CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9⁺ cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.     

Regulation of cancer stem cell properties by CD9 in human B-acute lymphoblastic leukemia

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9⁺ cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9⁻ cells. CD9⁺ cells were also serially transplantable in immunodeficient mice, indicating that CD9⁺ cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9⁺ population. Moreover, CD9⁺ cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.     

The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells

Background aimsCytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells.MethodsCIK cells were activated using IL-2 (CIK_IL-2) or IL-15 (CIK_IL-15) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay.ResultsCIK_IL-2 cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIK_IL-15 cells remained low. Further analysis of CIK_IL-15 cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIK_IL-15 cell-mediated cytotoxicity. In this context, CD3 ⁺ CD8 ⁺ CD25 ⁺ CD56^? CIK_IL-15 cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 ⁺ CIK_IL-15 cells, which showed the highest cytotoxic potential against ALL cells.ConclusionsThis study provides the first evidence that CIK_IL-15 cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT.     

IL-6 enhances the cytotoxic activity of thymocyte-derived CD56+ cells.

Thymocyte-derived lymphokine-activated killer (LAK) cells were used as a model for the study of the cytokine driven development of cytotoxicity. These cells are devoid of initial cytotoxic activity but upon culture in IL-2 they develop into cytotoxic effectors. The parameters of the response of thymocytes to IL-6 are similar to that of PBL in that IL-6, at concentrations as low as 1 mu/ml, increases cytotoxicity of thymocyte-LAK cells when generated in low doses (25-50 mu/ml) of IL-2. IL-6-enhanced thymocyte-LAK cytotoxicity is observed when tested against both NK-resistant and NK-sensitive tumor cell lines. IL-6 alone does not induce any cytotoxicity from thymocytes nor does IL-6 change the time course of thymocyte-LAK cell generation in IL-2 culture. IL-6 does not affect DNA synthesis, total cell number, proportion of CD56+ cells, or the expression of IL-2R (both P55 and P75 glycoproteins) in IL-2-cultured thymocytes. Instead, IL-6 used to treat mature thymocyte-LAK effector cells for as little as 1 hr prior to 51Cr-release assay increases LAK cytotoxicity. This enhancement is abrogated by pretreatment of effector cells with cycloheximide, suggesting that protein synthesis is required for IL-6 to enhance LAK cell activity. The precursor phenotypes of IL-6-responsive thymocyte-LAK cells are CD3-/CD5-. The effector phenotypes of IL-6-enhanced thymocyte-LAK cells are CD5-/CD56+. Thus, IL-6 depends on synthesis of rapid-turnover proteins to act on mature CD56+/CD5- LAK cells to increase their cytotoxic function.     

Restoration of CD3+CD56+ cell level improves skin lesions in severe psoriasis: A pilot clinical study of adoptive immunotherapy for patients with psoriasis using autologous cytokine-induced killer cells

Psoriasis is a chronic inflammatory skin disorder mediated by the cells and molecules of both the innate and adaptive immune systems. Autologous cytokine-induced killer (CIK) cell infusion is considered an effective and safe cancer treatment and is licensed for this use in China. Accumulated evidence indicating that CD3⁺CD56⁺ cells are significantly decreased in psoriatic patients prompted us to investigate if the restoration of CD3⁺CD56⁺ cells may be beneficial for psoriatic patients. We designed a clinical trial for psoriasis treatment that involved CIK cell infusion because CIK cells include a large amount of CD3⁺CD56⁺ T cells (NCT01894373 at www.clinicaltrials.gov). Six patients with severe psoriasis were initially enrolled, and four of them exhibited markedly lower levels of CD3⁺CD56⁺ cells in their peripheral blood (PB) relative to healthy donors. CIK cell infusion-associated toxicity was not observed in any infusion. The percentage of CD3⁺CD56⁺ cells in the PB markedly increased and the psoriasis area and severity index (PASI) synchronously decreased in four patients with lower CD3⁺CD56⁺ cell contents, and two of them obtained a more than 4-month PASI75 after completing a four-cycle treatment. However, a decrease in the CD3⁺CD56⁺ cells was observed concomitantly with disease recurrence after short-term amelioration. In contrast, no obvious improvement was observed in the two patients with nearly normal CD3⁺CD56⁺ cells in the PB before treatment. These observations suggest that the normalization of the CD3⁺CD56⁺ cell level may improve the skin lesions of severe psoriasis and warrant further clinical trials for severe psoriasis using repeated CIK adoptive immunotherapy.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

Human blood-derived fibrin releasates: Composition and use for the culture of cell lines and human primary cells

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-β1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1-10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.     

Expression of CD16, CD18 and CD56 in tributyltin-exposed human natural killer cells

Tributyltin (TBT) was produced in large quantities for use in wood preservation, marine antifouling paints, disinfection of circulating industrial cooling waters and slime control in paper mills. TBT is found in dairy products, meat and fish. We and others have shown that there are measurable levels of TBT in human blood. BTs appear to increase the risk of cancer and viral infections in exposed individuals. In previous studies, we demonstrated that the NK-cytotoxic function of lymphocytes was greatly diminished after a 1-h exposure to 300 nM TBT or a 24-h exposure to 200 nM TBT. Inhibition induced by a 1-h exposure to 300 nM TBT continues even after removal of the compound. There is also decreased ability of NK cells to bind to tumor target cells when they have been exposed to 200 nM TBT for 24 h. This loss of binding function is not seen when NK cells are exposed to 300 nM TBT for 1 h. However, NK cells exposed to 300 nM TBT for 1 h and then incubated in TBT-free media for 24, 48 or 96 h, show a significant loss of tumor-binding function by 96 h. The effects of TBT on cell surface molecules that are crucial to NK cell function is investigated. The data indicate there is a loss of expression of CD16 and CD56 on NK cells exposed to 200 nM TBT for 24 h. There is no decrease in expression of any of the markers studied when NK cells are exposed to 300 nM TBT for 1 h, consistent with the fact that a 1-h exposure has no effect on the ability of NK cells to bind to tumor targets. However, when NK cells are exposed to 300 nM TBT followed by 24, 48 or 96 h incubations in TBT-free media, there is a significant loss of CD16 and CD18 expression after 24 h and of CD16 and CD56 expression after 48 and 96 h.     

Dendritic cells reduce number and function of CD4+CD25+ cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma

Aim and background: CD4⁺CD25⁺ cells are described as professional regulatory/suppressor T cells that are crucial for the prevention of spontaneous autoimmune diseases. They play an important role in maintaining a balanced peripheral immune system. On the other hand, it has been suggested that regulatory T cells (Treg) suppress antitumor immune responses after tumor-specific vaccinations. Therefore, we determined the percentage of regulatory T cells in cytokine-induced killer (CIK) cells, an effector cell population with high impact for adoptive immunotherapeutic strategies. Results: CIK cells showed strong induction of CD4⁺CD25⁺ cells with high secretion of interleukin 10 (IL-10) after unspecific stimulation of the TCR complex and stimulation with interleukin 2. Depletion of CD25⁺ cells led to an increase in cytotoxic activity and a reduction of IL-10 release. A more pronounced reversal of suppression could be induced by coculture of CIK cells with dendritic cells (DCs). After coculture of CIK cells with DCs, the number of CD4⁺CD25⁺ cells as well as the IL-10 concentration in the supernatant decreased, and the cytotoxic activity against pancreatic carcinoma cells increased. This was shown for cells from healthy donors as well as for cells from patients with pancreatic carcinoma. Conclusion: Our established effector cells possess some regulatory features induced by unspecific TCR-activation that could be prevented by coculture with DCs. CIK cells have desirable properties for immunotherapeutical approaches, especially after coculture with DCs, which could be used additionally for induction of a specific immune response.     

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.     

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.     

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.     

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.