?(Fig.66c). Open in a separate window Figure 6 Comparison of the proportion of functional CD4+ T cell subsets between type 2 diabetes mellitis (T2DM) and control groups. capacity of myeloid cells to secrete proinflammatory cytokines was not diminished in terms of the sensitivity and magnitude of the response. Furthermore, cytolytic activity and interferon (IFN)\ production of natural killer (NK) cells and CD8+ T cells were not decreased in T2DM patients. Phenotypical maturation of dendritic cells, indicated by the up\regulation of major histocompatibility complex (MHC) proteins and co\stimulatory molecules in response to lipopolysaccharide (LPS), was slightly enhanced in T2DM patients. Finally, the functional differentiation profiles of CD4+ T cells did not differ between T2DM patients and the control group. These data indicate that patients with long\lasting T2DM do not have any gross functional defects in immune cells, at least in circulating monocytes, dendritic cells, NK cells and T lymphocytes. 22 males and 13 females). Body mass index (BMI) of the two groups MELK-IN-1 was also not different (DM, 234??32?kg/m2; controls, 242??22 kg/m2). Diabetes duration was 18.8??79 [mean??standard deviation (s.d.)] years, and fasting glucose concentration and HbA1c were 144??69?mg/dl and 937??18%, respectively. Among them, 30 patients (900%) had microvascular complications, such as retinopathy, neuropathy or cardiomyopathy. Table 1 Demographic and clinical characteristics of the study population 663??268%; 7445??918% 678??1019%, mean??s.d.), while the opposite was true of the memory B cell subset (2555??918% 322??1019%) (Fig. ?(Fig.2).2). Among the four CD4+ and CD8+ T cell subpopulations, the proportion of effector cells was higher in T2DM than the control group (285??148% 216??106%; 4333??1416% 3601??1364%), whereas the other three subpopulations did not show a difference (Fig. ?(Fig.2e).2e). There was no difference in the proportion of exhausted and senescent T cells between T2DM and the healthy control group (Fig. ?(Fig.2f).2f). The composition of PBMCs did not change in each individual during the period of a year (Fig. ?(Fig.2c2c and data not shown). Open in a separate window CD163 Figure 2 Comparison MELK-IN-1 of the cell composition of peripheral blood mononuclear cells (PBMCs) between type 2 diabetes mellitis (T2DM) patients and normal control subjects. Immune cell subsets were identified by immunofluorescent staining with lineage\defining antibodies. The gating strategy and a representative staining profile for determining the frequency of (a) various T cell subsets and (b) non\T cell populations [I, total B lymphocytes; II, memory B cells; III, naive B cells; IV, monocytes; V, natural killer (NK) cells; VI, myeloid dendritic cells (DCs)]. (c) Stability of the cell composition of PBMCs prepared at different time\points from an individual. Two T2DM patients (DM\16 and DM\26) and two control subjects (CO\01 and CO\02) were studied. *Not determined. (d) Cell composition of PBMCs, T and B cells. (e) The proportion of major T cell subsets in each CD4+ and CD8+ T cells. (f) The proportion of exhausted and senescent cell populations in each CD4+ and CD8+ T cells. Data are shown as box plots with 5C95 percentile whiskers. The two\tailed MannCWhitney test was used to compare the two groups. Proinflammatory cytokine production of leucocytes One of the major defence mechanisms of the innate immune system against microbial infection is the production of proinflammatory cytokines to eliminate the invading pathogens. We examined if this function is intact or not in patients with T2DM. When PBMCs were stimulated with different doses of LPS, a representative pathogen\associated molecular pattern (PAMP), the LPS doseCresponse of TNF\ secretion was not statistically different between the T2DM and normal control groups (Fig. ?(Fig.3a).3a). Similarly, the LPS concentration inducing half\maximal response of TNF\ secretion and the amount of TNF\ secreted at the half\maximal response were not different between the two groups (4.24??1.52 4.57??0.93?ng/ml; 9586??9310 7975??5916?pg/ml, mean??s.d.). IL\1 secretion of PBMCs from T2DM patients also did not differ from that of control individuals (Fig. ?(Fig.3b).3b). Notably, it was revealed that spontaneous secretion of TNF\ and IL\1 from unstimulated PBMCs was not different between the two groups (data not shown). In addition to proinflammatory cytokines, an anti\inflammatory cytokine (IL\10) secretion was not significantly different between the two groups upon stimulation with LPS. The cytokine secretion of PBMCs tended to be preserved across different time\points of blood sampling from the same subjects (Fig. ?(Fig.33c). Open in a separate window Figure 3 Comparable production of proinflammatory cytokines from peripheral blood mononuclear cells (PBMCs) between type 2 diabetes mellitis (T2DM) and the control group. PBMCs were stimulated with different doses of lipopolysaccharide (LPS), and the quantity of secreted cytokines was measured from culture supernatants. (a) DoseCresponse curves, concentration of LPS that induces half\maximal response (EC50), and the half\maximal response of tumour necrosis factor (TNF)\ production. Each line (left two graphs) and each dot (right two graphs) represents the values obtained MELK-IN-1 from each individual. The data in the centre graph are presented as mean??standard error of the mean (s.e.m.) of the group (= 33 for each). (b) Interleukin (IL)\1 or IL\10 production of PBMCs in response to increasing doses of LPS. The results are presented as mean??s.e.m. (= 33 for each group). (c) Reproducibility.