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Lymphocytes from insulin-dependent diabetic patients were previously shown to suppress insulin release from mouse islet cells in vitro. Glucagon release was not suppressed. In order to further analyze this phenomenon, lymphocytes from three insulin-dependent diabetic patients with associated autoimmune diseases were treated by using the panning method for cell separation before testing on islet cell suspensions. The OKT3+, OKT3-, and OKT4- cell subsets were obtained. Insulin release was suppressed by the OKT3+ (T lymphocyte-enriched) subset, but not by the OKT3- (T lymphocyte-depleted) subset. These results suggest that T lymphocytes are directly involved in the suppression of insulin release in this model. Furthermore, the OKT4- (T helper-depleted) subset also suppressed insulin release.

Unlabelled: Peripheral lymphocyte subsets were enumerated, using OKT monoclonal sera, in 56 diabetic (43 adults and 13 children) and 20 control subjects. Concomitantly, anti-islet humoral and cellular immunity was tested in vitro and serum thymulin level was measured. In the newly diagnosed patients (less than 30 days; 18 cases), the percent of OKT4+ and OKT8+ cells was reduced, the OKT8+ depletion being particularly pronounced in children. Tests for cellular immunity were positive in 83% of the newly diagnosed diabetic subjects and anti-islet cytotoxic antibodies were detected in 50%. The serum thymulin level was decreased in 2 children. Later on in the course of the disease, a marked reduction in OKT3+, OKT4+, and OKT8+ cell percentage was observed, the mean OKT4/OKT8 ratio being normal or lower than normal. The percent of antibody-positive sera rose to 64%, while anti-islet cellular immunity was detectable in 54%. When extrapancreatic manifestations of probable autoimmune nature were present, anti-islet cellular immunity was detected in 100% of cases, accompanied by cytotoxic antibodies in 54%., Conclusions: (1) the magnitude of T-cell depletion and/or imbalance in diabetic subjects depended mainly on the duration of the disease, (2) anti-islet cellular immunity was the anomaly most frequently detectable, and (3) a decrease in serum thymulin level was infrequently detected.

The non-obese diabetic (NOD) mouse is an animal model of insulin-dependent diabetes mellitus (IDDM), in which 80% of the females become diabetic after the age of 12 weeks. Using an in vitro assay we investigated the capacity of spleen lymphocytes from NOD mice to inhibit the insulin secretion of normal islet cells after stimulation by theophylline plus arginine. Spleen cells from diabetic NOD mice inhibited the insulin release of DBA/2 islet cells. Depletion experiments using monoclonal antibodies demonstrated that inhibitory cells belonged to the Lyt2 positive T lymphocyte subset. The phenomenon was not restricted by the MHC class I K region, shared by NOD and DBA/2 mice, since lymphocytes from diabetic NOD mice also inhibited the insulin secretion of normal Wistar rat islet cells. Inhibitory T cells were detected in overtly diabetic mice but also in non-diabetic females aged 5-11 weeks indicating that they are not secondary to metabolic disturbances and might contribute to their onset. Conversely they were not found in male NOD mice although some of these mice show insulitis. The presence of these inhibitory T cells might thus represent an early and sensitive marker of anti-islet cell-mediated autoimmunity.

We previously showed that circulating lymphocytes from more than 90% of insulin-dependent diabetics, block extra insulin secretion induced by stimulatory media in mouse pancreatic cells in vitro, without altering the secretion of glucagon. The present work demonstrates that this phenomenon depends on lymphocytes having the OKT3 marker, i.e., thymodependent lymphocytes. However, OKT4+ T helper cells are not required for the above phenomenon as proved by experiments using monoclonal sera against the OKT4 marker. When diabetes is associated with other autoimmune diseases, the pancreatic lymphocyte cytotoxicity observed in vitro is inhibited by the addition of a normal lymphocyte population; this could indicate that a "suppressor" factor is lacking in these patients. Conversely, addition of normal lymphocytes does not prevent lymphocyte cytotoxicity in diabetics without associated autoimmune diseases. Such a difference confirms the present trend to make a distinction between these two categories of diabetes.

The diabetic db/db mice of the C57 BL/KsJ strain display anti-islet immunity, thymic dysfunction, and lymphopenia. In the present work, lymphocytes, T-cells, and T-cell subsets were enumerated in thymus and spleen from diabetic db/db mice and their db/ + heterozygote littermates from the 10th day to the 10th month of life. A significant lymphopenia was detected in thymus and spleen from the second month on, involving specifically the T-cell compartment, as assessed by use of a monoclonal anti-Thy1 antibody in indirect fluorescence. The study of T-cell subsets by monoclonal anti-Lyt1 and anti-Lyt2 antibodies revealed a significant increase in Lyt1+ cells and a decrease in Lyt2+ cells, with a corresponding increase of the Lyt1+/Lyt2+ ratio. These anomalies appeared early in life, and were apparently linked neither with the degree of hyperglycemia nor with weight loss or infection. The T-cell depletion in thymus was more pronounced in young male (less than 3 mo) than in young female db/db mice. These alterations may correspond to an increase in the helper/suppressor-cytotoxic ratio and could be linked with the thymic anomalies present in these mice, contributing to the development of anti-islet autoimmunity.