TH17 CD4+ CELLS Disappear FROM BLOOD at the Onset of aGVHD
Authors: Lange Andrzej , Lach Antonina , Agnieszka Wojtowicz , Drabczak-Skrzypek Daria , Dorota Dlubek 
Published: October 2019

Abstract
Abstract 2307
IL-17 producing cells expand in the environment rich in IL-6 and TGF-beta. Our previous observation documented an increase in IL-6 and its reading protein CRP during the hematological recovery and later on at the time of frequent infectious complications. Therefore, HSCT patients are in a high risk of the expansion of Th17 lymphocytes which are potent pro-inflammatory lymphocytes. In this study we investigated the proportions of IL-17 producing cells in 7 days intervals beginning from hematological recovery covering the time of a risk of aGvHD and infectious complications post transplant. To illustrate the background to the generation of Th17+ lymphocytes serum CRP levels were measured and the manifestation of aGvHD was noted and followed. This study included also determination of NOD2/CARD15 gene mutation.
The cytoplasmic expressions of IL-17A were studied in stimulated PBMC (brefeldin A, Ionomycin and PMA) of alloHSCT pts (58 pts, median age: 42 yrs, range; 1.0–64 yrs), 14 patients manifested aGvHD at the time of hematological recovery and in 13 patients with aGvHD was clinically apparent later time post HSCT (from 18 to 93, median 36). The cells were labeled for CD4 (Becton Dickinson, San Jose, CA, USA) and intracellular IL-17A (e-biosciences, San Diego, CA, USA) detection. Fifty six transplanted cases were genotyped for 2104C-T (SNP8, Arg702W; rs.2066844), 2722G-C (SNP12, Gly908Arg; rs.2066845), and 3020insC (SNP13, Leu1007fsinsC; rs.2066847) mutations with the use of RFLP-PCR technique.
We found: (1) 14 pts with aGvHD seen at the time of hematological recovery (median 15.5 days, from 9 to 21 days post HSCT, when WBC > 500/ul and lymphocytes > 200/ul,) had lower percentages of IL-17A producing cells in CD4+ lymphocyte population as compared to those lacking aGvHD at the same stage of recovery (median 15 days, from 10 to 31), (0.29%±0.09 vs 0.78%±0.13, p=0.02, M-W U-Test). (2) 13 pts with aGvHD seen between 18 to 93 day post HSCT (median 36) had also lower percentage of IL-17A producing cells in CD4+ lymphocytes as compared to those lacking aGvHD and examined for the presence of IL-17A+ lymphocytes at the similar time post HSCT (median 35 day, from 27 to 43), (0.29±0.10 vs 0.99%±0.25, p=0.015 M-W U-Test), (2) manifestation of aGvHD at later time post HSCT allowed to examine the presence of IL-17A producing CD4+ lymphocytes prior to and then at the aGvHD manifestation. It was a significant decrease in proportions of IL-17A producing CD4+ lymphocytes at the day of aGvHD manifestation as compared to previous examinations performed 3 to 7 days before aGvHD became clinically apparent (1.02%±0.33 vs 0.29%±0.10, n=13, p=0.0009, Wilcoxon Test for pairs). Among these 13 pts 6 had only skin and 7 also gastrointestinal aGvHD, the latter pts had lower percentage of IL-17A producing CD4+ lymphocytes than those with only skin lesions (0.21%±0.08 vs 1.33%±0.47, for gut and skin aGvHD, respectively).
3) 5 out of 56 (8.9%) transplanted cases were positive for at least one out of three major NOD2/CARD15 mutations (SNP8, SNP12 and SNP13), notably all 3 patients with gut manifestation and investigated for NOD2/CARD15 mutation had this gene mutated what was seen only in 4 out of 17 pts with skin symptoms (p=0.017 Fisher exact test). In the present observation no correlation was found between CRP elevation and the proportion of IL-17 producing cells in CD4+ lymphocytes.

Conclusions;
1. Apparent clinical manifestation of aGvHD associates with a low proportion of Th17 lymphocytes in blood, 2. This is rather an active process as Th17+ cells proportions decline in blood during 3 to 7 days prior to overt aGvHD. 3. Gut manifestation associates with significantly lower number of blood Th17+ lymphocytes than seen in pts with only skin lesions. 4. NOD2 mutation may constitute a risk factor of gut aGvHD.
Supported by grant N R13 0082 06 from the Polish Ministry of Science & Higher Education.

Disclosures:
No relevant conflicts of interest to declare.