TLR3 ACTIVATION BY EBV-ENCODED SMALL RNA

Iwakiri D, Zhou L, Samanta M, Matsumoto M, Ebihara T, Seya T, Imai S, Fujieda M, Kawa K, Takada K.

Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates

signaling from Toll-like receptor 3. J Exp Med. 2009 Sep 28;206(10):2091-9. Epub 2009 Aug 31.

PubMed PMID: 19720839; PubMed Central PMCID: PMC2757889.

 

Epstein-Barr- virus encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by base pairing and giving rise to double-stranded RNA (dsRNA)-like molecules. EBER1 and EBER2 are 167 and 172 nucleotide long. EBER is the most abundant viral RNA in the latently infected cells. It binds to several cellular proteins including RNA activated protein kinase, ribosomal protein 22, lupus erythematosis-associated antigen and retinoic acid-inducible gene I.

In this interesting article, the investigators found that EBER exists in the sera of patients with active EBV infection and induces type I IFN and inflammatory cytokines through TLR-3 mediated signaling. The authors postulate that this may account for the pathogenesis of active EBV infections that are characterized by cytokinemia.

RESULTS:

• EBER is present in the culture supernatants of EBV-infected cells: The authors performed RT-PCR analysis of EBER1 in culture supernatants of Burkitt's lymphoma derived EBV-infected cell lines (Mutu+, Akata+) and EBV transformed lymphoblastoid cell lines (LCLs). They observed that EBER was detected at day 1 of culture and its expression increased and peaked on day 4. The release of EBER1 was higher than EBER2.
  

  
   

• EBER induces signaling from TLR3:
  
  • To investigate the role of EBER released from EBV infected cells, the authors first examined whether in vitro synthesized EBER1 could induce signaling from TLR3. They subjected total RNA from three LCL clones and EBV-infected and uninfected NU-GC-3 cells to RT PCR to detect TLR3. The results indicated that LCLs and gastric carcinoma NU-GC-3 cells expressed TLR3.
    
  • The authors then added in vitro synthesized EBER1 into culture medium and found the induction of IFN-beta in LCLs and EBV positive and negative NU-GC-3 cells. The authors performed similar experiments using poly (I:C) instead of EBER and observed similar results. On performing ELISA, the authors observed that IFN-beta production was dependent on the amount of EBER1 that was added to the culture.
    
  • Furthermore, when the authors pretreated LCLs with an anti-TLR3 antibody and then with EBER1, the expression of IFN-beta was markedly reduced as observed by both RT PCR and ELISA.
    
  • The authors then looked for the effect of TLR-3 knockdown on EBER-1 induced IFN-beta production. For this, negative control siRNA or TLR-3-siRNA were transfected into EBER knockout-EBV infected AGS cells and after 48 hrs, these cells were treated with EBER1 or poly (I:C) and IFN-beta production was analyzed by RT PCR. TLR3 knockout caused reduced induction of IFN. These results indicated that EBER1 induces IFN through TLR3.
    
  • IFN-regulatory factor 3 (IRF-3) and NF-kappa B function downstream of TLR3 signaling pathway. To analyze the effect of in vitro EBER1 on these downstream signals, the authors treated LCLs with in vitro synthesized EBER1 or poly(I:C) and cultured for 3 hrs after which they examined the phosphorylation of IRF3 and NF-kappa B by immunoblotting using antibodies against phosphorylated IRF3, total IRF3 and phosphorylated p65. They observed that both IRF-3 and NF-kappa B were phosphorylated upon treatment of the cells with EBER1 and poly(I:C).
    
  • Now, the authors examined whether EBER1 that was released in the culture supernatant could induce the expression of IFN. EBV positive and EBV negative Mutu cells, EBV-positive and EBV-negative Akata cells, and EBV-negative Akata cells that were stably infected with EBER-positive EBV or EBER-knockout EBV were cultured for 4 days and then the culture supernatants were harvested. LCLs were treated with 1ml of these culture supernatants or with RNA extracted from 1ml culture supernatant. The RNA extracted from the LCLs was subjected to RT-PCR to detect IFN-beta. The results indicated that IFN was induced in EBV-positive cells but not in EBV-negative cells or EBER-knockout EBV-infected cells.
    

    
     

• EBER1 is detected as a complex with La in the culture supernatants, and the complex can induce TLR3 signaling: The previous results demonstrated that EER1 was stably present in the culture supernatants which meant that it must be bound by some proteins and thus is protected from degradation by nucleases. The investigators therefore examined whether EBER1 existed as a complex with EBER-binding cellular proteins in culture supernatants. They transfected Mutu+ cells with Flag-tagged La, L22 and PKR expression plasmids, cultured them for 48 hrs and detected La, L22 and PKR by immunoblotting of the cell lysates and immunoprecipitation of culture supernatants using anti-Flag antibody. They observed that although Flag-tagged L22, La and PKR were expressed equally in transfected cells, only La could be strongly immunoprecipitated from the culture supernatant. The authors then detected EBER1 in the immunoprecipitates using RT PCR. For this, RNA was extracted from the immunoprecipitates and subjected to RT-PCR to detect EBER1. The results showed that EBER1 was preferentially co-precipitated with La. The presence of La in the culture supernatants indicated that La was actively secreted from the living cells rather than passively released from dead cells. The results further suggested that EBER1 is released from EBV-infected cells as a complex with La. To further investigate whether EBER can activate TLR3 in complexes with La, they transfected EBER positive- and EBER knockout- EBV infected AGS cells with Flag-tagged La and cultured for 48 hrs. The culture supernatants were immunoprecipitated with anti-Flag antibody. Both these EBER+ and EBER- immunoprecipitates were added into the media of EBER-knockout EBV-infected AGS cells previously transfected with TLR3 siRNA (TLR3 knockdown) or with control siRNA. IFN-beta induction was analyzed by RT PCR. IFN-beta induction was clearly induced by treatment with EBER+ immunoprecipitates, while no induction was seen upon treatment with immunoprecipitates from EBER negative cells. The induction was reduced by TLR3 knockdown. These results indicate that EBER1 can induce TLR3 signaling in complexes with La.
  

  
   

• EBER1 exists in sera from patients with active EBV infections and induces the production of type I IFN and inflammatory cytokines: The investigators next extracted RNA from sera or plasma of patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV positive and negative healthy donors and subjected to RT PCR for detection of EBER1. Results showed that EBER1 was detected in patient sera and in sera of healthy individuals. The level of EBER1 was much higher in patient sera than in sera of healthy individuals. No EBER1 was detected in sera of EBV negative healthy individuals. Furthermore, LCLs were treated with this RNA, incubated for 14hrs and subjected to RT PCR to detect IFN-beta and ELISA of the culture supernatant. The results showed that RNAs from those patient sera that contained a high amount of EBER1, induced the release of IFN-beta when added to the culture medium of LCLs. To confirm that IFN-beta production by patient sera was mediated through TLR3, the authors treated LCLs with anti-TLR3 anitbody before subjecting them to RNA from sera of patient. A marked reduction in the RNA-induced release of IFN-beta was observed. These results clearly indicated that RNA from serum induced the expression of IFN-beta through TLR3. The authors also tend to determine whether RNA from patients sera can cause induction of proinflammatory cytokines. Thus, they treated human PBMCs with RNA extracted from patients sera or in vitro synthesized EBER1 and cultured for 14hrs. Induction of IFN-gamma, and TNF-alpha was observed by RNA extracted from patient sera as well as by in vitro EBER1.
  

  
   

• EBER1 induces mature surface phenotype and antigen presenting capacity of DCs: To study the effect of EBER1 on DC phenotype, the investigators treated immature DCs with either poly(I:C) or EBER1 for 24 hrs and analyzed the surface markers of matured DCs by flow cytometry. They found that both the treatments caused increased levels of CD83 and CD86, indicating that EBER1 induces maturation of DCs. The treatment with si-TLR3 caused reduction in the up-regulation of CD86, indicating that EBER1 mediated DC maturation is TLR dependent. Next, the authors treated DCs with EBER1 and analyzed the production of IFN-beta and IL-12p40. The results indicated that IFN-beta and IL-12 production by DC was induced by EBER1 and thus EBER causes activation of DCs. To assess whether this activation of DCs by EBER is dependent on TLR3 or not, DCs were transfected with negative control siRNA or siRNA-TLR3 and then stimulated with sera from patients CAEBV containing high amounts of EBER1, low amounts of EBER1 and EBV-negative patients and IL-12 production was determined. The results indicated that the IL-12 production by DCs occurs through EBER1 mediated TLR3 signaling. To further determine whether DCs matured by EBER1 have the capacity for antigen presentation, the DCs treated with EBER1 or poly(I:C) were used for alloMLR assay. Here the authors compared the stimulatory properties of EBER1 or poly (I:C) treated DCs with those of untreated DCs. In this assay, after 24hr stimulation with EBER1 or poly(I:C), DCs were treated with mitomycin. Allo PBMCs were isolated from blood of healthy donors and cultured with mitomycin treated DCs. The PBMC proliferation was then measured using CellTiter 96 nonradioactive cell proliferation assay kit. The results indicated that EBER1-treated DCs were potent inducers of primary allo T cell responses.

CD1a-autoreactive T cells are a normal component of the human alpha-beta T cell repertoire

Source: de Jong A, Peña-Cruz V, Cheng TY, Clark RA, Van Rhijn I, Moody DB. CD1a-autoreactive T 

cells are a normal component of the human αβ T cell repertoire. Nat Immunol. 2010 Dec;11(12)

:1102-9. Epub 2010 Oct 31. PubMed PMID:21037579.

 

Members of CD1 system (CD1a, CD1b, CD1c and CD1d) bind and present lipid antigens from mammalian cells and bacteria. Based on results of several experimental studies, it is though that CD1 system is a system which allows T cells to survey CD1-expressing antigen presenting cells for changes in lipid content caused by infection, inflammation or malignancy. Studies suggest that human CD1a, CD1b and CD1c proteins and their non-human orthologs play important roles in immune response. Nearly all mammalian genomes have one or more genes encoding CD1a, and no mammalian species is known to have survived without genes encoding CD1 proteins. This retention of large gene families encoding CD1 proteins in most mammals suggests that each type of CD1 protein has a non-redundant and physiologically important role. Molecular and cellular evidences show that CD1a, CD1b, CD1c and CD1d differ in their transcriptional regulation, patterns of tissue expression, sub-cellular trafficking and antigen grove size. However, it is not known whether these differences also translate in to functional differences in the responding T cell population.

Human CD1-autoreactive T cells were discovered nearly two decades ago. Most of the insight into function of these cells has been obtained by several T cell clones rather than direct measurement of polyclonal T cells from blood and tissue. These studies have helped in assigning certain unchanging aspects of T cell phenotype such as TCR structure, expression of CD4 and CD8 co-receptors, antigens recognized and molecular mechanism of activation. However, long term culture of human T cells promotes growth of clones with in vitro growth advantage, which is not representative of in vivo repertoires. Even the best available reagents are limited in their ability to track fresh polyclonal T cells, especially CD1-autoreactive T cells. CD1-restricted T cells are able to recognize endogenous lipid antigens. The extent to which a lipid self antigen can be used to track larger pool of autoreactive T cells is unknown.

The authors sought to overcome these problems by designing a system in which cells provide a diverse pool of lipid antigens for loading onto CD1. The authors further wanted to minimize or remove the normally strong alloreactive responses to MHC class I and class II proteins that would confound with CD1-reactive responses. The authors thus transfected plasmids encoding human CD1 molecules into human myelogenous leukemia cells (K562 cells) that have low to absent expression of MHC. This resulted in MHCs that can be universally used with MHC-mismatched subjects. As these cells express a wide array of self lipid antigens, CD1 autoreacitivity can be detected broadly without prior knowledge of antigen structures. The authors observed that this method detected stronger and more frequent responses to CD1a than to any other human CD1 molecule. The authors also found that CD1a reactive T cells were fundamentally different from CD1d-reactive invariant NKT cells on the basis of their TCR patterns and effector functions. Another important observation that the authors made was that CD1a-autoreactive T cells expressed skin-homing markers and could be isolated from the skin and activated by epithelial CD1a-expressing Langerhans cells (LC). This study identified CD1a-autoreactive cells as subset of human T cells repertoire and defined CD1a as a target of the cells of Th22 helper T cell subset, which suggests a new model of LC-T cells interaction in epithelial homeostasis.

The results are described in detail below:

• CD1a-autoreactive T cells in peripheral blood: The investigators first developed a system that can measure the CD1 autoreactivity of non-clonal T cell population ex vivo. They transfected K562 cells, which have low surface density of MHC proteins, with genes encoding CD1a, CD1b, CD1c or CD1d. This resulted in high surface expression of these proteins. The investigators then confirmed the T cell-activating function of each transfected CD1 molecule by the presentation of known exogenous lipid antigens to CD1-restricted control T cell lines. For this experiment, IL-2 was analyzed in supernatant of K562 cells trasnfected with a particular CD1 molecule and incubated with T cell lines recognizing that particular CD1 molecule and exogenous lipid antigen specific for that CD1. For example, they used dideoxymycobactin (DDM) for CD1a, glucose monomycolate (GMM) for CD1b, mannosyl phosphomycoketide (MPM) for CD1c and alpha-galactosylceramide for CD1d. To determine whether CD1-restricted T cells can be detected in human peripheral blood, the investigators analyzed blood samples from 14 donors. Thus, the authors first stimulated polyclonal T cells with DCs expressing CD1a, CD1b, CD1c and CD1d and then analyzed IFN gamma secretion by T cells using CD1-trasnfected K562 cells as APCs. All the 14 donors responded to K562-CD1a cells and the mean responses were greater than those to CD1b, CD1c and CD1d. Three of the 14 donors responded to K562-CD1b, while only one of the 14 donors responded to CD1c or CD1d. Furthermore, the authors took five donors and expanded T cells in the presence of DCs and then tested the restriction of resulting short term T cell lines with K562-CD1a cells in the presence or absence of a CD1a blocking monoclonal antibody. They looked for IL-2 response in these cells. The results showed that a good response was observed when no antibody was present, however when blocking antibody was present , expression of Il-2 was reduced to background level. These results indicated that CD1a on the APCs was required for T cell response. The investigators then took a separate group of 14 donors, stimulated them with autologous DCs and analyzed clonal precursor frequency ex vivo. They generated a panel of 1291 T cell clones and the determined what percentage of autoreactive T cell clones secreted IL-2 in response to K562-CD1a cells relative to mock transfectants. They observed that one in every 50 T cell clones was CD1a autoreactive in 12 out of 14 samples. They analyzed co-receptor expression in 12 samples and found 11 CD4+ single positive clones, 1 CD8+ single positive clone, and no CD4- or CD8- clones. They next sequenced TCR alpha- and beta- chains and found that none of the CD1a-reactive clones expressed the invariant Valpha24 (TRAV10) or Vbeta11 (TRBV25), which are found on human NKT cells. The authors also observed that none of the clones expressed identical CDR3 sequences. Thus, they concluded that CD1a-autoreactive T cells are common in peripheral blood of humans and do not have the highly conserved TCR sequences seen in invariant NKT cells. The authors took eight random donors and purified CD45RO+ memory T cells, treated them with K562-CD1a cells and detected IFN-gamma response using ELISPOT assay. All the eight donors showed IFN-gamma response. Antibody blocking experiments showed that CD1a was necessary and sufficient for the response. The authors estimated the frequency of CD1a-autoreactive T cells to be between 0.02% and 0.4%. As not all CD1a-reactive T cells produce IFN-gamma, the authors performed ex vivo analysis of expanded cell population and found that T cell response to CD1a were of greater magnitude than those to other isoforms of CD1. Taken together these results indicate that CD1a-autoreactive T cells are abundant in blood without immune stimulation and were present in most or all donors. Thus, these cells can be considered to constitute a subset of the normal human alpha beta T cell repertoire.
  
• Homing of CD1a-autoreactive T cells to skin: In the periphery, CD1a is found predominantly in skin. Thus, the investigators hypothesize that CD1a-autoreactive T cells in the peripheral circulation might normally home to and localize in the skin. The investigators first determined whether CD1a-autoreactive T cells recovered from the blood expressed skin-homing markers. As it is known that surface expression of homing markers can be rapidly altered during in vitro culture, the authors sorted peripheral blood T cells ex vivo immediately after isolation on the basis of the expression of the membrane marker CD45RO and cutaneous lymphocyte antigen (CLA). The authors earlier made a chance finding of which they took advantage in these experiments. Donor 8 had an autoreactive T cell clone that was isolated from each of the three samples taken for the study. This indicated that these T cells had undergone clonal expansion in vivo. The authors confirmed the clonality of each isolate by expression of identical TCR alpha and TCR beta chains. They were than able to measure the presence of this CD1a specific clone in the naïve T cell fraction, memory T cell fraction and skin-homing memory T cell fraction of the peripheral blood. They found the PCR products for clonotypic primers almost exclusively in the memory T cell fraction (CD45RO+ CLA+). These results indicated that in donor 8, CD1a-autoreactive T cells were CLA+ memory T cells and that CD1a-autoreactive T cells can be a part of skin-homing T cell subset.
  
• CD1a-dependent IL-22 production: The investigators next stimulated CD1a-reactive T cell lines with K562-CD1a cells and measured cytokine mRNA by real time PCR. These cytokines included IFN-gamma, IL-2, those associated with T helper 1, T helper 2 and IL-17 producing subsets of MHC-restricted repertoire as well as cytokines produced by CD1d restricted NKT cells. The investigators compared the mRNA produced in response to K562-CD1a cells treated with control IgG with the mRNA produced in response to K562-CD1a cells treated with the CD1a-blocking mAb. Of the eight donors studies in this work, two showed an up-regulation of IFN-gamma alone while five showed an up-regulation of IL-22. The authors also detected secretion of IL-22 protein a dose dependent manner in response to CD1a. The authors observed a lack of substantial CD1a-dependent up-regulation of IL-17 mRNA in most donors and thus they considered that IL-22 and IL-17 are not coordinately upregulated in human CD1a-autoreactive T cells. The authors next performed intracellular cytokine staining of IL-17 and IL-22 in T cell lines from donors showing IL-22 upregulation. The experiments were performed without any stimulation and with stimulation with PMA and ionomycin. The results showed that even in the presence of stimulators, IL-17 production is not induced in IL-22 producing T cell lines. When the authors repeated same experiments in T cell fraction of total PBMCs, the presence of stimulus led to induction of production of both cytokines. These data support the idea that IL-17 and IL-22 represent the effector molecules produced by distinct T cell population in humans. These results also indicate that CD-1a-autoreactive T cells are the ones that produce IL-22. Thus, till now the authors found that a subset of CD1a-autoreactive T cell clones, selected on the basis of their production of IFN-gamma and confirmed for clonality by Vbeta PCR analysis, expressed either IFN-gamma alone or both IFN-gamma and IL-22 in various ratios in response to CD1a. These data provided direct evidence of dual cytokine-producing CD1a-autoreactive T cells at the clonal level and suggested that CD1a-dependent IFN-gamma responses observed by the authors in their previous study were probably both Th1 and Th22-Th1 cells.
  
• Skin-homing Th22 cells recognize CD1a: In addition to producing IL-22, Th22 cells are also characterized by expression of the aryl hydrocarbon receptor and expression of the chemokine receptors CCR6, CCR4 and CCR10, which promote skin homing. The authors observed that CD1a-autoreactive T cell lines expressed the aryl hydrocarbon receptor. The authors next sorted memory CD4+ T cells into the following fractions: CCR6+CXCR3+CCR4-CCR10-, CCR6+CXCR3-CCR4+CCR10- and CCR6+CXCR3-CCR4+CCR10+ and measured cytokine profiles in these fractions by RT-PCR. The results confirmed that these three populations were enriched in Th1, Th17 and Th22 cells, respectively. The authors then performed single in vitro expansion of sorted Th1 or Th22 T cell fractions with DCs and cultured them with K562-CD1a cells preincubated with mAb to CD1a or control IgG and observed that in vitro expansion caused twofold or greater CD1a-dependent up-regulation of cytokine mRNA in five of six donor. They also observed that Th22 cells showed the greatest and most frequent CD1a responses. IL-2 and IL-22 were the most dominantly upregulated cytokines in these cells. These results indicate that Cd1a-autoreactive T cells are found in human h22 subset and thus CD1a is a target for Th22 cells.
  
• CD1a-autoreactive responses from skin: To assess whether CD1a-autoreactive T cells are present in the skin, the investigators isolated T cells from human skin biopsy specimens and assessed CD1a recognition. During the initial screen, skin T cell clones were isolated whose activation was blocked by mAB to CD1a. The authors next assessed CD1a-dependent production of IL-22 in polyclonal T cells. In case of two samples (out of 3), lymphocytes isolated from human skin showed substantial production of IL-22 in response to K562-CD1a cells but not to K562-CD1b or K562-CD1c cells. These results indicated the dominance of CD1a-mediated responses in healthy human skin and their ability to produce IL-22.
  

 

Innate and Adaptive T cell Immunity to Campylobacter jejuni

Source:

Edwards LA, Nistala K, Mills DC, Stephenson HN, Zilbauer M, Wren BW, Dorrell

N, Lindley KJ, Wedderburn LR, Bajaj-Elliott M. Delineation of the Innate and

Adaptive T-Cell Immune Outcome in the Human Host in Response to Campylobacter

jejuni Infection. PLoS One. 2010 Nov 9;5(11):e15398. PubMed PMID: 21085698.

 

Campylobacter jejuni is one of the most common causes of gastroenteritis in the world. Infection with this bacteria results in clinical symptoms that can range from mild diarrhea to severe inflammatory enteritis. It has also been linked to subsequent development of Guillain-Barre syndrome (GBS). Due to significant morbidity caused by it in children in the developing world and due to emergence of antibiotic resistant clinical isolates, there is a need to further understand C. jejuni mediated pathogenesis.

There are some obstacles associated with the study of pathogenesis of and immunity to this pathogen. These are a) no convenient animal model of infection is available, b) it is unethical to perform human studies due to risk of volunteers developing GBS, c) C. jejuni mediated gastritis is self limited d) there is added risk of intestinal perforation due to which it is difficult to investigate immunity to infection in situ.

The present study is conducted by investigators at Institute of Child Health, London School of Hygiene and Tropical Medicine and Addenbrooke's hospital, Cambridge. In this study, the authors used an ex vivo model of infection which is based on human pediatric small intestine and colonic pinch biopsies in the co-culture system and looked for cytokine production in response to C. jejuni infection. This in vitro organ culture system (IVOC) has earlier been used to study enteropathogenic E. coli infection.

Previous studies have shown C. jejuni mediated effects on murine and human DC. However, the effect of this interaction of bacteria and DC on human T cells is not known. Thus, the second objective of this work was to look at the impact of bacterial-driven dendritic cell activation on T –cell mediated immunity.

Results:

1. Ex-vivo colonization of human intestine by C. jejuni: The investigators first sought to determine whether C. jejuni was able to colonize human gut explants tissue in the IVOC model of infection. Thus, they co-cultured human intestinal biopsies from the terminal ileum with wild type (WT) C. jejuni 11168H. Following this, bacteria were localized by immuno-labeling with primary un-labeled anti-Campylobacter antibody and secondary FITC-conjugated antibody. Confocal microscopy revealed that C. jejuni was routinely found in close association with the epithelial lining.
   
2. Ex-vivo release of mucosal cytokines in response to C. jejuni infection: Pediatric terminal ileum and colonic biopsy tissue were exposed to WT C. jejuni and post-infection production of cytokines was determined. The majority of cytokines were undetectable or minimally expressed in uninfected tissue. Post infection, IFN-gamma showed the most significant induction in both the tissues. IL-22 was secreted spontaneously by both tissues. IL-17 induction was modest. Among cytokines known to influence T cells, IL-23 showed the most significant increase. In contrast, increase in IL-12 and IL-6 was intermediate.
   
3. C. jejuni wild type strain drive an IL-23/Il-12 response in monocyte-derived dendritic cells: The authors next sought to determine the effect of C. jejuni infection on DC cytokine responses. They particular focused on IL-12 family members as they are critical mediators in defining the molecular nature of downstream T cell immunity. The IL-12 family members (IL-12, IL-23, IL-27 and IL-35) are known to share subunits. IL-12 is composed of p35 and p40, IL-23 is composed of p40 and p19, IL-27 is composed of p28 and Epstein Barr virus induced gene 3 (EBI3). The investigators exposed DCs to WT C. jejuni and investigated the expression of IL-12 family subunits by real time PCR. They observed that majority of individuals showed expression of p19, p35, p40 and EBI3 subunits in response to infection, but none of them showed induction of p28. The authors further quantified the cytokine response by ELISA and observed that IL-12, IL-23, IL-1beta and IL-6 were undetectable in DCs exposed to medium alone. In contrast, all four cytokines were induced in the presence of WT C. jejuni.
   
4. C. jejuni-infected DCs generate a cytokine milieu that favors single Th-17, Th-1 and double Th-17/Th-1 positive T cell responses: The authors further tested the ability of supernatants from infected DCs to propagate CD4+CD45RO+ T cell effector responses. They enriched the PBMCs for CD4+CD45RO+ memory T cells and stimulated with anti-CD3, anti-CD28 coated beads for 5 days in the presence of supernatants taken from DCs cultured with medium alone or with WT C. jejuni. They performed flow cytometric analysis and found an increase in single positive IL-17A and IFN-gamma producing T cells and a preferential increase in IL-17A/IFN-gamma double positive T cells. The authors also confirmed the increase in IFN-gamma and IL-17 protein levels by ELISA.
   
5. T-cell derived IL-22 expression in response to C. jejuni infection: The main source of IL-22 is innate immune cells. However, its expression has also been known to be associated with Th-1, Th-17 and a distinct Th-22-cell subtype. The authors therefore investigated whether C. jejuni infection results in T cells producing IL-22. They performed same experiments as mentioned above and observed that T cell mediated IL-22 expression was indeed observed in the presence of infected DC supernatants.
   
6. IL-17A and F modulate number of intracellular C. jejuni in intestinal epithelia: Based on the results, the authors hypothesized that in addition to well-established bactericidal and tissue repair functions of IFN-gamma, IL-22 and IL-17, these cytokines may also modulate adhesion and invasion of intestinal epithelial cells (IEC) by C. jejuni. To test this hypothesis, they incubated polarized Caco-2 cells with IFN-gamma, IL-17A, IL-17F or IL-22 for 24 hours and then infected the cells with C. jejuni. At three hours post infection, the investigators performed a standard bacterial adhesion and invasion assay. None of the cytokines were able to modify the number of C. jejuni bacterial cells that adhered to IEC. IL-17A and in particular IL-17F caused reduction in number of viable intracellular bacteria. In contrast, IFN-gamma or IL-22 had no effect on number of viable intracellular bacteria.

TLR agonist and Epstein Barr Virus

Source: Iskra S, Kalla M, Delecluse HJ, Hammerschmidt W, Moosmann A. Toll-like

receptor agonists synergistically increase proliferation and activation of B

cells by epstein-barr virus. J Virol. 2010 Apr;84(7):3612-23. Epub 2010 Jan 20.

PubMed PMID: 20089650; PubMed Central PMCID: PMC2838115.

 

EBV maintains itself in its host in latent state for prolonged times. The most preferred host cell type for EBV is B cell. Four modes of latent infection have been described. Mode 0, I, and II are characterized by resting B cell phenotype and expression of very few of EBV proteins. In contrast, mode III involves expression of 12 EBV proteins. The combined action of these proteins induces B cell activation and proliferation, alter receptor expression and enhance antigen presentation.

Three categories of exogenous physiological signals that lead to B cell activation have been recognized. These are signal 1, the stimulation of B cell receptor by antigen binding, signal 2, stimulation of CD40 by CD40L and signal 3, the stimulation of pattern recognition receptors, TLRs, by microbe associated molecular patterns (MAMP). It is known that combination of all three signals is required for maximal proliferation of naïve B cells. Studies have also reported that stimulation with TLR ligands alone causes transient activation of B cells.

Earlier studies have established that EBV's latent membrane proteins LMP2A and LMP1 mimic signaling by the BCR and CD40, respectively. However, in case of primary B cell infection by EBV, whether and how a potential signal 3 is generated is not known. Studies have reported that EBV infection of naïve B cells elevates expression of TLR7, thus, TLR7 pathway might play a role in generation of signal 3.

It is also known from previous studies that other microbial agents present at the site of EBV infection might influence EBV infection, B cell transformation and release of virus. Thus, in the present study, the authors investigate the effects of CpG DNA and other exogenous TLR ligands on EBV driven B cell proliferation, clonal outgrowth and induction of EBV associated cellular receptors and cytokines.

RESULTS:

• Controlled analysis of infection and transformation by EBV: EBV particles or their components might trigger TLR mediated activation of B cells. Thus, it was necessary to control for such effects by using virus like particles alongside transformation competent EBV. The investigators used a recombinant EBV system that expresses enhanced GFP. To produce transforming, GFP-encoding EBV, EBV lyric cycle was induced in human epithelial 293 cells that stably carry genomes of this recombinant EBV as episomes. Cells which carry a mutant version of EBV genome (lacking terminal repeats, 293/TR- cells), release virus like particle with empty capsids without DNA. These virus like particles are able to bind to B cells and are also taken up by B cells. When B cells are infected with EBV/TR- (pseudoinfection) there is sufficient uptake of virus particles including GFP and there is also recognition of B cells by CD4+ T cells specific for EBV-proteins. The authors next analyzed virus-associated GFP expression and de novo GFP expression in cells infected with EBV and EBV/TR-. GFP expression was detected in both set of cells on day 1 of post-infection. In contrast, a high increase in fluorescence was observed in cells infected with EBV and not with EBV/TR- on day 3 post-infection. These results indicate de novo expression of GFP from the viral genome. Later on, this GFP-high subpopulation of cells increased in number and dominated the cultures by day 7. These results are consistent with the EBV-mediated B cell transformation.
  
• Rapid activation and proliferation of B cells in response to EBV: When B cells are infected with EBV, their phenotype changes from resting to lymphoblastoid one. This phenotype is characterized by larger cell size, enlarged cytoplasm, expression of activation markers and proliferation. To reassess the timing of this process, the investigators infected primary B cells with EBV or EBV/TR- and analyzed cells' scatter characteristics by flow cytometry. On day 3 after infection with EBV and not with EBV/TR-, a distinct population of lymphoblastoid cells with high forward and side scatter levels emerged and continuously expanded thereafter. The authors also looked for surface expression of co-stimulatory surface molecule CD86 on these B cells and found that there was no expression of CD86 on B cells infected with EBV/TR-. In contrast, in cells infected with EBV, CD86 was detected at day 3, highly expressed at day 7 and remained highly expressed thereafter.
  
• CpG DNA synergistically enhances B cell transformation by EBV: The authors next infected primary B cells with EBV or EBV/TR- in the presence or absence of CpG DNA. CpG DNA is bacterial or viral DNA containing unmethylated CpG dinucleotides and it is a ligand to TLR9, and it causes activation and proliferation of B cells. On day 7 after infection, infection with EBV alone caused growth of two lymphoblastoid cell lines from resting B cells. EBV/TR- alone produced no lymphoblastoid cells and presence of CpG DNA along with EBV/TR- led to only week production. When CpG DNA was present along with EBV, several lymphoblastoid cell lines were produced. In addition, the immediate presence of CpG DNA was critical for production of lymphoblastoid cells. To further test whether other immune cells could affect the EBV-CpG synergism, the investigators repeated the same experiments with total PBMCs in the presence or absence of Cyclosporin (inhibits T cell activation). They observed that effect of CpG DNA on EBV-mediated production of lymphoblastoid cells from PBMCs and from B cells were similar. They also observed that cyclosporine had no effect. These results indicated that EBV-CpG synergism was mediated by direct effects of EBV and CpG DNA on B cells. The authors also tested whether lymphoblastoid cells detected after EBV infection in the presence of CpG DNA are transformed permanently or not. Their results showed that presence of CpG DNA at the time of EBV infection led to a long term increase in number of transformed B cell clones. The authors further determine live/dead cell ratios in EBV-CpG DNA treated versus EBV-infected B cell cultures. They observed that addition of CpG DNA together with EBV in early infection led to a six fold higher survival rate on the day when lymphoblastoid cell growth was initiated. Combined together, all these results indicate that the presence of a TLR ligand at the site of EBV infection increases the efficiency of infection and transformation of B cells.
  
• CpG DNA increases activation of B cells by EBV: To determine whether CpG modifies the expression of CD80 or CD86 molecules on the surface of B cells, the authors infected peripheral B cells with EBV or EBV/TR- in the presence or absence of CpG DNA and analyzed induction of CD80 by flow cytometry. They observed that CD80 induction was highest and fastest after EBV infection in presence of CpG DNA. The authors further looked for secretion of cytokines (IL-6 and IL-10) in these cells. IL-6 was rapidly induced by combined action of EBV and CpG DNA. In contrast, IL-10 secretion was delayed. However, when blocking antibodies against IL-6 or IL-10 were added, no effect on lymphoblastoid cell outgrowth was observed in the early days of infection. Thus, the authors concluded that enhanced IL-6 or IL-10 release is not a major factor in the rapid CpG-mediated increase of outgrowth in the days after EBV infection.
  
• Susceptibility of naïve and memory B-cell subsets to EBV transformation in the presence of CpG DNA: The authors isolated peripheral B cells from blood and separated them into naïve (CD27-) and memory (CD27+) B cells. These cells were then infected with EBV or EBV/TR- in the presence or absence of CpG DNA. The results obtained suggested that the synergistic effect of EBV-CpG is similar on both naïve and memory B cells while CpG alone was effective on memory cells only.
  
• Effect of CpG DNA on early activation of EBV latency genes: The authors infected primary B cells with EBV in the presence or absence of CpG DNA and analyzed expression levels of EBNA1, EBNA2 and LMP1 genes of EBV at different time points. The authors could see the induction of these genes on infection with EBV, but presence or absence of CpG DNA had little effect.
  
• Effect of CpG DNA on the induction of EBV's lytic cycle: The investigators cultivated lymphoblastoid cell lines in the presence or absence of CpG DNA and determined expression levels of five genes from EBV's immediate, early and late lytic stages by quantitative real-time PCR. They observed that most of these genes were downregulated by CpG DNA except BALF4. When the authors tested supernatants of these cells to determine whether they contain EBV that can infect and transform sensitive B cells, they found that supernatants from CpG DNA treated cells had this ability while those from untreated cells did not have the ability to transform B cells. These results suggested that CpG DNA might favor EBV release by latently infected B cells without up-regulation of lytic genes.
  
• Effects of various TLR ligands or bacteria on EBV transformation of tonsillar or peripheral B cells: The authors next used various other TLR liagnds e.g., Pam3CSK4 (TLR-2ligand), imiqiomod (TLR7 ligand), LPS (TLR4 lignad), LTA (TLR2 lignad) and whole fixed Staphylococcus aureus bacteria. The authors infected B cells with EBV in presence of these ligands or CpG DNA at various concentrations and analyzed lymphoblastoid cells growth 7 days after infection. The authors observed that several TLR ligands favored the outgrowth of EBV-infected B cells. Of these, CpG DNA had the strongest effect, followed by S. aureus and TLR2 ligand Pam3CSK4 and LTA. Only high concentrations of imiquimod were effective while LPS had no effect. In the presence of EBV TR-, effects of all TLR ligands were small. Not much difference was observed in tonsillar or peripheral B cells. These results suggest that not only agonists of TLR9 but also those of other TLRs, such as TLR2, support EBV-driven B cell activation and early transformation.
  

 

    

Bystander T cells in human immune responses to dengue antigens

Bystander activation of T cells refers to activation of T cells specific for an antigen X during an immune response against antigen Y. Such an activation of T cells is independent of TCR signaling and occurs through cytokines by novel activating receptors. It has been reported in models of viral infections such as herpes simplex virus, LCMV and HIV leading to proliferation of memory T cells and subsequent production of cytokines. Studies have also found bystander CD8+ T cell activation in response to intracellular bacteria. Dengue viral infection is the cause of dengue fever and dengue hemorrhagic fever. Previous studies have noted that in response to dengue infection, many pro-inflammatory cytokines are released during acute infection. Thus, bystander T cell activation may possibly occur during dengue infection.

This study was conducted by the lab of Lertmemongkolchai, The Center for Research and Development of Medical Diagnostic Laboratories, Thailand. The investigators aimed to investigate the activation of bystander T cell activation in healthy children living in endemic areas who might be vulnerable re-infection with dengue virus. Thus, they examined IFN-gamma production, which is the established indicator for bystander T cell activation, after restimulating with inactivated dengue viral antigen in vitro. Furthermore, the authors looked for bystander T cell activity by resistance to cyclosporine S (CsA), as CsA is known to inhibit T cell activation by via the TCR dependent pathway.

Source: Suwannasaen D, Romphruk A, Leelayuwat C, Lertmemongkolchai G. Bystander T

cells in human immune responses to dengue antigens. BMC Immunol. 2010 Sep

20;11:47. PubMed PMID: 20854672; PubMed Central PMCID: PMC2949776.

 

The major results of the study are as follows:

1. Healthy Thai school children could produce IFN-gamma in response to inactivated dengue virus serotype 2 in vitro: The investigator took blood samples from 55 healthy Thai school children and co-cultured them with control (medium alone), stimulators including Phytohemagglutinin (PHA) and combination of cytonies, IL12 and IL15, or Den2 in the presence or absence of CsA for 48 hrs. Cyclosporin A (CsA) is known to inhibit T cell activation via the TCR –dependent pathway. The culture supernatants were then assayed for IFN-gamma by sandwich ELISA. In the presence of positive control stimulators, a higher production of IFN-gamma was observed in compared to negative controls. In the presence of CsA, the production of IFN-gamma was inhibited. This inhibition was observed in case of stimulation by PHA and not with IL-15, IL-12. These results indicate that CsA sufficiently inhibit IFN-gamma production via the TCR-dependent but not the cytokine dependent pathway. The authors also observed that inhibition by CsA in response to Den2 and PHA treatment was very high in comparison to IL-12, IL-15 treatment indicating that IFN-gamma production by Den2 was mainly through TCR dependent pathway. To validate whether CsA does inhibit all TCR-stimulated IFN-gamma activation, the authors used a MHC class I restricted T cell epitope control of pooled peptides (CEF) of cytomegalovirus, EBV and influenza virus. The authors used five whole blood samples from healthy school children and cultured them with medium control, Den2, PHA, and CEF in the presence of absence of CsA. The results showed that CsA completely inhibited IFN-gamma production in response to CEF-stimulation but it only partially inhibits IFN-gamma production in response to stimulation by Den2. These results showed that CsA could inhibit all IFN-gamma production from TCR-dependent activation.
   
2. Identification of IFN-gamma+ cells that respond to Den2: Eighteen children who showed high IFN-gamma production were followed up to determine the IFN-gamma producing cells. Thus, the investigators co-cultured whole blood samples with medium and 18 HA units Den2 in the presence or absence of CsA. These cells were then analyzed by flow cytometry. The types of IFN-gamma+ cells were then analyzed by the combination of tri-CD3, FITC-CD8 and PE-CD4 or PE-CD56 and compared in the presence or absence of CsA. The results showed that percentage of IFN-gamma + cells in response to stimulation with Den2 was significantly higher than that in response to control (medium alone). In the presence of CsA, there was a change in IFN-gamma+ cells, which was due to decrease in number of NK cells and increase in number of T cells. The results suggested that dengue virus causes bystander T cell activation in vitro as some CD4+ and CD8+ T cells resisted the effect of CsA. The IFN-gamma + cells consisted of NK cells, CD4+ T cells and CD8+ T cells. The distribution of bystander or CsA-resistant CD4+ ranged from 10-59% and distribution of bystander or CD8+ cells ranged from 7-39%.
   
3. Kinetics of bystander T and NK cell activation: The authors next sought to describe characteristics of activated bystander T cells. Thus, the authors compared kinetics of IFN-gamma production by bystander T cells with those of NK cells and activated specific T cells. The authors studied blood samples from seven school children after 12h, 24h, 36h stimulation. The IFN-gamma+ cells were detected as early as 12h post stimulation. All the seven samples showed similar kinetics of bystander CD4+, CD8+ and NK cells. After 12h post stimulation, bystander CD4+/CD8+ cells were detected while specific CD4+/CD8+ cells were not detected. In contrast, after 24 h post stimulation the proportion of bystander and specific CD4/CD8 cells were equal.
   
4. IL-12 dependent pathway mediated T cell to produce IFN-gamma- The authors next treated whole blood samples from healthy donors with anti-cytokine antibodies, anti-IL-12, anti-IL-15 and anti-IL-18 and then treated these samples with heat –inactivated Burkholderia pseudomallei, which is a strong bystander or cytokine-dependent T cell inducer. After 48 h, culture supernatants were analyzed for IFN-gamma by ELISA. The results showed that all these three anti-cytokine antibodies could decrease IFN-gamma production by B. pseudomallei. To further investigate the production of IFN-gamma in response to dengue virus, the authors took eleven blood samples from healthy adult blood donors, treated with anti-cytokine antibodies against IL-12, IL-15 and IL-18 and then tested IFN-gamma production from culture supernatants. Anti-IL12 antibody but not anti-IL15 or anti-IL-18 antibody caused a statistically significant reduction in IFN-gamma production in response to dengue virus stimulation. The results suggest that IFN-gamma is mainly activated via IL-12 dependent pathways. When CsA was added in addition to anti-IL12, IFN-gamma production was completely inhibited. The authors then selected three representative blood samples to characterize the intracellular source of IFN-gamma by flowcytometry. They observed that major IFN-gamma producing cells were NK, CD4+ and CD8+ T cells. The authors next selected two representative samples to determine the effect of neutralizing antibodies on IFN-gamma production. Results showed that anti-IL12 and CsA cause a decrease in IFN-gamma producing cells derived from both CD4 and CD8 cells. The results also showed that CD4+ T cells were equally sensitive to anti-IL-12 and CsA, while CD8+ T cells were less sensitive to anti-IL12 than CsA. These results confirmed that bystander T cells are produced in response to dengue virus and these T cells produce IFN-gamma via IL-12 dependent pathway.
   

Pneumocystis infection enhances antibody-mediated resistance to a subsequent influenza infection

Source: Wiley JA, Harmsen AG. Pneumocystis infection enhances antibody-mediated

resistance to a subsequent influenza infection. J Immunol. 2008 Apr

15;180(8):5613-24. PubMed PMID: 18390746; PubMed Central PMCID: PMC2600725.

 

 

In the lung concurrent immune responses can occur. These occur due to

1. Simultaneous and/or consecutive exposure to multiple pathogens or immunogens
   
2. Exposure of asthamatic patients to pulmonary pathogens
   
3. When pathogen associated immune responses interact with responses to underlying chronic pulmonary diseases involving infections or COPD or emphysema
   

The progression of concurrent immune responses in the lung can lead to beneficial or detrimental outcomes. In the present paper, the authors tried to further look at this area by using co-infection with two different but regularly encountered pulmonary pathogens that elicit opposing immune responses. They used Pneumocystis murina, which elicits a type 2 immune response and influenza type A virus, which elicits a type 1 immune response.

The major results of the studies are as follows:

• Pathogen recovery from lungs: Mice were infected with Pneumocystis-infected lung homogenate and after 2-3 weeks they were infected with influenza. The control mice were given uninfected lung homogenate 2-3 weeks prior to influenza infection. Viral recovery by plaque assay was determined in co-infected mice after 1 week of influenza infection. In another set of experiments the sequence of co-infections was reversed. When Pneumocystis infection was initiated two weeks prior to influenza infection, the recovery of influenza virus decreased 100 fold in comparison to control mice. In contrast, when Pneumocystis infection was initiated three weeks prior to influenza infection, recovery of virus decreased to 10 fold. The authors further examined the rate of clearance of virus in two weeks apart co-infection model and control mice over a 10 day period following virus challenge. The greatest difference in viral clearance among the two groups was observed after seven days. Thus, the results suggested Pneumocystis infection two weeks prior to influenza infection caused reduction in influenza virus recovery.
  
• Analysis of cell recovery in BALF: The lungs of each animal were lavaged 1 week after co-infection with the influenza virus. Lymphocytes and macrophages were present in equal numbers among the two groups during the resolution of influenza infection. In contrast, neutrophils were significantly higher in airways of control mice. Among the co-infected models, neutrophils were same as those present at day 0 of influenza infection after Pneumocystis infection. The eosinophils remained at negligible levels in the influenza only control animals. The number of eosinophils was high both at the time of influenza and during resolution in co-infection models.
  
• Change in weight: The authors also examined change in weights of both groups of animals following influenza infection. Over the 10 days following the influenza infection, co-infected animals lost least amount of weight. In contrast, influenza only control animals continued to lose weight.
  
• Recovery of serum albumin and lactate dehydrogenase in BALF: Levels of albumin and LDH generally increase during lung damage and thus this increase has been used as an indicator of lung damage. In the present study, the authors observed that both albumin and LDH levels remained same at day 0 and day 7 in co-infected animals. In contrast, significant increase in albumin and LDH levels was observed in influenza only control animals.
  
• Recovery of inflammatory cytokines in BALF: The levels of inflammatory cytokines, TNFalpha, IL-10, IFN, IFN-gamma, MCP-1 and IL-6 were significantly reduced in co-infected animals compared to control animals.
  
• Germinal center B cell analysis: The investigators also performed FACS analysis of GC B cell proliferation on spleen and local tracheal-bronchial lymph node (TBLN). The authors observed that GC B cell proliferation was greater in both spleen and TBLN in co-infected animals and in control mice 1 week after influenza infection.
  
• Influenza antibody titers detected in the BALF and serum: In order to determine whether immune response to the Pneumocystis infection altered the local and/or systemic influenza specific antibodies levels, the authors also sought to determine the influenza specific antibodies. They observed that in lungs of co-infected animals, influenza specific IgA, IgG and IgM antibodies appeared earlier and remained at significantly higher levels following influenza infection.
  
• Absence of antibody negates enhanced viral clearance in co-infected mice: The authors found out that there is rapid appearance of influenza specific antibodies, which could be responsible for effective viral clearance. Thus, to determine whether the enhanced rate of viral clearance in case of co-infected animals was due to influenza specific antibodies, the authors co-infected uMT (mice generated by disruption of one of the membrane exons of the u-chain gene and have no detectable B cells or circulating antibody but have normal T cells), SCID and immunocompetent mice. They found that virus recovery in uMT co-infected mice and uMT- influenza only mice was same at seven days prior to influenza infection. The levels of viral recovery were equivalent between all of the SCID and uMT infection groups. Among the wild type mice, significant differences in virus recovery were observed among the co-infected mice and influenza only infected mice.