Niclas Setterblad

Oxidative stress–induced assembly of PML nuclear bodies controls sumoylation of partner proteins

PML multimerization into nuclear bodies following its oxidation promotes sumoylation and sequestration of partner proteins in these structures.

Intracytoplasmic domains of MHC class II molecules are essential for lipid-raft-dependent signaling

In addition to their role in antigen presentation, major histocompatibility complex (MHC) class II molecules have been widely described as signaling proteins in diverse antigen-presenting cells (APCs) including B cells and dendritic cells. By contrast, little is known of the signaling function of MHC class II molecules expressed in solid tumors. We describe the functional organization and signaling ability of I-Ak expressed in a sarcoma, and report the recruitment of I-Ak to lipid rafts after MHC class II engagement. Lipid raft integrity was required for I-Ak-mediated reorganization of the actin cytoskeleton and translocation of protein kinase C-α(PKC-α) to the precise site of stimulation via I-Ak. Truncation of the intracytoplasmic domains of I-Ak did not perturb I-Ak recruitment to lipid rafts but abrogated PKC-αtranslocation and actin rearrangement. PKC-αwas detected in lipid microdomains and enrichment of activated PKC-αin lipid rafts was induced by I-Ak signaling. Ordering of the molecular events following engagement of the MHC class II molecules revealed that I-Ak recruitment to lipid rafts precedes signaling. This is consistent with the absence of a requirement for the intracytoplasmic tails for localization to lipid rafts. These data reveal that lipid-rich microdomains play a key role in MHC class II-mediated signaling in a solid tumor.

B Cell Lipid Rafts Regulate Both Peptide-Dependent and Peptide-Independent APC-T Cell Interaction

Formation of an immunological synapse (IS) between APCs and T CD4+ lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.

Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells

Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP‐1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid‐rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide‐bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)‐DR induced rapid internalization in immature DCs. The proportion of cell‐surface detergent‐insoluble glycosphingolipid‐enriched microdomain‐clustered HLA‐DR was higher in immature DCs despite the higher surface expression of HLA‐DR in mature DCs. Constituents of HLA‐DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA‐DR‐containing microdomains to include protein kinase C (PKC)‐δ, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA‐DR redistributed HLA‐DR and ‐DM and PKC‐δ as well as enriching the actin content of mature DC microdomains. The increased expression of HLA‐DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA‐DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.

Signalling via MHC Class II Molecules Modifies the Composition of GEMs in APC

Major histocompatibility complex (MHC) class II molecules are responsible for peptide presentation to helper T lymphocytes and as such play an essential role in the immune response. These molecules transmit intracellular signals leading to diverse consequences in B lymphocytes including proliferation and apoptosis. Recent studies have revealed that glycolipid enriched membrane microdomains (GEMs) behave as signalling platforms for a variety of lymphocyte receptors. We have quantified human leucocyte antigen (HLA)‐DR molecules localized in GEMs in human B lymphocytes. Use of a model imitating the interaction of HLA‐DR with a T‐cell receptor (TCR) modified the constituents of the HLA–DR‐enriched GEMs. Confocal microscopy demonstrated a recruitment of HLA–DR and the ganglioside GM1 at the site of HLA–DR interaction with the stimulating ligand. Moreover, cholesterol depletion efficiently impaired this recruitment. Co‐localizing proteins detected in HLA–DR‐enriched GEMs include protein kinase C (PKC)‐δ and actin. These data reveal that MHC class II antigens are localized in GEMs in mature human B lymphocytes and indicates that the formation of the immunological synapse regulates the composition of HLA–DR enriched GEMs in the antigen presenting cell (APC).

MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells.

Dendritic cells (DC) play a central role in the immune response, linking innate and adaptative responses to pathogens. Myeloid DC (MDC) produce interleukin‐12 in response to bacterial stimuli, whereas plasmacytoid DC (PDC) produce high levels of type I interferon upon viral infection. Human leukocyte antigen (HLA)‐DR engagement has been shown to induce apoptosis in various antigen‐presenting cells (APC). We now report the consequences of HLA‐DR molecule engagement in human PDC, which had thus far not been studied as a result of the difficulty in isolating such cells. HLA‐DR engagement on PDC, obtained using a two‐step, immunomagnetic separation, led to recruitment of HLA‐DR molecules at the site of engagement in mature but not immature PDC. In contrast, relocalization of protein kinase C (PKC) isoenzymes, indicating PKC activation, was observed at the site of HLA‐DR engagement and was accompanied by relocalization of a lipid raft marker, the ganglioside M1 staining, in immature and mature PDC. Similar to MDC, HLA‐DR‐mediated apoptosis was regulated throughout PDC maturation. Freshly isolated PDC were resistant, whereas CD40 ligand‐matured PDC were sensitive to HLA‐DR‐mediated apoptosis. Neither caspase activation nor PKC activation was required for HLA‐DR‐mediated apoptosis. However, the intrinsic pathway of apoptosis was implicated as mature PDC underwent mitochondrial depolarization in response to HLA‐DR engagement. These data provide further arguments for considering HLA‐DR‐mediated apoptosis as a conserved mechanism of regulating survival of diverse APC and support the ongoing development of humanized ligands for HLA class II molecules as therapeutic tools for use in lymphoproliferative disease.

Cognate MHC–TCR interaction leads to apoptosis of antigen-presenting cells

Antigen presentation to T lymphocytes has been characterized extensively in terms of T lymphocyte activation and eventual cell death. In contrast, little is known about the consequences of antigen presentation for the antigen‐presenting cell (APC). We have determined the outcome of major histocompatibility complex class II‐restricted peptide presentation to a specific T cell. We demonstrate that specific T lymphocyte interaction with peptide‐presenting APCs led to apoptosis in the APC population. In contrast, T lymphocyte interaction with nonpeptide‐loaded APCs or APCs loaded with monosubstituted peptide failed to induce T lymphocyte secretion of interleukin‐2 and APC apoptosis. Phosphatidylserine externalization and mitochondrial depolarization were used to evaluate APC apoptosis. Fas/Fas ligand interactions were not required, but cytoskeletal integrity and caspase activation were essential for APC apoptosis. Antigen presentation leading to T lymphocyte activation is therefore coordinated with apoptosis in the APC population and could provide a mechanism of immune response regulation by eliminating APCs, which have fulfilled their role as specific ligands for T lymphocyte activation. This pathway may have particular importance for APCs, which are not sensitive to death receptor‐induced apoptosis.