V?Gene Family Usage in Spontaneous Lymphomas of AKR Mice: Evidence for Defective Clonal Deletion

T-cell receptor (TCR) ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4⁺8⁺) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V ß gene family usage, 2 out of 14 lymphomas expressed V ß6. Normally, V ß6⁺ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V ß6⁺ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.     

The in vivo induction of lymphocyte apoptosis in MRL-lpr/lpr mice treated with FTY720

The in vitro treatment of lpr thymocytes with FTY720 resulted in a dose-dependent reduction in cell viability due to apoptosis. In order to study the effect of FTY720 in vivo, lpr mice received an oral daily dose of 1 mg/kg FTY720 for 14 days, beginning at 16 weeks of age which was when the animals were developing massive lymphoadenopathy. Compared with untreated lpr mice, FTY720-treated lpr mice had significantly prolonged lives. At 24 weeks of age, treated mice demonstrated markedly reduced weights of the spleen and lymph nodes, and the proportion of CD3⁺B220⁺ and CD4⁻CD8⁻ cells in the thymus, spleen and lymph nodes decreased markedly. In addition, in these mice the percentage of CD4⁺CD8⁺ and CD3⁻B220⁻ cells in the thymus and the percentage of CD4⁺CD8⁻, CD4⁻CD8⁺, CD3⁺B220⁻ and CD3⁻B220⁺ cells in the spleen returned to almost the normal values observed in wild-type mice. Histological observation 1 day after the final administration of FTY720 revealed a remarkable infiltration of neutrophils in the lymphoid organs. Apoptotic cells were detected in all the lymphoid organs using in situDNA nick-end labelling. Electron microscopy showed that the apoptotic cells were ingested by phagocytes. FTY720 therapy is thus highly effective in Fas-mutant animals with abnormally expanding lymphocytes.     

Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab−/−) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab−/− mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab−/− mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.     

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0·01–0·001), whereas in BALB/c normal controls they increased significantly (P < 0·05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and anti-dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0·001 for both ssDNA and dsDNA and P < 0·01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.     

Heat shock protein 90 inhibition by 17-DMAG lessens disease in the MRL/lpr mouse model of systemic lupus erythematosus

Elevated expression of heat shock protein 90 (HSP90) has been found in kidneys and serum of systemic lupus erythematosus (SLE) patients and MRL/Mp-Fas^lpr/Fas^lpr (MRL/lpr) autoimmune mice. We investigated if inhibition of HSP90 would reduce disease in MRL/lpr mice. In vitro, pretreatment of mesangial cells with HSP90 inhibitor Geldanamycin prior to immune-stimulation showed reduced expression of IL-6, IL-12 and NO. In vivo, we found HSP90 expression was elevated in MRL/lpr kidneys when compared to C57BL/6 mice and MRL/lpr mice treated with HSP90 inhibitor 17-DMAG. MRL/lpr mice treated with 17-DMAG showed decreased proteinuria and reduced serum anti-dsDNA antibody production. Glomerulonephritis and glomerular IgG and C3 were not significantly affected by administration of 17-DMAG in MRL/lpr. 17-DMAG increased CD8⁺ T cells, reduced double-negative T cells, decreased the CD4/CD8 ratio and reduced follicular B cells. These studies suggest that HSP90 may play a role in regulating T-cell differentiation and activation and that HSP90 inhibition may reduce inflammation in lupus.     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.     

Accelerated induction of mycobacterial antigen-specific CD8+ T cells in the Mycobacterium tuberculosis-infected lung by subcutaneous vaccination with Mycobacterium bovis bacille Calmette–Guérin

Both CD4⁺ and CD8⁺ T cells are important in protection against Mycobacterium tuberculosis infection. To evaluate the effect of vaccination with Mycobacterium bovis bacille Calmette–Guérin (BCG) on the CD8⁺ T-cell response to pulmonary M. tuberculosis infection, we analyzed the kinetics of CD8⁺ T cells specific to the mycobacterial Mtb32a_309–318 epitope, which is shared by M. tuberculosis and M. bovis BCG, in the lung of mice infected with M. tuberculosis. The CD8⁺ T cells were detected by staining lymphocytes with pentameric major histocompatibility complex (MHC) class I H-2D^b–Mtb32a_209–318 peptide complex and were analysed by flow cytometry. Mtb32a-specific CD8⁺ T cells became detectable on day 14, and reached a plateau on day 21, in the lung of M. tuberculosis-infected unvaccinated mice. Subcutaneous vaccination with M. bovis BCG in the footpads induced Mtb32a-specific CD8⁺ T cells in the draining lymph nodes (LNs) on day 7 and their numbers further increased on day 14. When M. bovis BCG-vaccinated mice were exposed to pulmonaryinfection with M. tuberculosis 4 weeks after vaccination, the Mtb32a-specific CD8⁺ T cells in the infected lung became detectable on day 7 and reached a plateau on day 14, which was 1 week earlier than in the unvaccinated mice. The pulmonary CD8⁺ T cells from the BCG-vaccinated M. tuberculosis-infected mice produced interferon-γ in response to Mtb32a_209–318 peptide on day 7 of the infection, whereas those of unvaccinated mice did not. The results demonstrate that induction of mycobacterial antigen-specific protective CD8⁺ T cells in the M. tuberculosis-infected lung is accelerated by subcutaneous vaccination with M. bovis BCG.     

Vaccination with autoreactive CD4Th1 clones in lupus-prone MRL/Mp-Fas mice

We studied the efficacy of T cell vaccination with CD4⁺αβTh1 clones in lupus-prone MRL/Mp-Fas^lpr/lpr (MRL/lpr) mice. CD4⁺αβ Th1 clones, dna51 (Vβ8.3) and rnp2 (Vβ14), which stimulated anti-dsDNA or U1 ribonucleoprotein (U1RNP) antibody (Ab) production respectively, were isolated from splenocytes of MRL/lpr mice. Antinuclear Ab kinetics, renal function, renal histology, survival rate, and lymphocyte subpopulations in the spleen were monitored after intravenous adoptive transfer of IL-2-stimulated (s-) or irradiated (i-) clones to 3 week-old female MRL/lpr mice. Anti-idiotypic humoral and T cell responses against the transferred autoreactive Th1 clones were determined in parallel. Compared with PBS-treated MRL/lpr mice, anti-dsDNA Ab titers, and the activity index for lupus nephritis were all decreased in MRL/lpr mice vaccinated with i-dna51 cells, whereas survival rate was not improved. The numbers of CD4⁺Vβ8.3 T cells in the spleen were also significantly decreased in these mice. Anti-idiotypic Abs recognizing a 12 amino acid sequence of clone dna51 T cell receptor Vβ8.3-complementarity-determining region (CDR) 3 were detected in the MRL/lpr mice that received i-dna51 or s-dna51 cells. These Abs suppressed dna51 cell proliferation, as well as cytotoxicity of CD8⁺T cells against dna51. The present study suggests that vaccination with CD4⁺αβTh1 clone, dna51, elicits anti-idiotypic T cell and humoral responses against dna51 in MRL/lpr mice, although the immunoregulatory effects on lupus may be limited.     

Association of T cell dysfunction with the presence of IgG autoantibodies on CD4+ lymphocytes in haemophilia patients; results of a 10-year study

HIV induces progressive dysfunction followed by numerical depletion of CD4⁺ lymphocytes. IgG autoantibodies and gp120-containing immune complexes have been implicated in the pathogenesis of AIDS. We carried out a longitudinal study in 19 HIV^– and 72 HIV⁺ haemophilia patients over a 10-year period in order to investigate a possible relationship between the occurrence of autoantibodies and CD4⁺ lymphocyte changes. IgM, IgG, C3d and gp120 on the surface of CD4⁺ lymphocytes were determined in heparinized whole blood with flow cytometry and double-fluorescence. The in vitro response of autoantibody-coated cells was tested in cell cultures with concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PWM), anti-CD3 MoAb or pooled allogeneic stimulator cells (MLC). After a 10-year follow up, 12 of 71 HIV⁺ and 16 of 19 HIV^– haemophilia patients showed no evidence of immunoglobulins on circulating CD4⁺ lymphocytes. HIV^– haemophilia patients without autoantibodies had CD4⁺and CD8⁺ cell counts in the normal range (957 ± 642/μl and 636 ± 405/μl) and normal T cell responses in vitro (mean relative response (RR) ≥ 0.7). In contrast, HIV⁺ haemophilia patients showed immunological abnormalities which were associated with the autoantibody and immune complex load of CD4⁺ blood lymphocytes. HIV⁺ patients without autoantibodies had a mean CD4⁺ lymphocyte count of 372 ± 274/μl, a mean CD8⁺ lymphocyte count of 737 ± 435/μl, and normal T lymphocyte stimulation in vitro (mean RR ≥ 0.7). HIV⁺ patients with complement-fixing IgM on CD4⁺ lymphocytes had somewhat lower CD4⁺ (255 ± 246/μl, P= NS) and CD8⁺ (706 ± 468/μl, P= NS) lymphocyte numbers, and also normal T lymphocyte stimulation (mean RR ≥ 0.7) in vitro. However, patients with complement-fixing IgG autoantibodies showed a strong decrease of CD4⁺ (150 ± 146/μl, P< 0.02) and CD8⁺ (360 ± 300/μl, P< 0.02) lymphocytes and impaired CD4⁺ lymphocyte stimulation in vitro with a mean RR of 0.5 ± 0.5 for Con A (P= NS), 0.7 ± 0.8 for PHA (P< 0.03), 0.4 ± 0.4 for PWM (P= NS), 0.8 ± 1.2 for anti-CD3 MoAb (P< 0.04) and 0.7 ± 1.0 for pooled allogeneic stimulator cells (P= 0.05). Patients with gp120-containing immune complexes on CD4⁺ blood lymphocytes demonstrated strongly decreased CD4⁺(25 ± 35/μl, P< 0.0001) and CD8⁺ (213 ± 212/μl, P< 0.006) lymphocyte counts as well as strongly impaired T lymphocyte responses in vitro upon stimulation with PHA (RR 0.2 ± 0.1, P < 0.02), PWM (RR 0.2 ± 0.2, P= 0.05), anti-CD3 MoAb (RR 0.1 ± 0.1, P< 0.04), and allogeneic stimulator cells (RR 0.2 ± 0.1, P< 0.02). These data led us to speculate that autoantibody formation against CD4⁺ lymphocytes is an important mechanism in the pathogenesis of AIDS. We hypothesize that autoantibodies against circulating CD4⁺ lymphocytes inhibit CD4⁺ cell function, especially the release of cytokines, and induce CD4⁺ cell depletion. The reduction and dysfunction of CD4⁺ lymphocytes may be responsible for the CD8⁺ cell depletion observed in HIV⁺ patients.     

Fas Ligand-dependent and -independent mechanisms of toxicity induced by T cell lymphomas in lymphoid organs and in the liver

In the current study, we investigated the effect of growth of FasL⁺ tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL⁺ LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vβ3⁺ and Vβ11⁺ T cells. When injected into syngeneic mice, the FasL⁺ LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin β, and tumor necrosis factor β. Together, these data suggested that, in mice bearing FasL⁺ LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.     

Viral 5′‐triphosphate RNA and non‐CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR‐independent immune responses

Certain viral nucleic acids aggravate autoimmunity through nucleic acid‐specific TLR. Viral 5′‐triphosphate RNA (3P‐RNA) and double‐stranded non‐CpG DNA induce antiviral immunity via TLR‐independent pathways but their role in autoimmunity is unknown. Transient exposure of 16‐wk‐old MRL^lpr/lpr mice to 3P‐RNA aggravated lupus nephritis by increasing IFN signaling and decreasing CD4⁺CD25⁺ T cells. By contrast, transient exposure to non‐CpG DNA exacerbate lupus nephritis in association with splenomegaly, lymphoproliferation, hypergammaglobulinaemia and increased B220⁺CD138⁺ plasma cells. Both, 3P‐RNA and non‐CpG DNA increased glomerular complement factor C3c deposits but both nucleic acid formats were less potent in aggravating renal pathology as compared with CpG DNA. 3P‐RNA and non‐CpG DNA also localized to the glomerular mesangial cells and activated cultured mesangial cells to produce IL‐6. We conclude, 3P‐RNA or non‐CpG DNA both trigger autoimmune disease in MRL^lpr/lpr mice by specifically activating adaptive immunity but similarly enhance inflammation on the tissue level.     

Killer cell immunoglobulin receptor profile on CD4+ CD28− T cells and their pathogenic role in non‐dialysis‐dependent and dialysis‐dependent chronic kidney disease patients

There is a progressive increase in cardiovascular disease with declining renal function, unexplained by traditional risk factors. A CD4⁺ T-cell subpopulation (CD4⁺ CD28⁻), activated by human heat-shock protein 60 (hHSP 60), expands in patients with acute coronary syndrome and is associated with vascular damage. These cells exhibit cytotoxicity via expression of activating killer cell-immunoglobulin-like receptor KIR2DS2, mainly in the absence of inhibitory KIR2DL3. We investigated expansion of these cells and the pathogenic role of the KIR in non-dialysis-dependent chronic kidney disease (NDD-CKD) and end-stage haemodialysis-dependent renal disease (HD-ESRD) patients. CD4⁺ CD28⁻ cells were present in 27% of the NDD-CKD and HD-ESRD patients (8–11% and 10–11% of CD4⁺ compartment, respectively). CD4⁺ CD28⁻ cells were phenotyped for KIR and DAP12 expression. Cytotoxicity was assessed by perforin and pro-inflammatory function by interferon-γ expression on CD4⁺ CD28⁻ clones (NDD-CKD n = 97, HD-ESRD n = 262). Thirty-four per cent of the CD4⁺ CD28⁻ cells from NDD-CKD expressed KIR2DS2 compared with 56% in HD-ESRD patients (P = 0·03). However, 20% of clones expressed KIR2DL3 in NDD-CKD compared with 7% in HD-ESRD patients (P = 0·004). DAP12 expression in CD28⁻ 2DS2⁺ clones was more prevalent in HD-ESRD than NDD-CKD (92% versus 60%; P < 0·001). Only 2DS2⁺ 2DL3⁻ DAP12⁺ clones were cytotoxic in response to hHSP 60. CD4⁺ CD28⁻ cells exhibited increased KIR2DS2, reduced KIR2DL3 and increased DAP12 expression in HD-ESRD compared with NDD-CKD patients. These findings suggest a gradual loss of expression, functionality and protective role of inhibitory KIR2DL3 as well as increased cytotoxic potential of CD4⁺ C28⁻ cells with progressive renal impairment. Clonal expansion of these T cells may contribute to heightened cardiovascular events in HD-ESRD.     

T cell clones from a Sjögren's syndrome salivary gland biopsy produce high levels of IL-10

Sjögren''s syndrome (SS) is characterized by a focal periductal salivary gland infiltrate consisting mainly of T and B lymphocytes. Most of the T cells bear the memory of CD4⁺ Th-1-like phenotype and express high levels of class II, though CD8⁺ cells are also present. We have studied 17 labial salivary gland and 15 peripheral blood T cell clones from a patient with primary SS. The tissue clones were 71% CD8⁺ and 29% CD4⁺, and the peripheral blood-derived clones were 60% CD8⁺ and 40% CD4⁺. The CD4⁺ T cell clones from both the salivary gland and autologous peripheral blood were of the Th1 phenotype, in that they produced interferon-gamma (IFN-γ) and IL-2 but very little IL-4 after 24 h stimulation with phorbol myristate acetate and anti-CD3 antibody. The salivary gland-derived CD4⁺ clones produced 15 times more IL-10 (7·92 ng/ml) than peripheral blood-derived CD4⁺ clones (0·52 ng/ml, P≤0·02). The tissue CD8⁺ clones produced 1·2 times (P<0·04) more IFN-γ and CD4⁺ clones produced 3·5 times less IL-2 (P<0·02) than the respective PBM-derived clones. The accumulation of Th1-type cells producing high levels of IL-10 in the salivary gland suggests a specific immunoregulatory function at the site of inflammation in SS.     

Phenotypic Abnormalities of Splenic and Bone Marrow B Cells inlprandgldMice

Mice homozygous for the mutantFasgenelprdevelop generalized lymphoproliferation and produce autoantibodies resembling those found in human SLE. We have previously shown that these autoantibodies are produced by B2 cells rather than B1 cells and that the autoantibody-producing B cells are intrinsically abnormal. We investigated further thelprB cell with a large panel of antibodies to B-cell surface markers to identify phenotypic abnormalities. B cells from spleen and bone marrow of age-matched congenic mice differing only at thelprlocus were examined by flow cytometry. Two consistent phenotypic differences were identified. First, spleen cells from olderlprmice had an increase in the number and percentage of IgM⁺B cells expressing low levels of CD23. Second,lprbone marrow had decreased numbers of B220^h1IgM⁺-syndecan-1⁺CD23⁺B cells. All other markers tested, except the previously identified modest increase of Ia onlprspleen cells, showed no consistent differences. B cells fromgldmice showed the same phenotypic abnormalities as those fromlpr.Compared to T cells, the relative paucity of cell surface marker differences betweenlprand +/+ B cells suggests that B cells may have fewer regulatory mechanisms to silence autoreactive specificities. The phenotypic differences identified may provide clues to the mechanism of autoantibody production inlprmice, while the overwhelming phenotypic similarity betweenlprand +/+ B cells suggests that the major abnormality oflprB cells may lie in their specificity, that is, in their inability to delete autoreactive subsets.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

Lymphadenopathy induced by the cooperation between lprcg and gld genes is of lpr but not of gld phenotype

Mice homozygous for the lpr (lymphoproliferation), lpr^cg or gld (generalized lymphoproliferative disease) mutation develop strikingly similar lymphadenopathy with expansion of B220⁺ CD4^? CD8^? double-negative (DN) T cells and autoimmunity. To elucidate the roles of bone marrow (BM) and lymph node (LN) in lymphoproliferation, BM and LN were transplanted simultaneously into normal or +/+ mice in various genotype combinations. In lpr/lpr or lpr^cg/lpr^cg BM recipients grafted lpr/lpr and lpr^cg/lpr^cg LN swelled but +/+ and gld/gld LN atrophied. In gld/gld BM recipients all of LN swelled regardless of genotype. Thus, lpr and lpr^cg are phenotypically different from gld in the interaction of BM-derived DN T cells and +/+ LN. Compared with lpr the lpr^cg gene differs in its ability to complement with gld in induction of lymphadenopathy. To determine whether lymphoproliferation induced by the cooperation between lpr^cg and gld is of lpr or gld phenotype, LN of various genotypes were implanted into double heterozygous lpr^cg/+, gld/+ mice. Grafted lpr/lpr and lpr^cg/lpr^cg LN swelled but +/+ and gld/gld LN atrophied, indicating that it is of lpr phenotype. Moreover, grafted lpr^cg/+ LN swelled but lpr/+ LN atrophied, indicating that, in the heterozygous state, lpr^cg is phenotypically different from lpr as it allows for LN accumulation of DN T cells induced by lpr^cg-gld cooperation.     

Thymus-dependent effects of a traditional chinese medicine,ren-shen-yang-rong-tang (Japanese name; Ninjin-Youei-To), in autoimmune MRL/MP-Ipr/Ipr mice

Autoimmune MRL/lpr mice were i.p. treated with 200 mg/kg Ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), a traditional Chinese herbal medicine (Japanese name: Kampo), from 8 weeks of age every 3 days before the onset of autoimmune disease. Compared to age-matched control MRL/lpr mice, the serum IL-6 concentration in NYT-treated mice was decreased, their serum IFN-γ concentration was increased, and the proliferative responses of whole and enriched CD4⁺ cells in their spleen and lymph nodes stimulated with ConA in vitro were restored. FACS analysis revealed that the rate of decreased CD4⁺CD8⁺ T-cell population in the thymus was decreased in MRL/lpr mice but recovered by NYT treatment. Further, adult thymectomized (ATX) MRL/lpr mice were treated with 200 mg/kg NYT similarly. NYT treatment prolonged the survival of sham-operated MRL/lpr mice and ameliorated their proteinuria but did not improve such autoimmune manifestations in ATX-MRL/Ipr mice. These findings suggest that NYT plays an important role in the abrogation of autoimmune-prone T cell differentiation and that the therapeutic effect of NYT is dependent on the thymus in MRL/Ipr mice.