Benno Kuropka

CD4+ T Cells from Human Neonates and Infants Are Poised Spontaneously To Run a Nonclassical IL-4 Program

Senescence or biological aging impacts a vast variety of molecular and cellular processes. To date, it is unknown whether CD4+ Th cells display an age-dependent bias for development into specific subpopulations. In this study, we show the appearance of a distinct CD4+ T cell subset expressing IL-4 at an early stage of development in infant adenoids and cord blood that is lost during aging. We identified by flow cytometric, fluorescent microscopic, immunoblot, and mass spectrometric analysis a population of CD4+ T cells that expressed an unglycosylated isoform of IL-4. This T cell subpopulation was found in neonatal but not in adult CD4+ T cells. Furthermore, we show that the mRNA of the Th2 master transcription factor GATA3 is preferentially expressed in neonatal CD4+ T cells. The Th2 phenotype of the IL-4+CD4+ T cells could be reinforced in the presence of TGF-β. Although the IL-4+CD4+ T cells most likely originate from CD31+CD4+ T recent thymic emigrants, CD31 was downregulated prior to secretion of IL-4. Notably, the secretion of IL-4 requires a so far unidentified trigger in neonatal T cells. This emphasizes that cytokine expression and secretion are differentially regulated processes. Our data support the hypothesis of an endogenously poised cytokine profile in neonates and suggest a link between cytokine production and the developmental stage of an organism. The determination of the IL-4 isoform–expressing cells in humans might allow the identification of Th2 precursor cells, which could provide novel intervention strategies directed against Th2-driven immunopathologies such as allergies.

Quantitative analysis of the human T cell palmitome

Palmitoylation is a reversible post-translational modification used to inducibly compartmentalize proteins in cellular membranes, affecting the function of receptors and intracellular signaling proteins. The identification of protein “palmitomes” in several cell lines raises the question to what extent this modification is conserved in primary cells. Here we use primary T cells with acyl-biotin exchange and quantitative mass spectrometry to identify a pool of proteins previously unreported as palmitoylated in vivo.

Sortase A mediated site-specific immobilization for identification of protein interactions in affinity purification–mass spectrometry experiments

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein‐protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C‐terminal linker region. As a result of this site‐directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC‐based pull‐down experiments and demonstrate the suitability of the approach.

Emerging Roles of ADAP, SKAP55, and SKAP-HOM for Integrin and NF-κB Signaling in T cells

Adapter proteins are scaffolding proteins that lack enzymatic or transcriptional activity. They play a crucial role in the organization of signalosomes, which are molecular complexes involved in signal transduction. Here, we review some of the recent findings regarding the three cytosolic adapter proteins ADAP, SKAP55, and SKAP-HOM with respect to their role in T-cell adhesion, migration and proliferation.

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function

Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Combining enzymatic 18O-labeling and 2-D LC-MS/MS for a study of protein interactions in primary T cells

Affinity-MS experiments with primary cells require alternative proteomic approaches, as the widely used metabolic labeling method SILAC is impracticable. We now describe a novel application of a recently developed 2-D LC-MS/MS approach for identification of phosphorylation-dependent protein interactions in human primary T cells. Using this approach, we identified a set of Tyr 595-phosphorylated ADAP interaction partners which belong to the larger TCR proximal signaling complex. The results show that a combination of two-dimensional RP–RP LC-MS/MS and 18O-labeling is a powerful means for peptide-based affinity MS experiments.

Tyrosine-phosphorylation of the scaffold protein ADAP and its role in T cell signaling

ABSTRACT Introduction: The Adhesion and Degranulation promoting Adaptor Protein (ADAP) is phosphorylated upon T cell activation and acts as a scaffold for the formation of a signaling complex that integrates molecular interactions between T cell or chemokine receptors, the actin cytoskeleton, and integrin-mediated cellular adhesion and migration. Areas covered: This article reviews current knowledge of the functions of the adapter protein ADAP in T cell signaling with a focus on the role of individual phosphotyrosine (pY) motifs for SH2 domain mediated interactions. The data presented was obtained from literature searches (PubMed) as well as the authors own research on the topic. Expert commentary: ADAP can be regarded as a paradigmatic example of how tyrosine phosphorylation sites serve as dynamic interaction hubs. Molecular crowding at unstructured and redundant sites (pY595, pY651) is contrasted by more specific interactions enabled by the three-dimensional environment of a particular phosphotyrosine motif (pY571).

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)1. Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3N and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

Understanding the elusive protein corona of thermoresponsive nanogels

AIM We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels. MATERIALS & METHODS Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum. RESULTS Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona. CONCLUSION We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.

Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility

The major histocompatibility complex of class II (MHCII) immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU)] allowing the identification and label-free quantification (LFQ) of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAtEAU algorithm in facilitating robust and accessible quantitative analysis of immunopeptidomes across cellular contexts. In silico analysis of the peptides enriched for each HLA-DM expression conditions suggests a higher affinity of the pool of peptides isolated from the high DM expression samples. Interestingly, our analysis reveals that while for certain autoimmune-relevant epitopes their presentation increases upon DM expression others are clearly edited out from the peptidome.