Giuseppe Carra

Reciprocal activating interaction between natural killer cells and dendritic cells

We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-α. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2–activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4+ T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-γ and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.

The Reciprocal Interaction of NK Cells with Plasmacytoid or Myeloid Dendritic Cells Profoundly Affects Innate Resistance Functions

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-γ through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-α and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.

Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand β-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) γ strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by β-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor β, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4+ T cells, IL-1β, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans.

Summary: Interleukin‐12 (IL‐12) and IL‐23 share a common chain. Yet, their production in response to pathogens is differentially regulated, and their functions are distinct and often antithetic. IL‐12 is involved in the induction or amplification of the T‐helper (Th) type 1 response, whereas IL‐23 has been associated with the generation of the Th17 response and IL‐17 production. Mycobacterium tuberculosis and yeast zymosan induce IL‐23, but in the absence of other stimuli, no IL‐12 is induced in human dendritic cells (DCs). The stimulation of IL‐23 by M. tuberculosis was mostly explained by the triggering of Toll‐like receptor (TLR2) and the cytoplasmic receptor nucleotide oligomerization domain (NOD)‐containing protein 2, whereas zymosan induces IL‐23 primarily by stimulating the β‐glucan receptor dectin‐1 alone or in combination with TLR2. IL‐23, IL‐6, transforming growth factor (TGF‐β1), and IL‐1β in supernatants from activated human DCs induce human naive CD4+ T cells to produce IL‐17. These data are consistent with various recent reports that TGF‐β is an inducer of IL‐17 production both in human and in mouse cells. However, IL‐1 is necessary in combination with some or all of the other cytokines to induce IL‐17 production in human T cells. The ability of various stimuli to induce Th17 cells depends not only on their induction of IL‐23, IL‐6, and TGF‐β production in DCs but also on their ability to activate directly or indirectly the inflammasome and to induce IL‐1β.

Biosynthesis and Posttranslational Regulation of Human IL-12

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light α-chain, formerly defined as p35, disulfide-linked to a heavier β-chain, formerly defined as p40. The β-chain is also produced in large excess in a free form, and disulfide-linked β-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 α and β genes (P5-0.1). The IL-12 heterodimer and free β-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the β-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the α-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the α-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free β-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. β-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.

Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes

The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.

Different sensitivity to interleukin 4 of interleukin 2- and interferon α-induced CD69 antigen expression in human resting NK cells and CD3+, CD4−, CD8− lymphocytes

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.

Heterogeneity of lymphokyne-activated killer (LAK) populations at the clonal level: Both NK and CD3+, CD4−, CD8− clones efficiently mediate tumor cell killing

To investigate at the clonal level the phenotypic and functional properties of interleukin 2 (IL-2) activated killer cells (LAK), recombinant IL-2 activated peripheral blood lymphocytes were cultured under limiting conditions. Among 56 clones that lysed P815 in the presence of phytohemagglutinin (PHA) (22% of total proliferating microcultures) 36 clones lysed also the natural killer (NK)-sensitive K562 and the NK-resistant Hu126 glioma cell lines and one clone lysed only the K562 cell line. Several LAK clones were further assayed for both phenotype and functional activity. Of 22 clones, 10 were CD3-, CD4-, CD8-, and expressed the CD16 marker of NK cells; only one clone had the conventional phenotype of cytolytic T cells (CD3+, CD4-, CD8+), while 11 clones were CD3+, CD4-, CD8- and did not express alpha/beta heterodimer of T-cell antigen receptor as identified by WT31 monoclonal antibody. Only one of the latter clones was CD16+. Endogenous production of IL-2 after stimulation with PHA and phorbol myristate acetate was positive in 3/9 CD3- and in 8/8 CD3+, CD4-, CD8- clones. CD3- mediated strong antibody-dependent cellular cytotoxicity, a function exerted also by some CD3+, CD4-, CD8- T-cell clones to a lower extent. CD3+, CD4-, CD8- T-cell clones lysed different major histocompatibility complex unrelated tumor targets; moreover, this lytic activity seems to be CD3 dependent.

Selective association of a 22-38 kDa glycoprotein with MHC class II DP antigen on activated human lymphocytes at the plasma membrane.

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.