Michael Julius

A unique subset of self-specific intraintestinal T cells maintains gut integrity.

Lymphocytes residing in the intestinal epithelium are exclusively T cells and account for one of the largest collection of T cells in the organism. However, their function remains obscure. We and others have shown that the development of intestinal intraepithelial T cells is compromised in mutant mice prone to chronic intestinal inflammation. These results led us to directly assess their role in regulating the development of colitis secondary to transfer of primary splenic TCRαβ+CD4+CD45RBhi T cells into severe combined immunodeficiency (SCID) mice. Here we demonstrate that prior reconstitution of SCID recipients with intraintestinal TCRαβ+CD4−CD8α+β− T cells prevents disease, and does so in an interleukin (IL)-10–dependent fashion. In contrast, reconstitution with either TCRγδ+ or TCRαβ+CD4− CD8α+β+ intestinal T cells did not prevent colitis. TCRαβ+CD4−8α+β− T cells are unique to the intestinal epithelium of both rodents and humans. Previous repertoire analyses of TCRαβ+CD4−CD8α+β− T cells revealed a high proportion of cells expressing high affinity, self-specific TCR within this subset. We demonstrate that monoclonal, self specific TCRαβ+CD4−CD8α+β− cells derived from TCR transgenic mice also prevent the onset of colitis. Thus, intestinal TCRαβ+CD4−CD8α+β− T cells, selected based on their self-reactivity, maintain gut integrity in a IL-10–dependent fashion.

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2

Glycosylphosphatidylinositols (GPIs) are attached to the C‐termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI‐N‐acetylglucosaminyltransferase (GPI‐GnT), which transfers N‐acetylglucosamine from UDP‐ N‐acetylglucosamine to phosphatidylinositol. GPI‐GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG‐A, PIG‐H, PIG‐C and GPI1. Here, we report that GPI‐GnT requires another component, termed PIG‐P, and that DPM2, which regulates dolichol‐phosphate‐mannose synthase, also regulates GPI‐GnT. PIG‐P, a 134‐amino acid protein having two hydrophobic domains, associates with PIG‐A and GPI1. PIG‐P is essential for GPI‐GnT since a cell lacking PIG‐P is GPI‐anchor negative. DPM2, but not two other components of dolichol‐phosphate‐mannose synthase, associates with GPI‐GnT through interactions with PIG‐A, PIG‐C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI‐GnT; however, the enzyme activity is enhanced 3‐fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI‐GnT and imply co‐regulation of GPI‐GnT and the dolichol‐phosphate‐mannose synthase that generates a mannosyl donor for GPI.

Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinated, and their delivery of function integrated, is unknown. Here we show that lipid rafts function to segregate Lck and Fyn in T cells before activation. Coaggregation of TCR and CD4 leads to Lck activation within seconds outside lipid rafts, followed by its translocation into lipid rafts and the activation of colocalized Fyn. Genetic evidence demonstrates that Fyn activation is strictly dependent on receptor-induced translocation of Lck. These results characterize the interdependence of Lck and Fyn function and establish the spatial and temporal distinctions of their roles in the cellular activation process.

Interaction of the Wiskott–Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells

The Wiskott–Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9ΔPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation.

Intestinal inflammation observed in IL-2R/IL-2 mutant mice is associated with impaired intestinal T lymphopoiesis

BACKGROUND & AIMS Although interleukin (IL)-2(-/-) and IL-2Ralpha(-/-) mice develop inflammatory bowel disease, IL-2Rbeta(-/-) animals are apparently free of gut pathology. Intraintestinal T lymphopoiesis is reported to be impaired in IL-2Rbeta(-/-) mice; we have determined whether this characteristic correlated with the apparent resistance of this mutant strain to intestinal inflammation. This led us to reassess intraintestinal T lymphopoiesis in these 3 mutant strains. METHODS Intestinal histology and intraintestinal T lymphopoiesis were analyzed in unmanipulated mutant mice and in athymic and euthymic radiation chimeras reconstituted with bone marrow derived from IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) donors. RESULTS Intraintestinal T lymphopoiesis was ablated in the 3 mutant strains and was associated with cryptopatch abnormalities. The intestinal mucosa of mice reconstituted with lymphocytes from IL-2Rbeta(-/-) mice exhibited lesions of both the small and large bowel similar to those observed in the early stages of human gluten enteropathy and acute ulcerative colitis, respectively. Analysis of euthymic and athymic bone marrow radiation chimeras indicated that T cells located in the intestinal mucosa of unmanipulated IL-2(-/-), IL-2Ralpha(-/-), and IL-2Rbeta(-/-) mice are of thymic origin. CONCLUSIONS Null mutations at IL-2/IL-2Ralpha and beta loci differentially affect intraintestinal and intrathymic T lymphopoiesis. These conditions are associated with lesions of intestinal inflammation that are mediated by thymus-derived T cells.

Enrichment of Lck in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCRαβ

Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCRβ chain. The present study uses a family of murine IL-2-dependent CD4+ T cell clonal variants in which anti-TCRCβ signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRCβ responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCRζ:ζ-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4+ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRCβ signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRCβ proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.

Functional Requirements for Signaling through the Stimulatory and Inhibitory Mouse NKR-P1 (CD161) NK Cell Receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56lck. The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.

Synergistic regulation of immunoreceptor signaling by SLP-76-related adaptor Clnk and serine/threonine protein kinase HPK-1.

ABSTRACT Recently, the identification of Clnk, a third member of the SLP-76 family of adaptors expressed exclusively in cytokine-stimulated hemopoietic cells, has been reported by us and by others. Like SLP-76 and Blnk, Clnk was shown to act as a positive regulator of immunoreceptor signaling. Interestingly, however, it did not detectably associate with known binding partners of SLP-76, including Vav, Nck, and GADS. In contrast, it became complexed in activated T cells and myeloid cells with an as yet unknown tyrosine-phosphorylated polypeptide of ∼92 kDa (p92). In order to understand better the function of Clnk, we sought to identify the Clnk-associated p92. Using a yeast two-hybrid screen and cotransfection experiments with Cos-1 cells, evidence was adduced that p92 is HPK-1, a serine/threonine-specific protein kinase expressed in hemopoietic cells. Further studies showed that Clnk and HPK-1 were also associated in hemopoietic cells and that their interaction was augmented by immunoreceptor stimulation. A much weaker association was detected between HPK-1 and SLP-76. Transient transfections in Jurkat T cells revealed that Clnk and HPK-1 cooperated to increase immunoreceptor-mediated activation of the interleukin 2 (IL-2) promoter. Moreover, the ability of Clnk to stimulate IL-2 promoter activity could be blocked by expression of a kinase-defective version of HPK-1. Lastly we found that in spite of the differential ability of Clnk and SLP-76 to bind cellular proteins, Clnk was apt at rescuing immunoreceptor signaling in a Jurkat T-cell variant lacking SLP-76. Taken together, these results show that Clnk physically and functionally interacts with HPK-1 in hemopoietic cells. Moreover, they suggest that Clnk is capable of functionally substituting for SLP-76 in immunoreceptor signaling, albeit by using a distinct set of intracellular effectors.

Lck-dependent Fyn Activation Requires C Terminus-dependent Targeting of Kinase-active Lck to Lipid Rafts

Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active Y505FLck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of Y505FLck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.

T-Cell development and selection in the intestinal epithelium

Though it is now well established that T lymphocytes develop in the murine intestinal epithelium, many features of T lymphopoiesis occurring at this site remain controversial and poorly understood. One of the most contentious issues is whether all TcR alpha beta+ iIEL subsets, characterized by the differential expression of CD4, CD8 alpha and CD8 alpha, develop in situ, as suggested by the analysis of athymic radiation chimeras and parabionts. Athymic radiation chimeras have also been used to study positive and negative selection events imposed on developing iIEL expressing an MHC class I restricted transgenic TcR alpha beta. Results reveal that while distinct mechanisms may regulate these processes in the gut, the functional repertoire generated at this site is identical to that resulting from intra-thymic T-cell development. Strikingly, this is not the case for the development and selection of iIEL expressing an MHC class II restricted TcR alpha beta. Features peculiar to MHC class II molecules expressed by enterocytes are discussed in this context. In addition, two model systems are presented towards understanding the basis for the observed oligoclonal repertoire of TcR alpha beta+ iIEL. Specifically, the role of luminal antigen is addressed by comparing the repertoire of iIEL developing in the orthotopic gut with that of iIEL developing in an ectopic sterile intestine in the same athymic animal. The role of TcR alpha beta-mediated signals in support of the putative oligoclonal expansion of iIEL, is addressed using mice lacking the protein tyrosine kinase, Lck.