Fluorochrome Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 CTLA-4 CTLA-4 CD15 CD3 FITC. Bio) PD-1 (MIH4, BD) ICOS (C398.4A, Biolegend) PD-L1 (MIH1, BD)

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1 Additional file : Table S. Antibodies used for panel stain to identify peripheral immune cell subsets. Panel : PD- signaling; Panel : CD + T cells, CD + T cells, B cells; Panel : Tregs; Panel :, -T, cdc, pdc; Panel : []. Intracellular antibodies are underlined, and clones and company are listed under each antibody. Fluorochrome Panel Panel Panel Panel Panel CTLA- CTLA- CD CD FITC (A.H.H, (A.H.H, LS (HI, (HITa, BD) LS Bio) Bio) PE PerCP-Cy. PECy BV V BV Dapi APC AF APC Cy PD- (MIH, BD) EOMES (WD, PD-L (MIH, BD) TCR (IP, CD (OKT, Tbet (B, PD- (MIH, BD) CCR (, BD) PD-L (MIH, BD) Tim- (F-E, CD (HIB, CD (RPA-T, BD) BATF (MBMC, CDRA (HI, BD) CD CD (RPA-T, (RPA-T, PD- (MIH, BD) ICOS (C.A, PD-L (MIH, BD) FoxP (D, CDd (F, CD (RPA-T, BD) CD (M-A, CDRA (HI, BD) CD (ebiordr, PD- (MIH, BD) CD (A, PD-L (MIH, BD) Tim- (F-E, CD (HBe, BD) CD (HCD, CD (G, CDc (L, PD- (MIH, BD) PD-L (MIH, BD) CD (MOP, BD) CD (G, HLA DR (L, CD (WM, CDb (ICRF, BD) BATF, basic leucine zipper transcription factor ATF-like; cdc, conventional dendritic cells; CTLA-, cytotoxic T lymphocyte-associated protein-; EOMES, eomesodermin; FoxP, forkhead box P; HLA, human leukocyte antigen; ICOS, inducible T cell co-stimulator;, myeloid derived suppressor cells;, natural killer; pdc, plasmacytoid DC; PD-, programmed cell death-; PD-L, programmed cell death ligand-; Tbet, T box expressed in T cells; TCR, T cell receptor; Tim-, T cell immunoglobulin and mucin domain-; Tregs, regulatory T cells.

2 Additional file : Table S. Peripheral immune cell subsets analyzed by flow cytometry. Analysis of subsets using unique markers [Lepone, et al. Analyses of peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types. J Circ Biomark. ;].. CD + T cells: Helper T lymphocytes ( subsets). CD + T cells: Cytotoxic T lymphocytes ( subsets) Markers of PD- pathway and T cell activation (in CD and CD): EOMES: activation TCR: activation Tbet: activation BATF: activation/exhaustion Maturation status of T cells (in CD and CD): Naïve: CDRA + CCR + Central Memory (CM): CDRA - CCR + Effector Memory (EM): CDRA - CCR - Terminal (EMRA): CDRA + CCR - T cell markers (in CD and CD): CTLA-: inhibition PD-L: activation/cross-inhibition Tim-: inhibition ICOS: activation (only on CD). Tregs: Regulatory T lymphocytes (CD + CD + FoxP + CD - ) ( subsets) CDRA: Tregs highly expandable in vitro CTLA-: Treg suppression CDd - : suppressive Tregs ICOS: Treg suppression PD-L: cross-inhibition. B cells: CD + ( subsets) CTLA-: inhibition Tim-: inhibition PD-L: cross-inhibition. : Natural killer cells (CD + CD - ) ( subsets) CD + CD dim : Mature, lytic CD + CD br : Functional intermediate, lytic and cytokine production CD - CD br : Immature, cytokine production, abundant in placenta CD - CD dim : Unconventional, non-lytic, non-cytokine production Tim-: activation PD-L: cross-inhibition. -T: CD + CD + ( subsets) Tim-: activation PD-L: cross-inhibition. cdcs: conventional dendritic cells (DCs) (CD - CD - CDc + CD - ) ( subsets). pdcs: plasmacytoid DCs (CD - CD - CDc - CD + ) ( subsets) Markers of DC activation CD: activation Tim-: inhibition PD-: activation/inhibition PD-L: cross-inhibition. s: Myeloid derived suppressor cells (CDb + HLA-DR low/- CD + ) ( subsets) CD: common myeloid marker CD: granulocyte marker CD: immature s PD-L: cross-inhibition Frozen PBMC were thawed then stained using unique markers in immune flow cytometry panels to identify a total of peripheral immune cell subsets. 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. Nine classic immune cell types as well as refined subsets relating to maturation and function were examined BATF, basic leucine zipper transcription factor ATF-like; CTLA-, cytotoxic T lymphocyte-associated protein-; EOMES, eomesodermin; FoxP, forkhead box P; HLA, human leukocyte antigen; ICOS, inducible T cell costimulator; PBMCs, peripheral blood mononuclear cells; PD-, programmed cell death-; PD-L, programmed cell death ligand-; Tbet, T box expressed in T cells; TCR, T cell receptor; Tim-, T cell immunoglobulin and mucin domain-.

3 Additional file : Table S. PD-L clone MIH- used to detect surface expression of PD-L in immune cell subsets does not compete for binding with avelumab. PBMC isolated from a patient with metastatic prostate cancer were rested for hours, and then pre-incubated for minutes with the indicated concentrations of avelumab or IgG isotype control prior to multiparametric stains of PD-L to detect PD-L within the various immune cell types. Cells that were not pre-incubated with the isotype control or avelumab also served as controls. PD-L was detectable, and measured at a very similar frequency within immune cell subsets (data shown of CD, CD, cdc and B cells, both as a percentage of parent and percentage of total PBMC), regardless of whether the PBMC were preincubated with the IgG isotype control or avelumab. These results demonstrate that the PD-L clone (MIH-) used in the present study to assess surface expression of PD-L does not compete for binding with avelumab in PBMC, and can thus be used to measure PD-L expression in patients treated with avelumab. % of Parent % of Total PBMC Subset Not Pre- Incubated Pre-Incubated with IgG Isotype Control (ug/ml) Pre-Incubated with Avelumab (ug/ml).. CD CD cdc B cells Pre-Incubated with IgG Not Pre- Isotype Control (ug/ml) Incubated Pre-Incubated with Avelumab (ug/ml)

4 Additional file : Table S. Effect of cycles of avelumab on classic and PD-L+ classic immune cell subsets. Classic subsets (A) and PD-L+ classic subsets (B) were examined pre-therapy and post- cycles (n=) of avelumab. Results are displayed as the number of patients (percentage of total patients) with an increase of more than %, minimal change of less than %, and a decrease of more than % compared to pre-therapy. Unadjusted p-values (= indicates no change; arrows indicate direction of change compared to pre-therapy; and holm adjusted p-value is listed for subsets with significant unadjusted p-value) were calculated using the Wilcoxon matched-pairs signed rank test. n/a = not applicable as frequency of subset <.% PBMC; ^ = majority of patients with minimal change. cdc, conventional dendritic cells;, myeloid derived suppressor cell;, natural killer; pdc, plasmacytoid DC; PBMC, peripheral blood mononuclear cell; Tregs, regulatory T cells. A. B. Pre vs cycles Pre vs cycles Classic Subsets PD-L+ Classic subsets CD (%) (%) (%). CD (%) (%) (%). ( ^,.) CD (%) (%) (%). CD (%) (%) (%). Treg (%) (%) (%). Treg n/a n/a n/a n/a (%) (%) (%). (%) (%) (%). -T (%) (%) (%). -T (%) (%). B cells (%) (%). B cells (%) (%). cdc (%) (%) (%). cdc (%) (%) (%). pdc (%) (%). pdc (%) (%). (%) (%). (%) (%) (%).

5 Additional file : Table S. Effect of avelumab on PD-L+ refined immune cell subsets. PD-L+ refined subsets were examined pre-therapy and post- cycle (n=), cycles (n=) and cycles (n=) of avelumab. Results are displayed as the number of patients (percentage of total patients) with an increase of. more than %, minimal change of less than %, and a decrease of more than % compared to pretherapy. Unadjusted p-values (= indicates no change; arrow indicates direction of change compared to pretherapy; and holm adjusted p-value is listed for subsets with significant unadjusted p-value) were calculated using the Wilcoxon matched-pairs signed rank test. n/a = not applicable as frequency of subset <.% PBMC. ^ = majority of patients with minimal change; * = difference in medians pre- vs post-therapy <.. Pre vs cycle Pre vs cycles Pre vs cycles Subset Increas e PD-L+ ICOS+ CD (%) (%) (%). (%) (%) (%). (%) (%) (%). naïve CD (%) (%) (%). (%) (%) (%). (%) (%) (%). CM CD (%) (%). (%) (%) (%). ( *,.) (%) (%) (%). EM CD EMRA CD naïve CD CM CD EM CD EMRA CD mature functional intermediate immature unconventional (%) (%) (%) (%) (%) (%).. (%) (%) (%) (%) (%). ( ^,.). (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)... (%) (%) (%) (%) (%) (%) (%) (%)... (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%). (%) (%) (%). (%) (%) (%) m (%) (%) (%). (%) (%) (%). (%) (%) (%). g (%) (%) (%). (%) (%). (%) (%) (%). lin neg (%) (%) (%). (%) (%). (%) (%) (%). (,.)

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