Towards a better characterisation of leukemic cells in chronic lymphocytic leukaemia: Cell-size heterogeneity reflects their activation status and migratory abilities

cancers Article Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities Gayane Manukyan 1,2, Zuzana Mikulkova 1, Peter Turcsanyi 3, Jakub Savara 1,4 , Markéta Trajerová 1, Zuzana Kubova 3, Tomas Papajik 3and Eva Kriegova 1,*   Citation: Manukyan, G.; Mikulkova, Z.; Turcsanyi, P.; Savara, J.; Trajerova, M.; Kubova, Z.; Papajik, T.; Kriegova, E. Towards a Better Characterisation of Leukemic Cells in Chronic Lymphocytic Leukaemia: Cell-Size Heterogeneity Reflects Their Activation Status and Migratory Abilities. Cancers 2021,13, 4922. https://doi.org/10.3390/ cancers13194922 Academic Editor: Mary Frances McMullin Received: 17 August 2021 Accepted: 27 September 2021 Published: 30 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Immunology, Faculty of Medicine and Dentistry, PalackýUniversity and University Hospital, 77900 Olomouc, Czech Republic; [email protected] (G.M.); [email protected] (Z.M.); [email protected] (J.S.); Marketa.Trajer[email protected] (M.T.) 2Laboratory of Molecular and Cellular Immunology, Institute of Molecular Biology NAS RA, Yerevan 0014, Armenia 3Department of Hematology-Oncology, Faculty of Medicine and Dentistry, PalackýUniversity and University Hospital, 77900 Olomouc, Czech Republic; peter[email protected] (P.T.); [email protected] (Z.K.); [email protected] (T.P.) 4Department of Computer Science, Faculty of Electrical Engineering and Computer Science, VSB-Technical University of Ostrava, 70800 Ostrava, Czech Republic *Correspondence: [email protected] Simple Summary: Chronic lymphocytic leukaemia (CLL) is a heterogeneous chronic disease characterised by the clonal expansion of mature CD19 + CD23 + CD5 + B-cells in blood, bone marrow and lymphoid tissue. Despite the CLL tumour cell population showing considerable heterogeneity in cell size, the functional characteristics of leukemic cells that differ in size have not been explored. The results of our study demonstrate that differences in CLL cell size reflect their activation state, polarisation and migratory capacity, with large CLL cells being more activated, polarised and motile than the small CLL cells present in the CLL cell pool. Our data provide evidence of the importance of cell-size heterogeneity within the CLL cell pool and the dynamics of cell-size changes for disease pathogenesis. Abstract: 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 CD5 low CXCR4 hi phenotype, while the l-CLL cells were characterised by the CD5 hi CXCR4 dim 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. Keywords: chronic lymphocytic leukaemia; cell-size heterogeneity; pool of leukemic cells; polarisation; migration Cancers 2021,13, 4922. https://doi.org/10.3390/cancers13194922 https://www.mdpi.com/journal/cancers Cancers 2021,13, 4922 2 of 17 1. Introduction Chronic lymphocytic leukaemia (CLL) is characterised by the clonal expansion of mature CD19 + CD23 + CD5 + B-cells in the blood, bone marrow and lymphoid tissue [ 1 ]. CLL cells are characterised by their phenotypic, clonal and functional heterogeneity and contain a pool of resting and proliferating CLL subpopulation cells [ 2 – 6 ]. The inverted expression of CD5 and CXCR4 has been indicated as useful for identifying enriched fractions among recently born/divided and elderly/quiescent CLL cells [ 2 , 3 , 7 ]. This means that the fraction of proliferating, recently born/divided migrating CLL cells has been shown to preferentially express high levels of CD5, while low levels of CD5 are associated with resting, elderly/quiescent circulating cells [ 3 , 8 ]. The overlapping B-cell receptor (BCR) repertoires between CD5 high and CD5 low cells suggest a dynamic relationship between these two B-CLL cell subpopulations [ 4 ]. Studies have shown that CXCR4 dim CD5 bright ‘proliferative’ cells overexpress more ‘cell division’ genes, while CXCR4 bright CD5 dim ‘resting’ cells express higher levels of ‘antiproliferative’ genes, suggesting that the latter subset may represent a distinct self-renewing type from which all clonal members are derived [3,8]. In addition to immunophenotypic heterogeneity, CLL cells differ in size and morphology. Typical CLL cells are mature small and medium-sized lymphocytes with clumped chromatin surrounded by a thin ring of cytoplasm [ 9 ]. A small number of studies that have analysed the clinical significance of atypical CLL morphology have revealed the adverse prognostic significance of increased prolymphocyte numbers and their association with markers of poor prognosis, thus predicting a shorter progression-free survival [ 9 , 10 ]. However, no information is available on the characteristics of typical CLL cells based on their size. This study aimed to analyse the biology of small (s-CLL) and large (l-CLL) CLL cells delineated by forward scatter (FSC) flow cytometry, with a primary focus on activation status, immunophenotype, proliferation, polarisation and migration. 2. Materials and Methods 2.1. Patients and Sampling Peripheral blood samples were collected from 23 patients with CLL (16 males and seven females; a median age of 65 years). A diagnosis of CLL was established according to the International Workshop on Chronic Lymphocytic Leukaemia guidelines [ 11 ]. Of the enrolled patients, eight were treatment-naïve, six had been pre-treated with immunochemotherapy and nine had been treated with novel agents (ibrutinib (IBR), idelalisib). The detailed clinical characteristics of the enrolled patients are shown in Table 1. An additional eight CLL patients were evaluated before the initiation of the IBR treatment and after 8 weeks of therapy. Table 1. Characteristics of the CLL patients. Parameters CLL Patients (n= 23) Age in years: median (min–max) 65 (51–84) Gender: male/female 16/7 White blood cell count (×109/L): median (min–max) 112.3 (26.7–498.3) Percentage of lymphocytes in the peripheral blood: median (min–max) 92.8 (75.7–98.8) Time from diagnosis to sampling (in months): median (min–max) 96 (6–200) Binet stage: A/B/C 6/5/12 Bulky lymphadenopathy ≥5cm: yes/no 5/18 Splenomegaly: yes/no 4/19 IgHV mutational status: unmutated/mutated 15/8 Cancers 2021,13, 4922 3 of 17 Table 1. Cont. Parameters CLL Patients (n= 23) Genetic characteristics: TP53 disruption (deletion 17p and/or TP53 mutations): yes/no/n.a. 6/13/4 del(11q22): yes/no/n.a. 7/15/1 del(13q14): yes/no/n.a. 3/18/2 Treatment: yes/no 15/8 Previous immunochemotherapy: 6 Time from the end of treatment to sampling (in months): median (min–max) 22 (4–68) Current treatment with iBCR (ibrutinib/idelalisib): 9 (8/1) Time from the start of iBCR treatment to sampling (in months): median (min–max) 3 (1–10) Legend: n.a. = not available; iBCR = B-cell receptor (BCR) signalling inhibitors. Next-generation sequencing was used for the detection of TP53 mutations, Sanger sequencing for IgHV mutational status and cytogenetics and FISH analysis for deletion 17p and other aberrations, as previously reported [12–14]. 2.2. Cell-Size Evaluation of CLL Cells The CLL cells of the patients were evaluated by peripheral blood smears after staining using the May–Grünwald–Giemsa protocol. The smears were evaluated with cellSens Entry (Olympus Life Science) imaging software and the CellaVision DM96 (Sysmex Corporation, Kobe, Japan) automated image analysis system at magnifications of 600 × and 1000×, respectively. 2.3. Surface Expression of Markers on Small and Large CLL Cells The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)- FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5,15]. Isotype-matched antibodies (BioLegend) were used as negative controls. The determination of s-CLL and l-CLL cells was conducted using FSC data and a backgating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI) . 2.4. Intracellular ZAP-70 Staining Whole blood cells were labelled with CD3-FITC, CD5-PerCP-Cy5.5 and CD19-APCCy7 antibodies. Thereafter, they were treated with fixing and permeabilising reagents (BioLegend) according to the manufacturer’s instructions, and they were then stained with the PE-conjugated ZAP-70 protein (clone 1E7.2, BioLegend). The quantity of ZAP70-positive CLL cells was estimated using isotype control and residual ZAP-70-positive T-cells (T method). Through the T method, ZAP-70 was positioned close to the left edge of the T-cell cluster in a ZAP-70/CD3 plot, which was applied to calculate the percentage of CLL-positive cells [16]. 2.5. Cell-Culture Experiments Peripheral blood mononuclear cells were isolated using density-gradient centrifugation. CLL cells were then further separated by fluorescence-activated cell sorting (FACS) using a FACSAria Fusion cell sorter (BD Biosciences), which is used for culturing, apoptosis, Cancers 2021,13, 4922 4 of 17 migration, polarisation and proliferation studies. CLL cell sorting resulted in purity > 95% for CD19 + CD5 + cells, as determined by flow cytometry. Sorted and paired s-CLL cells (the smallest 15% of the CLL cells in the CLL pool) and l-CLL cells (the largest 15% of the CLL cells in the CLL pool) were cultured in the absence or presence of CpG (3 µ g/mL) and CD40L (400 ng/mL) in a complete RPMI-1640 medium, which was supplemented with 10% heat-inactivated foetal bovine serum, 2mM of L-glutamine, 100U/mL of penicillin and 100µg/mL of streptomycin for 24 and 48 h. 2.6. Migration Analysis Transmigration of the CLL cells was assessed using polycarbonate Transwell inserts with a 5 µ m pore size (Corning Costar). Briefly, the s-CLL and l-CLL cells sorted at 1×106/mL each were placed in the upper chamber in the RPMI-1640 medium containing 1% bovine serum albumin (BSA). Inserts were placed into the lower chamber containing RPMI-1640 with 1% BSA in the presence or absence of 200 ng/mL of CXCL12 (BioLegend). After 3 h at 37 ◦ C in 5% CO 2 , the cells migrated into the lower chamber and were counted using a BD FACSCanto II instrument. The migration rate was calculated as the ratio of CXCL12-treated to CXCL12-untreated cells that transmigrated through the insert. 2.7. Proliferation Assay The proliferation rate was measured using a colorimetric cell cytotoxicity assay (Cell Cytotoxicity Assay Kit; Abcam); 0.5 × 10 5 sorted s-CLL and l-CLL cells were separately cultured with CpG and CD40L in duplicates in a 96-well plate. The ratio of OD 570 to OD 605 was used to determine cell viability. 2.8. Assessment of Apoptosis The apoptotic rate of the cultured s-CLL and l-CLL cells was assessed by analysing the binding of annexin V-FITC and the incorporation of propidium iodide (PI). Annexin V/PI double-negative cells were considered viable cells, and annexin V+/PI-cells were classified as apoptotic cells. 2.9. Fluorescent Microscopy A 1:1 mixture of sorted s-CLL and l-CLL cells in a liquid suspension was fluorescently stained for microscopy (similar to the protocol for immunofluorescence in the case of suspension cells provided by ThermoFisher). The cells were fixed with 4% paraformaldehyde for 10 min and permeabilised by 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 4 min . Unspecific binding was blocked with 1% BSA in PBS for 30 min. Then, the cells were incubated for 90 min with primary antibodies, i.e., mouse anti-human CD5 (1:300, clone CD5/54/F6, Abcam) and rabbit anti-human CXCR3 (1:300, polyclonal, Sigma-Aldrich). This was followed by incubation with secondary antibodies, i.e., goat pre-absorbed antimouse IgG (H&L, 1:800, Cy5, polyclonal, Abcam) and goat anti-rabbit IgG (H&L, 1:800, Alexa Fluor488, polyclonal, Abcam) for 60 min. The CLL cells were washed with PBS and centrifuged following the completion of all of the above steps. Finally, the cell pellet was mounted using Fluoromount-G mounting media with 4 0 ,6-diamidino-2-phenylindole (DAPI; Invitrogen) to label the nuclear DNA. Images were taken at a final magnification of 600 × using Gen5 software (v.3.08.01; BioTec Instruments, Winooski, VT, USA) and a BioTec Cytation 5MV (BioTec Instruments, Winooski, VT, USA) cell-imaging multi-mode reader. 2.10. Quantification of IFN-γand IgM in Cell-Culture Supernatants by Enzyme-Linked Immunosorbent Assay The interferon gamma (IFNγ ) levels were measured in the supernatants of cultured s-CLL and l-CLL cells using the MAX ™ Deluxe Set Human IFNγ enzyme-linked immunosorbent assay (ELISA; BioLegend). The total IgM levels were determined using a human IgM ELISA kit (Mabtech AB, Sweden). Cancers 2021,13, 4922 5 of 17 2.11. Time-Lapse Video Microscopy The s-CLL and l-CLL cells were separately treated with (i) CpG (3 µ g/mL) and cultured in a climate-controlled chamber (37 ◦ C and 5% CO 2 ) for 24 h or (ii) CXCL12 ( 200 ng/mL ) and placed on a heated (37 ◦ C and 5% CO 2 ) microscope stage. Cell polarisation and motility were monitored by the time-lapse video microscopy of cells over 60 min and recorded using Gen5 software and the BioTec Cytation 5MV reader at a final magnification of 200 × . The time-dependent changes in cell shape and motility were visually evaluated. 2.12. Statistical Analysis The difference between the groups was analysed using the Mann–Whitney test. A Wilcoxon signed-rank test was used to estimate the effect of inducers within theinvestigated groups, and correlations were assessed using Spearman’s correlation analysis. The results are expressed as mean ± standard error of the mean. All statistical analyses and the unsupervised principal component analysis (PCA) were performed using R software (www.r-project.org, version 4.1.1, accessed on 8 August 2021). 3. Results 3.1. The Expression of Surface and Intracellular Markers The size of typical CLL cells ranged from 6.0–9.5 µ m; their morphology and size distribution within the CLL cell pool are shown in Figure 1. Cancers 2021, 13, x FOR PEER REVIEW 5 of 16 2.11. Time-Lapse Video Microscopy The s-CLL and l-CLL cells were separately treated with (i) CpG (3 µg/mL) and cultured in a climate-controlled chamber (37 °C and 5% CO2) for 24 h or (ii) CXCL12 (200 ng/mL) and placed on a heated (37 °C and 5% CO2) microscope stage. Cell polarisation and motility were monitored by the time-lapse video microscopy of cells over 60 min and recorded using Gen5 software and the BioTec Cytation 5MV reader at a final magnification of 200×. The time-dependent changes in cell shape and motility were visually evaluated. 2.12. Statistical Analysis The difference between the groups was analysed using the Mann–Whitney test. A Wilcoxon signed-rank test was used to estimate the effect of inducers within the investigated groups, and correlations were assessed using Spearman’s correlation analysis. The results are expressed as mean ± standard error of the mean. All statistical analyses and the unsupervised principal component analysis (PCA) were performed using R software (www.r-project.org, version 4.1.1 accessed on 08/08/2021). 3. Results 3.1. The Expression of Surface and Intracellular Markers The size of typical CLL cells ranged from 6.0–9.5 µm; their morphology and size distribution within the CLL cell pool are shown in Figure 1. Figure 1. The morphological characteristics of CLL cells. (A) Peripheral blood smears from patients with CLL (magnification 600×). (B) The s-CLL and l-CLL cells from a pool of typical CLL cells with scant cytoplasm, small round nuclei with condensed chromatin and inconspicuous nucleoli (magnification 1000×). During smear preparation, CLL cells are easily disrupted, creating smudge cells (P1 and P3). (C) The distribution of CLL cell sizes within a CLL cell pool based on the forward-scatter (FSC) data using a back-gating strategy. (D) The size distribution of CLL cells in a particular patient. Representative examples are shown for three CLL patients (P1–P3). In this study, two CLL cell subpopulations with different cell sizes were selected based on the back-gating strategy (Figure 2). Each s-CLL and l-CLL cell population sorted represented ~15% of the CLL cell pool. Figure 1. The morphological characteristics of CLL cells. ( A ) Peripheral blood smears from patients with CLL (magnification 600 × ). ( B ) The s-CLL and l-CLL cells from a pool of typical CLL cells with scant cytoplasm, small round nuclei with condensed chromatin and inconspicuous nucleoli (magnification 1000 × ). During smear preparation, CLL cells are easily disrupted, creating smudge cells (P1 and P3). ( C ) The distribution of CLL cell sizes within a CLL cell pool based on the forwardscatter (FSC) data using a back-gating strategy. ( D ) The size distribution of CLL cells in a particular patient. Representative examples are shown for three CLL patients (P1–P3). Cancers 2021,13, 4922 6 of 17 In this study, two CLL cell subpopulations with different cell sizes were selected based on the back-gating strategy (Figure 2). Each s-CLL and l-CLL cell population sorted represented ~15% of the CLL cell pool. Cancers 2021, 13, x FOR PEER REVIEW 6 of 16 Figure 2. The cytometric analysis for the identification of CLL subpopulations with small and large cell sizes. (A) The back-gating strategy for defining s-CLL and l-CLL cells (each constituted ~15% of the CLL cell pool). (B) Dot plots showing the positioning of CD5high and CD5low cells (defined by CD5/CXCR4 markers) on the SSC/FSC plot. The immunophenotyping of both cell populations revealed that s-CLL cells exhibited low levels of CD5 expression (p < 0.001) and high levels of CXCR4 expression (p < 0.05) compared with l-CLL cells. Additionally, the percentage of CXCR3+, CD20+ and HLA-DR+ cells was found to be lower in the group of s-CLL cells than in the l-CLL group (Figure 3A). The percentage of CD38+ and CD49d+ cells did not differ between the studied groups. The intracellular labelling of the cells revealed a lower expression of ZAP-70 in the s-CLL cells than in the l-CLL cells (p < 0.01; Figure 3B). Figure 2. The cytometric analysis for the identification of CLL subpopulations with small and large cell sizes. ( A ) The back-gating strategy for defining s-CLL and l-CLL cells (each constituted ~15% of the CLL cell pool). ( B ) Dot plots showing the positioning of CD5high and CD5low cells (defined by CD5/CXCR4 markers) on the SSC/FSC plot. The immunophenotyping of both cell populations revealed that s-CLL cells exhibited low levels of CD5 expression (p< 0.001) and high levels of CXCR4 expression (p< 0.05) compared with l-CLL cells. Additionally, the percentage of CXCR3 + , CD20 + and HLA-DR + cells was found to be lower in the group of s-CLL cells than in the l-CLL group (Figure 3A). The percentage of CD38 + and CD49d + cells did not differ between the studied groups. The intracellular labelling of the cells revealed a lower expression of ZAP-70 in the s-CLL cells than in the l-CLL cells (p< 0.01; Figure 3B). Correlation analyses revealed a significant positive association of CD5 high and CD5 low cells with the percentages of CXCR3-positive cells, as indicated in our previous study [ 5 ]; this was also visualised by immunofluorescent staining (Figure 3C). The percentage of CLL cells in the patients’ blood negatively correlated with the percentage of s-CLL and l-CLL cells that tested positive for HLA-DR (r = − 0.59, p< 0.01 and r = − 0.48, p< 0.05, respectively) and CD20 MFI (r = − 0.55, p< 0.01 and r = − 0.56, p< 0.01 respectively). In the s-CLL cells, CD5 MFI positively correlated with CXCR3 percentage (r = 0.51, p< 0.05); in the l-CLL cells, CD5 MFI positively correlated with both CXCR3 percentage (r = 0.51, p< 0.05) and ZAP-70 MFI (r = 0.46, p< 0.05). Cancers 2021,13, 4922 7 of 17 Cancers 2021, 13, x FOR PEER REVIEW 7 of 16 Figure 3. The immunophenotype of s-CLL and l-CLL cells from all the blood samples of the CLL patients. (A) The surface expression levels of CD5 and CXCR4, and the percentage of positive cells for CXCR3, CD20, CD38 and HLA-DR. (B) The percentage of ZAP-70-positive cells, and representative dot plots of the intracellular expression levels of ZAP-70 in s-CLL and l-CLL cells. (C) The double-immunofluorescence staining of s-CLL and l-CLL cells for CXCR3 and CD5; DAPI was used for nuclear staining. Correlation analyses revealed a significant positive association of CD5high and CD5low cells with the percentages of CXCR3-positive cells, as indicated in our previous study [5]; this was also visualised by immunofluorescent staining (Figure 3C). The percentage of CLL cells in the patients’ blood negatively correlated with the percentage of s-CLL and lCLL cells that tested positive for HLA-DR (r = −0.59, p < 0.01 and r = −0.48, p < 0.05, respectively) and CD20 MFI (r = −0.55, p < 0.01 and r = −0.56, p < 0.01 respectively). In the s-CLL cells, CD5 MFI positively correlated with CXCR3 percentage (r = 0.51, p < 0.05); in the lCLL cells, CD5 MFI positively correlated with both CXCR3 percentage (r = 0.51, p < 0.05) and ZAP-70 MFI (r = 0.46, p < 0.05). To determine if the s-CLL and l-CLL subpopulations were distinct within the expression profiles, PCA was applied. The analysis results revealed the dense clustering of the two subpopulations with few dispersed cases; 66.7% of the cases were correctly classified in the s-CLL and l-CLL subsets. Of the studied markers, the CXCR3, CXCR4, CD20 and CD5 markers had the most consistent ability to correctly classify s-CLL and l-CLL cells (Figure 4). Figure 3. The immunophenotype of s-CLL and l-CLL cells from all the blood samples of the CLL patients. ( A ) The surface expression levels of CD5 and CXCR4, and the percentage of positive cells for CXCR3, CD20, CD38 and HLA-DR. ( B ) The percentage of ZAP-70-positive cells, and representative dot plots of the intracellular expression levels of ZAP-70 in s-CLL and l-CLL cells. ( C ) The double-immunofluorescence staining of s-CLL and l-CLL cells for CXCR3 and CD5; DAPI was used for nuclear staining. To determine if the s-CLL and l-CLL subpopulations were distinct within the expression profiles, PCA was applied. The analysis results revealed the dense clustering of the two subpopulations with few dispersed cases; 66.7% of the cases were correctly classified in the s-CLL and l-CLL subsets. Of the studied markers, the CXCR3, CXCR4, CD20 and CD5 markers had the most consistent ability to correctly classify s-CLL and l-CLL cells (Figure 4). Cancers 2021,13, 4922 8 of 17 Cancers 2021, 13, x FOR PEER REVIEW 8 of 16 Figure 4. The PCA analysis of surface marker expression in s-CLL and l-CLL cells. Each dot represents a score for the s-CLL (blue) and l-CLL (pink) cell populations derived from the studied CLL patients (n = 23). The model loadings are represented by vectors and indicate how each surface marker contributes to the cell variability in a specific direction. The percentage given is the explained variance per principal component of a patient. 3.2. The Effects of CpG and CD40L on the Phenotype of Small and Large Chronic Lymphocytic Leukaemia Cells The sensitivity of CLL cells to stimulation may reflect distinctive phenotypic and functional cell subsets. To illustrate the phenotyping features of cultured s-CLL and l-CLL cells, they were left untreated or stimulated with CpG and CD40L. The cultured s-CLL and l-CLL cells retained their expression patterns, which were observed at the basal level and responded to the inducers in a similar manner (Figure 5). Figure 4. The PCA analysis of surface marker expression in s-CLL and l-CLL cells. Each dot represents a score for the s-CLL (blue) and l-CLL (pink) cell populations derived from the studied CLL patients (n= 23). The model loadings are represented by vectors and indicate how each surface marker contributes to the cell variability in a specific direction. The percentage given is the explained variance per principal component of a patient. 3.2. The Effects of CpG and CD40L on the Phenotype of Small and Large Chronic Lymphocytic Leukaemia Cells The sensitivity of CLL cells to stimulation may reflect distinctive phenotypic and functional cell subsets. To illustrate the phenotyping features of cultured s-CLL and l-CLL cells, they were left untreated or stimulated with CpG and CD40L. The cultured s-CLL and l-CLL cells retained their expression patterns, which were observed at the basal level and responded to the inducers in a similar manner (Figure 5). Cancers 2021,13, 4922 9 of 17 Cancers 2021, 13, x FOR PEER REVIEW 9 of 16 Figure 5. The immunophenotypes of sorted s-CLL vs. l-CLL cells cultured for 24 and 48 h with CpG and CD40L; CXCR4 MFI and the percentage of CXCR3, CD20 and CD38 on cultured CLL cells (n = 11). * p < 0.05, ** p < 0.01, *** p < 0.001. In particular, an increased percentage of positive l-CLL cells for CXCR3 and CD38 was observed for all stimulated and unstimulated samples compared with s-CLL cells. An increased percentage of CD20 in l-CLL cells was observed only when the cells were left untreated or when they were treated with CD40L at both 24 and 48 h of cultivation. The differences in CXCR4 expression between s-CLL and l-CLL cells were less significant. In contrast to other markers, l-CLL cells, which were left untreated for 24 h, exhibited a decreased expression of CXCR4 (p < 0.05) compared with s-CLL cells. 3.3. The Migration Rate of Small and Large Chronic Lymphocytic Leukaemia Cells Next, we estimated the migration rate of the sorted s-CLL and l-CLL cells towards CXCL12. We observed that l-CLL cells migrated faster than s-CLL cells in this context (Figure 6A). Figure 5. The immunophenotypes of sorted s-CLL vs. l-CLL cells cultured for 24 and 48 h with CpG and CD40L; CXCR4 MFI and the percentage of CXCR3, CD20 and CD38 on cultured CLL cells (n= 11). * p< 0.05, ** p< 0.01, *** p< 0.001. In particular, an increased percentage of positive l-CLL cells for CXCR3 and CD38 was observed for all stimulated and unstimulated samples compared with s-CLL cells. An increased percentage of CD20 in l-CLL cells was observed only when the cells were left untreated or when they were treated with CD40L at both 24 and 48 h of cultivation. 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