Th17 Cells Blood Proportions Are Influenced by Factors Associated with CRP Serum Level Increase and with NOD2/CARD15 Gene Mutation

Abstract 4487

HLA mismatch constitute the risk of aGvHD. However, environmental factors play a significant role what is exemplified by the protective effect of germ free environment. This association can be discussed on the ground of a significant role of cytokine milieu which determines lymphocytes subsets differentiation. Our previous work showed a decrease of proportions of Th17 cells in blood at the onset of aGvHD (Dlubek et al., Transplantation Proceedings 2010; 42(8):3277-9). These cells, however, were seen at the tissue site likely being marginalized during aGvHD process. In this study we looked at Th17 lymphocytes proportions in blood of patients post alloHSCT in the context of an increase of C-reactive protein in serum and NOD2/CARD15 gene mutations.

Eighty four patients (median age: 41 yrs, range: 1–64 yrs) entered the study. All of them were clinically followed for the presence of infections complications and aGvHD. Blood work was done at the very first day of the later complications and in addition routinely in one week intervals beginning from the day of transplantation. The cells were labeled for CD4 (Becton Dickinson, San Jose, CA, USA), intracellular IL-17A (e-biosciences, San Diego, CA, U04)SA) and FoxP3 (Becton Dickinson, San Jose, CA, USA) detection. CRP was measured in serum using a Behring BN Prospect nephelometer (Dade Behring Inc., Marburg, Germany). All studied cases were genotyped for NOD2/CARD15 mutations: 104C-T (SNP8, Arg702W; rs.2066844), 2722G-C (SNP12, Gly908Arg; rs.2066845), and 3020insC (SNP13, Leu1007fsinsC; rs.2066847) with the use of RFLP-PCR technique.

It was found:

1. Acute GvHD manifestation was seen in 34 (40%) patients among them 22 (26%) had only skin symptoms and 12 (14%) presented gut manifestation. Gut aGvHD was seen at later time post HSCT than skin aGvHD (median 33 vs. 21 days, M-W U test p=0.035).

2. Twelve patients had NOD2/CARD15 mutations. They had lower proportions of Th17 lymphocytes in blood as compared to those lacking these mutations (median 0.03 vs. 0.07%, M-W U test p=0.052) and 6 (50%) of them presented aGvHD.

3. CRP levels measured from 3 to 5 days before clinically apparent aGvHD were significantly higher in patients with gut symptoms as compared to those presented only skin lesions (median 66.8 vs. 7.61 mg/L, M-W U test p=0.020). Notably, patients having only skin symptoms had rather lower CRP levels in sera than those without aGvHD (median 7.61 vs. 27.6 mg/L, M-W U test p=0.028).

4. In patients with gut manifestation FoxP3+CD4+ cells proportions were negatively correlated with serum CRP levels (Spearman r −0.664, p=0.018) what was not seen in patients presented only skin symptoms aGvHD (0.068, p=0.769).

In conclusion:

CRP elevation constituted the risk of gut aGvHD and was associated with rather exhausted T regulatory cell function. NOD2/CARD15 mutations associated factors contributed to the lowering of Th17 cells in blood what was previously found to be associated with the overt manifestation of aGvHD (Dlubek et al., Transplantation Proceedings 2010; 42(8):3277-9). These both findings are in line with the hypothetical role of microbial pathogens which via inflammation favor differentiation of lymphocytes to Th17 positive cell subset.