TIM-1 signaling is required for maintenance and induction of regulatory B cells.

Apart from their role in humoral immunity, B cells can exhibit IL-10-dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain-1 (TIM-1) identifies over 70% of IL-10-producing B cells, irrespective of other markers. We now show that TIM-1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM-1 lacking the mucin domain (TIM-1(Δmucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Δmucin) mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL-10(+) Bregs, since a single transfer of WT TIM-1(+) B cells can restore long-term graft survival. These data suggest that TIM-1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM-1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM-1 signaling.

Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis.

Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1(-/-) hosts repopulated with Bim(-/-) conventional CD4(+) T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1(-/-) hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.

Interaction of dual-specific autoantibodies with dsDNA and a synthetic dimer peptide simulating the hinge region of IgG2a molecules.

Anti-dsDNA autoantibodies and immune complex formation are major factors in SLE pathogenesis. Understanding stable immune complex formation is critical in deciphering mechanisms of autoimmune pathogenesis. Previous studies identified a subpopulation of murine lupus monoclonal autoantibodies that exhibited dual specificity (anti-DNA and anti-IgG2a hinge) and formed stable immune complexes [J. Mol. Rec. 10(1997)225]. Two monoclonal autoantibodies, BV 17-45 and BV 16-13, were extensively studied because of their dual specificity. To quantitatively assess the role of each specificity in the formation of stable immune complexes, studies were performed to determine binding affinities for various sized dsDNA fragments (21, 43, 84, and 114 bp) and the covalent dimer of a nine amino acid hinge peptide. Results characterizing BV 17-45 showed that the affinity for dsDNA directly correlated with increased dsDNA size. Results with BV 16-13 revealed a generally lower affinity for the various dsDNA fragments. Binding inhibition studies, using a covalently linked dimer of a nine amino acid synthetic hinge peptide as an inhibitor of the antibody-43 bp dsDNA interaction, yielded relative affinities for the anti-hinge activity. Binding affinities for the synthetic hinge specificity were lower than affinities measured for the anti-dsDNA activity. Collectively, the binding and inhibition studies provided insight into the correlation between dual specificity and avid immune complex formation. A model was proposed based on the concept that large dsDNA fragments caused localization of the dual-specific antibodies through the anti-dsDNA activity, thereby facilitating subsequent binding and cross-linkage via the anti-hinge specificity. These synergistic interactions resulted in the formation of avid immune complexes.

Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region.

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17-45 and BV 16-13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17-45 and BV 16-13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25-100 degrees C suggested that the epitope recognized by BV 17-45 was the same as the IgG epitope recognized by BV 16-13. Further studies with papain- and pepsin-generated F(ab')2, Fab, and Fc fragments of BV 17-45 and BV 16-13 revealed that the dual-specific autoantibodies BV 17-45 and BV 16-13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.

Dual specificity and the formation of stable autoimmune complexes.

Since dual specificity at the antibody active-site level involves new principles relative to monospecific antigen-antibody interactions and may be a general property of autoantibodies, it was important to further characterize such antibodies. Four lupus derived autoantibodies were studied to understand parameters and mechanisms involved in the participation of dual-specific antibody molecules in the formation of highly stable immune complexes. Because the dual-specific binding properties of selected lupus-related murine autoantibodies had been previously described using a solid-phase polystyrene-based ELISA, a conformational sensitive membrane based assay (CSI) was used on a comparative basis to further characterize NZB/NZW F1 murine monoclonal anti-DNA autoantibodies BV 04-01 (anti-ssDNA), BV 16-19 (anti-ssDNA), BV 17-45 (anti-dsDNA), and BV 16-13 (anti-dsDNA). All four monoclonal autoantibodies exhibited anti-IgG binding in the solid-phase ELISA. However in the CSI assay, only anti-dsDNA monoclonal autoantibodies BV 17-45 and BV 16-13 demonstrated anti-IgG binding, while anti-ssDNA autoantibodies BV 04-01 and BV 16-19 did not. Upon subjection to time-dependent thermal denaturation, with and without thiol reduction at 100 degrees C in the CSI, the self-binding activities of BV 17-45 and BV 16-13 were abrogated demonstrating that the recognized IgG autoepitope(s) possessed conformational or discontinuous three-dimensional properties. The immunological implications of dual specificity are discussed on a structure-function basis and its correlation with formation of pathogenic immune complexes.

Detection of protease activity using a fluorescence-enhancement globular substrate.

Bovine 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 alpha-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 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 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.