Sidney H. Golub

Lymphokine-activated killer cell activity:Characteristics of effector cells and their progenitors in blood and spleen

Leukocytes in blood and spleen can be activated by interleukin 2 (IL-2) to become cytotoxic to certain tumor cell lines in vitro. Recent evidence suggests that such lymphokine-activated killer (LAK) cells can bring about the regression of solid tumors in animals and patients, under certain circumstances. Here, Ronald Herberman and colleagues from eight international laboratories, review what is known of the characteristics of LAK cell activity and conclude that most of it can be attributed to natural killer cells stimulated by IL-2.

Studies on Cytotoxicity Generated in Human Mixed Lymphocyte Cultures: II. Anti-K562 Effectors Are Distinct from Allospecific CTL and Can Be Generated from NK-Depleted T Cells

SummaryHigh levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.

Modulation of natural killer and lymphokine‐activated killer cell cytotoxicity by lactoferrin

Natural killer (NK) and lymphokine‐activated killer (LAK) cell cytotoxic functions can be strongly augmented by the iron‐carrier protein lactoferrin (LF). LF significantly enhances NK and LAK activities when added at the beginning of NK or LAK cytotoxicity assays. LF is effective in augmenting cytotoxic activities at concentrations as low as 0.75 μg/ml, and higher concentrations of LF induce greater augmentation of NK and LAK. Iron does not appear to be essential for LF to increase NK and LAK, as depleting iron from LF with the chelator deferoxamine does not affect the capacity of LF to increase cytotoxicity. LF is known to have RNase enzymatic activity, and LF enhancement of NK and LAK can be blocked by RNA. However, LFs from two different sources with over 100‐fold difference in RNase activity are equally effective in enhancing NK and LAK. Furthermore, purified non‐LF RNase does not modulate NK or LAK activity and DNA is as effective as RNA in blocking LF augmentation of NK or LAK cytotoxicity. Therefore, the RNase activity is unlikely to be responsible for LF enhancement of the cytotoxicities. Newborn infants are known to have low NK activity and NK and LAK cells have been implicated in host defense against microbial infections. Thus, maternal milk‐derived LF may have a role in boosting antimicrobial immunity in the early stages of life. In adults, LF released from neutrophils may enhance NK and LAK functions in the inflammatory process induced by microbial infections.

Interferon-α Primed Tumor-Infiltrating Lymphocytes Combined with Interleukin-2 and Interferon-α as Therapy for Metastatic Renal Cell Carcinoma

AbstractMurine models demonstrate therapeutic synergy for the combination of interleukin-2, interferon-α and tumor-infiltrating lymphocytes. We treated 11 patients with metastatic renal cell carcinoma with a novel regimen consisting of in vivo primed tumor-infiltrating lymphocytes, interferon-α and interleukin-2. Patients received interferon-α before radical nephrectomy; in vivo primed tumor-infiltrating lymphocytes were isolated and expanded in vitro. Low dose continuous infusion inter-leukin-2 at a dose of 2 × 106 units per m2 per day was administered for 96 hours during each treatment week and interferon-α was administered as a subcutaneous injection at a dose of 6 × 106 units per m.2 per day on days 1 and 4 of the interleukin-2 infusion. No therapy was given during the last 3 days of a treatment week. One course of therapy consisted of 3 weeks of therapy followed by 3 weeks of rest. Patients were treated until maximal response, disease progression or dose limiting toxicity. A maximum of 6 courses of t...

Human pulmonary macrophages utilize prostaglandins and transforming growth factor β1 to suppress lymphocyte activation

The ability to activate peripheral blood lymphocytes (PBLs) in vitro with interleukin‐2 (IL‐2) is suppressed by the presence of autologous human pulmonary alveolar macrophages (AMs). AMs suppress both IL‐2‐induced proliferation and the induction of lympho‐ kine‐activated killer cell (LAK) activity in a dose‐ dependent manner (79 ±6% suppression of LAK activity at a 0.25:1 AM/PBL ratio). Increasing the IL‐2 concentration increased baseline LAK activity but did not prevent AM‐mediated suppression. At least two different mechanisms of suppression were observed, one diffusible in nature and the other contact dependent. Indomethacin prevented the component of inhibition that diffused across porous polycarbonate membranes, indicating prostaglandins as the diffusible inhibitor. In contrast, indomethacin had no effect when added alone into conventional AM‐ PBL cocultures, but a combination of indomethacin and anti‐transforming growth factor β1 (TGF‐β1) antibody did prevent inhibition. This result suggests that TGF‐ß1 acts as an additional contact‐dependent inhibitor. PBLs that were rendered unresponsive to IL‐2 completely recovered their responsiveness within 4 days after removing AMs from the coculture. These features suggest that pulmonary macrophages have multiple mechanisms for locally suppressing IL‐2 responses and lymphocyte activation.

The Use Of Viable Frozen Lymphocytes For Studies In Human Tumor Immunology

Viable frozen lymphocytes displayed activity in blastogenesis assays that was indistinguishable from freshly prepared lymphoid cells. Similarly, cytotoxic activity of lymphocytes against melanoma target cells from melanoma patients was only slightly affected by the freezing procedure. Frozen lymphocytes provided a highly reproducible source of cells in these assays. The use of viable frozen peripheral blood lymphoid cells for the retrospective analysis of a cancer patients immune response is described.

Cell-Mediated Immunity Is Depressed Following Cardiopulmonary Bypass

Sequential in vitro lymphocyte function tests in 13 patients undergoing cardiac operation were performed to determine factors that contribute to depressed cell-mediated immunity following operation. Lymphocytes were stimulated with phytohemagglutinin (PHA), pokeweed mitogen, concanavalin A (Con A), and mitomycin-treated, pooled, allogeneic lymphocytes (MLC). Mitogen responses were measured by 3H-labeled thymidine incorporation. Circulating levels of T, B, and Fc-receptor lymphocytes were determined by counting E, EAC, and EA rosettes. Serum cortisol was measured by radioimmunoassay. The T-cell-dependent lymphocyte responses (PHA, Con A, and MLC) were significantly decreased 24 hours after operation, and this was accompanied by a 60% decrease in circulating T-cell levels. The PHA, Con A, and MLC responses, and circulating T-cell levels returned to preoperative values one week following operation. Lymphocyte responses to mitogens remained significantly decreased when the number of T cells in the postoperative cultures were adjusted to preoperative levels. This indicates that the T cells remaining after operation were functionally impaired. We conclude that lymphocyte proliferative responses and antigen recognition are significantly depressed following cardiac operation, and that these responses are related to decreased numbers of circulating T lymphocytes and depressed function of the remaining T lymphocytes.

The regulatory effect of adherent cells on lymphokine activated killer cells

We analyzed the effect of adherent cells on the induction of lymphokine-activated killer (LAK) cell activity by depleting adherent cells from peripheral blood lymphocytes (PBL) or by adding adherent cells to PBL before culture with interleukin-2. We found that adherent cells clearly down-regulate LAK cell induction. These inhibitory effects are dependent on the number of adherent cells added. Inhibitory effects of adherent cells are abolished by the addition of indomethacin to the LAK culture. Soluble factors derived from adherent cells, such as interferons and interleukin-1, have a slight enhancing effect on LAK induction. In contrast, adherent cells appear to inhibit LAK induction primarily by producing prostaglandin E2(PGE2). PGE2 in turn inhibits the induction of LAK effector cells by inhibiting the expression of the transferrin receptor on LAK cells. These effects are manifested most strikingly in the early phases of LAK induction.

Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19+ large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.