Extramedullary hematopoiesis: mesenchymal stromal cells from spleen provide an in vitro niche for myelopoiesis.

Murine spleen has been shown to harbour stromal cells that support hematopoiesis with production of myeloid antigen-presenting cells. Similar stromal lines have now been isolated from long-term cultures (LTC) of human spleen. When human progenitor populations from spleen, bone marrow and cord blood were employed as a source of progenitors for co-culture above splenic stromal lines, myelopoiesis was supported. Human splenocytes gave production of predominantly myeloid dendritic-like cells, with minor subsets resembling conventional dendritic cells (cDC) cells, and myeloid or monocyte-derived DC. Human bone marrow progenitors gave rise to myelopoiesis from hematopoietic progenitors, while human cord blood supported limited myelopoiesis from existing myeloid precursors. Transcriptome analysis compared two stromal lines differing in myelopoietic support capacity. Gene profiling revealed both stromal lines to reflect perivascular reticular cells with osteogenic characteristics. However, the 5C6 stroma which failed to support hematopoiesis uniquely expressed several inhibitors of the WNT pathway. Combined data now show that splenic stroma of both human and murine origin provides a mesenchymal stromal cell microenvironment which is WNT pathway-dependent, and which supports in vitro myelopoiesis with production of specific subsets of myeloid and dendritic-like cells.

Monoclonal antibody specific to the Dia blood group antigen generated by phage display technology.

BACKGROUND: Alloanti-Dia can be implicated in mild to severe blood transfusion reactions. Given the concomitance of a high prevalence of the Dia antigen and antibody circulating in some populations, an anti-Dia typing reagent is required in order to enable safe blood transfusions. Limitations of hybridoma technology to produce such a reagent led to the use of phage display technology to generate an anti-Dia monoclonal antibody. MATERIALS AND METHODS: A library of phages displaying murine single-chain variable fragment antibody (scFv-phages) was consecutively adsorbed with different panels of Di(a-b+) red cells to eliminate scFc-phages that potentially bind irrelevant blood group antigens. Thereafter, the subtractive library was specifically selected for the scFv-phages that bound Dia antigen by sequentially biopanning the library with Di(a+b+) cell ghosts and Di(a+b-) intact red cells. A specific interaction between the selected scFv-phages and Dia epitope was validated with the Dia peptide by a competitive haemagglutination inhibition assay and confirmed with the red cells by flow cytometry. RESULTS: An scFv-phage clone specifically bound the Dia epitope, as shown by its binding competition with the human anti-Dia to the Dia peptide in a haemagglutination inhibition test. Moreover, it was highly reactive to Di(a+b+) red cells but not to Di(a-b+) red cells, as determined by flow cytometry. DISCUSSION: In this study, a Dia-specific scFv-phage antibody was successfully produced. The selection protocol might be a prototypic platform for producing monoclonal antibodies to relevant blood group antigens. The scFv-phage produced in this way warrants further development for use as a reagent for Dia red cell typing.

A novel nonsense mutation found in the CD177 gene of Thai individuals with the HNA-2 null phenotype.

OBJECTIVES: The aim of this study is to explore the molecular basis and to develop a simple sequence-specific primer polymerase chain reaction (PCR-SSP) technique for screening genotypes associated with the human neutrophil antigen-2 (HNA-2) null phenotype among Thai blood donors. BACKGROUND: Single-nucleotide polymorphisms (SNPs) c.787A>T of the CD177 gene is well known to be primarily demonstrated as a genetic determinant for HNA-2 deficiency. METHODS: The SNPs in the CD177 gene (exons 7 and 9) of 49 Thai blood donors with the known percentage of CD177 expression by flow cytometry including 48 HNA-2 positive and 1 HNA-2 null individuals were identified by long-range PCR amplification and sequencing. Moreover, screening for the c.1254G>A mutation was developed using an in-house PCR-SSP technique and tested among 771 unrelated donor samples. RESULTS: A HNA-2 null sample from the first cohort was heterozygous for c.787A/T and homozygous for c.1291G/G, namely, a 787A-1291G/787T-1291G (AG/TG) genotype. Interestingly, we could identify SNP c.1254G>A (rs188387562, p. Trp418Ter) that caused a nonsense mutation of the CD177 gene in exon 9. This individual might have the 787A-1254A-1291G/787T-1254G-1291G genotype. From the second cohort (771 unrelated donors), the 1254GG homozygote was the most common (96.37%), followed by the 1254GA heterozygote (3.50%) and 1254AA homozygote (0.13%). Blood samples of two individuals with 787AT-1254GA-1291GG and 787AA-1254AA-1291GG genotypes were tested and the HNA-2 antigen expressions were 0.03% and 0.16% in rank. CONCLUSIONS: The c.787A>T is a primary genetic hallmark to determine the HNA-2 null phenotype. Additional screening of the novel c.1254G>A in combination with c.787A>T is a suitable, convenient and effective diagnosis among Thais.

Structural and biological features of a novel plant defensin from Brugmansia x candida.

Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78-85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded ß-sheet (ß1-ß3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 µM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.

The Frequency of SF3B1 Mutations in Thai Patients with Myelodysplastic Syndrome

Genetic mutations in genes encoding critical component of RNA splicing machinery including SF3B1 are frequently identified and recognized as the pathogenesis in the development of myelodysplatic syndrome (MDS). In this study, PCR sequencings specific for SF3B1 exon 13, 14, 15, and 16 were performed to analyse genomic DNA isolated from bone marrow samples of 72 newly diagnosed MDS patients. We found that 10 of 72 (14%) patients harbor SF3B1 missense mutations including E622D (1/72), R625C/G (2/72), H662Q (1/72), K666T (1/72), K700E (4/72) and G740E (1/72), respectively. Mutations were predominantly located on exon 14 and 15 of SF3B1 coding sequence. Interestingly, patients with SF3B1 mutations exhibited higher platelet counts (195×109/L VS. 140×109/L, p-value = 0.025) as well as lower hemoglobin levels (81 g/L VS. 92 g/L, p-value = 0.009) and associated with ring sideroblast phenotype (p-value < 0.001) when compared with patients without the SF3B1 mutation. In summary, we reported the frequency of SF3B1 mutations in Thai patients with different subtypes of MDS. SF3B1 mutations were predominantly occurred in MDS-RS and considered as favourable prognosis value. This study further highlighted the clinical important of SF3B1 mutations analysis for the classification of MDS.

MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis.

BACKGROUND: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II- dendritic-like 'L-DC', and CD11b+CD11c+MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished. RESULTS: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL-10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDC-like cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma. CONCLUSION: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF.

Flow-Cytometric Analysis of HNA-2 Expression and Phenotypes Among Thai Blood Donors.

BACKGROUND: Antibodies specific to human neutrophil antigen (HNA), especially HNA-2, are implicated in various conditions, including neonatal alloimmune neutropenia, febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The distribution of the HNA-2 phenotype frequencies in the Thai population remains unknown. This study aimed to investigate HNA-2 phenotype frequencies in Thai blood donors and to compare the relationships of sex and age with HNA-2 expression. METHODS: EDTA blood samples were collected from 220 unrelated healthy Thai blood donors, including 150 males and 70 females, with ages ranging from 20 to 57 years. Polymorphonuclear cells were isolated and stained with monoclonal antibodies clone MEM-166 and clone 2D1, which are specific to human CD177 (HNA-2) and CD45, respectively. HNA-2 expression according to sex and age was analyzed by flow cytometry. RESULTS: Among the 220 donors, HNA-2-positive and HNA-2-null-phenotype frequencies were 0.995 and 0.005, respectively. Mean antigen expression was significantly higher in women (71.01±15.46%) than in men (64.59±18.85%; P <0.05). No significant differences in HNA-2 expression were found between different age groups. HNA-2 phenotype frequencies were similar to those in Asian, African, American, and Brazilian populations, but were significantly different from those in eastern Japanese, Korean, and French populations (P <0.001). CONCLUSIONS: This is the first report of HNA-2 phenotype frequencies in a Thai population, and the data will be helpful in predicting the risk of HNA-2 alloimmunization and in recruiting granulocyte panel donors.

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development.

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.

Delineation of a novel dendritic-like subset in human spleen.

Dendritic cells (DCs) and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c(+)hCD11b(+)HLA-DR(-)hCD86(+) subset in the spleen, along with the previously described subsets of hCD1c(+) DCs, hCD123(+) plasmacytoid DCs (pDCs), hCD16(+) DCs and hCD141(+) DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c(+)HLA-DR(+) and hCD11c(+)HLA-DR(-) cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCD80 and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors.

Spleen stroma maintains progenitors and supports long-term hematopoiesis.

Hematopoietic stem/progenitor cells (HSPC) differentiate in the context of stromal niches producing cells of multiple lineages. Limited success has been achieved in the past with induction of hematopoiesis in vitro. Previously, spleen long-term stromal cultures (LTC) were shown to continuously support restricted hematopoiesis for production of novel dendritic-like cells (LTC-DC). An in vivo equivalent dendritic cell type was then described which is specific for spleen. The in vivo counterpart cell was termed 'L-DC' and represents a dendritic-like CD11c(lo)CD11b(hi)CD8α-MHC-II- cell which differs phenotypically and functionally from monocytes/macrophages and conventional and plasmacytoid DC. Splenic stroma is now shown to maintain HSPC and to support their restricted in vitro differentiation to give this 'L-DC' subset. In order to characterise progenitors of this distinct cell type, LTC were analysed for cell subsets produced, and these subsets sorted and assessed for hematopoietic potential in subsequent co-cultures over STX3 stroma. Progenitors were defined as a lineage (Lin)(-)ckit(lo) subset reflecting HSPC. Furthermore, when Lin(-)ckit(hi)Sca1(+)Flt3(-) HSPC were sorted from bone marrow, they colonised splenic stroma with long-term production of L-DC. The maintenance of HSPC by splenic stroma was confirmed when non-adherent cells collected from LTC showed oligopotent reconstitution of the hematopoietic compartment of lethally irradiated mice. All data support a model whereby spleen houses a niche for HSPC in the resting state, with production of progenitors, and their differentiation to give tissue-specific antigen presenting cells.

Characterisation of dendritic cells arising from progenitors endogenous to murine spleen.

Heterogeneity amongst dendritic cell (DC) subsets leads to a spectrum of immune response capacity against pathogens. Several DC subsets in spleen have been described which differ in terms of phenotype and function. We have previously reported a distinct population of CD11c(lo)CD11b(hi)MHC-II(-)CD8(-) dendritic-like "L-DC" in murine spleen, which can also be generated in splenic stromal longterm cultures. Here, the ontogeny of L-DC development in perinatal mice has been compared with other known splenic DC subsets. Flow cytometric analysis has revealed the presence of L-DC at embryonic age (E)18.5 spleen, while plasmacytoid (p)DC and conventional (c)DC appear at 2 and 4 days following birth. Co-cultures of E18.5 spleen above splenic stroma also showed production of only L-DC, while spleen cells from D0 through D5 neonates showed production of both L-DC and cDC-like cells. Addition of an M-CSFR inhibitor to co-cultures revealed that while the development of cDC-like cells depended on M-CSF, many L-DC developed independently of M-CSF. Furthermore, purified hematopoietic stem cells (HSC) and multipotential progenitors (MPP) isolated from neonatal D1 spleen are capable of developing into L-DC in co-cultures. These studies reveal a lineage of dendritic-like cells developing in the spleen microenvironment, and which appear to arise from endogenous progenitors laid down in spleen during embryogenesis.

Distinct progenitor origin distinguishes a lineage of dendritic-like cells in spleen.

The dendritic cell (DC) compartment comprises subsets of cells with distinct phenotypes. Previously this lab reported methodology for hematopoiesis of dendritic-like cells in vitro dependent on a murine splenic stromal cell line (5G3). Co-cultures of lineage-depleted bone marrow (Lin(-) BM) over 5G3 continuously produced a distinct population of dendritic-like "L-DC" for up to 35 days. Here the progenitor of L-DC is investigated in relation to known BM-derived hematopoietic progenitors. It is shown here that L-DC-like cells also derive from the CD150(+)Flt3(-) long-term reconstituting-hematopoietic stem cells (HSC), and also from the Flt3(+) multipotential progenitor subset in BM. Lin(-) BM co-cultures also produce a transient population of cells resembling conventional (c) DC. Production of cDC-like cells is shown here to be transient and M-CSF dependent, and also appears following co-culture of described common dendritic progenitors or monocyte dendritic progenitors over 5G3. BM cells from C57BL/6-flt3L(tm1lmx) and C57BL/6-Csf2(tm1Ard) mice which lack cDC precursors and monocytes, are shown here to contain L-DC progenitors which can seed 5G3 co-cultures. L-DC are functionally distinct cells, in that they arise independently of M-CSF, and by direct differentiation from HSC.

Development of two distinct dendritic-like APCs in the context of splenic stroma.

Murine splenic stroma has been found to provide an in vitro niche for hematopoiesis of dendritic-like APC. Two distinct cell types have been characterized. The novel "L-DC" subset has cross-presenting capacity, leading to activation of CD8(+) T cells, but not activating CD4(+) T cells, which is consistent with their CD11c(lo)CD11b(hi)MHC-II(-) phenotype. For L-DC, an equivalent tissue-specific APC has been found only in spleen. A second population of CD11c(hi)CD11b(lo)MHC-II(+) cells resembling conventional dendritic cells (cDC) can activate both CD4 and CD8 T cells. Production of L-DC but not cDC-like cells is now shown to be dependent on contact between the L-DC progenitor and stroma such that the presence of a Transwell membrane can prevent L-DC development. Since L-DC can be produced continuously in vitro in stromal co-cultures overlaid with bone marrow (BM) progenitors, it was hypothesized that L-DC progenitors are self-renewing. The L-DC progenitor is shown here to be defined by the Flt3(-)c-kit(+)Lin(-)Sca-1(+) (F(-)KLS) subset of adult BM which contains primitive HSC. Since the less primitive F(+)KLS HSC subset also contains L-DC progenitors, Flt3 does not appear to be a defining marker for this progenitor. Precursors of the cDC-like subset are found only within the F(+)KLS subset and seed production of a transient population of APC. All data identify differentiation of L-DC from HSC, and of cDC-like cells from DC precursors, which occurs independently of inflammatory signals and is dependent on a splenic stromal microenvironment.

Hematopoiesis leading to a diversity of dendritic antigen-presenting cell types.

Hematopoietic stem cells (HSCs) undergo expansion and differentiation, giving rise to all terminally differentiated blood cells throughout life. HSCs are found in distinct anatomical sites during development, and in adults, hematopoiesis occurs predominantly on the luminal side of the bone cavity in bone marrow. Millions of newly formed blood cells are generated per second to accommodate the short half-life of hematopoietic cells. For this to happen, HSCs must sustain their self-renewal capacity as well as their capability to commit and differentiate toward multiple cell lineages. Development of the hematopoietic system is finely regulated as the animal ages, so that it does not become exhausted or misdirected. This review covers aspects of hematopoietic development from the embryonic period through adult life in relation to development of dendritic cells. It also considers a role for HSCs in extramedullary sites and their possible role in myelopoiesis, with formation of tissue-specific antigen-presenting cells.

Spleen as a site for hematopoiesis of a distinct antigen presenting cell type.

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen.

Myelopoiesis related to perinatal spleen.

Adult murine spleen is known to have a major role in the development of dendritic cell (DC) subsets, including conventional DC and plasmacytoid DC. In this lab, long-term cultures (LTCs) established from murine spleen support continuous production of novel dendritic-like cells, termed LTC-DC. An in vivo equivalent subset also exists in spleen, namely L-DC. As co-cultures using LTC-derived splenic stroma support the outgrowth of L-DC from spleen and bone marrow sources, it is likely that spleen represents an important niche for DC development. To investigate the appearance of L-DC during ontogeny, spleen was isolated from embryonic and neonatal mice of different ages for analysis of myeloid and DC subsets. Perinatal spleen was also used to establish co-cultures for identification of progenitors, and LTCs were established from spleens for assessment of stromal competence. Although spleen from 16-day embryos (E16.5) contained myeloid cells, DC subsets did not appear until day 4 after birth (D4). However, murine spleen at D0 contained progenitors, which could seed co-cultures for L-DC production. LTC could not be established from spleen until D4. The appearance of L-DC after D4 in spleen is dependent on the formation of the appropriate stromal microenvironment which occurs in the early postnatal period.

Ex vivo generation of cytokine-induced killer cells (CD3+ CD56+) from post-stem cell transplant pediatric patients against autologous-Epstein-Barr virus-transformed lymphoblastoid cell lines.

EBV-PTLDs affect as high as 20% of SCT recipients especially those with T-cell depleted grafts while high mortality rates were also noted. Adoptive allogeneic and autologous CTLs have a therapeutic potential in this setting. However, the process of expansion of these cells is tedious and time consuming in both allogeneic and autologous CTL generation. For the allogeneic SCT, another major obstacle is unavailability of donors especially in an unrelated SCT setting. The aim of the present study was therefore to investigate the efficacy of autologous CIK cells (CD3+ CD56+) against autologous EBV-LCLs from post-SCT pediatric patients. We could demonstrate that CIK cells can be generated within two wk and did show the significant cytotoxicity against autologous EBV-LCLs. CIK cells may provide a potent tool for use in post-transplantation adoptive immunotherapy.

Bone marrow derived mesenchymal stem cells from chronic myeloid leukemia t(9;22) patients are devoid of Philadelphia chromosome and support cord blood stem cell expansion.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. It is characterized at cytogenetic level by the Philadelphia (Ph) chromosome and at the molecular level by the BCR/ABL gene rearrangement. Bone marrow derived mesenchymal stem cells (MSCs) are also pluripotent stem cells that can differentiate into several mesenchymal tissues. To date, no study has been performed to characterize whether MSCs from CML harbor the abnormal Ph chromosome similar to CML bone marrow cells. We isolated and characterized MSCs from diagnostic marrow samples (n=11) and showed that MSCs can be readily isolated from CML marrow and exhibit major expansion potential as well as intact osteogenic differentiation ability. Moreover, they do not harbor the Ph chromosome confirmed by fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). Thus, we demonstrated that CML marrow is an abundant source of MSCs appearing through both FISH and RT-PCR not to be involved by the malignant process of CML. Furthermore, these MSCs from a CML patient could support in vitro cord blood expansion as those MSCs from a normal donor. Since MSCs are able to support engraftment of hematopoietic stem cells in stem cell transplantation (SCT) as well as suppress alloreactive T cells causing graft-versus-host disease, this current report thus provides evidence that in a SCT setting of CML patients, autologous MSCs could be a source of stem cell support in future cell therapy applications.

Dendritic cells pulsed with total tumor RNA for activation NK-like T cells against glioblastoma multiforme.

Dendritic cells (DCs) are potent antigen presenting cells and play critical role in T cell-mediated immunity. DCs have been shown to induce strong anti-tumor responses both in vitro and in vivo. Their efficacies in tumor therapy are being investigated in clinical trials. Previous evidence has shown that these DCs enhance the cytotoxicity of NK cells. We generated NK-like T cells (CD3(+)CD56(+)), a novel type of effector cells differentiated from normal lymphocyte, which is now being used for adoptive immunotherapy in clinical trials. This study aimed to elucidate the effects of NK-like T cells after co-culturing with DCs against tumor cells. The result revealed that tumor-derived RNA-pulsed DCs can enhance the immune responses of NK-like T cells against glioblastoma multiforme cell line but these effector cells did not appear to have the cytotoxic effect against normal cells (human umbilical vein endothelial cells (HUVEC) and fibroblasts) in vitro. This study may be beneficial for the development of new immunologic effector cells for using in adoptive immunotherapy for glioblastoma multiforme in the future.

Generation of CD3+ CD56+ cytokine-induced killer cells and their in vitro cytotoxicity against pediatric cancer cells.

A certain number of pediatric cancer patients still succumb to relapse following conventional treatment of their malignancies. One of the mechanisms of relapse is escape from immunity. Adoptive cellular immunotherapy with effector cells has the potential to overcome this escape. In adults, the CD3+ CD56+ cell, a cytokine-induced killer (CIK) cell, appears to be a promising effector cell type with the greatest cytotoxicity. This effector cell type may work in children as well. No similar studies with children have been published. We speculated that expanded CD3+ CD56+ cells obtained from pediatric cancer patients during remission would act similarly against various pediatric tumor cell lines; therefore, we undertook the present study to find support for our speculation. This study was undertaken to generate and expand CD3+ CD56+ CIK cells from normal peripheral blood mononuclear cells (PBL) obtained from 6 children with cancer (2 with acute lymphoblastic leukemia, 2 with large cell lymphoma, and 2 with osteosarcoma) in remission after intensive chemotherapy and to study the cytotoxic activities of these cells against chronic myeloid leukemia cell line K562 t(9;22), 4 pediatric tumor cell lines [infant acute lymphoblastic leukemia RS4 t(4;11), TEL/AML acute lymphoblastic leukemia REH t(12;21), alveolar rhabdomyosarcoma Rh-Cr t(2;13), and Ewing sarcoma EW-Le t(11;22)], and 2 pediatric glioblastoma multiforme cultured cell lines (G74 and G77). CIK cells were generated and expanded in culture medium to which interferon gamma, monoclonal antibody against CD3, and interleukin 2 were added at appropriate times. Cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cell cytotoxicity against various pediatric tumor cell lines. The results show that after 21 days in culture CD3+ CD56+ CIK cells derived from the 6 pediatric patients accounted for a median of 28.3% of the entire culture (range, 10.7%-36.4%). Before expansion no such cells were found in any of the 6 children. Median lytic activity rates of CIK cells were 45.5% to 64.5%, rates that contrasted drastically to the lytic activity rates of PBL, which were only 8% to 12%. The findings of the present study are encouraging. They provide information for developing adoptive immunotherapy for future clinical trials with pediatric cancer patients, particularly those patients with minimal residual disease after intensive chemotherapy or stem cell transplantation (especially nonmyeloablative transplantation procedures).