Regulatory T cells‐derived IL‐35 promotes the growth of adult acute myeloid leukemia blasts

Tumor immune escape mechanism mediated by CD4+CD25+regulatory T cells (Tregs) is a key factor in the pathogenesis of acute myeloid leukemia (AML). IL‐35, as a novel inhibitory cytokine, is produced by Tregs specially and regulates functions of Tregs in murine. However, IL‐35 expression of Tregs in human is still disputed, and its role in AML is yet to be elucidated. In this study, we found that IL‐35 was expressed highly in peripheral blood plasma of adult patients with AML and significantly correlated with the clinical stages of malignancy. Tregs‐derived from adult AML patients produced IL‐35 in a stimulation‐dependent manner. IL‐35 promoted AML blasts immune escape by expanding Tregs and inhibiting CD4+CD25‐effector T cells (Teffs). Furthermore, IL‐35 directly promoted the proliferation of AML blasts and reduced the apoptosis of AML blasts. Together, our study demonstrates that IL‐35‐derived from Tregs promotes the growth of adult AML blasts, suggesting that IL‐35 has an important role in the pathogenesis of AML.     

The side population,as a precursor of Hodgkin and Reed‐Sternberg cells and a target for nuclear factor‐κB inhibitors in Hodgkin’s lymphoma

Although disturbed cytokinesis of mononuclear Hodgkin (H) cells is thought to generate Reed‐Sternberg (RS) cells, differentiation of Hodgkin’s lymphoma (HL) cells is not fully understood. Recent studies indicate that cells found in a side population (SP) share characteristics of cancer stem cells. In this study we identified an SP in the HL cell lines, KMH2 and L428. This SP almost entirely consists of distinct small mononuclear cells, whereas the non‐SP is a mixture of relatively large cells with H or RS cell‐like morphology. Culture of the small mononuclear cells in the SP from KMH2 generated a non‐SP. Single cell culture of the SP cells generated large cells with H or RS cell‐like morphology. We found that CD30 overexpression and constitutive nuclear factor‐κB (NF‐κB) activity, both of which are characteristics of HL cells, are shared between the SP and non‐SP cells for both KMH2 and L428. Inhibition of NF‐κB induced apoptosis in both fractions, whereas the SP cells were resistant to a conventional chemotherapeutic agent doxorubicin. The results show that HL cell lines contain an SP, that is enriched for distinct small mononuclear cells and generates larger cells with H and RS cell‐like morphology. The results also stress the significance of NF‐κB inhibition for eradication of HL cells. (Cancer Sci 2010; 101: 2490–2496)     

Expression of CCR5 receptors on Reed-Sternberg cells and Hodgkin lymphoma cell lines: involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions

The expression of CCL5/Rantes by Hodgkin (H) and Reed-Sternberg (RS) cells has been recently documented. In the present study we demonstrated that the CCL5 receptor (CCR5) is constitutively expressed by Hodgkin Lymphoma (HL)-derived cell lines (i.e. L-428, KM-H2, L-1236 and L-540) as shown by immunohistochemistry, flow cytometry and western blotting and also detected by immunohistochemistry on primary H-RS cells from lymph node tissues. sCD40L never significantly affected CCR5 expression, whereas a short exposure to doxorubicin down regulated its expression. CCR5 receptors on HL cell lines were functionally active, since neutralizing anti-CCL5 monoclonal antibodies inhibited basal proliferation of HL-derived cell lines and recombinant CCR5 ligands (CCL3/Mip-1 alpha, CCL4/Mip1 beta and CCL5/Rantes) increased their clonogenic growth. CCL5 secretion by L-1236, L-428 and KM-H2 cells was stimulated by CD40 engagement and also by coculturing L-1236 cells on primary stromal fibroblasts from HL-involved lymph nodes (HLF). Coculture experiments indicated that a direct contact of H-RS cells induces HLF cells to produce CCL5. Supernatants from L-1236, L-428 and KM-H2 cells stimulated migration of purified CD4+ T-cells and eosinophils in vitro. The migratory response to HL-cell lines supernatants was only partially neutralized (CD4+ cells: 70%; esinophils: 36%) by anti-CCL5 antibodies, reinforcing the notion that multiple chemokines are involved in the recruitment of nonmalignant reactive cells in HL tissues. Taken together, our results indicate a possible involvement of the CCR5/CCR5-ligands signaling in the regulation of H-RS cells growth and in the formation/maintenance of the typical tissue microenvironment of HL.     

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin''s disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration.     

The role of interleukin-3 in classical Hodgkin's disease

Classical Hodgkin's disease (HD) is a peculiar form of lymphoma characterized by a low frequency of tumor cells, the so-called Hodgkin (H) and Reed/Sternberg (RS) cells, embedded in a background of non-neoplastic (reactive) cells believed to be recruited and activated by H-RS cell-derived cytokines/chemokines. How these tumor cells can survive in such a seemingly hostile environment has confused researchers. We have previously identified interleukin (IL)-3 receptor (R) expression as a common feature of classical HD and unveiled the potential role of IL-3 as a growth and anti-apoptotic factor for H-RS cells. More then 90% of malignant cells of classical HD usually express the alpha chain of the IL-3R (IL-3R(alpha)), as evidenced by immunostaining of frozen sections and cell suspensions from neoplastic lymph nodes. Consistently, HD-derived cell lines (L428, KMH2, HDLM2 and L1236) express the alpha and beta chains that form IL-3R, both at the mRNA and protein level, with a molecular size of IL-3R(alpha) identical (70 kDa) to that expressed by human myeloid cells. Exogenous IL-3 promotes the growth of cultured H-RS cells, such an effect being potentiated by IL-9 and stem cell factor (SCF) co-stimulation, and is able to partially rescue tumor cells from apoptosis induced by serum deprivation. Finally, cultured H-RS cells are able to increase the production of IL-3 by pre-activated T cells, suggesting an involvement of IL-3/IL-3R interactions in the cellular growth of HD through paracrine mechanisms. This review will outline the biological activity of IL-3 and summarize the evidence indicating IL-3 as a growth and anti-apoptotic factor for H-RS cells in classical HD.     

Expression of CD40 ligand (CD154) in B and T lymphocytes of Hodgkin disease: potential therapeutic significance

BACKGROUND: The malignant Hodgkin and Reed-Sternberg (H/RS) cells of Hodgkin disease (HD) express CD30 and CD40 receptors that can activate nuclear factor kappa B and transduce survival signals. The authors have reported previously that the B lymphocytes of HD express CD30 ligand (CD30L, CD153). Furthermore, they and others have reported previously that the CD40L survival pathway is augmented in patients with B-cell malignancies, as CD40L was constitutively expressed by the malignant B cells and infiltrating T cells, and sera from those patients contained elevated levels of soluble CD40L. In this study, the authors investigated the hypothesis that the survival of H/RS cells was similarly promoted by an augmented CD40L signals in HD patients. METHODS: The expression of CD40L on lymphocyte subsets of patients with classic HD was determined by two-color fluorescent-activated cell sorter analysis. Serum soluble CD40L levels were determined by enzyme linked immunosorbent assay. RESULTS: CD40L was constitutively expressed on both the T and B cells of HD patients but was more prominently expressed on the B lymphocytes. Soluble CD40L was detected in the serum of 17 of 37 patients (45%) and was higher than 1 ng/mL in 4 patients (10%). Both interleukin (IL)-4 and IL-10, which are known to be secreted by H/RS cells and surrounding T cells, up-regulated CD40L expression on normal B cells. CONCLUSIONS: Thus, the expression of CD40L and CD30L on the B cells of HD patients suggests that B lymphocytes may play a role in the regulation of H/RS cell growth in vivo. Depriving H/RS cells from CD30L and CD40L survival signals by eliminating B cells from HD lesions may be of therapeutic value.     

Transactivation of the ICAM-1 gene by CD30 in Hodgkin's lymphoma

The ICAM-1/LFA-1 complex mediates cell-cell interaction. ICAM-1 is overexpressed in Hodgkin/Reed-Sternberg (H/RS) cells, and serum levels of its soluble form are higher in Hodgkin's lymphoma (HL) patients than in controls. There are no data, however, regarding the regulation of expression of ICAM-1 in H/RS cells. CD30 was identified in H/RS cells of HL and has attracted much interest as a molecular marker of HL. To analyze ICAM-1 expression in H/RS cells, we examined the expression of ICAM-1, LFA-1, CD30 and CD30L in HL-derived cell lines. All cell lines expressed ICAM-1 and CD30, but not LFA-1 or CD30L. CD30 induced ICAM-1 expression. Analysis of the ICAM-1 promoter showed the importance of NF-kappaB binding site for CD30-induced ICAM-1 gene expression. Coexpression of IkappaB, IKK, NIK and TRAF dominant-negative constructs with CD30 inhibited CD30-induced activation of ICAM-1 promoter, suggesting that CD30 induces ICAM-1 via NF-kappaB signalling. The ICAM-1 promoter was activated by the C-terminal region of CD30, which activated NF-kappaB signalling. A decoy CD30 lacking the cytoplasmic region inhibited ICAM-1 promoter activity in HL cell lines. Thus, in H/RS cells, ligand-independent activation of CD30 signalling activates NF-kappaB and this leads to constitutive ICAM-1 expression, suggesting a link between 2 well known phenotypic characteristics of HL, CD30 and ICAM-1 overexpression.     

Up‐regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration‐resistant prostate cancer

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor‐induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome‐wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration‐resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin‐2 (IL‐2) and transforming growth factor‐β (TGF‐β) pathways. Studies revealed that the levels of expression of genes responsible for T‐cell proliferation (C‐FOS, C‐JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4⁺CD25^highCD127^– Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.     

Therapy‐induced antitumor vaccination in neuroblastomas by the combined targeting of IL‐2 and TNFα

L19‐IL2 and L19TNFα are fusion proteins composed of L19(scFv), specific for the angiogenesis‐associated ED‐B containing fibronectin isoform and IL‐2 or TNFα. Because of the tumor targeting properties of L19, IL‐2 and TNFα concentrate at therapeutic doses at the tumor vascular level. To evaluate the therapeutic effects of L19‐IL2 and L19mTNFα in neuroblastoma (NB)‐bearing mice, A/J mice bearing Neuro2A or NIE115 NB were systemically treated with L19‐IL2 and L19mTNFα, alone or in combination protocols. Seventy percent of Neuro2A‐ and 30% of NIE115‐bearing mice were cured by the combined treatment with L19‐IL2 and L19mTNFα, and further rejected a homologous tumor challenge, indicating specific antitumor immune memory. The immunological bases of tumor cure and rejection were studied. A highly efficient priming of CD4⁺ T helper cells and CD8⁺ CTL effectors was generated, paralleled by massive infiltration in the tumor tissue of CD4⁺ and CD8⁺ T cells at day 16 after tumor cell implantation, when, after therapy, tumor volume was drastically reduced and tumor necrosis reached about 80%. The curative treatment resulted in a long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2 type of response. Concluding, L19‐IL2 and L19mTNFα efficiently cooperate in determining a high percentage of NB cure that, in our experimental models, is strongly associated to the generation of adaptive immunity involving CD4⁺ and CD8⁺ T cells.     

Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern

To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model. We used a novel designer peptide based self-organizing matrix. The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples. To validate our results we also included a gene expression data set of laser captured Hodgkin-Reed - Sternberg (H-RS) cells. The gene expression profiles were analyzed using Affymetrix technology. We found that the 3D culture affected gene expression of a HL derived cell-line inducing a more tumor-related expression profile. 3D culture affected the expression of 500 genes in L1236, upregulating genes involved in immune response and apoptosis and downregulating genes involved in cell division. It also affected genes involved in actin filament polymerization.     

IL‐8 induces exocytosis of arginase 1 by neutrophil polymorphonuclears in nonsmall cell lung cancer

Arginase 1 (ARG1) inhibits T‐cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs‐related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor‐infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor‐infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14? cell samples from NSCLC expressed interleukin (IL)‐8 mRNA, whereas TNFα mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL‐8, albeit at different levels. IL‐8 was as effective as TNFα in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL‐8 gene‐silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL‐8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL‐8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression. © 2009 UICC     

Interleukin 4 production by peripheral blood lymphocytes in patients with classical Hodgkin lymphoma

Although the production of interleukin (IL) 2 and interferon (IFN) gamma by peripheral blood lymphocytes in patients with Hodgkin lymphoma (HL) is well documented, the synthesis of IL4 has not been investigated before. The present study examines the production of IL4 by 2-day phytohaemaglutinin (PHA)-stimulated peripheral blood (PB) cells in HL and correlates the cytokine levels with the proportion of the different T-cell sub-populations. We observed a significant increase in the mean level of production of IL4 in patients with HL when compared with normal controls. The increased amount of IL4 in patients with HL correlated significantly with the proportion of the CD3(+)CD8(+) cells but not with CD3(+)CD4(+). The intensity of cytoplasmic IL4 (expressed as relative median fluorescence (RMF)) was significantly higher in the CD3(+)CD8(+) cells of the patients with HL compared with the CD3(+)CD4(+) sub-population, or with the normal CD3(+)CD8(+) cells and correlated with the levels of IL4 release in culture supernatants. In conclusion, there is increased production of IL4 by PHA-activated PB lymphocytes in HL. The CD3(+)CD8(+) T-cell population appears to be responsible for this increased synthesis.     

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.     

Proficient mismatch repair protein expression in Hodgkin and Reed Sternberg cells.

Hodgkin and Reed-Sternberg (H/RS) cells are characterized by chromosomal instability. Nevertheless, neither specific nor consistent chromosomal alterations could be characterized in H/RS cells. Microsatellite instability (MSI) is another form of genomic instability but its role in the pathogenesis of classical Hodgkin's disease (cHD) has not been investigated so far. We analyzed MSI and mismatch repair (MMR) protein expression in H/RS cells of cHD in order to assess genomic instability in these cells. Using a sensitive single cell approach, MSI-low was detected in a portion of single cells of the H/RS cell line L1236. Mutations of genes encoding for hMSH2 and hMLH1 were excluded by RT-PCR in L1236 cells. An analysis of pooled single H/RS cells of seven primary cases of cHD showed loss of heterozygosity for some allelic markers but absence of MSI in all 7 cases. Owing to a tight correlation between MSI-high, inactivating mutations of MMR genes and MMR protein expression in colon cancer, MMR protein expression commonly is used as a marker for MSI. In order to screen additional primary cases of cHD for MSI, we performed immunohistochemistry for hMSH2 and hMLH1 in 6 of the 7 cases analyzed by single cell PCR and 20 additional cases of cHD. H/RS cells from 25 out of 26 cases showed a nuclear staining pattern for hMSH2 and hMLH1 similar to germinal center B cells of non-malignant lymph nodes. These results indicate a proficient MMR system in most H/RS cells. It is concluded that a defect MMR system is unlikely to contribute to the malignant phenotype and genomic instability of H/RS cells in cHD.     

Cervical cancer cell‐derived interleukin‐6 impairs CCR7‐dependent migration of MMP‐9‐expressing dendritic cells

Cervical carcinogenesis is a consequence of persistent infection with high‐risk human papillomaviruses (HPVs). Recent studies indicate that HPV‐transformed cells actively instruct their microenvironment to promote carcinogenesis. Here, we demonstrate that cervical cancer cells activate monocytes to produce their own CCL2 for further monocyte recruitment and reprogram their function during differentiation and maturation to dendritic cells (DCs). Our data show that cervical cancer cells suppress the induction of the chemokine receptor CCR7 in phenotypically mature DCs and impair their migration toward a lymph node homing chemokine, required to initiate adaptive immune responses. We confirmed the presence of CD83⁺CCR7^low DCs in cancer biopsies. The second factor essential for DC migration, matrix‐metalloproteinase MMP‐9, which also has vasculogenic and protumorigenic properties, is not suppressed but upregulated in immature as well as mature DCs. We identified interleukin‐6 (IL‐6) as a crucial cervical cancer cell‐derived mediator and nuclear factor kappaB (NF‐κB) as the central signaling pathway targeted in DCs. Anti‐IL‐6 antibodies reverted not only NF‐κB inhibition and restored CCR7‐dependent migration but also blocked MMP‐9 induction. This is the first report demonstrating the dissociation of CCR7 and MMP‐9 expression in phenotypically mature CD83⁺ DCs by cancer cells. Our results show that cervical cancer cells actively shape the local microenvironment. They induce the accumulation of myeloid cells and skew their function from immune activation to local production of protumorigenic MMP‐9. Neutralizing anti‐IL‐6 antibodies can counteract this functional dysbalance and should therefore be considered for adjuvant cervical cancer therapy.     

Reduction of Foxp3+ T cell subsets involved in incidence of chronic graft‐versus‐host disease after allogeneic hematopoietic stem cell transplantation

Foxp3+ T cells (CD4+ Tregs and CD8+ Treg) have been demonstrated to play roles in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (Allo‐HSCT). We have found that Foxp3+ γδTCR+ Treg cells (γδTregs) exerted regulatory functions. In the current study, patients were recruited and divided as non‐cGVHD, limited cGVHD and extensive cGVHD groups. Healthy volunteers were recruited as healthy group. Treg cells were evaluated by flow cytometry. Serum cytokine levels of IL‐2, tumour necrosis factor‐α, interferon‐γ and transforming growth factor‐β1 (TGF‐β1) were evaluated by ELISA. The results showed that percentages of CD4+ Tregs, CD8+ Tregs and γδTregs were all significantly increased in non‐cGVHD group compared with those in healthy group, limited cGVHD group and extensive cGVHD group. Moreover, compared with extensive cGVHD group, percentages of these three types of Tregs were significantly increased in limited cGVHD group. The levels of TGF‐β1 increased dramatically in non‐cGVHD group compared with other groups. Spearman's correlation analysis revealed that the increased levels of TGF‐β1 and IL‐2 were positively associated with increased Treg subsets, indicating that TGF‐β1 and IL‐2 participated in the expansion process of Foxp3+ Tregs in vivo. Our findings support that increasing the number of Tregs following allo‐HSCT would be a preferential strategy for controlling cGVHD. Copyright © 2015 John Wiley & Sons, Ltd.