Sanja Milosavljevic, PhD
The pathological disease process of beta-cell destruction which leads to type 1 diabetes (T1D) onset begins several years prior to diagnosis. During that time, remaining beta-cells keep up with insulin requirements until only 20-30% of the beta-cell mass is left. At that point, the body's need for insulin surpasses production and clinical symptoms develop. After metabolic stabilization, in individuals experiencing a remission phase after the onset (called the "honeymoon phase"), the same process of autoimmune destruction of insulin-producing beta-cells continues until complete beta-cell depletion is achieved. Recent-onset T1D subjects (requiring lower dose of exogenous insulin after the diagnosis, compared to a longstanding T1D subject) represent an extremely important group of patients because of ongoing beta-cell destruction which can be studied using a variety of sophisticated approaches. Understanding the immunobiology of the disease will hopefully lead to improved clinical therapies and ultimately a long-term cure.
Since T1D is T-cell mediated disorder, we are interested in the role T-cells have in T1D pathogenesis. There are a variety of T cells types and the ones most interesting to us are T cells with regulatory properties. Careful isolation of these cells by flow cytometry is one of the most important steps in this type of research because it affects the conclusions we can make from subsequent experiments. In our FACS isolation, we always use Fluorochrome Minus One (FMO) to set a threshold and apply other rigorous controls appropriate to the staining we are doing (Figure 1).
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Figure 1. Example of FACS sorting. PBMCs were stained with a cocktail of monoclonal antibodies (Abs) where each Ab is conjugated to specific fluorochrome. The threshold for CD25 positivity was determined using FMO (left panel). However, only the top 1% of cells expressing the highest level of CD25 surface marker are isolated as CD4+CD25+high T cells. This cell population has high proportion of cells with regulatory properties, confirmed using both surface markers and in a standard in vitro suppression assay.
CD4+CD25+high regulatory T cells have a crucial role in maintaining immune homeostasis. Their role is to regulate proliferation, migration and the function of all other T cells in the body, including those with autoimmune specificities. We have developed a specific combination of stains (YOPRO1/7AAD) for highly sensitive detection of early apoptotic human PBMCs [1]. Using this stain we recently reported significantly higher apoptosis levels of CD4+CD25+high T-cells in individuals in the T1D honeymoon phase as well as in individuals with a strong likelihood of being diagnosed (relatives of individuals with T1D that are at high risk to develop T1D). However, longstanding T1D and T2D subjects failed to show the same trait [2], as presented in Figure 2.
Figure 2. Apoptosis of CD4+CD25- and CD4+CD25+high T cells determined by YOPRO1/7AAD staining across T1D-related study groups. T cell subsets were FACS sorted and stained simultaneously with YOPRO1/7AAD. CD4+CD25- (left panel) do not show differences between groups (ANOVA F=0.59, df (6, 78), NS). CD4+CD25+high T cells (right panel) showed significantly higher apoptosis in recent-onset T1D subjects as well as in group of high risk first degree relatives of T1D subjects that have 2 or 3 detected T1D-related auto-antibodies (ANOVA F=16.07 (df 6, 78), p<0.0001).
Apoptosis of regulatory T cells among CD4+CD25+high T cells has been for the first time reported for fresh ex vivo T cells and further investigation of this phenomenon (their antigen specificity, mechanism of apoptosis and life cycle) is one of our current projects. Our hypothesis is that by inhibiting ongoing apoptosis (using an appropriate agent), regulatory T cells could be rescued from death and able to successfully guard pancreatic beta-cells from destruction. Additionally, pre-diabetic individuals at high risk for developing disease could also potentially be selected for preventive therapy based on their levels of ongoing CD4+CD25+high T cell apoptosis.
An additional project of interest relates to the in vitro generation of higher number of functional regulatory T cells. We are exploring two ways to achieve this: a) expansion of naturally derived CD4+CD25+high T cells, and b) conversion of CD4+CD25- T cells into cells with regulatory properties (adaptive regulatory T cells). Other approaches could also be applied, such as the in vitro differentiation of stem cells.
Type 1 diabetes is a challenging disease with a worrisome increase in incidence, but we are convinced that working together with patients and their families and using our multidisciplinary approach, we will significantly contribute to a world-wide effort in fighting this disorder and discovering a much needed cure.
Selected manuscripts:
1. Glisic-Milosavljevic S, Waukau J, Jana S, Jailwala P, Rovensky J, Ghosh S: Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods. Cell Prolif 2005, 38:301-311.
2. Glisic-Milosavljevic S, Waukau J, Jailwala P, Jana S, Khoo H-J, Albertz H, Woodliff J, Koppen M, , Alemzadeh R, et al.: At risk and Recent-Onset Type 1 Diabetic Subjects Have Increased Apoptosis in the CD4+CD25+high T-cell fraction. PLoS ONE 2007, 2:1-7.
3. Glisic-Milosavljevic S, Wang T, Koppen M, Kramer J, Ehlenbach S, Waukau J, Jailwala P, Jana S, Alemzadeh R, Ghosh S.: Dynamic changes in CD4+CD25+high T cell apoptosis after the diagnosis of type 1 diabetes (submitted to Clin Exp Immunol)