Denis Hudrisier

Prevention of acute and chronic allograft rejection with CD4 + CD25 + Foxp3 + regulatory T lymphocytes

A major challenge in transplantation medicine is controlling the very strong immune responses to foreign antigens that are responsible for graft rejection. Although immunosuppressive drugs efficiently inhibit acute graft rejection, a substantial proportion of patients suffer chronic rejection that ultimately leads to functional loss of the graft. Induction of immunological tolerance to transplants would avoid rejection and the need for lifelong treatment with immunosuppressive drugs. Tolerance to self-antigens is ensured naturally by several mechanisms; one major mechanism depends on the activity of regulatory T lymphocytes. Here we show that in mice treated with clinically acceptable levels of irradiation, regulatory CD4+CD25+Foxp3+ T cells stimulated in vitro with alloantigens induced long-term tolerance to bone marrow and subsequent skin and cardiac allografts. Regulatory T cells specific for directly presented donor antigens prevented only acute rejection, despite hematopoietic chimerism. By contrast, regulatory T cells specific for both directly and indirectly presented alloantigens prevented both acute and chronic rejection. Our findings demonstrate the potential of appropriately stimulated regulatory T cells for future cell-based therapeutic approaches to induce lifelong immunological tolerance to allogeneic transplants.

Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell’s surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.

Preferential Recognition of Self Antigens Despite Normal Thymic Deletion of CD4 + CD25 + Regulatory T Cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4+CD25+ regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4+CD25+ regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4+CD25+ thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.

Intercellular transfer of antigen-presenting cell determinants onto T cells: molecular mechanisms and biological significance

Upon physiological stimulation, receptors with tyrosine kinase activity (RTK) are rapidly internalized together with their soluble ligands. T cell activation is the consequence of recognition by the T cell receptor (TCR) of specific peptide‐major histocompatibility protein complexes (peptide‐MHC) present at the membrane of antigen‐presenting cells (APC). The TCR belongs to the RTK family and is known to be endocytosed upon ligand recognition. It differs from most other RTK in that its ligand, the peptide‐MHC complex, is membrane bound and the TCR‐ligand interaction is quite weak. Recent experiments have shown that the TCR ligand becomes internalized by T cells upon stimulation. Here we review current knowledge on the molecular mechanisms by which the membrane‐bound MHC molecules can be transferred onto T cells, and propose hypotheses on the role this phenomenon could play in physio‐pathological situations involving T cells.—Hudrisier, D., Bongrand, P. Intercellular transfer of antigen‐presenting cell determinants onto T cells: molecular mechanisms and biological significance. FASEB J. 16, 477–486 (2002)

Acquisition of viral receptor by NK cells through immunological synapse.

Occasional EBV infection of human NK cells may lead to malignant diseases such as naso-pharyngeal NK lymphoma although NK cells do not express CD21, the primary receptor for EBV. Here we show that during early EBV infection in patients, NK cells attacked EBV-infected autologous B cells. In vitro, NK cells activated by conjugation to CD21+ B-EBV cell targets transiently acquired a weak CD21+ phenotype by synaptic transfer of few receptor molecules onto their own membrane. In the presence of viral particles, these ectopic receptors allowed EBV binding to the novel NK cell host. Hence, trans-synaptic acquisition of viral receptor from target cells might constitute an unsuspected mode of infection for otherwise unreachable lymphoid hosts.

Active trans‐synaptic capture of membrane fragments by natural killer cells

Prior to delivery of a lethal hit, NK cells form an immunological synapse to scan the target cells and engage their activatory and inhibitory receptors. Using freshly isolated NK cells, IL‐2‐activated polyclonal NK bulk or the NKL cell line, we report here that early during this recognition process, human NK cells actively capture target cell membrane fragments. This novel NK cell function occurs via the immunological synapse, is controlled by Src kinase, ATP, Ca2+ and PKC and involves rearrangements of the actin cytoskeleton. Furthermore, this process is down‐regulated bysignals emanating from inhibitory NK receptors recognizing protective MHC class I alleles.

Synaptic Transfer by Human γδ T Cells Stimulated with Soluble or Cellular Antigens

B, αβ T, and NK lymphocytes establish immunological synapses (IS) with their targets to enable recognition. Transfer of target cell-derived Ags together with proximal molecules onto the effector cell appears also to occur through synapses. Little is known about the molecular basis of this transfer, but it is assumed to result from Ag receptor internalization. Because human γδ T cells recognize soluble nonpeptidic phosphoantigens as well as tumor cells such as Daudi, it is unknown whether they establish IS with, and extract molecules from, target cells. Using flow cytometry and confocal microscopy, we show in this work that Ag-stimulated human Vγ9/Vδ2 T cells conjugate to, and perform molecular transfer from, various tumor cell targets. The molecular transfer appears to be linked to IS establishment, evolves in a dose-dependent manner in the presence of either soluble or cellular Ag, and requires γδ TCR ligation, Src family kinase signaling, and participation of the actin cytoskeleton. Although CD45 exclusion characterized the IS performed by γδ T cells, no obvious capping of the γδ TCR was detected. The synaptic transfer mediated by γδ T cells involved target molecules unrelated to the cognate Ag and occurred independently of MHC class I expression by target cells. From these observations, we conclude thatm despite the particular features of γδ T cell activation, both synapse formation and molecular transfer of determinants belonging to target cell characterize γδ T cell recognition of Ags.

Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells

Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4+ T, CD8+ T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger trogocytosis. On the basis of the analysis of a series of surface molecules on CD4+ T, CD8+ T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B cells, we identified only the BCR and MHC molecules as potentials triggers of trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger trogocytosis in T or B cells. Altogether, our results indicate that although trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via trogocytosis proposed for different types of cells.

Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells.

Objective:Compared with cell-free viral infection, virological synapses increase HIV capture by target cells, viral infectivity and cytopathicity, and are believed to be less sensitive to antibody neutralization. We have evaluated the impact of antibodies against HIV envelope glycoproteins (gp120 and gp41) on cell-to-cell HIV transmission. Methods:We analyzed the role of trogocytosis in cell-to-cell HIV transmission and the inhibitory mechanisms of antigp120 antibody IgGb12 and antigp41 antibodies 4E10 and 2F5 using cocultures of NL4-3 or BaL-infected MOLT/CCR5 cells with primary CD4 T cells. Results:Analysis of early steps of HIV transmission in these cocultures showed that IgGb12, but not 4E10 and 2F5, inhibited the formation of virological synapses. Consequently, IgGb12 but not antigp41 antibodies blocked the transfer of HIV particles from infected to target cells and the trogocytic transfer of CD4 molecules from target to infected cells. Interestingly, trogocytic transfer of membranes was not detected in the HIV transmission direction. Furthermore, analysis of late events of HIV transmission showed that all neutralizing antibodies blocked productive infection of target cells, suggesting that HIV infection between T cells is transmitted by a neutralization-sensitive mechanism involving HIV budding from infected cells and capture by target cells. Conclusion:Despite mechanistic differences, antigp120 and antigp41 antibodies block infectious cell-to-cell HIV transmission. Our data suggest that eliciting high titers of neutralizing antibodies in vivo should be maintained as a main end of HIV vaccine design.

Binding of Viral Antigens to Major Histocompatibility Complex Class I H-2Db Molecules Is Controlled by Dominant Negative Elements at Peptide Non-anchor Residues IMPLICATIONS FOR PEPTIDE SELECTION AND PRESENTATION

Binding of viral antigens to major histocompatibility complex (MHC) class I molecules is a critical step in the activation process of CD8+ cytotoxic T lymphocytes. In this study, we investigated the impact of structural factors at non-anchor residues in peptide-MHC interaction using the model of lymphocytic choriomeningitis virus (LCMV) infection of its natural host, the mouse. Altering viral genes by making reassortants, recombinants, and using synthetic peptides, CD8+ cytotoxic T lymphocytes were shown to recognize only three H-2Db-restricted epitopes, GP amino acids 33-41/43, GP 276-286, and NP 396-404. However, LCMV NP and GP proteins contain 31 other peptides bearing the H-2Db motif. These 34 LCMV peptides and 11 other known H2-Db-restricted peptides were synthesized and examined for MHC binding properties. Despite the presence of the H-2Db binding motif, the majority of LCMV peptides showed weak or no affinity for H-2Db. We observed that dominant negative structural elements located at non-anchor positions played a crucial role in peptide-MHC interaction. By comparative sequence analysis of strong versus non-binders and using molecular modeling, we delineated these negative elements and evaluated their impact on peptide-MHC interaction. Our findings were validated by showing that a single mutation of a favorable non-anchor residue in the sequence of known viral epitopes for a negative element resulted in dramatic reduction of antigen presentation properties, while conversely, substitution of one negative for a positive element in the sequence of a non-binder conferred to the peptide an ability to now bind to MHC molecules.