American Association for Cancer Research
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Supplementary Figure Legends, Figures 1 - 4 from Foxp3+ T Cells Inhibit Antitumor Immune Memory Modulated by mTOR Inhibition

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journal contribution
posted on 2023-03-30, 22:25 authored by Yanping Wang, Tim Sparwasser, Robert Figlin, Hyung L. Kim

PDF file - 308KB, Supplemental Figure 1. Tumor increases Tregs. The percent of CD4 cells expressing FoxP3 were examined from lymph nodes, spleen, bone marrow and peripheral blood lymphocytes (PBL) from tumor (~1 cm in diameter)bearing mice and control mice without tumor (n=5 per group). Flow cytometry results and summary data are shown. Histograms provide mean + SEM. *p<0.0,***p<0.005. Supplemental Figure 2. αCD4 antibody depletes effector CD4 subtypes. a. Splenocytes (n=3 per group) were collected before (day 0) and 1 or 10 days after administering αCD4 antibody, and analyzed by flow cytometry. The number of total splenocytes staining for CD4 and IL-2, IL-4 or IL-17 is shown. b. Percent of CD4 cells staining for IL-2, IL-4 or IL-17 is shown. Histograms provide mean + SEM. Supplemental Figure 3. CD4 depletion and mTor inhibition enhanced the antitumor effect of DC vaccine in B6 mice implanted with B16 tumor cells. B16 tumor cells were implanted into B6 mice (n=5 per group) on day 0. Tumor-lysate pulsed DCs were administered on day 3. CD4 lymphocytes were depleted with αCD4 antibody on days 6 and 9. Mice were treated with daily temsirolimus on days 9 to 23. Tumor growth was monitored. *p<0.05, **p<0.01,***p<0.005. Supplemental Figure 4. CD4 depletion prior to immune stimulation prevents formation of CD8 memory cells. The experimental scheme is similar to Figure 4 (n=5 per group). An additional group is included where αCD4 antibody is administered on day -1 and +1 (Group A). a. The percent of CD8 cells expressing Thy1.1 is shown from splenocytes harvested 5 days following rechallenge of memory cells. b. The percent of CD8 cells expressing Eomes is shown. Histograms provide mean + SEM. *p<0.05, **p<0.01,***p<0.005.



Inhibition of mTOR signaling enhances antitumor memory lymphocytes. However, pharmacologic mTOR inhibition also enhances regulatory T-cell (Treg) activity. To counter this effect, Treg control was added to mTOR inhibition in preclinical models. Tregs were controlled with CD4-depleting antibodies because CD4 depletion has high translational potential and already has a well-established safety profile in patients. The antitumor activity of the combination therapy was CD8 dependent and controlled growth of syngeneic tumors even when an adoptive immunotherapy was not used. Lymphocytes resulting from the combination therapy could be transferred into naïve mice to inhibit aggressive growth of lung metastases. The combination therapy enhanced CD8 memory formation as determined by memory markers and functional studies of immune recall. Removal of FoxP3-expressing T lymphocytes was the mechanism underlying immunologic memory formation following CD4 depletion. This was confirmed using transgenic DEREG (depletion of regulatory T cells) mice to specifically remove Foxp3+ T cells. It was further confirmed with reciprocal studies where stimulation of immunologic memory because of CD4 depletion was completely neutralized by adoptively transferring tumor-specific Foxp3+ T cells. Also contributing to tumor control, Tregs that eventually recovered following CD4 depletion were less immunosuppressive. These results provide a rationale for further study of mTOR inhibition and CD4 depletion in patients. Cancer Res; 74(8); 2217–28. ©2014 AACR.

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