Search

Numerous publications have addressed CD147 as a tumor marker and regulator of cytoskeleton, cell growth, stress response, or immune cell function; however, the molecular functionality of CD147 remains incompletely understood. Using affinity purification, mass spectrometry, and phosphopeptide enrichment of isotope-labeled peptides, we examined the dynamic of the CD147 microenvironment and the CD147-dependent phosphoproteome in the Jurkat T cell line upon treatment with T cell stimulating agents.We identified novel dynamic interaction partners of CD147 such as CD45, CD47, GNAI2, Lck, RAP1B, and VAT1 and, furthermore, found 76 CD147-dependent phosphorylation sites on 57 proteins. Using the STRING protein network database, a network between the CD147 microenvironment and the CD147-dependent phosphoproteins was generated and led to the identification of key signaling hubs around theG proteins RAP1B and GNB1, the kinases PKCβ, PAK2, Lck, and CDK1, and the chaperone HSPA5. Gene ontology biological process term analysis revealed that wound healing-, cytoskeleton-, immune system-, stress response-, phosphorylation- and protein modification-, defense response to virus-, and TNF production-associated terms are enriched within the microenvironment and the phosphoproteins of CD147. With the generated signaling network and gene ontology biological process term grouping, we identify potential signaling routes of CD147 affecting T cell growth and function. [ABSTRACT FROM AUTHOR]

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ+ macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ+ human primary macrophages and THP-1 cells to collagen in comparison with their FRβ- counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen. [ABSTRACT FROM AUTHOR]

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. [ABSTRACT FROM AUTHOR]

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century. [ABSTRACT FROM AUTHOR]

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions. [ABSTRACT FROM AUTHOR]

GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin β-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions. [ABSTRACT FROM AUTHOR]

The buildup of TCR signaling microclusters containing adaptor proteins and kinases is prerequisite for T cell activation. One hallmark in this process is association of the TCR with lipid raft microdomains enriched in GPI-proteins that have potential to act as accessory molecules for TCR signaling. In this study, we show that GPI-anchored CD48 but not CD59 was recruited to the immobilized TCR/CD3 complex upon activation of T cells. CD48 reorganization was vital for T cell IL-2 production by mediating lateral association of the early signaling component linker for activated T cells (LAT) to the TCR/CD3 complex. Furthermore, we identified CD2 as an adaptor linking the Src protein tyrosine kinase Lck and the CD48/LAT complex to TCR/CD3: CD2 associated with TCR/CD3 upon T cell activation irrespective of CD48 expression, while association of CD48 and LAT with the TCR/CD3 complex depended on CD2. Consequently, our data indicate that CD2 and CD48 cooperate hierarchically in the buildup of the early TCR signalosome; CD2 functions as the master switch recruiting CD48 and Lck. CD48 in turn shuttles the transmembrane adapter molecule LAT.

The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr(505) upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr(505) to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr(394) to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr(505) and Tyr(394) phosphorylation and conformational alterations.

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

Polyunsaturated fatty acids (PUFAs) are known to suppress inflammatory and autoimmune responses and, therefore, clinical applications of PUFAs as immunomodulatory substances are extensively studied. PUFAs are known to inhibit T cell responses, but with respect to TCR/CD3-mediated signal transduction only a block in CD3-induced phospholipase Cgamma1/calcium signaling has been shown so far. In this study, we investigated PUFA-mediated changes in downstream T cell signal transduction. We show that among the mitogen-activated protein kinase families activation of c-Jun NH(2)-terminal kinase, but not phosphorylation of extracellular signal-regulated kinase-1/-2 or p38 is inhibited. CD3/CD28-induced activity of NF-AT was markedly reduced by PUFA treatment, while activation of other nuclear receptors (AP-1 and NF-kappaB) remained unaltered. Furthermore, IL-2 promoter activity, IL-2 and IL-13 mRNA levels, IL-2 secretion, and IL-2R alpha-chain expression were significantly diminished by PUFA treatment, whereas the expression of IFN-gamma, IL-4, IL-10, and CD69 remained essentially unaffected by PUFAs. In conclusion, PUFA treatment of T cells inhibits selectively c-Jun NH(2)-terminal kinase and NF-AT activation, resulting in diminished production of IL-2 and IL-13.

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.