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Haematologica. 2022 Dec; 102(12): 2069–2076.
CD40 signaling instructs chronic lymphocytic leukemia cells to attract monocytes via the CCR2 centrality
Martijn H.A. van Attekum
iSection of Hematology, Academic Medical Eye, Academy of Amsterdam; the Netherlands
iiDepartment of Experimental Immunology, Academic Medical Center, University of Amsterdam; kingdom of the netherlands
Jaco A.C. van Bruggen
1Department of Hematology, Academic Medical Center, University of Amsterdam; holland
2Department of Experimental Immunology, Bookish Medical Center, Academy of Amsterdam; the Netherlands
Erik Slinger
1Department of Hematology, Bookish Medical Center, Academy of Amsterdam; kingdom of the netherlands
twoDepartment of Experimental Immunology, Academic Medical Center, University of Amsterdam; the Netherlands
Thou. Cristina Lebre
iiDepartment of Experimental Immunology, Academic Medical Center, University of Amsterdam; the Netherlands
Emilie Reinen
1Section of Hematology, Bookish Medical Eye, University of Amsterdam; kingdom of the netherlands
twoSection of Experimental Immunology, Bookish Medical Center, University of Amsterdam; the Netherlands
Sabina Kersting
3Department of Hematology, Haga Educational activity Hospital, The Hague, holland
Eric Eldering
threeSection of Hematology, Haga Teaching Hospital, The Hague, the Netherlands
4Lymphoma and Myeloma Center Amsterdam (LYMMCARE), the Netherlands
Arnon P. Kater
1Department of Hematology, Academic Medical Heart, University of Amsterdam; the netherlands
4Lymphoma and Myeloma Center Amsterdam (LYMMCARE), the Netherlands
Received 2022 Sep 27; Accepted 2022 Sep 22.
- Supplementary Materials
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van Attekum et al. Graphical Abstract
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van Attekum et al. Supplementary Appendix
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Disclosures and Contributions
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Abstract
Chronic lymphocytic leukemia (CLL) cells are provided with essential survival and proliferative signals in the lymph node microenvironment. Here, CLL cells engage in various interactions with bystander cells such as T cells and macrophages. Phenotypically distinct types of tumor infiltrating macrophages tin either be tumor supportive (M2) or play a role in tumor immune surveillance (M1). Although recent in vitro findings suggest a protective part for macrophages in CLL, the actual residual between these macrophage subsets in CLL lymphoid tissue is nonetheless unclear. Furthermore, the machinery of recruitment of monocytes towards the CLL lymph node is currently unknown. Both questions are addressed in this paper. Immunofluorescence staining of lymph node samples showed macrophage skewing towards an M2 tumor-promoting phenotype. This polarization probable results from CLL-secreted soluble factors, every bit both patient serum and CLL-conditioned medium recapitulated the skewing effect. Because that CLL cell cytokine secretion is affected by adjacent T cells, we adjacent studied CLL-mediated monocyte recruitment in the presence or absence of T-cell signals. While unstimulated CLL cells were inactive, T cell-stimulated CLL cells actively recruited monocytes. This correlated with secretion of various chemokines such equally C-C-motif-ligand-2,3,4,five,7,24, C-X-C-motif-ligand-five,10, and Interleukin-10. Nosotros also identified CD40L equally the responsible T-jail cell factor that mediated recruitment, and showed that recruitment critically depended on the C-C-motif-chemokine-receptor-2 centrality. These studies show that the shaping of a tumor supportive microenvironment depends on cytokinome alterations (including C-C-motif-ligand-2) that occur after interactions between CLL, T cells and monocytes. Therefore, targeted inhibition of CD40L or C-C-motif-chemokine-receptor-ii may be relevant therapeutic options.
Introduction
Chronic lymphocytic leukemia (CLL) cells strongly depend on interactions with eyewitness T cells and monocyte-derived cells (MDCs) inside the lymph node (LN) microenvironment for their survival and resistance to therapy. 1 The role of LN-residing T cells in the pathogenesis of CLL has gained much attention. It is suggested that interaction of neoplastic B cells with T cells results in skewing of the T-prison cell compartment towards CD40L-expressing CD4+ T cells. 2 These T cells, in turn, induce both CLL cell survival and proliferation via upregulation of several pro-survival molecules as well as increased secretion of cytokines. 3,4 The interaction between MDCs and CLL is less well understood, although in vitro experiments bear witness that MDCs, in the form of Nurse-like cells, can induce CLL jail cell survival v through C-X-C motif chemokine 12, B-prison cell activating cistron and A proliferation-inducing ligand signaling. 5,6
Based on data from different malignancies, there are two subgroups of tumor-associated macrophages (TAMs): one) M2-like CD68+CD163+/CD206+ macrophages are characterized by an immunosuppressive phenotype, whereas 2 M1-similar CD68+CD80+ macrophages display an immunesurveilling phenotype. seven Although there is large intratumoral and intertumoral heterogeneity, it has been suggested that M1 TAMs pb to a better and M2 TAMs atomic number 82 to a worse prognosis beyond different tumor types. viii Tumors that are associated with M2 TAMs include breast, 9 ovarian, vii and prostate x cancers, whereas colon carcinoma TAMs are of M1 phenotype. 11
With respect to CLL, ex vivo evidence shows that MDCs are present in the LN, 12 and information technology was recently shown that MDCs contribute to CLL progression, every bit MDC depletion by clodronate treatment in the TCL1 CLL mouse model leads to slower CLL progression. 13,fourteen Whether LN-residing macrophages in homo CLL are indeed of a protective M2 phenotype has, however, non been directly studied. It is as well not known whether circulating monocytes tin actively be recruited towards the tumor-infiltrated LN.
Migration of CLL cells to the LN microenvironment depends on chemotactic gradients through the CXCL12/CXCR4, 15 CXCL13/CXCR5 16 and CCL19,21/CCR7 17 axes. Upon interaction with LN-residing cells, such as T cells, CLL cells can alter their secretome, four,18,19 which, in plough, could potentially affect both skewing and migration of other cells, like MDCs. Branch or reciprocal signals betwixt the triad formed past CLL cells, T cells, and MDCs could, therefore, critically contribute to the supportive microenvironment for CLL cells.
Here, we investigated both the possibly supportive differentiation of MDCs and their recruitment equally a issue of CLL-secreted cytokines in the context of T-cell signals. We constitute that CLL-secreted factors were able to differentiate macrophages towards a supporting M2 phenotype. Secondly, T jail cell/CD40 stimulation of CLL cells induced CLL cells to recruit monocytes; an action which critically depends on CCR2 signaling.
Methods
Patients' samples, stimulation and conditioned medium collection
Patient fabric was obtained from CLL patients, afterward written informed consent according to the guidelines of the Medical Ethical Commission of the Bookish Medical Center, Amsterdam, the Netherlands, in accordance with Annunciation of Helsinki protocols. For T-jail cell stimulation, peripheral claret mononuclear cells (PBMCs) were isolated from either healthy donors (HDs) or from CLL patients using Ficoll gradient purification according to the manufacturer's instructions (Lucron, Dieren, the Netherlands). These PBMCs (either magnetically sorted or not to enrichen the T-prison cell fraction) were added to CLL cells (in either an allogeneic or autologous fashion, as indicated) in a i:1 ratio, each at a concentration of 1.0*10vi cells/mL. Stimulating antibodies directed against CD3 (ane mg/mL, clone 1XE, Sanquin, Amsterdam, the Netherlands) and CD28 (iii μg/mL, clone 15E8, Sanquin) were added for T-jail cell activation. Subsequently 72 hours (h), conditioned medium was collected. For stimulation with CD40L, CLL cells were cultured at a concentration of 1.5*10half-dozen cells/mL on CD40L transfected NIH-3T3 cells or on mock transfected 3T3 cells as described previously, 3 all in IMDM supplemented with x% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin-100 μg/mL streptomycin (Life Technologies, Austin, TX, Usa), two mM L-glutamine (Life Technologies), and 0.00036% β-mercaptoethanol (Sigma, St. Louis, MO, USA) (IMDM+/+) for 16 h, after which conditioned medium was nerveless. Jail cell-free conditioned medium was kept at −80°C until use.
Migration assays
Conditioned or command media were diluted 1:ii in chemotaxis medium (PBS with 1% albumin, low endotoxin; Sigma). Monocytes were freshly isolated from HDs afterwards obtaining written informed consent using negative MACS depletion equally described previously xx and resuspended in chemotaxis medium. The diluted media were added in the lower chambers of a 5 μm chemotaxis assay plate (96 well ChemoTX®, NeuroProbe, Gaithersburg, MA, The states) and 100,000 monocytes were transferred to the upper chamber. Later on 2 h, chemotaxis was quantified by measuring the DAPI (iv,6 diamidino-2-phenylindole) signal of migrated monocytes as described previously. xx
When measuring inhibitor effects, both media and monocytes were incubated for 30 minutes (min) on ice with the indicated inhibitors direct prior to the migration assay. The following chemokine receptor inhibitors were used: one μg/mL CCR1 inhibitor BX471 (Sigma), 1 μg/mL CCR2 inhibitor INCB3284 (Tocris Bioscience, Bristol, Uk), 1μM CCR3 inhibitor SB328437 (Tocris), 1 μM CCR5 inhibitor Maraviroc (Apexbio, Houston, TX, Us), 1μM CXCR4/seven inhibitor Plerixafor (Apexbio), and 0.1 μg/mL IL-10 neutralizing antibody (R&D Systems, Minneapolis, MN, USA).
Measurement of chemokine levels
Previously generated microarray profiles 4 of purified (>99%) CLL cells stimulated for 16 h with activated T cells (deposited under accession number {"type":"entrez-geo","attrs":{"text":"GSE50572","term_id":"50572"}}GSE50572) were normalized and analyzed using the R2 platform (http://r2.amc.nl) and information were extracted using its DataGrabber characteristic. When testing protein secretion, conditioned media were analyzed for the indicated chemokines by Luminex using the ProcartaPlex 9-plex chemokine immunoassay kit extended with CCL7, CCL24, CXCL5, and IL-10 (eBioscience, San Diego, CA, United states) co-ordinate to the manufacturer's instructions.
Supplementary methods
Information on monocyte isolation and in vitro differentiation, LN material and immunofluorescence, rhCD40L stimulation and intracellular CCL2 measurements, and statistical analyses can exist establish in the Online Supplementary Methods.
Results
LN-residing macrophages display an M2 phenotype, and both CLL cells and CLL serum induce M2 skewing
To study the phenotype of macrophages in the CLL LN, paraffin-embedded LN sections from CLL patients were stained for the pan-macrophage marking CD68 in combination with either the M1 mark CD80 or the M2 marker CD206 using immunofluorescence. The CD80/CD206 fluorescence signal per macrophage (CD68+) was then quantified using an automated prison cell identification pipeline in CellProfiler. CD68 positive cells were present in all samples tested and were dispersed throughout the CLL-infiltrated LNs (Figure 1A and B). Inside the CD68+ cells, a higher CD206 intensity was observed as compared to CD80 (1.89 vs. one.00 arbitrary units) (Figure 1B and C, and Online Supplementary Effigy S1A).
Chronic lymphocytic leukemia (CLL) cells differentiate monocytes towards an M2 phenotype. (A) Alkane-embedded lymph node (LN) material from 9 CLL patients (for patients' characteristics see Online Supplementary Table S1) was stained past immunohistochemistry with Hematoxylin & Eosin (HE), CLL markers CD5 and CD20, and T-jail cell marker CD3. One representative slide is shown. Yellow scale bars stand for to 20 μm. More information on image acquisition can be constitute in the Methods section. (B) The same 9 samples every bit in (A) were stained by immunofluorescence for pan-macrophage marker CD68 in combination with either M1 marker CD80 (elevation) or M2 marking CD206 (bottom). One representative slide is shown; stainings of the other slides can be found in Online Supplementary Effigy S1A. Yellow scale confined correspond to 20 mm. (C) CD80 and CD206 intensity levels (both red signal) were quantified per macrophage (light-green bespeak) for each slide presented in Figure 1B and Online Supplementary Figure S1A using automated epitome analysis (see Methods section). Per-patient (each line) average macrophage intensity of both CD80 and CD206 are indicated (each dot). The patient presented in Figure 1B is indicated in blue. **P<0.01, paired t-tests. (D) A triple immunofluorescence staining with antibodies directed against T-prison cell marking CD3, CLL jail cell mark CD20, and macrophage marking CD68 was performed on four of the slides used for (1A). Ane representative slide is shown (sample CLL LN 09). Calibration bars correspond to 100 mm (top) or 10 mm (lesser). (E) Healthy donor (Hd) monocytes were differentiated for 72 hours (h) with IMDM containing 25% CLL serum or 25% pooled Hard disk serum, or with consummate medium containing IFN-Y (M1) or IL-4 (M2) as controls (left). In a separate experiment, monocytes were differentiated for 72 h by direct contact with CLL cells in complete medium, or with complete medium containing IFN-Y (M1), IL-iv (M2) or recombinant man (rh)NAMPT as controls (right). Monocyte differentiation was and so tested by staining for M1 marker CD80 and M2 markers CD163 and CD206 using flow cytometry. Each bar represents the relative geometrical hateful (GeoMean) of the fluorescence signal compared to the control condition and error confined signal Standard Error of Mean (SEM) of n=22 (left) or north=3 (right) CLL samples.
In order to visualize spatial organization of both T cells and macrophages within CLL LN, CD3– CD68–CD20 combinatory staining was performed. CD68+ cells could be found scattered throughout the LN, and were always surrounded past CLL cells. No typical configuration of CD3 in relation to CD68 could be detected, although, on occasion, CD68+ cells were in close contact with T cells (Figure 1D).
Nosotros next studied whether the leukemic cells could account for the observed M2 polarization. Outset, we tested whether soluble factors nowadays in CLL serum differentiated monocytes towards an M2 phenotype. Freshly isolated monocytes from HDs were incubated with either sera from 22 different CLL patients or pooled serum from HDs, and differentiation status was measured using flow cytometry. IFN-Y (M1) and IL-4 (M2) differentiated monocytes were included for comparison. Both M2 markers CD163 [mean relative Geomean ane.55±Standard Fault of Mean (SEM) 0.16] and CD206 (2.14±0.21), but not M1 marking CD80 (i.00±0.11) were increased in CLL serum-differentiated monocytes compared to Hard disk drive serum-differentiated monocytes (Figure 1E, left, and Online Supplementary Figure S1B for a representative gating strategy). Notably, no difference between serum from CLL samples from patients with either mutated or unmutated Immunoglubulin Heavy gene, or with low (<twenty*109) versus loftier (>100*ten9/L) leukocyte counts was observed (data not shown).
As CLL-serum components resulted in M2 differentiation, nosotros next investigated whether the observed M2 differentiation in the LN was actuated by CLL cells. To this end, HD-isolated monocytes were differentiated for 72 h using CLL cells or positive control NAMPT. 21 IFN-Y (M1) and IL-4 (M2) differentiated monocytes were again included equally control. We found an upregulation of M2 markers afterward IL-4 stimulation. In line with the differentiation effect of CLL serum, both CLL cells and NAMPT induced an upregulation of the M2 markers, merely not of the M1 marker (Effigy 1E, right). Furthermore, the M2 differentiation depended on soluble factors, as conditioned medium from CLL cells likewise induced M2 differentiation (Online Supplementary Figure S1C).
Taken together these information signal that CLL-secreted factors are able to differentiate macrophages towards an M2 phenotype.
T-cell-stimulated CLL cells secrete monocyte-attracting chemokines
Next, we investigated whether CLL cells could directly monocyte migration. Using trans-well migration assays, we found no migration of Hard disk drive monocytes towards supernatants of unstimulated CLL cells (Figure 2A). As both in vitro and ex vivo CLL LN studies strongly suggest active interaction of CLL cells with residential T cells, such as CD40L expressing follicular helper T cells within the LN 22 we hypothesized that such interaction could affect CLL cytokine secretion. Therefore, supernatants of CLL cells cultured in direct contact with HD PBMCs that included αCD3/αCD28 activated T cells (Tact) (Online Supplementary Figure S2A) were compared to unstimulated CLL cells for induction of monocyte recruitment. Indeed, conditioned medium from CLL cells co-cultured in contact with activated T cells induced migration of monocytes (Figure 2A). To exclude the possibility that migration resulted from a mixed-lymphocyte reaction, we verified that autologous T cells enriched from CLL samples induced like migration (Online Supplementary Figure S2B). In addition, we tested the migration effect of T cells without CLL cells and plant some migration induction of T cells just. This effect probable results from T-cell stimulation of B/CLL cells present in these samples due to contamination, equally this effect was reduced when T cells were magnetically enriched (Online Supplementary Effigy S2C). To determine the candidate chemokines expressed by stimulated CLL cells that could underlie the recruitment of monocytes, nosotros analyzed our previously generated microarray dataset ({"blazon":"entrez-geo","attrs":{"text":"GSE50572","term_id":"50572"}}GSE50572) of purified CLL cells that were stimulated with Tact. 4 Expression of several monocyte-attracting chemokines such every bit CCL2, 3, 4, 5, vii, CXCL1, 5, ten and IL-10 23–26 was up-regulated in CLL cells later on contact with Tact (Effigy 2B). To mensurate chemokine secretion past CLL cells, a Luminex assay was performed on three conditioned media used in Effigy 2A. All chemokines that were up-regulated on the mRNA level were also significantly upwardly-regulated on the protein level (Figure 2C).
T cell-stimulated chronic lymphocytic leukemia (CLL) cells secrete monocyte-attracting chemokines. (A) Freshly isolated good for you donor (HD) monocytes were seeded in the upper chambers of a trans-well migration plate to drift towards conditioned media (cond med) obtained from PBMC samples from CLL patients (for patients' characteristics meet Online Supplementary Tabular array S1) that were unstimulated (unstim) or stimulated (stim) for 72 hours (h) past contact with Hd PBMC T cells that were activated using α-CD3/α-CD28 antibodies. Side by side, the corporeality of migrated monocytes was quantified using DAPI staining. Each dot represents the relative (compared to the unstimulated CLL status) DAPI signals of eight different CLL conditioned media or 3 control media in 3 independent experiments using monocytes from 3 different donors and mean±Standard Error of Mean (SEM) are shown. All measurements were performed in triplicate. *P<0.05 in t-tests. (B) CLL cells were stimulated with α-CD3/αCD28 activated T cells or not stimulated for sixteen h. RNA from CD5/CD19 FACS sorted CLL cells (>99% purity) was subjected to microarray analysis and tested for differential expression of chemokines involved in monocyte migration. 23–26 Dots represent expression levels and mean±SEM are shown for 5 paired CLL samples. **P<0.01, ****P<0.0001 in a two-way ANOVA test with Bonferroni post hoc analysis. (C) Protein levels of chemokines involved in monocyte migration 23–26 were determined in three conditioned media that were used to perform the migration assays in (A) by using Luminex. Dots stand for protein levels and mean+SEM are shown for three CLL conditioned media; *P<0.05, ***P<0.001, ****P<0.0001 in a ii-way ANOVA test with Bonferroni post hoc assay.
CD40L-stimulated CLL cells attract monocytes as a effect of CCR2 axis signaling
As Thuman activity -stimulated CLL cells accept highly similar gene expression profiles compared to CD40L-stimulated CLL cells, 4 we investigated if CD40L stimulation similarly endows CLL cells with monocyte recruiting capacity. Comparable to the Tact results, supernatants from CD40L-stimulated CLL cells induced migration of monocytes (Figure 3A). These data point that a co-operative signal from Tact cells is needed for CLL cells to induce monocyte migration. Furthermore, CD40L appears to be responsible for the Tact -mediated monocyte migration. Notably, by using this T-cell free CD40L organisation, these data bespeak that CLL-derived (rather than Thuman action -derived) chemokines induce recruitment of monocytes. Secreted proteins in the conditioned media from CD40L-stimulated CLL cells were measured. In line with the Thuman activity information, several monocyte-attracting chemokines such every bit CCL2, 3, 4, 5, 7, 24, CXCL5, 10 and IL-ten were secreted by the CLL cells after CD40L stimulation (Effigy 3B). None of the chemokines tested were detectable in supernatant from CD40L overexpressing NIH-3T3 cells alone (data not shown).
CD40L-stimulated chronic lymphocytic leukemia (CLL) cells attract monocytes as a upshot of CCR2 axis signaling. (A) Freshly isolated healthy donor (Hard disk) monocytes were seeded in the upper chambers of a trans-well migration plate to drift towards conditioned media (cond med) obtained from PBMC samples from CLL patients (for patients' characteristics come across Online Supplementary Table S1) that were cultured for 16 hours (h) on CD40L-overexpressing (CD40L stim) or parental NIH-3T3 cells (unstim). Next, the corporeality of migrated monocytes was quantified using DAPI staining. Each dot represents the relative [compared to the unstimulated (unstim) CLL condition] DAPI signals of 12 different CLL conditioned media or iii control media in 3 contained experiments using monocytes from iii different donors and mean±Standard Error of Mean (SEM) are shown. All measurements were performed in triplicate. ****P<0.0001 in t-tests. (B) Protein levels of chemokines involved in monocyte migration 23–26 were determined in the conditioned media that were used to perform the migration assays in (A) by using Luminex. Dots correspond poly peptide levels and mean±SEM are shown for 12 CLL conditioned media; **P<0.01, ***P<0.001, ****P<0.0001 in a two-style ANOVA test with Bonferroni postal service hoc analysis. (C) Freshly isolated monocytes and conditioned media were pre-incubated for 30 minutes (min) with individual small-molecule inhibitors directed against indicated chemokine receptors, with an IL-10 neutralizing antibiotic, or a combination of all inhibitors (combi), before performing migration assays every bit in (A). Each dot represents the relative (compared to the unstimulated CLL condition) DAPI signals obtained in 4 contained experiments using monocytes from 3 different donors and different CLL supernatants; mean±SEM are shown. All measurements were performed in triplicate; *P<0.05, ***P<0.001, in a i-way ANOVA test with Bonferroni mail service hoc analysis. (D) Monocytes were seeded in the upper chambers of a trans-well migration plate to migrate towards migration medium without or with 10 ng/mL recombinant human being CCL2 (rhCCL2 low) or 100 ng/mL rhCCL2 (rhCCL2 high). Side by side, the amount of migrated monocytes was quantified using DAPI staining. Each dot represents the relative (compared to condition without rhCCL2) DAPI signals of nine split up read-outs in iii independent experiments using monocytes from three different donors and hateful±SEM are shown; **P<0.01, ****P<0.0001 in t-tests.
To pinpoint which of the up-regulated candidate chemokines was responsible for the migration of monocytes, we applied selective small-scale molecule inhibitors for the relevant chemokine receptors. 23–26 Inhibition of CCR2 was sufficient to reduce migration to a background level. At that place was no additive result of inhibition of other chemokine receptors, as a combination of the different receptor inhibitors yielded similar inhibition to CCR2 inhibition alone (Figure 3C). In a command experiment, no direct cytotoxic effect of the CCR2 inhibitor was detected after 72 h stimulation of CLL cells (Online Supplementary Figure S3A). Furthermore, supernatants from unstimulated CLL cells in combination with the different chemokine receptor inhibitors showed migration comparable to background (data non shown). We besides tested if the CCR2 inhibitor could revert CLL cell-induced M2 differentiation, as observed in Figure 1, only no event was plant (information not shown). As macrophage activation depends on Bruton Tyrosine Kinase, 27 we tested if migration could be reverted by inhibition via ibrutinib, but establish no effect of this inhibitor (data not shown).
Every bit CCL2 is a stiff CCR2 ligand, 28 we verified its induction in CD40L-stimulated CLL cells by using (cell free) recombinant CD40L (Online Supplementary Figure S3B). Furthermore, recombinant CCL2 resulted in monocyte migration (Effigy 3D). Combined, these data suggest that CD40 signaling is responsible for T cell-mediated monocyte migration by CLL cells and that this migration depends on the CCL2-CCR2 centrality.
Discussion
It is widely accustomed that interactions with local bystander cells in the LN are critical for CLL maintenance. 1 Various reports have mechanistically elucidated how bystander cells can support CLL cells, only the active role of CLL cells in shaping this supportive microenvironment is still largely unclear. In this complex interplay betwixt the leukemic and various types of surrounding cells, nosotros functionally addressed two fundamental aspects: the chemo-attraction of monocytes, and the crosstalk between CLL cells and activated T cells herein. Our findings are compatible with a model (Figure four) in which stimulation by CD40L on T cells in the LN induces CLL cells to secrete several monocyte-attracting chemokines. Of these, nosotros found CCL2 to be the nigh potent chemo-attractor, suggesting that this chemokine potentially plays an important office in vivo by recruiting monocytes towards the malignant cells in the LN via CCR2. The immunofluorescence information suggest that, following engagement with CLL cells in the LN, monocytes undergo skewing towards a tumor-supportive M2 phenotype (see also below).
Model of chronic lymphocytic leukemia (CLL) T-cell macrophage triad in the formation of a supportive tumor microenvironment. Stimulation of CLL cells by activated T-cell-produced CD40L (1) induces them to secrete CCL2 (2), which in turn recruits monocytes towards the lymph node (LN) (3). As a result of CLL-secreted factors, monocytes differentiate towards a tumor supporting M2 phenotype (four). Mo: monocyte; Atomic number 82: peripheral claret.
Several reports that studied migration of monocytes in the context of inflammation have concluded that chemo-attraction tin can occur via activation of several unlike chemokine receptor signaling pathways. 23–26 We hither identified CCR2 as the receptor nigh probable to be responsible for monocyte recruitment towards CLL cells. The most potent chemokine that recruits monocytes via the CCR2 receptor is CCL2, 28 which indeed recruited monocytes in our experiments (Effigy 3D). These data are in line with the recent observation that adoptive transfer of leukemic TCL1-derived splenocytes into recipient mice that are deficient for CCR2 resulted in significantly lower percentages and numbers of monocytes in the spleen. thirteen
Besides its importance in CLL, CCL2 has been shown to recruit monocytes towards master tumors in prostate cancer. Furthermore, this recruitment resulted in enhanced tumor growth. 29 CCR2 antagonist PF-04136309 reduced the number of monocytes and restored chemo-sensitivity in a pancreas tumor mouse model, indicating the therapeutic potential of CCL2/CCR2 inhibition. xxx Our studies advise that, also in CLL, these inhibitors can be a relevant therapeutic option, although additional in vivo studies are required.
In the low-cal of the large number of potential interactions in the CLL LN, it is worth noting that specifically the T-cell co-stimulatory signal CD40L leads to induction of monocyte trafficking. The levels of chemokines secreted by unstimulated CLL cells are insufficient to induce migration higher up background (Figures 2A and 3A). Although CLL cells stimulated by monocyte-derived Nurse-like cells evidence increased production of CCL3 and CCL 4,19 these cytokines apparently play a subordinate part in monocyte recruitment; despite their presence in the conditioned media (Figure 3B), monocyte migration is not prevented by blocking their cognate receptors CCR1 or −5 (Figure 3C). In dissimilarity to the monocyte-attracting upshot by CLL cells, information technology has been shown that bystander cells such as CD3+ or CD68+ cells are unable to produce CCL2 themselves. 31 We take previously shown that CD40L accounts for virtually of the transcriptional effects of T cells on CLL cells 4 and, based on our information, CD40L is sufficient to induce CCL2 production and monocyte recruitment. In this context, others have shown that another primal T-cell cytokine, IL21, is not essential for CCL2 consecration. 32
Our observation that the large bulk of macrophages in the CLL LN are of an M2 phenotype (Effigy 1B and C) strongly suggests initiation of M2 differentiating signaling events one time monocytes enter the CLL lymph node environment. Factors that can account for this differentiation include NAMPT 21 or High mobility group box 1 (HMGB-i) 12 secreted by LN-residing CLL cells. We could confirm that addition of NAMPT indeed skews monocytes towards an M2 blazon (Effigy 1E). In addition, T-helper-2 cells that also reside in the LN 22 secrete diverse cytokines that induce M2 differentiation, including IL-4, IL-x, and IL-13. Notably, the production of IL-x could be complemented by CLL cells that are stimulated by T cells (Figures 2C and 3B). Together these findings signal that the LN provides an M2-inducing milieu, which likely results in a supportive macrophage phenotype that can induce CLL jail cell survival and immune suppression.
Indeed, the clan of M2 differentiation and tumor support has been pointed out in several other tumor types. 8–xi Functionally, the tumor-promoting furnishings of M2 macrophages have been attributed to an increased production of direct tumor-promoting cytokines 33 and a suppression of the immune response. 21 M2 macrophages can, for example, induce a suppression of cytotoxic T cells, as they tin upwards-regulate expression of PD-ane on T cells. 21 In improver, they inhibit T-prison cell proliferation. 21 Lastly, M2 macrophages suppress T-cell activation and promote the differentiation towards Treg cells. 34 In the calorie-free of the recent evolution of T-prison cell therapy against CLL neoantigens, 35 the subversion of T cells by macrophages is an important point to accost.
In conclusion, our studies provide insight into several aspects of the circuitous interactions that take place in the CLL LN, and bespeak how the triad of CLL cell, T cell, and macrophage potentially contributes to the shaping of the tumor-microenvironment in CLL. Finally, we identified CCR2 as a potential therapeutic target to interrupt the intercellular interplay.
Supplementary Material
van Attekum et al. Graphical Abstract:
van Attekum et al. Supplementary Appendix:
Disclosures and Contributions:
Acknowledgments
The authors would like to thank all volunteers that donated blood for this study. Nosotros furthermore thank Richard Volckmann for his help with the microarray analysis and Steven Pals for providing us with the CLL LN slides.
Footnotes
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: world wide web.haematologica.org/content/102/12/2069
Funding
This work was supported past Dutch Cancer Foundation grant number UVA 2011-5097 (APK).
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709106/
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