International Immunology - current issue

IN THIS ISSUE

2008-06-16

Potentiation of NK cell-mediated cytotoxicity in human lung adenocarcinoma: role of NKG2D-dependent pathway

2008-06-13

Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.

Bam32: a novel mediator of Erk activation in T cells

2008-06-13

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4⁺ T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4⁺ T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

CD4+ T cell hyper-responsiveness in CD45 transgenic mice is independent of isoform

Salmond, R. J., McNeill, L., Holmes, N., Alexander, D. R. — 2008-06-13

The CD45 tyrosine phosphatase is required for T cell development and function by virtue of its role as a positive regulator of src family kinase activity. In addition, recent data have highlighted that CD45 also acts as a negative regulator of Lck function by dephosphorylation of critical tyrosine residues. Lck functionality and TCR responsiveness are elevated in transgenic mice expressing the CD45RO isoform at ‘intermediate’ (10–40% of wild type) levels, indicating that the expression level of CD45 is critical in determining the sensitivity of T cells to TCR stimulation. However, it is unclear whether such a phenotype is specific for the CD45RO isoform, typically expressed by activated T cells. In the present work, the roles of three isoforms of CD45, RO, RB and RABC, in thymocyte development, T cell responses and TCR signalling pathways were directly compared. The data demonstrate that expression of CD45RB or CD45RABC at intermediate levels also results in CD4⁺ T cell hyper-reactivity, as previously published for CD45RO. These data emphasize the dual functions of CD45 as both a positive and a negative regulators of TCR signalling irrespective of specific isoform expression.

The probiotic Escherichia coli strain Nissle 1917 induces {gamma}{delta} T cell apoptosis via caspase- and FasL-dependent pathways

Guzy, C., Paclik, D., Schirbel, A., Sonnenborn, U., Wiedenmann, B., Sturm, A. — 2008-06-13

Human T cells play a vital role in the innate and adaptive immune response to microbial antigens by acting as antigen-presenting cells while at the same time being capable of directly activating CD4⁺ T cells. Pathogenic microbes or loss of tolerance toward the host's own microflora trigger many diseases including inflammatory bowel diseases. We previously demonstrated that Escherichia coli Nissle 1917 directly interacts with the adaptive immune system by regulating central T cell functions. Here we aimed to investigate whether E. coli Nissle regulates T cell function, thereby linking the innate and adaptive immune system. In our study, we demonstrate that, in contrast to the other probiotic strains tested, E. coli Nissle increased activation, cell cycling and expansion of , but not β T cells. In T cells, E. coli Nissle reduced tumor necrosis factor- secretion but increased IL-6 and CXCL8 release. However, after activation, only E. coli Nissle induced T cell apoptosis, mediated via Toll-like receptor-2 by caspase- and FasLigand-dependent pathways. T cells play an important role in the recognition of microbial antigens and the perpetuation of inflammatory processes. The demonstration that E. coli Nissle, but not the other bacteria tested, profoundly regulate T cell function contributes to explaining the biological function of this probiotic strain in inflammatory diseases and provides us with a better understanding of the role of T cells.

Alloreactivity and anti-tumor activity segregate within two distinct subsets of cytokine-induced killer (CIK) cells: implications for their infusion across major HLA barriers

2008-06-13

Donor-derived cytokine-induced killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild grafts versus host (GVH) events were observed. To extend this strategy across major HLA barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells' alloreactivity are needed. We hypothesized that alloreactivity and anti-tumor activity of CIK cells segregate within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from seven patients who underwent HCT as treatment of metastatic colorectal cancer. We found that CIK cells maintained their alloreactivity across major HLA barriers when tested as bulk population; after CD56-positive selection, anti-tumor activity was restricted to the CD3+/CD56+ cell fraction and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+/CD56– cell fraction. Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host- or donor-derived PBMC, confirming their low potential for GVH across minor HLA barriers. Moreover, we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor-derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+/CD56– cells might reduce the risk of GVH without affecting the tumor-killing capacity and could help extending CIK infusions across major HLA barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells.

Human Th1 differentiation induced by lipoarabinomannan/lipomannan from Mycobacterium bovis BCG Tokyo-172

2008-06-13

Mycobacterium tuberculosis (tubercle bacilli) and the related acid-fast bacteria including Mycobacterium bovis Bacille Calmett–Guerin (BCG) have a characteristic cell wall (CW) containing various lipoglycans and glycolipids. Such lipoglycans have been reported to activate type-I inflammatory responses via dendritic cells (DCs) through Toll-like receptor 2. In this study, lipoglycans, lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinositol mannoside (PIM), were purified from the CW fractions of M. bovis BCG Tokyo-172, and the effect on the differentiation of human peripheral blood naive CD4 T cells into T_h1 and T_h2 was examined. LAM/LM molecules enhanced T_h1 differentiation under both T_h1 and T_h2 conditions, whereas some other glycolipids and phospholipid enhanced T_h2 differentiation under T_h2 conditions. Other components had little effect under the given conditions. Even in highly purified CD4 T cell cultures, LAM/LM enhanced T_h1 generation only under T_h1 culture conditions. These results indicate that LAM/LM possesses a potent augmenting activity in T_h1 differentiation in human CD4 T cells. LAM/LM appeared to act directly on naive CD4 T cells to enhance T_h1 differentiation under T_h1 culture conditions, while acting indirectly to up-regulate the generation of T_h1 cells via IL-12/DCs under T_h1 and T_h2 conditions. Therefore, these results provide the first evidence indicating that LAM/LM from M. bovis BCG may possess a potent modulating activity in the human system, and thus supporting the strategy for the use of BCG components in the vaccine development for such T_h2 diseases as allergic asthma and rhinitis.

Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE)

2008-06-13

The objective of the study was that the regulatory T cells (Treg) that specialize in the suppression of immune responses might be critically involved in the pathogenesis of autoimmune disease. As for systemic lupus erythematosus (SLE), however, published data concerning Treg phenotype and function are partly conflicting. We therefore performed quantitative and qualitative analyses of naturally occurring CD4⁺CD25⁺ Treg from SLE patients as compared with healthy controls (HC) in order to further elucidate the role of Treg in this systemic autoimmune disease. The phenotype of peripheral blood CD4⁺CD25⁺ Treg was determined by flow cytometry (FACS) in SLE patients and HC. Treg were isolated from SLE patients and HC and their functional capacity was analyzed in suppression assays. Phenotypic and functional data were correlated with clinical data. Decreased proportions of CD4⁺ Treg with high-level expression of CD25 (CD4⁺CD25^hi) were observed in active and inactive SLE patients (0.96 ± 0.08 and 1.17 ± 0.08%, respectively) as compared with HC (2 ± 0.1%). In contrast to HC, Treg from SLE patients displayed an activated phenotype as determined by the expression of CD69, CD71 and HLA-DR. The suppressive capacity of isolated Treg from SLE patients, however, was significantly reduced as compared with HC. Proportions of CD4⁺CD25^hi T cells and the suppressive capacity of Treg were inversely correlated with the clinical disease activity in SLE patients. Our data describe quantitative and qualitative defects of Treg in SLE patients. These deficiencies might contribute to the breakdown of self-tolerance and the development of the autoimmune response in SLE patients.

Role of V{alpha}14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of V{alpha}14+ NKT cells promotes the breakage of CTL tolerance

2008-06-13

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (-GalCer), a ligand for V14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or -GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and -GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40–CD40L interaction mediate the enhancement of CTL induction caused by -GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection.

Tonic B cell activation by Radioprotective105/MD-1 promotes disease progression in MRL/lpr mice

2008-06-13

Toll-like receptors (TLRs) have a crucial role in sensing microbial products and triggering immune responses. Recent reports have indicated that TLR7 and TLR9 have an important role in activating autoreactive B cells. In addition to TLR7 and TLR9, mouse B cells express TLR2, TLR4 and structurally related Radioprotective105 (RP105). We have previously shown that RP105 works in concert with TLR2/4 in antibody response to TLR2/4 ligands. We here report that B cells are constitutively activated by TLR2/4 and RP105. Such B cell activation was revealed by the 3 germ line transcript and serum IgG3 production, both of which were impaired by the lack of RP105 or TLR2/4. Serum IgG3 was not altered in germ-free or antibiotics-treated mice, suggesting that the microbial flora hardly contributes to the continuous activation of B cells. The lack of RP105-dependent B cell activation ameliorated disease progression in lupus-prone MRL/lpr mice. RP105^–/– MRL/lpr mice showed less lymphoadenopathy/splenomegaly and longer survival than MRL/lpr mice. Whereas glomerulonephritis and auto-antibody production were not altered, improvement in blood urea nitrogen and lower incidence of renal arteritis indicated that renal function was ameliorated in the absence of RP105. Our results suggest that RP105-dependent tonic B cell activation has a pathogenic role in MRL/lpr mice.

In vivo modulation of antigen-experienced cells in response to high-dose oral antigen: deletion but no evidence for alterations in the cytokine phenotype

2008-06-13

Whether also antigen-experienced CD4⁺ T cell populations undergo modulations upon oral antigen uptake supporting systemic unresponsiveness is still not fully understood. Using an adoptive transfer model with chicken ovalbumin (OVA)-specific T cells, we demonstrated that absolute numbers of transferred ex vivo-isolated CD4⁺ memory T cells and in vitro-polarized T_h1 cells considerably decrease within spleen and liver upon repetitive OVA feeding. As a consequence, these mice did not mount a delayed-type hypersensitivity reaction after OVA challenge. OVA-specific T_h1 cells re-isolated from the liver showed augmented signs of apoptosis. However, there was no evidence that transferred effector or memory T cells acquired a regulatory phenotype, became anergic or underwent immune deviation. Our data suggest that oral antigen application does not induce alterations in the phenotype of CD4⁺ effector and memory T cells. Instead, deletion of antigen-experienced CD4⁺ T cells preferentially within the liver might be a major mechanism contributing to antigen-specific systemic unresponsiveness upon oral antigen uptake.

The NF-{kappa}B, p38 MAPK and STAT1 pathways differentially regulate the dsRNA-mediated innate immune responses of epidermal keratinocytes

2008-06-13

The epidermis is the primary boundary between the body and the environment, and it serves as the first line of defense against microbial pathogens. Production of chemokines and cytokines is an important step in the initiation of innate immune responses to viral infections. Epidermal keratinocytes produce IFN-, -β and macrophage inflammatory protein (MIP)-1 in response to double-stranded RNA (dsRNA) or viral infections. We showed that human keratinocytes produced cytokines [tumor necrosis factor (TNF)-, IL-1β and IL-15] and chemokines [MIP-1β, RANTES and liver and activation-regulated chemokine (LARC)] in response to dsRNA, with activation of the nuclear factor B (NF-B), p38 mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription 1 (STAT1) pathways. To study the roles of these pathways in their production, we transfected keratinocytes with adenoviral vectors (Ax) carrying a dominant-negative form of inhibitor B (IB) (IBM), a dominant-negative mutant form of STAT1 (STAT1F) or suppressors of cytokine signaling 1 (SOCS1). Transfection with AxIBM or addition of a p38 inhibitor (SB203580) significantly decreased the dsRNA-mediated production of TNF-, IL-1β and MIP-1, but not of IFN-β, IL-15, MIP-1β, RANTES or LARC. Transfection with AxSTAT1F or AxSOCS1 inhibited the dsRNA-mediated production of TNF-, IL-15, MIP-1, MIP-1β, RANTES and LARC, but not IFN-β or IL-1β. In conclusion, the NF-B, p38 MAPK and STAT1 pathways differentially regulate dsRNA-mediated innate immune responses in epidermal keratinocytes.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions

2008-06-13

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P₁)-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P₁ in human DCs and thus does not exert a functional antagonism at S1P₁. Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P–S1P₁ axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

ICAT expression disrupts {beta}-catenin-TCF interactions and impairs survival of thymocytes and activated mature T cells

Hossain, M. Z., Yu, Q., Xu, M., Sen, J. M. — 2008-06-13

T cell factor (TCF) family of transcription factors and β-catenin critically regulate T cell development as demonstrated by the deletion of the tcf gene, which results in a block early in development that becomes complete in mice bearing tcf/lef double deletion. However, the role of β-catenin, a major TCF cofactor, remains controversial. To directly address this, we have generated transgenic mice expressing Inhibitor of β-catenin and TCF (ICAT), a naturally occurring inhibitor that specifically disrupts TCF and β-catenin interactions. In this report, we demonstrate that disrupting the interaction of β-catenin with TCF renders adult thymocytes and activated T cells highly susceptible to apoptosis. In contrast to previously reported observations during fetal thymocyte development, these data show that in adult mice, survival and not differentiation of thymocytes, depends on transcription by TCF and β-catenin. Indeed, we demonstrate that expression of ICAT impedes thymocyte survival by reducing the expression of Bcl_xL in thymocytes below a critical threshold. Survival of activated mature T cells was also impaired due to diminished expression of activation-induced Bcl_xL. Accordingly, expression of transgenic Bcl-2 rescued activated ICAT-Tg CD4 T cells from apoptosis. Thus, disruption of TCF-β-catenin interactions specifically impairs the survival of thymocytes and activated T cells.

CD8+CD122+ regulatory T cells recognize activated T cells via conventional MHC class I-{alpha}{beta}TCR interaction and become IL-10-producing active regulatory cells

2008-06-13

CD8⁺CD122⁺ regulatory T cells (CD8⁺CD122⁺ Treg) are naturally occurring Treg that effectively suppress the proliferation and IFN- production of both CD8⁺ and CD4⁺ target cells. This study investigated the molecular mechanisms of the recognition of target cells by CD8⁺CD122⁺ Treg using an in vitro culture system that reconstitutes the regulatory action of these cells. Naive CD8⁺CD122⁺ Treg co-cultured with pre-activated T cells became active Treg that produced IL-10 and suppressed IFN- production from the target T cells. CD8⁺CD122⁺ Treg effectively suppressed the IFN- production of the target cells of syngeneic mouse strains but not of allogeneic mouse strains with incompatible MHC. By using MHC-congeneic mouse strains, MHC-restricted suppression by CD8⁺CD122⁺ Treg was further confirmed. The blockade of cell surface molecules either on the Treg or on the target cells by specific blocking antibodies indicated that H-2K, H-2D, βTCR and CD8 were involved in the regulatory action but I-A and Qa-1 were not. These results indicate that CD8⁺CD122⁺ Treg recognize already-activated T cells via the interaction of conventional MHC class I–βTCR and become active regulatory cells that produce IL-10 and suppress the target cells.