Prostaglandin D2 suppresses human NK cell function via signaling through D prostanoid receptor.

NK cells play critical roles in immune responses against tumors or virus infections by generating type 1 cytokine and cytotoxicity responses. In contrast, during type 2 dominant immune responses, such as allergic diseases, activities of NK cells are often impaired. These type 2 immune-mediated diseases have been reported to be closely associated with local production of PGD(2). PGD(2) is an eicosanoid primarily synthesized by mast cells and alveolar macrophages, and it functions through two major receptors, D prostanoid receptor (DP) and chemoattractant receptor-like molecule on the Th2 cell. Within the immune system, PGD(2) binding to DP generally leads to suppression of cellular functions. In the current study, we show that: 1) DP is expressed in human NK cells as detected by mRNA analysis and Western blot; 2) PGD(2) inhibits cytotoxicity, chemotaxis, and type 1 cytokine production of human NK cells via signaling through DP; 3) PGD(2) signaling via DP elevates intracellular cAMP levels and the inhibitory effects on NK cells are cAMP dependent; 4) PGD(2) binding to DP suppresses Ca(2+) mobilization triggered by the cross-linking of the activating receptor, CD16. Together, these data uncover a novel mechanism by which PGD(2) functions through DP to suppress type 1 and cytolytic functions of human NK cells, thus contributing to the promotion of a type 2 immune response.

Human peripheral CD2-/lo T cells: an extrathymic population of early differentiated, developing T cells.

We previously reported that a subset of human peripheral blood CD3+ T cells expresses low-to-null CD2 levels (CD2-/lo), produces type 2 cytokines and is inducible to differentiate to functionally mature IFN-gamma+ cells. Multiple-color immunofluorescence analysis indicated that this population, representing <0.1% of the T cells in fresh lymphocytes, contains subsets that are phenotypically immature, including CD4-CD8- and CD3+TCR- cells. Ex vivo, the CD2-/lo cells can proliferate (carboxyfluorescein diacetate succinimidyl ester analysis) independently from exogenous stimulation, respond to CD3-mediated stimulation with significantly greater proliferation than the autologous mature cells and their subsets are inducible to undergo in vitro a developmental sequence similar to that reported for the phenotypically similar thymic populations. This is especially evident for the CD4+CD8+ subset. CD2-/lo T-cell populations exhibit a TCR repertoire (Vbeta chain distribution) that is complete but different (complementarity determining region R3 analysis) from that of the autologous CD2+ T cells. These characteristics distinguish peripheral CD2-/lo T cells as possible early differentiated T cells that may undergo extrathymic maturation, and potentially contribute to maintain the peripheral naive T-cell pool. These findings define the existence of phenotypically immature T cells in the periphery. Also, given the high numbers of CD2-/lo T cells generated, upon ex vivo culture, from peripheral lymphocytes of all adult and neonatal individuals tested, they have relevance to clinical applications for immune reconstitution of T cells, as well as myeloid cells, via myeloid colony-stimulating factors and type 2 cytokines.

Peripheral NK cell phenotypes: multiple changing of faces of an adapting, developing cell.

We have defined the existence of developmental relationships among human peripheral NK cells with distinct phenotypic and functional characteristics. These findings closely parallel the changes that occur in vivo during NK cell development, and in vitro in experimental culture systems supporting NK cell generation from hematopoietic progenitors. These new insights provide a simplified framework to understand NK cell immunobiology and the cellular bases for their roles in innate immunity, initiation and maintenance of immune responses via regulation of adaptive and accessory cell functions, and immune pathologies.

The IL-12 signature: NK cell terminal CD56+high stage and effector functions.

We report that human peripheral NK cells expressing high CD56 levels (CD56(+high)) are terminally differentiated cells indistinguishable from mature NK cells recently activated in the presence of IL-12, and not a functionally distinct NK-cell subset or progenitors to mature CD56(+low) NK cells. CD56(+high) NK cells coexpress all differentiation Ags constitutive or inducible in mature (CD56(+)) NK cells, except CD16, present at lower level than on most mature NK cells. Also, activation markers, activating receptors and adhesion molecules, and most inducible receptors are expressed exclusively and constitutively and are inducible at higher levels on CD56(+high) than on CD56(+low) NK cells. Consistent with their activated phenotype, many CD56(+high) NK cells are cycling and mediate heightened effector functions (proliferation, IFN-gamma and IL-10 but not IL-13 production) in response to IL-12 and other NK cell-specific stimuli. Conversely, IL-12 induces on CD56(+low) NK cells all markers constitutively expressed on the CD56(+high) NK cells, concomitantly preventing the IL-2 (and IL-15)-inducible expression of NKp44 and CD16 re-expression after immune complex-induced down-modulation, and CD56(-/+low) NK cells acquire a CD56(+high) NK cell phenotype in short term in vitro culture with IL-12. The significance of these findings to the NK cell-mediated regulation of immune responses and NK cell development is discussed.

Differential regulation of NK cell proliferation by type I and type II IFN.

IFN, produced during viral infections by accessory (type I IFN) or NK cells (type II IFN), play a primary role in the regulation of immune and anti-viral NK cell effector functions. Because IFN have anti-proliferative effects on several cell types, including hematopoietic cells, we asked whether they modulate proliferation of human NK cells, and whether IFN-alpha and IFN-gamma mediate distinct effects on NK cells at different developmental stages. Analysis of proliferation at the single-cell level in human NK cells indicated that both IFN types inhibit IL-4-induced accumulation of immature CD56(-) IL-13(+) NK cells in freshly separated peripheral blood lymphocytes and in cells derived from them after short-term cultures. However, IFN-gamma inhibited specifically the IL-4-dependent proliferation of these cells without affecting the IL-2-dependent one or that of the IL-13(-) cells, whereas IFN-alpha attenuated proliferation of NK cells at any developmental stage (both immature CD56(-)IL-13(+) and mature CD56(+)IL-13(-) IFN-gamma(+) NK cells) and contributed to their monokine-induced differentiation to IFN-gamma-producing cells. Adding to our previous report that IL-13 inhibits accumulation of mature IFN-gamma(+) NK cells, the present data unravel a mechanism by which peripheral immature IL-13(+) and mature IFN-gamma(+) NK cells can negatively regulate each other's accumulation.

Multiple color immunofluorescence for cytokine detection at the single-cell level.

Detection of cytokines and identification of the producer cells are essential to define the interplay and the role of distinct leukocyte subsets in the development of immune and inflammatory responses. Several methods used to study cytokine expression are based on detection of the encoding mRNA (Northern blot, RNase protection assay, RT-PCR) or of protein in the supernatant from stimulated cells (ELISA, RIA, ELISPOT). These are simple and useful, but have limitations related to the need of using purified cell populations to precisely define the effector cells, and exception made for RT-PCR and ELISPOT assays, the requirement for relatively large numbers of cells for sufficient resolution. Here we present a method based on immunofluorescence (flow cytofluorimetry) to detect intracellular accumulation of cytokines in mixed leukocyte populations. It has the distinct advantage of: 1. identifying producing cells in mixed cell populations, 2. comparing quantitatively the levels of production within cells in a given population and among different cell subsets, and 3. defining simultaneous production of distinct cytokines by a single cell type. The detailed description/discussion of the method uses natural killer (NK) cells as an example, but this method can be applied to the study of other cell types, and is of special interest/use when analyzing subsets present in very low proportion.

Accumulation of type 2 cytokine+ T cells: differentiation-independent proliferation of pre-existing type 2 T cells.

Analysis of proliferation and cytokine production at the single-cell level indicated that proliferation of pre-existing type 2 cytokine(+) human peripheral naive T cells (CD4(+) and CD8(+)) accounts for the accumulation of type 2 T cells in lymphocytes cultured with IL-2, with or without IL-4, and independently from TCR-mediated stimulation. This is because: firstly,the number of cells progenitor to the type 2 cytokine(+) T cells accumulated in culture is lower than that of the original cytokine(+) cells; secondly, percentages and numbers of the accumulated type 2 cytokine(+) T cells depend on those in the original lymphocyte population; thirdly, no accumulation occurs in cultures of lymphocytes experimentally depleted of type 2 cells; and fourthly, naive T cells do not require proliferation before producing type 2 cytokines. In contrast, accumulation of IFN-gamma(+) T cells in cultures with IL-12 can not be explained with induced proliferation of pre-existing IFN-gamma(+) cells, but depends on differentiation from more-immature cells. These novel insights into the regulation of type 2 and type 1 cytokine(+) T cells provide a new understanding of the cellular bases for the regulation of immune responses and for manipulating the immune system in clinical settings.

NKT and T cells: coordinate regulation of NK-like phenotype and cytokine production.

A maturation-dependent change in phenotype and cytokine production from relatively immature CD161(-) or CD161(+), IL-13(+)IL-4(+), IFN-gamma(-), to mature CD161(+)CD56(+) IFN-gamma(+) cells occurs in primary human alpha-galactosyl ceramide-reactive CD1d-restricted natural killer T (NKT) cells under the control of IL-12 and other monokines. Modulation of this process upon alpha-galactosyl ceramide stimulation explains the opposite roles of NKT cells to drive type 1 and type 2 immune responses. Because the same developmental changes occurred and were similarly regulated in T cells, the data establish that NKT cells should no longer be considered a functionally unique regulatory subset. However, the results of their analysis can be taken as a model for immunotherapeutic approaches with T cells for which a nominal or surrogate antigen is defined.

Peripheral immature CD2-/low T cell development from type 2 to type 1 cytokine production.

Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2(-/low) T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN-gamma or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13(+)IFN-gamma(+) cells producing high IL-2 levels, and finally IL-13(-)IFN-gamma(+) cells. The monokine combination IL-12, IL-18, and IFN-alpha substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2(-/low) and primary mature CD2(+) IL-13(+) T cells. IFN-alpha is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-gamma(+) ones. Upon TCR-mediated stimulation, IFN-gamma(+) cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine(+) T cells are immature cells that can differentiate to effector IFN-gamma(+) cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity.

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-gamma and TNF-alpha-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-gamma-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-gamma. 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-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma 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.

Expression of type 1 (interferon gamma) and type 2 (interleukin-13, interleukin-5) cytokines at distinct stages of natural killer cell differentiation from progenitor cells.

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161(+)/CD56(+) NK cells from these cultures contained a tumor necrosis factor alpha (TNF-alpha)(+)/granulocyte macrophage-colony-stimulating factor (GM-CSF)(+) subset, an interferon gamma (IFN-gamma)(+) subset, mostly included within the former, and very few IFN-gamma(-)/IL-13(+) cells. Instead, most immature CD161(+)/CD56(-) NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-alpha, and GM-CSF, but not IFN-gamma, and contained an IL-5(+) subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-gamma. Part of these produced both IFN-gamma and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161(+) NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-gamma.

Distinction between IL-13+ and IFN-gamma+ natural killer cells and regulation of their pool size by IL-4.

The hypothesis that distinct subsets of NK cells produce type 2 and type 1 cytokines in resting naive lymphocytes was tested analyzing cytokine production at the single-cell level. Two non-overlapping IL-13+ and IFN-gamma+ subsets were identified in adult and neonatal NK cells. IL-2 maintained their relative proportion. Accumulation of the former was induced by IL-4, but not IL-13, and inhibited by IL-12; that of the latter was induced by IL-12 and inhibited by IL-4 and IL-13. IL-4 induced preferential proliferation of the pre-existing peripheral IL-13+ cells, whereas IL-12 had minimal effect on proliferation of the IFN-gamma+ NK cells. The IL-13+ cells (CD161+ only) are phenotypically distinct from the IFN-gamma+ ones (CD56+) before and after culture under any condition analyzed, and produce IL-13 in response to NK-sensitive target cells and PMA+Ca(2+) ionophore, whereas also FcgammaRIIIA and IL-2+IL-12 stimulate IFN-gamma production. These data define the existence and regulation of two distinct resting peripheral NK cell subsets producing type 1 and type 2 cytokines, and suggest possible roles for IL-13+ NK cells in allergy.