Trogocytosis is the uptake of membrane fragments from one cell by another and has been described for immune cells in mice and humans. Functional consequences of trogocytosis are emerging, but a dramatic immune function has still to be associated with it. Here we show that some resting, and most activated, CD4 ؉ and CD8 ؉ T cells acquire immunosuppressive HLA-G1 from antigen-presenting cells (APCs) in a few minutes. Acquisition of HLA-G through membrane transfers does not change the real nature of the T cells but immediately reverses their function from effectors to regulatory cells. These regulatory cells can inhibit allo-proliferative responses through HLA-G1 that they acquired. These data demonstrate that trogocytosis of HLA-G1 leads to instant generation of a new type of regulatory cells, which act through cell-surface molecules they temporarily display but do not express themselves. Such regulatory cells whose existence is most likely limited in space and time might constitute an "emergency" immune suppression mechanism used by HLA-G-expressing tissues to protect themselves against immune aggression. In addition, T cells acquire from HLA-Gexpressing APCs their HLA-G-dependent capability to induce the slower differentiation of regulatory cells that act independently of HLA-G. These data re-emphasize the significance of HLA-G expression in normal and pathologic situations. Introduction"Trogocytosis" is a new name for "fast, cell-to-cell contactdependent uptake of membranes and associated molecules." 1 Trogocytosis has been documented in T and B lymphocytes, natural killer (NK) cells, antigen-presenting cells (APCs), and tumor cells (reviewed in Hudrisier and Bongrand 2 ). Most of the work on trogocytosis by T cells was done in the murine system, in which it was shown that CD4 ϩ and CD8 ϩ T cells acquired APC major histocompatibility complex (MHC) class II and MHC class I molecules, respectively, in an antigen-specific manner [3][4][5][6][7] : for trogocytosis to occur, T-cell receptor (TCR) engagement was necessary, and activation or anti-CD3 increased trogocytosis efficiency. 8,9 Yet, there is no strict dependence of trogocytosis on TCR engagement: (1) in some systems, trogocytosis was shown to depend on CD28 engagement 5 ; and (2) transfer of MHC class II antigens from APCs to T cells can occur in an all-autologous system. 8 Even though most of the work on APC material acquisition by T cells was done on MHC molecules, it was shown that costimulatory molecules B7-1 (CD80), B7-2 (CD86), and ICAM-1 (CD54) are also acquired by murine antigen-specific T cells. 5,10 Trogocytosis is a transfer of membrane fragments, not of individual molecules. Consequently, all molecules contained within a certain membrane area are transferred from one cell to another during trogocytosis, including some that might have nothing to do with the acquirer cell, or participate in cell-to-cell cross-talk. Thus, during trogocytosis, some molecules transfer passively. This was clearly evidenced by showing that CD8 ϩ T cells can acquire MH...

Trogocytosis is a fast uptake of membranes and associated molecules from one cell by another. Trogocytosis between natural killer (NK) cells and tumors is already described, but the functional relevance of NK-tumor targets material exchange is unclear. We investigated whether the immunosuppressive molecule HLA-G that is commonly expressed by tumors in vivo and known to block NK cytolytic function, could be transferred from tumor cells to NK cells, and if this transfer had functional consequences. We show that activated NK cells acquire HLA-G1 from tumor cells, and that upon this acquisition, NK cells stop proliferating, are no longer cytotoxic, and behave as suppressor cells. Such cells can inhibit other NK cells' cytotoxic function and protect NK-sensitive tumor cells from cytolysis. These data are the first demonstration that trogocytosis of HLA-G1 can be a major mechanism of immune escape that acts through effector cells made to act as suppressor cells locally, temporarily, but efficiently. The broader consequences of membrane sharing between immune and non-immune cells on the function of effectors and the outcome of immune responses are discussed.

HLA-G is

The non-classical Human Leukocyte Antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α1–α2–α3 non-covalently bound to β-2-microglobulin (Β2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than Β2M-associated HLA-G1 and HLA-G5. In the present work we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α1–α3 structure, that constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for an HLA-G truncated isoform. Following up on this finding, we show that the α1–α3-Fc structure coated on agarose beds is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.

Trogocytosis is the uptake of membranes from one cell by another. Trogocytosis has been demonstrated for monocytes, B cells, T cells, and NK cells. The acquisition of the tolerogenic molecule HLA-G by T cells and NK cells makes them behave as regulatory cells. We investigated here whether HLA-G, which is expressed by tumor cells in vivo, could be acquired by monocytes and if this transfer could have functional consequences. We demonstrate that resting, and even more so, activated monocytes efficiently acquire membrane-bound HLA-G from HLA-G tumor cells by trogocytosis. However, we demonstrate that HLA-G quickly disappears from the surface of the monocytes in contrast to the HLA-G acquired by T cells. Consequently, HLA-G(acq+) monocytes do not reliably inhibit the on-going proliferation of autologous activated T cells and do not inhibit their cytokine production. Thus, we show that the acquirer cell may control the functional outcome of trogocytosis.

HLA-G is a natural tolerogenic molecule involved in the best example of tolerance to foreign tissues there is: the maternal-fetal tolerance. The further involvement of HLA-G in the tolerance of allogeneic transplants has also been demonstrated and some of its mechanisms of action have been elucidated. For these reasons, therapeutic HLA-G molecules for tolerance induction in transplantation are actively investigated. In the present study, we studied the tolerogenic functions of three different HLA-G recombinant proteins: HLA-G heavy chain fused to β2-microglobulin (B2M), HLA-G heavy chain fused to B2M and to the Fc portion of an immunoglobulin, and HLA-G alpha-1 domain either fused to the Fc part of an immunoglobulin or as a synthetic peptide. Our results demonstrate the tolerogenic function of B2M-HLA-G fusion proteins, and especially of B2M-HLA-G5, which were capable of significantly delaying allogeneic skin graft rejection in a murine in vivo transplantation model. The results from our studies suggest that HLA-G recombinant proteins are relevant candidates for tolerance induction in human transplantation.

The acquisition by T cells of exogenous ligands originally expressed by APC has been already described. However, reports essentially focused on the outward signaling of acquired ligands and their effects on surroundings cells. We investigated the function of transferred receptors (not ligands) on the T cells that acquired them (not on cells they interact with). We show that inhibitory Ig-like transcript 2 receptors efficiently transfer from monocytes to autologous T cells by trogocytosis and integrate within the plasma membrane of the acquirer T cells. Furthermore, the acquired receptors can access compatible signaling machinery within acquirer T cells and use it to signal and alter the functions of their new host cells. These data are a formal demonstration that a transferred molecule may send signals to its new host cell. We also provide evidence that sensitivity to modulatory molecules can be acquired from other cells and introduce the notion of intercellular transfer of sensitivities.

The human leukocyte antigen (HLA)-G is a tolerogenic molecule, whose expression by allografts is associated with better acceptance. An increasing interest in producing HLA-G as a clinical-grade molecule for therapy use is impaired by its complexity and limited stability. Our purpose was to engineer simpler and more stable HLA-G-derived molecules than the full-length HLA-G trimolecular complex that are also tolerogenic, functional as soluble molecules, and compatible with good manufacturing practice (GMP) production conditions. We present two synthetic molecules: (α3-L)x2 and (α1-α3)x2 polypeptides. We show their capability to bind the HLA-G receptor LILRB2 and their functions in vitro and in vivo. The (α1-α3)x2 polypeptide proved to be a potent tolerogenic molecule in vivo: One treatment of skin allograft recipient mice with (α1-α3)x2 was sufficient to significantly prolong graft survival, and four weekly treatments induced complete tolerance. Furthermore, (α1-α3)x2 was active as a soluble molecule and capable of inhibiting the proliferation of tumor cell lines, as does the full length HLA-G trimolecular complex. Thus, the synthetic (α1-α3)x2 polypeptide is a stable and simpler alternative to the full-length HLA-G molecule. It can be produced under GMP conditions, it functions as a soluble molecule, and it is at least as tolerogenic as HLA-G in vivo.