Visualizing CD4 T-cell migration into inflamed skin and its inhibition by CCR4/CCR10 blockades using in vivo imaging model.

BACKGROUND: Chemokines are critical mediators of T-cell homing into inflamed skin. The complex nature of this multicellular response makes it difficult to analyse mechanisms mediating the early responses in vivo. OBJECTIVES: To visualize directly T-cell homing into inflamed skin and its inhibition by blockades using a unique noninvasive confocal microscopy. MATERIALS AND METHODS: A mouse model of allergic contact dermatitis was used. T cells from oxazolone-sensitized and -challenged Balb/c mice were first analysed phenotypically in vitro. CD4 T cells were then labelled with a tracker dye and transferred into Balb/c-SCID mice. The recipient mice were challenged with oxazolone and CD4 T-cell homing into inflamed skin was visualized. RESULTS: T cells with the skin homing receptors CCR4 and CCR10 were increased in the affected skin and draining lymph nodes, and effectively attracted by their specific chemokines CCL17, CCL22 and CCL27 in vitro. Using in vivo imaging, T-cell migration into the inflamed skin was observed at 2 h after application, peaking at 12 h and continuing for 48 h. Simultaneous systemic administration of neutralizing antibodies against CCR4 ligands (CCL17 and CCL22) and CCR10 ligand (CCL27) led to a significant suppression of T-cell migration and skin inflammation. CONCLUSIONS: Our data indicate that these tissue-selective adhesion molecules and chemokine/receptor pathways act in concert to attract specialized T-cell populations to mediate cutaneous inflammation. The in vivo imaging technique can be applicable to other models of cutaneous diseases to help with better understanding of the pathogenesis and monitoring the therapeutic effects.

Increased expression of activation-induced cytidine deaminase is associated with anti-CCP and rheumatoid factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P

Cancer gene therapy using mesenchymal stem cells expressing interferon-beta in a mouse prostate cancer lung metastasis model.

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-beta, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-beta was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-beta or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-beta-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-beta therapy correlating the antitumor effect. Systemic level of IFN-beta was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-beta therapy for prostate cancer lung metastasis.

Phenotype of genetically regulated thymic involution in young BXD RI strains of mice.

Age-related thymic involution is a multifactorial process related to age-related changes in intrathymic T-cell development and cytokines. In contrast, early thymic involution, because of genetic differences that cause rapid or slow thymic involution at younger age, is less well characterized. Here, we analysed three representative rapid-involuting strains of mice, BXD 8, 18 and 32, compared with three representative slow-involuting strains, BXD 9, 19 and 29, all at 2 months of age. In rapid-involuting strains compared with slow involution strains, thymocyte production, as indicated by CD4+ and CD8+ T-cell receptor recombination excision circle (TREC), were decreased. Rapid-involution strains of mice exhibited a developmental block at the DN1 to DN2 and CD4-CD8- (DN) to CD4+CD8+ (double positive, DP) transition stages. There was also increased susceptibility to H2O2-induced apoptosis, decreased thymic expression of IL-7, decreased expression of an IL-7 downstream anti-apoptosis gene, Bcl-2, and increased expression of a pro-apoptotic gene, Bad. In contrast, IL-7R expression was higher on DN thymocytes of rapid-involution strains. The increased expression of IL-7R was associated with an increased thymocyte proliferation in response to anti-CD3 + IL-7 or anti-CD3 + IL-12 + IL-7. These findings indicate that, even at young age, genetic differences of IL-7/IL-7R regulation pathway in BXD strains of mice can lead to characteristic phenotypic changes that have been previously associated with age-related thymic involution.

Primary adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and liver enzyme elevation.

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.

Genetic segregation of spontaneous erosive arthritis and generalized autoimmune disease in the BXD2 recombinant inbred strain of mice.

The BXD2 strain of mice is one of approximately 80 BXD recombinant inbred (RI) mouse strains derived from an intercross between C57BL/6J (B6) and DBA/2J (D2) strains. We have discovered that adult BXD2 mice spontaneously develop generalized autoimmune disease, including glomerulonephritis (GN), increased serum titres of rheumatoid factor (RF) and anti-DNA antibody, and a spontaneous erosive arthritis characterized by mononuclear cell infiltration, synovial hyperplasia, and bone and cartilage erosion. The features of lupus and arthritis developed by the BXD2 mice segregate in F2 mice generated by crossing BXD2 mice with the parental B6 and D2 strains. Genetic linkage analysis of the serum levels of anti-DNA and RF by using the BXD RI strains shows that the serum titers of anti-DNA and RF were influenced by a genetic locus on mouse chromosome (Chr) 2 near the marker D2Mit412 (78 cm, 163 Mb) and on Chr 4 near D4Mit146 (53.6 cm, 109 Mb), respectively. Both loci are close to the B-cell hyperactivity, lupus or GN susceptibility loci that have been identified previously. The results of our study suggest that the BXD2 strain of mice is a novel model for complex autoimmune disease that will be useful in identifying the mechanisms critical for the immunopathogenesis and genetic segregation of lupus and erosive arthritis.

Defective clearance of adenovirus in IRF-1 mice associated with defects in NK and T cells but not macrophages.

A replication-defective adenovirus-LacZ recombinant virus (AdLacZ) was injected intravenously into IRF-1(-/-) mice and wild-type mice to characterize the contribution of IRF-1 to the immune-mediated clearance of Ad vector. Compared with wild-type mice, IRF-1(-/-) mice expressed higher levels of the LacZ gene product in the liver. After infusion of the AdLacZ, the expression of IRF-1 mRNA was upregulated in the liver of wild-type mice, but not in IRF-1(-/-) mice. Both spleen and liver mononuclear cells from IRF-1(-/-) mice initially exhibited a markedly lower number of NK, NK-T and CD8 T cells. At day 7 after the administration of AdLacZ, there was a significantly increased population of NK, NK-T and CD8 T cells in both spleen and liver, and also CD11b(+) cells in liver of IRF-1(-/-) mice, compared with the increased in wild-type mice. As IRF-1 is an important signal for production of IFN-gamma by CD8 T and NK cells as well as production of IL-12 by CD11b(+) cells, we determined whether there were lower levels of these cytokines in IRF-1(-/-) mice after Ad challenge. Surprisingly, there were lower levels of IL-12, but higher levels of IFN-gamma and IL-18 in IRF-1(-/-) compared with wild-type mice at day 7 after administration with AdLacZ. These results indicate that delayed clearance of Ad is associated with partial correction of defects of the NK, NK-T and CD8 T cells and increased production of IFN-gamma and IL-18 in IRF-1(-/-) mice.

Identification of multiple genetic loci that regulate adenovirus gene therapy.

A key aspect of the immune response to adenovirus (Ad) gene therapy is the generation of a cytotoxic T-cell (CTL) response. To better understand the genetic network underlying these events, 20 strains of C57BL/6 x DBA/2 (BXD) recombinant inbred (RI) mice were administered with AdLacZ and analyzed at days 7, 21, 30, and 50 for liver beta-galactosidase (LacZ) expression and CTL response. Sera levels of interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were analyzed at different times after AdLacZ. There was a distinct strain-dependent expression of LacZ, which was strongly correlated with the CTL response. Among the five BXD RI strains that exhibited significantly prolonged LacZ expression, four also exhibited a marked defect in the production of Ad-specific CTL. There was a strong correlation between the sera levels of IFN-gamma, TNF-alpha, and IL-6, but cytokine responses were not significantly correlated with LacZ expression or the CTL response. Quantitative trait loci regulating LacZ on day 30 were found on chromosome (Chr) 19 (33 cM) and Chr 15 (42.8 cM). Cytotoxicity mapped to Chr 7 (41.0 and 57.4-65.2 cM), Chr 15 (61.7 cM), and Chr X (27.8 cM). IFN-gamma production mapped to Chr 18 (22, 27, and 32 cM) and Chr 11 (64.0 cM). TNF-alpha and IL-6 production mapped to Chr 6 (91.5 cM) Chr 9 (42.0 cM) and Chr 8 (52 and 73.0 cM). These results indicate that different strains of mice exhibit different pathways for effective clearance of AdLacZ depending on genetic polymorphisms and interactions at multiple genetic loci.

Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.

A comprehensive analysis of initial thymus size and involution rate has not been quantitated for different genetic backgrounds of mice, thus genetic linkage analysis of thymic involution has not been possible. Here, we have used a mathematical method to analyze the age-related decline in thymocyte count in C57BL/6 and DBA/2 mice and have observed that thymic involution could be best fit with a negative exponential curve N(t)=beta(0) x exp(-beta(1)t), where t represents the age (day). This regression model was applied to C57BL/6 x DBA/2 (B x D) recombinant inbred strains of mice to identify the genetic loci influencing age-related thymic involution. There was a dramatic genetic effect of B and D alleles on thymocyte count at young age and the age-related thymic involution rate. The strongest quantitative trait loci (QTL) influencing the rate of thymic involution were mapped to mouse chromosome (Chr) 9 (D9Mit20 at 62 cM) and Chr 10 (D10Mit61 at 32 cM). The strongest QTLs influencing the initial thymocyte count were mapped to ChrX (DXMit324 at 26.5 cM) and Chr 3 (D3Mit127 at 70.3 cM). The present study suggests that the initial thymus size and the rate of thymic involution may be influenced by a relatively small number of genetic loci.

Cellular mechanism of thymic involution.

Involution of the thymus and alterations in the development of thymocytes are the most prominent features of age-related immune senescence. We have carried out a comparative analysis of thymocyte and stroma in rapid thymic involution DBA/2 (D2) strain of mice compared with slow involution C57BL/6 (B6) strain of mice. Analysis of mice at 15 months of age suggested an age-related decrease in the thymocyte cell count, a block in the development of T cells and cortical involution in D2 mice compared with 3-month-old mice. TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling) staining and fluorescence-activated cell sorter (FACS) analysis showed that there was a significant increase in apoptotic cells in the cortex region of thymus in 15-month-old D2 mice compared with the same aged B6 mice. The thymocyte proliferation rate, as assessed by bromodeoxyuridine (BrdU) staining and [3H]-thymidine incorporation assay, was lower in 3-month-old D2 mice compared with the same age B6 mice. Immunohistochemical staining showed that the arrangement of MTS (mouse thymus stromal)-10+ epithelial cells and MTS-16+ connective tissue staining pattern had become disorganized in 15-month-old D2 mice but remained intact in B6 mice of the same age. These results suggest that, in D2 mice, both the thymocytes and stromal cells exhibit age-related defects, and that the genetic background of mice plays an important role in determining age-related alterations in thymic involution.

Different role of Apaf-1 in positive selection, negative selection and death by neglect in foetal thymic organ culture.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1-/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4+CD8+ (DP) thymocytes from Apaf-1-/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1-/- mice were crossed with the T-cell receptor (TCR)-alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha-/-Apaf-1-/- and TCR-alpha-/-Apaf-1+/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1-/- Db/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33+ thymocytes and an increased percentage of the autoreactive CD8+M33+ thymocytes in male, but not female Apaf-1-/- Db/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-alpha production by Kupffer cells.

The elevation of soluble Fas (sFas) in the sera of patients with liver disease suggests a role for sFas in the disease process; whether it is protective or not is controversial. To determine the effects of sFas on Fas-induced liver apoptosis, we manipulated mice to produce sFas by transfecting them in vivo with different amounts of an adenovirus that produces mouse sFas driven by the CMV promoter (AdsFas). Fas-mediated apoptosis was induced by administration of anti-mouse Fas (Jo2; 10 microg/mouse) one week later. The administration of AdsFas (10(3), 10(7), or 10(9) pfu/mouse), which was associated with only minimal side-effects, resulted in a significant reduction in the liver transaminase levels and mortality of the mice on challenge with Jo2, as compared to control mice treated with AdLacZ. However, the protective effect of AdsFas was not complete. The possibility that Jo2-induction of TNF-alpha in the Kupffer cells of the liver contributes to the pathology was therefore tested. Although administration of soluble TNF receptor (sTNFRI) alone did not protect the mice from the lethal effects of Jo2, administration of sTNFRI (200 microg/mouse) after infection with AdsFas (10(9) pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-alpha by Kupffer cells produce the lethal effects of Jo2 that remained after treatment with AdsFas, these cells were selectively ablated by treatment of the mice with gadolinium chloride prior to challenge with Jo2. This treatment greatly reduced early mortality and hepatocellular damage as well as TNF-alpha production 6 h after injection of Jo2. These results indicate that: (1) AdsFas prevents Jo2-induced apoptosis of hepatocytes; (2) In addition to mediating Fas-mediated apoptosis of hepatocytes, Jo2 can separately induce TNF-alpha production by Kupffer cells resulting in early mortality, and (3) Optimal protection from Jo2-induced mortality can be achieved by protection of liver cells by pretreatment with both AdsFas and sTNFRI.

Defective Fas ligand-mediated apoptosis predisposes to development of a chronic erosive arthritis subsequent to Mycoplasma pulmonis infection.

OBJECTIVE: To determine whether defective T cell apoptosis is associated with the development of a chronic arthritis subsequent to mycoplasma infection, and to determine whether deletion of T cells can prevent the development of this arthritis. METHODS: B6 wild-type (B6-+/+), B6-lpr/lpr, and B6-gld/gld mice were infected with Mycoplasma pulmonis. The severity of lymphocytic infiltration and joint damage was evaluated, and the degree of recovery of viable mycoplasma from the spleen and joints was determined. Antigen-presenting cells derived from Fas mutant lpr mice (lpr-APC) were transfected ex vivo with an adenovirus (Ad) vector to yield lpr-APC expressing high levels of Fas ligand (lpr-APC-AdFasL), which in turn were transferred intraperitoneally into M pulmonis-infected B6-gld/gld mice. The development of arthritis subsequent to M pulmonis infection and the induction of apoptosis of cells within the synovial tissue and lymph nodes of lpr-APC-AdFasL-treated B6-gld/gld mice were determined. RESULTS: Infection of B6-lpr/lpr and B6-gld/gld mice with M pulmonis resulted in an acute-phase inflammation of the synovium that later developed into a chronic erosive arthritis. Similar infection of B6-+/+ mice resulted only in an acute joint inflammatory response that resolved. Chronic arthritis in B6-gld/gld mice and B6-lpr/lpr was not due to persistent infection, since there were no differences in the rates of clearance of M pulmonis from the joints of B6-gld/gld or B6-lpr/lpr mice compared with B6-+/+ mice. Treatment of infected B6-gld/gld mice with lpr-APC-AdFasL resulted in a significantly decreased incidence of chronic arthritis that was associated with a decrease in lymph node T cells, but not with apoptosis of synovial T cells or fibroblasts. CONCLUSION: Defective Fas/FasL-mediated apoptosis of T cells is an important factor that rendered arthritis-resistant B6 mice susceptible to the development of a chronic erosive arthritis subsequent to mycoplasma infection. In vivo lpr-APC-AdFasL cell-gene therapy is a safe and effective method for inhibiting the development of this arthritis.

Activated CD8(+) T cells from aged mice exhibit decreased activation-induced cell death.

To uncouple the defects of activation and apoptosis of T cells from aged mice, we used anti-CD3 plus IL-2 stimulation to induce an activation response and analyzed the subsequent activation-induced cell death (AICD) response of T cells from 16-month-old mice. The results herein demonstrate that T cells from 16-month-old mice could be activated by anti-CD3-induced activation signals but exhibited distinct phenotypic and functional features compared to young (2-month-old) mice. These include a decrease in AICD, a delayed entry into the cell cycle, and a decreased telomerase activity. The decreased AICD of T cells from 16-month-old mice is associated with a decreased expression of Fas and Fas ligand (FasL), decreased susceptibility to anti-Fas-induced apoptosis, and an increased expansion of a CD8(+) T-cell population. Prior to activation, these T cells exhibit a phenotype that is CD44(hi)CD62L(hi). After stimulation, these T cells produced high levels of the pro-inflammatory cytokine, IFN-gamma, and developed an increased population of IFN-gamma(+)IFN-gamma R(-) T cells. Our results suggest that there is a dysregulation in T-cell homeostasis in aged mice associated with a decrease in AICD of CD8(+) T cells.

Normal T-cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator-peptide-derived superparamagnetic nanoparticles.

The present study analyzed the feasibility of using magnetic resonance imaging (MRI) to monitor T-cell homing in vivo after loading T cells with superparamagnetic iron oxide (CLIO) nanoparticles derivatized with a peptide sequence from the transactivator protein (Tat) of HIV-1. T cells were isolated from C57BL/6 (B6) mice and loaded with 0, 400, 800, 1600, or 8000 ng/ml of FITC conjugated CLIO-Tat (FITC-CLIO-Tat). There was a dose-dependent uptake of FITC-CLIO-Tat by T cells. Stimulation of FITC-CLIO-Tat loaded T cells with anti-CD3 (0.1 microg/ml) plus IL-2 (5 ng/ml) elicited normal activation and activation-induced cell death (AICD) responses, and normal upregulation of CD69, ICAM-1 (CD54), L-selectin (CD62L), and Fas. The FITC-CLIO-Tat loaded T cells (3 x 10(7)) were transferred intravenously (i.v.) into B6 mice and the in vivo MRI of mice was acquired using a spin-echo pulse sequence at 4.7 T with a Bruker Biospec system. Homing of T cells into the spleen was observed by a decrease in MRI signal intensity within 1 h after the transfer, which remained decreased for 2-24 h after transfer. These homing data were confirmed by FACS analysis and biodistribution analysis using 125I-CLIO-Tat. Thus, T cells can be efficiently loaded with FITC-CLIO-Tat without interfering with their normal activation and AICD, or homing to the spleen, and the biodistribution of FITC-CLIO-Tat loaded T cells can be monitored in vivo over time by MRI.

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.

Genetic dissection of age-related changes of immune function in mice.

Understanding of the genetic basis of normal and abnormal development of the immune response is an enormous undertaking. The immune response, at the most minimal level, involves interactions of antigen presenting cells (APCs), T and B cells. Each of these cells produce cell surface and soluble factors (cytokines) that affect both autocrine and paracrine functions. A second level of complexity needs to consider the development of the macrophage/monocyte lineage as well as the production of the common lymphoid precursor which undergoes distinct maturation steps in the thymus and periphery to form mature T cells as well as in BM (BM) and lymphoid organs to form mature B cells. A third level of complexity involves the immune response to infectious agents including viruses and also the response to tumour antigens. In addition, there are imbalances that predispose to decreased responses (immunodeficiencies) or increased responses (autoimmunity). A fourth level of complexity involves attempts to understand the differences in the immune response that occurs at a very young age, in adults, and at a very old age. This review will focus on the use of C57BL/6 J X DBA/2 J (BXD) recombinant inbred (RI) strains of mice to map genetic loci associated with the production of lymphoid precursors in the BM, development of T cells in the thymus, and T-cell responses to stimulation in the peripheral lymphoid organs in adult and in aged mice. Strategies to improve the power and precision in which complex traits such as the age-related immune response can be mapped is limited with the current set of 35 strains of BXD mice. Strategies to increase these strains by generating recombinant intercross (RIX) strains of mice are being developed to enable this large set of lines to detect quantitative trait loci (QTLs) with a much higher consistency and statistical power. More importantly, the resolution with which these QTLs can be mapped would be greatly improved and, in many cases, adequate to carry out direct identification of candidate genes. It is likely that, given the complexity of the immune system development, the number of cells involved in an immune response, and especially the changes in the immune system with ageing, mapping hundreds of genes will be required to fully understand age-related changes in the immune response. This review outlines ongoing and future strategies that will enable the mapping and identification of these genes.

Decreased T cell stimulatory capacity of monocyte-derived human macrophages following herpes simplex virus type 1 infection.

Macrophages play a central role in establishing a specific immune response by acting as professional antigen presenting cells (APC) for T cells leading to a vigorous immune response. In order to analyze if Herpes simplex Virus (HSV) type 1 infection might affect the macrophage APC-function, monocyte-derived human macrophages were infected with HSV-1 strain F in vitro. Cocultures with allogeneic T cells revealed a strongly impaired stimulatory capacity of HSV-infected macrophages compared to uninfected controls which was not owing to a productive viral infection in macrophages. An increased expression of Fas ligand (FasL/CD95L) was detected in HSV-infected macrophages by FACS analysis. Although the majority of the macrophages expressed high levels of Fas (CD95/Apo-1), the HSV-induced upregulation of FasL did not result in an increased autocrine apoptosis of macrophages which might be related to endogenous expression of the apoptosis inhibitor FLICE inhibitory protein (FLIP). However, substantial apoptosis occurred in peripheral T cells as well as Fas-sensitive Jurkat T cells when cocultured with HSV-infected macrophages. These findings suggest that the paracrine killing of activated T cells by FasL expressing APC might be a novel strategy of immune evasion by HSV.

Regulation of tumor necrosis factor alpha-mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt.

OBJECTIVE: To determine if tumor necrosis factor alpha (TNFalpha)-driven proliferation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with up-regulation of the activity of serine/threonine kinase B/Akt and with survival of RASF. METHODS: Staining of phosphorylated Akt was done using anti-phosphorylated Thr308 Akt antibody. Levels of phosphorylated Akt were analyzed by Western blot and Akt activity was analyzed using a kinase assay. TUNEL staining was used to analyze the cytotoxicity of TNFalpha treatment or TNFalpha combined with either the Akt activity inhibitor wortmannin, an adenovirus expressing dominant-negative mutant (AdAkt-DN), or an adenovirus expressing phosphatase and tensin homolog deleted on chromosome 10 (AdPTEN). RESULTS: The levels of phosphorylated Akt were higher in RASF than in osteoarthritis synovial fibroblasts (OASF), as demonstrated by immunohistochemical staining, immunoblot analysis, and an Akt kinase assay. The levels of phosphorylated Akt and Akt kinase activity were increased by stimulation of primary RASF with TNFalpha (10 ng/ml). Treatment of RASF with the phosphatidylinositol 3-kinase inhibitor wortmannin (50 nM) plus TNFalpha resulted in apoptosis of 60 +/- 8% (mean +/- SEM) of RASF within 24 hours. This proapoptosis effect was specific for Akt, since equivalent levels of apoptosis were observed upon TNFalpha treatment of RASF transfected with AdAkt-DN and with AdPTEN, which opposes the action of Akt. CONCLUSION: These results indicate that phosphorylated Akt acts as a survival signal in RASF and contributes to the stimulatory effect of TNFalpha on these cells by inhibiting the apoptosis response. This effect was not observed in OASF and may reflect the pathophysiologic changes associated with the proliferating synovium in rheumatoid arthritis.