Xenogeneic Cell Lines Research Articles

Xenogeneic fibroblasts inhibit the growth of the breast and ovarian cancer cell lines in co-culture.

Cell-based therapies cure some hematologic malignancies, although little information exists on solid cancer cell responses. The study objective was to test the hypothesis that xenogeneic fibroblasts can inhibit the growth of human cancer cell lines in vitro. Seven human cell lines (pancreatic cancer HPAF II; brain cancer U-87 MG; fibrosarcoma; ovarian cancer OVCAR3 and SKOV3; and breast cancer MCF7 and MDA-MB231) were co-cultured with two xenogeneic fibroblast cell lines (CV-1; monkey, Cercopithecus aethiops and DF-1; chicken, Gallus gallus) in a Transwell culture system. Cancer cell proliferation was assessed colorimetrically. Different concentrations of breast and ovarian cancer cells were tested. Gene expression induced by DF-1 xenogeneic fibroblasts was assessed by RNAseq of MCF7 breast cancer cells. The proliferation of the majority of the cancer cell lines was altered by co-culture with xenogeneic fibroblasts. Cell proliferation was increased (4-17%) by CV-1; DF-1 increased brain cancer cell proliferation (16%), decreased breast and ovarian cancer cell growth (15 and 26% respectively) but did not affect fibrosarcoma and pancreatic cancer cells. When the initial cancer cell concentrations were lowered 4-fold, growth inhibition of breast and ovarian cancer increased more than 2-fold. DF-1 fibroblasts induced significant differential expression of 484 genes in MCF7 breast cancer cells; 285 genes were downregulated and 199 genes were upregulated compared to control. Genes involved in the immune response were the major downregulated entities. RNAseq results were validated by qRT-PCR of 12 genes. The results show that xenogeneic fibroblasts can alter the growth and gene expression of cancer cells in vitro. This suggests a potentially novel investigational approach to the control of cancer cell growth.

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032–1043. DOI: 10.1089/ten.tec.2014.0665

[This corrects the article DOI: 10.1089/ten.tec.2014.0665.].

Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform.

In vitro generation of red blood cells (RBCs) from human embryonic stem cells and human induced pluripotent stem cells appears to be a promising alternate approach to circumvent shortages in donor-derived blood supplies for clinical applications. Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or coculture with xenogeneic cell lines. However, most current methods for hPSC expansion and EB formation are not amenable for scale-up to levels required for large-scale RBC generation. Moreover, differentiation methods that rely on xenogenic cell lines would face obstacles for future clinical translation. In this study, we report the development of a serum-free and chemically defined microcarrier-based suspension culture platform for scalable hPSC expansion and EB formation. Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in a 80-fold improvement in the yield of RBC generation compared to a conventional EB-based differentiation method. In addition, we report efficient terminal maturation and generation of mature enucleated RBCs using a coculture system that comprised primary human mesenchymal stromal cells. The microcarrier-based platform could prove to be an appealing strategy for future scale-up of hPSC culture, EB generation, and large-scale generation of RBCs under defined and xeno-free conditions.

74 - Superior Red Blood Cell Generation From Human Pluripotent Stem Cells via a Novel Microcarrier Based Embryoid Body Platform

In vitro generation of red blood cells (RBCs) from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) appears to be a promising alternate approach to circumvent shortages in donor-derived blood supplies for clinical applications. Conventional methods for hematopoietic differentiation of hESC and iPSC rely on embryoid body (EB) formation and/or co-culture with xenogeneic cell-lines. However, these current methods are not amenable for scale-up and face obstacles for clinical translation due to the use of xenogeneic materials. Here we report the development of a serum-free and chemically defined platform using recombinant human Laminin-521 coated microcarriers for pluripotent stem cell expansion and hematopoietic differentiation of hESC and hiPSC. Improved survival and better quality EBs generated with the microcarrier-based method resulted in at least a 6-fold improvement in hemangioblast expansion, potentially culminating in a 80-fold improvement in the yield of RBC generation as compared to a conventional EB-based method. Using the improved microcarrier-based differentiation protocol, we demonstrated successful differentiation of O-negative (Rhesus factor D negative) RBCs from donor hiPSC generated from finger-prick blood. Universal donor RBCs (blood type O-RhD negative) could prove to be extremely useful for future cell therapy applications such as emergency blood transfusions and for treating poly-immunized patients with recurrent transfusion needs. Thus, our microcarrier-based differentiation platform could prove to be an appealing strategy for future scale-up of hESC and hiPSC culture and for hematopoietic differentiation under defined conditions for large-scale generation of universal donor RBCs.

Long-Term Viability of Transplanted Hybrid Cellular Spheroids within Chondrocyte Sheets

Encapsulation of transplanted cells within an immunoisolating membrane may provide a new strategy for protecting these cells from recipient immune responses without the use of immunosuppressive drugs. We have previously reported a novel concept of immunoisolation and immunodelusion using recipient cells instead of traditional artificial materials. We developed a chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas (CSI-BAP) that would enable transplantation of cells across allogeneic and xenogeneic barriers without the cells being recognized as donor cells and without the need for immunosuppression. Recently, we have constructed hybrid cellular spheroids (HCSs) containing cells from two different cell lines (RIN-5F, an insulin-secreting cell line, and Hep-G2, a hepatocellular carcinoma cell line) to enhance the function and biocompatibility of the HCSs. These HCSs were then encapsulated with multiple layers of chondrocyte sheets obtained from the auricular cartilage of Sprague-Dawley (SD) rats. The in vitro ability of the CSI-BAP to secrete insulin was tested before transplantation. Histological evaluation of CSI-BAP chondrocyte microencapsulated immunoisolated islet morphology and viability of allogeneic or xenogeneic cell lines was performed 100 days after the CSI-BAP was transplanted into SD rats. Morphological evaluations revealed good viability of the islets and progression of islet encapsulation. In vitro insulin secretion from the CSI-BAP was well maintained. Additionally, insulin and albumin secretion from the CSI-BAP was confirmed by in vivo immunohistochemical examination. Moreover, the cell lines transplanted into the subcutaneous space in the form of HCSs within the chondrocyte sheets showed good viability of more than 100 days and sustained insulin and albumin secreting ability.

A Novel Culture Method of Canine Peripheral Blood Lymphocytes with Concanavalin A and Recombinant Human Interleukin-2 for Adoptive Immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.

Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture

Expansion of hematopoietic progenitor cells (HPC) in the presence of endothelium has been shown to result in grafts capable of restoring hematopoiesis in a myeloablated host. However, the use of xenogeneic endothelium or cell lines may carry risks in a clinical transplantation setting. We explored the feasibility of cord blood progenitor cell expansion in vitro in an autologous coculture system using umbilical vein endothelial cells (HUVEC). CD34+ HPC and HUVEC were isolated from the same umbilical cord. For 3 days, HPC were maintained in serum-free medium supplemented with a single cytokine (SCF) or a cytokine combination (SCF, Flt3-ligand, IL-6). Meanwhile, adherent HUVEC cultures were established. After addition of VEGF and IL-1 at day 3, the cells were either added to HUVEC cultures or grown without endothelial cells for further 7 days. Total cells, CD34+ and clonogenic progenitors were significantly increased when coculture was compared to liquid culture. Long-term culture-initiating cells (LTC-IC) and cobble stone area-forming cells (CAFC, limiting dilution analysis) were detected more frequently after coculture with endothelial cells. Also precursors and mature myeloid cells were observed after expansion. We conclude that coculture with autologous HUVEC represents a feasable approach for ex vivo expansion of cord blood HPC.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

Freshly isolated bone marrow cells induce death of various carcinoma cell lines.

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma

Aims: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma.Materials and Methods: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis.Results: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3×106 vs 2.3×104 cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls).Conclusion: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Adjuvant Effect of a Flt3 Ligand (FL) Gene-Transduced Xenogeneic Cell Line in a Murine Colon Cancer Model

Background. Flt3 Ligand (FL) has been shown to elicit antitumor responses induced by tumor antigen stimulation. Allogeneic and xenogeneic cell lines transduced with cytokine genes may be used to augment the antitumor efficacy of tumor antigens. Objective. The objective was to evaluate the augmentation of tumor lysate-induced immunity by a more clinically applicable FL gene-transduced xenogeneic cell line in combination with interleukin-2 (IL-2) in a CC-36 murine colon cancer model. Methods. Human 143B osteosarcoma tumor cells were transduced with full-length FL cDNA (143B-FL). Secretion of FL from 143B-FL was analyzed in vivo in normal BALB/c mice transplanted with 143B-FL, and expansion of dendritic cells (DC) was also analyzed in the same mice by flow cytometry. Eight-week-old, male BALB/c mice were used in a prophylactic vaccination protocol utilizing tumor lysate (CLy), 143B-FL, and soluble IL-2. Prophylactic group designations ( n = 10/group) were as follows: ten million 143B-FL cells (alone, with tumor lysate, or with tumor lysate and IL-2), IL-2 with tumor lysate, IL-2 alone, or a no treatment control. The tumor lysate (200 μg of protein) and IL-2 (100,000 IU) injections were administered intraperitoneally. Mice were challenged subcutaneously with 10 3 CC-36 tumor cells. Tumor protection and tumor burden (TB), as mean tumor diameter, were determined. Peripheral blood lymphocytes (PBLs) from the 143B-FL + IL-2 + tumor lysate vaccinated group were analyzed for cytolytic activity in 4-h chromium release assays. In addition, plasma cytokine concentrations of interleukin-12 (IL-12) and interferon gamma (IFN-γ) were assessed by ELISA. Student's t tests were used for all statistical comparisons. Results. In vivo expression of FL was observed 24 h following the inoculation of 143B-FL, and a four fold increase in DCs was observed in the peripheral blood of these mice. Mice immunized with a combination of 143B-FL, tumor lysate and IL-2 showed statistically significant protection against tumor development (10%) for 100 days after tumor challenge; incidences in other groups ranged from 40 to 100% ( P < 0.05). Moreover, this immunization protocol produced the lowest TB at 3- and 6-week time points (0, 1.6 mm) when compared to all other groups (TB between 7.2 and 15.9 mm) ( P < 0.05). In addition, PBLs from vaccinated mice showed increased cytolytic activity against CC-36 target cells. This corresponded to increased levels of IL-12 and IFN-g in the plasma of mice following vaccination. Conclusion. These data suggest that FL gene-transduced xenogeneic tumor cells may augment the immunity induced by tumor antigens and systemic IL-2 through the activation of dendritic cells and T-cell-mediated mechanisms.

Antitumor Activity of Expanded Human Tumor-Infiltrating γδ T Lymphocytes

Background: The objective of this study was to investigate the antitumor activity of selectively expanded γδ T cells in tumor-infiltrating lymphocytes (γδTILs) or tumor ascites lymphocytes (γδTALs) from patients with colorectal and ovarian epithelial carcinoma (OEC) in vitro and in vivo. Methods: γδTILs/TALs were expanded by the solid-phase antibody method; their cytolytic and proliferative activities in vitro were detected by the MTT method and ³H-TdR incorporation and their effect in vivo was evaluated by the nude mice model. Results: Expanded γδTILs from colorectal tumors demonstrated marked cytotoxicities to allogeneic human colon adenocarcinoma HR8348 and lymphoma Daudi cells, as well as xenogeneic murine thymoma EL-4 cell lines. Cytokines, including IL-2, IL-4, IL-12, IL-15, TNF-α and INF-γ, could promote the cytotoxicities of γδTILs to tumor cells, whereas IL-10, GM-CSF and TFG-β had no effect on such killing activities. Rested γδTILs could proliferate strongly in response to mitomycin C-treated Daudi and EL-4 tumor cells, but not to HR8348 tumor cells, suggesting that the latter might possess only cytotoxicity-related antigen recognized by γδTILs. Either αβTILs or γδTILs from patients with OEC displayed cytotoxicities to allogeneic or autologous OEC cell lines at a similar strength in vitro. Transferring γδTILs into Daudi cell-bearing BALB/c nude mice with an injection of IL-2 was able to maintain a high survival rate of the mice for 30 days, when compared with mice treated with αβTILs or without any treatment (p < 0.05). Without coinjection of IL-2, after 3 months of Daudi tumor inoculation, a high survival rate was observed in γδTIL-treated mice. Similarly, adoptive γδTALs from the ascites of patients with OEC transferred into nude mice displayed a stronger antitumor response to OEC SKOV3 cells than αβTALs in vivo. Tumor volumes in γδTAL-treated mice were smaller than in αβTAL-treated or non-TAL-treated mice within the period from day 23 to day 50 after tumor inoculation (p < 0.05). Fifty days after SKOV3 tumor inoculation, a decreasing trend of carcinogenic rate was observed in γδTAL-treated nude mice. Conclusion: Taken together, our results suggest that γδT cells could be a new candidate for adoptive immunotherapy in the future treatment of patients with cancer.

TRANSPLANTATION OF FETAL TISSUES

The transplantation of fetal tissues offers a unique potential for the development of therapeutic strategies for many clinical disorders that have previously been difficult or impossible to treat. Because of its inherent bioplasticity, fetal tissue is able to successfully integrate into anatomical sites composed of adult host tissues that are terminally differentiated. Also, the multipotency of fetal tissue makes possible its utilization in stem cell replacement therapies. Thus, new hope is being created for the management and cure of many neurodegenerative diseases and congenital immunodeficiencies and metabolic diseases. Furthermore, prospects for the development and utilization of immortalized and/or genetically engineered xenogeneic fetal cell lines in clinical cell replacement therapies continue to improve as our knowledge of the physiologic and immunologic issues associated with fetal tissue transplantation increases with basic research in this field. Nevertheless, many questions and limitations remain. It is unknown to what extent successful engraftment of fetal tissue implants depends on extrinsic signaling from the supporting host tissue. Also, many issues concerning the immunogenicity of fetal tissues and the optimal method(s) of surgical transplantation are unique to specific therapies and must be individually addressed.

Cd8+ lymphocytes that kill allogeneic and xenogeneic major histocompatibility complex class I targets

CD8+ CTLs generated in a two-way MLR should lyse target cells only if these targets share a class I MHC allele with the original stimulators. Using cattle PBMCs in a two-way MLR, we generated CDS + CTLs that kill allogeneic and xenogeneic cell lines. We have named these cells MLK cells. PBMCs isolated from two unrelated animals were cultured together. After 14 days microfluorimetry analysis was performed on the MLK cells with results showing >90% CD8+ cells. RFLP analysis revealed these cells to be predominately of one animal. MLK cells were then used as effector cells in cytotoxicity assays with syngeneic, allogeneic, and xenogeneic target cells. MLK cells were able to kill all targets. Incubating MLK cells with mAbs to CD8 markedly reduced killing, suggesting a TCR-mediated cytolytic pathway. Effective cytolysis of these targets by MLK cells was dependent on class I expression. MHC class I expression-impaired mutants of allogeneic and xenogeneic tagets were not susceptible to cytolysis. Comparisons to LAK cells revealed similarities in phenotype and function to the NK1.1 — , CD8 + subset.

Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2 125

Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2 125 (rIL 2 125). The rIL 2 125 used in these experiments was purified to homogeneity from Escherichia coli , contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4×10 6 units/mg. Human rIL 2 125 was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species. Long-term cultures were highly sensitive to low levels of rIL 2 125 and showed dose-dependent responses when used in short-term IL 2 assays. Bovine and porcine PBMC preincubated with human rIL 2 125 for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.

In vitro cytotoxicity of mouse macrophages activated by coculture with syngeneic sarcoma cells.

Normal resident peritoneal macrophages from C3D2 (C3H/Tif X DBA/2) F1 mice were activated in vitro by culturing with semisyngeneic tumour cells. The tumour cells originated from a methylcholanthrene-induced sarcoma (MC1M) growing in vivo in ascites form. Macrophage-mediated cytotoxicity was evaluated after 5 days of in vitro culture, using five different target cells. Semisyngeneic (L 929), allogeneic (B16 melanoma), and xenogeneic (HeLa) tumour cell lines and normal allogeneic fibroblast cell lines (3T3, 3T6) were tested. The morphology and kinetics of the cytotoxicity reaction were studied by scanning electron microscopy and compared with release of radioactivity from 14C-thymidine-labelled target cells. The activated macrophages were able to kill the semisyngeneic, allogeneic, and xenogeneic tumour cell lines tested under conditions that did not affect normal fibroblasts. The requirement for T cells during activation of the macrophages was also tested. The cytotoxicity decreased markedly when T cells were removed from the macrophage cultures before activation or when macrophages from nude mice were used in the experiments.

Two distinct human cloned T cell lines that exhibit natural killer-like and anti-human effector activities.

Cloned T cell lines from mixed lymphocyte cultures stimulated with autologous Epstein Barr virus- (EBV) transformed lymphoblastoid cell line (LCL) cells were established using a limiting dilution technique in the presence of T cell growth factor (TCGF). The T cell lines included two distinct clones of cytotoxic T cells (Tc) in addition to EBV-specific Tc. A cytotoxic profile of one cloned line was similar to that of endogenous NK cells in peripheral blood. The other cloned Tc line showed an anti-human cytotoxicity. The susceptible targets for this latter Tc line were various human cells, including autologous LCL and peripheral blood lymphocytes (PBL), stimulated with pokeweed mitogen, along with NK-sensitive and NK-resistant cell lines. Weak cytotoxic activity was detected against various xenogeneic cell lines. Furthermore, autologous and allogeneic cloned T cell lines were resistant to killing by the anti-human effector clone. These t wo distinct cloned Tc lines expressed the Leu-1 and Leu-2a antigens, which are markers of suppressor/cytotoxic T cells.

Further characterization of natural killer cells induced by Kunjin virus.

The natural killer (NK) cell induced one to two days after Kunjin virus infection of BALB/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. It is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. Levels of lysis of non-tumour target cells are dependent on their genotype. Furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible again after culture in vitro. The virus-induced NK cell is elicited as readily in athymic (nude) as in normal mice. X-irradiation inhibits its development if administered prior to infection. It is labile on culture at 37 degrees. The cell carries Fc receptors but its NK activity is not antibody-dependent.

Spontaneous Cell-Mediated Cytotoxicity (SCMC) Associated with Lymphocytes Negative for Acid α-Naphthyl Acetate Esterase (ANAE) Activity

A modified histochemical procedure for nonspecific acid α-naphthyl acetate esterase (ANAE) activity in human lymphocytes was used to identify a subpopulation of E-rosette forming cells. Performing a one hour reaction at pH 6.5 the distinct dot-like staining pattern was almost exclusively observed on high affinity E-rosettes which sedimented readily in a regular Ficoll-Metrizoate gradient. By combining latex phagocytosis with staining for ANAE activity, a clear-cut distinction between mononuclear phagocytes and lymphocytes could be made. An attempt was undertaken to relate the ANAE marker on human lymphocytes to their functional capacity in spontaneous cell-mediated cytotoxicity (SCMC) reactions. Using as targets two allogeneic (K562,IGR3) and a xenogeneic cell line (L1210) it could be clearly demonstrated that high SCMC activity is mediated by ANAE negative mononuclear cells, whereas enrichment for ANAE positive lymphocytes resulted in a loss of SCMC.