SummaryNatural killer cell stimulatory factor (NKSF), or interleukin 12 (IL 12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35 . NKSFAL 12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of EpsteinBarr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSFAL 12 . Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBVtransformed cell lines with phorbol diesters . The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon y (IFN-y) production from T and NK cells, one of the effects of NKSFAL 12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSFAL 12 . Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC) . The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSFAL 12 (i.e., IFN-y induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSFAL 12 mediates the same functions . Moreover, these activities are significantly inhibited by anti-NKSF/IL12 antibodies . The neutralizing anti-NKSF/IL12 antibodies also inhibit 85% of the IFN-y production in response to SAC, an NKSF/IL 12 inducer, and approximately 50% of the IFN-y production in response to non-NKSF/IL12-inducers such as IL-2, phytohemaglutinin, and anti-CD3 antibodies . These results indicate that induced or constitutively produced NKSFAL12 has a major role in facilitating IFN-y production by peripheral blood lymphocytes . Our findings that NKSFAL 12 is both spontaneously produced and inducible in adherent PBMC and lymphocytes suggest that NKSFAL 12 might be a major physiological regulator of T and NK cell function during an immune response and inflammation .i A. D'Andrea and M . Rengaraju contributed equally to this work . 1387J . Exp. Med .

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV+ individuals have a significant decrease in the number of the Lin−HLA-DR+CD123+ and BDCA-2+ PDC compared with uninfected donors (p = 0.0001). HIV+ individuals also have a sustained impairment in viral-induced IFN-α production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-α secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.

Peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients, asymptomatic or with acquired immunodeficiency virus, produced 10-fold less interleukin 12 (IL-12) free heavy chain and fivefold less biologically active IL-12 heterodimer than PBMC from uninfected healthy donors when challenged in vitro with the common human pathogen Staphylococcus aureus. In contrast, PBMC from HIV-infected individuals and uninfected control donors produced similar levels of tumor necrosis factor alpha, IL-1 beta, and IL-10, and PBMC from HIV-infected individuals produced three- to fourfold more IL-6 compared with PBMC from uninfected control donors. The defect in IL-12 production is not due to hyperproduction of IL-10, a cytokine exerting an autocrine-negative feedback on IL-12 production, but was directly related to HIV infection, as suggested by the reduced ability of monocytes infected in vitro with HIV to produce IL-12. IL-12 deficiency may be an important component of the immunodeficiency associated with HIV infection.

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-γ and TNF-α-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV+ individuals had significantly lower proportions of mature CD3−/CD161+/CD56+ NK cells and of IFN-γ-producing NK cells compared with noninfected donors, independent of CD4+ T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3−/CD161+/CD56+ NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-γ. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-γ, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-γ secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.

Interleukin-12 (IL-12) is a disulfide-linked heterodimeric cytokine originally identified as a product of EBV-transformed B cell lines. Monocyte/macrophages are the physiologically most relevant producers of IL-12, in response to both Gram-positive and -negative bacteria, bacterial products, and intracellular parasites. Although IL-12 has an enhancing effect on the survival and growth of early hematopoietic progenitor cells, most of the IL-12 biological activity has been described on T and NK cells, on which it induces production of lymphokines, primarily IFN-gamma, enhances cytotoxic activity, and, in cooperation with other stimuli, increases proliferation. IL-12 is an inducer of development of T helper type 1 (Th-1) cells and the equilibrium between IL-12 and IL-4 is probably important for the balance in vivo between Th-1 and Th-2 responses. IL-12 has an important role in the host resistance to infection, in particular to intracellular pathogens, by activating macrophages through induction of IFN-gamma from NK and T cells and by enhancing cell-mediated immune responses, dependent on Th-1 cell development. Peripheral blood mononuclear cells from HIV-seropositive individuals are impaired in their ability to produce IL-12 in response to bacterial stimulation, and IL-12 restores in vitro some of the depressed immunological functions, suggesting that a defect in IL-12 production may have a pathogenic role in the immunodeficiency of HIV-infected individuals. Natural IL-12 appears to provide a regulatory link between innate resistance and the development of the antigen-specific adaptive immune response and the recombinant protein has therapeutic potential because of its activity against tumors and infections and its effectiveness as an adjuvant enhancing cell-mediated immunity in vaccination.

No abstract

SummaryNatural killer cell stimulatory factor (NKSF), or interleukin 12 (ID12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and ID2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5 -NK cells and does not confer cytotoxic activity to CD16-CD5 + T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumorderived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV-infected patients with NKSF and/or Ib2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon 3' in response to NKSF and/or II.-2, although at levels 5-or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/ID12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.N 'atural killer cell stimulatory factor (NKSF) 1 is a heterodimeric lymphokine composed of a heavy (p40) and a light chain (p35) covalently linked (1, 2). The NKSF p40 chain belongs to the recently defined cytokine receptor family of proteins and has structural homology with the cellular receptor for II.-6 (3). NKSF has also recently been described as cytotoxic lymphocyte maturation factor (CLMF); the name I1.-12 has been proposed for this lymphokine (4, 5).NKSF was originally purified from the cell-free supernarant fluid of phorbol diester-activated human B lymphoblastoid cell lines. The two genes coding for the p40 and p35 chains 1 Abbreviations used in this paper: n, natural; NKSE natural killer cell stimulatory factor. of NKSF have been molecularly cloned; simultaneous transfection of mammalian cells with the two genes is necessary for the production of biologically active NKSF (2, 5). However, B lymphoblastoid-cell lines produce an excess of free P40 chain, in addition to the biologically active heterodimer (1, 4).NKSF exerts a variety of biological functions on human T and NK cells and possibly on other cell types. In particular, it has been shown to: (a) induce IFN-3' production from both T and NK cells and synergize in this effect with I1.-2, mitogens, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens (1, 2, 6); (b) exert a comitogenic effect on fresh resting T cells together with lectins or phorbol diesters (1, 2); (c) mediate a direct mitogenic effect on activated T or NK cell blasts (5, 7...

We analyzed dendritic cell (DC) and NK cell compartments in relation to CD4 recovery in 21 HIV-infected subjects followed to <50 copies/ml once starting antiretroviral therapy (ART) and observed for 52 wk of sustained suppression. Although CD4 counts increased in all subjects in response to ART, we observed a restoration of functional plasmacytoid DC (PDC) after 52 wk of sustained suppression under ART (from 1850 cells/ml to 4550 cells/ml) to levels comparable to controls (5120 cells/ml) only in subjects with a low baseline viral load, which also rapidly suppressed to <50 copies/ml upon ≤60 days from ART initiation. Recovery of PDC at week 52 correlates with level of CD95 expression on CD8 T cells and PDC frequency following first ART suppression. NK cytotoxic activity increased rapidly upon viral suppression (VS) and correlated with PDC function at week 52. However, restoration of total NK cells was incomplete even after 52 wk on ART (73 cells/μl vs 122 cells/μl in controls). Direct reconstitution experiments indicate that NK cytotoxic activity against virally infected target cells requires DC/NK cooperation, and can be recovered upon sustained VS and recovery of functional PDC (but not myeloid DC) from ART-suppressed subjects. Our data indicate that viremic HIV-infected subjects may have different levels of reconstitution of DC and NK-mediated function following ART, with subjects with lower initial viremia and the greatest reduction of baseline immune activation at VS achieving the greatest level of innate effector cell reconstitution.