 and CD28 Dynabeads and rIL2 were co-injected with CLL cells. MiRNA analysis was performed by Agilent's Human V2 platform and by quantitative PCR. MirVANA microRNA mimics and inhibitors were purchased from Ambion, Inc. For 3'UTR luciferase reporter experiments, miRNA target reporter vectors were purchased from Origene. Results : By flow cytometry, circulating CLL cells of 281 cases variably expressed IL23R side chain while consistently lacking IL12Rß1 chain expression. The engagement of the uncoupled IL23R complex expression (i.e. IL23R but not IL12Rb1 expression) by IL23 did not activate downstream signaling pathways, such as the up-regulation of pSTAT3. The 3-year TTFT probability of patients with low IL23R expression (IL23R-low) was 91% as compared to 75% of IL23R-high cases [χ2 9.1, P=.003; HR=3.2, 95%CI (1.4-7.1)]; in a multivariate model, IL23R expression still remained independently associated with TTFT. We explored the potential control of IL23R expression in CLL cells by miRNA and found 15 miRNAs inversely associated with IL23R expression, five of which predicted as regulators (miRNA-146b-5p, miRNA-155, miRNA-324-5p, miRNA-532-3p and miRNA-630). Among these, miR-324-3p and miR-146b-5p were demonstrated to functionally regulate the expression of IL23R and IL12Rβ1 proteins in CLL cells, respectively. Within lymphoid tissues, in situ, CLL clones expressing IL23R side chain also showed expression of IL12Rß1, which varied according to the density of CD40L-expressing bystander elements suggesting a microenvironment-driven regulation of the IL-23R complex. To functionally test this hypothesis, CLL cells were co-cultured in the presence of NIH-3T3 transduced with CD40L or with AAT cells. A significant up-regulation was observed for both the IL12Rß1 and IL23R side chains, suggesting the environment co-stimulation as a mechanism of IL-23R complex regulation. Consistently, the IL-23R complex was upmodulated in CLL cells expressing IL-23R but not IL12Rß1, upon xenograft with autologous T cells into NOD-Scid mice. We then investigated the effect of IL-23R engagement by IL-23 in CLL cells and found that IL-23R activity correlated with CLL cell proliferation and survival in vitro via STAT3 phosphorylation. The trophic nature of IL-23-mediated stimuli over CLL cells was further demonstrated in vivo through the adoption of an anti-IL23p19 monoclonal antibody for clinical use, which proved to be effective in eradicating the xenografted CLL clone in the infiltrated tissues (spleen, liver and BM) by inhibiting proliferation and inducing apoptosis. Noteworthy, the therapeutic effect of IL-23 antagonism was demonstrated by histopathology, flow cytometry and BCR clonality. Conclusions : Overall, we demonstrated that IL-23/IL-23R axis is a novel microenvironment-regulated determinant in CLL pathobiology representing a strong prospect in disease prognostication and treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Situ Analysis of Cellular Proliferation in Canine, Feline and Equine Tumors by Immunohistochemistry: A Comparison of Bromodeoxyuridine, Proliferating Cell Nuclear Antigen, and Interchromatin-Associated Antigen Immunostaining Techniques

1994 ◽  
Vol 6 (4) ◽  
pp. 453-457 ◽  
Author(s):  
Alain Pierre Théon ◽  
Loretta Metzger ◽  
Stephen Griffey
Keyword(s):  
Cell Proliferation ◽  
Proliferating Cell Nuclear Antigen ◽  
Cellular Proliferation ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Immunohistochemical Detection ◽  
Proliferating Cells ◽  
Proliferating Cell

Cell proliferation in canine, feline, and equine tumors was evaluated using immunohistochemical detection of in vitro 5–bromodeoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen (PCNA), and interchromatin-associated antigen (p105). Ten tumors in each species were analyzed. The tumor proliferative fraction (PF) was defined as the percentage of labeled nuclei for 5,000 tumor nuclei counted. Immunoreactivity was observed with all techniques in all species. A good correlation was observed between the proliferative fractions measured with the BrdU (PFBrdU) and PCNA (PFPCNA) techniques ( rs = 0.523, P = 0.0026). There was no correlation between the PFs measured with the BrdU (PFBrdU) and p105 (PFP105) techniques. Using the median values obtained from the different approaches as cutoff points to define slowly and rapidly proliferating tumors, there was an 80% agreement ( P = 0.009) between PFBrdU and PFPCNA and no agreement between PFBrdU and PFP105 The results of this study indicate that both BrdU and PCNA labeling methods can be used reliably for identifying proliferating cells in animal tumors. In addition, PCNA could be used to replace the BrdU method to assess tumor proliferative fraction because it does not require pretreatment of tissues.

Low or Undetectable Numbers of Philadelphia Chromosome Positive (Ph+) Leukemia Cells in the Primitive (CD34posCD38neg) Stem Cell Fraction in Chronic Myeloid Leukemia (CML) Patients during Tyrosine Kinase Inhibitor Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1079-1079
Author(s):  
Satu Mustjoki ◽  
Peter Rohon ◽  
Katrin Rapakko ◽  
Sari Hernesniemi ◽  
Perttu Koskenvesa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Cell Fraction ◽  
Cytogenetic Remission ◽  
Cell Fractions ◽  
Cd38 Antigen

Abstract Targeted tyrosine kinase inhibitors (TKIs) efficiently induce rapid hematologic and cytogenetic remission in most chronic myeloid leukemia (CML) patients. However, in vitro experiments have suggested that the most primitive CML stem cells residing in the CD34posCD38neg fraction are relatively resistant to TKIs. The prevalence of these stem cells in vivo in patients under TKI therapy is unclear. The aim of this project was to analyze the effect of TKI therapy on Ph+ leukemia stem cell pool in patients and to analyze the proportion of Ph+ cells in different stem cell fractions. A total of 26 chronic phase CML patients were included in the study. 18 patients were treated with imatinib, 5 with dasatinib, and 3 with bosutinib. The median time of TKI treatment was 20 months (range 3–72 months). Large volume (median 30 ml, range 5–55 ml) of bone marrow (BM) aspirate was collected and mononuclear cells (MNC) were isolated. CD34pos cells were separated with paramagnetic beads and further sorted into CD34posCD38pos and CD34posCD38neg cell populations with multicolor flow cytometry in order to analyze progenitor cell fractions of different maturation stage. Proportion of Ph+ cells was determined with interphase FISH by counting 1000 cells in each fraction. The median yield of MNCs from 30 ml of BM aspirate was 280x106 cells resulting in a median of 32 000 CD34posCD38neg cells (range 1000–91000). High-sensitivity counting of the proportion of Ph+ cells was feasible with a median number of counted interphase nuclei of 1005. During TKI therapy the CD34pos cells expressing highest CD38 antigen level were already mostly differentiated into B-cell lineage (CD19 positive). The CD34pos cells expressing low CD38 antigen levels expressed markers of more primitive cells such as C-kit (CD117) and CD133. Of 26 patients with CML, 19 were in complete cytogenetic remission (CCyR) when assessed by metaphase FISH of non-fractionated BM cells (1000 cells analyzed). Only 3 patients had single Ph+ cells in CD34pos cell fractions (less than 1%). In remainder of patients, all progenitor cell fractions, including the most primitive CD34posCD38neg cells, were negative for Ph+ cells. 3 patients had 0–1% of Ph+ cells in non-fractionated BM sample. One of them had 0.2% of Ph+ cells in CD34posCD38neg fraction, but the other 2 patients had 0/1000 Ph+ stem cells. 4 patients had a partial cytogenetic response (5–20% of Ph+ cells in non-fractionated BM sample). Again, the proportion of Ph+ cells was not increased in the most primitive CD34posCD38neg cell fraction. Interestingly, patients who had discontinued imatinib treatment had lower level of Ph+ cells in different CD34pos fractions (median 0.1%) when compared to non-fractionated BM (median 9.3%). Based on our data, in chronic phase CML patients, TKI therapy eradicates most Ph+ CD34pos progenitor cells. Unexpectedly, leukemic stem cells were not enriched in the most primitive CD34posCD38neg cell fraction in vivo. These results differ from the in vitro studies, where CD34posCD38neg cells have been shown to be resistant to TKIs. This could be due to non-physiological conditions (growth factor sensitivity, other cytokines) in cell culture assays. In addition, leukemic stem cells in vivo may be located in the subcortical hypoxic stem cell niche in the BM and are less likely to be aspirated. Our data underline the tremendous proliferative potential of very rare stem cells in CML patients in CCyR, as is evident after discontinuation of TKI therapy. Future studies evaluating the kinetics of disappearance of Ph+ cells from stem cell fractions during TKI therapy and the location of residual Ph+ stem cells in the BM are warranted and may give important information on the depth of the therapy response. Furthermore, this knowledge may aid in targeting therapy to these cells and finding curative treatment strategies in CML.

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