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This study analyzes the little known Manchukuo Young Girl Envoys – Manchukuo's first government-appointed diplomats – and their official visit to Japan between June 22 and July 12, 1932. Existing studies on the Envoys tend to interpret them from an angle of contemporary Japanese people's national sentiment and deem their visit to Japan a show that the Japanese authorities in Manchukuo and Japan orchestrated together, to render Japan a strong world power. This study problematizes that view and considers the Envoys more of a product of intense power struggles inside Manchukuo's highest Japanese ruling strata, suggesting that Manchukuo's decision-making circle in 1932 was far from being a unified entity. Examining the Envoys' interactions in Japan and comparing relevant Japanese- and Chinese-language news coverage on them, this study argues for the possibility of tracing the intertwined national ideals of Manchukuo's Chinese and Japanese government leaders based on the inspiring example of the Envoys.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Supplementary Figure 1. Gemcitabine and CTLA-4 blockade synergize to create an inflammatory microenvironment and anti-tumor response. Mice with palpable tumors were treated with low dose gemcitabine followed by systemic CTLA-4 blockade. Survival was improved (A) and tumors with combination treatment had a significant influx in CD4 and CD8 Tcells (B). Elispot (C) demonstrated improved enhanced IFNγ in response to SPAS-1 peptide (SNC9H8). Supplemental Figure S2. Lack of significant change of TIM3 or LAG3 over time in peripheral blood mononuclear cells (PBMC) over time. Supplementary Table S1. Toxicities in the limb according to Wieberdink criteria Supplemental Table 2. Nanostring fold change results of all genes

Pharmacodynamic biomarkers can play an important role in understanding whether a therapeutic agent has “hit its target” to impact biologic function. A pharmacodynamic biomarker for anti-CTLA-4 therapy remains to be elucidated. We previously reported that anti-CTLA-4 therapy increases the frequency of CD4 T cells expressing the inducible costimulator (ICOS) molecule. To determine whether the frequency of ICOS+ CD4 T cells could be used as a pharmacodynamic biomarker for anti-CTLA-4 therapy, we carried out flow cytometric studies and statistical analyses on data from 56 individuals, which included 10 healthy donors, 36 patients who received anti-CTLA-4 monoclonal antibody (mAb), and 10 patients who received treatment with a different immunomodulatory agent (gp100 DNA vaccine). After treatment with anti-CTLA-4 mAb (ipilimumab; Bristol-Myers Squibb), we detected a statistically significant increase in the frequency of ICOS+ CD4 T-cells. After two doses of anti-CTLA-4 therapy, the assay was found to have an estimated specificity of 96% [95% confidence interval (CI), 88–100] and sensitivity of 71% (95% CI, 54–85), with positive expression defined as a frequency that is more than the upper bound of 95% CI among baseline samples from all subjects. Our data suggest that an increased frequency of ICOS+ CD4 T cells measured by flow cytometry can be used as a reproducible pharmacodynamic biomarker to indicate biologic activity in the setting of anti-CTLA-4 therapy, which should enable appropriate immune monitoring to determine whether patients receiving anti-CTLA-4 monotherapy or combination treatment strategies are having an adequate biologic response. Cancer Immunol Res; 1(4); 229–34. ©2013 AACR.

PDF file - 528K, Supplementary Figure 1. BMS908662 enhances human T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 2. BMS908662 inhibits the growth of BRAF mutant tumor cells in vitro. Supplementary Figure 3. BMS908662 enhances murine T cell activation in vitro in a concentration-dependent manner. Supplementary Figure 4. Detection of ERK phosphorylation in activated T cells. Supplementary Figure 5. Gating strategies for OT-1 and ex vivo phosphoflow. Supplementary Figure 6. PLX4720 enhances human T cell activation in vitro in a concentration-dependent manner.

RAF inhibitors selectively block extracellular signal–regulated kinase (ERK) signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T-cell activation, based on the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T-cell activation and signaling in vitro and in vivo. We observe that T-cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade in vivo potentiates T-cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Finally, this combination demonstrates superior antitumor activity, compared with either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T-cell function by potentiating T-cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models. Cancer Immunol Res; 2(1); 70–79. ©2013 AACR.

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been + and CD4+ T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade–based immunotherapy results in a durable response to cancer therapy. Cancer Immunol Res; 6(2); 189–200. ©2018 AACR.

PDF file - 88K, Supplemental Figure 3. PBMCs from metastatic melanoma patients treated with ipilimumab depleted of CD14-expressing cells were stimulated to proliferate with OKT-3 and IL-2. CFSE dilution of CD3+ T cells in the culture is measured in the absence of CD14+ cells or with CD14+ cells added back.

PDF file - 103K, Supplemental Figure 1. An increased frequency of ICOS+ CD4 T-cells can be consistently detected on activated T-cells. Supplemental Table 1. Frequency of ICOS+ CD4 T-cells before and after in vitro activation. Supplemental Table 2. Reproducibility of flow cytometric assay to measure frequency of ICOS+ CD4 T-cells before and after in vitro activation in fresh and frozen samples. Supplemental Table 3. Intra-assay variability assessed for ICOS+ CD4 T-cells measured in samples obtained from anti-CTLA-4 treated patients.

PDF file - 101K, Supplemental Figure 4. Different summary statistics can be applied to measure HLA-DR expression on lineage negative, CD14+CD11b+ cells and assess relationship to overall survival (using maximum logrank statistics as described in Methods). CV was used as a self-normalizing measurement that was preferred to eliminate non-biological variation in clinical measurements (e.g. day-to-day variation, differences in sample handling and FACS acquisition).

PDF file - 81K

Supplementary Table 1: TCRB sequencing sample IDs

PDF file - 103K, Supplemental table 1. Univariate analysis of relationship between m-MDSC and overall survival at pre-treatment baseline and week 6 after ipilimumab treatment at 10mg/kg and 3mg/kg.

Evaluation of myeloid-derived suppressor cells (MDSC), a cell type implicated in T-cell suppression, may inform immune status. However, a uniform methodology is necessary for prospective testing as a biomarker. We report the use of a computational algorithm-driven analysis of whole blood and cryopreserved samples for monocytic MDSC (m-MDSC) quantity that removes variables related to blood processing and user definitions. Applying these methods to samples from patients with melanoma identifies differing frequency distribution of m-MDSC relative to that in healthy donors. Patients with a pretreatment m-MDSC frequency outside a preliminary definition of healthy donor range (+ T-cell expansion following ipilimumab. Algorithm-driven analysis may enable not only development of a novel pretreatment biomarker for ipilimumab therapy, but also prospective validation of peripheral blood m-MDSCs as a biomarker in multiple disease settings. Cancer Immunol Res; 2(8); 812–21. ©2014 AACR.

PDF file - 58K, Supplemental Figure 2. PBMCs available in multiple aliquots were serially analyzed over 3 weeks to evaluate day-to-day reproducibility of CVHLA-DR measures. A standard error of measurement of 1.4% and 0.7%, respectively for QC sample 1 and 2 was detected.

PDF file - 142K, Supplemental Figure 1. m-MDSC gating strategy using various HLA-DR cutoffs based on expression of HLA-DR in lineage positive cells yields differing m-MDSC frequencies and is susceptible to inter-user variability.

Antibody panels used for flow cytometry* FoxP3 buffer (BD) was used to fix and permeabilize cells. Only frequencies of frequencies of CD4+, CD8+ T cells from Treg panel 1 and, CD14+ cells from MDSC panel 1 were included. 1 Panel 1 (n=14), 2 Panel 2 (n=68).

Calibration of combination models. Calibration was calculated after 12 and 24 months using the calibrate function in the rms package of R for combination model 1 (A) and combination model 2 (B). Bootstrapping (1000 repeats) was performed to obtain bias-corrected estimates of predicted vs. observed values. Non-convergence reduced the number of included bootstrapping steps for combination model 2 to 981 or 990 after 12 or 24 months, respectively. "Predicted" survival probabilities at 12 or 24 months are those predicted by the Cox model, and "observed" refers to the corresponding Kaplan-Meier survival estimate at the given time-point. Mean absolute error in predictions, the mean squared error, and the 0.9 quantile of the absolute error is reported. "Error" refers to the difference between the predicted values and the corresponding bias-corrected calibrated values. Mean error was

Impact of delayed increases in % of CD8+ T cells on overall survival. Kaplan-Meier analysis of overall survival according to delayed changes (increase vs. decrease) of % of CD8+ T cells observed during ipilimumab treatment within patients showing early increases in ALC and delayed increase in % of CD4+ T cells. Censoring is indicated by vertical lines; p-values were calculated by log rank statistics.

Increases of CD4+, CD8+ and CD3- cell counts according to changes in ALC and overall survival according to changes of CD4+, CD8+ and CD3- cell counts. Absolute lymphocyte counts (ALC). *Early defines changes between baseline (BL) and a second time-point, ranging from 2-8 weeks after start of ipilimumab. #Delayed defines changes between BL and a third time-point, ranging from 8-14 weeks after start of ipilimumab. The percentage of patients identified with an increase in absolute numbers of CD4+, CD8+ T cells or CD3- lymphocytes was defined within patients showing an early increase or decrease in ALC (A). Kaplan-Meier analysis of overall survival according to early and delayed changes (increase vs. decrease) of absolute CD4+ T cells (B, E), CD8+ T cells (C, F) and CD3- lymphocytes (D, G) observed during ipilimumab treatment. Censoring is indicated by vertical lines; p-values were calculated by log rank statistics.

Multivariate Models including only patients receiving 3 mg/kg ipilimumab. Lactate dehydrogenase (LDH), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), hazard ratio (HR). * Relative lymphocyte count, absolute monocyte count, absolute and relative eosinophil count

Discriminatory ability of combination models. The concordance index (c-index, y-axis) was calculated for the combination of factors with independent impact according to Cox regression analysis (model 6.1) and 13 alternative combination models considering 5, 7, or 8 factors (A). The numbers refer to the rows in A. The c-indices are presented according to the number of combined factors (B). The combination model with highest discriminatory ability (7.4), which considered regulatory T cells in addition to the 6 factors with independent impact according to Cox regression analysis was chosen as combination model 1. No further increase of the c-index compared to combination model 1 was observed if one of the 3 remaining factors was additionally considered (models 8.1, 8.2, 8.3). C-indices were calculated for different combination models accounting for the number of unfavorable values of all factors considered in the given model (C). All possible models derived from combinations of the five routine factors were considered. The c-indices are presented according to the number of considered factors (D). The model with highest discriminatory ability (4.1) was selected.

Purpose: We compared the efficacy of human Langerhans cells (LC) as tumor immunogens in vivo with monocyte-derived dendritic cells (moDC) and investigated how interleukin 15 (IL15) supports optimal DC-stimulated antitumor immunity.Experimental Design: American Joint Committee on Cancer stage III/IV melanoma patients participated in this first clinical trial comparing melanoma peptide-pulsed LC with moDC vaccines (NCT00700167, www.ClinicalTrials.gov). Correlative studies evaluated mechanisms mediating IL15 support of DC-stimulated antitumor immunity.Results: Both DC vaccines were safe and immunogenic for melanoma antigens. LC-based vaccines stimulated significantly greater tyrosinase–HLA-A*0201 tetramer reactivity than the moDC-based vaccines. The two DC subtypes were otherwise statistically comparable, in contrast to extensive prior data in vitro showing LC superiority. LCs synthesize much more IL15 than moDCs and stimulate significantly more antigen-specific lymphocytes with a cytolytic IFN-γ profile even without exogenous IL15. When supplemented by low-dose IL15, instead of IL2, moDCs stimulate 5 to 6 logs more tumor antigen–specific effector memory T cells (TEMRA) over 3 to 4 weeks in vitro. IL2 and IL15 can be synergistic in moDC stimulation of cytolytic T cells. IL15 promotes T-cell expression of the antiapoptotic bcl-2 and inhibits candidate regulatory T-cell (Treg) expansion after DC stimulation, countering two effects of IL2 that do not foster tumor immunity.Conclusions: MoDC-based vaccines will require exogenous IL15 to achieve clinical efficacy. Alternatively, LCs can couple the endogenous production of IL15 with potent T-cell stimulatory activity. Optimization of full-length tumor antigen expression for processing into multiple immunogenic peptides for presentation by both class I and II MHC therefore merits emphasis to support more effective antitumor immunity stimulated by LCs. Clin Cancer Res; 17(7); 1984–97. ©2011 AACR.

Changes associated with overall survival and their correlation with clinical responses in sub-cohorts. Analyses were limited to patients with available data at all three time points or limited to patients treated with ipilimumab monotherapy at 3 mg/kg bodyweight. *Early defines a second time-point, ranging from 2-8 weeks after start (BL) of ipilimumab. #Delayed defines a third time-point, ranging from 8-14 weeks after start (BL) of ipilimumab.

Purpose: Cytotoxic T lymphocyte associated antigen (CTLA-4) blockade is being explored in numerous clinical trials as an immune-based therapy for different malignancies. Our group conducted the first preoperative clinical trial with the anti–CTLA-4 antibody ipilimumab in 12 patients with localized urothelial carcinoma of the bladder.Experimental Design: Six patients were treated with 3 mg/kg/dose of anti–CTLA-4 and six patients were treated with 10 mg/kg/dose of antibody. Primary end points of the study were safety and immune monitoring.Results: Most drug-related adverse events consisted of grade 1/2 toxicities. All patients had measurable immunologic pharmacodynamic effects, consisting of an increased frequency of CD4+ICOShi T cells in tumor tissues and the systemic circulation. To determine if CD4+ICOShi T cells could be a correlative marker for clinical outcome after treatment with anti–CTLA-4, a cohort of metastatic melanoma patients was studied retrospectively for frequency of CD4+ICOShi T cells and survival. Data from this small cohort of patients indicated that an increased frequency of CD4+ICOShi T cells, sustained over a period of 12 weeks of therapy, correlates with increased likelihood of clinical benefit consisting of overall survival.Conclusions: Our trial shows that anti–CTLA-4 therapy has a tolerable safety profile in the presurgical setting and that a preoperative model can be used to obtain biological data on human immune responses, which can efficiently guide the monitoring of patients treated in the metastatic disease setting. Clin Cancer Res; 16(10); 2861–71. ©2010 AACR.

Impact of cell frequencies or counts on OS at single time points. Absolute (Abs.), Baseline (BL), median survival time (MST), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), Relative (Rel.). *Early defines a second time-point, ranging from 2-8 weeks after start (BL) of ipilimumab. #Delayed defines a third time-point, ranging from 8-14 weeks after start (BL) of ipilimumab.

Gating strategy for quantification of subsets of T cells and regulatory T cells (Tregs), monocytes and myeloid-derived suppressor cells (MDSCs). Total cells were selected by gating on Time vs. SSC-A. Duplicates were removed via progressive gating on FSC-H vs. FSC-A and SSC-H vs. SSC-A. Dead cells were excluded by considering only EMA-negative cells. (A) A morphological gate was used to identify the population of lymphocytes. Next, CD3+ cells were selected and further separated into CD4+ and CD8+ cells. Ki67 expression was investigated on CD4+ and CD8+ cells. CD8+ T cells with suppressive potential were defined as CD103+. Previously described phenotypes of Tregs were defined as CD4+CD25+FoxP3+ and CD4+CD127lowCD25+FoxP3+. These were further subdivided into proliferating (Ki67+CD45RA-) and non-proliferating Tregs (Ki67-CD45RA+). (B) A lineage cocktail (CD3, CD19, CD56) was used to avoid cross-contamination. Previously described MDSC populations were identified as Lin-CD14+HLA-DRlow and Lin-CD14-CD15+CD11b+ within the all-cell gate. Overall monocytes were defined as CD14+, while subsets were separated into classical monocytes (Lin-CD14+CD16-HLA-DR+), non-classical monocytes (Lin-CD14-CD16+HLA-DR+) and Lin- CD14-CD16dimHLA-DR+ monocytes within the all-cell gate.

Overall survival according to clinical response and biomarker signature 12 weeks after start of ipilimumab. Overall survival (OS) was calculated from week 12 (day 84) to the date of last follow-up or death. OS according to clinical response in the week 12 tumor assessment (A) and according to the number of increasing cell subsets considering early changes in ALC, delayed changes in CD4+ T cells and delayed changes in CD8+ T cells (B). Among patients with clinical benefit (CR, PR, SD) in the 12 weeks tumor assessment those with increases in all three cell subsets had an excellent outcome. In contrast, three patients with early decrease in ALC and/or decrease of CD4+ or CD8+ frequencies had a poor prognosis despite of clinical benefit (C).

Supplementary Data from Preoperative CTLA-4 Blockade: Tolerability and Immune Monitoring in the Setting of a Presurgical Clinical Trial

Supplementary Figures S1-S3.

Purpose: To investigate changes of peripheral blood biomarkers and their impact on clinical outcome following treatment with ipilimumab in advanced melanoma patients.Experimental Design: Changes in blood counts and the frequency of circulating immune cell populations analyzed by flow cytometry were investigated in 82 patients to compare baseline values with different time-points after starting ipilimumab. Endpoints were overall survival (OS) and best clinical response. Statistical calculations were done by Wilcoxon-matched pairs tests, Fisher exact test, Kaplan–Meier analysis, and Cox regression analysis.Results: Increases in absolute lymphocyte counts (ALC) 2 to 8 weeks (P = 0.003) and in percentages of CD4+ and CD8+ T cells 8 to 14 weeks (P = 0.001 and P = 0.02) after the first dose of ipilimumab were correlated with improved survival. These associations did not meet significance criteria, when conservatively adjusted for multiple testing, but were additionally correlated with clinical responses (all P < 0.05). However, validation is required. Increases in all three factors were observed in 36% of patients, who had a favorable outcome and survival probabilities of 93.3% and 63.8% at 12 and 24 months, respectively. A partial or complete response was observed in 71% of these patients compared with only 8% in patients with decreases in ≥1 of the 3 factors, respectively. Changes of regulatory T cells or myeloid-derived suppressor cells were not associated with OS.Conclusions: Increases of ALC observed 2 to 8 weeks after initiation of ipilimumab and delayed increases in CD4+ and CD8+ T cells reflect changes associated with positive outcome. These changes represent surrogate marker candidates and warrant further validation. Clin Cancer Res; 22(19); 4848–58. ©2016 AACR.

Spectrum of factors, cut-offs, and differences in overall survival according to biomarkers in the identification and the confirmation cohort. Absolute (Abs.), Relative (Rel.), Lactate dehydrogenase (LDH), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs). * Green characters indicate the category associated with better survival in the identification cohort. ** Green cells indicate significant differences in overall survival (p < 0.05). Red cells indicate non-significant findings.

Correlations between adverse events and overall survival, clinical response, or biomarker categories. Overall survival was not different between patients stratified according to the occurrence of adverse events (AEs) in general (A) or immune-related AEs (irAEs). (B). Kaplan-Meier analysis is presented and censoring is indicated by vertical lines; p-values were calculated by log rank statistics (A&B). No correlations were observed between the occurrence of irAEs during ipilimumab treatment and the best tumor response (C, D) nor with the proposed combination groups of baseline biomarkers according to the combination model 1 (E) or combination model 2 (F). The best overall tumor response according to immune-related response criteria (irRC) was analyzed either as the rate of patients with an irRC response (sum of those with complete or partial responses) or irRC benefit (sum of those with complete responses, partial responses and stable disease). Differences were not statistically significant.

Panels of antibodies used for flow cytometry. * Cells were fixed and permeabilized with FoxP3 buffer (BD). Only frequencies of CD14+ cells from MDSC panel 1 and frequencies of CD4+, CD8+ T cells, as well as their ratio, were included from Treg panel 1. 1 Panel 1 (n=25), 2 Panel 2 (n=184).

Purpose: To identify baseline peripheral blood biomarkers associated with clinical outcome following ipilimumab treatment in advanced melanoma patients.Experimental Design: Frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), serum lactate dehydrogenase (LDH), routine blood counts, and clinical characteristics were assessed in 209 patients. Endpoints were overall survival (OS) and best overall response. Statistical calculations were done by Kaplan–Meier and Cox regression analysis, including calibration and discrimination by C-statistics.Results: Low baseline LDH, absolute monocyte counts (AMC), Lin−CD14+HLA-DR−/low-MDSC frequencies, and high absolute eosinophil counts (AEC), relative lymphocyte counts (RLC), and CD4+CD25+FoxP3+-Treg frequencies were significantly associated with better survival, and were considered in a combination model. Patients (43.5%) presenting with the best biomarker signature had a 30% response rate and median survival of 16 months. In contrast, patients with the worst biomarkers (27.5%) had only a 3% response rate and median survival of 4 months. The occurrence of adverse events correlated with neither baseline biomarker signatures nor the clinical benefit of ipilimumab. In another model, limited to the routine parameters LDH, AMC, AEC, and RLC, the number of favorable factors (4 vs. 3 vs. 2–0) was also associated with OS (P < 0.001 for all pairwise comparisons) in the main study and additionally in an independent validation cohort.Conclusions: A baseline signature of low LDH, AMC, and MDSCs as well as high AEC, Tregs, and RLC is associated with favorable outcome following ipilimumab. Prospective investigation of the predictive impact of these markers following ipilimumab and other treatments, e.g., PD-1 antibodies, is warranted. Clin Cancer Res; 22(12); 2908–18. ©2016 AACR.

Detailed gating strategy for quantification of subsets of monocytes and myeloid-derived suppressor cells (MDSCs), T cells and regulatory T cells (Tregs). Total cells were selected by gating on Time vs. SSC-A. Duplicates were removed via progressive gating on FSC-H vs. FSC-A and SSC-H vs. SSC-A. Dead cells were excluded by considering only EMA-negative cells. (A) A lineage cocktail (CD3, CD19, CD56) was used to avoid cross-contamination. Previously described MDSC populations were identified as Lin-CD14+HLA-DRlow and Lin-CD14-CD15+CD11b+ within the all-cell gate. Overall monocytes were defined as CD14+, while subsets were separated into classical monocytes (Lin-CD14+CD16-HLA-DR+), non-classical monocytes (Lin-CD14-CD16+HLA-DR+) and Lin- CD14-CD16dimHLA-DR+ monocytes within the all-cell gate. (B) A morphological gate was used to identify the population of lymphocytes. Next, CD3+ cells were selected and further separated into CD4+ and CD8+ cells. Ki67 expression was investigated on CD4+ and CD8+ cells. CD8+ T cells with suppressive potential were defined as CD103+. Previously described phenotypes of Tregs were defined as CD4+CD25+FoxP3+ and CD4+CD127lowCD25+FoxP3+. These were further subdivided into proliferating (Ki67+CD45RA-) and non-proliferating Tregs (Ki67-CD45RA+).

Overall survival and distribution after first dose of ipilimumab according to subsequent treatments. Of 209 patients, 71 received at least one additional systemic line of treatment after ipilimumab. 137 individuals did not receive further therapy and data were not available for one patient. 47 (combination model 1) or 67 (combination model 2) of 71 patients had complete data for classification according to biomarker combination models. The representation of PD-1/PD-L1-treated patients in the biomarker groups was shifted towards favorable biomarker combination groups for both combination models compared to those without subsequent PD-1/PD-L1 treatment. Therefore, a confounding effect of subsequent treatment with PD-1/PD-L1 antibodies on the biomarker results of this study cannot be ruled out (A). To investigate the potential confounding impact on OS and biomarker findings, subsequent treatments were categorized into three different groups: BRAF/MEK inhibitors (n=24), PD-1/PD-L1 antibodies (n=28), and chemotherapy/other treatments (n=33) and analyzed by the Kaplan-Meier method (B). Patients treated with PD-1/PD-L1 antibodies had a significant better survival compared to all 71 patients (p=0.006), while no significant difference was observed for the other two groups. Kaplan Meier analysis of overall survival of patients classified according to combination model 1 (C) or combination model 2 (D) is presented after exclusion of individuals who received subsequent treatment with anti-PD-1 or PD-L1 antibodies, as a confounding effect could not be ruled out. However, the prognostic impact of the proposed biomarker combinations at baseline of ipilimumab treatment remained strong (p

Individual early changes in absolute lymphocyte counts and delayed changes in frequencies of CD4+ and CD8+ T cells in different outcome groups. Individual early changes of absolute lymphocyte counts (ALC; A-C), and delayed changes in frequencies of CD4+ (D-F) and CD8+ (G-I) T cells during ipilimumab are presented for patients who were assigned to three prognostic groups differing in survival after start of ipilimumab (A, D, G - left: {less than or equal to}6 months; A, D, G - middle: 7-18 months; A, D, G - right: >18 months). Patients censored at 18 months of less were excluded (A, D, G). Individual early changes in ALC, delayed changes in % of CD4+ and CD8+ are presented in patients who experienced progressive disease (B, E, H - left) or clinical benefit (complete response or partial response or stable disease; B, E, H - right) and in non-responders (stable or progressive disease; C, F, I - left) or in responders (complete or partial response; C, F, I - right) according to immune related response criteria (irRC). Changes were analyzed separately for all groups using Wilcoxon matched pairs tests. The p-value is presented in case of a significant change or strong trend within the respective group.