Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription–polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of α-galactosylceramide (α-galcer) and endogenous antigens in mouse splenic and bone marrow–derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of α-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells.

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a B-cell receptor-knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement-dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles and a greater propensity to form self-reactive germinal centers. Using mixed bone marrow chimeras we found that the myeloid compartment was sufficient to restore negative selection in the auto-reactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of germinal centers.

IntroductionInvariant natural killer T (NKT) cells represent a unique subpopulation of T cells with a highly restricted T-cell receptor (TCR) repertoire, expressing V␣14/J␣18 or V␣24/J␣18 rearranged genes in mice and humans, respectively. 1 On activation by glycolipid antigens presented on CD1d molecules, NKT cells respond rapidly, secreting high levels of Th1 and Th2 cytokines. 2 Despite their limited TCR repertoire, NKT cells can activate antibody responses against T celldependent and T cell-independent antigens by ␣-galactosylceramide (␣GalCer) coadministration. [3][4][5] It is plausible that NKT cells influence more than 1 step of the precisely regulated cascade of cellular networking events that gives rise to T celldependent B-cell immune responses directed against protein antigens. 6 NKT cells are also capable of providing cognate help for B cells, eliciting antibody production through extrafollicular plasma cell formation and atypical germinal center (GC) reaction. 7,8 Mutations of the SH2D1A gene, encoding the signaling lymphocyte activation molecule (SLAM) associated protein (SAP), impair T cell-dependent humoral responses in patients with X-linked lymphoproliferative syndrome (XLP), as well as in SAP Ϫ/Ϫ mice. [9][10][11][12] In addition to this defect of conventional CD4 ϩ T cells affecting T follicular helper cell-B cell interactions, both XLP patients and SAP Ϫ/Ϫ mice have reduced numbers of NKT cells. [13][14][15] NKT cells develop in the cortex of the thymus as a result of homotypic interactions between thymocyte precursors carrying the NKT cell-specific ␣␤TCR with thymocytes expressing CD1d/ligands. 16 In this process, homotypic interactions mediated by the cooperative engagement of the self-ligand receptors Slamf1 and Slamf6 provide "second signals" that drive maturation of NKT cells in the thymus followed by migration to the medulla and exit into the periphery. 17 Because GC formation collapses in XLP patients and SAP Ϫ/Ϫ mice during infections, especially in a recall response, the relationship between the absence of NKT cells and the dysfunctional antibody responses in XLP patients and SAP Ϫ/Ϫ mice remains unknown. To this end we used conventional SAP Ϫ/Ϫ mouse and a novel conditional SAP fl/fl mouse strain together with T-cell transfers to follow antigen-specific antibody responses in the absence of NKT cells because of SAP deficiency or in the presence of NKT cells lacking functional SAP, respectively. These experiments demonstrate that SAP expression in NKT cells is dispensable for their effective response to lipid antigens, including cytokine production and providing noncognate support to protein-specific antibody responses. By contrast, cognate NKT cell help for B cells in response to lipid-antigens requires SAP expression, corresponding to direct T-B cell interactions. These findings suggest a more fundamental role of SLAMfamily receptor signaling in providing cognate help to B cells that is not limited to CD4 ϩ T cells.Submitted November 30, 2011; accepted May 6, 2012. Prepublished onli...