Irina Fernandez

CD101 Surface Expression Discriminates Potency Among Murine FoxP3+ Regulatory T Cells

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25–30% of CD4+CD25+ Treg and ∼20% of conventional memory T cells. CD101high Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101low Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101high Treg, compared with CD101low Treg. Moreover, treatment with CD101high Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101high Treg all of the in vivo suppressor activity was contained within the CD62Lhigh subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.

Characterization of TLR4-mediated auto-antibody production in a mouse model of histidyl-tRNA synthetase-induced myositis.

We have previously shown that intramuscular immunization with a recombinant fragment of murine histidyl-tRNA synthetase (HRS) in the absence of exogenous adjuvant generates Ag-specific, IgG class switched Abs a murine model of myositis. Markedly diminished IgG anti-HRS auto-Ab responses in TLR4 signaling-deficient C3H/HeJ mice indicate that TLR4 is required for auto-Ab formation and/or class switching in this system. Comparative time course assessment of HRS-immunized C3H/HeOuJ (wild type) and C3H/HeJ (TLR4 mutant) mice shows here that despite significant impairment of class switched IgG anti-HRS responses in TLR4-deficient C3H/HeJ mice, production of IgM anti-HRS auto-Abs is relatively preserved—suggesting that TLR4-mediated signals modulate IgG class switching rather than auto-Ab formation in this genetic background. In C57BL/6-derived knockout mice lacking either MyD88 (B6.MyD88−/−) or TRIF (B6.TRIF−/−) adaptor molecules, immunization studies indicate that TRIF exerts a dominant role in the generation of HRS-specific IgG auto-Abs. Complementing these analyses, in vitro stimulation of unfractionated, as well as T cell-depleted, C3H/HeOuJ splenocytes with recombinant murine HRS reveals that TLR4-mediated generation of class switched auto-Abs can occur independently of T cell help. Overall, these findings support a broader role for TLR4 in the breakdown of immune tolerance and development of autoimmunity.

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.

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.

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.

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 (Fishers 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.

An Autoimmune Basis for Raynaud's Phenomenon: Murine Model and Human Disease

Raynauds phenomenon (RP) is common in anti‐RNP–positive patients with rheumatic diseases but is not itself known to be caused by autoimmunity. The aim of this study was to assess autoantibodies that could mediate this process.

P.14.9 Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-tRNA synthetase-induced myositis

We have previously shown that intramuscular 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 WT 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 amino acids 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.