T-cells recognize particular peptide/MHC antigenic complexes through expression of clonally distributed T-cell receptor (TCR) molecules generated by somatically rearranged gene sequences. In addition to those derived from infectious pathogens, APC also display to T-cells MHC class I- and class II-bound peptides generated from endogenous “self” proteins. A normal consequence of expressing a TCR that engages such “self-antigenic” complexes on APC at a critically high threshold level is the induction of signals triggering the deletion or permanent inactivation of potentially autoreactive T-cells, a process termed immunological tolerance induction. Defects in such immunological tolerance induction processes underlie susceptibility to T1D.
In both humans and NOD mice unusual MHC class II genes clearly contribute to T1D by inducing pathogenic CD4 T-cell responses. However, while representing common variants shared by many strains lacking autoimmune proclivity, the MHC class I molecules expressed by NOD mice also exert pathogenic functions essential to T1D development. These common class I variants aberrantly exert diabetogenic functions in NOD mice through interactions with some of the multiple Idd genes located outside the MHC. Previous combined linkage and congenic truncation analyses indicated a polymorphic gene(s) within a 5.4Mb region on Chromosome (Chr.) 7 co-localizing with the previously identified Idd7 locus exerts the strongest interactive effect in determining the extent to which MHC class I dependent diabetogenic T-cells undergo deletion during their development in the thymus. The combined approaches of congenic truncation analyses and mRNA transcript profiling have now identified a hyper-expression of the NFkB inhibitory Nfkbid gene to be a strong candidate within the Idd7 region for interactively determining the extent to which diabetogenic MHC class I restricted CD8 T-cells undergo thymic deletion. The candidacy of Nfkbid is bolstered by previous reports that induction of high levels of NFkB activity are required to trigger the thymic deletion of T-cells differentiating into the MHC class I dependent CD8 lineage. Thus, both gene knockout and transgenic strategies are being employed to test the hypothesis that a hypermorphic Nkbid variant contributes to impaired thymic deletion of diabetogenic MHC class I dependent CD8 T-cells in NOD mice. Potential translation significance of these efforts is provided by recent evidence that the human Chr. 19q.13 region which is syntenic to the murine Idd7 locus contains a gene(s) contributing to T1D susceptibility or resistance.
It is now known that in humans some common MHC class I variants such as HLA-A2.1 also contribute to T1D development when co-expressed with particular combinations of other genes. To initially test this possibility, we generated NOD mice transgenically expressing human HLA-A2.1, but no murine class I molecules (designated NOD.ß2mnull.HHD). The human HLA-A2.1 molecules in these mice mediate diabetogenic CD8 T-cell responses. With collaborators at the Albert Einstein College of Medicine, we determined the ß-cell proteins insulin (INS) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) each contain two peptide epitopes primarily targeted by diabetogenic HLA-A2.1-restricted T-cells in NOD.ß2mnull.HHD mice. Other investigators subsequently verified the INS and IGRP antigens we identified in NOD-HLA-2.1 transgenic mice are also targeted by autoreactive CD8 T-cells in human T1D patients expressing this class I variant.
T-cell tolerance can be efficiently induced to peptides coupled to syngeneic splenocytes by the cross-linking agent ethylenecarbododiimide (ECDI). Furthermore, the administration of autologous leukocytes bearing various ECDI coupled self-antigenic peptides is in clinical trials as a potential intervention for autoimmune mediated multiple sclerosis (MS). We previously found treatment with autologous leukocytes bearing ECDI bearing HLA-A2.1 restricted insulin and IGRP peptides inhibited progression to overt T1D in NOD.ß2mnull.HHD mice by attenuating pathogenic CD8 T-cells targeting these epitopes. However, there are significant hurdles to the clinical use of cell-based therapies. Another major clinical translation issue is at present humans a high future risk for T1D can only be identified after they have already developed high levels of ongoing pancreatic ß cell autoimmunity evidenced by the presence of markers such as insulin autoantibodies (IAA). Thus, to currently be clinically translatable, a potential T1D intervention must be effective when initiated at a late prodromal stage of disease development. A recent study found that immunological tolerance can also be induced to antigens that have been ECDI coupled to a type of synthetic microbeads already approved for clinical use in humans. We now have evidence that treatments with such microbeads bearing ECDI cross linked INS and IGRP peptides provides an effective means to inhibit progression to overt T1D development when initiated in older NOD.ß2mnull.HHD mice that have already become IAA positive. Efforts are ongoing to determine the combination of INS and/or IGRP peptides that must be ECDI coupled to microbeads to exert late disease stage T1D prevention effects in humanized NOD.ß2mnull.HHD mice.
Given they provide the expression site for the MHC class II molecules that are essential to pathogenesis, it is not surprising that APC functions play a key role in T1D development. Previous work indicated populations of B-lymphocytes expressing plasma membrane bound immunoglobulin (Ig) molecules specific for various pancreatic ß-cell proteins are a critical subtype of APC for T1D development in NOD mice, and also likely human patients. The expression of such autoreactive Ig molecules allows B-lymphocytes to more efficiently capture ß-cell autoantigens for subsequent MHC class II mediated display to diabetogenic CD4 T-cells than other APC subtypes.
Mechanisms are normally in place to prevent the development or functional activation of B-lymphocytes expressing autoreactive Ig molecules. We have now localized to a 6.2 Mb region on Chr. 4 a gene(s) within the previously identified over-lapping Idd9/11 loci that contributes to a failure of B-lymphocytes expressing autoreactive Ig molecules to be functionally anergized in NOD mice. Combined congenic truncation and mRNA transcript expression analyses have identified the Ephb2 and Padi2 genes located within this 6.2 Mb region on Chr. 4 to be strong candidates for contributing to the failure of autoreactive B-lymphocytes in NOD mice to be functionally anergized. Of potential translational significance, Ephb2 was found to possibly function in a Mapk3 (aka Erk1) centric lymphocyte activation pathway also influenced by a subset of GWAS identified candidates for T1D susceptibility genes in humans. The NOD Ephb2 gene is expressed at lower levels in B-lymphocytes than the differing allelic variant characterizing a closely related, but T1D resistant control strain. Thus, we are pursuing studies to determine if transgenically increasing Ephb2 expression levels in B-lymphocytes exerts a T1D protective effect in NOD mice. Such a finding would strongly support the possibility that Ephb2 is a Idd9/11 region gene contributing to the development of diabetogenic B-lymphocytes in NOD mice.
Given their significant pathogenic role, depletion of B-lymphocytes by the CD20 specific rituximab antibody is being tested as a possible T1D intervention approach in humans. Early results indicate that anti-CD20 treatment retards the rate, but does prevent the further erosion, of residual pancreatic ß-cell mass in T1D patients. We have also found that anti-CD20 treatment only inhibits progression to overt T1D development in NOD when initiated before, and not after the appearance of IAA. This appears to be related to our novel observation that B-lymphocytes in NOD mice rapidly down-regulate CD20 expression upon entry into pancreatic islets. These collective results indicate that B-lymphocyte targeting strategies beyond the use of CD20 specific antibodies be considered as a possible T1D intervention approach. In collaboration with investigators at Medimmune we are currently testing if a pharmacological agent they have developed that blocks activity of the B-lymphocyte survival factor BAFF can independently or in conjunction with anti-CD20 provide an improved means for preventing progression to overt T1D when initiated in NOD mice at a late prodromal stage of disease development than previously employed approaches.
Previous work by other investigators found that unexpectedly a sizeable proportion of B-lymphocytes infiltrating the pancreatic islets of NOD mice express an Ig molecule recognizing the neuronal protein peripherin (PRPH). Recent work from our group has found T1D development is greatly accelerated in NOD mice transgenically expressing a PRPH reactive Ig molecule in virtually all B-lymphocytes. These results indicate that developing means that specifically target PRPH reactive B-lymphocytes might ultimately represent a particularly attractive T1D intervention approach.