H.-C. Hsu

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.

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.

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)=β0 × exp(−β1t), where t represents the age (day). This regression model was applied to C57BL/6 × DBA/2 (B × 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.

Soluble Fas gene therapy protects against Fas-mediated apoptosis of hepatocytes but not the lethal effects of Fas-induced TNF-α 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 μg/mouse) one week later. The administration of AdsFas (103, 107, or 109 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-α 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 μg/mouse) after infection with AdsFas (109 pfu/mouse) resulted in 100% survival of the mice on challenge with Jo2. To confirm that the production of TNF-α 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-α 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-α 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.

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.

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 ≤ 0.0001) and anti‐cyclic citrullinated peptide (CCP) (P = 0.0005). Serum levels of IFN‐γ (P = 0.0005) and IL‐17 (P = 0.007), but not IL‐4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN‐γ and IL‐17, leading to the development of anti‐CCP and RF.

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 IFNγ. 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 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.

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‐γ 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‐γ 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‐γ and IL‐18 in IRF‐1–/– mice.