Protection of cultured human monocytes from lymphokine-activated killer-mediated lysis by IFN-gamma

We have recently reported that IL 2-activated killer (LAK) cells are capable of lysing cultured human monocytes. In an effort to protect autologous monocytes from lysis, we treated monolayer cultures of adherent PBMC with various doses of human rIFN-gamma and assessed their susceptibility to LAK cells. IFN-gamma was shown to lessen the sensitivity of monocytes to lysis in a dose-dependent manner. Similar treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 h incubation with IFN-gamma was sufficient for the protective effects to take effect. Additionally, monocytes that were pulsed with IFN-gamma for 2 h, washed, and then cultured in medium alone retained their resistance to lysis for at least 3 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Furthermore, binding studies demonstrated that there was no significant difference between the number of conjugates formed by using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal. From these studies, it was apparent that treatment of monocytes with IFN-gamma lessened their sensitivity to LAK-mediated lysis. Thus, it may be possible through a specific sequence of IFN-gamma and IL-2 treatment that LAK activity could be manipulated against some tumor cells, but not normal cells, to abrogate some of the toxicity seen with this type of cancer therapy.     

Enhancement of human monocyte function against Candida albicans by the colony-stimulating factors (CSF): IL-3, granulocyte-macrophage-CSF, and macrophage-CSF

The effect of IL-3, granulocyte-macrophage (GM)-CSF and macrophage (M)-CSF on Candida albicans growth inhibition by human peripheral blood monocytes was investigated. By using a radiolabel microassay developed in our laboratory that makes use of the incorporation of [3H]glucose into residual C. albicans, we demonstrated that rGM-CSF and rIL-3 effectively enhanced human monocyte-mediated anticandidal activity. Incubation for 24 h with either GM-CSF or IL-3 significantly enhanced monocyte antifungal responses down to 0.01 U/ml. M-CSF, at higher concentrations of 10 U/ml, could also enhance monocyte function but to a smaller degree. None of the CSF interfered directly with fungal growth, even up to 1000 U/ml. Because IFN-gamma is also a known monocyte activator, its effect on monocytes was also assessed. Monocytes were first cultured in medium for several days and then further incubated with each of the cytokines. Monocytes aged in medium were found to lose their spontaneous anticandidal activity. Such aged monocytes did not develop anticandidal activity in response to IFN-gamma but did in response to GM-CSF or IL-3. To further elucidate this difference, fresh monocytes were continuously cultured with or without cytokines for 1 to 5 days before assessing their anticandidal activity. Monocytes cultured in IFN-gamma progressively lost their activity by 2 days but monocytes in GM-CSF or IL-3 maintained their high level of anticandidal activity throughout the whole length of culture. Therefore, GM-CSF and IL-3 not only enhanced fresh monocyte anticandidal activity, but maintained monocyte function for a longer period. These results suggest that GM-CSF and IL-3 may act on monocytes via a different pathway than does IFN-gamma.     

TNF-alpha and IFN-gamma reverse IL-4 inhibition of lymphokine-activated killer cell function

Recombinant IL-4 inhibits IL-2-induced lymphokine-activated killer (LAK) cell development of PBMC. We evaluated the effect of various cytokines in reversing IL-4-mediated LAK inhibition. PBMC were cultured in IL-2 (10-1000 u/ml) with or without IL-4 (2-100 u/ml) and tested for cytotoxicity against the NK-sensitive K562 cells and NK-resistant UCLA-SO-M14 cells. Addition of IL-4 at the beginning of culture suppresses LAK activity in a dose-dependent fashion. Addition of IFN-gamma or TNF-alpha partially reverses IL-4-mediated inhibition (30-100%) in a dose-dependent fashion. IFN-gamma and TNF-alpha must be added within the first 24 hr of initiating culture in order to reverse IL-4 inhibition. Furthermore, IFN-gamma and TNF-alpha are most effective at reversing IL-4 inhibition at low concentrations of IL-2 (less than 100 u/ml). Addition of other IL-2-induced cytokines such as GM-CSF (50 u/ml), M-CSF (250 u/ml), and IFN-alpha (10-10,000 u/ml) fails to reverse IL-4 inhibition. In addition to suppression of LAK induction, IL-4 also inhibits IL-2-induced IFN-gamma and TNF-alpha protein production in PBMC. The reversal of IL-4-mediated LAK inhibition by TNF-alpha and IFN-gamma may therefore be due to resupply of these endogenously suppressed cytokines.     

Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Effects of human alveolar macrophages on the induction of lymphokine (IL 2)-activated killer cells

Normal human alveolar macrophages (AM) significantly and reproducibly suppress induction of IL 2-activated killer (LAK) cell activity against allogeneic Burkitt's lymphoma (Daudi) cells. Incubation of purified peripheral blood lymphocytes for 4 days with autologous AM and 1 U/ml of IL 2 resulted in AM-mediated suppression of LAK activity, whereas peripheral blood monocytes isolated freshly by centrifugal elutriation from the same donor potentiated induction of LAK activity by IL 2. The suppression of LAK cell induction by human AM was dependent on the density of AM added to the lymphocyte cultures. Recombinant IFN-gamma did not affect AM-mediated suppression of LAK cell induction by IL 2. Both AM and monocytes stimulated with lipopolysaccharide markedly suppressed LAK cell induction by IL 2. AM-mediated down-regulation was seen only when AM were added immediately after the start of incubation of lymphocytes with IL 2; AM potentiated LAK activity when added 1 day later. Similar AM-mediated suppression of LAK cell induction was observed with four lines of allogeneic lung cancer cells as targets for LAK activity. These results indicate that AM may be important in regulation of in situ induction of LAK activity in the lung.     

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells.     

Lysis of mycobacteria-infected monocytes by IL-2-activated killer cells: role of LFA-1

Mycobacterium avium-intracellulare (MAI) is a ubiquitous soil contaminant that rarely causes disseminated disease in adults regardless of immunological status. In AIDS patients, however, this organism invades virtually every tissue and organ, and most conventional chemotherapeutic agents are usually ineffective against MAI. We report here that monocytes, in which MAI has established an intracellular parasitic stage, are under the control of natural killer (NK) cells. Autologous large granular lymphocytes (LGL), purified from human peripheral blood leukocytes, were capable of efficiently lysing autologous MAI-infected monocytes in a 5-hr 51Cr release assay. More importantly, interleukin 2 (IL-2) was able to activate the LGL to a higher degree of lysis of infected monocytes. LGL cultured in medium alone could not kill normal monocytes, but showed some degree of lysis of MAI-infected cells. IL-2 activated killer (LAK) cells, on the other hand, lysed normal monocytes to a moderate degree and this activity was makedly enhanced if the monocytes were infected with MAI. The sensitivity of monocytes was directly proportional to the inoculating number of bacteria, indicating that increased bacterial burden would enhance susceptibility to LAK-mediated lysis. Finally, the addition of monoclonal antibodies to LFA-1 (both alpha and beta chains), but not LFA-2 or LFA-3, blocked lysis of both infected and uninfected monocytes when added directly to the cytotoxicity assays, indicating that this adhesion protein is involved in the lysis of autologous, infected monocytes. Thus, NK/LAK cells may be important in containment of infection by lysis of infected monocytes before the bacteria can multiply and spread to other sites.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.     

Interleukin-10 contributes development of macrophage suppressor activities by macrophage colony-stimulating factor, but not by granulocyte-macrophage colony-stimulating factor

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.     

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     

IL-4 inhibits H2O2 production and antileishmanial capacity of human cultured monocytes mediated by IFN-gamma

The effect of IL-4 on the IFN-gamma-induced state of activation of cultured human monocytes was investigated with regard to their ability to produce hydrogen peroxide and their antileishmanial capacity towards the intracellular parasite Leishmania donovani. IL-4 was found to inhibit the IFN-gamma-dependent hydrogen peroxide production of monocytes. Treatment of monocytes with IFN-gamma (200 to 600 U/ml) for 48 h increased the hydrogen peroxide production fourfold above background. Coincubation of the monocytes with IL-4 (1 to 1000 U/ml) and IFN-gamma (200 to 600 U/ml) inhibited this increase by 50 to 100%. IL-4 alone did not modulate the hydrogen peroxide production of monocytes. Pretreatment of monocytes with IL-4 for 20 min to 3 h was already effective in preventing the IFN-gamma response. Addition of IL-4 not later than 6 h after the start of incubation with IFN-gamma was necessary for an optimal inhibitory effect. IL-4 also inhibited the IFN-gamma-induced antileishmanial capacity of monocytes: IFN-gamma (1000 U/ml) induced a 54 +/- 10% reduction in the number of parasites. Monocytes treated with combinations of IL-4 (100 to 1000 U/ml) and IFN-gamma (1000 U/ml) were unable to reduce the parasite numbers. IL-4 alone did not alter the uptake of Leishmania donovani nor induce antileishmanial activity. These results demonstrate that IL-4 disables human cultured monocytes to respond to IFN-gamma activation.     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Prostaglandin E2-mediated suppression of murine lymphokine-activated killer cell activity generated from tumor-bearing hosts by interferon-gamma

The effect of mouse recombinant interferon-gamma (IFN-gamma) on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, LAK-sensitive, spontaneously developed, weakly immunogenic, syngeneic murine mammary adenocarcinoma, a tumor model mimicking that of human disease. When all of the splenocytes prepared from tumor-bearing mice were cultured with recombinant interleukin-2 (IL-2) and IFN-gamma, LAK cell activity was suppressed in an IFN-gamma dose-dependent manner. An increase in the prostaglandin E2 (PGE2) content in the corresponding culture media was detected, as was IFN-gamma dose dependent. The suppression of generation of LAK cell activity by IFN-gamma was abrogated, accompanied by the elimination of the increase in PGE2 content, when plastic dish and nylon wool-treated nonadherent macrophage-depleted splenocytes were used. These results indicated that IL-2-induced LAK cell activity generated from the splenocytes of tumor-bearing mice was suppressed by IFN-gamma, and that PGE2 secreted from the macrophages of the splenocyte cultures served as the mediator in this IFN-gamma dose-dependent suppression of IL-2-induced LAK cell activity.     

Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.     

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.     

Monocyte-dependent,serum-borne suppressor of induction of lymphokine-activated killer cells in lymphocytes from melanoma patients

Summary Both phytohemagglutinin-induced cytotoxicity and recombinant-interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) activity against noncultured melanoma cells were significantly reduced when peripheral blood mononuclear cells (PBMC) from patients with metastatic melanoma were incubated in RPMI medium 1640 and 10% autologous human serum instead of 10% fetal calf serum, while serum from either healthy donors or patients with primary melanoma did not affect the level of cytotoxicity. The serum-mediated suppression was not restricted by major histocompatibility complex and was time-dependent. Addition of 10% human serum from the patients with metastatic melanoma [HS-Pt(m)] to the culture of PBMC with rIL-2 at the same time or 1 day after incubation significantly inhibited LAK activity. However, addition of 10% HS-Pt(m) 2 or 3 days after incubation did not inhibit LAK activity. Incubation of PBMC for 2 h with a high dose (10⁴ U/ml) of rIL-2 in the presence of 10% HS-Pt(m), followed by incubation in the absence of either rIL-2 or HS-Pt(m), did not affect LAK cell activity. These results suggest that HS-Pt(m) inhibits the early stage of LAK cell differentiation, rather than the binding of rIL-2 to PBMC or a later stage in the differentiation. In contrast to PBMC, monocyte-depleted peripheral blood lymphocytes exhibited comparable levels of LAK activity when cultured with rIL-2 either in 10% fetal calf serum, 10% human serum from healthy donors or 10% HS-Pt(m). Addition of purified autologous monocytes to the culture of monocyte-depleted peripheral blood lymphocytes with rIL-2 suppressed LAK cell induction when 10% HS-Pt(m) was present. Thus serum-mediated suppression of LAK cell induction is largely dependent on the presence of monocytes, which may produce a secondary inhibitor that acts on lymphocytes. Addition of indomethacin to the culture did not reverse this monocyte-dependent serum-mediated suppression in a majority of cases, suggesting that prostaglandin E₂ does not have a major role in the suppression.This work was supported in part by NIH grant RR5511-25 and grants from The Council for Tobacco Research USA Inc., The Meadows Foundation, the Erwin Zaban Melanoma Research Foundation, and the Gillson-Longenbaugh Foundation