International Immunology - recent issues

IN THIS ISSUE

2009-06-21

Synthetic methylated CpG ODNs are potent in vivo adjuvants when delivered in liposomal nanoparticles

2009-06-21

Although it is well documented that the immunological activity of cytosine–guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine–guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.

Constitutively active aryl hydrocarbon receptor expressed in T cells increases immunization-induced IFN-{gamma} production in mice but does not suppress Th2-cytokine production or antibody production

2009-06-21

The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) has been implicated in various immune functions. Our previous studies have shown that AhR activation by exposure of ovalbumin (OVA)-immunized mice to the potent ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases immunization-induced IFN- production in the spleen and suppresses the production of T_h2 cytokines and OVA-specific antibodies. In the present study, we used transgenic (Tg) mice that express a constitutively active mutant of aryl hydrocarbon receptor (CA-AhR) specifically in T-lineage cells to clarify the role of AhR activation in T cells in these reactions. The results of this study clearly demonstrated that AhR activation only in the T cells augments IFN- production upon OVA immunization. By contrast, production of T_h2 cytokines and antibodies were not significantly suppressed by CA-AhR in the T cells. These results suggest that suppression of T_h2 cytokines and antibodies production require AhR activation not only in T cells but also in other cell types as caused by TCDD exposure. Alternatively, these results may indicate that IFN- augmentation and T_h2 cytokines and antibodies suppression depend on different ways of functions of AhR in the T cells and that CA-AhR does not replicate the suppressive effect of TCDD-activated AhR on T_h2 cytokines and antibodies. Expression of CA-AhR in the T cells was also shown to increase the percentage of CD25⁺ cells among CD4⁺ cells in the thymus and spleen. Thus, studies using T-cell-specific CA-AhR Tg mice provide a way to dissect the role of AhR in individual cell types and how the AhR functions.

Functionally relevant decreases in activatory receptor expression on NK cells are associated with pulmonary tuberculosis in vivo and persist after successful treatment

2009-06-21

Correlates for the initiation of Mycobacterium tuberculosis hominis (Mth) replication from latency are needed in order to improve Mth control. In order to analyze if perturbations of peripheral NK cells may be associated with exit from Mth latency, sequential patients with newly diagnosed lung tuberculosis (TB) were studied. Peripheral NK cells were analyzed by cytofluorometry, in vitro culture and functional assays. At the onset of lung TB, imbalances in NK cell subsets were evident. Decreased CD56^brightCD16^+/– subsets with significantly compromised NKp30 and NKp46 expression and with specifically decreased -IFN production upon triggering were evident. These features were not completely restored when purified NK cells were cultured in vitro. Culture supplementation with -IFN increased only NKp30 expression in TB and healthy donors. Extensive peripheral NK cell triggering was evident in these patients, as shown by the expression of NK cell activation markers and of the lymph node-homing chemokine receptor CCR7 on CD16⁺ CD56^dull cells. Significant persistence of decreased NKp30 and NKp46 after successful treatment with a standard four-drug regimen was detected after full recovery. NK cell function is deeply affected in patients at the onset of pulmonary TB. The involvement of multiple activatory receptors may provide a relevant contribution to the spread of mycobacteria exiting from latency.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells

2009-06-21

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133⁺ cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

Roles of PU.1 in monocyte- and mast cell-specific gene regulation: PU.1 transactivates CIITA pIV in cooperation with IFN-{gamma}

2009-06-21

Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN- was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr^–/– BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN- stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBP in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.

Nasal vaccination with troponin reduces troponin specific T-cell responses and improves heart function in myocardial ischemia-reperfusion injury

2009-06-21

Myocardial ischemia with subsequent reperfusion (MI/R) can lead to significant myocardial damage. Ischemia initiates inflammation at the blood–microvascular endothelial cell interface and contributes significantly to both acute injury and repair of the damaged tissue. We have found that MI/R injury in mice is associated with a cellular immune response to troponin. Myocardial cells exclusively synthesize troponin and release the troponin into the bloodstream following injury. Mucosally administered proteins induce T cells that secrete anti-inflammatory cytokines such as IL-10 and transforming growth factor β at the anatomical site where the protein localizes. We found that nasal administration of the three subunits of troponin (C, I and T isoforms), given prior to or 1 h following MI/R, decreased infarct size by 40% measured 24 h later. At 1.5 months following MI/R, there was a 50% reduction in infarct size and improvement in cardiac function as measured by echocardiography. Protection was associated with a reduction of cellular immunity to troponin. Immunohistochemistry demonstrated increased IL-10 and reduced IFN- in the area surrounding the ischemic infarct following nasal troponin. Adoptive transfer of CD4+ T cells to mice from nasally troponin-treated mice 1 h after the MI/R decreased infarct size by 72%, whereas CD4+ T cells from IL-10–/– mice or nasally BSA-treated mice had no effect. Our results demonstrate that IL-10-secreting CD4+ T cells induced by nasal troponin reduce injury following MI/R. Modulation of cardiac inflammation by nasal troponin provides a novel treatment to decrease myocardial damage and enhance recovery after myocardial ischemia.

Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution

2009-06-21

B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3–/– mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3–/– mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3–/– bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3–/– mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.

The analysis of the functions of human B and T cells in humanized NOD/shi-scid/{gamma}cnull (NOG) mice (hu-HSC NOG mice)

2009-06-21

‘Humanized mice’ are anticipated to be a valuable tool for studying the human immune system, but the reconstituted human immune cells have not yet been well characterized. Here, we extensively investigated the differentiation and functions of human B and T cells in a supra-immunodeficient mouse strain, NOD/shi-scid/c^null (NOG) reconstituted with CD34⁺ hematopoietic stem cells obtained from umbilical cord blood. In these hu-HSC NOG mice, the development of human B cells was partially blocked, and a significant number of B-cell progenitors accumulated in the spleen. The mature CD19⁺IgM⁺IgD⁺ human B cells of the hu-HSC NOG mice could produce IgG in vivo and in vitro by antigenic stimulation. In contrast, although human T cells with an apparently normal phenotype developed, most of them could neither proliferate nor produce IL-2 in response to antigenic stimulation by anti-CD3 and anti-CD28 antibodies in vitro. The positive selection of human T cells in the thymus was sufficiently functional, if not complete, and mainly mediated by mouse class II, suggesting that the human T cells lost their function in the periphery. We found that multiple mechanisms were involved in the T-cell abnormalities. Collectively, our results demonstrate that further improvements are necessary before humanized mice with a functional human immune system are achieved.

Activation of invariant NKT cells confers protection against Chlamydia trachomatis-induced arthritis

Bharhani, M. S., Chiu, B., Na, K.-S., Inman, R. D. — 2009-06-21

The role of invariant NKT (iNKT) cells in reactive arthritis is unknown. We explored the functional role of NKT cells in reactive arthritis using an established murine model of Chlamydia trachomatis-induced arthritis (CtIA). CtIA in wild-type and CD1d knockout (KO) mice was induced by intra-articular injection of C. trachomatis. The effect of -galactosylceramide (-GalCer) activation of iNKT cells was investigated by intra-peritoneal administration of -GalCer. Histopathological and phenotypic changes, chlamydial clearance and cytokine and chemokine production in synovial tissue of the knee joint were investigated after onset of the arthritis. The severity of CtIA was significantly increased in CD1d KO mice, which was associated with decrease in bactericidal cytokine IFN-, regulatory cytokines IL-4 and IL-10 and increase in pro-inflammatory chemokines macrophage inflammatory protein-2 (MIP-2) and IFN--inducible protein-10 (IP-10). Local clearance of the pathogen from the joint was also decreased. Prior treatment of mice with -GalCer, a potent activator of iNKT cells, significantly reduced the severity of CtIA in mice. The amelioration of CtIA was associated with decrease in chlamydial load and induction of cytokines IFN-, IL-4 and IL-10 and significant suppression of MIP-2 and IP-10. Treatment of established CtIA with -GalCer also demonstrated modulation of CtIA and decrease in chlamydial load. These results suggest that iNKT cells are protective against CtIA and -GalCer-activated iNKT cells have an immunoregulatory role not only in preventing the induction of reactive arthritis but also in modulating established disease.

PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation

2009-06-21

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic–polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

Gfi1 negatively regulates Th17 differentiation by inhibiting ROR{gamma}t activity

2009-06-21

T_h cells have long been divided into two subsets, T_h1 and T_h2; however, recently, T_h17 and inducible regulatory T (iTreg) cells were identified as new T_h cell subsets. Although T_h1- and T_h2-polarizing cytokines have been shown to suppress T_h17 and iTreg development, transcriptional regulation of T_h17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T_h2 development, was repressed in T_h17 and iTreg cells compared with T_h1 and T_h2 lineages. Gfi1 expression was enhanced by the IFN-/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-β1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor t to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T_h17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T_h17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T_h17 differentiation, which represents a novel mechanism for the regulation of T_h17 development by cytokines.

IN THIS ISSUE

2009-05-26

Murine bone marrow-derived mast cells express chemoattractant receptor-homologous molecule expressed on T-helper class 2 cells (CRTh2)

2009-05-26

Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D₂. We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD₂ receptor; chemoattractant receptor-homologous molecule expressed on T_h class 2 cells (CRT_h2). Activation of CRT_h2 on BMMC by PGD₂ or the CRT_h2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D₂ (DK-PGD₂), resulted in signaling response including Ca²⁺ mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT_h2, Compound A. Activation of CRT_h2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD₂ and DK-PGD₂ induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD₂ injection. Both these processes were inhibited by the CRT_h2 antagonist. These results raise the possibility that the functional consequences of the PGD₂–CRT_h2 interaction on mast cells may be relevant in allergic inflammation.

Expression of survivin in lung eosinophils is associated with pathology in a mouse model of allergic asthma

Tumes, D. J., Connolly, A., Dent, L. A. — 2009-05-26

Humans vary markedly in their propensity to develop asthma, despite often being exposed to similar environmental stimuli. Similarly, mouse strains vary in susceptibility to airways pathology in experimental asthma. Sensitization and aerosol challenge with ovalbumin (OVA) induces eosinophil accumulation, mucus production and airways obstruction in BALB/c and C57BL/6 mice. In contrast, CBA/Ca mice show relatively little pathology. Allergen-induced production of IL-4, IL-5, IL-10 and IFN- was detected in all three strains, with BALB/c mice generating the highest levels of IL-4, IL-5 and IL-10. Microarray analysis was used to identify genes differentially regulated in lung tissue after OVA challenge. Differentially regulated genes in the lungs of the asthma-susceptible C57BL/6 and BALB/c strains numbered 242 and 145, respectively, whereas only 42 genes were differentially expressed in the resistant CBA/Ca strain. In C57BL/6 mice, transcripts were enriched for adhesion molecules and this was associated with high levels of eosinophil recruitment. Differentially regulated genes in the lungs of only the asthma-susceptible strains numbered 64 and several of these have not previously been associated with asthma. Many of the genes differentially regulated in the susceptible strains were enzymes involved in inflammation. Using network analysis, mRNA for the anti-apoptotic protein survivin was found to be up-regulated in the lungs following allergen challenge. Survivin mRNA and protein were also expressed at high levels in eosinophils recovered by bronchoalveolar lavage from BALB/c and C57BL/6 mice. We propose that rapid apoptosis of lung eosinophils due to low expression of survivin contributes to the limitation of pathology in CBA/Ca mice.

Dopamine released by dendritic cells polarizes Th2 differentiation

Nakano, K., Higashi, T., Takagi, R., Hashimoto, K., Tanaka, Y., Matsushita, S. — 2009-05-26

A major neurotransmitter dopamine transmits signals via five different seven transmembrane G protein-coupled receptors termed D1–D5. It is now evident that dopamine is released from leukocytes and acts as autocrine or paracrine immune modulator. However, the role of dopamine for dendritic cells (DCs) and T_h differentiation remains unclear. We herein demonstrate that human monocyte-derived dendritic cells (Mo-DCs) stored dopamine in the secretary vesicles. The storage of dopamine in Mo-DCs was enhanced by forskolin and dopamine D2-like receptor antagonists via increasing cyclic adenosine 3',5'-monophosphate (cAMP) formation. Antigen-specific interaction with naive CD4⁺ T cells induced releasing dopamine-including vesicles from Mo-DCs. In naive CD4⁺ T cells, dopamine dose dependently increased cAMP levels via D1-like receptors and shifts T-cell differentiation to T_h2, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, we demonstrated that dopamine D2-like receptor antagonists, such as sulpiride and nemonapride, induced a significant DC-mediated T_h2 differentiation, using mixed lymphocyte reaction between human Mo-DCs and allogeneic naive CD4⁺ T cells. When dopamine release from Mo-DCs is inhibited by colchicines (a microtubule depolymerizer), T-cell differentiation shifts toward T_h1. These findings identify DCs as a new source of dopamine, which functions as a T_h2-polarizing factor in DC-naive T-cell interface.

Irradiated CIITA-positive mammary adenocarcinoma cells act as a potent anti-tumor-preventive vaccine by inducing tumor-specific CD4+ T cell priming and CD8+ T cell effector functions

Mortara, L., Frangione, V., Castellani, P., De Lerma Barbaro, A., Accolla, R. S. — 2009-05-26

In the present study, we investigated the possibility to use irradiated, non-replicating class II transcriptional activator (CIITA)-transfected tumor TS/A cells as a cell-based vaccine. Eighty-three percent of TS/A-CIITA-vaccinated mice were completely protected from tumor growth and the remaining 17% displayed significant reduction of tumor growth. In contrast, only 30% of mice injected with irradiated TS/A parental cells were protected from tumor growth, whereas the remaining 70% of animals remained unprotected. Immunity generated in the TS/A-CIITA-vaccinated mice correlated with an efficient priming of CD4⁺ T cells and consequent triggering and maintenance of CD8⁺ CTL effectors, as assessed by adoptive transfer assays. Important qualitative differences were observed between the two cell-based vaccines, as TS/A-CIITA-vaccinated mice developed a CTL response containing a large proportion of anti-gp70 AH1 epitope-specific cells, completely absent in TS/A-vaccinated mice, and a mixed T_h1/T_h2 type of response as opposed to a T_h2 type of response in TS/A-vaccinated mice. Finally, in TS/A-CIITA-vaccinated mice, a statistically significant reduction in the percentage and absolute number of CD4⁺ CD25⁺ T regulatory cells as compared with those of untreated mice with growing tumors (P < 0.001) or mice vaccinated with TS/A parental cells were observed. These results let to envisage the use of CIITA-transfected non-replicating tumor cells as a vaccination strategy for prevention and, possibly, adjuvant immunotherapy in human settings.

Lysophosphatidic acid inhibits the cytotoxic activity of NK cells: involvement of Gs protein-mediated signaling

2009-05-26

Lysophosphatidic acid (LPA) is an activator and chemoattractant of NK cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of LPA on the interaction of human NK cells with tumor cells such as the Burkitt lymphoma cell line Raji and the human melanoma cell line A2058. Thereby we found that LPA inhibits the release of perforin and cytotoxic activity of NK cells. Analysis of signal transduction showed that LPA induces common signaling pathways of chemotaxins such as G_i protein-dependent actin re-organization, activation of the mitogen-activated protein kinase p38 as well as phosphatidylinositol-3-kinase-dependent signal molecules [protein kinase B/Akt and glycogen synthase kinase-3β (GSK-3β)]. In contrast to most chemotaxins, LPA is also able to activate G_s-dependent signaling molecules. This signaling cascade involves the LPA receptor type-2, increase cAMP levels and protein kinase A (PKA) activation, which in turn are responsible for the modulatory effect of LPA on NK cell-mediated cytotoxicity. Moreover, blocking the regulatory subunits of PKA I abrogates the inhibitory effect of LPA, whereas the catalytic subunits are not involved. Based on our data, one can assume that LPA contributes to the tumor escape from the immunological surveillance machinery.

Protein geranylgeranylation regulates the balance between Th17 cells and Foxp3+ regulatory T cells

2009-05-26

Recent studies have suggested that statins, the inhibitors for 3-hydroxy-3-methyglutaryl (HMG)-CoA reductase in the mevalonate pathway, exhibit anti-inflammatory effects. However, the immune modulatory effects of statins on the differentiation of CD4⁺ T cells and their underlying mechanisms are still largely unknown. To address these issues, we examined the effect of simvastatin and inhibitors for protein farnesylation and geranylgeranylation on the differentiation of IL-17-producing T cells (T_h17 cells) and Foxp3⁺ CD4⁺ T cells. Simvastatin inhibited the differentiation of T_h17 cells through the inhibition of HMG-CoA reductase activity but enhanced the differentiation of Foxp3⁺ CD4⁺ T cells. Geranylgeranyltransferase I inhibitor, GGTI-298, but not farnesyltransferase inhibitor, FTI-277, mimicked the effects of simvastatin, indicating that the inhibition of protein geranylgeranylation is responsible for the effects. Moreover, Foxp3⁺ CD4⁺ T cells developed in the presence of transforming growth factor-β and GGTI-298 functioned as regulatory T cells (Tregs) in in vitro T cell proliferation assay as well as in an autoimmune colitis model. Finally, GGTI-298 induced SOCS3 expression and inhibited IL-6-induced signal transducers and activators of transcription3 phosphorylation in CD4⁺ T cells. Taken together, these results indicate that protein geranylgeranylation enhances the differentiation of T_h17 cells and inhibits the differentiation of Foxp3⁺ Tregs partly via the inhibition of SOCS3 expression.

Mammalian nitrilase 1 homologue Nit1 is a negative regulator in T cells

Zhang, H., Hou, Y.-J., Han, S.-Y., Zhang, E. C., Huebner, K., Zhang, J. — 2009-05-26

The mammalian Nit1 protein is homologous to plant and bacterial nitrilases. In flies and worms, Nit1 is fused to the 5' end of Fhit, suggesting that Nit1 may functionally interact with the Fhit pathway. Fhit has been shown to play a role of a tumor suppressor. Somatic loss of Fhit in human tissues is associated with a wide variety of cancers. Deletion of Fhit results in a predisposition to induced and spontaneous tumors in mice. It has been suggested that Nit1 collaborates with Fhit in tumor suppression. Similar to mice lacking Fhit, Nit1-deficient mice are more sensitive to carcinogen-induced tumors. It was previously shown that ectopic expression of Nit1 or Fhit led to caspase activation and apoptosis, and that both proteins may play a role in DNA damage-induced apoptosis. In this study, we analyzed the physiological function of Nit1 in T cells using Nit1-knockout mice. Nit1-deficient T cells can undergo apoptosis induced by DNA damage due to irradiation and chemical treatment. However, apoptosis induced by Fas or Ca⁺⁺ signals appeared to be compromised. Additionally, Nit1 deficiency resulted in T cell hyperproliferative responses induced by TCR stimulation. The expressions of T cell activation markers were elevated in Nit1^–/– T cells. There was a spontaneous cell cycle entry and enhanced cell cycle progression in Nit1^–/– T cells. These data indicate that Nit1 is a novel negative regulator in primary T cells.

Prediction of HLA-DQ8 {beta} cell peptidome using a computational program and its relationship to autoreactive T cells

Chang, K. Y., Unanue, E. R. — 2009-05-26

The goal was to identify HLA-DQ8-bound β cell epitopes important in the T cell response in autoimmune diabetes. We first identified HLA-DQ8 (DQA1*0301/DQB1*0302) β cell epitopes using a computational approach and then related their identification to CD4 T cell responses. The computational program (TEA-DQ8) was adapted from one previously developed for identifying peptides bound to the I-A^g7 molecule and based on a library of naturally processed peptides bound to HLA-DQ8 molecules of antigen-presenting cells. We then examined experimentally the response of NOD.DQ8 mice immunized with peptides derived from the Zinc transporter 8 protein. Log-of-odds scores on peptides were experimentally validated as an indicator of peptide binding to HLA-DQ8 molecules. We also examined previously published data on diabetic autoantigens, including glutamic acid decarboxylase-65, insulin and insulinoma-associated antigen-2, all tested in NOD.DQ8 transgenic mice. In all examples, many peptides identified with a favorable binding motif generated an autoimmune T cell response, but importantly many did not. Moreover, some peptides with weak-binding motifs were immunogenic. These results indicate the benefits and limitations in predicting autoimmune T cell responses strictly from MHC-binding data. TEA-DQ8 performed significantly better than other prediction programs

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim sustains B lymphopoiesis in the absence of IL-7

2009-05-26

IL-7 is pivotal for B cell development. Proteins of the Bcl-2 family are essential regulators of lymphocyte survival. Particularly, the pro-apoptotic BH3-only members Bim and Puma mediate lymphocyte apoptosis provoked by cytokine deprivation. Herein, we addressed whether the absence of Bim or Puma within the hematopoietic compartment could bypass the requirement for IL-7-driven B cell development in adult mice. We found that deficiency of Bim, but not Puma, partially rescued B cell development in the absence of IL-7. The numbers of both sIgM^– and sIgM⁺ B cells were markedly increased in the bone marrow of recipients lacking IL-7 upon reconstitution with Bim-deficient hematopoietic progenitors, compared with their control or Puma-deficient counterparts. The augmentation of B cell lymphopoiesis in the absence of Bim was reflected in the mature peripheral compartment by an increase in both the number of immature and mature B cells in the spleen and in the circulating IgM levels. Bim-deficient B cells were also increased in IL-7-sufficient recipients suggesting that peripheral B cells homeostasis is governed by a Bim-dependent and IL-7-independent mechanism. Our data highlight the role of Bim as a key regulator of cell survival during B lymphocyte development in vivo.

Notch3 and pT{alpha}/pre-TCR sustain the in vivo function of naturally occurring regulatory T cells

2009-05-26

Dysregulated generation and/or function of naturally occurring ‘CD4⁺CD25⁺ regulatory T cells’ (T_regs) play key role in the development of autoimmune diseases, including type 1 diabetes. Recent findings suggest that Notch3 signaling activation promotes thymic generation and peripheral expansion and in vivo function of naturally occurring T_regs, thus preventing autoimmune diabetes progression in mouse models. However, the mechanisms underlying these effects have remained elusive, thus far. Here, we show that the expression of pT gene is up-regulated in naturally occurring T_regs, at both mRNA and protein levels. Moreover, by using double mutant mice, with T cell-targeted constitutive activation of Notch3 in a pT^–/– background, we demonstrate that pT deletion significantly counteracts the Notch3-dependent expansion, the increased FoxP3 expression and the enhanced in vitro activity of naturally occurring T_regs. Notably, the absence of pT also impairs the Notch3-dependent protection against experimentally induced autoimmune diabetes. Finally, by adoptive cell transfer experiments, we demonstrated that this failure is directly related to the impaired in vivo function of naturally occurring T_regs bearing pT deletion. Collectively, our data suggest that pT expression is required for the in vivo function of naturally occurring T_regs and that the activation of Notch3 signaling may positively regulate the function of this population, through the pT/pre-T cell receptor pathway.

IL-6 produced by immune complex-activated follicular dendritic cells promotes germinal center reactions, IgG responses and somatic hypermutation

2009-05-26

Reports that follicular dendritic cells (FDCs) produce IL-6 prompted the hypotheses that immune complexes (ICs) induce FDCs to produce IL-6 and that FDC–IL-6 promotes germinal center (GC) reactions, somatic hypermutation (SHM) and IgG production. FDCs were activated in vitro by addition of ICs and FDC–IL-6 production was determined. Wild-type (WT) and IL-6 knockout (KO) mice, as well as chimeras with WT and IL-6 KO cells, were immunized with (4-hydroxy-3-nitrophenyl)-acetyl (NP)–chicken gamma globulin (CGG) and used to study anti-(4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) responses, GC formation and SHM in the VH186.2 gene segment in Ig-gamma. FDC–IL-6 increased when FDCs encountered ICs. At low immunogen dose, 1 µg NP–CGG per mouse, the IgG anti-NIP response in IL-6 KO mice was low and immunohistochemistry revealed a reduction in both the number and size of GCs. The physiological relevance of FDC–IL-6 was apparent in the chimeric mice where total splenocytes from WT mice were unable to provide the IL-6 needed for normal IgG and GC responses in IL-6 KO animals with IL-6-defective FDCs. Moreover, the rate of mutation decreased from 18 to 8.9 mutations per 1000 bases (P < 0.001) in WT versus IL-6 KO mice. Addition of anti-IL-6 to GC reactions in vitro reduced antibody levels and SHM from 3.5 to 0.65 mutations per 1000 bases (P < 0.02). Thus, the absence of FDC–IL-6 correlated with a reduction in SHM that coincided with the reduction in GCs and specific anti-NIP. This is the first study to document that ICs induce FDC–IL-6 and that FDC-derived IL-6 is physiologically relevant in generating optimal GC reactions, SHM and IgG levels.

IN THIS ISSUE

2009-04-28

Th17 cells: from precursors to players in inflammation and infection

Awasthi, A., Kuchroo, V. K. — 2009-04-28

Upon activation, naive CD4⁺ T cells differentiate into different lineages of effector T_h subsets. Each subset is characterized by its unique cytokine profile and biological functions. T_h17, a newly described T_h subset that produces IL-17, IL-17F and IL-22 in preference to other cytokines, has been shown to play an important role in clearing specific pathogens and in inducing autoimmune tissue inflammations. Over the last 2–3 years, significant progress has been made to understand the development and biological functions of T_h17 subset. Transforming growth factor β (TGF) together with IL-6 or IL-21 initiates the differentiation while IL-23 stabilizes the generation of T_h17 cells. The transcription factors of T_h17 cells [retinoid-related orphan receptor (ROR) t, ROR- and signal transducer and activator of transcription-3] have been described recently. Since TGF-β is essential for the generation of both T_h17 and regulatory T (T_reg) cells from naive T cells, which suggests a developmental link between T_h17 and T_reg cells. Functions of these two subsets of T cells are, however, opposite to each other; T_h17 cells are highly pathogenic during the inflammatory process while T_reg cells are crucial for inhibiting tissue inflammation and maintaining self-tolerance. Here, we review the recent information on differentiation and effector functions of T_h17 cells during inflammatory conditions.

Identification of QTLs that modify peripheral neuropathy in NOD.H2b-Pdcd1-/- mice

2009-04-28

The non-obese diabetic (NOD) mouse strain is prone to developing various autoimmune syndromes including type I diabetes mellitus (T1DM), sialadenitis, thyroiditis and pancreatitis. Although the genetic basis of T1DM has been extensively analyzed, genetic factors that modify the other autoimmune phenotypes are largely unknown. We have recently reported that NOD mice with anti-diabetogenic MHC haplotype (H-2^b) and programmed cell death 1 (PD-1) deficiency (NOD.H2^b-Pdcd1^–/– mice) are protected from T1DM but develop various tissue-specific autoimmune diseases including peripheral neuropathy due to autoimmune neuritis, sialadenitis and gastritis. In the present study, we generated [(C57BL/6 x NOD.H2^b)_F1 x NOD–H2^b]_BC1–Pdcd1^–/– mice to screen non-MHC quantitative trait loci (QTLs) that modify autoimmune phenotypes other than T1DM. We identified seven QTLs for peripheral neuropathy and neuritis, one QTL for insulitis, four QTLs for gastritis, two QTLs for sialadenitis and seven QTLs for vasculitis throughout the genome and designated them as Annp loci for autoimmunity due to polymorphisms of non-MHC genes in NOD mice and PD-1 deficiency. Annp1, 5, 6 and 7 overlapped with reported loci for T1DM (Idd3, 9, 15 and 2, respectively), suggesting that these loci modify not only T1DM but also other autoimmune phenotypes. NOD allele was promotive at 9 of 14 Annp loci, while NOD allele was protective at the other loci. Half of Annp loci associated with a single phenotype, while the other seven loci associated with more than two phenotypes. These results indicate that NOD genetic background harbors various QTLs that modify autoimmune phenotypes either by organ-specific or by organ-non-specific manner.

Th1/Th17 polarization and acquisition of an arthritogenic phenotype in arthritis-susceptible BALB/c, but not in MHC-matched, arthritis-resistant DBA/2 mice

Boldizsar, F., Tarjanyi, O., Nemeth, P., Mikecz, K., Glant, T. T. — 2009-04-28

Proteoglycan (PG) aggrecan-induced arthritis (PGIA) is a murine model of rheumatoid arthritis (RA). Although BALB/c and DBA/2 mice share the same MHC (H-2d) haplotype, the BALB/c strain is susceptible to PGIA, while DBA/2 mice are resistant. Therefore, these two inbred mouse strains provide an opportunity to study arthritis susceptibility factors excluding the effects of MHC-associated genetic components. The goal of this study was to monitor changes in the cellular composition and activation state following intra-peritoneal (i.p.) immunization to induce PGIA; additionally, we sought to identify new susceptibility factors by comparing PG-induced immune responses in BALB/c and DBA/2 mice. Upon i.p. PG injection, resident naive B1 cells are replaced by both T cells and conventional B cells in the peritoneum of BALB/c mice. These peritoneal T cells produce IFN and IL-17, cytokines shown to be important in RA and corresponding arthritis models. Moreover, peritoneal cells can adoptively transfer PGIA to SCID mice, demonstrating their arthritogenic properties. Our results indicate that repeatedly injected antigen leads to the recruitment and activation of immune cells in the peritoneum; these cells then trigger the effector phase of the disease. The migration and activation of T_h1/T_h17 cells in the peritoneal cavity in response to PG immunization, which did not occur in the arthritis-resistant DBA/2 strain, may be critical factors of arthritis susceptibility in BALB/c mice.

Human memory CCR4+CD8+ T cell subset has the ability to produce multiple cytokines

Kondo, T., Takiguchi, M. — 2009-04-28

The CC chemokine receptor (CCR)4 is associated with trafficking of specialized cutaneous memory type 2 T_h cells in the skin. However, a CD8⁺ T cell population expressing CCR4 still remains uncharacterized. In the present study, we investigated the expression and function of CCR4 on human CD8⁺ T cells and characterized CCR4⁺CD8⁺ human T cells. Multi-color flow cytometric analysis revealed that CCR4⁺CD8⁺ T cells were predominantly found in the CD27⁺CD28⁺CD45RA^– memory subset and expressed the CCR7^+/–CCR5^– phenotype. CCR4⁺CD8⁺ T cells expressed neither perforin (Per) nor granzymes (Gra) A/B, suggesting that they were more immature memory T cells than the CCR6⁺CD8⁺ early effector memory T cells that express GraA and Per. CCR4⁺CD8⁺ T cells effectively produced IL-4, IFN-, IL-2 and tumor necrosis factor-, indicating that they are memory T cells having the ability to secrete type 1 and type 2 cytokines. These cells also showed chemotaxic activity in response to CC chemokine receptor ligand (CCL)17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine. These results suggest that CCR4⁺CD8⁺ T cells are in an immature memory T cell subset in the differentiation pathway of human CD8⁺ T cells and that they migrate to inflammatory sites in the skin where they are involved in cutaneous immunity.

Enhanced capture of extramembranous IgM and IgG on B cells in the NOD mouse--implications for immune complex trapping

Ekici, R., Sundstrom, M., Thay, B., Lejon, K. — 2009-04-28

Binding of various antibody isotypes to B cells through either FcRs or complement receptors has been attributed to play several roles, e.g. in immune complex (IC) transportation and regulation of B cell receptor signaling. We have revealed a novel B cell intrinsic receptor for IgM and IgG which is present in C57BL/6 (B6) mice and is more abundant in non-obese diabetic (NOD) mice. As a consequence, the level of extramembranous IgG monomers and IgM pentamers on peripheral blood B cells from NOD mice was significantly higher compared with B6 mice. The effect of this aberration was that all B cells in peripheral blood of (NOD.IgH^a x B6(IgH^b))F₁ mice carried both IgM allotypes on their surface. In addition, analysis of IC binding using IgG- or IgM-opsonized bacterial particles revealed a higher degree of binding in NOD mice compared with B6. We hypothesize that this novel Ig-binding receptor is part of the normal immune system function. The aberrant function in the NOD mouse could contribute to the development of Type 1 diabetes by altering normal B cell functions such as activation, IC transportation and B cell homeostasis.

Hydrolysis of tumor cell lipids after CTL-mediated death

Alves, B., Leong, J., Tamang, D. L., Elliott, V., Lowe, M., Hudig, D. — 2009-04-28

Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [³H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2^+/+ and PLRP2^–/– CTLs were compared, there was more killing by the PLRP2^+/+ CTLs. However, [³H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze–thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.

Differential IL-23 requirement for IL-22 and IL-17A production during innate immunity against Salmonella enterica serovar Enteritidis

2009-04-28

Early activation of the IL-12/IFN- axis has been shown following Salmonella enterica serovar Enteritidis (S. Enteritidis) infection. We were interested to study whether IL-22 and IL-17A production is initiated early in response to S. Enteritidis. We demonstrate here that IL-22 was strongly elevated in the peritoneal lavage fluid and in serum already 1 day post-intraperitoneal infection (d.p.i.) of mice; not only IL-22 but also IL-17A was produced ex vivo by activated peritoneal exudate cells (PEC). Peritoneal T cells were identified as cellular source of IL-17A. The early IL-22 production was completely IL-23-dependent. In contrast, IL-17A production was only partially IL-23-dependent. To investigate the local production of upstream cytokines important for induction of IL-22, IL-17A and IFN- during salmonellosis, the production of IL-23 and IL-12 was studied. Elevated p19 and p40 mRNA levels were found in PEC at 1 d.p.i., whereas p35 mRNA levels were not changed. Besides, the T_h17-promoting cytokines IL-6, IL-1β and transforming growth factor-β were produced in response to S. Enteritidis. However, IL-6 was not required for IL-22 or IL-17A production by PEC. By ex vivo analysis of PEC at 1 d.p.i., we show that the major producers of early IL-12/23p40 in the peritoneal cavity were dendritic cells (DC), whereas macrophages notably contributed to IL-6 production. Taken together, these data suggest that DC initiate early IL-22 production at the site of infection which may contribute to resistance against salmonellosis. Furthermore, we provide evidence that production of IL-22 and IL-17A is differentially regulated during infection.

T cell sensitivity to TGF-{beta} is required for the effector function but not the generation of splenic CD8+ regulatory T cells induced via the injection of antigen into the anterior chamber

2009-04-28

The introduction of antigen into the anterior chamber (AC) of the eye induces the production of antigen-specific splenic CD8⁺ regulatory T cells (AC-SPL cells) that suppress a delayed-type hypersensitivity (DTH) reaction in immunized mice. Because the generation of these regulatory T cells is also induced by exposure to transforming growth factor (TGF)-β and antigen or F4/80⁺ cells exposed to TGF-β and antigen in vitro, we investigated (i) whether these cells are produced in dominant negative receptor for transforming growth factor β receptor type II (dnTGFβRII) or Cbl-b^–/– mice whose T cells are resistant to TGF-β, (ii) whether DTH is suppressed by wild type (WT) CD8⁺ AC-SPL cells in Cbl-b^–/– and dnTGFβRII mice and (iii) the effect of antibodies to TGF-β on the suppression of DTH by CD8⁺ AC-SPL cells. DnTGFβRII immunized and Cbl-b^–/– mice produced splenic CD8⁺ regulatory cells after the intracameral injection of antigen and immunization. The suppression of a DTH reaction by CD8⁺ AC-SPL cells in WT mice was blocked by the local inclusion of antibodies to TGF-β when WT splenic CD8⁺ AC-SPL cells were injected into the DTH reaction site. Moreover, the DTH reaction in immunized dnTGFβRII and Cbl-b^–/– mice was not suppressed by the transfer of WT CD8⁺ AC-SPL cells to the site challenged with antigen. In aggregate, these observations suggest that T cell sensitivity to TGF-β is not an obligate requirement for the in vivo induction of CD8⁺ AC-SPL T cells but the suppression of an in vivo DTH reaction by CD8⁺ AC-SPL cells is dependent on TGF-β.

The effects of c-Abl mutation on developing B cell differentiation and survival

Brightbill, H., Schlissel, M. S. — 2009-04-28

c-Abl is a widely expressed Src family protein tyrosine kinase that is activated by chromosomal translocation in certain human leukemias. While shown in various experimental systems to regulate cell division and stress responses, its biological functions remain poorly understood. Although expressed at similar levels throughout B cell development, we found that the fraction of phosphorylated, active c-Abl peaks at the pro-B stage. We went on to perform a detailed analysis of B cell development in c-Abl-deficient mice. We confirmed a striking but variable decrease in pro- and pre-B cell numbers, a decrease in pre-B cell growth and an increase in pre-B cell apoptosis. This phenotype was not rescued by transgenic expression of a functional IgHC transgene and only partially rescued by the anti-apoptosis gene Bcl-x. Unlike their wild-type counterparts, c-Abl-deficient pre-B cells show a defect in Ca²⁺ flux upon cross-linking of CD19, a co-receptor known to be involved in pre-B cell receptor signaling and failed to express CD25 on the cell surface. Despite these pre-B cell-signaling defects, selection for in-frame heavy-chain rearrangements was intact in the mutant mice. Remarkably, we were able to rescue the proliferative defect by culturing cells in vitro with large amounts of rIL-7. We conclude that c-Abl is required for normal B cell differentiation and survival.

FasL cross-linking inhibits activation of human peripheral T cells

Paulsen, M., Mathew, B., Qian, J., Lettau, M., Kabelitz, D., Janssen, O. — 2009-04-28

Activation of resting T cells in vitro is triggered by combined TCR and CD28 engagement and can be modulated by simultaneous ligation of various other surface receptors. Although the Fas ligand (FasL) is best known for its capacity to initiate cell death in Fas-bearing cells, it has recently been implicated in the regulation of T cell activation. Thus, a cross-talk between the TCR and FasL is likely, but far from being biochemically elucidated. We now report that FasL engagement by immobilized but not soluble FasFc fusion protein and anti-FasL polyclonal antibody blocks the activation of human peripheral T cells even in the presence of CD28 co-stimulation. The data presented here stress the importance of the Fas/FasL system for signal initiation via the TCR–CD3 complex and provide further arguments for a retrograde signaling capacity of FasL or a crucial role of Fas as a co-stimulatory molecule.

NK cells provide helper signal for CD8+ T cells by inducing the expression of membrane-bound IL-15 on DCs

2009-04-28

NK cell recognition of cells that do not express or express low amounts of MHC class I molecules results not only in direct killing of target cells but also in the generation of specific T cell responses consequent to the induction of dendritic cell (DC) activation. While IL-12 production by NK cell-activated DCs is generally thought to play a critical role, a similar DC-mediated NK cell help has been reported also in IL-12-knockout mice. Here, we show that human NK cells can induce on DC surface membrane, via IFN- secretion, the expression of high levels of IL-15. Remarkably, we show that DC expression of this membrane-bound form of IL-15, which is only partially associated with IL-15R molecules, is essential to promote specific CD8⁺ T lymphocyte response in the absence of DC-derived IL-12.

Sequences derived from self-RNA containing certain natural modifications act as suppressors of RNA-mediated inflammatory immune responses

2009-04-28

The ability of the host to distinguish between self and foreign nucleic acids is one of the critical factors contributing to the recognition of pathogens by Toll-like receptors (TLRs). Under certain circumstances, eukaryotic self-RNA may reach TLR-containing compartments allowing for self-recognition. Specific modifications were previously demonstrated to suppress immune activation when placed at several positions in an immune stimulatory RNA or silencing RNA (siRNA). However, we show that even a simple natural modification such as a single 2'-O-methylation at different nucleotide positions throughout a sequence derived from a self-RNA strongly interferes with TLR-mediated effects. Such a single modification can even have an inhibitory effect in vitro and in vivo when placed in a different than the immune stimulatory RNA strand acting as suppressive RNA. Several safeguard mechanisms appear to have evolved to avoid cellular TLR-mediated activation by self-RNAs that may under other circumstances result in inflammatory or autoimmune responses. This knowledge can be used to include as few as a single 2'-O-methyl modification at a specific position in a siRNA sense or anti-sense strand to avoid TLR immune effects.