Supplementary Figures from Age Correlates with Response to Anti-PD1, Reflecting Age-Related Differences in Intratumoral Effector and Regulatory T-Cell Populations
Authors: Kugel Curtis H. , Douglass Stephen M. , Webster Marie R. , Kaur Amanpreet , Liu Qin , Yin Xiangfan , Weiss Sarah A. , Darvishian Farbod , Al-Rohil Rami N. , Ndoye Abibatou , Behera Reeti , Alicea Gretchen M. , Ecker Brett L. , Fane Mitchell , Allegrezza Michael J. , Svoronos Nikolaos , Kumar Vinit , Wang Daniel Y. , Somasundaram Rajasekharan , Hu-Lieskovan Siwen , Ozgun Alpaslan , Herlyn Meenhard , Conejo-Garcia Jose R. , Gabrilovich Dmitry , Stone Erica L. , Nowicki Theodore S. , Sosman Jeffrey , Rai Rajat , Carlino Matteo S. , Long Georgina V. , Marais Richard , Ribas Antoni , Eroglu Zeynep , Davies Michael A. , Schilling Bastian , Schadendorf Dirk , Xu Wei , Amaravadi Ravi K. , Menzies Alexander M. , McQuade Jennifer L. , Johnson Douglas B. , Osman Iman , Weeraratna Ashani T. 
Published: April 2023

<p>PDF containing all supplementary figures. Supplementary Figure Legends. Supplemental Figure 1. A, graph indicating the number of patients for which best response data to pembrolizumab was obtained, and from which institution. B, various response groups of patients treated with pembrolizumab, separated by gender, and using 62 years of age as the most statistically significant cut-off. Supplemental Figure 2. A, B Change in mouse weight on final day of experiment from start in female and male mice bearing BSC9AJ2 tumors, after treatment with 3 doses of 300µg rat IgG2AK (N=5) or 4 doses of 300µg anti-PD1 (N=5) every 5 days, starting on day 0. Supplemental Figure 3. A, CD45+ cells within Yumm1.7 and B. BSC9AJ2 tumors. Err=95%CI. B, CD45+ cells within in tumors of Yumm1.7 and Yumm2.1 tumors. C, CD8b+ cells following 5 hour incubation with PMA and ionomycin analyzed by FACS analysis. Err=SD. D, E CD45+CD8b+ cells expressing TNFα and IFNγ within spleens from mice harboring Yumm1.7 or BSC9AJ2 tumors. Err=95%CI. Significance was determined using individual two-tailed student's t-test assuming unequal variance for 1-2 factors and using a 2-way ANOVA for 3 or more factors. Supplemental Figure 4. A 10X view with 40X zoom of FOXP3+ foci staining in melanoma patient biopsies. B, FOXP3+ staining from patient tumors across various age groups. C, CD8+ staining from patient tumors across various age groups. D, percentages of patients in the indicated age groups, whose CD8:FOXP3 ratio is low (< median - 1), med (median +/- 1), or high (> median + 1). Supplemental Figure 5. A. Mouse weight on final day of experiment from start in mice treated with anti-CD25 or an IgG1A isotype 5 days prior to sub-dermal of BSC9AJ2 cells with treatments continuing every 5 days until sacrifice. Anti-PD1 was administered every 3-4 days, starting on day 9 post-tumoral injection. Err=95%CI. Statistical significance determined by 2 way ANOVA. Err bars = SEM. B. Tumor growth for individual mice, per condition in A. Supplemental Figure 6. A. Ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ in BSC9AJ2 tumors from young female mice 4 days following cyclophosphamide intraperitoneal injection at indicated doses. 5 mice in PBS group, and 3 mice per dose. Err=SEM. B, intra-tumoral CD3+CD4+FoxP3+ populations. Err=SD. C, tumor growth in young female mice bearing BSC9AJ2 tumors were injected 6 days post tumor implantation with either 100µL PBS control, or 100µL PBS containing 25 mgs/kg cyclophosphamide intra-peritoneally. Four days later, mice were also given either 300µg anti-PD1 or vehicle control. A total of 4 doses of PD1 was given on day 0, 5, 10, and 14. Statistical significance was determined using a linear mixed-effect model. Err.=SEM. D, fold change in mouse weight from C. Err=SD. E. Intra-tumoral ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ at the end of the experiment. Err.=SEM.</p>