Role of microbiota in evolution of adaptive immune system

Source: Science 24 December 2010: 
Vol. 330 no. 6012 pp. 1768-1773 
DOI: 10.1126/science.1195568

Has the Microbiota Played a Critical Role in the Evolution of the Adaptive Immune System?

1. Yun Kyung Lee and Sarkis K. Mazmanian^*
   

   
    

• Microbes reside in several anatomical sites of human body including the skin, vagina, and mouth.
  
• Lower gastrointestinal tract of mammals harbor the greatest density and diversity of commensal microorganisms (including bacteria, archaea, fungi, viruses, protozoans, and helminthes.
  
• It has been observed that the development of gut associated lymphoid tissue (GALT), the first line of defense for the intestinal mucosa, is defective in germ free animals.
  
• It has also been observed that germ free mice display fewer and smaller Peyer's patches, smaller and less cellular mesenteric lymph nodes and less cellular lamina propia of small intestine relative to animals with a microbiota. Furthermore, these mice have been shown to exhibit reduced expression of TLRs and MHC class II molecules.
  
• Number of CD4+ T cells is also reduced in lamina propria of germ free mice. Development of isolated lymphoid follicles is also defective in the absence of microbes.
  
• Multiple proportions of intestinal immune cells require the microbiota for their development and function.
  
• In addition to its effect on intestinal immunity microbiota also effects extra intestinal immunity.
  
• It has also been observed that germ free mice are more susceptible to microorganisms like Shigella, Bacillus and Leishmania. Thus, in addition to development microbiota also effects functional aspects of intestinal and systemic immunity.
  
• Host mechanisms and the microbiota may have evolved to collaborate against infectious agents.
  
• Studies have shown an antagonistic relationship between microbiota and overt pathogens.
  
• Harnessing the immunomodulatory capabilities of microbiota may offer new avenues for development of antimicrobial therapies for infectious diseases.
  
• Microbiota play important roles in effector CD4+ T cell differentiation. During infection microbial and host signals provide cues to naïve CD4+ T cells to differentiate into various proinflammatory and anti-inflammatory subsets.
  
• Microbiota has been shown to affect the TH1-TH2 balance in the systemic immune compartments.
  
• Studies have shown that TH17 cell development in the gut is specifically affected by commensal bacteria.
  
• Of the various microbial species that constitute the microbiota of mice, only segmented filamentous bacteria (SFB) have been shown to direct intestinal T helper cell development.
  
• Researchers from around the world have proposed that life style changes have caused a fundamental alteration in the association of humans with the microbial world. The alteration in composition of healthy microbiota leading to altered microbial colonization is known as dysbiosis.
  
• Although it is not known yet whether dysbiosis causes any human diseases, it may affect autoimmunity by altering the balance between toleragenic and inflammatory members of the microbiota.
  
• In a healthy microbiome, there is an optimal proportion of both pro- and anti-inflammatory organisms (represented here by SFBs and B. fragilis), which provide signals to the developing immune system (controlled by the host genome), leading to a balance of Treg and TH17 cell activities.
  
• Altered community composition of the microbiome due to life-style, known as dysbiosis, may represent this disease-modifying component. An increase in proinflammatory microbes (for example, SFBs in animalmodels)may promote TH17 cell activity to increase and thus predispose genetically susceptible people to TH17-mediated autoimmunity.
  
• The imbalance between TH17 cells and Tregs ultimately leads to autoimmunity.
  
• We propose that certain microbes, such as SFBs, that can peacefully coexist with a healthy host but still retain pathogenic potential be termed "pathobionts" to distinguish them from opportunistic pathogens that are acquired from the environment and cause acute infections.
  
• The adaptive immune system distinguishes between self and foreign antigens and mounts an appropriate response to clear invading pathogens by recognizing non-self molecules.
  
• The adaptive immune system must either tolerate or ignore the microbiota.
  
• Several studies suggest that symbiotic bacteria have evolved the mechanisms to suppress unwanted inflammation toward the microbiota by actively inducing mucosal tolerance.
  
• The authors propose a model for co-evolution of adaptive immune system with the microbiota.
  
• According to this model, The adaptive immune system develops under the control of the vertebrate genome to produce various cell types. The evolutionarily ancient molecule TGFb directs the differentiation of Foxp3+ Treg cells.
  
• Over millennia of coevolution, commensal microbes (B. fragilis used as an example here) produced molecules that networked with the primordial immune systemto help expand various Treg cell subsets (for example, IL-10–producing Foxp3+ Treg cells).
  
• Pro-inflammatory pathobionts (such as SFBs) may have induced TH17 cell differentiation to increase mucosal defenses against enteric pathogens.
  
• The modern adaptive immune system may have arisen from two distinct events: Tregs and Th17 cell types evolved independently or through the sequential development of TH17 cells from Treg cell precursors.
  
• Taken together, the modulation of Tregs and TH17 cells by commensal microorganisms and pathobionts, respectively, appears to shape the immune status of the host and thus represents a possible risk factor for autoimmune diseases that appears to depend on balanced Treg-Th17 proportions.
  

  
   

Loss of Th17 is associated with CD4 T activation in 2009 H1N1 patients

Source: Jiang TJ, Zhang JY, Li WG, Xie YX, Zhang XW, Wang Y, Jin L, Wang FS, Zhao M.

Preferential loss of Th17 cells is associated with CD4 T cell activation in

patients with 2009 pandemic H1N1 swine-origin influenza A infection. Clin

Immunol. 2010 Dec;137(3):303-10. Epub 2010 Oct 12. PubMed PMID: 20943443.

 

H1N1 swine-origin influenza A virus (S-OIV) is a novel influenza H1N1 strain that first emerged in humans in Mexico during March 2009. The incubation period is 1-7 days. Past studies have reported that host immune response can be a critical factor in determining various outcomes of influenza infection. These studies have reported that while moderate increase in proinflammatory responses may favor viral clearance, hyper activated inflammatory response can have detrimental effects on the host. It has also been noted that after innate immunity activation there could be an abundance of virus induced inflammatory cytokines which can lead to subsequent antigen non-specific T cell activation in mice and human with viral infection.

Among the T cells, helper CD4+ T cells release a number of distinct cytokines. One such cytokine is IFN-gamma which is released by Th1 cells and which is conventionally thought to exacerbate tissue damage and control viral infection. Regulatory T cells (Tregs) are immunosuppressive and play an important role in the regulation of immune responses. In contrast, IL-17 producing CD4+ T cells (Th17) are known to play role in both chronic inflammation and in host defense against pathogens. Many studies have found that Th17 cells can play important role in protecting mice against influenza challenge. Studies have also observed Th1 and Th17 hypercytokinemia as early host response in severe pandemic influenza.

In authors' words, "whether the change of T helper subsets could bridge the inflammatory activation and T cell activation during host-pathogen interactions at an early stage of A/H1N1 infection has not been well defined".

Thus, in the present work, the authors tend to study peripheral T cells subsets in acute S-OIV-infected patients.

Study subjects: For this study, investigators collected blood samples from 53 confirmed S-OIV-infected patients and 21 healthy controls. The patients were divided into three groups according to the day of first clinical manifestation. These three groups included early stage group (Patients enrolled within 3 days after clinical onset of symptoms), intermediate stage group (patients enrolled between 4-7 days after onset) and late stage group (patients enrolled after 8 days).

RESULTS:

1. S-OIV infection results in generalized T cell depletion and T cell activation: Using flow cytometry the authors compared the absolute numbers of circulating CD3+, CD4+ and CD8+ T cells in 53 patients at the acute and convalescent stages of S-OIV infection. They observed that mean absolute CD3+, CD4+ and CD8+ T cell counts in healthy individuals were much higher than in patients. Among the three groups of patients, the patients in the early stage showed lowest counts and the counts progressively increased in intermediate and late stage patients. These data suggested that S-OIV infection leads to a rapid depletion of CD3+, CD4+ and CD8+ T cells at the early stage (1-3 days), followed by a rapid and significant restoration of CD3+, CD4+ and CD8+ T cells at 4 days after the onset of illness. Among the patients with early stage infection, T cell counts almost doubled in the convalescent phase. Among the intermediate stage patients, T cells counts were decreased at the convalescent stage and among the late stage patients no difference in T cell counts was observed in the convalescent phase. The authors also compared the expression of CD38 and HLA-DR on CD4+ and CD8+ T cells in different patient groups and healthy controls. They found that expression of CD38 and HLA-DR was higher in all CD4+ T cells than in healthy controls in early stage patients. Furthermore, they observed that expression of CD38 was upregulated in CD8+ T cells in early stage patients but there was no significant difference in HLA-DR expression on CD8+ T cells in early stage patients. The expression of CD38 and HLA-DR on CD4+ and CD8+ T cells gradually decreased in patients at the intermediate stage and late stage when compared to early stage patients. Interestingly, CD38 and HLA-DR expression were higher in convalescent stages among different groups.
   
2. Preferential loss of IL-17 expressing Th17 cells after S-OIV infection: The authors then compared the frequencies of Th1 (IFN-gamma producing CD4+ T cells), Th17 and Tregs in peripheral blood from healthy controls and patients and observed that the absolute T cell counts of Th1, Th17 and Tregs cells were significantly decreased in patients in comparison to controls. They also observed that percentage of Th1 cells was significantly increased in S-OIV infected patients in comparison to controls. They further observed that percentage of Th1 cells was more at the convalescent phase in early stage patients. All these data indicate that Th1 cells play important roles in viral clearance. In contrast to Th1 cells, the frequency of Th17 cells was significantly reduced in S-OIV infected patients in comparison to controls. However, the Th17 cells showed a gradual increase from early to late phase. Tregs did not show any significant difference in frequency among patients and controls. Thus, it can be derived that Th17 cells are more prone to be depleted at an early stage after S-OIV infection.
   
3. Th17 cells and CD4 depletion at early clinical onset is associated with sustained CD4 T cell immune activation: The authors next sought to determine the impact of depletion of Th17 cells and CD4 on T cell activation. They found that frequency of CD38+ T or HLA-DR+ T cells was negatively correlated with CD4 T cell counts or Th17 cell frequency. The authors did not observe any negative correlation with Th1 or Treg frequency. Taken together, all these data indicate that the CD4 depletion and selective loss of Th17 cells, not Th1 or Treg cells, were strongly associated with increased CD4+ T cell activation at the early stage of S-OIV infection.
   
4. S-OIV infection induced IFN-α constricts Th17 responses: The authors also analyzed the serum concentrations of IFN-α to examine its association with decrease of Th17 cells in virus infected patients. They found that serum IFN- α was highly upregulated in patients in comparison to controls. They also found that patients of early stage had lowest concentration of IL-17 among the three groups. To determine the effect of IFN- α on the production of IL-17 from Th17 cells in vitro, the authors treated PBMCs from healthy controls with IFN- α and looked for expression of IL-17 and IFN-λ. The experiment showed that IL-17 production from CD4+ T cells was significantly reduced in the presence of IFN- α. In contrast, the production of IFN-λ was significantly increased in the presence of IFN- α. These results indicate that S-OIV infection –induced IFN- α may partly constrict the function of Th17 cells.

Integral role of integrins in Th17 development

Source: 

• Pociask DA, Kolls JK. Integral role of integrins in Th17 development. J 
  

Clin Invest. 2010 Dec 1;120(12):4185-7. doi: 10.1172/JCI45450. Epub 2010 

Nov 22. PubMed PMID: 21099101; PubMed Central PMCID: PMC2993609.

 

• Acharya M, et al. αv Integrin expression by DCs is required for Th17 cell 
  

differentiation and development of experimental autoimmune 

encephalomyelitis in mice. J Clin Invest. 2010;120(12):4445–4452.
						

 

• Melton AC, Bailey-Bucktrout SL, Travis MA, Fife BT, Bluestone JA, 
  

Sheppard D. Expression of αVβ8 integrin on dendritic cells regulates Th17 

cell development and experimental autoimmune encephalomyelitis in mice. 

J Clin Invest. 2010;120(12):4436–4444. 

 

Th17 cells are a lineage of CD4+ T cells and are supposed to be derived by exposure of naïve CD4+ T cells to IL-6 and TGF-beta. They have been recently identified (2007). These cells secrete IL-17A and IL-17F as well as IL-21 and IL-22. Recent studies have shown that Th17 cells are critical for host defense against bacterial, fungal and viral infections at mucosal surfaces. In additions, Th17 cells have also been implicated in autoimmune diseases such as multiple sclerosis, psoriasis and rheumatoid arthritis.

Several studies have shown that naïve CD4+ T cells differentiate into Tregs in the presence of TGF-beta. However they differentiate into Th17 cells in the presence of IL-6 and TGF-beta. TGF-beta is a multifunctional cytokine involved in many aspects of immunology, angiogenesis and epithelial growth as well as in pathogenic states such as fibrosis. TGF-beta is secreted from CD4+ T cells in an inactive form. In this form, TGF-beta is present in a complex with the latency associated peptide (LAP) through non-covalent bonds. Recent studies have shown that DCs can activate TGF-beta through integrins, suggesting that activation of TGF-beta occurs at the DC/T cell synapse. This activation of TGF-beta then drives the differentiation of Th17 T cells.

Integrins are a family of heterodimeric cell surface receptors consisting of an alpha and a beta subunit. There are total 24 integrin subunits including 18 alpha and 6 beta. Five of these integrins share the αν subunit (ανβ1, ανβ3, ανβ5, ανβ6 and ανβ8) and can bind to the RGD tripeptide sequence on the LAP of TGF-beta. Two mechanisms have been proposed to explain integrin mediated activation of TGF-beta. According to first mechanism the binding of integrins, which are bound to the cytoskeleton such as integrin ανβ6, to the TGF-beta induces a conformational change upon the latent complex of TGF-beta. This conformational change allows the active portion of TGF-beta to be exposed to its receptor without breaking the TGF-beta/LAP bond. In the second mechanism proposed, integrin ανβ8, which lacks cytoskeleton attachment acts as an anchor for TGF-beta, allowing proteolysis by membrane bound MMP-14 (also known as mt1-MMP).

Two recent papers have demonstrated the requirement of integrin ανβ8 activation of TGF-beta in the differentiation of Th17 cells (Acharya et al., 2010 and Melton et al., 2010). Both of these studies used experimental autoimmune encephalitis (EAE) diseases model. In this EAE diseases model, EAE was induced by immunization with MOG_35–55 peptide emulsified in CFA (containing Mycobacterium tuberculosis H37Ra).

One of these studies considered the common requirement of TGF-beta in the development of Tregs and Th17 cells and found out that conditional knockout mice (αν-tie2 mice) that lack integrin αν on all hematopoietic cells have reduced proportions of Th17 cells in the lamina propria. When CD4+ T cells from these mice were treated with exogenous TGF-beta, they were able to differentiate into Th17 cells. The authors crossed mice with a floxed itgav allele (the allele that encodes αv) to LysM-cre, which allowed expression of αν integrins on lymphoid cells but not on macrophages and DCs. The authors showed that expression of αν integrins on LysM expressing cells was required for activation of TGF-beta, which is further required for Th17 cells generation in αν-tie2 mice. These results demonstrate the useful of αν in activation of TGF-beta and generation of Th17 cells. But, these data do not identify which αν integrins is responsible here. Mice lacking αν integrins are incapable of producing ανβ1, ανβ3, ανβ5, ανβ6 and ανβ8. So, any one of them can play a role in activation of TGF-beta.

In another such recent study, the authors show similar reduction in number of Th17 cells in lamina propria of mice lacking ανβ8 expression on DCs (β8^fl/fl × CD11c-cre mice). In both studies, mice did not develop experimental autoimmune encephalitis (EAE), a condition which is Th17 dependent. Both the studies looked at the cytokines involved in Th17 polarization. They found that there were no differences in IL-6, IL-23, TGF-beta expression after immunization for EAE. Both group of investigators showed that DCs were required to activate TGF-beta. When naïve CD4+ T cells were cultured in the presence of latent TGF-beta, they did not differentiate into Th17 cells unless DCs were also present in vitro.

These two studies show a novel mechanism of development of Th17 cells. Th17 cells are important in autoimmune diseases and thus a lot of research has been going on to explore their generation. According to the mechanism proposed by these studies, naïve CD4+ t cells recognize antigens presented by DCs in MHC-classII dependent manner and also get induced to Th17 cells by activation of TGF-beta -through integrin ανβ8. One question that is still unanswered is how IL-17 is produced by gamma-delta T cells. A recent study has shown that Th17 differentiation can occur in the absence of TGF-beta signaling. They showed that IL-6, IL-23 and IL-1beta efficiently generated IL-17 production in naïve T cells independent of TGF-beta. All these studies are very important as they through light on Th17 differentiation. In order to develop therapeutic strategies for autoimmune diseases, it is critical to understand origin and development of Th17 cells.

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.

NKT cells and SAP

Source: Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R. Signaling lymphocytic

activation molecule-associated protein controls NKT cell functions. J Immunol.

2005 Mar 15;174(6):3153-7. PubMed PMID: 15749842.

 

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency disorder, which is caused by mutation in the gene Src homology 2 (SH2) domain containing gene 1A (SH2D1A). This gene encodes signaling lymphocyte activating molecule (SALM)-associated protein (SAP). There are many evidences that indicate that SAP plays a role in regulation of lymphocytes.

• The clinical course of XLP consists of uncontrolled proliferation of B and T cells.
  
• Studies have observed that SAP knock out (SAPKO) mice generate abnormally large virus specific CD8+ and CD4+ T cell following infection with LCMV.
  
• It has also been observed that SAP binds to intracellular domains of SLAM and B4, both of these are immuno-regulatory molecules, which are primarily expressed on T cells and NK cells, respectively.
  
• SAPKO mice are unable to generate memory B cell response.
  

Despite all these evidences about role of SAP in controlling lymphocyte proliferation, a role for SAP in NKT cells development and function, a type of regulatory cells, is not known. It has been previously reported that CD1d restricted NKT cells control EBV-specific lymphocyte proliferation [Ho et al., 2004 (CD4-CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion)]. This can be due to a possible link between NKT cell function and large T cell expansions seen in XLP patients following EBV infection.

In a beautiful paper published in 2005 from the labs of Dr. Rusung Tan, the investigators sought to determine the role of NKT cells in the immune dysregulation of SAPKO mice.

Their findings are briefly described below:

1. SAPKO mice completely lack CD1d restricted NKT cells.
   
2. When SAPKO mice were injected with potent NKT agonist alpha-GalCer, the mice did not develop or activate NKT cells that produce cytokines (IFN-gamma and IL-4).
   
3. When SAPKO mice were co-injected with OVA and alpha-GalCer, the mice did not show OVA specific CTL responses.
   
4. Patients with XLP did not have a population of CD1d restricted NKT cells in their peripheral blood.
   

All these findings indicated that SAP is critical for normal CD1d restricted NKT cell development and function.

 

Performance of experiments:

• SAPKO mice and wild type mice were taken and SAP gene expression was confirmed by PCR and immunoprecipitaion. Lymphocytes were stained with Abs to CD3 and NK1.1, these two molecules are the markers present on murine NKT cells, and flow-cytometric analyses were performed. The investigators observed that frequency of CD3+ NK1.1+ NKT cells from liver, thymus, spleen and lymph nodes was significantly low in SAPKO mice in comparison to wild type. An earlier study (Hammond et al., 2004) has shown that NK1.1 expression may not be the ideal marker for CD1d restricted NKT cells as many of the CD1d restricted cells fail to express NK1.1. Thus, Dr. Tan and his team used CD1d tetramer and Ab to TCRVbeta8 to stain lymphocytes from spleen, liver, thymus and lymph nodes from SAPKO and wild type mice. The data obtained confirmed that SAPKO mice lack CD1d-restrcited NKT cells.
  
• Up on activation NKT cells rapidly produce cytokines. The investigators thus hypothesized that since SAPKO mice lack CD1D-restricted NKT cells, they would not produce these cytokines upon activation. Thus, SAPKO and wild type mice were intra-peritoneally injected with alpha-GalCer and liver cells were isolates after 2 hrs. These cells were stained with CD1d tetramer to identify CD1d-restricted NKT cells and intracellular cytokine staining was performed using anti-IFN-gamma-allophycocyanin or anti-IL4-allophycocyanin. The authors observed no CD1d restricted NKT cells and no IFN-gamma or IL-4 production in SAPKO mice. In contrast, wild type mice produced significant amount of IFN-gamma and IL-4. These findings indicate that SAPKO mice are unable to mount a rapid NKT-induced cytokine response.
  
• A previously conducted study showed that NKT cells not only regulate LCMV-induce cytokine production, but they also controlled the magnitude of the cell-mediated immune response to an acute viral infection. Thus, NKT cells are important for generation and regulation of antigen specific T cells. Thus, to determine whether NKT cells are important for activation or proliferation of antigen specific T cells, the authors immunized SAPKO and wild type mice with vehicle (a control for alpha-GalCer) and OVA, CFA or alpha-GalCer. At day 14, PBMCs were isolated and stained with Kb-OVA254-267 tetramers and CD8 Ab to measure OVA-CTL expansion. Wild type mice showed significant CTL-expansion in comparison to SAPKO mice immunized with OVA and alpha-GalCer. These data indicate that the generation of Ag-specific CTL response is promoted by CD1d restricted NKT cells and SAPKO mice are unable to generate CTL in response to immunized Ag and NKT cell agonist.
  
• The scientists also took peripheral blood from two XLP patients, who had mutations in the second exon of SAP, and stained the PBMCs with CD1d tetramer and anti-CD3 Ab. They followed the same experiments in eight healthy individuals. The healthy controls showed significant percentage of CD1d-restricted NKT cells in comparison to XLP patients, indicating that CD1d-restricted NKT cells are absent in XLP patients.
  

 

Conclusions:

NKT cells act as a bridge between innate immune response and adaptive immune response by rapidly activating NK cells and helping in maturation of DCs. Thus, the authors hypothesized that in the absence of NKT cells, the immune response to virus is sub-optimal. Thus, viral replication occurs unchecked leading to formation of a chronic viral stage. It has been observed that boys with XLP disease show continuous viremia with EBV during the course of diseases. Such uncontrolled virus infection may lead to chronic stimulation of virus specific CTLs that are not efficient to clear virus-infected cells.

NKT cells suppress EBV-associated tumors

EBV, a first discovered human tumor virus, is considered to be one of the most successful human pathogen. It is involved in the development of many malignancies, such as Hodgkin's lymphoma and nasopharyngeal carcinoma in immunocompromised individuals. Majority of the individuals infected with EBV remain symptom free. Thus, it is believed that EBV has co-evolved with the human immune system to a peaceful co-existence.

It is known that natural killer T cells (NKT) are a special type of T cells that express NK lineage receptors including CD1d. They respond to glycolipid antigen presented by CD1d. CD1d is mainly expressed on antigen presenting cells (APCs, dendritic cells, macrophages and B cells). Thus, NKT cells primarily interact with APCs rather than tissues. Upon activation, these NKT cells release both Th1 and Th2 cytokines. However, it is present not known whether there are subsets of NKT that specialize in Th1 and Th2 cytokine production. Furthermore, how these cytokines lead to a regulated immune response in vivo is not known. Earlier studies have suggested that cytokine profile of NKT cells might be influenced by quality of TCR signal. The quality of TCR signal is in turn dependent on different antigens.

The present paper is from the lab of Dr. Yuling He, Wuhan University School of Medicine, China. In an earlier paper, the authors observed that CD8+ NKT cells drive syngenic T cells into a Th1-bias response to suppress EBV-associated malignancies and that IL-4-biased CD4+ NKT cells do not affect T cell cytotoxicity. In the present work, the authors sought to determine the mechanism of the EBV-induced CD8+ NKT cell response to EBV-associated malignancies.

Source: Xiao W, Li L, Zhou R, Xiao R, Wang Y, Ji X, Wu M, Wang L, Huang W, 

Zheng X, Tan X, Chen L, Xiong T, Xiong J, Jin Y, Tan J, He Y. EBV-induced 

human CD8(+) NKT cells synergise CD4(+) NKT cells suppressing EBV-associated

tumours upon induction of Th1-bias. Cell Mol Immunol. 2009 Oct;6(5):367-79.

PubMed PMID:19887050.

RESULTS:

1. Circulating mDC1cells from patients with EBV-associated malignancies impaired IFN-gamma production of CD8+ NKT cells: In another previous study, the authors observed that the frequencies of total and CD8+ NKT cells in PBMCs from Latent EBV infection (LEI) and control negative subjects (CN) were significantly higher than those from Hodgkin lymphoma (HL) patients and nasopharyngeal carcinoma (NPC) patients. To determine the interaction of CD8+NKT cells with APCs, the authors first determined frequencies and CD1d expression of circulating DCs, which included myeloid dendritic cells 1 and 2 (mDC1 and mDC2) and plasmacytoid dendritic cells (pDC), in PBMCs from NC and LEI subjects , and HL and NPC patients by staining with different markers, BDCA1 for mDC1, BDCA2 for pDC and BDCA3 for mDC2 and CD1d. The frequencies of mDC1 cells were comparable among HL and NPC patients, and LEI and NC subjects. CD1d was found to be predominant expressed on mDC1 cells than mDC2 and pDC cells. The expression level of CD1d was comparable on LEI and NPC subjects and HL and NPC patients. The authors next determined cytokine expression by CD4+ and CD8+ NKT cell lines. These cell lines were generated from PBMC from LEI subjects. Type 1 mDCs were purified from LEI and NC subjects and HL and NPC patients. These mDCs were than co-cultured with CD4+ or CD8+ NKT cell lines in the presence of alphaGalCer or alphaGalCer with CD1d mAb for 1 hr. The mDCs were then re-purified using staining with alpha-GalCer loaded CD1d tetramer. Then, the expression of IFN-gamma and IL-4 was assessed by Q-PCR. Both IFN-gamma and IL-4 were detected in CD4+ and CD8+ NKT cell lines cultured with mDCs from different groups of patients and subjects. The authors observed that both NKT cell lines from LEI and CN subjects highly expressed IFN-gamma upon stimulation with alphaGalCer. Although, not much effect was seen on IL-4 expression among various patients and subjects. Furthermore, using the same set of experiments the authors determined IFN-gamma and IL-4 expression by using ELISA. They observed that prestimulation with alphaGalCer caused significant increase in expression level of IFN-gamma in CD8+ NKT cell lines incubated with PBMCs from LEI and CN than from HL and NPC patients. IL-4 levels were in CD4+ NKT cells cultured with PBMCs from both patients and subjects. These results indicate that circulating mDC1 cells of HL and NPC patients cause a decrease in IFN-gamma production by CD8+ NKT cell line and have no effect on IL-4 production.
   
2. EBV induced CD8+ NKT cells promote a Th1 biased response that suppresses EBV-associated tumor cells in vitro: The authors observed in one of their early study that CD4+ NKT cells in PBMCs from HL and NPC patients and LEI and CN subjects produced similar low levels of IFN-gamma and high levels of IL-4, IL-13, IL-10 and TGF-beta1. In contrast, CD8+ NKT cells from control subjects produced expressed high levels of IFN-gamma and IL-2 but low levels of IL-4, IL-13, IL-10, and TGF-beta1. They also found that CD8+ NKT cells from LEI and CN subjects efficiently killed alpha-GalCer loaded EBV-associated HL and NPC tumor cell lines but not non-tumor cells. CD4+ from various patients and subjects either did not show such killing or showed very weak toxicity to both tumor and non-tumor cells. In the same study, the authors also observed that EBV-induced human NKT cells rapidly responded to EBV-associated tumor cells by secreting cytotoxic cytokines such as IL-10 and IFN-gamma. In the present study, the authors tried to verify whether this killing was specific, the authors first demonstrated present of EBV through the use of southern blot and QPCR in these cell lines. Thus, EBV-genes, LMP1, EBNA1, BZLF1, BALF2, and RAZ were examined in human EBV-associated tumor cell lines and non-tumor cell lines. The authors were able to detect these viral genes and their mRNA transcripts in tumor cell lines but not in non-tumor cell lines. The next task was to determine the mechanism by which NKT cells inhibit EBV-associated malignancies in vivo. Thus, the authors established hu-thym-SCID chimaeras. These chimaeras were implanted with human Hodgkin´s-derived EBV-associated B-cell lymphoma cell lines (LBC) or NPC. The animals were sacrificed 12 weeks post tumor implantation. Various organs including thyms, liver spleen and peripheral blood were collected and single cell suspensions were prepared. These cells were stimulated with alphaGalCer and cytokine expression (IL-4, IL-10 and IFN-gamma) was determined, using cytokine Ab, CD4 Ab, CD8 Ab and CD1d tetramer. The authors observed that a large number of CD8+ NKT cells expressed IFN-gamma and IL-2, but few cells expressed IL-4, IL-10 and IL-13. In contrast, CD4+ NKT cells expressed moderate levels of IL-4, IL-10 and IL-13 and low levels of IFN-gamma.
   
3. EBV- induced CD4+ NKT cells synergized with CD8+ NKT cells to promote a Th1 bias response against EBV-associated tumors: In their earlier work, the authors observed some kind of synergy between EBV-exposed thymic CD4+ and CD8+ NKT cells to suppress EBV-associated malignancies and prolong animal survival. To further investigate the mechanism of this synergistic effect, the SCID mice were adoptively transferred iv with different combinations of immune cells (CD4+/CD8+ NKT cells or both and CD3+CD56-Cd161- T cells) purified from EBV-exposed hum-thym-SCID chimaeras. After, three days these chimaeras were re-challenged with EBV. The scientists observed that in chimaeras transferred with CD4+ NKT cell alone, the re-challenge with EBV caused inhibition in proliferation of CD3+ T cell in comparison to chimaeras transferred with CD8+ NKT cell alone. Interesting, the chimaeras transferred with both CD4+ and CD8+ NKT cells showed a vigorous proliferation of CD3+ T cells and a high frequency of IFN-gamma producing cells.
   

To be continued……………….

ESCRT factors restrict mycobacterial growth

Mycobacterium tuberculosis is considered as one of the most successful pathogen. Some of the reasons that are responsible for its success include its ability to survive within the macrophages. But how does the bacteria survive intra-cellularly is not known. In the present paper, the authors have tried to address this question.

Source: Philips JA, Porto MC, Wang H, Rubin EJ, Perrimon N. ESCRT factors 

Restrict mycobacterial growth. Proc Natl Acad Sci U S A. 2008 Feb 26;105

(8):3070-5. Epub 2008 Feb 19. PubMed PMID: 18287038; 

PubMed Central PMCID: PMC2268586.

 

It is known that virulent mycobacterial are able to alter the maturation of phagosome inside the macrophages. They reside in a cellular compartment that resembles an early endosome. This endosome retains early endosome markers such as Rab5 and fails to recruit mature enodosomal markers such as Rab7.

In an earlier study, the authors performed a functional genomic screening to identify host factors, which can influence the uptake and growth of Mycobacteria. The authors used Drosophilla S2 cells, a macrophage like cell line that is open to RNAi and M. fortuitum, which like M. tuberculosis restricts phagosome fusion with lysosomes. M. fortuitum was able to induce the expression macrophage activated promoters (maps), which are the bacterial genes that are specifically induced when the bacteria grows intra-cellularly. Thus, by visualizing GFP (Green fluorescent proteins, proteins that produce free fluorescence when exposed to blue light) produced under the control of the map24 promoter, the authors were able to monitor the bacterial response to the intracellular environment. In another previous study, the authors identified 86 dsRNAs, which were able to diminish bacterial GFP production. As production of GFP reflects both the bacterial response to the phagosomal environment and bacterial growth, these dsRNAs can affect the infection by altering bacterial uptake, intra-cellular bacterial growth or induction from the map24 promoter.

In the present study, the authors further characterized three host cell activities Rab7, CG8743, and the ESCRT machinery. First to check the affect of dsRNAs, they used M. smegmatis, as it is difficult to measure intra-cellular growth of M. fortuitum. Thus, they examined two possibilities, if dsRNA treatment results in less intra-cellular growth of M. fortuitum and then it should have little effect on M. stegmatis because they do not grow in S2 cells. The other possibility was if the dsRNA altered phagosome environment and caused diminished map induction, then treatment with dsRNA should cause increase in permissiveness of phagosome for bacterial growth and M. stegmatis should grow. They treated S2 cells with dsRNA and then infected them with M. stegmatis. After two days, cells were examined by microscopy. They observed that four dsRNAs out of 86 strongly increased the percentage of heavily infected cells. These dsRNAs targeted Rab7, dVps28, CG8055 and CG8743. To confirm that bacterial growth was occurring intra-cellularly, they further blocked the bacterial uptake by using a dsRNA which targets Pes, a receptor required for entry of bacteria into the cell. The investigators observed that blocking did not cause any increase in bacterial growth and thus the results indicated that growth must be occurring intra-cellularly. Based on these results, the authors suggested that Rab7, dVps28, CG8055 and CG8743 might be playing important roles in restricting the intracellular growth of M. stegmatis.

Rab7 is a GTP binding protein and a marker for late enodosome. But the role of dVps28, CG8055 and CG8743 are not known. dVps28 and CG8055 are members of ESCRT machinery, which is made up of three proteins ESCRTI, ESCRTII and ESCRTIII. The authors thus, tested the effect of other dsRNAs that target additional ESCRT components on M. stegmatis growth. The authors observed that all dsRNAs, which robustly disrupt ESCRT machinery, caused increased growth of M. stegmatis and also altered the phagosome environment resulting in map24 and map49 induction by M. fortuitum and increased growth of M. stegmatis.

It is known that the ESCRT machinery acts on the membrane of the endosome. its function is to deliver ubiquitinated receptors into intraluminal vesicles of multivesicular bodies. These bodies are finally delivered to the lysosome for degradation. In Drosophila, a lack of ESCRT functioning causes accumulation of enlarged endosomes with accumulation of ubiquitin.

The authors next sought to determine whether ESCRT machinery acts directly on mycobacterial phagosome. They examined the localization of bacteria in ESCRT-depleted cells. S2 cells, which were depleted of dTsg101 and Vps28 (part of ESCRTI) were infected with M. stegmatis. After three hrs of infection, bacteria were found within the heavily ubiquinated vesicular compartments. These results showed that bacteria reside in the compartment on which ESCRT machinery acts. These results indicate that ESCRT machinery directly acts on the bacterial phagosome.

To test whether ESCRT machinery affects mycobacterial growth infection in mammalian macrophages, the authors depleted dTsg101 and Vps28 from murine macrophages RAW264.7 using SiRNA. These cells were then infected with M. fortuitum map24:GFP. The depletion caused less GFP production when cells were infected with M. fortuitum map24:GFP in comparison to controls. To further determine whether phagosome in ESCRT depleted cells was also less restrictive for mycobacterial growth, Raw264.7 cells were depleted of dTsg101 and Vps28 and infected with M. smegmatis hsp60::GFP. In control cells there was little growth 24 hrs post infection. In contrast, substantial growth was observed in depleted cells. Thus, the authors concluded that ESCRT machinery is required to restrict the growth of M. stegmatis in mammalian macrophages.

The diminished GFP production in cells depleted of ESCRT and infected with M. fortuitum map24:GFP is due to diminished expression and not due to decreased bacterial growth. To validate this, the authors used an M. fortuitum strain that expresses both red fluorescent protein (dsRed2) and GFP under control of two separate promoters. dsRed2 is under the control of msp12 promoter and is constitutively expressed. GFP is expressed under the control of map49, which is induced during intracellular growth. When ESCRTI depleted cells were infected with this bacteria, the authors observed diminished GFP expression as early as 3 hrs post infection. Examination of red fluorescence however, revealed that bacterial uptake was similar in both depleted and control cells and bacterial growth was similar or slightly increased in controls. These results suggested the phagosomal environment in ESCRT depleted cells was different from the non-depleted cells. The effect of this difference is such that the induction from the map24 and map49 promoters was decreased and bacterial growth is not efficiently restricted.