1. “Analyses of Pre-therapy Peripheral Immunoscore and Response to
Vaccine Therapy,” by Farsaci et al.
SUPPLEMENTARY FILES
Materials and Methods: Custom software for the calculation of the peripheral immunoscore
Supplementary Figure S1: Gating strategy to identify classic peripheral immune cell types and refined immune cell subsets with phenotypes reflecting immune function that were used to generate the peripheral immunoscores of patients treated with docetaxel plus or minus PANVAC.
Supplementary Figure S2: Gating strategy to identify classic peripheral immune cell types and refined immune cell subsets with phenotypes reflecting immune function that were used to generate the peripheral immunoscores of patients treated with Quadramet plus or minus PROSTVAC.
Supplementary Figure S3: Point assignment method used in generating peripheral immunoscores.
Supplementary Table S1: Antibodies and panels used in flow cytometry analysis to identify immune cells.
Supplementary Table S2: PBMC frequencies (min, median, max) and bin cutoffs used in generating peripheral immunoscores in breast cancer patients treated with docetaxel +/- PANVAC vaccine and prostate cancer patients treated with Sm-153 (Quadramet) +/- PROSTVAC vaccine.
Supplementary Table S3: Summation of points assigned to individual subsets to generate peripheral immunoscore.

2. Supplementary Figure S1
lympho mono
CTLA-4+ CD8
PD-1+ CD8
Tim-3+ CD8
2B4+ CD8 CM
CD8
CD8+ T cells
CTLA-4+ CD4
PD-1+ CD4
Tim-3+ CD4
2B4+ CD4
CD4+
T cells CM
CD4 B
lympho mono
CD4+ T cells
CD25+
Treg
CD49d- Treg C
CD3-CD16-
CD19-CD20-CD56-
MDSC
Lin-
MDSC
lympho mono NK
CD56brCD16- NK
Legend follows

3. Supplementary Figure S1: Gating strategy to identify classic peripheral immune cell types and refined immune cell subsets with phenotypes reflecting immune function that were used to generate the peripheral immunoscores of patients treated with docetaxel plus or minus PANVAC. One vial of frozen peripheral blood mononuclear cells (PBMC) per subject was thawed and then stained in 3 flow cytometry panels using antibodies indicated in Supplementary Table 1. Panel 1 (A) identified parental cell types of CD4+ and CD8+ T cells as well as markers involved in maturation (CCR7, CD45RA), and suppressive function (CTLA4, PD-1, Tim-3, 2B4). Panel 2 (B) identified the parental cell type of Tregs as well as CD49d- Tregs. Panel 3 (C) identified the parental cell types of MDSCs and NK cells, as well as Lin- MDSC (CD14-CD15-) and immature NK cells (CD56brCD16-). Samples were collected on an LSR II flow cytometer equipped with UV, red, blue, and violet lasers, and analyzed using FlowJo with gating set using fluorescence minus one controls.

4. Supplementary Figure S2
CTLA-4+ CD8
PD-1+ CD8
Tim-3+ CD8 CM
CD8
lympho mono
CD8+ T cells
CTLA-4+ CD4
PD-1+ CD4
Tim-3+ CD4
CD4+
T cells CM
CD4 B
CD25+
ICSO+ Treg
CD4+ T cells
Treg
lympho mono C
Non-lympho mono
CD3-CD16-
CD56-
PD-L1+ cDC
cDC
lympho mono NK
Tim3+ NK D
PD-L1+ MDSC
MDSC
CD11b+
Legend follows

5. Supplementary Figure S2: Gating strategy to identify classic peripheral immune cell types and those refined subsets with phenotypes reflecting immune function that were used to generate the peripheral immunoscores of patients treated with quadramet plus or minus PROSTVAC. One vial of frozen peripheral blood mononuclear cells (PBMC) per subject was thawed and then stained in 4 flow cytometry panels using antibodies indicated in Supplementary Table 1. Panel 1 (A) identified parental cell types of CD4+ and CD8+ T cells as well as markers involved in maturation (CCR7, CD45RA), and suppressive function (CTLA4, PD-1, Tim-3). Panel 2 (B) identified the parental cell type of Tregs as well as ICOS+ Tregs. Panel 3 (C) identified the parental cell types of conventional DCs (cDCs) and natural killer (NK) cells, as well as PD-L1+ cDCs and Tim-3- NK cells. Panel 4 (D) identified the parental cell type of myeloid-derived suppressor cells (MDSCs) as well as PD-L1+ MDSCs. Samples were collected on an LSR II flow cytometer equipped with UV, red, blue, and violet lasers, and analyzed using FlowJo with gating set using fluorescence minus one controls.

6. Supplementary Figure S3C B D
Legend follows

7. Supplementary Figure S3: Point assignment method used in generating peripheral immunoscores. The distribution of predetermined classic peripheral blood mononuclear cell (PBMC) immune cell types at baseline, indicated as a percentage of total PBMC, were divided in mathematical tertile bins: low frequency, middle frequency, and high frequency bins. Points were assigned to each classic PBMC immune cell type based on the tertile distribution. If a classic PBMC immune cell type (e.g., CD4) was expected to positively correlate with progression-free survival (PFS), zero (0) points were assigned to the low bin, one (1) point for the middle bin, and two (2) points if in the high bin. If a subset (e.g., Treg) was expected to negatively correlate with PFS, zero (0) points were assigned to the high bin, one (1) point if in the middle bin, and two (2) points if in the low bin. CD4 (A) and Treg (B) in patients with breast cancer treated with docetaxel +/- PANVAC vaccine. CD4 (C) and Treg (D) in prostate cancer treated with Sm-153 (Quadramet) +/- PROSTVAC.

8. Docetaxel +/- PANVAC vaccine Quadramet +/- PROSTVAC vaccine
Supplementary Table S1: Antibodies and panels used in flow cytometry analysis to identify
classic and refined immune cell types
Docetaxel +/- PANVAC vaccine
Quadramet +/- PROSTVAC vaccine
Panel 1:
T lymphocytes
Panel 2:
Tregs
Panel 3:
MDSC & NK
NK & DC
Panel 4:
MDSC
CD4 – AF700
RPA-T4
Biolegend
CD3 – V500
UCHT1
BD Biosciences
CD4 – BV605
CD3 – APC-Cy7
CD33 – APC
WM53
CD8 – PE-Cy7
RPA-T8
CD25 – APC
M-A251
CD56 – PE-Cy7
MEM-188
CD8 – APC
CD56 – APC
HLA-DR – BV605
L243
CD45RA – FITC
HI100
FoxP3 – PE-Cy7
PCH101
eBioscience
CD16 – PerCP-Cy5.5
3G8
CD45RA – AF700
H100
FoxP3 – Pacific Blue
206D
CD16 – FITC
CD11b – APC-Cy7
ICRF44
CCR7 – APC-Cy7
G043H7
CD127 – FITC
A019D5
CD19 – FITC
HIB19
CCR7 – PerCP-Cy5.5
150503
CD127 – APC-eF780
eBioRDR5
CD11c – BV605
3.9
PDL1 – PE-Cy7
MIH1
CTLA4 – PE*
L3D10
CD49d – PerCP-Cy5.5
9F10
CD20 – FITC
2H7
CTLA4 – FITC
ICOS – PerCP-Cy5.5
C398.4A
CD303 – PerCP-Cy5.5
201A
PD-1 – PerCP-Cy5.5
EH12
CD33 – AF700
PD-1 – PE
MIH4
Tim-3 – BV421
F38-2E2
2B4 – APC
Jan-65
CD14 – APC-Cy7
MφP9
CD15 – Pacific Blue
W6D3
Clones and company are listed under each antibody. Intracellular antibodies are underlined.
*Stained both extra- and intracellular.
Tregs, T regulatory lymphocytes; MDSC, myeloid-derived suppressor cells; NK, natural killer cells; DC, dendritic cells.

9. Supplementary Table S2 A B C D
Criterion 1: Docetaxel +/- PANVAC
Subset (% of PBMC)
CD4
CD8 NK
Treg
MDSC
Ratio CD4:Treg
Ratio CD8:Treg
Ratio CD4:MDSC
Ratio CD8:MDSC
min
3.48
3.49
2.91
0.31
0.18
8.85
3.41
1.88
1.85
median
27.13
15.71
9.40
0.85
1.21
31.92
19.88
22.44
11.43
max
52.40
40.45
57.60
2.17
7.13
150.89
90.09
243.15
202.25
Cutoffs for Low Bin
≤ 22.97
≤ 12.86
≤ 7.22
≥ 0.972
≥ 2.00
≤ 23.99
≤ 14.65
≤ 11.88
≤ 5.34
Cutoffs for Mid Bin
>22.97, <31.02
>12.86, <20.12
>7.22, <11.60
>0.676, <0.972
>0.707, <2.00
>23.99, <41.54
>14.65, <24.36
>11.88, <40.30
>5.34, <21.92
Cutoffs for High Bin
≥ 31.02
≥ 20.12
≥ 11.60
≤ 0.676
≤ 0.707
≥ 41.54
≥ 24.36
≥ 40.30
≥ 21.92 B
Criterion 2: Docetaxel +/- PANVAC
Subset (% of PBMC)
CD4 central memory
CD8 central memory
CD4 ≥2 suppressive markers
CD8 ≥2 suppressive markers
Treg CD49d-
Lin- MDSC
NK CD56hi CD16-
Ratio CM CD4:Treg CD49d-
Ratio CM CD8:Treg CD49d-
Ratio CM CD4:Lin- MDSC
Ratio CM CD8:Lin- MDSC
Ratio CD4 CM:NK CD56br CD16-
Ratio CD8 CM:NK CD56br CD16-
min
0.300
0.000
0.008
0.009
0.298
0.059
0.023
0.920
0.850
0.890
median
2.500
0.031
0.074
0.797
0.322
0.389
3.160
0.350
7.760
0.690
5.130
0.530
max
9.600
1.900
0.401
1.644
1.989
2.105
1.580
12.840
3.360
8.990
39.130
Cutoffs for Low Bin
≤ 1.9
≤ 0.100
≥ 0.062
≥ 0.252
≥ 0.932
≥ 0.427
≥ 0.561
≤ 1.99
≤ 0.17
≤ 4.82
≤ 0.38
≤ 2.91
≤ 0.39
Cutoffs for Mid Bin
>1.900, <3.000
>0.100, <0.500
>0.0230, <0.062
>0.048, <0.252
>0.652, <0.932
>0.189, <0.427
>0.250, <0.561
>1.990, <4.100
>0.170, <0.520
>4.820, <11.380
>0.380, <2.080
>2.910, <10.540
>0.390, <2.000
Cutoffs for High Bin
≥ 3.000
≥ 0.5
≤ 0.023
≤ 0.048
≤ 0.652
≤ 0.189
≤ 0.250
≥ 4.100
≥ 0.520 ≥
≥ 2.08
≥ 10.54
≥ 2 C
Criterion 1: Quadramet +/- PROSTVAC
Subset (% of PBMC)
CD4
Ratio CD4:MDSC
Ratio CD4:Treg
CD8
Ratio CD8:MDSC
Ratio CD8:Treg
MDSC NK
Treg
min
12.04
0.83
4.04
8.71
0.99
2.79
1.75
5.56
0.78
median
26.39
4.75
10.73
22.79
3.34
8.35
5.91
10.64
2.45
max
53.01
26.94
17.49
42.32
19.87
54.11
20.48
20.45
4.96
Cutoffs for Low Bin
≤ 23.67
≤ 2.53
≤ 9.36
≤ 17.88
≤ 2.87
≤ 6.38
≥ 10.19
≤ 8.70
≥ 3.03
Cutoffs for Mid Bin
>23.67, <30.01
>2.53, <5.78
>9.36, <12.64
>17.88, <26.19
>2.87, <5.51
>6.38, <12.41
>4.06, <10.19
>8.70, <12.54
>2.12, <3.03
Cutoffs for High Bin
≥ 30.01
≥ 5.78
≥ 12.64
≥ 26.19
≥ 5.51
≥ 12.41
≤ 4.06
≥ 12.54
≤ 2.12 D
Criterion 2: Quadramet +/- PROSTVAC
Subset (% of PBMC)
Central Memory CD4
Ratio Central Memory CD4 CM:Treg ICOS+
CD4 expressing ≥2 suppressive markers
Central Memory CD8
Ratio Central Memory CD8: Treg ICOS+
CD8 expressing ≥2 suppressive markers
cDC PDL1+
MDSC PDL1+
NK TIM3-
Treg ICOS+
min
2.51
0.0371
0.68
0.0297
median
0.51
0.0729
2.43
max
0.282
5.6271
10.2
0.142
7.0376
Cutoffs for Low Bin
≤ 0.445
≤ 0.37
≥ 0.107
≤ 2.095
≤ 1.71
≥ 0.077
≥ 0.118
≥ 2.775 ≥
≥ 1.525
Cutoffs for Mid Bin
>0.445, <0.975
>0.37, <0.79
>0.067, <0.107
>2.095, <3.738
>1.71, <3.59
>0.052, <0.077
>0.025, <0.118
>0.897, <2.775
>6.535, <10.606
>0.976, <1.525
Cutoffs for High Bin
≥ 0.975
≥ 0.79
≤ 0.067
≥ 3.738
≥ 3.59
≤ 0.052
≤ 0.025
≤ 0.897
≤ 6.535
≤ 0.976
Peripheral blood mononuclear cell (PBMC) frequencies (min, median, max) and bin cutoffs used in generating peripheral immunoscores in breast cancer patients treated with docetaxel +/- PANVAC vaccine (A, B), and prostate cancer patients treated with Sm-153 (Quadramet) +/- PROSTVAC vaccine (C, D). As bins were determined using tertiles within each of the patient populations, the cutoffs for a given immune cell type assessed in the breast and prostate cancer trials differed.

10. Supplementary Table S3
Points assigned to a given patient for each immune cell type included within the peripheral immunoscore
TOTAL Score PT
CD4
CD8 NK
Treg
MDSC
Ratio eff CD4:Treg
Ratio CD8:Treg
Ratio CD4:MDSC
Ratio CD8:MDSC 1 5 3 2 11 6 7 9 10 14 16 12 18 4 19 22 23 8 25
201
203
204
207
210
214
216 17
218
219
221
Summation of points assigned to individual subsets to generate peripheral immunoscore. The peripheral immunoscore for a given patient was the sum of points assigned to the individual peripheral blood mononuclear cell (PBMC) immune cell types that were included in a specific criterion used to build the immunoscore. Representative example of summation of points (Criterion 1) in breast cancer patients treated with Docetaxel + PANVAC vaccine.