• Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice

      Prajeeth, Chittappen K; Beineke, Andreas; Iskandar, Cut D; Gudi, Viktoria; Herder, Vanessa; Gerhauser, Ingo; Haist, Verena; Teich, René; Huehn, Jochen; Baumgärtner, Wolfgang; et al. (2014-11-13)
      Abstract Background Theiler’s murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3+ CD4+ regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains. Methods In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3+ Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler’s murine encephalomyelitis (TME). Results As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. Conclusion We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.
    • Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice.

      Prajeeth, Chittappen K; Beineke, Andreas; Iskandar, Cut Dahlia; Gudi, Viktoria; Herder, Vanessa; Gerhauser, Ingo; Haist, Verena; Teich, René; Huehn, Jochen; Baumgärtner, Wolfgang; et al. (2014)
      Theiler's murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3(+) CD4(+) regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains.
    • Longitudinal proliferation mapping in vivo reveals NADPH oxidase-mediated dampening of Staphylococcus aureus growth rates within neutrophils.

      Seiß, Elena A; Krone, Anna; Formaglio, Pauline; Goldmann, Oliver; Engelmann, Susanne; Schraven, Burkhart; Medina, Eva; Müller, Andreas J; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-04-05)
      Upon the onset of inflammatory responses, bacterial pathogens are confronted with altered tissue microenvironments which can critically impact on their metabolic activity and growth. Changes in these parameters have however remained difficult to analyze over time, which would be critical to dissect the interplay between the host immune response and pathogen physiology. Here, we established an in vivo biosensor for measuring the growth rates of Staphylococcus aureus (S. aureus) on a single cell-level over days in an ongoing cutaneous infection. Using intravital 2-photon imaging and quantitative fluorescence microscopy, we show that upon neutrophil recruitment to the infection site and bacterial uptake, non-lethal dampening of S. aureus proliferation occurred. This inhibition was supported by NADPH oxidase activity. Therefore, reactive oxygen production contributes to pathogen containment within neutrophils not only by killing S. aureus, but also by restricting the growth rate of the bacterium.
    • A mathematical model of the impact of insulin secretion dynamics on selective hepatic insulin resistance.

      Zhao, Gang; Wirth, Dagmar; Schmitz, Ingo; Meyer-Hermann, Michael; Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106, Germany. (2017-11-08)
      Physiological insulin secretion exhibits various temporal patterns, the dysregulation of which is involved in diabetes development. We analyzed the impact of first-phase and pulsatile insulin release on glucose and lipid control with various hepatic insulin signaling networks. The mathematical model suggests that atypical protein kinase C (aPKC) undergoes a bistable switch-on and switch-off, under the control of insulin receptor substrate 2 (IRS2). The activation of IRS1 and IRS2 is temporally separated due to the inhibition of IRS1 by aPKC. The model further shows that the timing of aPKC switch-off is delayed by reduced first-phase insulin and reduced amplitude of insulin pulses. Based on these findings, we propose a sequential model of postprandial hepatic control of glucose and lipid by insulin, according to which delayed aPKC switch-off contributes to selective hepatic insulin resistance, which is a long-standing paradox in the field.
    • Memantine potentiates cytarabine-induced cell death of acute leukemia correlating with inhibition of K1.3 potassium channels, AKT and ERK1/2 signaling.

      Lowinus, Theresa; Heidel, Florian H; Bose, Tanima; Nimmagadda, Subbaiah Chary; Schnöder, Tina; Cammann, Clemens; Schmitz, Ingo; Seifert, Ulrike; Fischer, Thomas; Schraven, Burkhart; et al. (BMC, 2019-01-16)
      Treatment of acute leukemia is challenging and long-lasting remissions are difficult to induce. Innovative therapy approaches aim to complement standard chemotherapy to improve drug efficacy and decrease toxicity. Promising new therapeutic targets in cancer therapy include voltage-gated K We analyzed acute lymphoid (Jurkat, CEM) and myeloid (HL-60, Molm-13, OCI-AML-3) leukemia cell lines and patients' acute leukemic blasts after treatment with either drug alone or the combination of cytarabine and memantine. Patch-clamp analysis was performed to evaluate inhibition of K Our study demonstrates that memantine inhibits K Our study underlines inhibition of K
    • Mesenteric lymph node stromal cell-derived extracellular vesicles contribute to peripheral de novo induction of Foxp3(+) regulatory T cells.

      Pasztoi, Maria; Pezoldt, Joern; Beckstette, Michael; Lipps, Christoph; Wirth, Dagmar; Rohde, M; Paloczi, Krisztina; Buzas, Edit Iren; Huehn, Jochen; Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-18)
      Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-β when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-β-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.
    • Microbiome Dependent Regulation of Tregs and Th17 Cells in Mucosa.

      Pandiyan, Pushpa; Bhaskaran, Natarajan; Zou, Mangge; Schneider, Elizabeth; Jayaraman, Sangeetha; Huehn, Jochen; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Mammals co-exist with resident microbial ecosystem that is composed of an incredible number and diversity of bacteria, viruses and fungi. Owing to direct contact between resident microbes and mucosal surfaces, both parties are in continuous and complex interactions resulting in important functional consequences. These interactions govern immune homeostasis, host response to infection, vaccination and cancer, as well as predisposition to metabolic, inflammatory and neurological disorders. Here, we discuss recent studies on direct and indirect effects of resident microbiota on regulatory T cells (Tregs) and Th17 cells at the cellular and molecular level. We review mechanisms by which commensal microbes influence mucosa in the context of bioactive molecules derived from resident bacteria, immune senescence, chronic inflammation and cancer. Lastly, we discuss potential therapeutic applications of microbiota alterations and microbial derivatives, for improving resilience of mucosal immunity and combating immunopathology.
    • miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

      Łyszkiewicz, Marcin; Winter, Samantha J; Witzlau, Katrin; Föhse, Lisa; Brownlie, Rebecca; Puchałka, Jacek; Verheyden, Nikita A; Kunze-Schumacher, Heike; Imelmann, Esther; Blume, Jonas; et al. (PLOS, 2019-03-01)
      The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.
    • Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells.

      Weiss, Jonathan M; Bilate, Angelina M; Gobert, Michael; Ding, Yi; Curotto de Lafaille, Maria A; Parkhurst, Christopher N; Xiong, Huizhong; Dolpady, Jayashree; Frey, Alan B; Ruocco, Maria Grazia; et al. (2012-09-24)
      Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-β. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.
    • Neuroprotective intervention by interferon-γ blockade prevents CD8+ T cell-mediated dendrite and synapse loss.

      Kreutzfeldt, Mario; Bergthaler, Andreas; Fernandez, Marylise; Brück, Wolfgang; Steinbach, Karin; Vorm, Mariann; Coras, Roland; Blümcke, Ingmar; Bonilla, Weldy V; Fleige, Anne; et al. (2013-09-23)
      Neurons are postmitotic and thus irreplaceable cells of the central nervous system (CNS). Accordingly, CNS inflammation with resulting neuronal damage can have devastating consequences. We investigated molecular mediators and structural consequences of CD8(+) T lymphocyte (CTL) attack on neurons in vivo. In a viral encephalitis model in mice, disease depended on CTL-derived interferon-γ (IFN-γ) and neuronal IFN-γ signaling. Downstream STAT1 phosphorylation and nuclear translocation in neurons were associated with dendrite and synapse loss (deafferentation). Analogous molecular and structural alterations were also found in human Rasmussen encephalitis, a CTL-mediated human autoimmune disorder of the CNS. Importantly, therapeutic intervention by IFN-γ blocking antibody prevented neuronal deafferentation and clinical disease without reducing CTL responses or CNS infiltration. These findings identify neuronal IFN-γ signaling as a novel target for neuroprotective interventions in CTL-mediated CNS disease.
    • The NF-κB transcription factor c-Rel controls host defense against Citrobacter rodentium.

      Luu, Maik; Romero, Rossana; Bazant, Jasmin; Abass, Elfadil; Hartmann, Sabrina; Leister, Hanna; Fischer, Florence; Mahdavi, Rouzbeh; Plaza-Sirvent, Carlos; Schmitz, Ingo; et al. (Wiley-VCH, 2019-11-14)
      Mice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen.
    • Phosphorylation of Atg5 by the Gadd45β-MEKK4-p38 pathway inhibits autophagy.

      Keil, E; Höcker, R; Schuster, M; Essmann, F; Ueffing, N; Hoffman, B; Liebermann, D A; Pfeffer, K; Schulze-Osthoff, K; Schmitz, I; et al. (2013-02)
      Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45β)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45β-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45β/MEKK4 negatively regulates the autophagic process.
    • Phosphorylation of Atg5 by the Gadd45β-MEKK4-p38 pathway inhibits autophagy.

      Keil, E; Höcker, R; Schuster, M; Essmann, F; Ueffing, N; Hoffman, B; Liebermann, D A; Pfeffer, K; Schulze-Osthoff, K; Schmitz, I; et al. (2013-02)
      Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45β)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45β-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45β/MEKK4 negatively regulates the autophagic process.
    • Polymicrobial sepsis and non-specific immunization induce adaptive immunosuppression to a similar degree.

      Schmoeckel, Katrin; Mrochen, Daniel M; Hühn, Jochen; Pötschke, Christian; Bröker, Barbara M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
      Sepsis is frequently complicated by a state of profound immunosuppression, in its extreme form known as immunoparalysis. We have studied the role of the adaptive immune system in the murine acute peritonitis model. To read out adaptive immunosuppression, we primed post-septic and control animals by immunization with the model antigen TNP-ovalbumin in alum, and measured the specific antibody-responses via ELISA and ELISpot assay as well as T-cell responses in a proliferation assay after restimulation. Specific antibody titers, antibody affinity and plasma cell counts in the bone marrow were reduced in post-septic animals. The antigen-induced splenic proliferation was also impaired. The adaptive immunosuppression was positively correlated with an overwhelming general antibody response to the septic insult. Remarkably, antigen "overload" by non-specific immunization induced a similar degree of adaptive immunosuppression in the absence of sepsis. In both settings, depletion of regulatory T cells before priming reversed some parameters of the immunosuppression. In conclusion, our data show that adaptive immunosuppression occurs independent of profound systemic inflammation and life-threatening illness.
    • Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

      Franckaert, Dean; Dooley, James; Roos, Evelyne; Floess, Stefan; Huehn, Jochen; Luche, Herve; Fehling, Hans Joerg; Liston, Adrian; Linterman, Michelle A; Schlenner, Susan M; et al. (2015-04)
      Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.
    • Recirculating IL-1R2 Tregs fine-tune intrathymic Treg development under inflammatory conditions.

      Nikolouli, Eirini; Elfaki, Yassin; Herppich, Susanne; Schelmbauer, Carsten; Delacher, Michael; Falk, Christine; Mufazalov, Ilgiz A; Waisman, Ari; Feuerer, Markus; Huehn, Jochen; et al. (Springer Nature, 2020-01-27)
      The vast majority of Foxp3+ regulatory T cells (Tregs) are generated in the thymus, and several factors, such as cytokines and unique thymic antigen-presenting cells, are known to contribute to the development of these thymus-derived Tregs (tTregs). Here, we report the existence of a specific subset of Foxp3+ Tregs within the thymus that is characterized by the expression of IL-1R2, which is a decoy receptor for the inflammatory cytokine IL-1. Detailed flow cytometric analysis of the thymocytes from Foxp3hCD2xRAG1GFP reporter mice revealed that the IL-1R2+ Tregs are mainly RAG1GFP- and CCR6+CCR7-, demonstrating that these Tregs are recirculating cells entering the thymus from the periphery and that they have an activated phenotype. In the spleen, the majority of IL-1R2+ Tregs express neuropilin-1 (Nrp-1) and Helios, suggesting a thymic origin for these Tregs. Interestingly, among all tissues studied, the highest frequency of IL-1R2+ Tregs was observed in the thymus, indicating preferential recruitment of this Treg subset by the thymus. Using fetal thymic organ cultures (FTOCs), we demonstrated that increased concentrations of exogenous IL-1β blocked intrathymic Treg development, resulting in a decreased frequency of CD25+Foxp3+ tTregs and an accumulation of CD25+Foxp3- Treg precursors. Interestingly, the addition of IL-1R2+ Tregs, but not IL-1R2- Tregs, to reaggregated thymic organ cultures (RTOCs) abrogated the IL-1β-mediated blockade, demonstrating that these recirculating IL-1R2+ Tregs can quench IL-1 signaling in the thymus and thereby maintain thymic Treg development even under inflammatory conditions.
    • Regulation of neuroinflammatory properties of glial cells by T cell effector molecules.

      Prajeeth, Chittappen K; Huehn, Jochen; Stangel, Martin; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02)
    • The role of c-FLIP splice variants in urothelial tumours.

      Ewald, F; Ueffing, N; Brockmann, L; Hader, C; Telieps, T; Schuster, M; Schulz, W A; Schmitz, I; Institute of Molecular and Clinical Immunology, Otto-von-Guericke-University Magdeburg and Department of Immune Control, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. (2011)
      Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP(long) (c-FLIP(L)) and c-FLIP(short) (c-FLIP(S)), which can have opposing functions. We observed diminished expression of the c-FLIP(L) isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP(S) was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP(L) to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP(L) isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.
    • Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ.

      Trsan, Tihana; Busche, Andreas; Abram, Maja; Wensveen, Felix M; Lemmermann, Niels A; Arapovic, Maja; Babic, Marina; Tomic, Adriana; Golemac, Mijo; Brinkmann, Melanie M; et al. (2013-10-08)
      Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.
    • TCR signalling network organization at the immunological synapses of murine regulatory T cells.

      van Ham, Marco; Teich, René; Philipsen, Lars; Niemz, Jana; Amsberg, Nicole; Wissing, Josef; Nimtz, Manfred; Gröbe, Lothar; Kliche, Stefanie; Thiel, Nadine; et al. (2017-08-17)
      Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.