I. G. H. Schmidt-Wolf

PROPAGATION OF LARGE NUMBERS OF T CELLS WITH NATURAL KILLER CELL MARKERS

Summary. Previously, a subset of T cells co‐expressing the NK cell antigen CD56 has been described. These CD3+CD56+ cells are rare in peripheral blood collections and have been poorly characterized. We have developed culture conditions which allow for the rapid expansion of CD3+CD56+ cells. The protocol for cellular expansion includes the addition of interferon‐gamma on day O, interleukin‐1, interleukin‐2 and a monoclonal antibody against CD3 on day 1 to peripheral blood lymphocytes. Cells of the CD3+CD56+ phenotype increased up to 6000‐fold using this protocol after 16 d in culture. These cells have been characterized by flow cytometry and have been found to express the alpha, beta T cell receptor, co‐express the CD5 and CD8 antigens and do not express the CD16 antigen. Morphologically, these cells cannot be distinguished from NK cells. CD3+CD56+ killer cells lyse a variety of tumour cells with intermediate activity between CD3−CD56+ NK cells and CD3+CD56− T cells.

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12

Abstract Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.

Lymphocyte apoptosis: induction by gene transfer techniques.

Efficient gene transfer of lymphocytes has been shown to be extremely difficult. The molecular background for this gene transfer resistance is not completely understood. We reasoned that apoptosis may play a role in this gene transfer resistance of lymphocytes. We show that transfection of lymphocytes via nonviral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via the TNFα and TNFα receptor pathway, we studied the amount of TNF secreted by transfected lymphocytes. The percentage of apoptotic lymphocytes correlated well with TNFα secretion. TNF secretion was dependent on the gene transfection method used. High amounts of TNF secretion were detected using receptor-mediated gene transfer and lipofection. In con- trast, only low amounts of TNF were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF secretion was due to the use of anti-CD3 antibody. Induction of apoptosis and increase in necrosis was blocked using an anti-TNF antibody. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with the interleukin-2 or interleukin-7 gene. In conclusion, gene transfer techniques led to TNF secretion, apoptosis and necrosis of lymphocytes. This could be blocked using an anti-TNF antibody. Blockage of apoptosis after gene transfer should have an impact on the use of lymphocytes transfected with cyto- kine genes as immunologic effector cells in cancer gene therapy protocols.

Lymphokine-activated killer cell activity after cryopreservation.

The effect of cryopreservation on the cytotoxic activity of lymphokine-activated killer (LAK) cells was studied. LAK cells were generated by incubating peripheral blood lymphocytes for 3-5 days with recombinant interleukin-2 (rIL-2) and then cryopreserved using a programmed freezer. Cytotoxicity was determined in a 51Cr release assay. After thawing, the LAK cells had reduced cytotoxicity (25.5-39.1% as compared to the original lytic units). Cytotoxic activity could be restored to pre-cryopreserved levels by reincubation with rIL-2 for 2 days after thawing. Thus, maximal cytotoxicity of cryopreserved LAK cells could be achieved by incubation with rIL-2 before and after the freezing process. The level of cytotoxicity was comparable to that of LAK cells from fresh peripheral blood lymphocytes. Cryopreserved LAK cells may have potential in adoptive immunotherapy.

In vitro and in vivo activity of murine lymphokine‐activated killer cells after cryopreservation

The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine‐activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin‐2 (rIL‐2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL‐2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre‐B‐cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival > 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival >120 days, 50% survival > 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.

Potential of Autologous Immunologic Effector Cells for Prediction of Progression of Disease in Patients with Chronic Myelogenous Leukemia

In autologous bone marrow transplantation, immunologic effector cells such as lymphokine activated killer (LAK) cells may be useful for purging of bone marrow since these cells might have an additional in vivo effect on tumor cells in contrast to other purging protocols. Recently, immunologic effector cells termed cytokine-induced killer (CIK) cells have been shown to be more useful than LAK cells for purging of autologous BM in the context of autologous BMT. Here, we show that the expression of bcr/abl in CIK cells generated from patients with CML correlates with progression of disease in individual patients. In addition, progression of disease from chronic phase to accelerated phase could be predicted in two patients by studying the expression of bcr/abl in CIK cells generated from CML patients. Thus, it might be possible to use CIK cell generation for the prediction of progression of disease in CML patients.

Coexpression of Lymphoid and Myeloid Markers on Cell Surfaces

Cells coexpressing lymphoid and myeloid cell surface markers have been described for various leukemias and non-Hodgkins lymphomas. It is unclear whether these mixed lineage characteristics are due to malignancies of early progenitor cells or alternatively to malignant cells with lineage infidelity. Recently, it has been shown that cells coexpressing lymphoid and myeloid markers can be generated from peripheral blood lymphocytes from normal individuals as well. In this review, consequences of this surprising fact are discussed.