Lymphocyte Cell-Cycle Inhibition by HLA-G Is Mediated by Phosphatase SHP-2 and Acts on the mTOR Pathway

Human leukocyte antigen G (HLA-G) is involved in regulating T-cell responses through its interaction with inhibitory receptors belonging to the immunoglobulin-like transcript family (ILT). In this context, we investigated the pathways involved in the control of cell-cycle entry of T cells following HLA-G interaction with its inhibitory receptor. We show that HLA-G acts through its interaction with the LILRB1 receptor expressed on T lymphocytes. Both HLA-G and LILRB1 antibodies block the inhibitory effect of HLA-G and restore T-cell proliferation. The interaction of HLA-G with T lymphocytes is associated with phosphorylation of SHP-2 phosphatase, but not SHP-1. In addition, in activated T cells, their incubation with HLA-G is not associated with a decrease in the TCR or CD28 downstream pathways, but is associated with dephosphorylation of the mTOR molecule and p70S6K. In contrast, Akt, which acts upstream of mTOR, is not affected by HLA-G. The inhibition of SHP-2 by NSC-87877(5 µM), a chemical inhibitor of SHP-2, or the use of siRNA, abrogates dephosphorylation of mTOR and impairs the overexpression of p27kip in the presence of HLA-G. Together, these results indicate that HLA-G is associated with activation of phosphatase SHP-2, which inhibits the mTOR pathway and favors the inhibition of the cell-cycle entry of human-activated T cells

Bacillus Calmette–Guérin-Induced Human Mast Cell Activation Relies on IL-33 Priming

Bacillus Calmette–Guérin (BCG) vaccine is an attenuated strain of Mycobacterium bovis that provides weak protection against tuberculosis (TB). Mast cells (MCs) are tissue-resident immune cells strategically that serve as the first line of defence against pathogenic threats. In this study, we investigated the response of human MCs (hMCs) to BCG. We found that naïve hMCs exposed to BCG did not secrete cytokines, degranulate, or support the uptake and intracellular growth of bacteria. Since we could show that in hMCs IL-33 promotes the transcription of host-pathogen interaction, cell adhesion and activation genes, we used IL-33 for cell priming. The treatment of hMCs with IL-33, but not IFN-γ, before BCG stimulation increased IL-8, MCP-1 and IL-13 secretion, and induced an enhanced expression of the mycobacteria-binding receptor CD48. These effects were comparable to those caused by the recombinant Mycobacterium tuberculosis (Mtb) 19-KDa lipoprotein. Finally, stimulation of hMCs with IL-33 incremented MC-BCG interactions. Thus, we propose that IL-33 may improve the immunogenicity of BCG vaccine by sensitising hMCs

Human Melanoma-Associated Mast Cells Display a Distinct Transcriptional Signature Characterized by an Upregulation of the Complement Component 3 That Correlates With Poor Prognosis

Cutaneous melanoma is one of the most aggressive human malignancies and shows increasing incidence. Mast cells (MCs), long-lived tissue-resident cells that are particularly abundant in human skin where they regulate both innate and adaptive immunity, are associated with melanoma stroma (MAMCs). Thus, MAMCs could impact melanoma development, progression, and metastasis by secreting proteases, pro-angiogenic factors, and both pro-inflammatory and immuno-inhibitory mediators. To interrogate the as-yet poorly characterized role of human MAMCs, we have purified MCs from melanoma skin biopsies and performed RNA-seq analysis. Here, we demonstrate that MAMCs display a unique transcriptome signature defined by the downregulation of the FcεRI signaling pathway, a distinct expression pattern of proteases and pro-angiogenic factors, and a profound upregulation of complement component C3. Furthermore, in melanoma tissue, we observe a significantly increased number of C3$^{+}$ MCs in stage IV melanoma. Moreover, in patients, C3 expression significantly correlates with the MC-specific marker TPSAB1, and the high expression of both markers is linked with poorer melanoma survival. In vitro, we show that melanoma cell supernatants and tumor microenvironment (TME) mediators such as TGF-β, IL-33, and IL-1β induce some of the changes found in MAMCs and significantly modulate C3 expression and activity in MCs. Taken together, these data suggest that melanoma-secreted cytokines such as TGF-β and IL-1β contribute to the melanoma microenvironment by upregulating C3 expression in MAMCs, thus inducing an MC phenotype switch that negatively impacts melanoma prognosis

Generation of a Novel Regulatory NK Cell Subset from Peripheral Blood CD34+ Progenitors Promoted by Membrane-Bound IL-15

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells

Régulation de la fonction lymphocytaire T allogénique par la molécule HLA-G et son implication en transplantation

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

Ectonucleotidase CD38 demarcates regulatory, memory-like CD8+ T cells with IFN-γ-mediated suppressor activities.

Regulatory CD8(+) T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8(+) T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38(high) T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8(+)CD38(high) (CD44(+)CD122(+)CD62L(high)) lymphocytes suppress CD4(+) effector T-cell proliferation in an antigen-non specific manner via IFN-γ. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-β and granzyme B release. IL-15 potentiates the suppressive activity of CD8(+)CD38(high) T cells and controls their survival and expansion. In humans CD8(+)CD38(high) T cells inhibit CD4(+) effector T cell proliferation. In vivo, CD8(+)CD38(high), but not CD8(+)CD38(-) T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8(+)CD38(high) T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8(+)CD38(high) T cells as potential inhibitors of excessive immune responses

P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33

MCs are tissue-resident immune cells that strategically reside in barrier organs and respond effectively to a wide range of stimuli, such as IL-33, a mediator released upon epithelial damage. Adenosine triphosphate (ATP) accumulates at sites of tissue injury and is known to modulate MC activities. This study investigated how an inflammatory tissue environment rich in IL-33 modulatesthe ATP-mediated activation of MCs. Human primary MCs primed with IL-33 displayed a strongly increased response to ATP but not ADP. This resulted in increased degranulation, IL-8 release, and pERK1/2 signalling. Such effects are unique to IL-33 stimulation and not shared by the epithelial alarmin, TSLP. MC exposure to IL-33 also increased membrane expression of purinergic and ATP-binding P2X receptors. The use of selective P2X receptor inhibitors identified P2X7 receptor as the key mediator of the enhanced ATP-induced ERK1/2 signalling and degranulation in IL-33-primed MCs. Whilst the inhibition of P2X1 and P2X4 receptors had no effect on MC degranulation, inhibiting these receptors together with P2X7 resulted in further decreased MC-mediated degranulation. These data therefore point toward the potential mechanisms by which IL-33 contributes to the modulation of ATP-mediated activation in human MCs

Mast cell activation test in the diagnosis of allergic disease and anaphylaxis

Background. Food allergy is an increasing public health issue and the commonest cause of life-threatening anaphylactic reactions. Conventional allergy tests assess for the presence of allergen-specific IgE, significantly overestimating the rate of true clinical allergy resulting in over-diagnosis and adverse impact on health-related quality of life. Objective. To undertake initial validation and assessment of a novel diagnostic tool, the mast cell activation test (MAT). Methods. Primary human mast cells (hMCs) were generated from peripheral blood precursors, and sensitized using patient sera and then incubated with allergen. Mast cell degranulation was assessed by flow cytometry and mediator release. We compared the diagnostic performance of MAT to existing diagnostic tools to assess in a cohort of peanut-sensitized individuals undergoing double-blind, placebo-controlled challenge. Results. hMCs sensitized with sera from peanut, grass pollen and hymenoptera- (wasp venom) allergic patients demonstrated allergen-specific and dose-dependent degranulation by both expression of surface activation markers (CD63 and CD107a) and functional assays (prostaglandins D2 and ß-hexosaminidase release). In this cohort of peanut-sensitized individuals, MAT was found to have superior discrimination performance compared to other testing modalities including component-resolved diagnostics and basophil activation test. Using functional principle component analysis, we identified 5 clusters or patterns of reactivity in the resulting dose-response curves, which at preliminary analysis corresponded to the reaction phenotypes seen at challenge. Conclusion. MAT is a robust tool which may confer superior diagnostic performance compared to existing allergy diagnostics, and may be useful to explore differences in effector cell function between basophils and mast cells during allergic reactions