The role of the different CD3γ domains in TCR expression and signaling.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ-) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ- lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ- T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ- patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.

Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors.

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Cooperative Interaction of Nck and Lck Orchestrates Optimal TCR Signaling.

The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαß heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding motifs for the Src family kinase, Lck, and the adaptor protein, Nck. Lck binds to a receptor kinase (RK) motif and Nck binds to a proline-rich sequence (PRS). Both motifs only become accessible upon ligand binding to the TCR and facilitate the recruitment of Lck and Nck independently of phosphorylation of the TCR. Mutations in each of these motifs cause defects in TCR signaling and T cell activation. Here, we investigated the role of Nck in proximal TCR signaling by silencing both Nck isoforms, Nck1 and Nck2. In the absence of Nck, TCR phosphorylation, ZAP70 recruitment, and ZAP70 phosphorylation was impaired. Mechanistically, this is explained by loss of Lck recruitment to the stimulated TCR in cells lacking Nck. Hence, our data uncover a previously unknown cooperative interaction between Lck and Nck to promote optimal TCR signaling.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αß and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αß T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αß T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome.

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.

Optogenetic control of integrin-matrix interaction.

Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix-OptoIntegrin system may serve as a blueprint for rendering matrix-receptor interactions amendable to precise control with light.

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors.IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

Kidins220/ARMS binds to the B cell antigen receptor and regulates B cell development and activation.

B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D-interacting substrate of 220 kD (Kidins220)/ankyrin repeat-rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase-independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell-specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca(2+), and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain-positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning.

A novel thymoma-associated immunodeficiency with increased naive T cells and reduced CD247 expression.

The mechanisms underlying thymoma-associated immunodeficiency are largely unknown, and the significance of increased blood γδ Τ cells often remains elusive. In this study we address these questions based on an index patient with thymoma, chronic visceral leishmaniasis, myasthenia gravis, and a marked increase of rare γδ T cell subsets in the peripheral blood. This patient showed cutaneous anergy, even though he had normal numbers of peripheral blood total lymphocytes as well as CD4(+) and CD8(+) T cells. Despite his chronic infection, analyses of immunophenotypes and spectratyping of his lymphocytes revealed an unusual accumulation of naive γδ and αß T cells, suggesting a generalized T cell activation defect. Functional studies in vitro demonstrated substantially diminished IL-2 and IFN-γ production following TCR stimulation of his "untouched" naive CD4(+) T cells. Biochemical analysis revealed that his γδ and αß T cells carried an altered TCR complex with reduced amounts of the ζ-chain (CD247). No mutations were found in the CD247 gene that encodes the homodimeric ζ protein. The diminished presence of CD247 and increased numbers of γδ T cells were also observed in thymocyte populations obtained from three other thymoma patients. Thus, our findings describe a novel type of a clinically relevant acquired T cell immunodeficiency in thymoma patients that is distinct from Good's syndrome. Its characteristics are an accumulation of CD247-deficient, hyporresponsive naive γδ and αß T cells and an increased susceptibility to infections.

The CD3 conformational change in the γδ T cell receptor is not triggered by antigens but can be enforced to enhance tumor killing.

Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the αßTCR, antigen induces a conformational change at the CD3 subunits (CD3 CC) that is absolutely required for αßTCR activation. Here, we demonstrate that the CD3 CC is not induced by antigen stimulation of the mouse G8 or the human Vγ9Vδ2 γδTCR. We find that there is a fundamental difference between the activation mechanisms of the αßTCR and γδTCR that map to the constant regions of the TCRαß/γδ heterodimers. Enforced induction of CD3 CC with a less commonly used monoclonal anti-CD3 promoted proximal γδTCR signaling but inhibited cytokine secretion. Utilizing this knowledge, we could dramatically improve in vitro tumor cell lysis by activated human γδ T cells. Thus, manipulation of the CD3 CC might be exploited to improve clinical γδ T cell-based immunotherapies.

Non-overlapping functions of Nck1 and Nck2 adaptor proteins in T cell activation.

BACKGROUND: Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Nck1 and Nck2 are two highly related adaptor proteins downstream of the TCR that each contains three SH3 and one SH2 domains. Their individual functions and the roles of their SH3 domains in human T cells remain mostly unknown. RESULTS: Using specific shRNA we down-regulated the expression of Nck1 or Nck2 to approximately 10% each in Jurkat T cells. We found that down-regulation of Nck1 impaired TCR-induced phosphorylation of the kinases Erk and MEK, activation of the AP-1 and NFAT transcription factors and subsequently, IL-2 and CD69 expression. In sharp contrast, down-regulation of Nck2 hardly impacts these activation read-outs. Thus, in contrast to Nck2, Nck1 is a positive regulator for TCR-induced stimulation of the Erk pathway. Mutation of the third SH3 domain of Nck1 showed that this domain was required for this activity. Further, TCR-induced NFAT activity was reduced in both Nck1 and Nck2 knock-down cells, showing that both isoforms are involved in NFAT activation. Lastly, we show that neither Nck isoform is upstream of p38 phosphorylation or Ca2+influx. CONCLUSIONS: In conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells.

Kidins220/ARMS associates with B-Raf and the TCR, promoting sustained Erk signaling in T cells.

The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D-interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αßTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220-TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.

γδ T-cell conference 2012: close encounters for the fifth time.

The fifth international γδ T-cell conference was held in Freiburg, Germany, from May 31 to June 2, 2012, bringing together approximately 170 investigators from all over the world. The scientific program covered topics such as thymic development and the mechanisms of ligand recognition and activation, the interaction of γδ T cells with other immune and non-immune cells and its implications for homeostasis, infection, tissue repair and autoimmunity, and the role of γδ T cells in malignancy and their potential for novel immunotherapies. Here we discuss a selection of the oral communications at the conference, and summarise exciting new findings in the field regarding the development, mode of antigen recognition, and responses to microorganisms, viruses and tumours by human and mouse γδ T cells.

Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRß chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRß chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

Synthetic immune signaling.

A successful immune response against pathogens requires the activation of different cell types of the immune system. These activation processes are difficult to study by classical biochemical and genetic methods alone. In this review we describe how approaches of synthetic biology, such as rebuilding of minimal functional signaling systems and the design of new molecules acting as signaling switches, can be used to get a deeper insight into the signaling mechanism of immune cells. In particular, the interaction of receptors with signal-transducing elements can be studied in detail with these new methods. In addition, sophisticated synthetic immune receptors are being tested in the clinic for gene therapy against certain cancer types.

Innate, antigen-independent role for T cells in the activation of the immune system by Propionibacterium acnes.

Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL-12-mediated IFN-gamma production. We show that P. acnes elicits IL-12p40 and p35 mRNA expression in macrophages, and IFN-gamma mRNA in liver CD4(+) T cells and NK cells. After priming with P. acnes, CD4(+) T cells serve as the major IFN-gamma mRNA source. In the absence of CD4(+) T cells, CD8(+) T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN-gamma to induce the P. acnes-driven immune effects. Moreover, in the absence of alpha beta T cells, gamma delta T cells also enable the development of strongly enhanced TNF-alpha and IFN-gamma responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T-cell types, independent of their antigen specificity, exert NK-cell-like functions, which contribute decisively to the activation of the innate immune system.

Models of antigen receptor activation in the design of vaccines.

Vaccination techniques have developed rapidly over the last several decades from the immunization with live attenuated pathogens to the use of peptide and DNA subunit vaccines, from the use of classical adjuvants to cell-directed delivery. Vaccination techiques are also under investigation for the treatment of tumors and autoimmune diseases. However, profound knowledge of activation mechanisms of the immune cells on a molecular level is prerequisite for a better understanding of the immune response, and for the development of effective immunomodulatory tools. In this review we discuss the models of BCR and TCR activation, and using the example of some vacciantion technologies, we show, how the understanding of these models could help in the design of a new generation of vaccines.

Target-dependent T-cell activation by coligation with a PSMA x CD3 diabody induces lysis of prostate cancer cells.

Recently, we have described a bispecific PSMA x CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA x CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme- based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca(2+)-influx and Erk, IkappaB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.

T cell receptor engagement triggers its CD3epsilon and CD3zeta subunits to adopt a compact, locked conformation.

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3epsilon subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3epsilon and CD3zeta subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3epsilon and CD3zeta, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.