Nonadherent PBMC Research Articles

Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12.

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL+IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21day) NK cell proliferation. The expanded NK cells are predominantly CD56(bright), and we show that isolated CD56(dim)CD16(+) NK cells can switch to a CD56(bright)CD16(-) phenotype and proliferate in response to 4-1BBL+IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required 'help' from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL+IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56(bright) and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.

Characterization of optimal T Cell/Dendritic Cell (DC) Co-Culture Conditions for Ex Vivo Expansion of Antigen-Specific Human T Cells.

Adoptive T cell immunotherapy has demonstrated clinical efficacy in controlling reactivation of human herpesviruses (e.g., cytomegalovirus, CMV and Epstein-Barr Virus, EBV) that are known etiologic agents of life-threatening illness in immunocompromised populations. Early approaches utilized in the expansion of large numbers of human virus-specific CD4+ and CD8+ T cell often involved laborious culture techniques impractical for widespread clinical use in populations at risk, including stem cell transplant (SCT) recipients. Advances in our understanding of the biology of myeloid dendritic cells (DC) can now facilitate more practical approaches for ex vivo antigen-specific T cell expansion. These advances have included novel approaches to isolate human peripheral blood monocytes (e.g., CD14 selection) and the optimization of cytokine cocktails inducing DC maturation. To define the optimal culture conditions for human antigen-specific T cell expansion, we conducted an iterative series of pairwise CMV-specific T cell expansions, in which important determinants of the expansion process could be directly compared. T cells, derived from apheresis products obtained from CMV-seropositive healthy donors, were expanded under GLP-compliant conditions, following incubation with autologous myeloid DC pulsed with overlapping pentadecapeptide pools spanning the CMV pp65 protein. In each case, we assessed the expanded CMV-specific T cell populations by immunophenotyping of the generated DC, and by phenotypic analysis, cytokine flow cytometry (CFC), ELISPOT analysis and HLA-peptide tetramer staining of the expanded T cells. Specifically, we compared:DC generated from monocytes isolated via plastic adherence to those isolated via positive selection of CD14+ T cells using magnetic beads;DC matured using tumor necrosis factor-α (TNFα) alone or in combination with other cytokines (ITIP, containing TNFα, IL-1β, IL-6 and PGE2);the quality of expansions derived from a starting lymphocyte population selected by non-adherence to plastic vs. the CD14-negative fraction in the monocyte selection process.From this series of experiments, we can conclude that while CD14 selection of monocytes results in a more phenotypically homogeneous population of DC, that this does not improve the quantity or quality of expanded virus-specific T cells. When we compared TNFα to ITIP maturation of DC, we found that while ITIP induced significantly higher CD83 expression on DC, TNFα-matured DC favored the maintenance of CMV-specific CD4+ T cells in culture resulting in ultimately greater numbers and functional proportions of tetramer-stained CD8+ T cells. Finally, we consistently found that the use of non-adherent PBMC resulted in dramatically better quantitative expansions of CMV-specific CD4+ and CD8+ T cells, vs. a lymphocyte population obtained following depletion of monocytes using CD14 positive selection. These results suggest a set of optimal T cell/DC co-culture conditions that lead to the generation of the largest numbers of expanded, functional CD4+ and CD8+ T cells, and are likely to have relevance for human trials wherein adoptive immunotherapy and/or DC immunization is planned.

Monocyte/Macrophage Infection Is Associated with the Development of Simian Immunodeficiency Virus Encephalitis in Pigtailed Macaques

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It is hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the CNS during end stages of immunosuppression. The relationship between development of encephalitis and circulation of infected monocytes and why only a fraction of infected hosts develop lentiviral encephalitis is unknown. We proposed to examine the whether monocyte/macrophages from pigtailed macaques that develop simian immunodeficiency virus (SIV) encephalitis (SIVE) contain more replication competent virus than macaques that do not develop SIVE during the course of infection and at necropsy. Compared to macaques that did not develop SIVE, the monocyte associated SIV-DNA load of monocytes and the capability of monocyte-derived macrophages and nonadherent PBMC to produce virus ex vivo was increased in macaques that developed SIVE. Macaques with SIVE had more infected macrophages in peripheral organs with the exception of lymph nodes. Brains with SIVE had greater numbers of T cells and NK cells with cytotoxic potential than brains without encephalitis. However, T cell and NK cell infiltration in SIVE was more modest than observed in classical acute viral encephalitides. These findings support the hypothesis that inherent differences in host monocyte/macrophage viral production are associated with the development of encephalitis.

Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells

Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), pre-stimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5–9 days, induced CD3−/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover, we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies.

The susceptibility of macrophages to human immunodeficiency virus type 1 X4 isolates dependson their activation state

The demonstration that macrophages express CXCR4 has led to a reexamination of their susceptibility to human immunodeficiency (HIV)-1 X4 strains. Here, we examined the susceptibility to X4 HIV-1Lai of two previously characterized macrophage populations, obtained either as 1) adherent cells of five-day cultures of blood mononuclear cells (PBMC), followed by two days without nonadherent PBMC nor added cytokines (MDM-5d); or 2) as adherent cells recovered from one-hour incubation of PBMC, which were cultured for seven days with macrophage colony-stimulating factor (MDM-MCSF). Exposing MDM-5d or MDM-MCSF to HIV-1Lai did not lead to productive infection, as indicated by a lack of (MDM-MCSF) or low (MDM-5d) viral p24 levels in culture supernatants. However, MDM-5d vigorously transmitted HIV-1Lai to autologous T lymphocytes, which was not the case of HIV-1Lai-exposed MDM-MCSF. PCR analysis of the LTR RU5 region showed that X4 HIV-1Lai entered into both types of macrophages in the same manner as R5 HIV-1BaL. However, in contrast to MDM-5d, there was a block of HIV-1Lai retrotransciption in MDM-MCSF. Cytokine profile analysis of the two types of macrophages showed that TNF-α, IL-6 and RANTES levels were higher in MDM-5d than in MDM-MSCF, while the IL10 level was higher in MDM-MCSF, both producing similar IL16 levels. Altogether, these data indicate that HIV-1 X4 strains enter into macrophages but that their replication is blocked thereafter in a different manner according to the activation status of the cells.

Soluble Glycosaminoglycans Do Not Potentiate RANTES Antiviral Activity on the Infection of Primary Macrophages by Human Immunodeficiency Virus Type 1

Macrophages play an important role in human immunodeficiency virus (HIV)-1 infection. They exist in various differentiation and activation states in vivo, a heterogeneity that may affect their interactions with HIV-1 and susceptibility to drugs. Here, we found that RANTES and MIP-1β, heparin, or soluble chondroitin sulfate B, but not chondroitin sulfate A, inhibited HIV-1BaL infection of macrophages obtained as the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without either nonadherent PBMC or added cytokines (MDM-5d), whereas they did not affect infection of macrophages obtained as the adherent cells recovered from 1-h incubation of PBMC and subsequent 7-day culture with macrophage colony-stimulating factor (MDM-MCSF). Such different behavior was not related to differences in HIV-1 binding but rather to postbinding steps, as HIV-1BaL attached similarly to MDM-5d and MDM-MCSF, a binding that was affected by soluble glycosaminoglycans but not by RANTES. Of note, CCR5 expression on both types of MDM was comparable, and it was not downregulated by RANTES on either. Mixing RANTES with each of the glycosaminoglycans did not restore inhibition of MDM-MCSF infection by HIV-1; however, heparin at concentrations that had low antiviral activity for MDM-5d counteracted RANTES anti-HIV-1 activity for these cells, whereas chondroitin sulfate B had no additive effect on that of RANTES. Both glycosaminoglycans affected RANTES binding to MDM. Thus, in contrast to cell surface proteoglycans that contribute to the attachment of RANTES to macrophages and enhance its anti-HIV-1 activity, soluble glycosaminoglycans do not facilitate, and may even offset, the anti-HIV-1 activity of RANTES.

G-CSF reduces IFN-gamma and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function.

Despite a 10-fold increase of T cell dose, the incidence and severity of acute GVHD following allogeneic transplantation of G-CSF-mobilized PBSC is not increased compared to BMT. Experimental murine studies demonstrate that G-CSF polarizes donor T cells toward a type 2 cytokine response. To determine whether G-CSF alters T cell cytokine responses, we investigated the effects of G-CSF administration on T cell proliferative and cytokine responses to alloantigen and Con A in nonadherent PBMC (NAC) and CD3+ T cells obtained from normal individuals before and after G-CSF administration (10 microg/kg x 4 days). Although T cell proliferative and cytokine (IFN-gamma and IL-4) responses to alloantigen stimulation and Con A were significantly reduced in post-G-CSF NAC, they were restored by the removal of non-T cells from post-G-CSF NAC. Furthermore, there was less T cell alloreactivity in MLR in the presence of autologous post-G-CSF monocytes than in the presence of pre-G-CSF monocytes. This alteration was not replicated in vitro by culturing PBMC with G-CSF. These results suggest that G-CSF administration suppresses T cell proliferative and cytokine (IFN-gamma and IL-4) responses to allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplantation (2000).

The Inhibitory Effect of RANTES on the Infection of Primary Macrophages by R5 Human Immunodeficiency Virus Type-1 Depends on the Macrophage Activation State

We investigated whether culture conditions could affect the RANTES antiviral effect on human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages. Monocyte-derived macrophages (MDM) were obtained either as (1) the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without nonadherent PBMC or added cytokines (MDM-5d), or (2) as the adherent cells recovered from 1-h incubation of PBMC, which were cultured for 7 days with macrophage colony-stimulating factor (M-CSF; MDM-MCSF). Infection of MDM-5d from different donors with HIV-1 R5 strains was reproducibly inhibited by RANTES (IC50≤ 10 nM), but infection of MDM-MCSF was not inhibited by ≥100 nM RANTES, even when added at initiation of cultures, although it was still inhibited by a CD4 antibody. RANTES had no antiviral effect when MDM-5d were treated with physiological concentrations of M-CSF or GM-CSF before infection. CCR5 and CXCR4 expression as well as that of other cell surface molecules, including adhesion molecules, was not affected by the cytokines. MDM-MCSF from Δ32CCR5 homozygous individuals did not render them permissive to HIV-1, suggesting that it is unlikely that the virus uses another coreceptor. RANTES binding to MDM was chondroitin sulfate, but not heparan sulfate, dependent, and RANTES bound more efficiently to MDM-5d than to MDM-MCSF. Chondroitin sulfate removal partially offset the RANTES antiviral effect for MDM-5d. Thus RANTES anti-HIV-1 activity for primary macrophages depends on culture conditions and their consequent activation status, which may lead to differences in proteoglycan surface expression. These data may be relevant for the development of chemokine-based therapy for HIV-1 infection.

A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis.

The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF-alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.

Natural killer (NK) cells as effectors of antibody‐dependent cytotoxicity with chimeric antibodies reactive with human squamous‐cell carcinomas of the head and neck

In patients with cancer, antibody-dependent cellular cytotoxicity (ADCC) may be used as a laboratory test or for enhancing immunotherapy with murine monoclonal or chimeric mouse/human anti-tumor antibodies (mMAbs or cMAbs, respectively). We have established an ADCC assay with IgG1 cMAb SF-25, using human squamous-cell carcinoma of the head and neck (SCCHN) cell lines as targets. By flow cytometry, all SCCHN cell lines tested expressed the antigen recognized by cMAb SF-25. Trypsinization of the cell monolayers facilitated binding of cMAb SF-25 to the antigen on the cell surface of SCCHN targets. Using the PCI-50 SCCHN cell line as a target coated with this cMAb at the optimal concentration of 1.0 micrograms/ml, normal human peripheral blood mononuclear cells (PBMC, n = 28) were found to mediate ADCC at a mean level of 283 +/- 42 (SEM) lytic units (LU20/10(7) effector cells). Non-adherent monocyte-depleted PBMC and natural killer (NK) cells purified by negative selection mediated significantly higher levels of ADCC than unseparated PBMC against SCCHN targets. NK cells, defined as CD3-CD56+ cells, could be effectively armed by cMAb SF-25, as confirmed by flow cytometry and ADCC assays. IL2-activated armed NK cells mediated higher levels of ADCC than non-armed NK cells. Binding of cMAb SF-25 to NK cells and their ADCC were enhanced by pre-incubation with polyethylene glycol. Arming of NK cells with chimeric antibodies should be considered in developing novel strategies for treatment of human SCCHN, especially in the adjuvant setting.

Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1.

Recent evidence indicates that integrins do transduce signals that are involved in the regulation of a number of cellular processes. We have shown previously that human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their adhesion to fibronectin (FN). Here we investigate whether cross-linking of beta 1 FN receptors on human NK cells stimulates tyrosine kinase activation. Our results indicate that cross-linking of beta 1 integrins on NK cells, either freshly isolated from PBL or generated from 10-day coculture of nonadherent PBMC with an irradiated EVB+ lymphoblastoid B cell line (long-term activated NK cells), altered the tyrosine phosphorylation pattern. In particular, we found stimulation of tyrosine phosphorylation of two proteins migrating with an apparent mass of 105 and 115 kDa, which was not observed after CD16 engagement. Phosphorylation of pp105-115 was already observed at 1 min, raised maximal values at 3 to 5 min, and persisted until 20 min after stimulation. Tyrosine phosphorylation of pp105-115 was also observed upon engagement of alpha 4 beta 1 and alpha 5 beta 1 FN receptors either with mAb directed against the alpha subunits or after NK cell adhesion to FN or its 120- and 40-kDa fragments. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of phosphorylation stimulated through alpha 4 beta 1 and alpha 5 beta 1 integrins, indicating that ligation of FN receptors on NK cells activates tyrosine kinase(s). Overall our results suggest that beta 1 integrins on NK cells play a major role as signaling molecules in the regulation of NK cell functions.

Long term activation of natural killer cells results in modulation of beta 1-integrin expression and function.

Integrin expression and function is largely modulated by cell activation. Here we provide evidence that long term activation of human NK cells results in a marked modulation of beta 1-integrin expression and adhesive functions. By flow cytometry and immunochemical analysis we have detected induction of alpha 1 beta 1 and alpha 2 beta 1, increased expression of alpha 4 beta 1 and alpha 5 beta 1, and decline of alpha 6 beta 1 on CD3-CD56+ NK cells generated from 10-day coculture of nonadherent PBMC with irradiated RPMI 8866 EBV+ lymphoblastoid B cell line. Adhesion assays performed on extracellular matrix-coated plates showed that, unlike fresh NK cells, long term-activated NK cells bind to native collagen I via alpha 2 beta 1 and to heat-denatured collagen I in an RGD-dependent manner, although they lose the ability to bind to laminin. In regard to the adhesion to FN, no major quantitative changes are observed after long term NK cell activation. However, whereas alpha 4 beta 1 and alpha 5 beta 1 completely mediate the adhesion of fresh NK cells to fibronectin, binding of activated NK cells is only partially beta 1-dependent and seems to involve also non-beta 1-integrin(s) recognizing and RGD sequence. The modulation of beta 1-integrin expression and the acquisition of new adhesive properties on long term-activated NK cells may be relevant for their traffic and tissue localization during inflammation and immune response.

In-vitro effects of cyclosporin A, FK506, 6-mercaptopurine, and prednisolone on lymphokine-activated killer cells

In transplant recipients, immunosuppressive regimens are deleterious on natural killer (NK) and lymphokine-activated killer (LAK) cells, which beyond their well-known antitumoral activity display many important biological functions. In order to find which regimens could preserve NK and LAK functions we tested the influence of CsA, FK506, 6-mercaptopurine and prednisolone on HLA-unrestricted cytotoxicity during an in-vitro IL-2 activation. For each drug we obtained peripheral blood samples from 11 healthy volunteers. Non-adherent PBMC were incubated 2 days with either CsA, FK506, 6-mercaptopurine or prednisolone, whose concentrations ranged from 0 to 10 micrograms/ml, in order to screen infratherapeutic, therapeutic, and supratherapeutic doses. Thereafter, 100 IU of IL-2 were added for a further 3-day culture. Before and after the culture, we analysed (1) the cell subsets by direct immunofluorescence staining with anti-CD3/CD16/CD56 antibodies, (2) the LAK activity with the lysis of Daudi cells, (3) the cell proliferation with a 24-h incorporation of thymidine. Cyclosporin and FK506 did not impair the LAK cytotoxicity nor the number of LAK cells, whereas both prednisolone and 6-mercaptopurine decreased the LAK cytotoxicity, the number of CD3- CD16+ CD56+ cells, and the thymidine uptake. As a whole, the LAK cytotoxicity was correlated with the number of CD3- CD16+ CD56+ cells but not with the number of CD3+ CD16- CD56- cells, and it also increased with the incorporation of thymidine. This latter was correlated with the number of CD3- CD16+ CD56+ cells, but not with the number of CD3+ CD16- CD56- cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of IFN-alpha in peripheral blood mononuclear cells by HIV-infected monocytes. Restricted antiviral activity of the HIV-induced IFN.

PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV-infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN-alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20-fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease.

Immunosuppression by human gangliosides: II. Carbohydrate structure and inhibition of human NK activity

Gangliosides shed by tumors enhance tumor formation, possibly by suppressing host antitumor immune function, and gangliosides purified from animal tissues and cultured cells inhibit human cellular immune function in vitro. Determination of immunosuppressive activity of highly purified gangliosides, to uncover structure-activity relationships, is therefore important. Here we have studied a series of gangliosides obtained from human tissue and determined their effects on human natural killer (NK) activity. Total gangliosides from human brain tissue were moderately inhibitory; 100 nmol/ml reduced NK activity of human nonadherent PBMC by 43%. The influence of carbohydrate structure upon inhibitory activity was determined by study of eight highly (HPLC) purified individual gangliosides. Of these, we unexpectedly found that the two minor brain gangliosides with the simplest carbohydrate structures, G M2 and G M3 , were very active inhibitors (75 and 47%, respectively, at 50 nmol/ml). In contrast, the structurally more complex major species, G M1 , G D1a , G D1b , G T1b , and two other minor gangliosides, G D2 and G D3, were inactive. Reduced effector-target binding in a single-cell binding assay by G M2 but not G M3 suggests different mechanisms of inhibition by these two active gangliosides. Since G M2 and G M3 are present in high concentrations in, and are shed by, several common human tumors (e.g., neuroblastoma, melanoma, and glioma), their ability to inhibit NK cytotoxicity supports the hypothesis of a role of shed tumor gangliosides in the enhancement of tumor formation.

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury.

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.

Inhibition of cytokine production by cyclosporin A and transforming growth factor beta.

We investigated the ability of cyclosporin A (CsA) and transforming growth factor beta (TGF-beta) to modulate the production of TNF-alpha and TNF-beta and IFN-gamma by unseparated, nonadherent, and adherent PBMC. Treatment of unseparated PBMC with CsA resulted in a significant dose-dependent inhibition of all three cytokines ranging from greater than 90% inhibition for IFN-gamma and TNF-beta, to approximately 70% for TNF-alpha. Pretreatment of unseparated or nonadherent PBMC with TGF-beta inhibited the production of IFN-gamma by 60-70%. However, the inhibition of TNF-alpha and TNF-beta production by these cells was only minimally affected, and at 0.1-1 ng/ml TGF-beta could enhance TNF-alpha production by unseparated PBMC. In contrast, pretreatment of adherent PBMC with TGF-beta inhibited the production of TNF-alpha by approximately 60%. TGF-beta also inhibited both TNF-alpha production and tumor cell cytotoxicity mediated by murine peritoneal-derived macrophages. These observations indicate that the biological effects of CsA and TGF-beta on immune functions are of a wider range than previously reported.

Requirement for HLA-DR+ accessory cells in natural killing of cytomegalovirus-infected fibroblasts.

The role of HLA-DR+ cells in NK activity against CMV-infected FS4 foreskin fibroblasts and K562 erythroleukemia cells was examined. When nonadherent PBMC were depleted of either HLA-DR+ or Leu-11b+ cells by treatment with mAbs plus C, NK activity against CMV-FS4 target cells was markedly reduced. In contrast, depletion of HLA-DR+ cells had no effect on NK activity against K562 target cells. When HLA-DR-depleted cells were added to Leu-11b-depleted cells, NK activity against CMV-FS4 was restored. Negative selection experiments indicated that the HLA-DR+ cells contributing to NK activity against CMV-FS4 are not B or T cells, while negative and positive selection experiments excluded a role for monocytes. Experiments in which HLA-DR- and Leu-11b- cells were mixed in varying proportions indicated that NK(CMV-FS4) is mediated by Leu-11b+ cells, while HLA-DR+ cells provide an accessory function. Irradiation (50 GY) abolished the NK effector function of Leu-11b+ cells, but not the accessory function of HLA-DR+ cells. The NK activity against CMV-FS4 of HLA-DR- cells was restored by the addition of rIFN-alpha or of cell-free supernatants generated by coculturing PBMC or Leu-11b- cells with CMV-FS4. The ability of these supernatants to restore NK activity of HLA-DR- cells was completely abrogated by the addition of neutralizing amounts of antibody to IFN-alpha. In related experiments, neutralization of IFN-alpha in NK assays had little or no effect on NK activity against CMV-FS4, suggesting that the accessory function of HLA-DR+ cells might be mediated by alternative mechanisms in addition to the secretion of extracellular IFN-alpha.

Induction of lymphokine activated killer cells in serum-free medium

Lymphokine activated killer (LAK) cells may play a role in immunosurveillance against spontaneous neoplasms. To date, LAK cells have been grown in medium supplemented with human serum (HS). Formulation of a defined medium that supports LAK cell generation would be useful to delineate the mechanisms that regulate LAK cell induction. When compared to HS medium, optimal induction of LAK cells required medium containing transferrin, insulin, bovine serum albumin, fatty acids (linoleic, oleic, palmitic), pyruvate and indomethacin. In addition, when 5% autologous monocytes were added to PBMC cultured in serum-free medium without indomethacin, marked suppression of LAK cell induction occurred. Addition of indomethacin abrogated suppression and resulted in enhanced cytotoxicity compared to non-adherent PBMC cultured in HS medium.

Discrepancy in the expression of autoantibodies in healthy aged individuals

Sera from 50 healthy old subjects and from 51 young controls were tested by ELISA assays for a panel of autoantibodies, including IgM RF, anti-DNA, anti-F(ab′) 2, anti-thyroglobulin, anti-human albumin, anti-human hemoglobin, anti-secretory component from human IgA, and anti-gliadin. In vitro production of anti-DNA as well as anti-F(ab′) 2 antibodies were measured after stimulation of PBMC by pokeweed mitogen (PWM) in 12 healthy elderly subjects and 11 young controls. Sera from elderly donors contained threefold higher amounts of IgM RF than young controls ( P < 0.001). On the contrary, the levels of anti-DNA as well as anti-F(ab′) 2 antibodies were similar in both groups ( P < 0.3 for the two determinations). Anti-DNA and anti-F(ab′) 2 antibodies were also measured in supernates of PWM-stimulated glass nonadherent PBMC cultures from both old and young healthy donors without finding any significant difference between the two groups. Additional ELISA tests were also performed in both elderly and young control sera to detect antibodies against six other different antigens. No significant difference was found in the percentages of positive sera between the two groups. This discrepancy in production of IgM RF compared to other autoantibodies in healthy elderly subjects does not provide support for a general increase of autoantibodies with aging. The biological significance of an increase in IgM RF with aging remains to be determined.