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A novel CD93 polymorphism in non-obese diabetic (NOD) and NZB/W F1 mice is linked to a CD4+ iNKT cell deficient state.

Zekavat G, Mozaffari R, Arias VJ, Rostami SY, Badkerhanian A, Tenner AJ, Nichols KE, Naji A, Noorchashm H - Immunogenetics (2010)

Bottom Line: This allele carries a coding polymorphism in the first epidermal growth factor-like domain of CD93, which results in an amino acid substitution from Asn-->His at position 264.This polymorphism does not appear to influence protein translation or ecto-domain cleavage, as CD93 is detectable in bone-marrow-derived macrophage and B-cell precursor lysates and in soluble form in the serum.These data suggest that Cd93 may be an autoimmune susceptibility gene residing within the Idd13 locus, which plays a role in regulating absolute numbers of CD4(+) NKT cells.

View Article: PubMed Central - PubMed

Affiliation: Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
In the present study, we characterize a polymorphism in the CD93 molecule, originally identified as the receptor for the C1q complement component (i.e., C1qRp, or AA4.1) in non-obese diabetic (NOD) mice. This allele carries a coding polymorphism in the first epidermal growth factor-like domain of CD93, which results in an amino acid substitution from Asn-->His at position 264. This polymorphism does not appear to influence protein translation or ecto-domain cleavage, as CD93 is detectable in bone-marrow-derived macrophage and B-cell precursor lysates and in soluble form in the serum. The NOD CD93 isoform causes a phenotypic aberrancy in the early B-cell developmental stages (i.e., pro-, pre-, immature, and transitional), likely related to a conformational variation. Interestingly, the NZB/W F1 strain, which serves as a murine model of Lupus, also expresses an identical CD93 sequence polymorphism. Cd93 is located within the NOD Idd13 locus and is also tightly linked to the NZB/W F1 Wbw1 and Nkt2 disease susceptibility loci, which are thought to regulate natural killer T (NKT) cell homeostasis. Consistent with this genetic linkage, we found B6 CD93(-/-) and B6.NOD(Idd13) mice to be susceptible to a profound CD4(+) NKT cell deficient state. These data suggest that Cd93 may be an autoimmune susceptibility gene residing within the Idd13 locus, which plays a role in regulating absolute numbers of CD4(+) NKT cells.

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iNKT cell analysis in B6, B6 CD93−/−, B6.NODIdd13 and NOD mice. a The left panel shows the frequency of iNKT cell staining (CD3lo, PBS57-loaded CD1d Tetramer+) in the liver of B6 (n = 9, mean 15.9 ± 5.79%) B6 CD93−/− (n = 7, mean 9.78 ± 4.20%), B6.NODIdd13, (n = 5, mean 7.71 ± 4.29%), and NOD (n = 5, mean 8.15 ± 3.11%) mice. The right panel further resolves CD4+ iNKT and CD4− iNKT cells. b The bar graph indicates the frequency of CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice. c The bar graph shows the absolute number of thymic DN and CD4SP iNKT cells (CD3lo, PBS57-loaded CD1d Tetramer+) in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. d The bar graph indicates the total number of splenic CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. Error bars indicate ± SD, *p < 0.05 compared with B6 mice
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Fig4: iNKT cell analysis in B6, B6 CD93−/−, B6.NODIdd13 and NOD mice. a The left panel shows the frequency of iNKT cell staining (CD3lo, PBS57-loaded CD1d Tetramer+) in the liver of B6 (n = 9, mean 15.9 ± 5.79%) B6 CD93−/− (n = 7, mean 9.78 ± 4.20%), B6.NODIdd13, (n = 5, mean 7.71 ± 4.29%), and NOD (n = 5, mean 8.15 ± 3.11%) mice. The right panel further resolves CD4+ iNKT and CD4− iNKT cells. b The bar graph indicates the frequency of CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice. c The bar graph shows the absolute number of thymic DN and CD4SP iNKT cells (CD3lo, PBS57-loaded CD1d Tetramer+) in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. d The bar graph indicates the total number of splenic CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. Error bars indicate ± SD, *p < 0.05 compared with B6 mice

Mentions: The Cd93 locus maps to a region of chromosome 2, which regulates iNKT cell number and/or function (Chen et al. 2007; Esteban et al. 2003). Therefore, we undertook an analysis of the iNKT cell compartments of B6 CD93−/− (Norsworthy et al. 2004), B6.NODIdd13, and NOD mice. iNKT cells were analyzed in the liver, thymus and spleen using an alpha-GalCer analog, PBS57-loaded CD1d tetramer. iNKT cells were further resolved into CD4+ and CD4− subsets in the spleen and liver and CD4 single positive (CD4SP) and double-negative (DN) subsets in the thymus. Flow cytometric analysis of lymphoid cells in the liver of non-autoimmune B6 mice revealed 15.9 ± 5.79% of the population staining for iNKT cells (Fig. 4a). In contrast, there was a global deficiency of iNKT cells in lymphoid populations in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice (9.7 ± 4.20%, 7.71 ± 4.29%, and 8.15 ± 3.11%, respectively, p < 0.05 compared with B6 mice, Fig. 4a). We further resolved the population of iNKT cells into the CD4+ iNKT and CD4− iNKT cell subsets (Fig. 4a, right column), which allowed us to determine the frequencies of CD4+ iNKT and CD4− iNKT cells in the liver (Fig. 4b). There is a twofold reduction in the frequency of CD4+ hepatic iNKT cells in B6 CD93−/−, B6.NODIdd13, and NOD, compared with B6 mice (p < 0.05, Fig. 4b). Conversely, the frequency of CD4− iNKT cells in the liver is similar across each of the tested strains of mice (p > 0.05). Figure 4c shows that the total number of thymic DN and CD4SP iNKT cells is up to twofold less in B6 CD93−/−, B6.NODIdd13, and NOD mice, compared with B6 (p < 0.05). Similarly, there is a two- to threefold reduction of total splenic CD4+ iNKT cells in B6 CD93−/−, B6.NODIdd13, and NOD compared with B6 mice (p < 0.05), while the CD4− iNKT cell numbers is comparable in all strains of mice tested (p > 0.05, Fig. 4d). To exclude the possibility that the reduction of CD4+ iNKT cells was due to an overall lymphopenia, we analyzed the total numbers of thymic and splenic lymphocytes and found that the total number of lymphoid cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice was comparable (p > 0.05, data not shown). Our studies thus confirm that NOD mice possess a CD4+ iNKT cell deficiency (Baxter et al. 1997; Godfrey et al. 1997; Hammond et al. 2001) and show that B6 CD93−/− and B6.NODIdd13 mice exhibit a CD4+ iNKT cell deficiency similar to NOD mice.Fig. 4


A novel CD93 polymorphism in non-obese diabetic (NOD) and NZB/W F1 mice is linked to a CD4+ iNKT cell deficient state.

Zekavat G, Mozaffari R, Arias VJ, Rostami SY, Badkerhanian A, Tenner AJ, Nichols KE, Naji A, Noorchashm H - Immunogenetics (2010)

iNKT cell analysis in B6, B6 CD93−/−, B6.NODIdd13 and NOD mice. a The left panel shows the frequency of iNKT cell staining (CD3lo, PBS57-loaded CD1d Tetramer+) in the liver of B6 (n = 9, mean 15.9 ± 5.79%) B6 CD93−/− (n = 7, mean 9.78 ± 4.20%), B6.NODIdd13, (n = 5, mean 7.71 ± 4.29%), and NOD (n = 5, mean 8.15 ± 3.11%) mice. The right panel further resolves CD4+ iNKT and CD4− iNKT cells. b The bar graph indicates the frequency of CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice. c The bar graph shows the absolute number of thymic DN and CD4SP iNKT cells (CD3lo, PBS57-loaded CD1d Tetramer+) in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. d The bar graph indicates the total number of splenic CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. Error bars indicate ± SD, *p < 0.05 compared with B6 mice
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Fig4: iNKT cell analysis in B6, B6 CD93−/−, B6.NODIdd13 and NOD mice. a The left panel shows the frequency of iNKT cell staining (CD3lo, PBS57-loaded CD1d Tetramer+) in the liver of B6 (n = 9, mean 15.9 ± 5.79%) B6 CD93−/− (n = 7, mean 9.78 ± 4.20%), B6.NODIdd13, (n = 5, mean 7.71 ± 4.29%), and NOD (n = 5, mean 8.15 ± 3.11%) mice. The right panel further resolves CD4+ iNKT and CD4− iNKT cells. b The bar graph indicates the frequency of CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice. c The bar graph shows the absolute number of thymic DN and CD4SP iNKT cells (CD3lo, PBS57-loaded CD1d Tetramer+) in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. d The bar graph indicates the total number of splenic CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. Error bars indicate ± SD, *p < 0.05 compared with B6 mice
Mentions: The Cd93 locus maps to a region of chromosome 2, which regulates iNKT cell number and/or function (Chen et al. 2007; Esteban et al. 2003). Therefore, we undertook an analysis of the iNKT cell compartments of B6 CD93−/− (Norsworthy et al. 2004), B6.NODIdd13, and NOD mice. iNKT cells were analyzed in the liver, thymus and spleen using an alpha-GalCer analog, PBS57-loaded CD1d tetramer. iNKT cells were further resolved into CD4+ and CD4− subsets in the spleen and liver and CD4 single positive (CD4SP) and double-negative (DN) subsets in the thymus. Flow cytometric analysis of lymphoid cells in the liver of non-autoimmune B6 mice revealed 15.9 ± 5.79% of the population staining for iNKT cells (Fig. 4a). In contrast, there was a global deficiency of iNKT cells in lymphoid populations in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice (9.7 ± 4.20%, 7.71 ± 4.29%, and 8.15 ± 3.11%, respectively, p < 0.05 compared with B6 mice, Fig. 4a). We further resolved the population of iNKT cells into the CD4+ iNKT and CD4− iNKT cell subsets (Fig. 4a, right column), which allowed us to determine the frequencies of CD4+ iNKT and CD4− iNKT cells in the liver (Fig. 4b). There is a twofold reduction in the frequency of CD4+ hepatic iNKT cells in B6 CD93−/−, B6.NODIdd13, and NOD, compared with B6 mice (p < 0.05, Fig. 4b). Conversely, the frequency of CD4− iNKT cells in the liver is similar across each of the tested strains of mice (p > 0.05). Figure 4c shows that the total number of thymic DN and CD4SP iNKT cells is up to twofold less in B6 CD93−/−, B6.NODIdd13, and NOD mice, compared with B6 (p < 0.05). Similarly, there is a two- to threefold reduction of total splenic CD4+ iNKT cells in B6 CD93−/−, B6.NODIdd13, and NOD compared with B6 mice (p < 0.05), while the CD4− iNKT cell numbers is comparable in all strains of mice tested (p > 0.05, Fig. 4d). To exclude the possibility that the reduction of CD4+ iNKT cells was due to an overall lymphopenia, we analyzed the total numbers of thymic and splenic lymphocytes and found that the total number of lymphoid cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice was comparable (p > 0.05, data not shown). Our studies thus confirm that NOD mice possess a CD4+ iNKT cell deficiency (Baxter et al. 1997; Godfrey et al. 1997; Hammond et al. 2001) and show that B6 CD93−/− and B6.NODIdd13 mice exhibit a CD4+ iNKT cell deficiency similar to NOD mice.Fig. 4

Bottom Line: This allele carries a coding polymorphism in the first epidermal growth factor-like domain of CD93, which results in an amino acid substitution from Asn-->His at position 264.This polymorphism does not appear to influence protein translation or ecto-domain cleavage, as CD93 is detectable in bone-marrow-derived macrophage and B-cell precursor lysates and in soluble form in the serum.These data suggest that Cd93 may be an autoimmune susceptibility gene residing within the Idd13 locus, which plays a role in regulating absolute numbers of CD4(+) NKT cells.

View Article: PubMed Central - PubMed

Affiliation: Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
In the present study, we characterize a polymorphism in the CD93 molecule, originally identified as the receptor for the C1q complement component (i.e., C1qRp, or AA4.1) in non-obese diabetic (NOD) mice. This allele carries a coding polymorphism in the first epidermal growth factor-like domain of CD93, which results in an amino acid substitution from Asn-->His at position 264. This polymorphism does not appear to influence protein translation or ecto-domain cleavage, as CD93 is detectable in bone-marrow-derived macrophage and B-cell precursor lysates and in soluble form in the serum. The NOD CD93 isoform causes a phenotypic aberrancy in the early B-cell developmental stages (i.e., pro-, pre-, immature, and transitional), likely related to a conformational variation. Interestingly, the NZB/W F1 strain, which serves as a murine model of Lupus, also expresses an identical CD93 sequence polymorphism. Cd93 is located within the NOD Idd13 locus and is also tightly linked to the NZB/W F1 Wbw1 and Nkt2 disease susceptibility loci, which are thought to regulate natural killer T (NKT) cell homeostasis. Consistent with this genetic linkage, we found B6 CD93(-/-) and B6.NOD(Idd13) mice to be susceptible to a profound CD4(+) NKT cell deficient state. These data suggest that Cd93 may be an autoimmune susceptibility gene residing within the Idd13 locus, which plays a role in regulating absolute numbers of CD4(+) NKT cells.

Show MeSH
Related in: MedlinePlus