L Van Kaer

Distinct Roles of Dendritic Cells and B Cells in Va14Ja18 Natural T Cell Activation In Vivo

Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as α-galactosylceramide (αGalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-γ or IL-4 production by these cells. We hypothesized that αGalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in αGalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, αGalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells.

Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural TCR

Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor β-chain repertoire and how α-galactosylceramide (α-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-αGalCer- and αGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional αβ TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.

Invariant natural killer T cells: innate‐like T cells with potent immunomodulatory activities

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that react with glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. Although iNKT cells express an antigen-specific receptor of the adaptive immune system, they behave more like cells of the innate immune system. A hallmark of iNKT cells is their capacity to produce copious amounts of immunoregulatory cytokines quickly after activation. The cytokines produced by iNKT cells can influence the level of activation of many cell types of the innate and adaptive immune systems as well as the quality of an adaptive immune response. As such, iNKT cells have emerged as important regulators of immune responses, playing a role in microbial immunity, autoimmunity, tumor immunity, and a variety of inflammatory conditions. Although several endogenous and exogenous glycolipid antigens of iNKT cells have been identified, how these glycolipids orchestrate iNKT-cell functions remains poorly understood. Nevertheless, iNKT cells hold substantial promise as targets for development of vaccine adjuvants and immunotherapies. These properties of iNKT cells have been investigated most extensively in mouse models of human disease using the marine sponge-derived agent alpha-galactosylceramide (alpha-GalCer) and related iNKT-cell antigens. While these preclinical studies have raised enthusiasm for developing iNKT-cell-based immunotherapies, they also showed potential health risks associated with iNKT cell activation. Although alpha-GalCer treatment in humans was shown to be safe in the short term, further studies are needed to develop safe and effective iNKT-cell-based therapies.

Targeted Colonic Claudin-2 Expression Renders Resistance to Epithelial Injury, Induces Immune Suppression and Protects from Colitis

Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naive Cl-2TG mice harbored significantly increased numbers of regulatory (CD4+Foxp3+) T cells than WT littermates. Furthermore, macrophages isolated from Cl-2TG mouse colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared with WT littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities.

Effect of high fat diet on NKT cell function and NKT cell-mediated regulation of Th1 responses.

Diet is one of the important factors that modulate immune responses. In the present study, we have examined the capacity of dietary lipids to modify immune responses in mice and we have investigated the contribution of glycolipid‐reactive natural killer T (NKT) cells in this process. Mice fed, high fat diet (HFD; 21.2% fat, 0.20% cholesterol) for 3 weeks, as compared with mice fed standard fat diet (SFD; 4.3% fat, 0.03% cholesterol), showed significantly reduced interferon‐γ production in sera at 6 or 12 h after intraperitoneal injection of an NKT cell ligand, α‐galactosylceramide. In contrast, production of interleukin‐13 was significantly higher at 2 and 6 h in HFD fed mice compared with mice on SFD. No difference was detected in the serum interleukin‐4 levels between these two groups of animals. The proportion of NKT cells in spleen and liver was reduced in mice fed HFD compared with those on SFD. In addition, activation of NKT cells assessed by up‐regulation of CD69 was suppressed specifically in liver from mice fed HFD. Recall responses of conventional T cells and delayed‐type hypersensitivity (Th1 type) against ovalbumin were significantly suppressed in mice fed HFD in comparison with those fed SFD. This suppression was not observed in CD1d−/− mice, suggesting that NKT cells in mice fed HFD played a role in suppressing Th1 responses. Taken together, our findings suggest a critical link between NKT cells, dietary lipid and adaptive immune responses.

Lymphocytes Lacking IκB-α Develop Normally, But Have Selective Defects in Proliferation and Function

NF-κB has been implicated in the development, activation, and function of B and T lymphocytes. We have evaluated the in vivo effects of deletion of IκB-α, a major inhibitor of NF-κB, on lymphocyte development, proliferation, and function. To elucidate the long term role of IκB-α in lymphocytes, fetal liver cells of 14.5-day-old IκB-α−/− or wild-type embryos were transplanted into irradiated recombinase-activating gene-2-deficient mice. Within 4 wk, the IκB-α−/− fetal liver cells reconstitute mature B and T cell populations in the recipients comparable to those produced by wild-type fetal liver cells. However, the proliferative responses of IκB-α−/− B cells are enhanced, whereas those of IκB-α−/− T cells are reduced. The levels of IgG1, IgG2a, IgA, and IgE produced by IκB-α−/− B cells are elevated relative to those produced by IκB-α+/+ or IκB-α+/−. Moreover, the specific immune responses to OVA and the generation of germinal centers are impaired in recipients of IκB-α−/− fetal liver cells. These results indicate that IκB-α plays a vital role in signal transduction pathways regulating lymphocyte proliferation and also in the production of specific Ig isotypes.

TAP1 Mutant Mice Reject Heart Grafts From Donors With No MHC Disparity

AP1 AND TAP2 (transporter associated with antigen presentation) proteins are responsible for the translocation of peptides from the cytosol into the endoplasmatic reticulum (ER), where they are loaded into newly synthesized major histocompatibility (MHC) class I proteins, allowing their assembly and expression at the cell surface. Evidence that this process is the most important mechanism of peptide delivery to MHC class I molecules comes from TAP1 mutant mice, which are defective in normal MHC class I expression and display a drastic deficiency of CD8 T cells. 1 However, some CD8 T cells are positively selected in these mutant mice. Therefore, some TAP-independent peptides, maybe derived from signal sequences or cleaved by proteases in the lumen of ER, probably occupy the MHC class I groove and permit some class I surface expression. 2 Despite the drastic deficiency of CD8 T cells, TAP1 mutant mice are capable of rejecting allografts. Moreover, it has been shown that both b2m 3 and TAP1/b2m mutant mice 4 reject skin grafts from H2 b syngeneic wild-type donors. It has been suggested that the residual CD8 population would react with TAP-dependent peptides plus class I molecules in the wild-type mice, triggering rejection. 5 However, the mechanisms involved in this type of rejection have not been elucidated. We used TAP1 mutant mice in an experimental model of heart transplantation to investigate the immune mechanisms involved in the rejection of heart grafts, particularly grafts with no MHC disparity. MATERIALS AND METHODS

NK cells inhibit T-bet-deficient, autoreactive Th17 cells.

The differentiation and maintenance of Th17 cells require a unique cytokine milieu and activation of lineage‐specific transcription factors. This process appears to be antagonized by the transcription factor T‐bet, which controls the differentiation of Th1 cells. Considering that T‐bet‐deficient (T‐bet−/−) mice are largely devoid of natural killer (NK) cells due to a defect in the terminal maturation of these cells, and because NK cells can influence the differentiation of T helper cells, we investigated whether the absence of NK cells in T‐bet‐deficient mice contributes to the augmentation of autoreactive Th17 cell responses. We show that the loss of T‐bet renders the transcription factors Rorc and STAT3 highly responsive to activation by stimuli provided by NK cells. Furthermore, reconstitution of T‐bet−/− mice with wild‐type NK cells inhibited the development of autoreactive Th17 cells through NK cell‐derived production of IFN‐γ. These results identify NK cells as critical regulators in the development of autoreactive Th17 cells and Th17‐mediated pathology.

NF-κB Protects NKT Cells from Tumor Necrosis Factor Receptor 1-induced Death.

Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-xL-coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1—the two signal transducers at the NKT TCR proximal signalling node—only partially recapitulated the NKT cell deficiency observed in IκBΔNtg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔNtg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase —which blocks downstream NF-κB activation— sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.