Supplemental Figure 1: Flow cytometry gating strategy for CCR/CXCR homing receptors

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Sabrina Cannon
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1 Supplemetal Figure 1: Flow cytometry gatig strategy for CCR/CXCR homig receptors First, we discrimiated cells based o size scatter ad, after exclusio of doublet cells ad dead cells, as well as o cells usig the dump chael (staiig for CD14, CD15, CD16 ad CD19), we aalyzed cell markers. Hece, we gated o CD4 + ad CD8 + cells to aalyze distict subsets of aïve or effector/memory cells usig CD45RA ad CCR7 mabs. I each subset, we evaluated the percetage of positive cells for each homig receptor. o evaluate these 9 receptors, we staied cells with oe of three 3 differet staiig cocktails, the first oe cotaiig CRH2, CCR6, CCR, CXCR3 mabs (9 colors), ad the secod ad third, with CXCR5, CD3, CCR9 mabs ad CLA, CCR, CXCR4 mabs respectively (each 8 colors). A exemplificatio of the adopted appropriate gatig strategy is depicted. Supplemetal Figure 2: Overall survival of 57 MMel studied for CCR ad CXCR markers. Kapla Meier curves of the three idepedet cohorts pooled together (patiets characteristics ad statistics for progosis parameters preseted i Suppl. able 2) ad aalyzed for overall survival from diagosis (A) or from blood samplig for CCR ad CXCR studies (). Supplemetal Figure 3: Correlatios betwee Ns, CMs, EMs, EMRAs CD4 + ad CD8 + cells. A. Distributios of correlatio coefficiets betwee the 4 subpopulatios (Ns, CMs, EMs ad EMRAs) of the idicated flow cytometry parameters (idicated o y-axis). Dashed lies correspod to the.5,.1 ad.1 P-sigificace levels. Correlatio matrices from the 2 groups were foud to differ from a idetity matrix (P < -2, artlett's test).. Distributios of correlatio coefficiets betwee CD4 + ad correspodig CD8 + lymphocytes. Upper pael: 4 subpopulatios (Ns, EMRAs, EMs, CMs) for each family. Lower pael: double positive cells. Dashed lies correspod to the.5,.1 ad.1 P-sigificace levels. he RV coefficiet (give i paretheses) betwee the CD4 + ad CD8 + sub-populatio matrices idicates a poor correlatio idex betwee CXCR4 ad CD3 markers. Of ote, each sample was split ito two batches that were staied with the same CCR mab but differet additioal markers ad the subjected to flow cytometry. herefore, CCR was measures twice ad featured o the graphs as CCR ad CCR.2. Note that these two CCR measuremets correlated amog each other. Supplemetal Figure 4: Icrease i circulatig proportios of CD4 + CD3 + Ns associated with liver metastases. A. ROC curves depictig the predictive properties of CD3 o CD4 + Ns determied i patiets presetig liver metastases (N=19) i multimetastatic patiets (N=36) ad associated area uder the curve (AUC).. Distributio of the expressio of CD3 o CD4 + Ns i each subset of

2 patiets, stratified accordig to the diagosis of liver metastases. C-D. Distributios of CD3 + CD4 + cells coexpressig CXCR5 (C) or CCR9 (D) receptors i MMel patiets who were kow for the ulceratio status (N=18). Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by logistic (A) ad beta regressio (-D) modelig. P values are idicated. Supplemetal Figure 5: CLA expressio o cells is weakly associated with disease dissemiatio. A-. CLA expressio o CD8 + (A) ad CD4 + () EMRAs (upper paels) ad CMs (lower paels) is depicted for HV, for patiets presetig with oly cutaeous/ln metastases (Cut+LN), with additioal lug ivolvemet (), with dissemiated diseases () ad with distat metastases plus lug ivolvemet (+) at the time of iclusio i oe of the three protocols described i M&M. C-D. Match-paired compariso betwee CLA expressios (performed i flow cytometry o fresh tissues) i all CD8 + (C) ad CD4 + (D) cell subsets from blood () ad tumors () at surgery i the prospective cohort of 2 patiets with MMel. Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by beta regressio (A-) ad liear mixed effects (C-D) modelig. Raw P values are idicated. Supplemetal Figure 6: CD4 + CXCR4 + EMRAs accumulated i tumor LN. A-. CXCR4 expressio o CD4 + EMRAs (A) ad CMs () is depicted for HV, for patiets presetig with oly cutaeous/ln metastases (Cut+LN), with additioal lug ivolvemet (), with dissemiated diseases () ad with distat metastases plus lug ivolvemet (+) at the time of iclusio i oe of the three protocols described i M&M C. Matchpaired compariso betwee CXCR4 expressios (performed by flow cytometry o fresh tissues) i all CD4 + cell subsets from blood () ad tumors () at surgery i the prospective cohort of 2 patiets with MMel. Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by beta regressio (A-) ad liear mixed effects (C) modelig. Raw P values are idicated. Supplemetal Figure 7: CXCR5 expressio decreases durig lug metastases. A-. CXCR5 expressio o CD4 + EMRAs (A) ad CD4 + CCR9 + cells () is depicted for HV, for patiets presetig with oly cutaeous/ln metastases (Cut+LN), with additioal lug () ivolvemet, with dissemiated diseases () ad with metastases i lug ad other distat orgas (+) at the time of iclusio i oe of the three protocols described i M&M C. Match-paired compariso betwee CXCR5 expressios (performed i flow cytometry o fresh

3 tissues) i all CD4 + cell subsets from blood () ad tumors () at surgery i the prospective cohort of 2 patiets with MMel D. CD4 + CXCR5 + cell cytokie profile. Flow cytometry-guided sortig based o CXCR5/CD3 expressio i blood CD4 + cells i oe represetative patiet (out of two yieldig similar results, meas from duplicate wells) to aalyze cytokie release after a 4 hr CD3/CD28 beads-drive stimulatio. Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by beta regressio (A-) ad liear mixed effects (C) modelig. Raw P values are idicated. Supplemetal Figure 8. CRH2 expressio ad distat metastases. Flow cytometric aalyses of double positive CRH2 + CCR + (A), CCR + CXCR3 + () ad CRH2 + CXCR3 + (C) CD4 + cells i 57 MMel patiets accordig to their metastatic patter at samplig. Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by beta regressio modelig. Raw P values are idicated. Supplemetal Figure 9. CD3 expressio by CD4 + CMs associated with distat metastases. A. CD3 expressio o CD4 + CMs is depicted for healthy voluteers (HV), for patiets presetig with oly cutaeous/lymph ode (Cut+LN) metastases, with additioal lug () ivolvemet, with dissemiated diseases () ad with distat metastases plus lug ivolvemet (+) at the time of iclusio i oe of the three protocols described i M&M.. CD4 + CD3 + cell cytokie profile. Flow cytometry-guided sortig based o the CXCR5/CD3 expressio of blood CD4 + cells i oe represetative patiet (out of two yieldig similar results, meas from duplicate wells) to aalyze cytokie release after a 4 hr CD3/CD28 beads-drive stimulatio.. C. Match-paired compariso betwee CD3 expressio (performed by flow cytometry o fresh tissues) i all CD4 + cell subsets from blood () ad tumors () of patiets at surgery i the prospective cohort of 2 patiets with MMel. D. Flow cytometry aalysis of CD3 ad Foxp3 expressio gatig o CD4 + cells i oe represetative example out of three. Each poit represets oe patiet specime ad the total umber is idicated for all subpopulatios studied. Statistical aalyses were performed by beta regressio (A) ad liear mixed effects (C) modelig. Raw P values are idicated.

4% 25.2% CCR7 CCR 2.65%.316% 2.39%.9% CXCR3 25% 3.95% 19.8% 18.4% 18.3% 13.6% 33.9% 41.5% CCR6 CD3.34%.

4 Suppl. Figure 1 A SS 66.6% 98.7% 69.1% 7.5% 1.2% 25.2% Dump SS CD4 chael CD45RA FS CRH2 Pulse width FS CD8 EMRAs Ns CMs EMs 7.19% 2.99% 3.92% 7.67% 48.4% 25.2% CCR7 CCR 2.65%.316% 2.39%.9% CXCR3 25% 3.95% 19.8% 18.4% 18.3% 13.6% 33.9% 41.5% CCR6 CD3.34%.2%.3%.7% 1.12%.51% 1% 1.47% CCR9 9.19%.24% 6.75% 24.9% CLA CXCR4.5% 8.8% 4.2% 2.61% 14.6% 6.21% 12.8% 8.75% CXCR5 Gated o CD4 CXCR5 % Max % Max CD4 ICOS PD1

5 Suppl.%Figure%2 A.. Overall%survival%from%diagostic Overall%survival%from%samplig Survivig%patiets%() Survivig%patiets%() ime%from%diagostic%(yr) ime%from%blood%samplig%(yr)

6 Suppl. Figure 3 A..

7 Suppl6tFigurety A6 (6- RtoftCDy P CD(-[ P Ns 6 ruetpositivetratet[sesitivity] -68-6: -6y -6) -6- AUCtootstrapt:t-67- RtoftCDy P CD(-[ P tns y [6] [ )6] ) (6] ( -6] - Pt=t-6--(: Other localizatios Liver localizatio (7 (9 C6 RtoftCDy P CXCR] P CD(-[ P )6] ) (6] ( -6] ) -6y -6: -68 (6- (9druetegativetrate7t[(9Specificity] Pt=t-6-y(y D6 RtoftCDy P CCR9 P CD(-[ P ( -68-6: -6y -6) Pt=t-6-(-( - No ulcerated 7 Ulcerated (( - No ulcerated 7 Ulcerated (-

8 Suppl-3Figure35 A- CD87CLA7 - H3of3CD8 7 CLA 7 3EMRAs H3of3CD4 7 CLA 7 3EMRAs CD47CLA7 P3=3-357 HV Cut7LN HV Cut7LN P3=3-323 P3= P3=3-8 H3of3CD8 7 CLA 7 3CMs H3of3CD4 7 CLA 7 3CMs C- 6 5 HV Cut7LN 24 9 lood umor 7 D- 6 5 HV Cut7LN 24 9 lood umor 7 P3=3-35 H3of3CD8 7 CLA P3<313x3 x4 H3of3CD4 7 CLA P3<313x3 x4 P3<313x3 x Ns CMs EMs EMRAs Ns CMs EMs EMRAs

9 Suppl.2Figure A. P2=2.75. P2= P2=2.3 7 C. V2of2CD4 = CXCR4 = 2EMRAs HV Cut=LN 24 9 lood P2=2.31 = V2of2CD4 = CXCR4 = 2CMs HV Cut=LN 24 9 P2=2.57 P2=2.1 P2=2.252 P2=2.65 = umor P2= V2of2CD4 = CXCR4 = P2=2.121 P2= Ns CMs EMs EMRAs

10 Supplf5Figure5< Af P5<5s5x5sM %P P5<5s5x5sM %P f t5of5cdp / CXCR= / 5EMRAs Cf t5of5cdp / CXCR= / <M 8M =M PM VM HM sm M =M PM VM HM sm M HV HP s8 lood umor P5=5MfMMPx s< P5<5s5x5sM %P Cut/LN 9 P5=5MfMMxM P5<5s5x5sM %P s8 sx P5=5MfMPs9 ss s8 sm sx P5=5MfMMMV / H= sv s8 Ns CMs EMs EMRAs t5of5cdp / CCR9 / CXCR= / Df Cytokie5cocetratio5cpgEmla < 8 = P V H s M smmmm smmm smm sm s IL%sHp<M IFN%γ HV s9 P5<5s5x5sM %P Cut/LN x P5=5MfMMV< ss P5<5s5x5sM %P P5<5s5x5sM %P sm CDP / CXCR= / / H= CDP / CXCR= % CDsMV % IL%s<A IL%H IL%sM IL%9 IL%HH IL%8 IL%sV IL%P IL%= IL%sβ NF%α

11 Suppl.LFigureL8 A. PL=L.3. C. 5LofLCD4 x CRH2 x CCR x HV CutxLN 19 PL<L1LxL -4 PL=L.21 PL=L.49 PL=L.45 PL<L1LxL -4 PL=L.29 PL=L.7 PL=L.37 x 5LofLCD4 x CCR x CXCR3 x PL=L.187 HV CutxLN 22 PL=L.256 PL=L.427 PL=L.4 PL=L.434 x 5LofLCD4 x CRH2 x CXCR3 x HV CutxLN 2 PL=L.61 x

12 SupplsRFigureR< As RofRCDV ) CDf%H ) RCMs Cs RofRCDV ) CDf%H ) MP M% fp f% P % fv fm f% E = V M % HV MV f8 lood umor fe PR=R%s%%fH Cut)LN < f8 PR<RfRxRf% dv PR=R%s%VMP f< PR=R%s%%V% ff f8 PR=R%s%f8H f% f< PR<RfRxRf% dv ) M= fv f8 Ns CMs EMs EMRAs s Ds CytokieRcocetratioR(pgxml/ CDf%H f%%% f%% f% f 4 fsv8 %sfm <Vs%% GatedRoRCDV ) RRcells FoxpH ILdfMp8% IFNdγ ILdf8A ILdM ILdf% ILd< ILdMM ILd= ILdfH ILdV ILdP ILdfβ NFdα VsH8 CDV ) CDf%H ) CDV ) CDf%H d CXCRP d

13 Supplemetal able 1: Characteristics of mabs used i this study Specificity Fluorochrome Ab cloe Compay Referece CXCR5 AF488 RF82 D 5582 CLA FIC HECA-452 D CRH2 FIC M16 D CD3 PE er-ac8 D 55 CCR PE R&D FA3478P CD4 PE-CF594 RPA-4 D CD8 PerCP SK1 D CCR9 PerCP Cy5.5 L/CCR9 ioleged CXCR4 PerCP Cy5.5 12G5 ioleged CXCR3 PE Cy7 1C6/CXCR3 D CD4 PE Cy7 SK3 D CCR7 V421 G43H7 ioleged CD14 V5 M5E2 D CD15 V5 HI98 D CD16 V5 3G8 D CD19 V5 HI19 D 5621 CD8b APC 2S8.5H7 D 6458 CCR6 AF647 A9 D CD45RA APC-H7 HI D Dead Cells Yellow -- Ivitroge L34959

14 Supplemetal able 2: Retrospective aalysis performed o 3 cohorts gatherig 57 MMel patiets; impact of cliical parameters o the overall survival from blood samplig N [%] HR [95 %CI] Age (yrs) A 44 [13;72].99 [.97;1.1], P =.5368 Geder reslow (mm) o primary tumor A Stage Metastases Cohort LDH (UI/L) A A Mea[Mi;Max] LR, P value.38, P =.5368 M 29 [5.9] 1 1., F 28 [49.1] 1.34 [.77;2.32], P =.2924 P = ,.95 [.84;1.9], P =.4568 [.;12.] P =.4568 III [.5] , IV 47 [82.5] 2.58 [1.16;5.76], P =.97 P =.97 Ski+LN [.5] 1 g [19.3] 1.94 [.75;5.4], P =.1589 Miscellaeous [.5] 5.5 [2;15.], P =.7 Miscellaeous+ g [45.6] 2.65 [1.13;6.24], P =.148 IMAIL-2 5 [8.8] 1 dwig 13 [22.8].64 [.23;1.79], P =.3989 SORAFEM 39 [68.4].8 [.32;2.2], P = [98;27] 1 [1;1], P = , P =.59.87, P = , P =.7187

15 Supplemetal able 3: Chemokie receptor aalysis performed o 4 differet cohorts of 47 MMel patiets treated with ipilimumab; descriptio of mai cliical parameters Geder M F All patiets - N [%] 28 [59.6] 19 [4.4] Frech Cohort - N [%] 12 [54.5] [45.5] Italia Cohort N [%] 6 [6.] 4 [4.] America Cohort N [%] 7 [63.6] 4 [36.4] Germa Cohort N [%) 3 [75.] 1 [25.] Age (yrs) A 61 [24;91] 6 [82;37] 55.6 [81;24] 65 [91:41] 66.8 [75;53] Stage III IV 5 [.6] 42 [89.4] 4 [18.2] 18 [81.8] 1 [.] 9 [9.] [.] 4 [.] LDH (UI/L) A 356 [25;128] 434 [25;135] 347 [54;144] 238 [477;135] 279 [558;128] Dose of Ipilimumab (mg/kg) 3 41 [87.2] 6 [12.8] 16 [72.7] 6 [27.2] [.] [.] 4 [.] Cocomitat therapy No Yes 34 [72.3] 13 [27.7] 13 [59.1] [.] 9 [4.9] [.] 4 [.] C Prior therapy No Yes Radiotherapy/Chemotherapy yrosie kiase ihibitor D Immuotherapy E [21.3] 37 [78.7] 29 [61.7] [21.3] 12 [25,5] 9 [4.9] 13 [59.1] 7 [31.8] 5 [22.7] 6 [27.2] 1 [.] 9 [9.] 8 [8.] 3 [3.] 3 [3.] [.] [.] 2 [18.2] 2 (18.2] 4 [.] 3 [75.] 1 [25.] 2 [5.] Cliical respose at 3 moths NR R Complete Respose Partial Respose Stable Disease 29 [61.7] 18 [38.3] 1 [2.1] 7 [14.9] [21.3] 12 [54.5] [45,5] 1 [4.6] 7 [31.8] 2 [9.1] 6 [6.] 4 [4.] (.] 4 (4.] 7 [63.6] 4 [36.4] [.) 4 [36.4] 4 [.] (.] A Mea[Mi;Max] Patiets were treated with local radiotherapy. C Patiets received betwee 7 ad 8 itratumoral ijectio of IL-2 (9MUI/ijectio). D Vemurafeib, Dabrafeib, Pazopaib, Levatiib. E IL-2, Iterfero, ati-pd1, ati-cla-4

16 Supplemetal able 4: Summary of the pricipal fidigs of this paper Surface marker CCR6 CXCR3 CXCR4 CXCR5 CCR9 CD3 Subset of cells CD4 EMs, CMs,EMRAs CD8 Ns, EMs, CMs CD4, CD8, Ns CD4, CD8, EMs CD4 CMs CD4 EMRAs CD4 EMs,CMs CD8 Ns, EMs, CMs, EMRAs CD4 Ns, CMs, EMRAs CD8 Ns, CMs CD4 Ns Correlatio with metastatic dissemiatio patters Reductio correlates with cutaeous ad lymph ode metastases Reductio correlates with cutaeous ad lymph ode metastases Reductio correlates with lug metastases Reductio correlates with lug metastases Reductio correlates with lug metastases Icrease correlates with hepatic metastasis Progostic impact Poor progosis if expressed by CD8 EMs Good progosis if expressed by CD4 EMs Good progosis if expressed by CD8 Ns Predictive impact CD4 EMs, EMRAs, CMs, Icrease correlates with visceral metastasis Ns CCR9/CD3 CD4 Icrease i ulcerated melaoma CRH2 CCR CD4 EMs CD8 CMs, EMs CD4 EMs Icrease correlates with visceral metastases Icrease Poor progosis CD4 ad CD8 Ns, CMs Icrease correlates with visceral metastases CLA CD8 EMs Icrease after oe ijectio of ipilimumab CCR/CLA CD4 EMs Poor progosis Icrease predicts ipilimumab respose

Practical Basics of Statistical Analysis

Practical Basics of Statistical Aalysis David Keffer Dept. of Materials Sciece & Egieerig The Uiversity of Teessee Koxville, TN 37996-2100 dkeffer@utk.edu http://clausius.egr.utk.edu/ Goveror s School