Zuzana Hroteková

CD 127- and FoxP3+ expression on CD25+CD4+ T regulatory cells upon specific diabetogeneic stimulation in high-risk relatives of type 1 diabetes mellitus patients.

Abnormalities in CD4+CD25+ regulatory T cells (Treg) may contribute to type 1 diabetes (T1D) development. First‐degree relatives of T1D patients are at increased risk especially when they carry certain HLA II haplotypes. Using two novel markers of CD4+CD25+ Treg (CD127− and FoxP3+ respectively), we evaluated number and function of Treg after specific stimulation with diabetogeneic autoantigens in 11 high‐risk (according to HLA‐linked risk) relatives of T1D patients and 14 age‐matched healthy controls using a cytokine secretion assay based on interferon‐γ (IFN‐γ) production. High‐risk relatives of T1D patients had significantly lower pre‐ and post‐stimulatory number of CD127− Treg than that of healthy controls (P < 0.05). Labelling Treg with FoxP3+ demonstrated similar trend but did not reach statistical significance. Although the stimulation with diabetogenic autoantigens did not lead to a significant change in number of Treg in both groups, the defective function of Treg was performed by significantly higher activation of diabetogeneic T cells in high‐risk relatives of T1D patients compared to healthy controls (P ≤ 0.02). Individuals at increased HLA‐associated genetic risk for T1D showed defects in Treg.

Immune regulatory T cells in siblings of children suffering from type 1 diabetes mellitus

Patients with type 1 diabetes are suffering from defects in immune regulatory cells. Their siblings may be at increased risk of type 1 diabetes especially if they are carriers of certain human leucocyte antigen (HLA) alleles. In a prospective non‐randomized study, we intended to evaluate 31 healthy siblings of paediatric patients with type 1 diabetes and explore immune regulatory populations of CD4+CD25+ T cells and natural killer (NK) T cells. Tested siblings of type 1 diabetes patients were stratified according to the HLA‐associated risk of possible diabetes development. Immune regulatory function of CD4+CD25+ T cells was tested in vitro. Significant differences in CD4+CD25+ but not in NK T cells have been identified. Siblings of type 1 diabetes patients carrying high risk HLA alleles (DQA1*05, DQB1*0201, DQB1*0302) had significantly lower number of immune regulatory CD4+CD25+ T cells than the age‐matched healthy controls or siblings carrying low‐risk HLA alleles (DQB1*0301, DQB1*0603, DQB1*0602). Regulatory function of CD4+CD25+ T cells demonstrated a dose‐escalation effect. In siblings of type 1 diabetes patients, the defect in immune regulatory CD4+CD25+ T cells exists in association with genetic HLA‐linked risk for type 1 diabetes.

Selective Depletion of Alloreactive T Cells and Study of Anti-Tumor Activity of Specific T Cell Clones in Patients with Leukemia and Renal Carcinoma

BACKGROUND Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patients PBMC. CONCLUSION We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.