Coculture Of Peripheral Blood Lymphocytes Research Articles

T Cell Hyperreactivity in Obese Strain (OS) Chickens. Different Mechanisms Operative in Spleen and Peripheral Blood Lymphocyte Activation

The enhanced T cell reactivity (ConA hyperresponsiveness and IL 2 hypersecretion) of spleen lymphocytes of Obese strain (OS) chickens with spontaneous autoimmune thyroiditis has recently been shown to be due to a defect in macrophage-derived non-specific suppressor factors that regulate IL 2 secretion and IL 2-promoted T lymphoblast proliferation in normal healthy animals. In the present study, we present several lines of evidence that the increased T cell response of peripheral blood lymphocytes (PBL) of OS chickens is due to mechanisms entirely different from the described dysregulation of splenic T cells: 1) In contrast to the splenic macrophages, peripheral blood monocytes of OS animals are not deficient in the production of IL 2 antagonistic activity (IAA); 2) therefore, cocultivation of PBL from OS and Normal White Leghorn (NWL) chickens in communicating culture chambers did not abrogate the difference in Con A response as previously observed with spleen lymphocytes. 3) Immunofluorescence with a monoclonal antibody (INN CH 16) against the chicken IL 2 receptor revealed enhanced numbers of mitogen activatable T cells in OS PBL but not OS spleen lymphocytes. 4) After prolonged Con A stimulation of PBL, OS and NWL lymphoblasts did not differ from each other in functional aspects. In contrast to this, Con A lymphoblasts from OS spleens exhibited enhanced staining with INN CH 16 in parallel with an increased proliferative response to IL 2. Thus, the primary T cell dysfunction involved in the development of autoimmune disease in OS chickens is the result of at least two separate regulatory defects.

Cellular immune response to human sarcomas: cytotoxic T cell clones reactive with autologous sarcomas. I. Development, phenotype, and specificity.

Human T cell clones cytotoxic for autologous sarcoma cell lines have been developed from patient JM with an osteogenic sarcoma, and from patients EG and RM with malignant fibrohistiocytoma. These clones were derived from the cocultivation of peripheral blood lymphocytes (PBL) with the respective patient's autologous irradiated established tumor cell lines (AIT). After two cycles of stimulation for 5 days in bulk culture, these "educated" lymphocytes were seeded at a density of 1 X 10(6) cells/well in 24-well plates and were cultured in the presence of highly purified natural IL 2 and AIT, the latter serving as a feeder layer. Cell numbers were reduced from the initial seeding density by one log each week until reaching a density of 10(2) cells. These cells were found to be stable in viability and cytotoxic activity, after which limiting dilution was then performed. Within 4 to 6 wk, clones were isolated with unique specificities. These clones were capable of proliferating to a total density of 10(9) cells/ml and maintained their specific cytotoxicity for more than 6 mo. Testing with a panel of target cells of various histotypes, cold-target inhibition assays, and blocking of cytotoxicity with anti-HLA monoclonal antibodies showed that the T cell clones recognize a common sarcoma-associated antigen and that the lysis is HLA restricted. Phenotypically, cytotoxic clones derived from JM were Leu-1+, Leu-2+, and Leu-3-, whereas those derived from EG exhibited either Leu-24 or Leu-3+ markers, the latter phenotype lacking cytotoxicity. RM exhibited mainly Leu-3+ clones with strong cytotoxicity. All were HNK-1- and HLA class II+, with less than 1% of cells of each clone stained by anti-TAC monoclonal antibody. The clones from each patient did not lyse autologous or allogeneic PBL, mitogen-induced T lymphoblasts, normal fibroblasts, cells isolated from benign neoplasms, carcinoma cells, Daudi B lymphoid cells, or K562 cells. With the exception of EG, all clones produced immune interferon in a range from 12 to 50 U/ml. The generation of long-term specific T cell clones can be used to further dissect the cellular immune response to sarcomas. Cytotoxic T cell clones have potential application for tumor immunotherapy.

Transmissible Lymphoma and Simian Acquired Immunodeficiency Syndrome in Rhesus Monkeys<xref ref-type="fn" rid="FN1">2</xref>

Four rhesus monkeys (Macaca mulatta) were inoculated with a homogenate of a cutaneous lepromatous leprosy lesion from a mangabey monkey (Cercocebus atys). One died of B-cell lymphoma, and another died of an immunodeficiency syndrome. Cell suspensions prepared from the tumor and spleen of the monkey with lymphoma induced lymphoma or an immunodeficiency syndrome when inoculated into additional young rhesus monkeys. The immunodeficiency syndrome was similar to simian acquired immunodeficiency syndrome and consisted of opportunistic infections, lymphoid hyperplasia or atrophy, wasting, and syncytial cell formation. Mitogen responses and percentages of T4- and T8-positive lymphocytes were normal until the animals were moribund. Lymphoblastoid cell lines became established in vitro from tumor cell suspensions. These cells were infected with a herpesvirus related to Epstein-Barr virus. In addition, a retrovirus morphologically similar to human T-cell lymphotrophic virus type III (HTLV-III) and simian T-lymphotrophic virus type III (STLV-III) was isolated from one of the lymphoblastoid cell lines (LCL). Type D retroviruses could not be demonstrated in the monkeys in the transmission study; however, a retrovirus similar to that in the LCL was isolated from 4 animals by coculture of peripheral blood lymphocytes with the human cell line H9. These results suggest that this retrovirus, STLV-III/Delta, may be associated with the immunodeficiency syndrome in these macaques and may be of mangabey origin.

Analysis of a cytostatic lymphokine produced by incubation of lymphocytes with tumor cells: relationship to leukoregulin and distinction from recombinant lymphotoxin, recombinant tumor necrosis factor, and natural killer cytotoxic factor.

Supernatants from the coculture of peripheral blood lymphocytes and the NK-susceptible cell line K562 were highly growth inhibitory for a variety of tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by NK cells. Rather, the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin, a recently described lymphokine. The supernatants of tumor-lymphocyte cultures contained only low levels of IFN-alpha and IFN-gamma, and antibodies to interferons did not affect the observed growth inhibition. The pattern of target cell susceptibility to growth inhibition by this factor was also quite distinct from that seen with purified recombinant LT or TNF. Furthermore, monoclonal antibodies to these cytokines also had no effect on the cytostasis, arguing against a requirement for, or synergistic interaction with, low levels of these cytokines. Some of the targets susceptible to the factor were only growth inhibited but not lysed, thereby distinguishing it from NKCF. Furthermore, the cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which have been reported to block NKCF. Therefore, the results show that a cytostatic factor is released in tumor-lymphocyte incubation that is quite distinct from interferons, LT, and TNF but has characteristics that resemble those of leukoregulin.

The role of interferon in spontaneous cell-mediated cytotoxicity in pigs

Specific release of 51Cr and the production of interferon (IFN) increased in parallel in a spontaneous cell-mediated cytotoxicity (SCMC) assay in which uninfected PK-15 cells or PK-15 cells persistently infected with transmissible gastroenteritis virus (PK-15-TGE cells) were used as targets, and peripheral blood lymphocytes (PBL) from a young adult pig were used as effector cells. Higher levels of both specific 51Cr release and IFN were obtained in the assays containing PK-15-TGE cells. Co-cultivation of PBL from newborn piglets with PK-15-TGE cells yielded similar levels of IFN to those produced by co-cultivation of adult PBL and PK-15-TGE cells, but lower levels of IFN were produced by co-cultivation with uninfected PK-15 cells. Pretreatment of adult PBL with IFN augmented their SCMC effector activity for both PK-15 and PK-15-TGE cells in the 51Cr release assay. Pretreatment of the PK-15-TGE target cells with IFN did not affect their release of 51Cr in the SCMC reaction, while IFN pretreatment of PK-15 targets protected them against SCMC. In a single cell cytotoxicity assay the effects of IFN pretreatment on the effector adult PBL and on the PK-15 and PK-15-TGE target cells were confirmed, and SCMC incompetent PBL from neonatal piglets were rendered cytotoxic by pretreatment with IFN. PBL from newborn piglets bound to either target cell with the same frequency as PBL from SCMC competent adult pigs, and IFN pretreatment of either effector or target cells had no effect on target-binding frequency.

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line.

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells.

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.

Stimulation of human NK-cell activity by cultured cells. I. Response of normals.

Natural cell-mediated cytotoxicity, observed on co-cultivation of peripheral blood lymphocytes (PBL) with malignant melanoma tumor cells (M4), is described. The activity was inhibited when PBL were cultured in autologous serum. Studies using competitive inhibition with cold targets and stimulation of PBL with other tumor cell lines failed to detect specificity.