Effector and regulatory events during natural killer–dendritic cell interactions

Summary:  The different cell types of the innate immune system can interact with each other and influence the quality and strength of an immune response. The cross talk between natural killer (NK) cells and myeloid dendritic cells (DCs) leads to NK cell activation and DC maturation. Activated NK cells are capable of killing DCs that fail to undergo proper maturation ('DC editing'). Encounters between NK cells and DCs occur in both inflamed peripheral tissues and lymph nodes, where both cell types are recruited by chemokines released in the early phases of inflammatory responses. Different NK cell subsets (CD56^brightCD16⁻ versus CD56⁺CD16⁺) differ in their homing capabilities. In particular, CD56^brightCD16⁻ NK cells largely predominate the lymph nodes. In addition, these two subsets display major functional differences in their cytolytic activity, cytokine production, and ability to undergo proliferation. NK cell functions are also greatly influenced by the presence of polarizing cytokines such as interleukin (IL)-12 and IL-4. The cytokine microenvironment reflects the presence of different cell types that secrete such cytokines in response to microbial products acting on different Toll-like receptors (TLRs). Moreover, NK cells themselves can respond directly to microbial products by means of TLR3 and TLR9. Thus, it appears that the final outcome of a response to microbial infection may greatly vary as a result of the interactions occurring between different pathogen-derived products and different cell types of the innate immunity system. These interactions also determine the quality and strength of the subsequent adaptive responses. Remarkably, NK cells appear to play a key role in this complex network.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

Case report: natural transmission of an AZFc Y-chromosomal microdeletion from father to his sons

Y-chromosomal microdeletions, associated with oligozoospermia or azoospermia, are usually de novo deletions in the affected patients. We report here the rare case of an affected father who transmitted a Y-chromosomal microdeletion to at least two of his three sons naturally and who also fathered a daughter. The extent of the deletion, which was determined with new STS-primers and covers 3.5 Mb, was identical in the father and his azoospermic sons. To determine any possibly modifying influence of other genes involved in spermatogenesis, we analysed two polymorphisms of the DAZL gene, the autosomal homologue of the deleted DAZ gene. DAZL and DAZ might be functionally related to each other. However, we found identical polymorphisms in exon 2 and 3 of the DAZL gene, in both father and his sons, corresponding to the most prevalent genotype in fertile men. Thus, other genes or environmental factors must modify spermatogenesis in men with identical Y-chromosomal microdeletions.     

Expression of Intercellular Adhesion Molecule-1 (ICAM-1) on Cultured Human Endometrial Stromal Cells and Its Role in the Interaction With Natural Killers

PROBLEM: Recent evidence emphasizes the role of natural killer cells (NKs) as potential effectors of peritoneal immune surveillance directed against the outgrowth of endometrial cells, refluxed with menstrual debris, in ectopic sites. This NK-mediated cytotoxicity toward autologous endometrial antigens seems to be significantly decreased in endometriosis patients. METHOD: We set up experiments to clarify which molecules are involved in NK-endome-trial cell interaction. In particular, we evaluated the surface expression and functional activity of intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein that has been identified as one of the ligands for lymphocyte function-associated antigen-1 (LFA-1), present on almost all leucocyte cell types. Immunofluorescence flow cytometry was used to assess ICAM-1 expression on resting and IL 1β-activated endometrial stromal cells in culture. Dermal fibroblasts were used as control cells. Cytotoxicity and binding assays by ⁵¹Cr release in presence and absence of a specific monoclonal antibody (mAb) against ICAM-1 were then performed in order to determine the effect of this molecule on NK-mediated cytotoxic and binding activity toward endometrial stromal cells. RESULTS: The results of this study indicated that ICAM-1 expression on endometrial stromal cells seems to be constitutively higher than on dermal fibroblasts and can be up-regulated upon exposure to IL 1β. Furthermore, a mAb against ICAM-1 strongly inhibits the binding but not the cytotoxicity of NKs toward endometrial cells. No difference in the expression of this molecule was observed throughout the cycle. CONCLUSIONS: The presence of ICAM-1 on human endometrium might relate to the action of the immunocompetent cells in human specific reproductive events.     

Regulation of immune responses by CD1d-restricted natural killer T cells

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share receptor structures and properties with conventional T lymphocytes and natural killer (NK) cells. NKT cells are specific for glycolipid antigens such as the marine sponge-derived agent α-galactosylceramide (α-GalCer) presented by the major histocompatibility complex (MHC) class I-like molcule CD1d. My laboratory has evaluated the function of NKT cells by generating and analyzing CD1d-deficient mice. These studies showed that CD1d expression is required for NKT cell development, but not absolutely necessary for the generation of polarized T helper (Th) cell responses. Further, we have studied the in vivo response of NKT cells toα-GalCer stimulation and the capacity of α-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of α-GalCer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. Our findings indicate that NKT cells play a regulatory role in the immune response and that specific activation of these cells may be exploited for therapeutic purposes.     

Association between HLA and Japanese patients with rheumatoid arthritis

Japanese patients with rheumatoid arthritis (RA) were observed to have a statistical association with HLA-DR4, MT3. Strong association between the clinical severity of RA and HLA was also observed. Male patients had a stronger association with HLA than female patients. Males are more resistant to RA than females. This suggested that the threshold of liability for RA is higher in males than in females. Japanese patients with RA with systemic vasculitis were negative for HLA-Bw44 and had antilymphocytotoxic autoantibody, indicating that RA with systemic vasculitis is different in etiology from RA without systemic vasculitis.     

Thymic stroma is required for the development of human T cell lineages in vitro

Development of the T cell lineage is characterized by the homingof hematopoietic precursors to thymus, followed by their acquisitionof receptors for antigen. T cell receptors are ß or heterodimers associated with CD3 (TCR-CD3). Very early T cellprecursors in humans have been characterized as CD7+45+ cellswhich lack the T cell differentiation antigens CD1, CD2, CD3,CD4, and CD8. A phenotypically equivalent early thymocyte populationalso occurs in postnatal life, and we have previously shownthat interleukin 2 (IL2) promotes the development in vitro ofboth the ß and the T cells from these early thymocytes.Here we have analyzed the requirements of the induction of theIL2 pathway in early thymocytes, and their developmental potential.We show that: (I) thymic stromal cells, which are present inthymocyte suspensions, are necessary to induce the IL2 pathwayand the development of ß or T cell lineages fromearly thymocytes in vitro; and (II) when removed from the invivo environment, early thymocytes can develop in vitro intoTCR-CD3^– cells of the natural killer (NK) lineage. Weconclude that CD7+45+, CD1–2–3–4–8–early thymocytes are multipotential progenitors that, at least,have the capacity to develop into ß or T cell andNK lineages. The analysis of the mechanisms of generation andselection of human T and NK cell diversity, not feasible inbone marrow cultures, is now possible.     

Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2.

Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma.     

Characterization of the natural killer cell activity in Hashimoto's and Graves' diseases

Natural killer (NK) cell activity and blood mononuclear cell subpopulations were characterized in patients with Hashimoto's thyroiditis ( n = 11), Graves' disease ( n = 20), non-toxic goitre ( n = 10) and in normal controls ( n = 22). NK cell activity against K 562 target cells and the capability of IFN-α, Il-2, and indomethacin to enhance NK cell activity in vitro did not differ significantly between the groups. The percentages of large granular lymphocytes, CD5 +, CD4 +, CD8 + and CD16 + cells were normal in patients with non-toxic goitre, Hashimoto's and Graves' diseases. There was no correlation between NK cell activities and TgAb, MAb and TSAb. Although NK cell activity is suppressed in several autoimmune diseases, NK cell function is normal in patients with autoimmune thyroid disorders.