1 Somatic Mutations in Clonally Expanded T-lymphocytes in Patients with Chronic Graft-Versus- 2 Host Disease 3 4 Giljun Park1,2, Daehong Kim1,2, Jani Huuhtanen1,2, Sofie Lundgren1,2, Rajiv K. Khajuria1, Ana M. 5 Hurtado3, Cecilia Muñoz-Calleja4, Laura Cardeñoso5, Valle Gómez-García de Soria6, Tzu Hua Chen- 6 Liang3, Samuli Eldfors7, Pekka Ellonen7, Sari Hannula7, Oscar Bruck1,2, Anna Kreutzman1,2, Urpu 7 Salmenniemi8, Tapio Lönnberg9, Andres Jerez3, Maija Itälä-Remes8, Mikko A. I. Keränen1,2,10, and 8 Satu Mustjoki, MD1,2 9 10 Supplementary Appendix 11 12 Contents: 13 Supplementary results page 2 14 Supplementary Figure S1 page 3 15 Supplementary Figure S2 page 4, 5 16 Supplementary Figure S3 page 6 17 Supplementary Figure S4 page 7 18 Supplementary Figure S5 page 19 Supplementary Table S1 page 20 Supplementary Table S2 page 21 Supplementary Table S3 page 22 Supplementary Table S4 page 23 Supplementary Table S5 page 24 Supplementary Table S6 page 25 Supplementary Table S7 page 26 Supplementary Table S8 page 27 Supplementary Table S9 page 28 Supplementary Table S10 page 29 Supplementary Table S11 page 30 31 32 33 1 34 Supplementary results 35 Clinical Characteristics of the Index cGvHD Patient 36 The index patient was a 56-year-old male, who was diagnosed with chronic phase chronic myeloid 37 leukemia (CML) in the fall 1999. The clinical status and treatment history are described in detail in 38 Supplemental Figure 1. After short leukocyte reduction with hydroxyurea and the patient received 39 allo-HSCT from his HLA-matched sibling donor after myeloablative conditioning. Double 40 immunosuppression with cyclosporine and methylprednisone was started at the time of the allo- 41 HSCT, and methotrexate was also given on day +1, +3 and +6 after the transplantation. Within the 42 first 100 days the patient had a human herpesvirus 6 (HHV6) infection and the first 43 cytomegalovirus (CMV) reactivation. At the end of that period, the first symptoms of GvHD 44 emerged in the form of elevated transaminases. Since the beginning of 2001, the patient suffered 45 from cGvHD affecting his liver, eyes, nails and skin including sclerodermatous skin lesions that 46 presented with a varying degree of severity over time. Mycophenolate was added to the 47 immunosuppressive regimen in the spring 2001. In the course of the cGvHD, the patient was 48 treated with photopheresis in 2002 as well as with a low-dose irradiation of his lymph nodes in 49 2003. The immunosuppression was continuously adjusted according to the clinical presentation of 50 the cGvHD. In recent years, the patient has mostly received mycophenolate and varying 51 supplementation with methylprednisone and cyclosporine depending on the manifestation of 52 sclerodermatous skin lesions. The patient haven’t had a relapse of CML nor received any donor 53 lymphocyte infusions. He was sampled (blood) for the first time in 2013 and in the disease course 54 thereafter (Supplementary Figure S1). 55 2 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 3 82 Supplementary Figure S2. A Lymphocyte population in peripheral blood CD19+APC CD16/CD56+PE CD45+PerCP CD3+FITC CD3+FITC B Phenotype of donor and index patient’s T cells CD4+ CD8+ 3.60 12.7 5 19.9 15.4 10 4 10 4 10 CCR7 CCR7 3 10 3 10 CCR7+PE Dono CCR7+PE 0 0 3 3 -10 32.4 51.3 -10 59.6 5.00 C Naiv 3 3 4 5 3 3 4 -10 0 10 10 10 -10 0 10 10 CD45RA CD45RA CD45RA+Alexa700 CD45RA+Alexa700 EM TEMRA 6.22 1.38 2.01 2.17 5 5 10 10 4 4 10 10 CCR7 CCR7 Inde 3 3 10 10 CCR7+PE CCR7+PE 0 0 3 3 -10 75.0 17.4 -10 16.0 79.9 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 CD45RA CD45RA CD45RA+Alexa700 CD45RA+Alexa700 4 83 C Gating strategy for flow-assisted cell sorting 84 250K 250K 250K 200K 200K 200K monocytes 4.89 150K 150K CD3- 150K 44.9 SSC-A SSC-A SSC-A 100K 100K 100K CD3+ 50K 50K 50K 53.9 0 0 0 0 50K 100K 150K 200K 250K 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 FSC-A CD3+APC CD3+APC CD8+ 5 5 CD4+CD8+ 10 12.6 10 4 4 10 10 CD4+CD8+ 11.3 3 3 10 10 CD8+PECy7 CD4+ Vb+ 0 59.3 0 57.3 Vb- 3 3 40.0 -10 -10 1 2 3 4 3 3 4 10 10 10 10 -10 0 10 10 CD4+PerCP CFITCAVβ 20+FITC 5 CD4+ 10 4 10 3 10 Vb+ 0 39.5 Vb- 3 55.6 -10 3 3 4 -10 0 10 10 Vβ 20+FITC Supplementary Figure S2. Flow cytometry analysis of the index patient. (A) Basic lymphocyte immunophenotyping was performed to identify T (CD3+), natural killer (NK) (CD3-CD16+CD56+), and B (CD3-CD19+) cells by flow cytometry. (B) Multicolor-flow cytometry was applied to identify the detailed immunophenotype of donor and index patient’s memory T cell subtypes. Central memory (CM), naive, effector memory (EM) and terminal effector-memory (TEMRA) cells. (C) Gating strategy for flow based cell sorting. Monocytes were gated based on morphological features using SSC-A and FSC-A plots. CD3- and CD3+ cells were sorted from lymphocytes based on antibody staining, and CD3+ cells were separated to CD4+, CD4+CD8+ and CD8+ fractions. Finally, TCR Vβ20+ and Vβ20- cells were sorted. 5 85 86 87 Supplementary Figure S3. 88 89 90 91 TLR2 p=0.0054 p=0.0048 20 CD4-GVHD CD4-control 15 CD8-GVHD CD8-control 10 VAF (%) VAF 5 92 0 93 94 95 96 97 98 99 100 101 102 103 Supplementary Figure S3. Variant allele frequencies of TLR2 mutations 104 in cGVHD patients’ and healthy controls’ CD4+ and CD8+ T cells TLR2 mutation analyzed by amplicon sequencing in cGVHD patients (CD4+ T, n=21 and CD8+ T, n=22) and healthy controls (CD4+ T, n=5 and CD8+ T, n=4). Mononuclear cells (MNCs) were separated from whole blood with Ficoll-Paque PLUS (GE Healthcare, UK) followed by magnetic beads sorting of both CD4 and CD8+ T cells. The purity of sorted fractions was evaluated by flow cytometry (FACsVerse) and confirmed to be >98%. The analysis indicated 10-fold higher variant allele frequency in cGVHD patients compared to healthy controls. Unpaired t-test was performed with Welchi's correction to calculate p value using Graphpad Software (San Diego, USA). Error bar present Mean ± SD. 6 105 Supplementary Figure S4. 106 107 108 109 110 111 A B 112 6 p=0.0293 113 100000 114 10000 mock TLR2_WT 115 4 p=0.0020 1000 TLR2_W558L CT) CT) 116 ∆∆ ∆∆ ( 117 100 ( 2 118 10 119 Relative expression level expression Relative 1 120 level expression Relative 0 121 ELK1 FOS 122 123 124 125 Supplementary Figure S4. Functional analysis of wild type and TLR2 mutants in HEK293 cell line To identify the effect of TLR2 mutant on the downstream gene expression, RT-qPCR was performed for (A) TLR2 and (B) ELK1 and FOS1 genes using stably expressed TLR2_WT (n=7) and TLR2_W558L (n=7) in HEK293 cells. TLR2_W558L increased expression level of downstream target genes, ELK1 and FOS1. Unpaired t-test was performed with Welchi's correction to calculate p value (TLR2_WT vs TLR2_W558L) using Graphpad Software (San Diego, USA). Error bar present Mean ± SD. 7 126 Supplementary Figure S5. 127 128 129 130 Supplementary Figure S5. Gene expression fold change within CD4+ T cell clusters between 2015 131 and 2017 132 8 133 Supplementary Table S1. Summary of Study Cohorts 134 135 A. Patients with cGvHD 136 137 Patient characteristics N % 138 Total 135 139 Age at sampling (mean, range) 48 (16-70) 140 Patient sex 141 Female 58 43,0 142 Male 77 57,0 143 Donor sex* 144 Female 53 39,3 145 Male 81 60,0 146 Sex mismatch* 147 No 73 54,1 148 Yes 61 45,2 149 Diagnosis 150 AML, MDS 63 46,7 151 ALL 17 12,6 152 NHL 17 12,6 153 CLL 2 1,5 154 HL 4 3,0 155 MPD 15 11,1 156 MM 11 8,1 157 Other 6 4,4 158 Donor type 159 Sibling 79 58,5 160 MUD 56 41,5 161 Stem Cell Source 162 Peripheral blood 122 90,4 163 Bone marrow 13 9,6 164 HLA match** 165 Match 114 84,4 166 Mismatch 13 9,6 167 Haplo 7 5,2 168 Conditioning* 169 MAC 64 47,4 170 RIC 70 51,9 171 Prophylaxis of acute GVHD 172 Calcineurin-based 125 92,6 173 mTOR-inhibitor based 10 7,4 174 Acute GVHD** 175 No 66 48,9 176 Grade <2 17 12,6 177 Grade >2 50 37,0 178 Chronic GVHD* 179 Mild 28 20,7 180 Moderate-Severe 106 78,5 181 Delay from tx to sampling 182 < 12 m 67 49,6 9 183 12-36 m 53 39,3 184 > 36 m 15 11,1 185 186 B. Patients without cGVHD 187 188 Patient characteristics N % 189 Total 38 190 Age at sampling (mean, range) 48 (16-70) 191 Patient sex 192 Female 23 60,5 193 Male 15 39,5 194 Donor sex* 195 Female 12 31,6 196 Male 26 68,4 197 Sex mismatch* 198 No 20 52,6 199 Yes 18 47,4 200 Diagnosis 201 AML, MDS 23 60,5 202 ALL 6 15,8 203 NHL 2 5,3 204 CLL 2 5,3 205 HL 3 7,9 206 MPD 1 2,6 207 MM 0 0 208 Other 1 2,6 209 Donor type 210 Sibling 23 60,5 211 MUD 15 39,5 212 Stem Cell Source 213 Peripheral blood 33 86,8 214 Bone marrow 5 13,2 215 HLA match* 216 Match 29 76,3 217 Mismatch 2 5,3 218 Haplo 6 15,8 219 Conditioning 220 MAC 20 52,6 221 RIC 18 47,4 222 Prophylaxis of acute GVHD 223 Calcineurin-based 37 97,4 224 mTOR-inhibitor based 1 2,6 225 Acute GVHD 226 No 18 47,4 227 Grade <2 8 21,1 228 Grade >2 12 31,5 229 Delay from tx to sampling 230 < 12 m 14 36,8 231 12-36 m 22 57,9 232 > 36 m 2 5,3 10 233 234 AML; acute myeloid leukemia, MDS; myelodysplastic syndrome, NHL; non-hodgkin lymphoma, 235 CLL; chronic lymphocytic leukemia, HL; Hodgkin’s lymphoma, MPD; myeloproliferative 236 syndrome, MM; multiple myeloma.