Increased Interleukin-17 Proportion in Helper T Cell (Th17) in Patients Post Allogeneic Hematopoietic Stem Cell Transplantation.

Cytokines belonging to interleukin -17 family (IL-17A, B, C, D, E and F) are produced mainly by activated and memory CD4+ cells which are described as Th17. These cells (IL-17+CD4+) promote autoimmune reaction (mouse model), were found in tissues involved in autoimmune process in man and produce IFNgamma. Therefore, Th17 cells constitute a population which may contribute to aGvHD together with Treg cells (FoxP3+). In this study we examined the cytoplasmic expression of IL-17A and FoxP3 in CD4+ lymphocytes in 6 pts post HSCT (3 MUD and 3 SIB, median (mean) age: 35 (33,9) years, 3 AML, 2 ALL-T, 1 SCID). Three of them developed aGvHD. All consecutive pts were included to the study. Peripheral blood lymphocytes were examined or at the very first day of aGvHD manifestation either at the beginning of hematological reconstitution and thereafter in one week intervals. Nine healthy individuals served as controls. PBMC were stimulated with BD Leukocyte Activation Cocktail containing mixture of PMA, Ionomycin and brefeldin A in the presence of BD GolgiStop then stained with CD4, IL-17A and FoxP3 monoclonal antibodies and analyzed in CD4+ and CD4- lymphocytes gates by three colour flow cytometry. Therefore, all results reflect the situation in stimulated cells. FoxP3 mRNA was measured in unstimulated PBMC with the use of qPCR. We found:

1. Patients post HSCT (during 6 weeks period post transplant) had higher percentages of IL-17A+ cells in CD4+ lymphocytes as compared to healthy controls (2.0%±1.08 vs 0.2%±0.06, p=0.006) independently whether they developed or not aGvHD.

2. In contrast to the lower proportion of total CD4+ cells in pts post HSCT then in controls (18.5%±2.1 vs 34.9%±0.7, p=0.0006) proportions of a subset of CD4+ lymphocytes which produced IL-17A+ were rather higher in pts post HSCT as compared to controls (0.2%±0.06 vs 0.07%±0.02, ns).

3. IL-17A+ cells contributed rather to CD4+ then CD4- lymphocytes in controls (0.2%±0.06 vs 0.05%±0.01, p=0.007) as well as in pts post HSCT (2.0%±1.08 vs 0.05%±0.01, p=0.00004)

4. FoxP3 positive cells in stimulated CD4+ lymphocytes were in lower proportions in pts post HSCT then in healthy individuals (51.6%±3.9 vs 62.2%±2.9, ns).

5. Percentages of FoxP3+CD4+ in stimulated lymphocytes (cytometry) correlated with quantities of FoxP3 mRNA in unstimulated PBMC (qPCR) (R=0,42, p=0,065).

6. Notably, there was a negative correlation between proportions of FoxP3 and IL-17A positive cells in CD4+ lymphocytes in patients post HSCT (R= − 0,55; p=0,012).

In conclusion, HSCT was associated with:

1. an increase in proportion of Th17 cells,

2. a higher contribution of IL-17A+ cells to the pool of CD4+ cells as compared to healthy individuals and

3. a network in the immune system in which CD4+ FoxP3 + cells and Th17 cells play an opposite role.