Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease

Abstract

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.

Figure 1

GAD65 transgenic construct and Southern blot of tail DNA. (A) Map of the transgene, comprising the enhancer and promoter regions of the MHC class I K^b gene, ligated 5′ of the murine GAD65 coding region, ligated 5′ of a series of noncoding exons from the HGH gene. The introduced mutations replaced the pyridoxal phosphate binding motif NPHK with NPLA. The black bar represents the 700-bp probe used on Southern blots to detect a predicted 2-kb BglII fragment. (B) Southern analysis of BglII-digested genomic DNA extracted from mouse tails: lanes 1, 3, 4, 5, and 10 are tg(+) line 14; lanes 2, 6, 7, 8, and 9 are non-tg(+) littermates. The 7-kb doublet and 2-kb band correspond to endogenous and transgenic bands, respectively. Tg(+) mice of lines 12 and 13 gave essentially the same results as those shown for line 14.

Figure 2

Transgenic mRNA and protein expression. (A) Design of primers to detect spliced hybrid GAD-HGH RNA (see text). (B) Reverse transcription–PCR analysis of tissues from tg(+) mice (+) and non-tg(+) mice (−) mice, in the presence (RT+) or absence (RT−) of avian myeloblastosis virus reverse transcriptase (AMV-RT) by using transgene-specific primers (Upper) and hypoxanthine-guanine phosphoribosyltransferase-specific primers (Lower). M, 100-bp molecular marker (GIBCO/BRL); B, brain; K, kidney; L, liver; T, thymus; H, heart; I, islets. The origin of the different bands is discussed in the text. Tissue lysates from adult (C and D) or day 2 (E) tg(+) mice (+) and non-tg(+) mice (−) were immunoprecipitated with Stiff Man Syndrome patient serum and Western blotted with mAb mN65 against GAD65 (C and E). The membrane in C was stripped and reprobed with GAD65-specific rabbit polyclonal antisera, 7673 (D). Sizes of proteins were estimated from coelectrophoresed, prestained markers (not shown).

Figure 3

Histological analysis of tissues from transgenic mice. Paraffin-embedded tissue sections stained with hematoxylin/eosin revealed the presence of infiltrating leukocytes in the pancreas (A) and occasionally salivary gland (G), but not in kidney (E) or liver (F). (B-D) Consecutive frozen sections of the pancreas showing the same islet stained with antibody against CD4 (B), CD8 (C), and B220 (D). Arrows in A-D and G indicate infiltrates.

Figure 4

Frequency and severity of insulitis and diabetes. Insulitis in 7-week-old female (A) and 10-week-old male (B) tg(+) (filled bar) and non-tg(+) (empty bar) mice. (A) tg(+), n = 6; non-tg(+), n = 7. (B) Non-tg(+), n = 10; tg(+), n = 10. Severity of insulitis on a scale of − to ++++ was assessed as described in the text. Incidence of spontaneous diabetes in female (C) and male (D) tg(+) and non-tg(+) mice. Blood glucose levels were measured weekly starting from 10 weeks of age. ⧫ and □ represent weekly assessments of tg(+) mice and non-tg(+) littermates, respectively.

Figure 5

Establishment of MHC class I-GAD65 FvB transgenic mice. (A) Southern blot of tail genomic DNA. Lanes 1, 2, 4, and 5 were tg(+); lane 3 was non-tg(+). Lanes 1 and 2 are from line 27; lanes 4 and 5 from line 9. (B) Reverse transcription–PCR analysis of tg(+) (+) and non-tg(+) (−) tissues for transgene-specific RNA expression. M, 100-bp marker (GIBCO/BRL); B, brain; K, kidney; L, liver; S, spleen; T, thymus; I, pancreatic islets of Langerhans; C, water. (C) Immunoprecipitation/Western blot analysis of thymus lysates of 2-day-old non-tg(+) (lane 1), line 9 tg(+) (lane 2), and line 27 tg(+) (lane 3) mice. Immunoprecipitation was by mN65, Western by 7673 (see text). (D) GAD65 immunoprecipitation/Western blot analysis of lysates from adult tg(+) (+) or non-tg(+) (−) mice. Immunoprecipitation was by Stiff Man Syndrome patient serum, Western by mN65 (see text). B, brain; K, kidney; L, liver; S, spleen; T, thymus.

Figure 6

Proliferative responses to antigen of splenocytes from individual NOD or FvB mice immunized with GAD65 or ovalbumin (ova). (A and B) 4 × 10⁵ splenocytes per well from three tg(+) NOD mice (closed symbols) and three non-tg(+) NOD mice (open symbols) were stimulated in vitro with GAD65 9 days after immunization with GAD65 (A) or ova (B). (C) 4 × 10⁵ splenocytes per well from two sets of three tg(+) NOD mice were stimulated in vitro with ova, 9 days after immunization with either GAD65 (closed symbols) or ova (open symbols). (D) 4 × 10⁵ cells per well from spleen (squares) or draining lymph nodes (circles) of FvB tg(+) (closed symbols) and FvB non-tg(+) mice (open symbols), and from the spleen of non-tg(+) NOD mouse (x) were stimulated in vitro with GAD65, 9 days after immunization with GAD65. FvB symbols represent data from three mice. (E) 4 × 10⁵ splenocytes per well from two sets of three FvB mice [two tg(+), 1 non-tg(+)] were stimulated in vitro with ova, 9 days after immunization with GAD65 (closed symbols) or ova (open symbols). Proliferation assessed as uptake of [³H]thymidine, added during the last 18 hr of a 90-hr culture. Data are mean cpm from triplicate wells. SDs were less than 10%.