Studies on the relationship of human natural killer and lymphokine-activated killer cells with lysosomal staining and analysis of surface marker phenotypes

We have previously demonstrated that natural killer (NK) cells are lysosome-rich and stain more intensely with lysosomotropic agents such as neutral red and quinacrine (Qu) than do non-NK cells. In this study we combined the quantitation of Qu staining with surface marker staining to define subpopulations of NK cells. While all NK activity was contained within the Qu+ population, most but not all NK cells expressed the surface marker CD16. A subpopulation of NK cells was found to be Qu+CD16- composed of medium- to large-sized cells with a granular appearance on Giemsa staining. Culture with interleukin-2 (IL-2) induced enhanced cytotoxicity in peripheral blood lymphocytes (PBL) against NK-sensitive and NK-resistant tumor cells. Like NK cells, these lymphokine-activated killer (LAK) cells were predominantly Qu+CD16+. However, some LAK cells were Qu+CD16-. The Qu+CD16+ cells were typical large granular lymphocytes (LGL). The Qu+CD16- cells were also large lymphocytes, more than 50% of which were proliferating. However, the granulation in some Qu+CD16- cells, as detected by Giemsa staining, was more prominent and numerous than others in the same population. No LAK activity was ever detected in Qu- cells, which were uniformly small lymphocytes. Quantitation of Qu staining in effector cells was therefore demonstrated to have a good correlation with NK and LAK functions, and with surface markers can help to characterize both types of cells. Moreover, these results indicate that both NK and LAK populations include a small subset of CD16- cells in each.     

Similarities between LAK cells derived from human thymocytes and peripheral blood lymphocytes: expression of the NKH-1 and CD3 antigens

Human thymocytes are devoid of NK cells but develop lymphokine-activated killer (LAK) activity after culture with recombinant interleukin-2 (rIL-2). The most active precursor for this activity appears to be a CD3-negative cell. The purpose of these studies was to compare the phenotype and functional activities of thymocyte and peripheral blood lymphocyte (PBL) LAK cells. Following culture, rIL-2-activated thymocytes resemble PBL-generated LAk and PBL NK cells. For each of these populations, lytic activity is highest in NKH-1-positive cells. Two-color fluorescence of each population also indicates that NKH-1+ cells are highly granular, as measured by staining with the lysosomotropic vital dye quinacrine. PBL, PBL-derived LAK cells, and thymus-derived LAK cells have a portion of cells that express both CD3 and NKH-1. However, approximately 60-80% of NKH-1+ cells lack detectable CD3. This suggests that both CD3+ and CD3- cells may be capable of LAK activity. Thymic-derived LAK cells respond to interferon in a manner very similar to NK and PBL-derived LAK cells, but lack the NK-associated CD16 antigen. Thus, despite the absence of NK cells in the thymus, it is possible to generate thymocyte LAK activity which bears a strong resemblance to LAK activity derived from peripheral blood lymphocytes.     

Human lymphokine-activated killer cells: further isolation and characterization of the precursor and effector cell

In a series of experiments we have demonstrated the progressive enrichment (5- to 40-fold) in lymphokine-activated killer (LAK) precursor activity by adherence depletion, sheep red cell rosetting, and depletion of CD3- and DR-positive lymphocytes. The LAK precursor cell thus appears to fall within the 'null' cell population. CD16 and CD11 are cell surface antigens expressed on the surface of the LAK precursor as demonstrated in sorting experiments. A 6- to 100-fold enrichment compared to unseparated peripheral blood was noted when sorted cells positive for CD16 and CD11 were tested. The LAK effector has been identified as being primarily CD3- and CD2+. Similar sorting equipment demonstrated a 7- to 500-fold difference in lytic activity for fresh tumor when comparing CD2+/CD3- and CD2+/CD3+ cells. The CD16+/CD11+ lymphocyte can proliferate in response to interleukin-2 (IL-2) alone in the absence of accessory cells and can be expanded in IL-2 alone with maintenance of lytic activity.     

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44^lo) or elevated (CD44^hi) expression of CD44 are generated and that the CD44^hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.     

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells

We analyzed the antigenic phenotype of lymphokine-activated killer (LAK) effector cells. Human blood lymphocytes were cultured for 3 days with 100 U/ml recombinant interleukin 2 (rIL 2), subpopulations isolated with monoclonal antibodies and a fluorescence-activated cell sorter (FACS) and assayed for cytotoxic activity against 51chromium labeled noncultured melanoma tumor cells. Initial experiments compared the LAK effector function of CD5+ T lymphocytes vs CD5- cells (predominantly CD16+ NK cells). The mean percent specific release at a 10:1 effector:target (E:T) ratio was 25% +/- 16 for CD5- cells, 10% +/- 6 for CD5+ cells, and 22% +/- 9 for unsorted cells. In contrast, when lymphocyte subpopulations were isolated before rIL 2 culture (LAK precursors), CD5- cells but not CD5+ cells developed LAK activity (28% +/- 12 vs 1% +/- 1, mean percent specific release, 10:1 E:T ratio), confirming our previous results showing that only CD16+ cells were LAK precursors. The discrepancy between LAK effector and precursor phenotypes suggested that LAK precursors acquired CD5 determinants during rIL 2 culture; however, double label immunofluorescence of rIL 2 cultured CD16+ cells showed that this was not the case. The data suggested that in the presence of other cell types, some T lymphocytes may develop LAK activity, but purified blood T lymphocytes do not develop LAK function when cultured with rIL 2 alone. We also analyzed LAK effector function in lymphocyte subpopulations defined by CD4 and CD8 antigens. The data showed that lymphocytes with a low density expression of CD8 and no expression of CD4 were enriched for LAK effector cells, whereas CD4+ and CD8- had less activity than unsorted cells. Lymphocytes with a high density expression of CD8 had activity similar to unsorted cells. We also assessed the contribution of Leu-7 (HNK-1) granular lymphocytes to LAK effector function. After culture with IL 2, lymphocytes were depleted of Leu-7+ cells by antibody and complement treatment and then were sorted into CD5+ and CD5- fractions. The cytotoxic activity of Leu-7-CD5+ cells was a mean 5% +/- 5 vs a mean 14% +/- 8 for the total CD5+ population (20:1 E:T ratio). The activity of Leu-7- CD5- was slightly less than the total CD5- fraction (21% +/- 9 vs 28% +/- 14, 10:1 E:T ratio). In conclusion, LAK effector function was highest in non-T cell (CD5- CD16+) populations and some activity was also present in T cell populations (CD5+ and predominantly Leu-7+).     

Murine lymphokine-activated killer (LAK) cells: phenotypic characterization of the precursor and effector cells

Murine and human lymphocytes incubated in recombinant interleukin 2 (RIL 2) generate a population of cytotoxic cells (lymphokine-activated killer cells [LAK]), which are able to lyse a wide array of fresh tumor cells but do not lyse fresh normal cells. Intravenous administration of these cells with the concomitant administration of RIL 2 can eliminate established pulmonary and hepatic metastases in mice. To characterize the cell that has in vitro LAK activity, we subdivided murine lymphocytes by lysing select subpopulations with the use of complement and antibodies against lymphocyte surface markers or by fluorescence-activated cell sorting. Thy-1.2-negative splenocytes were found to generate near normal amounts of LAK activity after RIL 2 incubation. Small and inconsistent LAK cell activity was generated from Thy-1.2-positive splenocytes. Ia-positive and surface immunoglobulin-positive splenocytes had little or no LAK precursor capability and did not appear to be necessary for LAK activation. Treatment of splenocytes with anti-asialo GM1 (anti-ASGM1) heterosera and complement markedly decreased their ability to generate LAK activity. At the effector stage, cytotoxic cells were of the Thy-1.2-positive, Ia-negative phenotype. Ia-depleted cells were separated into subpopulations bearing or not bearing the gamma Fc receptor (gamma FcR). The majority of cytotoxicity resided in gamma FcR-positive cells. Thus the precursors of murine LAK cells are "null" lymphocytes bearing neither T nor B cell surface markers but develop the Thy-1.2 cell surface marker in vitro, in association with the development of lytic activity for fresh tumor cells after stimulation by RIL 2.     

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.     

Lysis of murine macrophages by lymphokine-activated killer-like cells induced by BCG in vitro.

The lymphokine-activated killer (LAK)-like activity was found to be induced in mouse splenocytes cultured together with Bacillus Calmette-Guérin (BCG). The killer cells induced by BCG were capable of killing both NK-sensitive (YAC-1, P388D1) and NK-resistant (P815) tumor cells. As an important finding, they also lysed syngeneic macrophages (M phi). The anti-M phi killer activity appeared on day 2, and reached a peak on day 5 of culture. Phenotype analysis of the killer cells by depletion techniques using monoclonal antibody (mAb) and complement indicated that the majority of these anti-M phi killer cells were Thy-1+ and asialo GM1+. This M phi cytolysis could be inhibited by the addition of cold M phi, YAC-1 tumor cells, and P815 tumor cells, suggesting that the same population of the effector cells recognize M phi and tumor cells. The addition of anti-MHC class I, anti-MHC class II, anti-L3T4, or anti-Ly-2 mAb directly to assay cultures did not affect anti-M phi cytolysis, suggesting that the MHC molecules are not involved in the cytolysis of M phi by the BCG-induced killer cells. The addition of anti-LFA-1 mAb partially inhibited the cytotoxicity, suggesting importance of the contact between targets and effectors in the cytolysis. Our present data suggest that activation of murine lymphocytes with BCG induces LAK-like cells capable of killing a wide variety of tumor cells as well as M phi and this anti-M phi cytolysis is mediated by nonspecific killer cells.     

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.     

Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease

Prompted by our recent finding that lymphokine-activated killer (LAK) cells mediate both veto and natural suppression, we tested the ability of adoptively transferred LAK cells to block two in vivo alloreactions which complicate bone marrow transplantation: resistance to transplanted allogeneic bone marrow cells, and lethal graft-vs-host disease. Adoptive transfer of either donor type B6D2 or recipient-type B6 lymphokine-activated bone marrow cells, cells found to have strong LAK activity, abrogated or inhibited the resistance of irradiated B6 mice to both B6D2 marrow and third party-unrelated C3H marrow as measured by CFU in spleen on day 7. The ability of lymphokine-activated bone marrow cells to abrogate allogeneic resistance was eliminated by C lysis depletion of cells expressing asialo-GM1, NK1.1, and, to a variable degree, Thy-1, but not by depletion of cells expressing Lyt-2, indicating that the responsible cells had a LAK cell phenotype. Similar findings were obtained by using splenic LAK cells generated by 3 to 7 days of culture with rIL-2. Demonstration that allogeneic resistance could be blocked by a cloned LAK cell line provided direct evidence that LAK cells inhibit allogeneic resistance. In addition to inhibiting allogeneic resistance, adoptively transferred recipient-type LAK cells prevented lethal graft-vs-host disease, and permitted long term engraftment of allogeneic marrow. Irradiation prevented LAK cell inhibition of both allogeneic resistance and lethal graft-vs-host disease. These findings suggest that adoptive immunotherapy with LAK cells may prove useful in preventing graft rejection and graft-versus-host disease in human bone marrow transplant recipients.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Distinct and non-cross-reactive epitopes are recognized on B16 melanoma by LAK cells and anti-B16 monoclonal antibodies.

Two rat anti-B16 melanoma monoclonal antibodies (MoAb), designated IB16-6 and IB16-8, recognize an epitope expressed with high density on the surface of B16 parental cells and B16-F1, F10, F10FLR, and BL6 sublines. The purpose of this study was to define by means of cytolytic and clonogenic assays whether these MoAbs reacted with the same or distinct determinants as those recognized on B16 targets by lymphokine-activated killer (LAK) cells. Using 125I-labeled antibody and Scatchard analysis, the affinity constant (KA) of IB16-6 was determined to range from 5.6 to 9.4 x 10(8) liter/M and the number of receptor sites per B16 cell was 4.8 x 10(4) to 2.5 x 10(5). The effects of anti-B16 MoAb on LAK activity were determined by either preincubating 51Cr-labeled B16 target cells with varying concentrations of MoAb, followed by the cytolytic assay, or exposing unlabeled B16 cells to MoAb, and then carrying out a 10-day clonogenic assay. Over a wide range of antibody concentrations, IB16-6 and IB16-8 had minimal effects on LAK activity, and even at MoAb concentrations up to 1 mg there were no changes in target cell sensitivity or colony-forming ability. Enzymatic treatment of B16 melanoma cells with either trypsin or pronase completely removed the epitope recognized by MoAb IB16-6 but did not alter B16 sensitivity to LAK cells. These observations indicate that the LAK recognition unit was distinct from the epitope reactive with MoAb IB16-6 and that the B16 determinant(s) recognized by LAK cells is resistant to proteolytic enzymes. The molecular structure of each of these remains to be determined.     

Mouse tumors are heterogeneous in their susceptibility to syngeneic lymphokine-activated killer cells and delineate functional subsets in such effectors

We have analyzed whether lymphokine-activated killer (LAK) cells, generated from C57BL/6J (B6) spleen cells at different times after recombinant interleukin-2 (rIL-2) culture, could be heterogeneous in their ability to lyse a variety of tumor targets. When tested 3 days after exposure to 250 U/ml rIL-2 (day-3 LAK cells) a significant lysis was detected with the natural-killer(NK)-sensitive YAC lymphoma, the NK-resistant P815 mastocytoma, three different syngeneic melanomas and a syngeneic fibrosarcoma (group 1 targets), whereas no lysis was observed with a reticulum cell sarcoma, two different lymphomas or concanavalin A blasts, all of B6 origin (group 2 targets). LAK cells cultured for 5 days, however, lysed group 2 targets and showed a parallel increase of cytotoxic activity against group 1 targets. At day 7, LAK activity declined on all targets examined. In cold-target inhibition studies, the lysis of group 1 tumor targets by day-3 or day-5 LAK cells could be inhibited only by group 1 and not by group 2 unlabelled tumor cells. All group 1 tumors could effectively compete each other. Conversely, the lysis of group 2 tumor targets by day-5 LAK cells was inhibited by both group 1 and group 2 targets. These data indicate the presence of separate LAK effectors that appear to arise with different time kinetics and have different recognition structures. In vitro antibody depletion at the effector level showed that day-3 LAK cells with cytotoxic activity against group 1 tumors were ASGM1+. Day-5 LAK cells included both ASGM1+ and Lyt2+ effectors and both populations, although to a different extent, contributed to the lysis of all targets. Our results indicate that LAK cells are functionally heterogeneous. This heterogeneity is defined by their susceptible target cells and cannot be ascribed to different (Lyt2+ versus ASGM1+) lineages.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.