Using four independent Idd10 haplotypes, NOD, CAST, B6, and A/J, we have demonstrated correlations between T1D susceptibility and 1) CD101 protein sequence variants and variants that potentially influence the expression of Cd101, 2) decreased CD101 expression on FoxP3+ CD4 T cells, 3) increased expression of CD101 on CD4+ CD11b+ CD11c+ dendritic cells and 4) decreased CD101 expression on Gr1+ myeloid cells. The bone marrow cells of the T1D susceptible NOD and NOD.CAST Idd10 strains also had a lower percentage of Gr1+ cells as compared to the two congenic strains protected from T1D, NOD.B6 Idd10 and NOD.A/J Idd10. Our data are consistent with the hypothesis that genetic variation of Cd101 is responsible for mediating Idd10’s T1D-modifying effects.
CD101 expression differences between susceptible and protective Cd101
haplotypes are modest, which is consistent with most variant genes that contribute to complex traits such as autoimmune disease where a 2-fold difference in expression or function of a disease associated protein is considered large (17
). It is also notable that the direction of the expression differences is not the same in all tissues and in some cell types no difference in CD101 expression has been observed. This suggests that regulation of CD101 expression is mediated at least in part by tissue-specific transcription factors or that an extrinsic effect of Cd101
haplotypes influences CD101 expression in other cell types in a complex manner. Detailed analyses of mixed bone marrow chimeras will be required to determine if CD101 expression differences are extrinsic or intrinsic to each cell type.
In comparing the Cd101
genomic sequence of the two T1D-susceptible haplotypes, NOD and CAST, with the two T1D-protective haplotypes, B6 and A/J, the number of disease associated SNPs in Cd101
was reduced from 125 (B6/NOD variation only) to 62 disease associated SNPs (). Notably, only 4 of the 10 B6/NOD amino acid differences previously reported (1
) were shown to be disease associated in the haplotype analysis (); the highest density of disease-associated SNPs was found in introns 3, 4 and 5 (). Although no disease-associated Cd101
SNPs alter nucleotides known to be critical for splicing (consensus sequences at the donor, acceptor and branch sites), it is possible that the observed disease-associated CD101 expression changes are caused by one or more SNPs altering splicing efficiency and therefore total protein production. Mechanisms that could account for a reduction in splicing efficiency include one or more causal SNPs altering intronic or exonic splicing enhancer or silencer motifs as well as SNPs changing the secondary structure of the pre-mRNA in a manner by which the ordered removal of introns is made less efficient. Alternatively, disease-associated SNPs could alter the efficiency of Cd101
pre-mRNA transcription by influencing regulatory regions in the 5’ and 3’ UTR as well as transcriptional enhancer regions that are present in introns. The additional complexity of genotype-dependent cell type-specific expression seen in this and a related study (6
) suggests the possibility that the binding efficiency of tissue-specific transcription factors that influence splicing/transcription are influenced by polymorphisms that define distinct Cd101
haplotypes. Although none have been described, CD101 isoforms created by alternative splicing could be expressed in a genotype-dependent manner. However, the CD101-specific monoclonal antibody used in the current study could fail to recognize one or more of the putative isoforms if the epitope recognized is missing due to splicing out of the sequence encoding the epitope or there is a conformational change induced by the deletion of nearby amino acids. If this scenario is true, we could be underestimating the differential expression of CD101. Although we favor the hypothesis that the disease-causing sequence variation in Cd101
is causing an alteration in gene expression, it is conceivable that the four disease-associated amino acid changes in CD101 () are causal, and the functional difference mediated by changing one or more of these amino acids drives the observed expression differences.
Results from B6 CD101 knockout mice support the hypothesis that CD101 expression changes in Idd10 congenics strains are directly functional: in both models reduced CD101 expression on myeloid cells in the bone marrow is correlated with a reduction in the percentage of Gr1+ bone marrow cells. Since in the CD101 KO model a change of amino acids is not required for the phenotypic change in the Gr1+ bone marrow population, this argues that it is the CD101 expression change in the Idd10 congenic mice that influences the generation of Gr1+ cells. The role of CD101 in myeloid cell development could extend beyond Gr1+ cells; myeloid lineage cells in the bone marrow are progenitors of multiple cell subsets in the periphery, including dendritic cells, macrophages, myeloid suppressor cells and granulocytes. The detailed effect of Cd101 genotype on various precursor populations within the bone marrow and peripheral cell subsets awaits further definition, as does the question of how CD101 contributes to the generation of Gr1+ cells in the bone marrow. It is possible that CD101 influences the efficiency of maturation of Gr1+ cells via signaling or through CD101-mediated cell-cell interactions.
Although the data present do not prove the hypothesis that Cd101
is the Idd10
gene influencing the development of T1D, the observed differential expression of CD101 on cell types important in immune regulation invites speculation on potential causal pathways influenced by the Idd10
antigen presenting cells and FoxP3+
Tregs are known to be essential for eliciting (28
) and regulating (17
), respectively, the autoimmune response to islets. It is important to consider that these same cell types may or may not contribute to N. aro
-mediated liver autoimmunity described by Mohammed et al
). As opposed to T1D pathogenesis, there is a likely causal role for differentially-expressed CD101 on Gr1+
cells in a disease process that is partially dependent on the clearance rate of infectious bacteria.
As is the case for CD101, expression of most immune molecules is not restricted to one subset of cells. In regard to increased expression of CD101 on FoxP3+
CD4 T cells found in mice having the disease-protective genotypes, it is notable that the expression of CD101 on FoxP3+
CD4 T cells is correlated with an increased ability to suppress effector T cells (3
). Punkosdy et al
) recently demonstrated that CD101 expression on FoxP3+
Tregs is greatly increased during a chronic viral infection extending the observation that there is dynamic regulation of CD101 on this T cell subset. Multiple studies have demonstrated that FoxP3+ CD4 T cells can suppress T1D in the NOD model (17
). It is tempting to speculate that NOD.B6 Idd10
and NOD.A/J Idd10
CD4 T cells have an increased ability to mediate suppression since they express higher levels of CD101 on this important regulatory cell population as compared to NOD and NOD.CAST Idd10
Although it is likely, as argued above, that the genetic variation in CD101 accounts for T1D susceptibility, we have also demonstrated that other genes within the ~800 kb Idd10
region have potentially causal disease-associated sequence differences; thus, the gene responsible for the phenotypic effects of Idd10
must still be considered to be unknown. No genes in the syntenic region on human Chromosome 1p13-1p12 show evidence of association with T1D, including CD101
). We are currently developing a NOD.B6 Idd10
mouse lacking CD101 expression by backcrossing B6 CD101−/−
mice to the NOD parental strain. If variants of Cd101
do indeed modify T1D progression by altering CD101 expression or function, a prediction would be that the frequency of T1D in NOD.B6 Idd10
mice would differ from that of NOD.B6 Idd10
mice. Furthermore, if the reduction of CD101 expression on cell types such as Tregs contributes to the susceptibility phenotypes of the NOD and CAST Idd10
haplotypes, we would predict that NOD.B6 Idd10
and NOD.B6 Idd10
mice will be more susceptible to T1D than NOD.B6 Idd10
mice. However, if the increased expression of CD101 on a subset of CD11c+
cells which is observed in mice having a susceptible Idd10
haplotype is causal for T1D susceptibility, an opposite prediction would be made: NOD.B6 Idd10
mice would be expected to be protected from T1D. If CD101 expression on both Tregs and CD11c+
cells contribute to T1D pathogenesis, the disease status of NOD.B6 Idd10
mice would be difficult to predict.
The importance of CD101 in the immune response has been recently demonstrated in a model of infection-induced liver autoimmunity where disease can be induced on both the B6 and NOD backgrounds (6
). NOD Idd10
congenic mice having the B6 and A/J T1D-protective haplotypes showed delayed clearance of Novosphingobium aromaticivorans
in the liver and subsequently developed more severe liver autoimmunity than NOD and NOD.CAST Idd10
mice. On the B6 background, reduction of CD101 expression decreased bacterial clearance in the liver following infection and increased the autoimmune response.
Further elucidation of the roles of particular cell subsets expressing CD101 in T1D and infection-induced liver autoimmunity will increase our understanding of the cellular functions that are modulated by this molecule. It is likely that the pleiotropic expression and function of CD101 during immune homeostasis and immune challenge contributes to a complex functional balance of innate and adaptive effector and regulatory cells populations, a balance that likely differs depending on the target tissue. Thus, it would not be surprising if the pivotal CD101-expressing cell type in T1D differs from that critical for influencing the progression of infection-induced liver autoimmunity.