Supplementary Data

SUPPLEMENTARY DATA

Supplementary Table 1. Chemokine transcripts modulated in human islets by IL-1β exposure.

Fold-Change (IL-1b vs. Gene Symbol p-value (IL-1b vs. Cont.) Cont.)

CCL Motif CCL3/CCL3L1/CCL3L3/CCL3P1 0.00264286 33.6703 CCL5 3.28E-05 17.4667 CCL8 0.00275959 10.9838 CCL20 0.00025476 7.44651 CCL7 0.0397251 2.73854 CCL2 0.00474325 2.52303 CCL28 0.0187363 2.41523 CCL4 0.166808 2.33024 CCL22 0.127037 2.18022 CCL11 0.671377 1.17468 CCL16 0.764476 1.06454 CCL26 0.378284 1.02353 CCL27 0.896528 1.00506 CCL1 0.925227 1.00202 CCL14 / CCL15 0.997963 1.00008 CCL25 0.845303 -1.01019 CCL19 0.53194 -1.01633 CCL23 0.374854 -1.0179 CCL24 0.507378 -1.02171 CCL17 0.834009 -1.02651 CCL21 0.297808 -1.0284 CCL13 0.545427 -1.02955 CCL18 0.369049 -1.93245

CX3CL Motif CX3CL1 0.000219617 8.1873

CXCL Motif CXCL3 4.77E-05 12.4706 CXCL5 0.114982 11.9567 CXCL6 0.000991316 8.16419 CXCL10 0.0115036 8.07591 CXCL11 0.0110674 7.04154 CXCL2 0.000953461 5.76661 CXCL1 1.08E-06 5.76188 CXCL7 0.0684859 2.9263 CXCL16 0.325227 1.2783 CXCL9 0.500492 1.26333 CXCL13 0.410061 1.01463 CXCL14 0.837539 1.00591 IL8 (CXCL8) 0.131298 -1.32621

CXCL12 0.317907 -1.40545 CXCL17 0.389805 -1.57848

XCL Motif XCL1 0.040432 1.02243 XCL1 / XCL2 0.464888 -1.00954

Supplementary Table 2: Chemokine transcripts modulated in human islets by TNFα exposure.

p-value (TNFa vs. Gene Symbol Cont.) Fold-Change (TNFa vs. Cont.)

CCL Motif CCL5 6.96E-06 21.2476 CCL3/CCL3L1/CCL3L3/CCL3P1 0.0448851 6.92144 CCL20 0.000414905 5.71495 CCL2 0.00717359 2.21738 CCL8 0.308226 1.88492 CCL4 0.498378 1.4471 CCL7 0.745327 1.1438 CCL28 0.761113 1.09821 CCL25 0.635856 1.02293 CCL11 0.96347 1.01604 CCL13 0.865768 1.00746 CCL21 0.858509 1.00432 CCL14 / CCL15 0.983076 1.00065 CCL19 0.924795 -1.00223 CCL27 0.816689 -1.00831 CCL26 0.689016 -1.00963 CCL23 0.591641 -1.0098 CCL1 0.568419 -1.0114 CCL17 0.894878 -1.01526 CCL22 0.943463 -1.03226 CCL24 0.278159 -1.0333 CCL16 0.617253 -1.10091 CCL18 0.715319 -1.27433

CX3CL Motif CX3CL1 0.00961706 3.61172

CXCL Motif CXCL5 0.0710959 14.1789 CXCL11 0.0232147 8.42051 CXCL3 0.00264677 4.22678 CXCL10 0.0466351 4.19985 CXCL6 0.0339153 2.9504 CXCL1 6.99E-05 2.93754 CXCL9 0.0116585 2.48067 CXCL2 0.0132979 2.03215 CXCL16 0.0973236 1.48306 CXCL7 0.67473 1.23758 CXCL13 0.846394 1.0031 CXCL14 0.958412 -1.00138 CXCL17 0.752148 -1.16418

CXCL12 0.27451 -1.40974 IL8 (CXCL8) 0.0451573 -1.42879

XCL Motif XCL1 0.775143 1.00261 XCL1 / XCL2 0.3157 -1.01216

Supplementary Table 3. Chemokine transcripts modulated in human islets by IFNγ exposure.

Fold-Change (IFNg vs. Gene Symbol p-value (IFNg vs. Cont.) Cont.)

CCL Motif CCL8 0.0000009074980 283.475 CCL5 0.0000776013000 13.7006 CCL7 0.0132835000000 3.53503 CCL3/CCL3L1/CCL3L3/CCL3P1 0.3375530000000 2.57284 CCL22 0.1474650000000 2.0885 CCL2 0.3187820000000 1.32603 CCL25 0.1147210000000 1.08989 CCL11 0.9100650000000 1.04365 CCL4 0.9603710000000 1.02969 CCL17 0.8339260000000 1.02652 CCL26 0.8034920000000 1.0065 CCL19 0.8757090000000 1.00403 CCL27 0.8702990000000 -1.00636 CCL14 / CCL15 0.6947940000000 -1.01318 CCL21 0.5981750000000 -1.01404 CCL13 0.7785540000000 -1.01479 CCL23 0.4549000000000 -1.01491 CCL1 0.1876210000000 -1.02975 CCL24 0.1759530000000 -1.04613 CCL18 0.9220580000000 -1.07611 CCL16 0.7031190000000 -1.08291 CCL28 0.7538350000000 -1.11094 CCL20 0.5190870000000 -1.30676

CX3CL Motif CX3CL1 0.00001285 16.8165

CXCL Motif CXCL9 0.00000000 2691.41 CXCL10 0.00000011 1672.4 CXCL11 0.00000016 796.145 CXCL12 0.52326600 1.87727 CXCL17 0.37663800 1.59946 CXCL16 0.25804600 1.32851 CXCL3 0.56737100 1.2822 CXCL1 0.39580000 1.19713 CXCL2 0.65746400 1.13011 CXCL13 0.86807200 1.00289 CXCL14 0.58166900 -1.01605 CXCL7 0.96396700 -1.02521 IL8 (CXCL8) 0.02331040 -1.56905

CXCL5 0.61560500 -2.12879 CXCL6 0.09139810 -2.47395

XCL Motif XCL1 0.70662500 1.00376 XCL1 / XCL2 0.17658500 -1.01819

Supplementary Table 4. Chemokine transcripts modulated in human islets by exposure to cytokine cocktail (Mix: IL1β + TNFα + IFNγ).

p-value (Mix vs. Gene Symbol Cont.) Fold-Change (Mix vs. Cont.)

CCL Motif CCL8 1.44E-07 760.352 CCL5 9.31E-09 376.663 CCL22 1.02E-06 60.6901 CCL3/CCL3L1/CCL3L3/CCL3P1 0.000855022 59.4942 CCL7 0.000195725 9.56682 CCL20 0.000424883 6.63474 CCL4 0.017054 4.8511 CCL2 0.00106579 3.12556 CCL11 0.051998 2.21085 CCL17 0.0887852 1.25241 CCL28 0.63701 1.17199 CCL14 / CCL15 0.116011 1.0565 CCL13 0.407494 1.04097 CCL24 0.273685 1.03667 CCL27 0.443164 1.03057 CCL19 0.294559 1.02793 CCL26 0.606151 1.01356 CCL1 0.669881 1.00924 CCL23 0.785113 1.00539 CCL25 0.971775 -1.00184 CCL21 0.511491 -1.01758 CCL16 0.567486 -1.12741 CCL18 0.49447 -1.67679

CX3CL Motif CX3CL1 4.00E-06 32.9726

CXCL Motif CXCL9 2.90E-12 3877.66 CXCL10 7.47E-08 2124.52 CXCL11 5.63E-08 1454.11 CXCL3 1.78E-05 16.1993 CXCL5 0.141368 9.97514 CXCL2 0.000508253 6.64056 CXCL1 7.72E-07 6.07301 CXCL6 0.00322939 5.9842 CXCL16 0.029834 1.79618 CXCL12 0.91643 1.10818 CXCL14 0.160439 1.04285 CXCL7 0.954808 1.03172

CXCL13 0.442073 1.01362 CXCL17 0.767973 -1.16716 IL8 (CXCL8) 0.147481 -1.30999

XCL Motif XCL1 0.241592 1.01203 XCL1 / XCL2 0.688429 -1.00519

Supplementary Table 5. Case descriptions for pancreas samples acquired through the nPOD program.

Case Group AutoAb Age (years) Gender Ethnicity C- BMI Clinical History Histopathology HiRes HLA Transplant Cause of (ng/ml)

6036 T1D mIAA+ 49 (34 w/ Female African <0.05 25.5 Diabetes, Ins- islets, plentiful A*6802, 6802 DRB1*07/-, Anoxia diabetes) American multiple and mostly large; B*1401, 1401 DQB1*02/- hospitalizations mixed C*0802, 0802 for ketoacidosis, inflammation most DRB1*0701, pancreatitis, regions with occ. 0701 alcohol use and acinar atrophy DQA1*0301 , drug addict 15 0301, years ago, GI DQB10201, 0201 ulcers

6052 T1D ICA512+ 12 (1 w/ Male African 0.18 20.3 Diabetic Ins+ islets (reduced A*0201, 6801 DRB1*09/16, Cerebral mIAA+ diabetes) American ketoacidosis numbers); Insulitis, B*0602, 1502 DQB1*05/09 edema IA2ic+ (DKA), CMV esp. peri-islet. C*2705, 5001 positive Heterogenous islet DRB1*0901, distribution and 1602 size. DQA1*0102, 0301 DQB1*0303, 0502

6087 T1D mIAA+ 17.5 (4 w/ Male Caucasian <0.05 21.9 Toe amputations, Ins-/Gluc+ islets- A*2601, 3201 DR*4/17 Gun shot ZnT8+ diabetes) colon resection, reduced frequency. B*0801, 4001 wound cataracts, Mild, focal islet C*0304, 0701 glaucoma, infiltrates- some are DRB1*0301, 0404 hypertension, CD3+ while others DQA1*0301 , congestive heart CD3-negative. No 0501, DQB1*0201, failure pancreatitis or 0302 exocrine atrophy.

6112 No Negative 6.3 Female Hispanic 5.11 18.4 Diabetes Normal- excellent A*2601 , 3101 A*26/31, Head diabetes insipidus islet density. No B*5101 , 5801 B*51/58. trauma secondary to inflammation or fatty C*0701 , 1502 DR*4/13, head trauma infiltrates. DRB1*0407 , 1302 DQ*5/7 DQA1*0102 , 0301, DQB1*0301,0501

6115 No Negative 0.4 Male Caucasian 4.59 17.1 Twin, six days in Ins+ normal islets. A*0101, 6801 DR*04/07 Anoxia diabetes ICU, recently Very high Ki67+ B*3501 , 5701 vaccinated for acinar primarily. C*0401 , 0602 rotavirus and No infiltrates. PLN DRB1*0401 , others in blocks. 0701 DQA1*0201 , 0301, DQB1*0301, 0303

6117 No Negative 0.33 Male Caucasian 3.27 18.4 N/A Ins+ islets, normal. A*0101, 1101 A*01/11, Head diabetes High Ki67 in acinar B*1801, 5701 B*18/57, Trauma and moderate in C*0602, 0701 DRB1*07/-, islets. Very mild DRB1*0701 , DQB1*02/09 infiltrates per- 0701 acinar/capsular DQA1*0201 , region. 0201, DQB1*0201, 0303

mIAA: micro insulin autoantibodies

Supplementary Figure 1. Genomic organization of the major human chemokine clusters and their transcription in response to cytokine stimulation. 31 human chemokine genes are organized in 4 major clusters on chromosome 4 (GRO and IP10 regions) and chromosome 17 (MCP and MIP regions). The majority of cytokine-induced chemokine genes are found within these clusters and comprise genes transcribed by human islet cells in response IL-1β and/or TNFα (red arrows) or in response IFNγ (yellow/red arrows since all genes induced by IFN [yellow] were also upregulated by IL-1β and/or TNFα [red]). Black arrows indicate chemokine genes not differentially regulated by individual cytokine treatments and gray arrows refer to pseudogenes; the direction of the arrows indicates transcriptional orientation. Please note that CXCL5 is included here although its 11-14-fold upregulation after stimulation with IL- 1β or TNFα did not reach statistical significance (Tables S1-3). The diagram constitutes a modified version of a figure taken from Zlotnik et al., Genome Biology 2006 (1).

Supplementary Figure 2. Modulation of cytokine-induced chemokine mRNA transcription by anti-inflammatory agents and under conditions of iNOS or IDO blockade. Human islets obtained from healthy donors were cultured for 24h in the absence (control) or presence of a cytokine cocktail (Mix) as detailed in Methods. A., effects of 5mM acetylsalicylic acid (Aspirin) and B., 40μ L-methyl tryptophan (MT) or 1mM NMMA added to cytokine cocktail-stimulated cultures as indicated. Chemokine mRNA species were quantified by qRT-PCR using a 5’ nuclease assay and FAM® dye labeled TaqMan MGB probes with two PCR primers, and data normalization to endogenous HPRT1. Data (mean ± SE; 4 donors) was quantified using 2–∆∆ CT method and expressed relative to an islet sample incubated in medium alone. Asterisks indicate significant differences between control and cytokine treated islets (p value <0.05), # denotes significant differences between absence and presence of Aspirin or pathway blockers in cytokine cocktail-treated (Mix) islets (p value <0.05).

Supplementary Figure 3. Validation of human chemokine antibody specificity. A., HEK 293T cells were transfected with individual pIRES2-AcGFP1 vectors co-expressing GFP and the indicated human chemokine cDNAs (CCL5, CCL8, CCL22, CXCL9, CXCL10 or CX3CL1). 18-36 hours later, cells were stained for the presence of corresponding chemokine proteins by flow cytometry as detailed in Methods. B., HEK 293 T cells transfected with CXCL9 or CXCL10 vectors as indicated were stained in parallel with αCXCL9 (top) and αCXCL10 (bottom). Note the crossreactivity of αCXCL10 with CXCL9 but absence of crossreactivity for αCXCL9 with CXCL10 (Plots are gated on GFP+ HEK cells).

Supplementary Figure 4. Pancreatic chemokine expression by uninfected RIP-mice. Pancreata were obtained from uninfected RIP-GP mice and processed for immunohistological analysis as detailed in Methods. Note the lack of chemokine expression in the absence of LCMV infection (also compare to LCMV-infected RIP-GP mice as shown in Fig.4).