NIT-1, a Pancreatic β-Cell Line Established From a Transgenic NOD/Lt Mouse

Abstract

NOD/Lt mice harboring a hybrid rat insulin-promoter/SV40 large T-antigen gene spontaneously develop beta-cell adenomas. NIT-1 is a pancreatic beta-cell line established from one of these transgenic mice. Immunocytochemical staining of passage 18 cells showed most contained insulin, with less than 5% containing glucagon, and none containing pancreatic polypeptide or somatostatin. Glucagon content radioimmunoassayed in cell extracts was only 0.27% of the insulin content. Two-hour insulin secretion at 16.5 mM glucose was 638 ng/10(6) cells (41% of intracellular content) compared to only 1.3 ng glucagon (32% of intracellular content). Stimulated insulin secretion was consistently observed in response to 11 and 16.5 mM glucose between passages 11 and 19. At passage 19, both theophylline and tolbutamide stimulated insulin secretion at 5.5 mM glucose. Northern-blot analysis confirmed high levels of insulin mRNA but only trace glucagon mRNA and undetectable somatostatin mRNA. Interferon-gamma (IFN-gamma)-induced MHC class I RNA expression was correlated with markedly increased antigen expression at the cell surface. Similarly, a MHC-linked "occult" class I-like antigen detected by Cr release assay only after exposure of standard NOD/Lt islet cells to IFN-gamma was strongly induced by IFN-gamma in NIT-1 cells. Cell surface MHC class II antigen was not constitutively expressed on NIT-1 cells and could not be detected after IFN-gamma incubation, despite demonstration of IFN-gamma-induced Aa, Ab, and Li invariant-chain RNA transcripts. Similarly IFN-gamma induction of intercellular adhesion molecule 1 (Icam-1) transcripts was not accompanied by demonstrable cell surface expression of ICAM-1 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

“…The sources of naturally processed peptides were two cell lines: NIT-1, a NOD-derived insulinoma, and NOD.C3, an APC line generated from NOD splenocytes [14,32]. Both cell lines expressed high levels of cell surface Kd molecules, which facilitated the identification of large numbers of naturally processed peptides ( Supplementary Fig.…”

“…The NIT-1 insulinoma (a kind gift from Dr. E. Leiter [32]) and RMAsKd cells were grown in DMEM supplemented with 10% fetal calf serum (FCS). The T2Kd cells, used for binding analyses and isolation of peptides, were maintained in DMEM supplemented with 10% FCS.…”

Characterization of a Ca2+ Release-activated Nonselective Cation Current Regulating Membrane Potential and [Ca2+] Oscillations in Transgenically Derived β-Cells

“…Eighteen hours prior to experiments, medium was changed to RPMI 1640 medium containing 15% dialyzed horse serum, 2.5% dialyzed fetal calf serum, 1 mM glucose, and antibiotics as above. MIN6 cells were cultured essentially as described (30). Islets of Langerhans were isolated from 8 -10-week-old C57BL/KsJ (ϩ/ϩ) mice (Jackson Laboratories) as described (5,9,12 ] i was expressed as the ratio change in fluorescence (340/380) as detailed in full elsewhere (9,20).…”

First Signature of Islet β-Cell-Derived Naturally Processed Peptides Selected by Diabetogenic Class II MHC Molecules

“…To this end, we made use of the Nit-1 insulinoma cell line, an SV40 transformed insulinoma generated by Leiter's laboratory (15). Nit-1 cells, which are known not to express I-A g7 , were infected with a lentivirus encoding for the murine CIITA gene (referred to as 'NitCIITA') and then screened by FACS analysis for induction of I-A g7 molecules on their cell surface.…”