ALBERT

Description: 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: 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).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.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).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. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4079 Alloreactivity strongly influences the course post HSCT being a primary force in pathomechanism of aGvHD, shapes the immunological reconstitution post-transplant and mounts graft vs leukemia effect. Therefore the understanding of the interplay between the main subsets of CD4 positive cells may help in understanding the mechanism which facilitates the activity of the immune system post HSCT. For that we investigated the presence of cells with a potential to generate IL-17, IFN-gamma and FoxP3 lymphocytes appearing in blood to post HSCT. The cytoplasmic expressions of IL-17A, FoxP3 and IFN-gamma were studied in stimulated PBMC (brefeldin A, Ionomycin and PMA) of alloHSCT patients (63 patients, median age: 43 yrs, range: 5.5 –60 yrs), 12 patients manifested aGvHD at the time of hematological recovery and in 17 patients aGvHD was clinically apparent after hematological reconstitution (from 17 to 93, median 31). The cells were labeled for CD4 (Becton Dickinson, San Jose, CA, USA) and intracellular IL-17A (e-biosciences, San Diego, CA, USA), FoxP3 and IFN-gamma (Becton Dickinson, San Jose, CA, USA) detection. 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. Patients post HSCT studied at the beginning of hematological recovery (1st observation point) had lower blood levels of IL-17 producing lymphocytes in CD4+ cells (Th17) independently whether they developed aGvHD at the hematological recovery or (later time) post-transplant as compared to those lacking aGvHD (median 0.090% (0.195%) vs 0.425%, p=0.019 (p=0.126)). Primarily low Th17 blood level in aGvHD cases normalized (increased) along the observation time, when a positive response to the therapy was noticed, being in one week intervals in the levels as follow (1st vs 2nd (3rd) time point: median 0.090% vs 0.355% (0.355%), p=0.049 (p=0.061)). Of note, patients lacking aGvHD at any time post HSCT had similar blood levels of Th-17 cells during 30 days post-transplant observation time. The first quartile of Th17 blood values of the whole group was 0.1% and this value was chosen as a cut-off point dividing the whole group into patients with low and higher blood Th17 levels. It became apparent that survival of patients having low Th17 blood levels at the beginning of hematological recovery, irrespective of the presence of absence of aGvHD, had poorer survival (2 yrs survival 30% vs 61%, p=0.045). When fatal cases were analyzed patients with low Th17 blood levels died rather due to aGvHD and those with higher levels due to infectious complications or relapse (7/10 vs 4/16, p=0.043). FoxP3+ cells and IFN-gamma producing lymphocytes were determined in blood of HSCT patients at the same time as it was done for Th17 cells. It was shown that the blood levels of FoxP3+ cells were well correlated with the levels of IFN-gamma producing cells (r=0.405, p=0.003) but not to the similar extent with Th17 cells (r=0.253, p=0.047). Th17 plays an important role in the mucous membrane barrier against microbial infections. Mutations in NOD2/CARD15 gene are described as a risk factor of aGvHD. We found that mutations in the NOD2/CARD15 gene is not more frequent in aGvHD cases but influences the level of Th17 in blood in such a way that patients with mutations had lower blood values of Th17 at the hematological recovery in the aGvHD group (median 0.080% vs 0.210%, p=0.051) as well as in the whole alloHSCT patient group (median 0.080% vs 0.310%, p=0.027). Conclusions: 1.Th17 lymphocytes being lower at the beginning of aGvHD manifestation likely to marginalize at the sites of inflammation. 2.Low levels of Th17 were associated with rather poor survival of HSCT pts with aGvHD as a primary cause of death. 3.NOD2/CARD15 mutations associate with lower values of Th17 in blood. Supported by the grants N N402 430039 and N R13 0082 06 from the Polish Ministry of Science & Higher Education. Disclosures: No relevant conflicts of interest to declare.