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).
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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.