Early Effector Research Articles

Host Resistance to Plasmodium-Induced Acute Immune Pathology Is Regulated by Interleukin-10 Receptor Signaling.

ABSTRACTThe resolution of malaria infection is dependent on a balance between proinflammatory and regulatory immune responses. While early effector T cell responses are required for limiting parasitemia, these responses need to be switched off by regulatory mechanisms in a timely manner to avoid immune-mediated tissue damage. Interleukin-10 receptor (IL-10R) signaling is considered to be a vital component of regulatory responses, although its role in host resistance to severe immune pathology during acute malaria infections is not fully understood. In this study, we have determined the contribution of IL-10R signaling to the regulation of immune responses during Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM). We show that antibody-mediated blockade of the IL-10R during P. berghei ANKA infection in ECM-resistant BALB/c mice leads to amplified T cell activation, higher serum gamma interferon (IFN-γ) concentrations, enhanced intravascular accumulation of both parasitized red blood cells and CD8+ T cells to the brain, and an increased incidence of ECM. Importantly, the pathogenic effects of IL-10R blockade during P. berghei ANKA infection were reversible by depletion of T cells and neutralization of IFN-γ. Our findings underscore the importance of IL-10R signaling in preventing T-cell- and cytokine-mediated pathology during potentially lethal malaria infections.

Identification of innate lymphoid cells in single-cell RNA-Seq data.

Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

Transient FOXP3 upregulation protects human conventional T cells from restimulation-induced cell death during activation-induced proliferation

Abstract An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, followed by timely disposal of those cells in order to avoid damage to host tissues. Effector T cell proliferation is constrained by the action of forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) and restimulation-induced cell death (RICD), an apoptotic pathway triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Constitutive FOXP3 expression renders Tregs resistant to RICD, in part through repression of the signaling adaptor molecule SLAM-associated protein (SAP) and FAS ligand (FASL). Interestingly, FOXP3 is also expressed transiently in human conventional T cells (Tcons) during initial rounds of activation-induced proliferation, but its function in this context remains unclear. Here we uncover a new role for FOXP3 induction in protecting both CD4+ and CD8+ human Tcons from premature RICD during the expansion phase. SiRNA-mediated silencing of FOXP3 sensitized early effector Tcons to RICD. This increase in RICD was completely dependent on de novo transcription, but FOXP3 knockdown did not alter SAP or FASL expression in these cells. Instead, FOXP3 silencing increased glycolysis and reduced autophagy in Tcons, which we and others have linked to changes in apoptosis sensitivity. Surprisingly, FOXP3-dependent autophagy specifically protected CD4+ but not CD8+ Tcons from RICD. Our results suggest a fundamentally different mechanism connecting transient FOXP3 expression to metabolic activity and RICD resistance in CD4+ versus CD8+ T cells.

A Human Lin- CD123+ CD127low Population Endowed with ILC Features and Migratory Capabilities Contributes to Immunopathological Hallmarks of Psoriasis.

Innate lymphoid cells (ILC) are members of a heterogeneous family with a lymphoid origin that mimics the T helper (Th) cytokine profile. ILC are involved in early effector cytokine-mediated responses during infections in peripheral tissues. ILC also play an important role in chronic skin inflammatory diseases, including psoriasis. Although classical ILC express CD127, it has been recently reported that the presence of non-classical CD127− ILC populations and an early ILC precursor (EILP) CD127low. ILC development has predominately been investigated in mouse models. However, in humans, different transcription factors have been described for ILC identification. NFIL3 (nuclear factor, IL-3 regulated) is crucial for ILC development in response to IL-7. CD123 (IL-3Rα) is usually used to exclude basophils during ILC identification, however, it is unknown if in response to IL-3, NFIL3 could be relevant to induce ILC features in Lin− CD123+ populations in addition, is also unknown whether peripheral blood (PB) population with ILC features may have skin-homing potential to participate in skin inflammatory chronic diseases. Here, we report a Lin− CD123+ CD127low CD7+ CLA+ population that share some phenotypic properties with basophils, but expresses several transcription factors for ILC commitment such as inhibitor of DNA binding 2 (Id2), NFIL3, promyelocytic leukemia zinc finger (PLZF), thymocyte selection-associated high-mobility group box protein (TOX), and T cell factor-1 (TCF-1). In addition, this population expresses different ILC markers: CD132, CD90, CD161, α4 integrin, c-Kit, CRTH2, AhR, and IL-23R. IL-3 prevents apoptosis and increases their NFIL3, TOX, and PLZF expression. In PB, the CD123+ CD127low population is predominantly a conspicuous population that expresses T-bet and RORγt. The Lin− CD123+ CD127low population in PB has a limited Th type cytokine expression and highly expresses IL-8. The Lin− CD123+ CD127low population expresses skin-homing receptors (cutaneous lymphocyte antigen and CXCR4) and transmigrates through endothelial cells in response to SDF-1. An equivalent Lin− CD123low population was identified in control skin, which shows a broader phenotypic diversity and cytokine production, including IL-22 and IL-17. Remarkably, the CD123low population in the lesion and non-lesion skin of psoriasis patients expresses IL-17 and IL-22. Our findings suggest the identification of an alternative Lin− CD123+ CD127low population with ILC features endowed with migratory capabilities that might contribute to immunopathological hallmarks of psoriasis.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome ofimmunization regimens in terms of memory cell numbers generated.

Early Effector T Lymphocytes Coexpress Multiple Inhibitory Receptors in Primary Non-Small Cell Lung Cancer.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.

Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation

ABSTRACTCombination of oncolytic virotherapy with immunomodulators is emerging as a promising therapeutic strategy for numerous tumor entities. In this study, we developed measles Schwarz vaccine strain vectors encoding immunomodulators to support different phases in the establishment of antitumor immune responses. Therapeutic efficacy of the novel vectors was evaluated in the immunocompetent MC38cea tumor model. We identified vectors encoding an IL-12 fusion protein (MeVac FmIL-12) and an antibody against PD-L1 (MeVac anti-PD-L1), respectively, as the most effective. Treatment of established tumors with MeVac FmIL-12 achieved 90% complete remissions. Profiling of the tumor immune microenvironment revealed activation of a type 1 T helper cell-directed response, with MeVac FmIL-12 ensuring potent early natural killer and effector T cell activation as well as upregulation of the effector cytokines IFN-γ and TNF-α. CD8+ T cells were found to be essential for the therapeutic efficacy of MeVac FmIL-12. Results of this study present MeVac FmIL-12 as a novel approach for targeted IL-12 delivery and elucidate mechanisms of successful immunovirotherapy.

Proteomic Analysis of Shigella Virulence Effectors Secreted under Different Conditions.

A series of novel effector molecules secreted by the type three secretion system (T3SS) of Shigella spp. have been reported in recent years. In this study, a proteomic approach was applied to study T3SS effectors systematically. First, proteins secreted by theS. flexneri wild-type strain after Congo Red induction were separated and identified using two-dimensional electrophoresis to display the relative abundance of all kinds of early effectors for the first time. Then, a gene deletion mutant of known virulence repressor (OspD1) and a gene overexpressed mutant of two known virulence activators (MxiE and IpgC) were constructed and analyzed to discover potential late effectors. Furthermore, the supernatant proteins of gene deletion mutants of two known translocators (IpaB and IpaD), which would constantly secrete effectors, were also analyzed. Among all of the secreted proteins identified in our study, IpaH1.4, IpaH_5, and IpaH_7 have not been reported before. These proteomics data of the secreted effectors will be valuable to understand the pathogenesis of S. flexneri.

Bruton Tyrosine Kinase Inhibition Attenuates Liver Damage in a Mouse Warm Ischemia and Reperfusion Model.

Bruton tyrosine kinase (Btk) is a central player in multiple signaling pathways of lymphoid and myeloid cells. Myeloid cells are crucial early effectors in organ ischemia-reperfusion (IR) injury. BTKB66 is a selective, irreversible inhibitor of Btk. In this study, we hypothesized that Btk inhibition would reduce hepatocellular injury in a murine model of liver warm hepatic IR. First, BTKB66 was tested in in vitro models of lipopolysaccharide-mediated neutrophil and macrophage activation. Then, to assess its efficacy in vivo, BTKB66 was administered orally to mice for 7 days before subjecting them to 90 minutes of warm hepatic ischemia followed by reperfusion for 6 or 24 hours. Clinical and pathologic features in the livers, including AST, ALT, and a panel of cytokines and chemokines, were examined. BTKB66 potently inhibited lipopolysaccharide-mediated activation of bone marrow-derived neutrophils and macrophages in vitro. It also reduced the severity of IR injury as determined by AST and ALT levels, as well as immune cell infiltrates. BTKB66 significantly decreased hepatic markers of sterile inflammation, such as C-X-C motif chemokine 1, C-X-C motif chemokine 2, and C-X-C motif chemokine 10, in parallel with depression of serum markers of the myeloid cell activation, such as CCL5, CCL11, and C-X-C motif chemokine 5. BTKB66 treatment ameliorated hepatocellular injury in a well-established model of liver partial warm ischemia and in situ reperfusion. These findings confirm that neutrophil recruitment and activation play an essential role in IR stress, and that targeting Btk activity may provide a useful approach for preventing hepatocellular damage and improving outcomes in liver transplantation.

Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages.

MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation.

Prolonged PD1 Expression on Neonatal Vδ2 Lymphocytes Dampens Proinflammatory Responses: Role of Epigenetic Regulation.

A successful pregnancy depends on the maintenance of tolerance at the fetal-maternal interface; strong inflammation in the placental bed is generally associated with adverse fetal outcomes. Among the mechanisms that foster tolerance and limit inflammation, the fetal immune system favors Th2 or regulatory responses over Th1 responses. The unintended consequence of this functional program is high susceptibility to infections. Human Vδ2 T cells mount innate-like responses to a broad range of microorganisms and are poised for Th1 responses before birth. In infants they likely play a key role in protection against pathogens by exerting early Th1 effector functions, improving function of other innate cells, and promoting Th1 polarization of adaptive responses. However, their propensity to release Th1 mediators may require careful regulation during fetal life to avoid exaggerated proinflammatory responses. We investigated molecules with the potential to act as a rheostat for fetal Vδ2 cells. Programmed death 1 (PD1) is a negative regulator of T cell responses and a determinant of tolerance, particularly at the fetal-maternal interface. Neonatal Vδ2 cells upregulate PD1 shortly after activation and, unlike their adult counterparts, express this molecule for at least 28 d. Engagement of PD1 by one of its ligands, PDL1, effectively dampens TCR-mediated responses (TNF-α production and degranulation) by neonatal Vδ2 cells and may thus help maintain their activity within safe limits. PD1 expression by neonatal Vδ2 cells is inversely associated with promoter DNA methylation. Prolonged PD1 expression may be part of a functional program to control Vδ2 cell inflammatory responses during fetal life.

De novo DNA methylation by DNA methyltransferase 3a controls early effector CD8+ T-cell fate decisions following activation

DNMT3a is a de novo DNA methyltransferase expressed robustly after T-cell activation that regulates plasticity of CD4(+) T-cell cytokine expression. Here we show that DNMT3a is critical for directing early CD8(+) T-cell effector and memory fate decisions. Whereas effector function of DNMT3a knockout T cells is normal, they develop more memory precursor and fewer terminal effector cells in a T-cell intrinsic manner compared with wild-type animals. Rather than increasing plasticity of differentiated effector CD8(+) T cells, loss of DNMT3a biases differentiation of early effector cells into memory precursor cells. This is attributed in part to ineffective repression of Tcf1 expression in knockout T cells, as DNMT3a localizes to the Tcf7 promoter and catalyzes its de novo methylation in early effector WT CD8(+) T cells. These data identify DNMT3a as a crucial regulator of CD8(+) early effector cell differentiation and effector versus memory fate decisions.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung.

ABSTRACTPigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67+) CD8+ T cells with an early effector phenotype (perforin+ CD27+) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4+ and CD8+ T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4+ and CD8+ T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4+ and CD8+ memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs.IMPORTANCE Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

IgG subclass co-expression brings harmony to the quartet model of murine IgG function.

A model of murine IgG function is presented in which the co-expression of the IgG subclasses is a central feature, class switching occurs before the commencement of somatic hypermutation, and there is little switching between subclasses. It is named the quartet model to emphasize the harmony that comes from the simultaneous presence of the four subclasses. In this model, IgG3 and IgG2b antibodies are particularly important early in the response, when T-cell help may be limiting. IgG3 initiates inflammation through complement fixation, whereas IgG2b provides early FcγR-mediated effector functions. As T-cell help strengthens, IgG2a antibodies increase the power of the response, whereas IgG1 production helps limit the inflammatory drive and limits immunopathology. The model highlights the fact that murine IgG subclasses function quite differently to human IgG subclasses. This allows them to serve the special immunological needs of a species that is vulnerable because of its small size.

Identification of Inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-Ribosyltransferase Activity

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen associated with drug resistance complications and, as such, an important object for drug discovery efforts. One attractive target for development of therapeutics is the ADP-ribosyltransferase Exotoxin-S (ExoS), an early effector of the type III secretion system that is delivered into host cells to affect their transcription pattern and cytoskeletal dynamics. The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N6-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β–stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development.

Intracellular glycogen reserves fuel early glycolytic metabolism associated with dendritic cell maturation and immune function.

Abstract Dendritic cells (DCs) serve as professional antigen presenting cells of the immune system. DCs activated by toll-like receptor (TLR) ligands undergo a process of maturation that is coupled with an increase in energy demand fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and cellular energy required for DC effector function. Inhibition of glycolysis impairs both the survival and effector function of activated DCs. According to the prevailing model in the field, TLR-driven glycolysis in DCs is sustained primarily by increases in glucose uptake. We have characterized that DCs store glucose in the form of glycogen in the immature state and that glycogen functions as an intracellular energy reserve that is critical for early effector responses by these cells. Our data show that both human and mouse DCs express enzymes essential for glycogen metabolism and that glycogen synthesis is upregulated by inflammatory signals. In addition, we have observed that inhibition of glycogen metabolism significantly impairs key maturation parameters of these cells. Our findings indicate that glycogen metabolism in DCs plays an important role in supporting the effector function of these cells. This work uncovers a novel pathway for metabolic homeostasis in DCs and provides a new paradigm for how the metabolic requirements of DC activation are regulated.

Regulatory T cells evade restimulation-induced cell death by FOXP3-mediated repression of the SAP promoter.

Abstract An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, paired with timely disposal of those cells in order to avoid damage to host tissues and maintain immune homeostasis. Restimulation-induced cell death (RICD) is one programmed apoptotic pathway that ensures effector T cell expansion remains in check, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). CD4+ regulatory T cells (Tregs) require IL-2 and experience frequent TCR stimulation, but are highly resistant to RICD. Although the mechanism remains unclear, previous work has shown that RICD resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcriptional repressor, the master regulator of Treg function. We have previously shown that T cells from patients with X-linked lymphoproliferative disease (XLP-1), caused by null mutations in the signaling adaptor molecule SLAM-associated protein (SAP), are resistant to RICD. In fact, SAP is required for optimal RICD in effector T cells. We now demonstrate that Tregs express very low levels of SAP compared to conventional T cells (Tconv). Using chromatin immunoprecipitation and reporter assays, we show that FOXP3 reduces SAP expression in Tregs by binding to and repressing the SH2D1A (SAP) promoter. Indeed, knockdown of FOXP3 restores SAP-driven RICD sensitivity in Tregs through a FAS ligand-dependent mechanism. Intriguingly, our preliminary data suggest that FOXP3 also confers RICD resistance in Tconv during initial rounds of activation-induced proliferation. Hence our results provide a mechanism by which FOXP3 confers resistance to RICD to help preserve Tregs and protect early effector Tconv cells.

Children diagnosed with both Type 1 diabetes and Celiac disease - An Immunological challenge

Type 1 diabetes (T1D) and celiac disease are both characterized by an autoimmune feature. As T1D and celiac disease share several common risk factors such as environment, genetics and immune dysregulation, patients have risk of developing the other disease subsequently. Patients with manifest T1D may have had a latent celiac disease, which is activated parallel to the anti-islet immune reactivity during the development of T1D. Contrary, a low prevalence of β-cell autoimmunity is found in young patients with celiac disease. The role of antigen-specific T cells and their relation to cytokines and chemokines is not well characterized in children with combination of T1D and celiac disease. Defective regulation and an impaired ability of responder T cells to be suppressed are suggested to contribute. We have previously shown that children suffering from these two immunological diseases in combination have a suppressed immune response to several antigens for example food antigens like gluten. Low percentages of both early and late effector memory CD8 + cells together with observations of immune aberrancies seen in the gut, in children who are prone to T1D, may suggests poor development of oral tolerance that may predispose for development of celiac disease. This review highlights the immunological complexity in these two common pediatric immunological disorders that indicates that the combination of type 1 diabetes and celiac disease is an immunological challenge. It is obvious that we are far from understanding the immunological impact of these two autoimmune diseases in combination. This immunological challenge therefore needs to be elucidated to be able to predict and prevent these autoimmune diseases.

Multiparametric flow cytometric analysis of whole blood reveals changes in minor lymphocyte subpopulations of multiple sclerosis patients

Objective: The objective of this study is to characterise the functionally relevant minor lymphocyte subpopulations in whole blood of multiple sclerosis (MS) patients and their potential utility as biomarkers for treatment follow up. Material and methods: Peripheral blood from 40 healthy donors (HD) and 66 MS patients [23 relapsing–remitting (RRMS) without treatment, 27 RRMS undergoing treatment (16 IFN-β, 11 natalizumab), and 16 progressive forms (eight secondary progressive and eight primary progressive)] was analysed by multiparametric flow cytometry. Results: Untreated MS patients showed a decrease in early effector memory (CD45RA−CCR7−CD27+) CD4+ and CD8+ T cells and an increase in Th17 lymphocytes in peripheral blood compared with HD. Regarding the effect of treatment, whereas no differences in relative percentages of cellular subpopulations were observed in patients under IFN-β treatment, those under treatment with natalizumab had an increased percentage of early effector memory CD4+ (CD45RA−CCR7−CD27+), central memory CD8+ (CD45RA−CCR7+CD27+) T cells, recent thymic emigrants (CD4+ CD45RA+CCR7+CD27+CD31+PTK7+) and transitional B cells (CD19+CD27−CD24hiCD38hi). Conclusions: Multiparametric flow cytometry analysis of whole blood is a robust, reproducible, and sensitive technology to monitor the effect of MS treatments even in minor lymphocyte subpopulations that might represent useful biomarkers of treatment response.

Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

BackgroundChlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS.ResultsIn this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166–171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells.ConclusionThe inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.