In this study, we have performed haplotype mapping of Idd5.1 and have generated new Idd5.1 congenic and NOD liCTLA-4 tg transgenic mice to address the genetic and functional role of Ctla4 as the causative gene in Idd5.1 that regulates susceptibility to T1D. Through haplotype analysis, we discovered that the SWR haplotype is NOD-like at most of the sequence polymorphisms in and near Ctla4, including the exon 2 SNP at residue 77 that is associated with changes in liCTLA-4 expression, but is B10-like at Icos, including a SNP in Icos exon 1 that causes a non-conservative amino acid change in the putative leader sequence. Since alterations in the expression of either liCTLA-4 or ICOS could conceivably alter the T1D frequency, the hybrid SWR haplotype allowed us to test whether the T1D protection conferred by the B10 Idd5.1 haplotype would be lost or retained in the SWR Idd5.1 haplotype. In a frequency study, the SWR Idd5.1 haplotype was shown to be unable to protect NOD mice from T1D, clearly in support of the hypothesis that increased liCTLA-4 expression in the B10 haplotype is required for T1D protection.
In contrast to the SWR haplotype, the CAST Idd5.1
haplotype was shown to be protective, which is consistent with its high liCTLA-4 expression () and again supports the hypothesis that increased liCTLA-4 protects from T1D. However, since with further sequencing, we discovered that the CAST haplotype had additional CAST-specific SNPs in Ctla4
, our study took on an added dimension. We propose that the additional 2 CAST-specific SNPs in exon 2 of Ctla4
are responsible for even higher expression of liCTLA-4 as compared to the protected B10 haplotype () and that the molecular basis for this increase is probably due to alteration of exon splicing enhancer motifs (Supplemental Figure 2
). The selection and retention of a second, distinct haplotype that produces high levels of liCTLA-4 suggests that an important function for this isoform has evolved in mice. On a practical level, the CAST allele at Ctla4
is a tool, that in conjunction with the B10 and NOD alleles, creates a hierarchy of liCTLA-4 expression levels with over a 10-fold difference at the mRNA level between the highest and lowest producing alleles. Although the CAST and B10 Idd5.1
haplotypes have yet to be compared directly in a T1D frequency experiment, the increased expression of liCTLA-4 in activated splenocytes from NOD.CAST Idd5.1
congenic mice, as compared with splenocytes from NOD.B10 Idd5.1
mice, is correlated with an increased level of protection from T1D by the CAST versus B10 Idd5.1
alleles, since (NOD.CAST Idd5.1
x NOD) F1 mice remain protected from T1D () whereas (NOD.B10 Idd5.1
x NOD) F1 are not (31
). Future experiments addressing the downstream molecular and cellular events determined by differing levels of liCTLA-4 should benefit from a comparison of Idd5.1
congenic strains. The apparent ability of exonic sequences to alter splicing preferences in the case of liCTLA-4 can also be made use of to model the expression changes by developing exon 2 knock-in mice having the CAST, B10, or NOD exon 2 SNPs.
Since in addition to Ctla4 and Icos, Pard3b and Nrp2 are within the Idd5.1 interval, we generated liCTLA-4 transgenic mice on the NOD background to test the hypothesis that increased liCTLA-4 expression is sufficient to explain the protective effect of the B10 Idd5.1 allele on the frequency of type 1 diabetes. Indeed, when liCTLA-4 is expressed transgenically, it confers protection from diabetes as compared to non-transgenic littermates and with an incidence remarkably similar to NOD.B10 Idd5.1 congenic mice (). Together with the haplotype analyses in the congenic strains discussed above, these positive results from mice with transgenic overexpression of liCTLA-4 make it unlikely that non-NOD alleles at any of the other Idd5.1 region genes are required to regulate the diabetes phenotype.
liCTLA-4 has been hypothesized to have a major role in the downregulation of T-cell responses (19
). Our present data demonstrate that NOD T-cells secrete high levels of IL-17 upon anti-CD3 stimulation, which is reduced by expression of the liCTLA-4 transgene (). IFN-γ levels are also decreased in the liCTLA-4 transgenic NOD mice. In addition to increased IL-17 production by NOD T cells, it has also been hypothesized that NOD mice have a defect in the regulatory T cell repertoire resulting from abnormalities in thymic selection (45
). Since liCTLA-4 has been shown to be highly expressed in the activated/memory T cells of diabetes resistant strains (21
), higher levels of liCTLA-4 may function to reduce effector and memory cell function presumably by raising the activation threshold of such cells. The naturally high expression of liCTLA-4 on activated/memory T cells of mice having a resistance allele at Ctla4
may serve to keep the immune system from responding following exposure to weak antigens or low affinity self-antigens, thus providing an important checkpoint for inhibiting T cell activation and maintaining self-tolerance.
We have also demonstrated that transgenic expression of liCTLA-4 even in the absence of flCTLA-4 is capable of negatively regulating T cell activation in vivo
. However, a functional signaling domain alone is not sufficient to completely reverse the autoimmunity and inflammation present in CTLA4-/-
mice (Figs. and ). Chikuma et al
) reached a similar conclusion when they studied CTLA4-/-
mice receiving a CTLA-4 transgene mutated in the B7 binding domain. CTLA-4-/-
T cells are spontaneously activated and readily secrete massive amounts of cytokines such as IFN-γ□ and IL-10 upon further activation with anti-CD3 and anti-CD28. Expression of liCTLA-4 inhibited the production of IFN-γ but not IL-10 or IL-17 from the CTLA-4-/-
T cells. Interestingly, we observed a reproducible, increased production of IL-17 by CTLA-4-/-
liCTLA-4-tg T cells as compared to CTLA-4-/-
T cells. Recently, McGeachy et al
) demonstrated that cells co-producing IL-17 and IL-10 acquire a non-pathogenic phenotype that could potentially confer these cells with regulatory function. This raises the possibility that the appearance of IL-17 producing T cells that co-produce IL-10 in the CTLA-4-/-
liCTLA-4 tg mice may be regulatory and not pathogenic T cells, thereby increasing the number of cells capable of restraining pathogenic effectors in the CTLA-4-/-
liCTLA-4 tg mice. Alternatively, there is some support for the hypothesis that there is antagonism between IFN-γ (Th1) and IL-17 (Th17) producing cells (48
), suggesting that the inhibition of IFN-γ by liCTLA-4 expression in CTLA4-/-
T cells could lead to the expansion of Th17 cells. Since the decrease in IFN-γ production by cells from CTLA-4-/-
liCTLA-4 tg is enough to reduce inflammation and prolong the survival of these mice, this hypothesis suggests that the inflammatory phenotype of CTLA-4-/-
mice is partly mediated by Th1 cells.
As detailed above, in the NOD liCTLA-4 transgenic T cells where liCTLA-4 is expressed in the presence of flCTLA-4 as well as the other isoforms of CTLA-4, IFN-γ and IL-17 are both inhibited. Considering these data, one might speculate that the inhibition of IFN-γ and increased production IL-17 in CTLA4-/- liCTLA-4 tg mice is due to the non-physiologic situation in which liCTLA-4 is present in the absence of flCTLA-4. Thus, the lack of complete protection and the presence of residual inflammation observed in the CTLA-4-/- liCTLA-4 tg mice may be due to the increased number of IL-17-producing Th17 cells even though disease in CTLA-4-/- mice is possibly Th1-driven.
liCTLA-4 has been reported to heterodimerize with flCTLA-4 and recruit SHP-2 to dephosphorylate the TcRς chain (30
). However, when liCTLA-4 is expressed in the absence of flCTLA-4, it interacts with the TcRς chain independently of flCTLA-4 and the recruitment of SHP-2 (30
). The inability to recruit SHP-2 when liCTLA-4 and flCTLA-4 are expressed separately could pose an impediment to the complete negative signaling of T cell responses by flCTLA-4 when liCTLA-4 levels are low because of genetically-determined expression levels. The formation of liCTLA-4 and flCTLA-4 heterodimers could stabilize the lattice resulting in an increased affinity in binding of SHP-2 to the TcRς complex. In the present study undertaking a detailed clinical and histopathological analysis on a large cohort of animals, we observed that CTLA4-/-
liCTLA-4 tg mice were only partially protected from the lethal autoimmune lymphoproliferative disease observed in CTLA-4-/-
mice. This partial protection from disease is consistent with the results of Chikuma et al
), who generated mice transgenic for a point-mutated CTLA-4 lacking B7 binding capacity and demonstrated that this ligand-nonbinding mutant CTLA-4 delayed lethal lymphoproliferative disorder of CTLA-4-/-
). Overall, our combined results indicate that CTLA-4 needs to bind to B7 molecules to completely abrogate the abnormal T cell activation present in CTLA-4-/-
mice, however, the signaling domain alone is sufficient to provide some protection from the activated phenotype present in CTLA-4-/-
mice. These results have an interesting parallel with the results from Masteller et al
) where only partial protection from disease in CTLA-4-/-
mice was provided by a transgene encoding a CTLA-4 molecule lacking the signaling domain. Thus, perhaps not surprisingly, both the B7 binding IgV domain and the signaling domain in the cytoplasmic tail are critical for the full functioning of the CTLA-4 molecule.
Overall, our present data suggest that both the ligand-independent and the ligand binding, B7-dependent, CTLA-4 isoforms are required for delivering maximal negative signal into T cells. These previous findings (38
) support our current data that when liCTLA-4 is expressed in wild type NOD mice where flCTLA-4 is also present, liCTLA-4 is able to inhibit T-cell expansion and production of both inflammatory cytokines, IFN-γ and IL-17. In contrast, expression of liCTLA-4 in CTLA-4-/-
mice only inhibited production of IFN-γ.
In this paper, using multiple approaches, we have determined that the SNP in exon 2 (position 77) of Ctla4 is the most likely SNP causing the differential expression of the liCTLA-4 isoform. Formal proof that the exon 2 SNP of Ctla4 alone causes the full protective effect of Idd5.1, rather than a model in which variation in ICOS or another gene within the Idd5.1 interval contributes a portion of the protective effect from T1D, will require the knock-in of the NOD allele at residue 77 of exon 2 into the B6 Ctla4 allele with subsequent backcrossing of the Idd5.1 region to the NOD background for the analysis of T1D. For example, until such an experiment is done it remains a possibility that the B6 Icos allele could be required in conjunction with the B6 allele at Ctla4 for full Idd5.1-associated protection to be observed, even though we have demonstrated that the B6 Icos allele in the absence of the B6 Ctla4 allele is not sufficient on its own to mediate T1D protection. If the residue 77 SNP is the sole cause of protection for the B6 allele of Idd5.1, this NOD.B6 Idd5.1 residue 77 knockin congenic mouse strain will have a T1D frequency equal to that of the NOD parental strain. If the knockin strain still has protection from T1D as compared to the NOD strain, then another B6-derived gene within the interval contributes to the Idd5.1-mediated T1D effect. Finally, we have shown by congenic and transgenic approaches that higher expression of liCTLA-4 in NOD mice correlates with protection from T1D and that overexpression of liCTLA-4 in CTLA-4-/- mice can partially rescue them from early lethality. Overall, our data suggest liCTLA-4 contributes to preventing activation and maintaining tolerance to self-antigens thus, preventing autoimmunity.