Thanks to the Department of Animal Care for their assistance in maintaining the mice. Anti-insulin B cells can acquire a GC phenotype in vitro in the absence of T cells..86 III-11. The presence of IgG autoAbs in insulin in patients with type 1 diabetes correlates with a role for anti-insulin B cells in the breakdown of tolerance that mediates disease pathogenesis.
B cell central tolerance in the developing repertoire is characterized by receptor editing and clonal deletion (38–41). When expression of autoAg was removed from the bone marrow, the self-reactive B cells were found in the peripheral repertoire. Stem cells in the bone marrow that commit to the T cell lineage enter the thymus to begin the development of T cells.
VH125Tg was also bred into the NOD background to study anti-insulin B cells in the context of a mouse genetically predisposed to develop type 1 diabetes. Since T cell activation is largely dependent on interaction with B cells, loss of B cell tolerance against insulin represents one of the earliest stages in the initiation of autoimmunity. Infection and inflammation may play a major role in breaking tolerance in anti-insulin B cells.
Accordingly, an anti-insulin H chain (VDJH125) was targeted to the H chain locus of B6 mice to generate anti-insulin B cells competent to undergo all native B cell functions, including isotype switch ( VH125SD B6).
Insulin- binding
Anti-insulin B cells can be identified in the spleen of WT recipients after adoptive transfer Anti-insulin B cells in the monoclonal repertoire of VH125SD/Vκ125Tg B6 mice do not undergo developmental arrest and escape to peripheral subsets in the spleen. Therefore, I determined whether anti-insulin B cells could populate the spleen of unirradiated WT B6 mice after adoptive transfer. Recipient WT mice were not irradiated to allow maximal competition between endogenous WT B cells and donor anti-insulin B cells.
One disadvantage of this approach is that non-irradiated recipient mice already have populated peripheries that can significantly limit the ability of donor anti-insulin B cells to settle into niches. Four days later, the presence of IgMa+ anti-insulin B cells in the spleens of recipient mice was determined by flow cytometry (Fig. II-3). Four days after adoptive transfer, a small population of anti-insulin B cells was detected in the spleen of the recipient WT B6 mouse (0.103%).
While the frequency was low and only a fraction of the number of injected cells, the presence of anti-insulin IgMa+ B cells demonstrates that these tolerant B cells actually compete quite well with non-insulin-binding B cells for peripheral residence. This experiment shows that anti-insulin B cells can compete for residence in the periphery of WT mice, revealing that the anti-insulin B-tolerant state does not limit their entry into the competitive repertoire of polyclonal B cells.
Anergy in B cells to insulin is reversed by TLR4 but not by CD40 costimulation in vitro. Impaired BCR-mediated Ca2+ mobilization in anti-insulin B cells is not restored after TLR4 costimulation. Previously, in another model, anti-insulin B cells were observed to have impaired Ca2+ mobilization when stimulated with insulin (102).
Neither stimulation with BRT alone nor the insulin-BRT conjugate restored Ca2+ flux in anti-insulin B cells (Fig. II-6, lower panels). I first wanted to understand the function of anti-insulin B cells in the new BCR Tg mouse model. Surprisingly, LPS plus insulin worked together to promote robust proliferation in anti-insulin B cells (Fig. II-4E).
In addition, different NFATs have different functions, exemplified by the restoration of proliferation in anti-insulin B cells in NFAT1−/− (NFATc2) mice ( 107 ). These data suggest that anti-insulin B cells in the peripheral repertoire of VH125SD B6 mice are predominantly mature follicular B cells and do not undergo developmental arrest. Anti-insulin B cells in the polyclonal repertoire of VH125SD B6 mice do not upregulate activation markers when stimulated with antigen.
These data suggest that anergy in B cells against insulin extends to impaired antigen-specific activation. IgM and IgG antibodies to insulin are produced in VH125SD B6 mice after immunization with TI but not with TD. Antibody responses of IgDa+ anti-insulin B cells in VH125SD B6 mice were assessed following two different immunization strategies.
IgMa+ and IgG+ anti-insulin hybridomas after immunization of VH125SD B6 mice with insulin-BRT. Immunization of VH125SD B6 mice with insulin/CFA failed to elicit IgG anti-insulin antibodies (Fig. III-3A). However, tolerance imposed on anti-insulin B cells in VH125SD B6 mice completely limits their ability to participate in TD immune responses.
Immunization with insulin-BRT promotes clonal expansion and restoration of surface IgM for anti-insulin B cells. The restoration of IgM on the surface is consistent with the return of anergy in the anti-insulin B cells of the CSR.
IgMa
Insulin-BRT has previously been used to detect rare anti-insulin B cells in the repertoires of normal mice independent of T cell help (95). Similarly, B cell anergy to insulin is reversed by BCR/TLR costimulation in an insulin-BRT response that is relatively TI. We present evidence that B cell anti-insulin defiance can occur in the absence of normal T cell help.
Can insulin BRT-primed anti-insulin B cells respond to TD immunization or T cell help in vitro? The loss of anergy provided by BCR/TLR costimulation stimulates the clonal expansion of anti-insulin B cells. Anergy in anti-insulin B cells is reversed by TLR-4, but not by CD40 costimulation in vitro.
These data indicate that anti-insulin B cells in the peripheral repertoire of VH125SDB6 mice are mainly mature FO B cells. Accordingly, we assessed whether the observed impairment of peripheral tolerance for B cells to insulin in response to insulin-BRT extends to GC formation. These data indicate that anti-insulin B cells acquire a GC phenotype and localize to GC structures in the spleen after immunization with insulin-BRT.
Insulin-BRT has previously been used to uncover rare anti-insulin B cells in the repertoires of normal mice, independent of T cell help ( 33 ). Similarly, anergy in anti-insulin B cells is reversed by BCR/TLR costimulation in the insulin-BRT response that is relatively TI. The small population of anti-insulin B cells present in the mature VH125Tg/NOD B cell repertoire is sufficient to promote T1D ( 12 , 25 ).
The increased frequency of anti-insulin B cells is first seen among immature B cells in the BM sinusoids. Anti-insulin B cells remain in the repertoire throughout development and are enriched in the pancreas of VH125TgNOD mice. The presence of anti-insulin B cells in. reactive with human insulin was identified by ELISA.
Anti-insulin B cells that escape central tolerance are enriched at the site of autoimmune attack.
