Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or K2EDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺, or CD56⁺CD16⁺ cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4⁺ cells were in general less affected by isolation and cryopreservation than diff CD8⁺ cells. Terminally diff effector CD4⁺ and CD8⁺ cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4⁺ and CD8⁺ cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4⁺CD25^hi expressing CD101 or CD129, CD4⁺CD25hiCD127⁻, and CD4⁺CD25^hiCD127⁻FOXP3⁺, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4⁺CD25⁺CD127⁻ cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4⁺, CD8⁺, and CD25^hi lymphocytes are in general not influenced by isolation and long-term cryopreservation.

Type 1 diabetes (T1D) and coeliac disease are both characterized by an autoimmune feature. As T1D and coeliac disease share the same risk genes, patients risk subsequently developing the other disease. This study aimed to investigate the expression of T helper (Th), T cytotoxic (Tc) and regulatory T cells (T_reg) in T1D and/or coeliac disease children in comparison to healthy children. Subgroups of T cells (Th:CD4⁺ or Tc:CD8⁺); naive (CD27⁺CD28⁺CD45RA⁺CCR7⁺), central memory (CD27⁺CD28⁺CD45RA^-CCR7⁺), effector memory (early differentiated; CD27⁺CD28⁺CD45RA^-CCR7^- and late differentiated; CD27^-CD28^-CD45RA^-CCR7^-), terminally differentiated effector cells (TEMRA; CD27^-CD28^-CD45RA⁺CCR7^-) and T_reg (CD4⁺CD25⁺FOXP3⁺CD127^-) cells, and their expression of CD39, CD45RA, CD101 and CD129, were studied by flow cytometry in T1D and/or coeliac disease children or without any of these diseases (reference group). Children diagnosed with both T1D and coeliac disease showed a higher percentage of TEMRA CD4⁺ cells (P<0·05), but lower percentages of both early and late effector memory CD8⁺ cells (P<0·05) compared to references. Children with exclusively T1D had lower median fluorescence intensity (MFI) of forkhead box protein 3 (FoxP3) (P<0·05) and also a lower percentage of CD39⁺ and CD45RA⁺ within the T_reg population (CD4⁺CD25⁺FOXP3⁺CD127^-) (P<0·05). Children with exclusively coeliac disease had a higher MFI of CD101 (P<0·01), as well as a higher percentage of CD129⁺ (P<0·05), in the CD4⁺CD25^hi lymphocyte population, compared to references. In conclusion, children with combined T1D and coeliac disease have a higher percentage of differentiated CD4⁺ cells compared to CD8⁺ cells. T1D children show signs of low CD39⁺/CD45RA⁺ T_reg cells that may indicate loss of suppressive function. Conversely, children with coeliac disease show signs of CD101⁺/CD129⁺ T_reg cells that may indicate suppressor activity.

Children diagnosed with a combination of type 1 diabetes (T1D) and celiac disease (CD) show a dysregulated T helper (Th) 1/Th17 response. Besides the cellular involvement, several soluble immune markers are involved in the autoimmune process of both T1D and CD. Only few studies have examined the peripheral pattern of different cytokines, chemokines, and acute phase proteins (APP) in children with combined T1D and CD. To our knowledge no studies have evaluated the serum levels of adipocytokines and matrix metalloproteinases (MMPs) in this context. The purpose of the present study was to acquire more knowledge and to gain deeper understanding on peripheral immunoregulatory milieu in children with both T1D and CD. The study included children diagnosed with both T1D and CD (n=18), children with T1D (n=27) or CD (n=16), and reference children (n=42). Sera were collected and analysis of twenty-eight immune markers (cytokines, chemokines, APPs, adipocytokines and MMPs) was performed with Luminex technique. The major findings showed that children with double diagnosis had lower serum levels of interleukin (IL)-22, monocyte chemoattractant protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, procalcitonin, fibrinogen, visfatin and matrix metalloproteinase (MMP)-2. These results indicate a suppressed immune profile in children with combined T1D and CD, including Th17 cytokines, chemokines, acute phase proteins, adipocytokines and MMPs. We conclude that besides cytokines and chemokines, other immune markers, e.g. APPs, adipocytokines and MMPs, are of importance for further investigations to elucidate the heterogeneous immune processes present in patients diagnosed with T1D in combination with CD.