Michael G. Kattah

Protein microarrays with carbon nanotubes as multicolor Raman labels

The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM—a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegeners granulomatosis, diluted up to 107-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure 12C and 13C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.

Differential Regulation of Chemokines by IL-17 in Colonic Epithelial Cells

The IL-23/IL-17 pathway plays an important role in chronic inflammatory diseases, including inflammatory bowel disease. In inflammatory bowel disease, intestinal epithelial cells are an important source of chemokines that recruit inflammatory cells. We examined the effect of IL-17 on chemokine expression of HT-29 colonic epithelial cells. IL-17 strongly repressed TNF-α-stimulated expression of CXCL10, CXCL11, and CCL5, but synergized with TNF-α for induction of CXCL8, CXCL1, and CCL20 mRNAs. For CXCL10, IL-17 strongly inhibited promoter activity but had no effect on mRNA stability. In contrast, for CXCL8, IL-17 slightly decreased promoter activity but stabilized its normally unstable mRNA, leading to a net increase in steady-state mRNA abundance. IL-17 synergized with TNF-α in transactivating the epidermal growth factor receptor (EGFR) and in activating ERK and p38 MAPK. The p38 and ERK pathway inhibitors SB203580 and U0126 reversed the repressive effect of IL-17 on CXCL10 mRNA abundance and promoter activity and also reversed the inductive effect of IL-17 on CXCL8 mRNA, indicating that MAPK signaling mediates both the transcriptional repression of CXCL10 and the stabilization of CXCL8 mRNA by IL-17. The EGFR kinase inhibitor AG1478 partially reversed the effects of IL-17 on CXCL8 and CXCL10 mRNA, demonstrating a role for EGFR in downstream IL-17 signaling. The overall results indicate a positive effect of IL-17 on chemokines that recruit neutrophils (CXCL8 and CXCL1), and Th17 cells (CCL20). In contrast, IL-17 represses expression of CXCL10, CXCL11, and CCR5, three chemokines that selectively recruit Th1 but not other effector T cells.

The U1-snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases

Summary:  The U1 small nuclear ribonucleoprotein particle (snRNP) is a target of autoreactive B cells and T cells in several rheumatic diseases including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). We propose that inherent structural properties of this autoantigen complex, including common RNA‐binding motifs, B and T‐cell epitopes, and a unique stimulatory RNA molecule, underlie its susceptibility as a target of the autoimmune response. Immune mechanisms that may contribute to overall U1‐snRNP immunogenicity include epitope spreading through B and T‐cell interactions, apoptosis‐induced modifications, and Toll‐like receptor (TLR) activation through stimulation by U1‐snRNA. We conclude that understanding the interactions between U1‐snRNP and the immune system will provide insights into why certain patients develop anti‐U1‐snRNP autoimmunity, and more importantly how to effectively target therapies against this autoimmune response.

A new two-color Fab labeling method for autoantigen protein microarrays.

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.

Making Sense of the Diverse Ligand Recognition by NKG2D

NKG2D recognizes multiple diverse ligands. Despite recent efforts in determining the crystal structures of NKG2D-ligand complexes, the principle governing this receptor-ligand recognition and hence the criteria for identifying unknown ligands of NKG2D remain central issues to be resolved. Here we compared the molecular recognition between NKG2D and three of the known ligands, UL16 binding protein (ULBP), MHC class I-like molecule, and retinoic acid early inducible gene as observed in the ligand-complexed crystal structures. The comparison shows that while the receptor uses a common interface region to bind the three diverse ligands, each ligand forms a distinct, but overlapping, set of hydrogen bonds, hydrophobic interactions, and salt bridges, illustrating the underlying principle of NKG2D-ligand recognition being the conservation in overall shape complementarity and binding energy while permitting variation in ligand sequence through induced fit recognition. To further test this hypothesis and to distinguish between diverse recognition and promiscuous ligand binding, four ULBP3 interface mutations, H21A, E76A, R82M, and D169A, were generated to each disrupt a single hydrogen bond or salt bridge. All mutant ULBP3 displayed reduced receptor binding, suggesting a specific, rather than promiscuous, receptor-ligand recognition. Mutants with severe loss of binding affect the receptor interactions that are mostly buried. Finally, a receptor-ligand recognition algorithm was developed to assist the identification of diverse NKG2D ligands based on evaluating the potential hydrogen bonds, hydrophobic interactions, and salt bridges at the receptor-ligand interface.

HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules

We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-β identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention.

Naive CD4 T Cell Proliferation Is Controlled by Mammalian Target of Rapamycin Regulation of GRAIL Expression

In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement, CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 costimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and proliferation of primary naive CD4 T cells.

The Ubiquitin Binding Protein TAX1BP1 Mediates Autophagosome Induction and the Metabolic Transition of Activated T Cells

SUMMARY During immune responses, naive T cells transition from small quiescent cells to rapidly cycling cells. We have found that T cells lacking TAX1BP1 exhibit delays in growth of cell size and cell cycling. TAX1BP1‐deficient T cells exited G0 but stalled in S phase, due to both bioenergetic and biosynthetic defects. These defects were due to deficiencies in mTOR complex formation and activation. These mTOR defects in turn resulted from defective autophagy induction. TAX1BP1 binding of LC3 and GABARAP via its LC3‐interacting region (LIR), but not its ubiquitin‐binding domain, supported T cell proliferation. Supplementation of TAX1BP1‐deficient T cells with metabolically active L‐cysteine rescued mTOR activation and proliferation but not autophagy. These studies reveal that TAX1BP1 drives a specialized form of autophagy, providing critical amino acids that activate mTOR and enable the metabolic transition of activated T cells. HIGHLIGHTSTax1bp1−/− T cells exhibit biosynthetic defects, delaying initial cell cyclingTAX1BP1 supports autophagic flux and activation of mTORCL‐cysteine rescues mTORC activation, bypassing the need for autophagic fluxTAX1BP1 supports T cell autophagy in response to TCR activation but not starvation &NA; Naive T cells undergo major bioenergetic and biosynthetic metabolic transitions as they initiate proliferation in response to T cell activation. Whang et al. now show that the ubiqutin binding protein TAX1BP1 is critical for autophagic flux and L‐cysteine‐dependent activation of mTORC in newly activated T cells.

Tetramers reveal IL-17–secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease

Significance Patients with the autoimmune disease systemic lupus erythematosus (SLE) develop pathogenic antibodies against their own “self”-antigens. The development of such autoantibodies is thought in part to require help from CD4+ T cells that also recognize the same self-antigens and direct autoantibody production. In this study, antigen-specific CD4+ T cells in lupus are identified and tracked for the first time using tetramers containing peptides from the spliceosomal protein U1-70. The proportion of circulating, tetramer-positive CD4+ cells correlates with the presence of U1-70–specific autoantibodies, and antigen-specific T cells are directly implicated in disease activity by their production of the proinflammatory cytokine IL-17. The role of these cells in disease pathology makes them candidate targets for therapeutic intervention. Antigen-specific CD4+ T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-Ek–containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4+ T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine140. The frequency of CD4+ T cells specific for U1-70(131-150):I-Ek (without phosphorylation) correlates with disease severity and anti–U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70–specific CD4+ T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4+ T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease.

Medicine on a need-to-know basis.

Disease-oriented, introductory medical curricula can help overcome educational and institutional barriers that separate aspiring translational scientists in PhD programs from the world of medicine.