Chiara Vitale

Transforming growth factor-β-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes

Different HLA class I‐specific killer inhibitory receptors (KIR) are expressed in vivo by a fraction of activated T cells, predominantly CD8+ , in which they may inhibit TCR‐mediated cell functions. In an attempt to identify mechanisms leading to KIR expression in T cells, we analyzed the effect of transforming growth factor‐β (TGF‐β) in T cells responding to bacterial superantigens in vitro. We show that TGF‐β induces the expression of CD94/NKG2A in cells responding to toxic shock syndrome toxin 1 or to other staphylococcal superantigens. Remarkably, maximal CD94 expression occurred at (low) TGF‐β concentrations which have no substantial effect on lymphocyte proliferation. Maximal CD94 expression occurred when TGF‐β was added shortly after the cells were placed in culture. No expression could be induced in CD94/NKG2A‐negative T cell clones. Although both CD4+ and CD8+ expressed CD94, the simultaneous expression of NKG2A was mostly confined to CD8+ cells. Monoclonal antibody‐mediated cross‐linking of CD94/NKG2A led to an impairment of T cell trigger ing via CD3, as determined in a redirected killing assay using the Fcγ receptor‐positive P815 murine target cells.

CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells

Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34+ hematopoietic precursors in human decidua (dCD34+). We show that dCD34+ cells differ from cord blood- or peripheral blood-derived CD34+ precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34+ cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56brightCD16−KIR+/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34+ cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment.

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

The corticosteroid-induced inhibitory effect on NK cell function reflects down-regulation and/or dysfunction of triggering receptors involved in natural cytotoxicity.

Corticosteroids are known to inhibit NK cell functions. However no information is available on whether such inhibition may affect the expression and/or the function of receptors involved in NK cell activation. In an attempt to analyze this point, we studied peripheral blood NK cells isolated from pediatric patients undergoing allogeneic BM transplantation. NK cells were analyzed before, during and after methylprednisolone administration to treat acute graft‐versus‐host disease. In NK cells freshly isolated from peripheral blood during methylprednisolone treatment, the surface expression of activating receptors, particularly NKp46 and NKp30, was consistently reduced. Such impaired expression could also be detected after 5 days of culture in IL‐2. Such cultured NK cells also failed to express the IL‐2‐inducible NKp44 receptor. Accordingly, cytotoxicity against different tumor target cell lines was sharply reduced. The effect on NK cells isolated from healthy individuals and cultured in the presence of methylprednisolone was also analyzed. A similar inhibitory effect occurred in the expression of activating NK receptors. In addition, a sharp impairment of NK cytotoxicity against different tumor target cell lines or immature DC was detected.

Expression of HLA class I-specific inhibitory receptors in human cytolytic T lymphocytes: A regulated mechanism that controls T-cell activation and function

Different families of major histocompatibility complex (MHC)-specific inhibitory receptors (NKRs) play a major role in natural killer (NK) cell function, allowing discrimination between normal cells and cells that do not express adequate amounts of MHC class I antigens. This occurs in most instances as a consequence of viral infection or tumor transformation. In T lymphocytes, expression of NKR is mostly confined to activated CD8+ cytolytic T lymphocytes (CTLs). While NKR expression by CTLs may be viewed as a mechanism preventing damages to normal cells by those CTLs that have acquired NK-like activity, it may also down regulate TCR-mediated T cell activation, thus, impairing CTL functions. The finding that certain cytokines can modulate killer inhibitory receptor expression in CTLs is of major interest and might be instrumental in novel therapeutic approaches aimed at the down regulation ofT-cell function in transplantation or autoimmunity.

Analysis of the activating receptors and cytolytic function of human natural killer cells undergoing in vivo differentiation after allogeneic bone marrow transplantation

Patients undergoing allogeneic bone marrow transplantation offer a unique system to analyze NK cell development in vivo. We analyzed NK cells from 23 such patients to assess the acquisition of activating receptors. Four patients displayed an immature NK cell surface phenotype at engraftment, as their cells were CD16–KIR– and NKG2D– but expressed low levels of NKp46, NKp30, 2B4 and NKG2A. These NK cells had particularly low cytolytic activity against the HLA‐class‐I– melanoma F01 cell line and the 721‐221 EBV‐infected B cell line. Moreover, cytoxicity was inhibited upon mAb‐mediated crosslinking of 2B4. Analysis of NK cells at day 30 after bone marrow transplantation revealed the occurrence of both phenotypic and functional maturation. These data are in agreement with a previous in vitro study showing that immature NK cell precursors express CD16, NKG2D and KIR only at a late stage of differentiation and also express inhibitory 2B4. Our present study allows a better understanding of the NK cell differentiation in vivo.

p75/AIRM1 and CD33, two sialoadhesin receptors that regulate the proliferation or the survival of normal and leukemic myeloid cells

Summary: Inhibitory receptors originally identified in natural killer (NK) cells have also been detected in other leukocyte types, thus suggesting that they may play a more general role in the control of leukocyte function. Here we report data on p75/adhesion receptor molecule 1 (AIRM1), a surface inhibitory receptor of the sialoadhesin family originally identified in NK cells that is also expressed by normal and leukemic myeloid cells. Given the homology between p75/AIRM1 and CD33, we also reanalyzed CD33, a major myeloid marker of still unknown function. We discuss recent data indicating that engagement of p75/AIRM1 or CD33 sharply inhibits the in vitro proliferation/differentiation of CD34+ myeloid precursors induced by stem cell factor and granulocyte–macrophage colony‐stimulating factor. Importantly, a similar in vitro inhibitory effect occurs in monocyte/macrophages as well as in chronic or acute myeloid leukemias. While CD33 appears to act via the induction of apoptosis, p75/AIRM1 blocks cell proliferation but does not appear to induce apoptosis. A synergistic effect in the induction of apoptosis has also been documented between antibodies specific for CD33 and the chemotherapic agent etoposide. Taken together, the use of appropriate ligands against CD33 or p75/AIRM1 may represent a new therapeutic tool for treatment of myeloid leukemias or diseases characterized by overwhelming macrophage activation.

Human NK cells at early stages of differentiation produce CXCL8 and express CD161 molecule that functions as an activating receptor

Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34(+) umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161(+)CD56(+)LFA-1(-) and more differentiated cytolytic CD161(+)CD56(+)LFA-1(+) NK cells. CD161(+)CD56(+)LFA-1(-) NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161(+)CD56(+)LFA-1(-) NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161(+)CD56(+)LFA-1(-) subset, because it did not induce cytokine release or CD107a expression in CD161(+)CD56(+)LFA-1(+) cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.

Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells

Previous studies showed that methylprednisolone (MePDN) down-regulates the surface expression of activating NK receptors and sharply inhibits the NK cytotoxicity both in vitro and in vivo. Since MePDN is administered to patients undergoing hemopoietic stem cell transplant to treat acute graft versus host disease (GvHD), we analyzed whether it could also inhibit the NK cell differentiation from CD34(+) hemopoietic cell precursors, thus interfering with the development of effector cells with anti-leukemic potential. We show that MePDN promotes the in vitro differentiation of CD161+CD56+/- immature NK cells by inducing a rapid expression of NKp46, NKG2D, DNAX-accessory molecule 1 (DNAM-1), leukocyte function-associated antigen-1 and NKG2A and an efficient cytolytic activity. This phenotypic and functional NK cell maturation occurred more rapidly than in parallel control cultures performed in the absence of MePDN. In addition, MePDN induced CD33+CD161-CD56- myeloid precursors to switch toward NK cells. It is also of note that immature NK cells when cultured in the absence (but not in the presence) of MePDN produced high amounts of IL-8. These data indicate that MePDN can accelerate the in vitro NK cell differentiation, thus revealing a dichotomous effect on immature versus mature NK cells; in addition, interference with the in vitro development of myeloid cells occurred. These effects should be further investigated in hemopoietic stem cell transplanted patients receiving steroids to treat GvHD.

Human innate lymphoid cells

The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed toward NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development.