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. 1997 Aug 5;94(16):8670-4.
doi: 10.1073/pnas.94.16.8670.

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

F Colucci  1 , M L Bergman, C Penha-Gonçalves, C M Cilio, D Holmberg
Affiliations

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

F Colucci et al. Proc Natl Acad Sci U S A. .

Abstract

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

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Figures

Figure 1
Figure 1
Apoptosis resistance trait scored by flow cytometry and analysis of phenotypic variance and distribution. (A) Apoptosis was induced by i.p. injection of CY. Lymph node cells were fixed and stained with TUNEL for detection of apoptosis, measured as percent of FITC positive cells. NOD and nonobese resistant (NOR) lymphocytes showed relative resistance, whereas B6 lymphocytes were four fold more sensitive. In absence of the terminal deoxynucleotidyltransferase (TdT) enzyme the nonspecific staining was <1%. The figure shows one representative of six independent experiments. (B) Phenotypic variance. T-distribution curves predicted by the apoptosis resistance scored in 8 NOD, 8 B6, 6 F1, and 78 F2 mice. (C) Phenotypic distribution. Comparison between the predicted T-distribution in the F2 progeny and the observed frequency distribution.
Figure 2
Figure 2
QTL analysis of the lymphocyte apoptosis resistance in the F2 intercross. The logarithmic of odds (lod) score curve along chromosome 1 represents the maximum likelihood estimates for the presence of a QTL at each point. The dotted line indicates a threshold level for statistical significance of lod score >3.0. The fitness of the QTL to four genetic modes of action of the NOD allele is indicated. Plots were generated by using the qtl 1.1 software (14). The locus accounts for 21% of the phenotypic variation observed, which would explain 50% of the genetic variance in the F2 progeny.
Figure 3
Figure 3
Chromosomal locations of the regions including the apoptosis-resistant trait locus and the Idd5 locus. The two regions partially overlap the two recombinant segments that in the NOR chromosome 1 are of DBA/2 and B6 origin. NOR lymphocytes display the same apoptosis resistance as NOD lymphocytes (Fig. 1A), whereas the B6 and DBA/2 lymphocytes are highly susceptible to apoptosis. Therefore, the locus controlling apoptosis resistance must be included in the 20 cM intervening region (from D1Mit72 to D1Mit19) that in NOR mice is composed of NOD alleles. Such a region encompasses both Ctla4 and Cd28. Capop, CY-apoptosis resistance.
Figure 4
Figure 4
Defective expression of CTLA-4 and reduced up-regulation of CD28 in activated NOD T cells. Splenic T cells of NOD and B6 mice were stimulated in vitro with soluble anti-CD3. Fresh resting T cells (A) and 48-h cultured splenocytes (B) were stained for either CTLA-4 or CD28. Mean fluorescence values are shown for CD4+ and CD8+ T cells of resting cells and electronically gated blasts of both NOD mice (open bars) and B6 mice (filled bars). (C) The expression of the activation marker CD69 on resting and activated T cell blasts is similar in NOD (dotted line) and B6 (solid line). Filled histograms show background levels of fluorescence detected with an isotype matched control antibody. Data are representative of two independent experiments.
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