Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities.

Chronic lymphocytic leukaemia (CLL) is a genetically, morphologically and phenotypically heterogeneous chronic disease with clinical variability between patients. Whether the significant heterogeneity of cell size within the CLL population contributes to the heterogeneous features of this disease has not been investigated. The present study aimed to characterise the phenotypic and functional properties of two subpopulations of typical CLL cells that differ in cell size: small (s-CLL) and large (l-CLL) CLL cells delineated by forward scatter cytometry. The s-CLL cells were characterised by the CD5lowCXCR4hi phenotype, while the l-CLL cells were characterised by the CD5hiCXCR4dim phenotype and indicated a higher expression of CXCR3, CD20, CD38 and HLA-DR. The l-CLL cells displayed higher migration activity towards CXCL12, a tendency towards a higher proliferation rate and an increased capacity to produce IgM in the presence of CpG compared with s-CLL cells. When stimulated with CpG and CXCL12, l-CLL cells were characterised by a higher polarisation phenotype and motility than s-CLL cells. Our study revealed that the differences in CLL cell size reflected their activation status, polarisation and migratory abilities. Our data provide evidence of the importance of cell-size heterogeneity within a CLL pool and the dynamics of cell-size changes for disease pathogenesis, thus deserving further investigation.

Deciphering the complex circulating immune cell microenvironment in chronic lymphocytic leukaemia using patient similarity networks.

The tissue microenvironment in chronic lymphocytic leukaemia (CLL) plays a key role in the pathogenesis of CLL, but the complex blood microenvironment in CLL has not yet been fully characterised. Therefore, immunophenotyping of circulating immune cells in 244 CLL patients and 52 healthy controls was performed using flow cytometry and analysed by multivariate Patient Similarity Networks (PSNs). Our study revealed high inter-individual heterogeneity in the distribution and activation of bystander immune cells in CLL, depending on the bulk of the CLL cells. High CLL counts were associated with low activation on circulating monocytes and T cells and vice versa. The highest activation of immune cells, particularly of intermediate and non-classical monocytes, was evident in patients treated with novel agents. PSNs revealed a low activation of immune cells in CLL progression, irrespective of IgHV status, Binet stage and TP53 disruption. Patients with high intermediate monocytes (> 5.4%) with low activation were 2.5 times more likely (95% confidence interval 1.421-4.403, P = 0.002) to had shorter time-to-treatment than those with low monocyte counts. Our study demonstrated the association between the activation of circulating immune cells and the bulk of CLL cells. The highest activation of bystander immune cells was detected in patients with slow disease course and in those treated with novel agents. The subset of intermediate monocytes showed predictive value for time-to-treatment in CLL.

High CXCR3 on Leukemic Cells Distinguishes IgHV mut from IgHV unmut in Chronic Lymphocytic Leukemia: Evidence from CD5high and CD5low Clones.

Despite the shared pattern of surface antigens, neoplastic cells in chronic lymphocytic leukemia (CLL) are highly heterogeneous in CD5 expression, a marker linked to a proliferative pool of neoplastic cells. To further characterize CD5high and CD5low neoplastic cells, we assessed the chemokine receptors (CCR5, CCR7, CCR10, CXCR3, CXCR4, CXCR5) and adhesion molecules (CD54, CD62L, CD49d) on the CD5high and CD5low subpopulations, defined by CD5/CD19 coexpression, in peripheral blood of CLL patients (n = 60) subgrouped according to the IgHV mutational status (IgHV mut, n = 24; IgHV unmut, n = 36). CD5high subpopulation showed a high percentage of CXCR3 (P < 0.001), CCR10 (P = 0.001), and CD62L (P = 0.031) and high levels of CXCR5 (P = 0.005), CCR7 (P = 0.013) compared to CD5low cells expressing high CXCR4 (P < 0.001). Comparing IgHV mut and IgHV unmut patients, high levels of CXCR3 on CD5high and CD5low subpopulations were detected in the IgHV mut patients, with better discrimination in CD5low subpopulation. Levels of CXCR3 on CD5low subpopulation were associated with time to the next treatment, thus further confirming its prognostic value. Taken together, our analysis revealed higher CXCR3 expression on both CD5high and CD5low neoplastic cells in IgHV mut with a better prognosis compared to IgHV unmut patients. Contribution of CXCR3 to CLL pathophysiology and its suitability for prognostication and therapeutic exploitation deserves future investigations.

Dynamic changes in HLA-DR expression during short-term and long-term ibrutinib treatment in patients with chronic lymphocytic leukemia.

There is the first evidence of changes in the kinetics of B cell antigen receptor (BCR) internalisation of neoplastic cells in chronic lymphocytic leukemia (CLL) after the short-term and long-term administration of ibrutinib. We aimed to assess the influence of short-term and long-term ibrutinib treatment on the HLA-DR expression on CLL cells, T cells and monocytes. The immunophenotyping of CLL and immune cells in peripheral blood was performed on 16 high-risk CLL patients treated with ibrutinib. After early ibrutinib administration, the HLA-DR expression on CLL cells reduced (P = 0.032), accompanied by an increase in CLL cell counts in peripheral blood (P = 0.001). In vitro culturing of CLL cells with ibrutinib also revealed the reduction in the HLA-DR expression at protein and mRNA levels (P < 0.01). The decrease in HLA-DR on CLL cells after the first month was followed by the gradual increase of its expression by the 12th month (P = 0.001). A one-month follow-up resulted in elevated absolute counts of CD4+ (P = 0.002) and CD8+ (P < 0.001) T cells as well as CD4+ and CD8+ cells bearing HLA-DR (P < 0.01). The long-term administration of ibrutinib was associated with the increased numbers of CD4+ bearing HLA-DR (P = 0.006) and elevation of HLA-DR expression on all monocyte subsets (P ≤ 0.004). Our results provide the first evidence of the time-dependent immunomodulatory effect of ibrutinib on CLL and T cells and monocytes. The clinical consequences of time-dependent changes in HLA-DR expression in ibrutinib treated patients deserve further investigation.

Neutrophils in chronic lymphocytic leukemia are permanently activated and have functional defects.

A growing body of studies highlights involvement of neutrophils in cancer development and progression. Our aim was to assess the phenotypic and functional properties of circulating neutrophils from patients with chronic lymphocytic leukemia (CLL). The percentage of CD54+ and CD64+ neutrophils as well as CD54 expression on these cells were higher in CLL patients than in age-matched healthy controls. Neutrophils from CLL produced more reactive oxygen species (ROS) compared to controls in both resting and activated conditions. Lipopolysaccharide-induced production of IL-1ß and TNF-a as well as reduced TLR2 expression in neutrophils from CLL than in neutrophils from controls suggesting their tolerant state. Finally, phenotypic alterations of neutrophils, particularly elevation of CD64 and CD54 markers, correlated with disease activity and treatment, and low percentage of neutrophils. Taken together, the alterations in percentage and functional characteristics of neutrophils reflect the clinical course of CLL. Our data provide first evidence that neutrophils in CLL are permanently primed and have functional defects.

Immunoregulatory T cells in multiple sclerosis and the effect of interferon beta and glatiramer acetate treatment on T cell subpopulations.

INTRODUCTION: Multiple sclerosis (MS) is a chronic disease characterized by demyelination and chronic inflammation of the central nervous system (CNS). Many of the immune cells including T and B cells seem to be involved in disease pathogenesis by inducing or controlling the immune responses in the nervous system of MS patients. The objective of this study was to evaluate the differences in subpopulations of T cells between MS patients and healthy controls and the effects of interferon beta (INF-beta) and glatiramer acetate (GA) treatment on T cell subpopulations. MATERIAL AND METHODS: We have investigated the frequency of subpopulations of T cells using flow cytometry in 84 relapsing-remitting MS patients; forty-five patients started treatment with INF-beta and eighteen patients with GA, twenty-one patients were not treated. We collected blood samples at the beginning and after 6 and 12 months. RESULTS: We observed a significant decrease in CD4(+)CD25(+) Treg cells (p=0.03) and a significant increase in T helper cells (p=0.002) and central memory T cells (p=0.03) in MS patients compared to healthy controls. After INF-beta therapy, we demonstrated a significant increase in naive T cells (p=0.008), a decline in central memory T cells (p=0.01). After GA therapy, we observed a significant increase in naive T cells (p=0.04), a decrease in central memory T cells (p=0.03) and an increase in T-suppressor cells (p=0.008). CONCLUSION: In conclusion, we demonstrated the imbalance of T-cell subpopulations in MS patients and the potential benefit of DMD (disease modifying drugs) treatment on its restoration.