95 research outputs found
NZB/NZW F1-Derived Monoclonal Anti-Dna Autoantibodies: Dual-Specificity and Immune Complex Formation
No full textThe glomerulonephritis associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) is mediated by immune complexes composed of DNA, complement components and anti-DNA autoantibodies, with the primary pathogenic antibodies being dsDNA specific. Recent studies had identified a subpopulation of anti-DNA autoantibodies that possessed dual-specificity and appeared to form very avid immune complexes. The dual-specific autoantibodies were derived from the NZB/NZW F1 murine model of lupus. Further characterization of four anti-DNA (two anti-ssDNA and two anti-dsDNA) monoclonal autoantibodies revealed that only the two anti-dsDNA autoantibodies, BV 17-45 and BV 16-13, exhibited dual-specificity (anti-dsDNA and anti-IgG) under native conditions. Temperature-dependent denaturation studies and SDS denaturation/CD analysis identified the IgG epitope recognized by both BV 17-45 and BV 16-13 as non-linear. Enzymatic digests and native Western blot analysis localized the anti-IgG epitope to the hinge region of IgG2a. By utilizing synthetic hinge peptides and a 12 amino acid phage display library, the location of the anti-hinge autoepitope was further defined to the upper hinge region of IgG2a murine immunoglobulin. To examine the role of each specificity (anti-dsDNA and anti-IgG2a hinge) in the formation of immune complexes, affinities and Krel values were determined for both BV 17-45 and BV 16-13 employing various lengths of dsDNA fragments (21bp, 43bp, 84bp, and 114bp) and a synthetic nine amino acid hinge peptide dimer. The results showed that the affinities for both BV 17-45 and BV 16-13 for dsDNA was higher than for the synthetic nine amino acid hinge peptide dimer. It was also observed that the affinities of the two monoclonal antibodies for dsDNA was directly dependent upon the length of dsDNA. This trend was a direct consequence of dual-specificity. The larger dsDNA fragments localized several autoantibody active sites, in close proximity, thereby causing binding to both self (via the hinge specificity) and dsDNA resulting in the formation of avid immune complexes. The overall effect was a significant increase in functional affinity of ∼ 4000 to 8000-fold. A dual-specificity autoimmune complex model was proposed and described to explain the role of the anti-dsDNA and anti-IgG2a specificity in the formation of avid immune complexes.Made available in DSpace on 2015-09-28T15:17:46Z (GMT). No. of bitstreams: 2
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Previous issue date: 1998Embargo set by: Seth Robbins for item 88025
Lift date: Forever
Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDsRestricted to the U of I community idenfinitely during batch ingest of legacy ETDsU of I Only172 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998
Interaction of dual-specific autoantibodies with dsDNA and a synthetic dimer peptide simulating the hinge region of IgG2a molecules
No full textTwo dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region
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Detection of Protease Activity Using a Fluorescence-Enhancement Globular Substrate
No full textBovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase® resulted in fluorescence enhancement of 4300%. Both α-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 × 10−6 units for 1 ng proteinase K, 1 × 10−3 units for 1 ng α-chymotrypsin and 10 × 10−3 units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity
The extent of metalloproteinase-mediated LAG3 cleavage limits the efficacy of PD1 blockade
No full textInhibitory receptors control immune responses preventing exacerbated T cell activation and the onset of autoimmunity; however, they also limit antitumor immunity. Enhanced co-expression of PD1 and LAG3 phenotypically mark functionally exhausted tumor-specific T cells, with dual PD1/LAG3 blockade synergistically limiting tumor growth in murine models. Like PD1, LAG3 expression is induced on activated T cells to negatively regulate T cell activation and proliferation and LAG3 is also required for maximal regulatory T (Treg) cell function. However, LAG3 expression and function is itself regulated by cell surface cleavage of the transmembrane domain connecting peptide by ADAM10 and ADAM17 metalloproteinase-disintegrins. This releases soluble LAG3, of which no biological function has been found to date. To investigate the impact of LAG3 cleavage on T cells within tumors, a non-cleavable LAG3 mouse (LAG3.NC) was generated in which exons 7 and 8 of Lag3, including the connecting peptide, is deleted in Cre-expressing cells. These exons are replaced and modified so that the connecting peptide is absent preventing LAG3 cleavage. LAG3.NC CD4Cre mice (with non-cleavable LAG3 expressed on all CD8+ and CD4+ T cells, including Tregs) and LAG3.NC E8ICre mice (restricted to CD8+ T cells only) exhibit enhanced expression of LAG3 on the respective T cell subsets in B16-F10 or MC38 tumors, co-expressing with PD1. Despite increased LAG3 expression, no difference in B16-F10 or MC38 tumor growth was observed in either LAG3.NC CD4Cre or LAG3.NC E8ICre mice compared with wild-type littermates. Upon therapeutic administration of anti-PD1 antibody (clone G4), MC38 tumor-bearing wild-type mice show significant tumor regression and 40% become tumor-free resulting in long-term survival. LAG3.NC CD4Cre mice were resistant to anti-PD1 therapy and succumb to tumor growth. However, anti-PD1 mediated tumor regression and long-term survival in LAG3.NC E8ICre mice. Analysis of re-stimulated CD8+ TILs isolated from LAG3.NC CD4Cre mice did not show enhanced IFN-gamma and TNF-alpha production following anti-PD1 therapy, which was observed with LAG3.NC E8ICre mice or wild-type littermates. Moreover, reduced proliferation was observed for all T cell subsets in LAG3.NC CD4Cre mice compared with LAG3.NC E8ICre and wild-type littermates following anti-PD1 treatment. As LAG3.NC CD4Cre, but not LAG3.NC E8ICre mice, are resistant to the favorable antitumor immune effects induced by anti-PD1, this suggests that enhanced LAG3 expression on CD4+ T cells or Tregs may act as a barrier to effective anti-PD1 immunotherapy. LAG3.NC mice crossed with Cre that restricts non-cleavable LAG3 to Tregs (Foxp3yfpiCre) or CD4+ T cells (ThPOKCre) are currently under analysis
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