Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors

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1 SUPPLEMENTARY INFORMATION Articles In the format provided by the authors and unedited. Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors Pakawat Chongsathidkiet 1,2,3,16, Christina Jackson 4,16, Shohei Koyama 5,16, Franziska Loebel 6, Xiuyu Cui 1,2, S. Harrison Farber 1,2,7, Karolina Woroniecka 1,2,3, Aladine A. Elsamadicy 1,2, Cosette A. Dechant 1,2, Hanna R. Kemeny 1,2, Luis Sanchez-Perez 1,2, Tooba A. Cheema 8, Nicholas C. Souders 9, James E. Herndon 1, Jean-Valery Coumans 11, Jeffrey I. Everitt 3, Brian V. Nahed 11, John H. Sampson 1,2,3,12,13, Michael D. Gunn 3,13,14, Robert L. Martuza 11, Glenn Dranoff 15, William T. Curry 11 and Peter E. Fecci 1,2,3 * 1 Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA. 2 Department of Neurosurgery, Duke University Medical Center, Durham, NC, USA. 3 Department of Pathology, Duke University Medical Center, Durham, NC, USA. 4 Department of Neurosurgery, The John Hopkins University School of Medicine, Baltimore, MD, USA. 5 Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita City, Osaka, Japan. 6 Department of Neurosurgery, Charité Medical University, Berlin, Germany. 7 Department of Neurosurgery, Barrow Neurological Institute, St. Joseph s Hospital and Medical Center, Phoenix, AZ, USA. 8 Unum Therapeutics, Cambridge, MA, USA. 9 Dana Farber Cancer Institute, Boston, MA, USA. 1 Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC, USA. 11 Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. 12 Department of Radiation Oncology, Duke University Medical Center, Durham, NC, USA. 13 Department of Immunology, Duke University Medical Center, Durham, NC, USA. 14 Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC, USA. 15 Novartis Institutes for Biomedical Research, Cambridge, MA, USA. 16 These authors contributed equally: P. Chongsathidkiet, C. Jackson, S. Koyama. * peter.fecci@duke.edu Nature Medicine

2 SUPPLEMENTARY INFORMATION Table 1 Retrospective Study: Patient and Control Characteristics Characteristics No. of controls (%) N = 46 No. of patients (%) N = 3 Age Median Range Sex Male Female Steroid status Naive Experienced Unknown (61) 18 (39) 46 (1) () () (54) 138 (46) 187 (63) 97 (32) 16 (5) Table 2 Prospective Study: Patient and Control Characteristics Characteristics No. of controls (%) N = 13 No. of patients (%) N = 15 Age Median Range Sex Male Female (54) 6 (46) (73) 4 (27)

3 a *** p=.8 b c 15 *** p= % 3 * p= % **** p< % 5 1.9% 1 % Lymphopenic d Controls All Patients Patients Dex Patients No Dex cells / L Control Naive CD4 Patient Naive CD4 Control Memory CD4 Patient Memory CD4 Naive : Memory Ratio 5 p= Controls Patients Spleen Volume (ml) 8 p= No Dex Dex Supplementary Fig 1. a, Frequency of lymphopenia (lymphocyte counts < 1, cells/μl) in n=3 newly diagnosed G patients and n=46 age-matched controls. G patients are also categorized into n=97 Dexamethasone-experienced (Dex) and n=187 Dexamethasone-naïve (No Dex) groups. b, Comparison of naïve (CD45RA + CD27 + ) and memory (CD45RA - ) CD4 + T-cell counts in n=13 G patients versus n=11 controls. Both naïve and memory CD4 + T-cell counts are reduced in patients, with the naïve T-cell loss being proportionately more severe. c, Ratio of naïve to memory CD4 + T-cell counts in the same cohorts of n=13 G patients versus n=11 controls. Disproportionate naïve T cell loss resulted in trend towards lower ratios in patients. d, Spleen volume on CT scans in n=176 dexamethasone-naïve (No Dex) and n=91 dexamethasone-experienced (Dex) G patients. All data in b-d are shown as mean ± s.e.m. P values were determined by two-tailed, Fisher s exact test (a), two-tailed, unpaired Student s t-test (b, d), and twotailed, Mann Whitney test with Gaussian approximation (c).

a T-cells / L 25 2 15 1 5 Control Naive * p=.153 p=.5295 CT2A IC Naive Control Memory CT2A IC Memory b Spleen 5. 1 7 **** p<.1 4. 1 7 3. 1 7 2.

1 Control VM/Dk VM/Dk + SMA-56 IC c d Unimplanted C57BL/6 C57BL/6 + IC CT2A Unimplanted C57BL/6 C57BL/6 + IC CT2A Supplementary Fig. 2.

b, Spleen T-cell counts in n=1 control C57BL/6 and n=6 control VM/Dk mice or n=14 IC CT2A gliomabearing C57BL/6 and n=8 IC SMA-56 glioma-bearing

c, Gross image depicting thymuses taken from unimplanted or IC CT2A gliomabearing C57BL/6 mice.

Histopathologic examination of thymus from IC CT2A mice showed loss of normal corticomedullary architecture.

4 a T-cells / L Control Naive * p=.153 p=.5295 CT2A IC Naive Control Memory CT2A IC Memory b Spleen **** p< **** p<.1 Control VM/Dk VM/Dk + SMA-56 IC c d Unimplanted C57BL/6 C57BL/6 + IC CT2A Unimplanted C57BL/6 C57BL/6 + IC CT2A Supplementary Fig. 2. a, Blood naïve (CD44 - CD62L + ) and memory (CD44 + ) CD4 + T-cell counts depicted for n=7 control C57BL/6 and n=9 IC CT2A IC mice. b, Spleen T-cell counts in n=1 control C57BL/6 and n=6 control VM/Dk mice or n=14 IC CT2A gliomabearing C57BL/6 and n=8 IC SMA-56 glioma-bearing VM/Dk mice. Data in a-b are shown as mean ± s.e.m. P values were determined by two-tailed, unpaired Student s t-test. c, Gross image depicting thymuses taken from unimplanted or IC CT2A gliomabearing C57BL/6 mice. d, H&E staining (upper panel) or IHC for CD3 (lower panel) of FFPE thymus taken from unimplanted or IC CT2A glioma-bearing C57BL/6 mice. Histopathologic examination of thymus from IC CT2A mice showed loss of normal corticomedullary architecture. These findings accompanied marked organ lymphopenia and lymphoid necrosis. IHC confirmed thymus of IC CT2A mice has marked T-cell lymphopenia, for H&E, scale bar = 5 μm; for IHC, scale bar = 2 μm. All data in a-d are representative findings from one of at least three independently repeated experiments with similar results. Both blood draw (a) and spleen/thumus harvest (b-d) were performed at 18 days following tumor implantation.

a b Frequency: % Total Cells 3 2 1 p=.5911 Frequency: % Total Cells 2.5 2. 1.5 1..5. p=.6576 Frequency: % Total Cells 6 4 2 p=.698 B cells NK cells Gran/Monos Supplementary Fig. 3. a, Sample flow cytometry plot examining bone marrow T-cells in control C57BL/6 mice (top), or the same mice bearing IC CT2A (bottom).

5 a b Frequency: % Total Cells p=.5911 Frequency: % Total Cells p=.6576 Frequency: % Total Cells p=.698 B cells NK cells Gran/Monos Supplementary Fig. 3. a, Sample flow cytometry plot examining bone marrow T-cells in control C57BL/6 mice (top), or the same mice bearing IC CT2A (bottom). b, Frequency of additional leukocyte populations in the bone marrow of n=5 control C57BL/6 or n=3 IC CT2A glioma-bearing C57BL/6 mice. Data in a are representative findings from one of at least three independently repeated experiments with similar results. Data in b are cumulative results from two experiments. Data in b are shown as mean ± s.e.m. P values were determined by two-tailed, unpaired Student s t-test.

c 5. 1 5 *** p=.3 4. 1 5 3. 1 5 2. 1 5 1. 1 5 ** p=.69 ** p=.34 5 1 15 2 25 Day after Tumor Implantation d Supplementary Fig. 3. (Cont.

6 c *** p= ** p=.69 ** p= Day after Tumor Implantation d Supplementary Fig. 3. (Cont.) c, Time course of T-cell accumulation in the bone marrow of mice bearing IC CT2A after tumor implantation. Bone marrow were harvest from n=3 IC CT2A glioma mice on day, 9, 15 and n=4 IC CT2A glioma mice on day 21 after tumor implantation. Data in c are representative findings from one of at least three independently repeated experiments with similar results. Data in c are shown as mean ± s.e.m. P values were determined by two-tailed, unpaired Student s t-test. d, Sample flow cytometry plot examining bone marrow T-cells. The relative proportions of central memory (CM), naïve (N), effector memory (EM), and terminal effector (TE) populations in a patient blood (top) and bone marrow (bottom). CD4 + T-cells are depicted. Similar results were obtained for CD8 + T-cells.

a 3. 1 5 p=.188 2. 1 5 1. 1 5 C57BL/6 + Sham IC b Supplementary Fig. 4.

7 a p= C57BL/6 + Sham IC b Supplementary Fig. 4. a, Bone marrow T-cell counts were compared among n=5 control un-operated C57BL/6 mice and n=5 C57BL/6 mice receiving sham IC injections of a saline/methylcellulose mixture. No difference in bone marrow T-cell counts was observed. Data depicted are from Day 18 post-injection. Data in a are representative findings from one of two independently repeated experiments with similar results. Data are shown as mean ± s.e.m. P values were determined by two-tailed, unpaired Student s t-test. b, Pictorial schematic for the experiments producing the data depicted in Figs 4 d-f

8 a p=.178 p=.1551 b T cells: % S1P *** p=.2 T-cells: % S1P p=.1538 c S1Pr1 mrna Relative Expression p=.2347 Control + AMD CT2A IC CT2A IC + AMD. d Spleen CLN e f Brain/Tumor S1P Ligand Concentration (pmol per mg of tissue) Control Brain * p=.185 CT2A IC Tumor Plasma S1P Ligand Concentration ( M) p=.1631 Supplementary Fig. 5. a. Bone marrow T-cell counts are shown for n=9 control tumor-naïve C57BL/6 and n=13 IC CT2A-bearing mice, or n=5 control tumor-naïve C57BL/6 and n=8 IC CT2A-bearing mice administered the CXCR4 antagonist AMD31 (AMD). b, Frequency of S1P1 + T-cell populations in the spleen (left) and cervical lymph nodes (CLN) (right) of n=12 control C57BL/6 or n=6 CT2A IC mice. c, S1Pr1 mrna expression levels in T-cells sorted from spleens of n=3 control C57BL/6 or n=3 CT2A IC mice assessed by qrt-pcr and normalized to GAPDH expression. d, Histograms showing expression levels of CD69, KLF2, and STAT3 in the T-cells of bone marrow of control C57BL/6 (gray) or CT2A IC (black) mice assessed by RNA prime flow. Data in a-d are representative findings from one of two independently repeated experiments with similar results. e, Concentration of S1P ligand in the plasma of n=5 control C57BL/6 or n=6 IC CT2A-bearing mice, as assessed by LC-MS/MS. f, Concentration of S1P ligand in the brain or brain tumor of n=5 control C57BL/6 or n=7 IC CT2A-bearing mice, as assessed by LC-MS/MS. Data in f are normalized to tissue weight. Data in a-c, e, f are shown as mean ± s.e.m. All P values were determined by two-tailed, unpaired Student s t-test.

9 g T-Cell Counts *** p< r = CT-2A IC CT-2A SC E771 IC E771 SC B16F1 IC B16F1 SC LLC IC LLC SC Spleen Weight (mg) h *** p<.1 r = Thymus Weight (mg) i * p=.39 CFSE+ T-Cell Counts (per milion injected) Control->CT2A IC 2 hrs FTY72->CT2A IC 2 hrs Supplementary Fig. 5. (Cont.) g,h. Negative correlation between bone marrow T-cell counts and either spleen (g) or thymus (h) weight across IC and SC murine tumor models. Data in g were obtained from n=6 IC CT2A, n=9 IC E771, n=6 IC B16F1, n=7 IC LLC, n=6 SC CT2A, n=1 SC E771, n=11 SC B16F1, and n=7 SC LLC tumor-bearing mice. N=21 control C57BL/6 were also included. Data in h were obtained from n=6 IC CT2A, n=5 IC E771, n=6 IC B16F1, n=7 IC LLC, n=6 SC CT2A, n=7 SC E771, n=6 SC B16F1, and n=7 SC LLC tumor-bearing mice. N=21 control C57BL/6 were also included. Data in g, h are cumulative results from a minimum of two experiments with each tumor type. Two-tailed, p values and Pearson coefficients for g, h are depicted. i, Accumulation of adoptively transferred CFSE-labeled T-cells in the bone marrow of CT2A IC recipients that were treated with either vehicle control (n=3 recipient mice) or FTY72 (n=3 recipient mice) 2 hours prior to receiving transfers. Transferred cells were splenocytes from control C57BL/6 donors. Data in i are shown as mean ± s.e.m. The p value was determined by two-tailed, unpaired Student s t-test.

10 a b Percent survival ** p=.69 S1P1 KI + Iso S1P1 KI + -PD-1 S1P1 KI + 4-1BB agonist S1P1 KI + 4-1BB + -PD-1 c Day ** p=.35 d *** p<.1 Blood CT2A IC+Control CT2A IC+G-CSF CT2A IC+Control CT2A IC+G-CSF e Percent survival ** p=.21 Control G-CSF 4-1BB agonist G-CSF+4-1BB agonist Day Supplementary Fig. 6. a, Representative flow cytometry plot depicting the frequency of S1P1 on the surface of T-cells in the bone marrow of C57BL/6 mice and S1P1 KI bearing IC CT2A tumor. b, S1P1 KI mice were implanted with IC CT2A tumors and treated with anti (α)-pd-1, 4-1BB agonist, or the combination of both (n=8 per group). Bone marrow (c) and blood (d) T-cell counts in n=8 control mice and n=8 IC CT2A-bearing mice administered control treatment or G-CSF intraperitoneally every 3 days following tumor implantation (Days 3-18). Counts were assessed 18 days following tumor implantation. e, IC CT2A IC mice were administered G- CSF, 4-1BB agonist, or the combination regimen of both drugs (n=8 per group). All data in a-e are representative findings from one of two independently repeated experiments with similar results. Data in c, d are shown as mean ± s.e.m. P values in c, d were determined by two-tailed, unpaired Student s t-test. Survivals in b, e were assessed by two-tailed generalized Wilcoxon test. P values for overall comparison are depicted.

Supplementary Figure S1. PTPN2 levels are not altered in proliferating CD8+ T cells. Lymph node (LN) CD8+ T cells from C57BL/6 mice were stained with

Supplementary Figure S1. PTPN2 levels are not altered in proliferating CD8+ T cells. Lymph node (LN) CD8+ T cells from C57BL/6 mice were stained with CFSE and stimulated with plate-bound α-cd3ε (10µg/ml)