Conservation of pathogenic TCR homology across class II restrictions in anti-ribonucleoprotein autoimmunity: extended efficacy of T cell vaccine therapy.

T cells have been shown to mediate aspects of anti-ribonucleoprotein (RNP) autoimmunity, and are a potential target of therapy in lupus and related diseases. In this study, we assessed the relevance of a conserved class of anti-RNP T cells to autoimmune disease expression and therapy. Our data show that anti-RNP T cell selection induced a limited set of homologous CDR3 motifs at high frequency. Homologous CDR3 motifs have been reported in other autoimmune diseases. Vaccination with irradiated anti-RNP (but not anti-tetanus toxoid) CD4(+) cells induced remission of anti-RNP-associated nephritis in ≥ 80% of treated mice, even with donor/recipient MHC class II mismatch, and in both induced and spontaneous autoimmunity. Vaccine responder sera inhibited anti-70k T cell proliferation and bound hybridomas expressing the conserved CDR3 motifs. Our data indicate that a limited set of TCR CDR3 motifs may be important for the pathogenesis of anti-RNP lupus and other autoimmune diseases. The ability to target a consistent set of pathogenic T cells between individuals and across class II restrictions may allow for the more practical development of a standardized anti-RNP T cell vaccine preparation useful for multiple patients.

Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-transfer RNA synthetase-induced myositis.

We have previously shown that i.m. administration of bacterially expressed murine histidyl-tRNA synthetase (HRS) triggers florid muscle inflammation (relative to appropriate control proteins) in various congenic strains of mice. Because severe disease develops even in the absence of adaptive immune responses to HRS, we sought to identify innate immune signaling components contributing to our model of HRS-induced myositis. In vitro stimulation assays demonstrated HRS-mediated activation of HEK293 cells transfected with either TLR2 or TLR4, revealing an excitatory capacity exceeding that of other bacterially expressed fusion proteins. Corresponding to this apparent functional redundancy of TLR signaling pathways, HRS immunization of B6.TLR2(-/-) and B6.TLR4(-/-) single-knockout mice yielded significant lymphocytic infiltration of muscle tissue comparable to that produced in C57BL/6 wild-type mice. In contrast, concomitant elimination of TLR2 and TLR4 signaling in B6.TLR2(-/-).TLR4(-/-) double-knockout mice markedly reduced the severity of HRS-induced muscle inflammation. Complementary subfragment analysis demonstrated that aa 60-90 of HRS were absolutely required for in vitro as well as in vivo signaling via these MyD88-dependent TLR pathways--effects mediated, in part, through preferential binding of exogenous ligands capable of activating specific TLRs. Collectively, these experiments indicate that multiple MyD88-dependent signaling cascades contribute to this model of HRS-induced myositis, underscoring the antigenic versatility of HRS and confirming the importance of innate immunity in this system.

T cell vaccination therapy in an induced model of anti-RNP autoimmune glomerulonephritis.

To establish the relevance of targeting disease-associated T cells in anti-RNP-associated glomerulonephritis, mice developing nephritis following immunization with U1-70-kd small nuclear ribonucleoprotein (snRNP) were treated with a single dose of irradiated antigen-selected T cell vaccine. T cell receptor usage in nephritic kidneys revealed oligoclonal use of T Cell Receptor V Beta (TRBV) genes as previously found in spleens and lungs of immunized mice with pulmonary disease. The CDR3 regions from T cell isolates showed sequence homology to those in humans with anti-RNP autoimmunity. Following T cell vaccination, urinalysis returned to normal in 5/7 treated mice (71% response rate) whereas all mock-treated mice continued to have an active urinary sediment (Fisher's Exact p=0.02). An oligoclonal population of T cells homologous to those identified in humans with anti-RNP autoimmunity is implicated in disease pathogenesis, and T cell vaccination is associated with a high rate of clinical improvement in established nephritis.

Tissue targeting of anti-RNP autoimmunity: effects of T cells and myeloid dendritic cells in a murine model.

OBJECTIVE: To explore the role of immune cells in anti-RNP autoimmunity in a murine model of pneumonitis or glomerulonephritis, using adoptive transfer techniques. METHODS: Donor mice were immunized with 50 mug of U1-70-kd small nuclear RNP fusion protein and 50 mug of U1 RNA adjuvant. Whole splenocytes as well as CD4+ cell and dendritic cell (DC) subsets from the immunized mice were infused into naive syngeneic recipients. Anti-RNP and T cell responses were assessed by immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry. Development of renal or lung disease was assessed by histology and urinalysis. RESULTS: Unfractionated splenocytes from donor mice without proteinuria induced predominantly lung disease in recipients (8 [57%] of 14 versus 2 [14%] of 14 developing renal disease; P = 0.046). However, infusion of CD4+ cells from donors without proteinuria induced renal disease more frequently than lung disease (7 [70%] of 10 versus 2 [20%] of 10; P = 0.01); adoptive transfer of RNP+CD4+ T cells from short-term culture yielded similar results (renal disease in 8 [73%] of 11 recipients versus lung disease in 3 [27%] of 11). Cotransfer of splenic myeloid DCs and CD4+ T cells from immunized donors prevented induction of renal disease in all 5 recipients (P = 0.026 versus recipients of fresh CD4+ cells alone), although lung disease was still observed in 1 of 5 mice. Transfer of myeloid DCs alone from immunized donors induced lung disease in 3 (60%) of 5 recipients, without evidence of nephritis. Cotransfer of splenocytes from mice with and those without nephritis led to renal disease in 4 of 5 recipients, without evidence of lung disease. CONCLUSION: These findings indicate that RNP+CD4+ T cells are sufficient to induce anti-RNP autoimmunity, tissue targeting in anti-RNP autoimmunity can be deviated to either a renal or pulmonary phenotype depending on the presence of accessory cells such as myeloid DCs, and DC subsets can play a role in both propagation of autoimmunity and end-organ targeting.

Differential tissue targeting of autoimmunity manifestations by autoantigen-associated Y RNAs.

OBJECTIVE: To assess the Y RNAs, a family of homologous RNAs that bind to the Ro autoantigen, for the ability to contribute to autoimmune disease by activating RNA-responsive Toll-like receptors (TLRs). METHODS: Using cell lines expressing or stably transfected with TLR-3, TLR-7, or TLR-8, we determined the patterns of RNA-specific TLR activation by in vitro transcripts of all of the known murine and human Y RNAs. Next, 8-10-week-old female mice were exposed to a single 50-microg subcutaneous injection of mouse Y1 or mouse Y3 RNA, and the effects were observed. RESULTS: Y RNA family members differed in their TLR reactivities. Both human and mouse Y3 RNAs, but not other human or mouse Y RNAs, prominently induced TLR-3 activation. Although most human and mouse Y RNAs activated TLR-7 efficiently, mouse Y3 RNA and human Y5 RNA did not. Single subcutaneous injections of mice with either mouse Y1 RNA or mouse Y3 RNA induced or inhibited lymphoid infiltrates in different target organs based on the Y RNA and TLR status of the mouse used. Mouse Y1 RNA induced kidney lesions in TLR-3-intact mice but not in TLR-3-knockout mice. In contrast, mouse Y3 RNA treatment was associated with nephritis in TLR-3-knockout mice but not in TLR-3-intact mice. Sialoadenitis developed in untreated TLR-3-/- mice and in TLR-3-/- mice treated with mouse Y3 RNA, but sialoadenitis was not present in TLR--/-) mice treated with mouse Y1 RNA. CONCLUSION: Y RNAs can induce innate immune responses and influence clinical manifestations of autoimmunity, suggesting that they are relevant to syndromes of anti-Ro autoimmunity. Distinct patterns of tissue targeting can be seen after exposure to different Y RNAs, in a pattern that correlates with the innate immune signals they induce. Thus, the balance of innate immune signals induced by exposure to endogenous Y RNAs may help determine the nature of the clinical syndrome in anti-Ro autoimmunity.

A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen.

OBJECTIVE: To test whether immunizing mice with autoantigens closely linked to mixed connective tissue disease (MCTD) could induce an MCTD-like clinical syndrome distinguishable from systemic lupus erythematosus (SLE). METHODS: Transgenic and knockout C57BL/6-derived mice were immunized subcutaneously at age 8-12 weeks with U1-70-kd small nuclear RNP (70K) fusion protein along with either Freund's complete adjuvant (CFA) or U1 RNA. After 2 months, mice were killed and analyzed histologically and serologically. RESULTS: Immunization of C57BL/6-derived mice transgenic for human HLA-DR4 with 70K and either CFA or U1 RNA led to anti-70K antibodies in 62% of mice (21 of 34), and diversified anti-RNP immune responses. MCTD-like lung disease also developed in 50% of immunized mice (17 of 34), and anti-70K antibodies were strongly correlated with lung disease. CFA and U1 RNA were comparably able to induce this syndrome. Mice deficient in Toll-like receptor 3 (TLR-3) also developed this same syndrome when immunized with 70K and CFA. However, TLR-3(-/-) mice failed to develop MCTD-like lung disease when treated with 70K and U1 RNA. Rather, TLR-3(-/-) mice immunized with 70K and U1 RNA developed an autoimmune syndrome characterized by glomerulonephritis typical of SLE. CONCLUSION: Exposure to 70K in an appropriate context is sufficient to induce autoimmunity and target organ injury consistent with MCTD. This system represents a new model of autoimmune interstitial lung disease, and establishes a closer link between anti-70K immunity and MCTD-like lung disease. Of note, changes in innate immune signaling can cause the same trigger to lead to the development of SLE-like nephritis rather than MCTD-like lung disease.

Development of albuminuria and glomerular lesions in normoglycemic B6 recipients of db/db mice bone marrow: the role of mesangial cell progenitors.

The pathologic hallmarks of diabetic nephropathy are excess mesangial extracellular matrix (ECM) and mesangial cell proliferation. We previously showed that mesangial cell phenotypic changes play an important role in the pathogenesis of diabetic nephropathy. We concluded that phenotypic changes were present in bone marrow (BM)-derived mesangial cell progenitors, as transplantation of BM from db/db mice, a model of type 2 diabetic nephropathy, transferred the db genotype and a nephropathy phenotype to naive B6 mice recipients. The recipients did not develop diabetes; however, they did develop albuminuria and glomerular lesions mirroring those in the donors (i.e., glomerular hypertrophy, increased ECM, and increased cell number with cell proliferation). We found that matrix metalloproteinase 2 (MMP-2) facilitated invasion of the mesangial cells into ECM and proliferation in vitro. Thus, increased MMP-2 activity in db/db mesangial cell progenitors may partially explain increased mesangial cell repopulation and proliferation in B6 recipients of db/db BM. In summary, BM-derived mesangial cell progenitors may play a crucial role in the development and progression of ECM accumulation and mesangial cell proliferation in this model of diabetic nephropathy in type 2 diabetes.

OX40 ligation enhances primary and memory cytotoxic T lymphocyte responses in an immunotherapy for hepatic colon metastases.

Previously, we showed that the eradication of murine hepatic metastatic colon carcinomas was achieved by using a combination therapy with adenovirus-mediated gene delivery of interleukin 12 (IL-12) and anti-4-1BB agonistic antibody. However, the therapeutic efficacy was compromised severely when mice with tumors larger than 8 x 8 mm(2) were treated. In this report, we studied the effect of OX40 costimulation through agonistic anti-OX40 in combination with the IL-12 + anti-4-1BB immunotherapy on primary and memory anti-tumor cytotoxic T lymphocyte responses in a large-tumor setting (8 x 8 to 12 x 12 mm(2)). Tumor-infiltrating leukocytes (TILs) isolated from mice treated with the combination therapy of IL-12, anti-4-1BB, and anti-OX40 showed a significantly higher ex vivo direct cytotoxic T lymphocyte (CTL) activity against parental tumors, as compared with those from mice treated with IL-12 and anti-4-1BB. In vivo depletion of CD4(+)T cells during combination therapy significantly decreased the number of tumor-infiltrating CD8(+)T cells and their cytotoxic activity in mice treated with IL-12 + anti-4-1BB + anti-OX40 combination therapy but not in the group treated with IL-12 + anti-4-1BB, which indicated that in vivo OX40 engagement on CD4(+) T cells led to the higher CTL responses observed in animals treated with IL-12 + anti-4-BB + anti-OX40 combination therapy. Furthermore, the combination therapy of IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in treated mice, as compared with treatment with IL-12 and anti-4-1BB. More importantly, long-term surviving mice from the combination therapy with IL-12, anti-4-1BB, and anti-OX40 exhibited higher memory CTL responses against parental tumor cells. The results demonstrated that OX40 ligation of CD4(+) T cells facilitated the development of primary and memory CTL responses against tumorcells and that coordinated immune activation by IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in mice with large tumor burdens. Thus, the combination therapy with the adenovirus encoding IL-12 (Adv.mIL-12) + anti-4-1BB + anti-OX40 antibodies may provide a better treatment modality for patients with advanced cancers, often associated with a state of immune suppression or tolerance.