Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset.

Antigen-specific T cells acquire a distinctive phenotype during activation, with characteristic acquisition of surface markers and patterns of gene expression. Early after antigen stimulation, CD4(+) T lymphocytes increase their surface density of the CD4 marker, a trait which has been used to identify antigen-activated cells. The recent development of MHC tetramer technologies has greatly improved the ability to detect HLA class I-restricted T cells specific for known antigen epitopes. We have recently extended these studies to human class II-restricted CD4(+) T cell responses and now describe antigen-specific T cell responses from human peripheral blood in which elevated CD4 expression levels in human T cells following antigen stimulation identify the activated and proliferating subset of cells. The CD4(high) population is substantially enriched in epitope-specific cells identified by class II tetramer staining and almost all tetramer-positive cells are contained within the CD4(high) population. T cell clones derived from the tetramer-positive, CD4(high) population demonstrate antigen specificity and maintain tetramer staining, while the substantial number of CD4(high) cells which fail to stain with tetramer appear to proliferate as a result of bystander activation. Epitope-specific components of a polyclonal immune response are directly visualized and quantitated by tetramer detection, providing a direct measure of the heterogeneity of the human immune response.

Prediction of diabetes with body mass index, oral glucose tolerance test and islet cell autoantibodies in a regional population.

OBJECTIVE: Our aim was to test the hypothesis that a combination of markers for Type 1 diabetes (glutamate decarboxylase and IA-2 autoantibodies) and for Type 2 diabetes [oral glucose tolerance test (OGTT) and body mass index (BMI)], would predict clinical diabetes in a regional population. DESIGN: A population-based follow-up cohort study. SETTING: Participants visited the primary health care centre in Lycksele, Sweden in 1988-92. PARTICIPANTS: A cohort of 2278 subjects (M/F 1149/1129) who were studied at follow-up in 1998. At base line there were 2314 subjects (M/F 1167/1147) who participated in the Västerbotten Intervention Program on their birthday when turning either 30, 40, 50 or 60 years of age. Main outcome measurements. A clinically diagnosed diabetes at follow-up when the medical records were reviewed for diagnosis of diabetes. At base line, the participants were subjected to a standard OGTT and their BMI determined along with the autoantibodies. RESULTS: At follow-up, 42/2278 (1.8%, 95% CI 1.2-2.3) (M/F 23/19) had developed diabetes: 41 subjects were clinically classified with Type 2 and one with Type 1 diabetes. There was no significant relation between autoantibody levels at base line and diabetes at follow-up. Stepwise multiple logistic regression showed that the odds ratio for developing diabetes was 10.8 (95% CI 6.3-18.9) in subjects in the fourth quartile of BMI (BMI > 27) compared with 7.8 (95% CI 4.8-12.6) in the fourth quartile of 2-h plasma glucose (>7.5 mmol L(-1)) and 7.2 (95% CI 4.8-11.4) in the fourth quartile of the fasting plasma glucose (>5.6 mmol L(-1)). CONCLUSION: Islet cell autoantibodies did not predict diabetes at follow-up. BMI measured at base line was as effective as 2-h plasma glucose and fasting plasma glucose to predict diabetes in this adult population.

Low-dose streptozotocin induces sustained hyperglycemia in Macaca nemestrina.

The potential for using macaques to create a nonhuman primate diabetic model was investigated. The significant objectives were to determine a) prognosis of STZ induced permanent beta cell destruction in nonhuman primates, and b) the potential to use STZ treated animals in a model of autoimmune diabetes by following adoptively transferred lymphocytes into MHC identical macaques. Beta cell impairment was achieved by a single intravenous, low dose (10-40 mg/kg body weight) streptozotocin injection in a majority of pigtailed macaques (Macaca nemestrina). Multiple injections, even at low doses at close intervals affected liver and kidney functions in addition to beta cell destruction. Abnormal IVGTT were observed in all streptozotocin-treated animals, in some within a week to 10 days. The fasting blood glucose levels rose from <70 mg/dl in pre-STZ stage to above 400 mg/dl in severely diabetic macaques. Histological evidence suggests loss of beta cells when animals were euthanized within two to four weeks post-STZ treatment. Near complete destruction of beta cells was observed in animals maintained longer than three months on insulin. Donor T cells from STZ-treated animals were incubated overnight with 10U/ml IL-2 and 2.5 ug/ml PHA and then injected iv into a MHC-identical non-diabetic sibling. Three weeks later a second injection of donor PMBC labeled with vital dye Cell Tracker Green was given and the animal was euthanized after 24 hours. The recipient showed labeled donor T cells in the pancreas, spleen and peripheral blood, consistent with specific homing of activated lymphocytes from the diabetic donor.

Glucose-regulated insulin expression in diabetic rats.

Retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an amplified, glucose-regulated secretion of insulin. We constructed LhI*TFSN virus to encode a glucose-regulatable transforming growth factor alpha promoter controlling furin expression with a viral LTR promoter driving constitutive expression of furin-cleavable human proinsulin. Autologous BB rat vascular smooth muscle cells transduced with LhI*TFSN virus and cultured in 1.7 and 16.7 mM glucose secreted 50.7 +/- 3.2 and 136.0 +/- 11.0 microU (mean +/- SD) of insulin per 10(6) cells per day, respectively. After the onset of diabetes spontaneously diabetic congenic DR lyp/lyp BB rats received stomach implants containing 2 x 10(6) LhI*TFSN-transduced primary rat vascular smooth muscle cells. In eight treated rats there was a major reduction in insulin requirement to as low as 25% of pretreatment level for up to 3 months and one rat became insulin free without hypoglycemia. Intraperitoneal glucose tolerance tests (IPGTTs) in diabetic rats receiving control implants did not show the characteristic decline in blood glucose of normal rats after glucose administration. In contrast, diabetic rats receiving LhI*TFSN-transduced cells showed significant clearances of blood glucose. These data suggest clinically significant levels of glucose-regulated insulin delivery from implanted vascular smooth muscle cells transduced with LhI*TFSN vector.