Supplement Figures S1-S4 from T-cell Homing Therapy for Reducing Regulatory T Cells and Preserving Effector T-cell Function in Large Solid Tumors

Fig. S1. CD4+ T cell infiltration in mouse and human tumor sections. Fig. S2. Correlations between the numbers of infiltrated murine T cells and the ratios of costimulatory and coinhibitory receptors. Fig. S3. Quantitative PCR analysis of CXCL9 and CXCL10 mRNA expression in tumor cells that were treated with doxorubicin. Fig. S4. Induction of CXCL9 and CXCL10 at mRNA levels in LLC and Mel2549 tumors after the treatment with IL-12 plus doxorubicin plus T cell infusion.</p

Supplementary Figures 1 through 9 and Supplementary Materials and Methods from Induction of NKG2D Ligands on Solid Tumors Requires Tumor-Specific CD8⁺ T Cells and Histone Acetyltransferases

Fig. S1. Gradual reduction of Rae-1 during tumor progression. Fig. S2. Induction of Rae-1 by chemotherapy in vitro but not in vivo. Fig. S3 Effector molecule perforin expression in tumors after indicated treatments. Fig. S4 Rae-1 expression is not induced by IL-12 plus doxorubicin in normal tissues. Fig. S5 Accumulation of NKG2D+CD8+ T cells in tumors after IL-12 plus doxorubicin treatment. Fig. S6 Immunochemistry staining of Rae-1 and TUNEL assay in tumor sections. Fig. S7 Rae-1 induction by IL-12 plus doxorubicin only occurs on tumor cells in vivo. Fig. S8 Antitumor cytotoxicity of IL-12 plus doxorubicin abolished by depletion of CD8+ T cells, but not CD4+ T cells or NK cells. Fig. S9 Enrichment of NK cells, CD4+ T cells, or CD8+ T cells in splenocytes collected from BALB/c mice treated with IL-12 plus doxorubicin.</p

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8⁺ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

<div><p>Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8⁺ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8⁺ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8⁺ T cells, on the yield, phenotype, and functional activity of expanded CD8⁺ T cells during the REP. We found that CD8⁺ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG₄ (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8⁺ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8⁺ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8⁺ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity <i>in vivo</i> after adoptive transfer into patients.</p> </div

Addition of anti-4-1BB antibody increased Granzyme B (GB) and Perforin (Perf) expression in CD8⁺ T cells during the REP in multiple patient samples.

<p>TIL from a representative patient was subjected to the REP with or without the addition of anti-4-1BB antibody and the cells stained for CD8 and Perf (<b>A</b>) or CD8 and GB (<b>B</b>) and analyzed by flow cytometry both before the REP (pre-REP) and after the control (IL-2) REP or REP with anti-4-1BB (IL-2+4-1BB). Viable cells were gated and the frequency of Perf⁺ (<b>A</b>) or GB⁺ (<b>C</b>) in the CD8⁺ subset analyzed. The change in Perf or GB expression in the CD8⁺ T-cell subset during the REP with or without added anti-4-1BB was determined in larger cohort of patients TIL samples (<b>B</b> and <b>D</b>). 4-1BB co-stimulation during the REP significantly increased Perf expression in CD8⁺ T cells in 34 separate TIL line tested (<b>B</b>) and increased GB expression in 11 separate TIL lines tested (<b>D</b>). Statistical analysis was done using Wicoxon signed rank sum test using biological relevance occurring when p<0.05.</p

Supplementary Tables 1 - 5 from A New Approach to Simultaneously Quantify Both TCR α- and β-Chain Diversity after Adoptive Immunotherapy

PDF file, 111K, Supplementary Table S1. Samples from 14 patients that received autologous TILs were studied. Supplementary Table S2.TCR Vα primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S3. TCR Vβ primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S4. House keeping genes used in DTEA analysis. Supplementary Table S5. DTEA can detect the low-frequency TCRVβ genes that cannot be readily identified by classical cloning.</p

Addition of anti-4-1BB antibody maintained CD28 expression on CD8⁺ T cells during the REP in a large cohort of patient samples (n = 34).

<p>TIL from a representative patient was subjected to the REP with or without the addition of anti-4-1BB antibody and the cells stained for CD8 and CD28 (<b>A</b>) or CD8 and CD27 (<b>B</b>) and analyzed by flow cytometry both before the REP (pre-REP) and after the control (IL-2) REP or REP with anti-4-1BB (IL-2+4-1BB). Viable cells were gated and the frequency of CD28⁺ (A) or CD27⁺ (<b>C</b>) in the CD8⁺ subset analyzed. The change in CD28 or CD27 expression in the CD8⁺ T-cell subset during the REP with or without added anti-4-1BB was determined in 34 independent TIL lines (<b>B</b> and <b>D</b>). The left hand panels in B and D show the overall median difference in the frequency of CD28⁺ or CD27⁺ cells in the CD8⁺ subset over the entire patient population. The right hand panels in <b>B</b> and <b>D</b> show the fold expansions in the CD28⁺ or CD27⁺ populations. Statistical analysis was done using Wicoxon signed rank sum test using biological relevance occurring when p<0.05.</p

Supplemental Figure 1 from Manipulating the Tumor Microenvironment <i>Ex Vivo</i> for Enhanced Expansion of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy

Supplemental Figure 1. Detection of 4-1BB on CD8+ T cells within fresh melanoma tumor fragments and in early tumor fragment cultures.</p

Addition of 4-1BB antibody in the REP increases CD8⁺ T-cell frequency and yield in a large cohort of patient samples (n = 34).

<p>The effects of addition of an optimal dose of anti-4-1BB (500 ng/ml), as determined previously, were tested in rapid expansions of TIL from 34 separate patient tumors. Addition of anti-4-1BB significantly increased the frequency of CD8⁺ T cells in the final TIL product in the patient population (<b>A</b>). CD8⁺ T-cell yield (<b>B</b>), and the fold expansion of CD8⁺ cells (<b>C</b>), was significantly increased when anti-4-1BB was added to the REP over the patient population. However, 4-1BB stimulation did not alter the total T-cell yield (<b>B</b>) or the total TIL fold expansion (<b>C</b>). Statistical analysis was done using the Wilcoxon signed rank test using biological relevance occurring when p<0.05.</p

CD8⁺ MART-1-reactive TIL provided 4-1BB co-stimulation during the REP have a greater proliferative response to re-stimulation with MART-1 peptide.

<p>TIL from HLA-A0201⁺ patients were rapidly expanded with or without added anti-4-1BB antibody. The cells were labeled with eFluor670 dye and re-stimulated with HLA-A0201⁺ MART-1 peptide-pulsed DC, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060031#pone.0060031-Li1" target="_blank">[8]</a>. The post-REP TIL were labeled with the cell division dye eFluor670 (Invitrogen) and re-stimulated with HLA-A0201-matched mature DC pulsed with MART-1 peptide for 7 days. The cells were harvested and stained for CD8 and MART-1 tetramer and analyzed by flow cytometry for the frequency of divided (eFluor670^low) in the gated CD8⁺ MART-1 tetramer⁺ subset. One representative experiment out of two is shown. As shown in <b>A</b>, CD8⁺ TIL isolated from 4-1BB costimulated REP cultures (#2183) had an enhanced response to MART-1 peptide re-stimulation, as shown by the increased number of cell divisions measured by eFluor670 dilution in the CD8⁺ MART-1 tetramer⁺ gated cells. TIL isolated from REP cultures that received anti-4-1BB also exhibited an increase in the fold expansion of CD8⁺ MART-1 tetramer⁺ cells following re-stimulation of the post-REP TIL with MART-1 peptide pulsed DC as shown in experiments with two separate TIL lines (#2183 and #2559) (<b>B</b>).</p

Supplementary Figures and Tables from Hypoxia-Driven Mechanism of Vemurafenib Resistance in Melanoma

Figure S1. Chemical Structure of MSC2156119J (Tepotinib, EMD 1214063); Figure S2. Morphology of melanoma cell 2D ambient air cultures and spheroids; Figure S3. The immunofluorescent staining of DAPI and LOX-1 in A375 2D ambient air cultures; Figure S4. The immunofluorescent staining of LOX-1 in MEL1617 3D spheroids on day 3 and 8 of cultures; Figure S5. The human phospho-kinase antibody array of 451Lu spheroids and 2D ambient air cultures; Figure S6. The human phospho-kinase antibody array of A375 spheroids and 2D ambient air cultures; Figure S7. Effect of PLX4032 on BRAF-ERK pathway in melanoma cells under hypoxic conditions. Supplementary Table S1: Primers for RT-PCR and Real-time PCR; Supplementary Table S2: Patient Characteristics; Supplementary Table S3: List of cutaneous melanoma cell lines in CCLE project</p