Tumors attenuating the mitochondrial activity in T cells escape from PD-1 blockade therapy
- Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Japan
Figures
Figure 1 with 3 supplements
PD-1 blockade significantly enhances the number and function of effector CD8+ T cells in mice with responsive, but not in those with unresponsive tumors.
(A) Schematic diagram of the experimental schedule. (B) Absolute number of lymphocytes per draining lymph node (DLN) was calculated and compared among mice with different responsive or unresponsive …
Figure 1—figure supplement 1
Stratification of responsive and unresponsive tumors in C57BL/6N and BALB/c genetic backgrounds.
(A) Schematic diagram of the PD-1 blockade therapy. Tumor size was measured on every alternative day. (B-C) Tumor growth was measured in anti-PD-L1 mAb treatment model (top panels) or genomic PD-1-/-…
Figure 1—figure supplement 2
Increment of CD44+ CD8+ T cells in DLN after PD-1 blockade in the hosts with responsive tumor.
Related with Figure 1C. CD8+ DLN cells were gated for CD44+. Representative FACS pattern for each group is shown in upper panel. CD44+ frequency (lower panel, left figure) and absolute number (lower …
Figure 1—figure supplement 3
Higher immune responses in responsive tumor-bearing host after PD-1 blockade compared to unresponsive group in BALB/c background.
Following the experimental schedule as mentioned in Figure 1A, mice were sacrificed on day 12 for analysis of immune responses. (A) Absolute number of lymphocytes per LN were compared between …
Figure 2 with 1 supplement
PD-1 blockade significantly enhances mitochondrial activity in CD8+ T cells in mice with responsive, but not in mice with unresponsive tumors.
(A) DLN CD8+ T cells were purified from the pool of five mice per group from the experiment of Figure 1. OCR of DLN CD8+ T cells was measured from responsive and unresponsive tumor groups (left). …
Figure 2—figure supplement 1
CD8+ T cells from mice with sensitive tumor have higher mitochondrial activity after PD-1 blockade than those with unresponsive tumors in BALB/c background.
(A) Representative OCR plot by Seahorse XFe analyzer, showing basal respiration, maximal respiration, spare respiratory capacity, ATP turnover, proton-leak, and non-mitochondrial respiration, is …
Figure 3 with 1 supplement
Unresponsive tumors can be classified into systemically immunosuppressive or non-immunosuppressive tumors.
(A) Unresponsive tumor cells (LLC, Pan02 and B16) were inoculated on the left flank of C57BL/6N mice. On day 6, responsive tumor (MC38) cells were inoculated on the right flank of the same mice. On …
Figure 3—figure supplement 1
Unresponsive tumors can be classified into systemically immunosuppressive or non-immunosuppressive tumors.
(A) Unresponsive tumor (B16) and responsive tumor (GL261) were injected and therapy were given as per the schedule mentioned in Figure 3A. Tumor growth of right responsive tumor (GL261) with or …
Figure 4
Unresponsive tumor-derived immune suppressive factor inhibits the mitochondrial responses in CD8+ T cells in vivo.
(A) Mice were treated in the same way as Figure 3A and sacrificed on day 14 for the analysis of DLN CD8+ T cells. (B) Absolute number of lymphocytes per LN from the MC38 side was calculated. (C) DLN …
Figure 5
The absence of MHC class I expression in B16.
(A–B) Tumor growth of responsive and unresponsive tumors was observed in wild type or immune-compromised (Rag2-/-) mice. Tumor sizes of responsive tumors (A) and unresponsive tumor (B) are shown. …
Figure 6 with 4 supplements
Small soluble factors released from SIP-positive tumors inhibit the T cell proliferation and mitochondrial function in vitro.
(A) Naïve CD8+ T cells (CD44- CD8+ T cells) were purified from spleen and LNs of C57BL/6N mice. Naïve CD8+ T cells were stimulated with anti-(CD3+CD28) mAbs-coated dynabeads for 48 hrs with or …
Figure 6—figure supplement 1
Tumor-derived suppressive factor inhibits proliferation and mitochondrial function of CD8+ T cells in vitro.
As per schedule mentioned in Figure 6A, naïve CD8+ T cells were stimulated in the presence of culture supernatant. (A-B) Naïve CD8+ T cells were stimulated in the presence of culture supernatant of …
Figure 6—figure supplement 2
Some human cancer cell lines release suppressive factors which inhibit T cells proliferation in vitro.
(A) Schedule of in vitro T cell proliferation assay (upper panel). Murine naïve CD8+ T cells were stimulated for 48 hrs with anti-(CD3+CD28) mAbs in the presence of culture supernatants collected …
Figure 6—figure supplement 3
Soluble suppressive factors inhibit mitochondria.
(A) In continuation with Figure 6F, mRNA level of PGC-1α and PGC-1β were quantified in the tumor supernatant (sup) treated naïve CD8+ T cells. (B) B cells (B220+ lymphocytes) were purified from …
Figure 6—figure supplement 4
Previously reported factors are not related with the nature of SIP- positive tumors.
Expression levels of immune suppression-associated genes were examined by quantitative reverse transcription PCR (qRT-PCR) in different tumor cell lines. Tumor names in red and blue are responsive …
Figure 7 with 1 supplement
Enhancing mitochondrial activation by bezafibrate partially overcomes suppression and improves survival of SIP-positive tumor-bearing hosts in vivo.
(A) Naïve CD8+ T cells purified from spleen and LNs of C57BL/6N mice were stimulated for 48 hrs with anti-(CD3+CD28) mAb along with LLC culture supernatant and Bezafibrate (5 μM). Following …
Figure 7—figure supplement 1
Enhancing mitochondrial activation by bezafibrate chemicals improves the anti-tumor effect for SIP-positive tumor in BALB/c background.
SIP-positive CT26 tumor was injected in mice and therapy was given as per schedule mentioned in Figure 7B. Tumor size (left) and survival (right) of CT26 tumor-bearing host are shown. Data represent …
Tables
Table 1
List of mouse cell lines from different genetic backgrounds used in this study.
| Cell line | Background | Response to PD-1 blockade therapy | Particulars | Source |
|---|---|---|---|---|
| GL261 | C57BL/6N | Responsive | Glioblastoma cell line | As a gift from Dr. Toda, Keio University, Japan |
| MC38 | C57BL/6N | Responsive | Colon carcinoma cell line | As a gift from Dr. James P. Allison, Memorial Sloan-Kettering Cancer Center (New York, NY, USA) |
| LLC | C57BL/6N | Unresponsive | Lewis lung carcinoma cell line | American Type Culture Collection |
| B16 | C57BL/6N | Unresponsive | Melanoma cell line | As a gift from Dr. Nagahiro Minato, Graduate School of Medicine, Kyoto University |
| Pan02 | C57BL/6N | Unresponsive | Pancreatic ductal adenocarcinoma cell line | National Cancer Institute |
| MethA | BALB/c | Responsive | 3-methylcholanthrene (MCA)-induced fibrosarcoma cell line | Cell Resource Center for Biomedical Research (Sendai, Japan) |
| CT26 | BALB/c | Unresponsive | N-nitroso-N-methylurethane-(NNMU) induced colon carcinoma cell line | National Cancer Institute |
Additional files
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Supplementary file 1
List of primers for quantifying mouse gene transcripts by qRT-PCR (related to Figure 6—figure supplements 3 and 4).
- https://cdn.elifesciences.org/articles/52330/elife-52330-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52330/elife-52330-transrepform-v2.docx
