Marie-Agnès Doucey

Recruitment of TNF Receptor 1 to Lipid Rafts Is Essential for TNFα-Mediated NF-κB Activation

Engagement of TNF receptor 1 by TNFalpha activates the transcription factor NF-kappaB but can also induce apoptosis. Here we show that upon TNFalpha binding, TNFR1 translocates to cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts, where it associates with the Ser/Thr kinase RIP and the adaptor proteins TRADD and TRAF2, forming a signaling complex. In lipid rafts, TNFR1 and RIP are ubiquitylated. Furthermore, we provide evidence that translocation to lipid rafts precedes ubiquitylation, which leads to the degradation via the proteasome pathway. Interfering with lipid raft organization not only abolishes ubiquitylation but switches TNFalpha signaling from NF-kappaB activation to apoptosis. We suggest that lipid rafts are crucial for the outcome of TNFalpha-activated signaling pathways.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide–major histo-compatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen. These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2Dd ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2Dd in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2Dd on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl‐inositol (GPI)‐anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI‐anchored green fluorescent proteins (GFP‐GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP‐GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL‐R3, GFP‐GPI(TRAIL‐R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP‐GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI‐anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP‐GPIs was observed irrespective of whether the GPI‐anchored molecules were painted or transfected. Furthermore, we show that painted GFP‐GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft‐associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl‐phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP‐ GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

CD3δ Establishes a Functional Link between the T Cell Receptor and CD8

T cells expressing T cell receptor (TCR) complexes that lack CD3δ, either due to deletion of the CD3δgene, or by replacement of the connecting peptide of the TCRα chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8β or tailless CD8β, we examined whether CD3δ serves to couple TCR·CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252–260 peptide in the context of H-2Kd. We report that, in thymocytes and hybridomas expressing the T1 TCR·CD3 complex, CD8αβ associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8αα or a CD3δ− variant of the T1 TCR. CD3δ was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3δ. Because CD8αβ is a raft constituent, due to this association a fraction of TCR·CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3δ couples TCR·CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8+ T cells.

Functional Avidity of Tumor Antigen-Specific CTL Recognition Directly Correlates with the Stability of MHC/Peptide Multimer Binding to TCR

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

C-Mannosylation of Human RNase 2 Is an Intracellular Process Performed by a Variety of Cultured Cells

C 2-α-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) isC-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was notC-mannosylated. These results not only form the basis for further studies on the biochemical aspects ofC-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins.

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.

Fluorescence-labeled soluble major histocompatibility complex class I-peptide “tetramers” constitute a powerful tool to detect and isolate antigen-specific CD8+ T cells by flow cytometry. Conventional “tetramers” are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and “tetramerization” by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252–260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys259 in the context of H-2Kd. We report that mutation of the CD8 binding site of Kd greatly impairs the binding of tetrameric but not octameric or multimeric Kd-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono‐and multimeric H‐2Kd‐peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252–260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd‐PbCS(ABA) complexes co‐engaging TCR and CD8. Using TCR ligand photo‐cross‐linking, we find that monomeric Kd‐PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross‐linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR‐associated Kd molecules, indicating that CTL activation is initiated by cross‐linking of CD8‐associated p56lck. They are also inhibited by methyl‐β‐cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross‐linking of engaged TCR.

Leishmania major-Specific B Cells Are Necessary for Th2 Cell Development and Susceptibility to L. major LV39 in BALB/c Mice

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (μMT). Whereas BALB/c μMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c μMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c μMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c μMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.