Function of miR-146a in controlling Treg cell mediated regulation of Th1 responses

Function of miR-146a in controlling Treg cell mediated regulation of Th1 responses
I recently had the pleasure of listening an interesting talk on the function of micro RNAs in controlling the function of Tregs. The presenter was Dr. Li-Fan Lu from Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY. Presented here is a brief blog on one of his paper.
Regulatory T cells (Tregs) have a suppressive function in body. They limit “collateral damage” resulting from protective immunity to infection and suppress sterile inflammation as well as unwanted immune responses to “self” and allergens. Tregs express FoxP3, an X chromosome encoded transcription factor which plays an important role in differentiation, homeostasis and function of Treg cells. It has been observed that FoxP3 directly or indirectly controls several thousand genes in Tregs. These genes are called as FoxP3 amplified genes many genes that serve as hallmark of Tregs and play important roles in their homeostasis and function. Also included among the FoxP3 amplified genes are those that code for noncoding RNAs called micro RNA (miRNA). These miRNAs serve as important regulators of Treg homeostasis and function both in basal and inflammatory settings. One such miRNA is miRNA-155, the cancer associated miRNA, which is constitutively expressed in high amounts in Treg cells in a FoxP3 dependent manner. It is transiently upregulated in T cells lacking FoxP3, B cells and myeloid cells up on activation. The investigators of this paper have previously shown that miR-155 causes increased response in Tregs to their key survival and growth molecule, IL-2 and is thus responsible for maintaining the numbers of Tregs. Another miRNA constitutively expressed in high amounts in Tregs is miR-146a. Like miR-155, it is induced up on activation of effector T cells and myeloid cells. In the present paper the authors tend to determine the role of miR-146a in Tregs. Their results are as follows:
• Elevated miR-146a expression in Tregs:
miRNA expressions were compared among FoxP3+ Treg cells and FoxP3- non Treg CD4+ cells---miR-146a was more expressed in Tregs in comparison to non-Tregs.
• miR-146a deficiency resulted in increased numbers but impaired function of Tregs:
Firstly, the authors examined FoxP3+ Treg cell subsets in the thymus and in the peripheral lymphoid organs of miR-146a deficient mice. They observed comparable sizes of different thymocyte and peripheral lymphoid and myeloid cell subsets and similar T cell activation status among the wild type and mutant mice. The number of FoxP3+ Tregs in mutant mice was higher in the periphery (but not in thymus) in comparison to wild type mice. These FoxP3+ Tregs also showed heightened proliferative activity as shown by increased expression of Ki67 and increase in other activation markers.
In next set of experiments, the authors transferred mixed bone marrow (1:1ratio of miR 146a deficient or sufficient Tregs mixed with Ly5.1 B6 or Ly5.1 FOXP3 KO) into Rag2-/- (or TCRβδ -/-) mice. They observed that in the mice group Mirn 146a-/-/FOXP3 KO, 40% of the mice died before the day of analysis. In these mice, all Tregs lack mir 146a as FOXP3 KO BM cells fail to generate Tregs; all other BM derived cells originate from both miR 146a deficient and sufficient BMs. The purpose of these experiments was to generate a miR 146a sufficient environment and then study the function of miR146a deficient Tregs in that environment. These mice showed severe clinical conditions and only 40% survived. Histologicaly also, these mice showed massive lymphocyte activation and tissue infiltraion. In contrast, mir 146a-/-/B6 mice had both miR 146a deficient and sufficient Tregs and they showed no clinical or histological signs of immune mediated lesions. So what was the cause of the adverse clinical and histological conditions in the mir 146a-/-/FOXP3 KO mice? The authors observed that the cause of these effects was not due to decrease in number of Tregs as the number of Tregs in KO mice was actually more than in control mice (mir 146a +/+/FOXP3 KO). The mir 146a -/-/ B6 mouse also showed an increase in miR 146a deficient Tregs than in controls (mir 146+/+/B6). These results suggested that miR146a deficiency increases the number of Tregs.
The authors next studied the activation of miR 146a sufficient T effector cells and observed a highly proliferative activity (Ki67), an increased CD62L low cell subset and high levels of activation markers ICOS and CTLA4 in FOXP 3 KO mice. However, both miR 146a deficient or sufficient cells showed control activation in a miR 146 a sufficient environment (B6 Ly5.1+ MICE). These results suggest that miR146 a play an important role in Treg mediated immunological tolerance.

• miR-146a deficiency in Treg cells resulted in dysregulated IFNγ responses: In the next set of experiments the authors isolated miR-146a deficient or sufficient Tregs from mir 146a-/-/B6 or mir146a-/-/FOXP3 KO mice and co-cultured them with wild type responder CD4+ T cells (in the presence of CD3 antibody and irradiated T cell depleted splenocytes). They observed that miR 146a deficient Tregs from mir 146a-/-/B6 mice were as suppressive as miR 146a sufficient Tregs from control animals (mir146a+/+/B6). However, miR 146a deficient Tregs from knock-outs were more suppressive than miR146a sufficient Tregs from controls. These results indicate that deficiency of miR 146a did not diminish the overall suppressive activity of Tregs. They also observed that loss of miR146a in Tregs resulted in increase production of pro-inflammatory cytokine IFNγ by both miR 146a sufficient and deficient Cd4 and CD8 cells. This increase was not observed in the presence of miR146a sufficient Tregs.

• IFNγ blockade prevents the autoimmune disease in mice harboring miR-146a deficient Tregs: The next question was weather this increased production of IFNγ is responsible for clinical condition in Mirn 146a-/-/FOXP3 KO mice. To answer this question, the investigators performed experiments in which IFNγ was blocked by treating these mice with IFNγ blocking antibody. The increase in number of miR 146a deficient Tregs was not affected in the presence of blocking antibody. However, the blocking of IFNγ prevented disease in deficient mice. The blockade of IFNγ with blocking antibody did not cause simultaneous increase in IL4 and IL-17 (Th2 responses). These results suggest that the diseases observed in the miR146a deficient Tregs was IFN-gamma dependent and blockade of IFN-gamma can control Th2 and Th17 responses but not Th1 response.

• miR 146a regulates stat1 in Tregs: Stat 1 is a key transcription factor downstream of IFN-gamma signaling. Thus, the authors transfected CD4+ T cells isolated from miR146a sufficient or deficient with a luciferase reporter construct containing wild type or mutant Stat1 and assessed luciferase activity. miR146a sufficient T but not deficient cells transfected with wild type Stat1 showed suppression of reporter activity. The authors also performed immunoblot analysis of Stat1 protein in Tregs and non-Tregs. There was an increase in production of Stat1 in Mir146a deficient Tregs and non-Tregs.

• miR 146a controls Treg cell mediated regulation of IFNγ responses through targeting Stat1: Based on previous experiments the authors hypothesized that miR146a deficiency causes dysregulated IFN-gamma production, which in turn causes increased expression of Stat1, which can then lead to failure of Tregs to control Th1 response. To test this hypothesis, the authors generated miR146a deficient mice harboring a single function stat1 allele (Mirn 146a-/-/Stat1+/-). The BM cells from these mice were mixed with Ly5.1+ FOXP3 KO mice and transferred into Rag2-/- mice. Thus, the recipients are Mirn 146a-/-/Stat1+/-/FOXP3 KO. Then they measured the amount of Stat1 phosphorylated in Tregs and non-Tregs from these and from control mice (Mirn 146a-/-/Stat1+/+/FOXP3 KO). Both Tregs and non-Tregs from test mice showed nearly 50% reduction in phosphorylated stat1 in comparison to controls. The test mice also developed a much milder and delayed disease. The authors also observed significant reduction in production of IFN-gamma in tests mice

Source: Lu LF, Boldin MP, Chaudhry A, Lin LL, Taganov KD, Hanada T, Yoshimura A, Baltimore D, Rudensky AY. Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses. Cell. 2010 Sep 17;142(6):914-29. PubMed PMID: 20850013; PubMed Central PMCID: PMC3049116.

Viral danger signal control CD1d de novo synthesis and NKT cell activation

Viral danger signal control CD1d de novo synthesis and NKT cell activation


Source: Raftery MJ, Winau F, Giese T, Kaufmann SH, Schaible UE, Schönrich G.
Viralndanger signals control CD1d de novo synthesis and NKT cell activation.
Eur J Immunol. 2008 Mar;38(3):668-79. PubMed PMID: 18253929.

Results:
1. Effect of IFNs on CD1D transcripts: Untreated DCs were treated with interferons. It was observed that type I IFN (IFN alpha) up regulates the number of CD1D transcripts. In contrast to this, IFN-gamma had no effect where as TNF-alpha down regulates the amount of CD1D transcripts. In another set of experiments, the authors found that IFN-alpha and beta diminished CD1A and CD1B mRNA in a dose dependent manner. In addition, they increased the levels of CD1D mRNA transcripts. Taken together these data indicate that type I IFN (signature cytokines of innate anti-viral immune response) enhance the expression of CD1D mRNA in human DC.
2. Differential regulation of CD1 expression in human DC exposed to TLR ligands: The authors next exposed DCs to various TLR ligands (Zymosan, TLR2; LPS, TLR4; Imiquimod, TLR7; CpG, TLR9) and looked for expression of CD1 genes. They observed that all TLR ligands reduced the amount of group 1 CD1 (A to C) transcripts and group 3 CD1 (E) transcripts. The only exception was CpG (TLR9 ligand) which did not cause any change in expression as TLR9 is not expressed in myeloid DCs. In contrast to group 1 and 3 CD1 molecules, group 2 CD1 molecules (CD1D) was upregulated by TLR7 ligand (Imoquimod, viral) and downregulated by TLR4 ligand LPS. These results indicate that CD1D is upregulated in response to viral pathogen associated molecular patterns in myeloid DCs.
3. Effect of viral infection on level of CD1D transcripts: The authors next infected immature DCs with virus (CMV or HSV-1). They observed that the number of CD1D transcripts was drastically enhanced in virus infected DCs. The other CD1 molecules were downregulated in response to infection. These results suggest that in response to virus infection, myeloid DCs downregulate group 1 and group 3 CD1 genes and upregulates expression of CD1D.
4. Effect of viral danger signals on CD1d protein: The investigators treated the UV-inactivated HSV-1, IFN-alpha, - beta, -gamma and imiquimod and investigated the surface expression of CD1 molecules and CD83, a marker for maturation of DCs by FACS. They observed that the surface expression of CD1d molecules was enhanced in presence of HSV-1 or type 1 IFN in comparison to untreated DCs. They also observed similar results with Western blot analysis. The DCs secreted type I IFN in response to UV-inactivated HSV-1. These results indicated that human myeloid DCs synthesize CD1d molecules induced by IFN-alpha in response to viral danger signals.
5. Effect of viral danger signal on NKT cells: As invariant NKT cells recognized only molecules presented by CD1d, the authors next investigated the effect of upregulation of CD1d molecules on DCs on NKT cells. Thus, they treated PBMCs with alpha-GalCer or UV-inactivated HSV-1. They looked for CD4+ NKT cells and IFN-gamma secretion and observed an increase in percentage of IFN-gamma secreting CD4+ NKT cells in virus treated cells compared to untreated ones. Blocking of CD1d caused reduction in NKT cells. These data indicate that activation and expansion of NKT cells in human PBMCs exposed to viral pathogen requires CD1d expressing cells.
6. What is the nature of NKT cell response, Th1 or Th2? Activated NKT cells secrete IFN-gamma, IL-4 and IL-10. The investigators observed that DCs treated with IFN-alpha, poly (I:C)(TLR3 ligand), imiquimod (TLR7) or UV inactivated HSV-1 could induce IFN-gamma secretion by NKT cells more efficiently than DCs treated with LPS (TLR4 ligand). Similar results were observed when IL-4 secretion was investigated. However, when IL-10 production was analyzed, it was observed that co-culture of DCs treated with virus or other signals with NKT cells caused reduced production of IL-10. Taken together these data indicate that DCs treated with viral danger signals cause NKT cells to induce a more Th1 like response in comparison to LPS treated DCs.

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.