Organ transplantation represents a unique therapeutic option for irreparable organ dysfunction and rejection of transplants results from a breakdown in operational tolerance. Although endothelial cells (ECs) are the first target in graft rejection following kidney transplantation, their capacity to alloactivate and generate particular T lymphocyte subsets that could intervene in this process remains unknown. By using an experimental model of microvascular endothelium, we demonstrate that, under inflammatory conditions, human ECs induced proliferation of memory CD4 + CD45RA −

Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into "neutrophil-like" cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 upregulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders.granulocytes |

To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 3 10 5 -fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 3 10 3 -fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents "signal 1," CD137 represents "signal 2," and cytokines represent "signal 3." The precise nature of signal 1 remains to be defined.

Human leukocyte antigen‐G (HLA‐G) expression by tumors has been evidenced in numerous malignancies in association with poor prognosis and resistance to immunotherapy in humans. Particularly, soluble form of HLA‐G was measured at high concentrations in malignant effusions and plasma from cancer patients, and inhibits antitumor immune cells in vitro through interaction with immunoglobulin‐like transcript (ILT) receptors. Nevertheless, in vivo study demonstrating that HLA‐G secretion by tumor cells allows their escape from immunosurveillance remained to be established. Despite nondescribed murine homolog, direct functional interaction of HLA‐G with murine paired immunoglobulin‐like receptor (PIR)‐B, ortholog of human ILT receptors, enables to investigate its role in vivo. Immunocompetent mice were injected either with syngeneic tumor cells co‐expressing HLA‐G5, the main soluble HLA‐G isoform, and the conformation stabilizer human β2‐microglubulin (hβ2m), or with hβ2m+HLA‐G5− tumor cells. hβ2m expressed at both tumor cell surface acted as a tumor antigen triggering a specific humoral response. Interestingly, although hβ2m+HLA‐G5− tumors were rejected, secreted HLA‐G5 provided hβ2m+HLA‐G5+ tumors a protection against hβ2m‐elicited immune rejection, enabling such immunogenic tumors to grow similarly to a poorly immunogenic tumor. HLA‐G5 tumor expression was associated with local and peripheral immunosuppression, characterized by dampened anti‐hβ2m B‐cell response, quantitative and functional T‐and B‐cell defects, accumulation of myeloid‐derived suppressor cells able to inhibit T‐cell proliferation and reduced T‐ and B‐cell tumor infiltrate. Our study provides the first in vivo proof that soluble HLA‐G counteracts tumor rejection and reinforces the importance to consider HLA‐G as a promising target to optimize current cancer immunotherapies.

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Vγ9Vδ2 T cells play a crucial role in the antitumoral immune response through cytokine production and cytotoxicity. Although the expression of the immunomodulatory molecule HLA-G has been found in diverse tumors, its impact on Vγ9Vδ2 T-cell functions remains unknown. Here we showed that soluble HLA-G inhibits Vγ9Vδ2 T-cell proliferation without inducing apoptosis. Moreover, soluble HLA-G inhibited the Vγ9Vδ2 T-cell production of IFN-γ induced by phosphoantigen stimulation. The reduction in Vγ9Vδ2 T-cell IFN-γ production was also induced by membrane-bound or soluble HLA-G expressed by tumor cell lines. Finally, primary tumor cells inhibited Vγ9Vδ2 T-cell proliferation and IFN-γ production through HLA-G. In this context, HLA-G impaired Vγ9Vδ2 T-cell cytotoxicity by interacting with ILT2 inhibitory receptor. These data demonstrate that HLA-G inhibits the anti-tumoral functions of Vγ9Vδ2 T cells and imply that treatments targeting HLA-G could optimize Vγ9Vδ2 T-cell-mediated immunotherapy of cancer.

Trogocytosis is a rapid transfer between cells of membranes and associated proteins. Trogocytic exchanges have been investigated between different cell types, mainly in two-cell systems, involving one donor and one acceptor cell type. Here, we studied trogocytosis in a more complex system, involving not only several immune cell subsets but also multiple tumor cells. We show that CD4 + T cells, CD8 + T cells and monocytes can acquire membrane patches and the intact proteins they contain from different tumor cells by multiple simultaneous trogocytoses. The trogocytic capabilities of CD4 + and CD8 + T cells were found to be similar, but inferior to that of autologous monocytes. Activated peripheral-blood mononuclear cells (PBMCs) may also exchange membranes between themselves in an all-autologous system. For this reason, monocytes are capable of acquiring membranes from multiple tumor cell sources, and transfer them again to autologous T cells, along with some of their own membranes (serial trogocytosis). Our data illustrate the extent of membrane exchanges between autologous activated immune effector cells and their environment, and how the cellular content of the local environment, including "bystander" cells, may impact the functions of immune effector cells.

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.