Supplemental Materials Regulation of T cell proliferation by JMJD6 and PDGF-BB during chronic hepatitis B infection Cai-Feng Chen 1#, Xia Feng 2#, Hui-Yu Liao 2#, Wen-Jing Jin 1, Jian Zhang 3, Yu Wang 4 Lu-Lu Gong 1, Jing-Jun Liu 1, Xiao-Hui Yuan 1, Bin-Bin Zhao 1, Ding Zhang 1, Guo-Feng Chen 3 Ying Wan 5, Jian Guo 4 Hui-Ping Yan 2, and You-Wen He 4 1
Table S1. Characteristics of treatment-naïve CHB patients and healthy donors. Characteristic Value CHB patients Healthy donors Number 39 39 %Men 67% 51% Median Age 35 (20-69) 32(24-54) Median HBV DNA(copies/ml) 1.2 10 6 (<500-2.0 10 8 ) <500 Median ALT level(u/l) 56.8(17.9-423) 18(8.3-45.0) Median AST level(u/l) 49.2(12.6-225) 22(10-42) Median TBIL level(µm/l) HBeAg postive (number) 16.1(4.7-425.1) 29 7.2(2.8-25.4) 0 ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin. 2
Table S2 List of primer sequences for qpcr detection of 32 cell cycle regulators. Primers for real time PCR Official Symbol Forward Reverse BAX GACATGTTTTCTGACGGCAAC AAGTCCAATGTCCAGCCC PCNA CCGAAACCAGCTAGACTTTCC GATGAGGTCCTTGAGTGCC RAD9A AGAAGTTCCGCTCACTGTTC AGGCTTCAGGTTCTTGGTTC TP53 GCCATCTACAAGCAGTCACAG TCATCCAAATACTCCACACGC RBL2 GAACCTGGGAACTTTGGAGAG GCGTTCAGACACCTTGAGAG CDKN2B GTTAAGTTTACGGCCAACGG ACCTTCTCCACTAGTCCCC TFDP1 ACTCACTTTGCCTCTCAGAAC CTTTCCTCTGCACCTTCTCG CCND2 CCTCCAAACTCAAAGAGACCAG TTCCACTTCAACTTCCCCAG BRCA2 TTCATGGAGCAGAACTGGTG AGGAAAAGGTCTAGGGTCAGG BCL2 ACTGGAGAGTGCTGAAGATTG AGTCTACTTCCTCTGTGATGTTG CDK8 AAGAGGAAAGATGGGAAGGATG GAAGAGAAATGACGTTTGGATGC MKI67 AAAAGAATTGAACCTGCGGAAG AGTCTTATTTTGGCGTCTGGAG BIRC5 CTTCATCCACTGCCCCAC ACTTTCTCCGCAGTTTCCTC SUMO1 GGGAAGGGAGAAGGATTTGTAA GTCCTCAGTTGAAGGTTTTGC UBE1 GTCACAAAGTTTTCGGCAGTC AATGTGCCAAGTCCAGATCC CDKN3 ACAATATCACCAGAGCAAGCC GCAGCTAATTTGTCCCGAAAC CDK6 TCGATGAACTAGGCAAAGACC AGGTGGGAATCCAGGTTTTC CDK5R1 AGATAAATGCCGACCCACAC TGAATCCTTGAGCCATGACG DNM2 CAGTAAGCTCAGTTCCTACCC AGTCCTCATGGTTCGTGTTG CDC20 GATGTAGAGGAAGCCAAGATCC AAGGAATGTAACGGCAGGTC GTF2HI TCCACATCCAATCATAAGCAGG CTTAGACCGTTACAGCCATCAG ARHI ACGTATCTCCCCTCCGAATC GCGGTAATCTCTGATCTTCCTG RPA3 TGTGGAAGTGGTTGGAAGAG AGGATAAAACTGAGGGAAGTCATG MNAT1 CTTGAAGCTGATGGTGAATGTG AGTTGTACCCTGAAGTTGCTC GTSE1 AAGTACTGCCACAGAAGTAGC CGATGAGAGGAAGGTCAATGAG GADD45A GGGAAAGTCGCTACATGGATC GTGTAGGGAGTAACTGCTTGAG ATM ATTCCGACTTTGTTCCCTCTG CATCTTGGTCCCCATTCTAGC MAD2L1 GACAGATCACAGCTACGGTG GGCGGACTTCCTCAGAATTG NBN AGACCAACTCCATCAGAAACTAC AATGAGGGTGTAGCAGGTTG MRE GTAACCCAAGCCATACAAAGC ACCTCCACTATAGTCCACTCG P21 TGTCACTGTCTTGTACCCTTG GGCGTTTGGAGTGGTAGAA CDK4 TTCCCATCAGCACAGTTCG TCTACATGCTCAAACACCAGG Forward, sense primer; Reverse, anti-sense primer. 3
Table S3. Characteristics of treatment responsive and unresponsive CHB patients. Characteristic Value HBeAg seroconversion HBeAg non-seroconversion Baseline EOT Baseline EOT Number 5 5 5 5 %Men 80% 80% 80% 80% Age 32(25-47) 33(26-49) 45(27-61) 46(28-62) HBV DNA(copies/ml) 3.2 10 5 (1.1 10 4-3.2 10 6 ) <500 8.2 10 5 (7.2 10 2-6.4 10 6 ) <500 ALT level(u/l) 82.16(41.8-191.8) 26(15.2-40.5) 73(45.9-130.9) 19(9.0-43.2) AST level(u/l) 51.96(25.9-114.7) 32(11.2-46) 49.1(32-62.1) 28(12-45) TBIL level(µm/l) 64.68(4.3-242.2) 6.8(2.6-10.6) 31.6(20.2-42.9) 7.2(2.8-11.2) HBeAg 1150(604-1888) 0.21(0-0.616) 814(660-1164) 118.9(15.11-446 ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin. 4
Table S4. Treatment details of 10 CHB patients. HBV1-5 were patients who underwent HBeAg seroconversion and HBV6-10 were patients who did not undergo HBeAg seroconversion Groups Drug names Course(months) HBV1 AdefovirDipivoxil Tablets 18 HBV2 AdefovirDipivoxil Tablets 15 HBeAg serological Conversion Non-HBeAg serological Conversion HBV3 AdefovirDipivoxil Tablets 17 HBV4 Peginterferon alfa-2a 15 HBV5 Peginterferon alfa-2a 18 HBV6 AdefovirDipivoxil Tablets 17 HBV7 Peginterferon alfa-2a 23 HBV8 AdefovirDipivoxil Tablets 12 HBV9 AdefovirDipivoxil Tablets 17 HBV10 AdefovirDipivoxil Tablets 13 5
Fig. S1. Regulation of T cell function by CDKN3 and JMJD6. (a-c) Effect of CDKN3 sirna treatment on proliferation, cell cycle status and cytokine production of JMJD6- silenced CD4 + and CD8 + T cells. PBMCs from HDs were transfected with NC sirna, JMJD6 sirna, JMJD6 sirna plus CDKN3 sirna, or CDKN3 sirna alone. Transfected 6
PBMCs were analyzed for CFSE dilution (n=5) (a), Ki67 expression (n=4) (b) and intracellular IL-2 production (c) of CD4 + and CD8 + T cells. (d) IL-2R expression on T cells upon JMJD6 sirna treatment. (e) IL-2R expression on T cells upon PDGF-BB treatment. PBMCs were cultured in the presence of PDGF-BB for 48 h and CD132, CD122 and CD25 were detected by FACS on CD4 + T cells. Labels in d and e are: NC si, non-specific control sirna; JMJD6 si, JMJD6 specific sirna; Iso, isotype control. 7
Fig. S2. CDKN3 isoform expression in human PBMCs with or without JMJD6 sirna treatment and effect of CHB sera on the proliferation of CD8 + T cells from healthy donors. (a) PBMCs were separately transfected with NC sirna or JMJD6 sirna and cultured for 2 d. CDKN3 isoforms were detected in the treated cells by PCR using CDKN3 isoform specific primers. GAPDH serves as a loading control. (b) The effect of sera from ALT(high) or ALT(low) CHB patients on CD8 + T cell proliferation. T cells from healthy donors were cultured with CHB patients sera and measured for proliferation as described in the Method. 8
Fig. S3. Inverse correlation between JMJD6 mrna expression and liver function in CHB patients (n=23). (a) Correlation between JMJD6 mrna expression in total PBMCs and patients ALT and AST levels. (b) Correlation between JMJD6 mrna expression in total PBMCs and patients HBV DNA load. (c) Correlation between JMJD6 mrna expression in total PBMCs and patients TBIL and DBIL. (d) Correlation between patients ALT or AST and TBIL in CHB patients. P values and r-square values are indicated. 9
Fig. S4. Expression of JMJD6 mrna in CD4 + T cells and PDGF-BB in the plasma of CHB patients was correlated with their treatment outcome. (a) JMJD6 mrna expression in purified CD4 + T cells from treatment-responsive and unresponsive CHB patients. Frozen PBMCs from CHB patients before treatment (Baseline) and at the end of the treatment period (EOT) were used for CD4 + T cell isolation and measurement of JMJD6 mrna expression by qpcr. (b) PDGF-BB levels in the plasma of treatment-responsive and unresponsive CHB patients as measured by ELISA. 10
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