Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab′)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies.

Significance:

Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature.

Regulatory T cells (Tregs) inhibit antitumor immunity at different phases and locations (e.g., priming phase in lymph nodes and effector phase in tumor tissue) by suppressing the function of T, natural killer (NK), and antigen-presenting cells (1–3). Tumors recruit and accumulate Tregs in tumor-microenvironment to evade antitumor immune activities (4, 5). Nevertheless, our knowledge regarding roles of Tregs in tumor immunity is largely derived from analyses after systemic depletion of Tregs (6–9) or systemic manipulation of Treg function (10). The specific roles that intra- and extratumoral Tregs play remain unclear due to technical difficulties in depleting or manipulating Tregs in a spatially specific manner.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel technology to selectively target and deplete cells using antibodies, against surface antigens, conjugated with a phthalocyanine dye, IR700. Once the antibody-IR700 conjugate is exposed to NIR-light, the targeted cells rapidly undergo necrosis (11, 12). Currently, EGFR-targeting NIR-PIT is in phase III clinical trial in head and neck cancer (ClinicalTrials.gov Identifier: NCT02422979). To achieve spatially specific depletion of Tregs, we have previously developed anti-CD25-F(ab′)2 NIR-PIT (13). An injection of anti-CD25-F(ab′)2-IR700 conjugate followed by NIR irradiation on the tumor induces rapid, selective depletion of pre-existing intratumoral Tregs, without depleting T cells that upregulated CD25 after the NIR-PIT, and suppresses tumor growth (13). This therapeutic effect depends on the presence of CD8 T and NK cells and their production of IFNγ (13). However, the exact target of IFNγ and the mechanisms of the consequent tumor regression remain to be elucidated.

In this study, we sought to understand the roles played by intratumoral Tregs by selectively depleting these Tregs utilizing anti-CD25-F(ab′)2 NIR-PIT. We found that depletion of tumor-infiltrating Tregs caused rapid regression of tumor vessels, intratumoral ischemia, and necrosis/apoptosis of tumor cells in an IFNγ-dependent manner. IFNγ receptor expression on vascular endothelial cells was required for these changes. We further examined a combination therapy with IL15 administration to induce complete eradication of the tumors. Our study sheds light on the understanding of the roles that intratumoral Tregs play in maintaining tumor microenvironment that supports tumor growth.

Mice

C57BL/6 (expressing either Ly5.1 or Ly5.2) mice, BALB/c mice, Ifngr1−/−, Ifngr1flox/flox, Tie2Cre, Foxp3DTR, and albino mice on a C57BL/6 background were purchased from Jackson Laboratory. Il15ra−/− mice (Jackson Laboratory) were backcrossed to C57BL/6 mice for 10 generation. All mouse experiments were performed in accordance with animal protocols approved by National Institutes of Health Animal Care and Use Committee. Males and females at 6- to 10-week-old were used.

Tumor cell lines and subcutaneous and lung metastasis tumor models

MC38, LL/2, and 4T1 tumor cell lines (ATCC) and EO771 tumor cell line (CH3 BioSystems) were maintained in RPMI1640 medium supplemented with 10% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.05 mmol/L 2-mercaptoethanol. All cell lines were tested negative for Mycoplasma (PCR at Frederick National Laboratory for Cancer Research) and authenticated (ATCC) before use. Subcutaneous tumors were generated by inoculating 4.0 × 105 MC38 cells, 8.0 × 105 EO771 cells, or 2.0 × 105 LL/2 cells in 100 μL PBS in right dorsum of C57BL/6-background mice, and 1.0 × 105 4T1 cells in BALB/c mice. Tumor size (V), calculated as V = (major axis) × (minor axis)2 × 1/2, was followed up until it reached 2,500 mm3. Mice with approximately 100 mm3 tumor were randomly grouped for experiments unless otherwise stated. For lung metastases generation, 1.0 × 106 MC38 or EO771 cells in 300 μL PBS were intravenously injected.

NIR-PIT

IR700-conjugated anti-CD25-F(ab′)2 and isotype matched control-F(ab′)2 were prepared as described in Supplementary Information. Subcutaneous tumor-bearing mice received 50 μg IR700-conjugated F(ab′)2 in 200 μL PBS intravenously, were shaved for irradiation, and, on the next day, underwent NIR-PIT (λ = 690 ± 5 nm) on tumor at 50 J/cm2 (ML7710 Laser System; Modulight Inc.).

Flow cytometry analysis

Single cell suspensions of tissues were incubated with anti-CD16/32 antibody, followed by fluorophore-conjugated antibodies. For intracellular-staining, minced tumors were incubated with 3 μg/mL brefeldin A (4 hours) without exogenous stimulation, made into single cell suspensions, fixed and permeabilized, and stained with antibodies. Where indicated, ex vivo stimulation with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μmol/L ionomycin (4 hours) was performed. See Supplementary Information for details.

Histological analysis

Lectin perfusion assay by DyLight 594-conjugated Tomato-lectin intravenous injection, vessel leakiness assay by FITC-conjugated dextran sulfate intravenous injection, pimonidazole-binding assay by pimonidazole intravenous injection, multiplex IHC including phopho-STAT1 staining, propidium-iodide intravenous injection assay, TUNEL assay using Click-iT TUNEL Alexa Fluor Imaging Assay, and transmission electron microscopy (TEM) studies are described in detail, along with quantification methods, in Supplementary Information.

Statistical analysis

P values between unpaired variables were calculated using Student t test. Where indicated, paired t test and χ2 test were used. P values less than 0.05 were considered significant. The results were shown as mean ± SEM.

Additional materials and methods

Additional details and product catalog numbers are described in the Supplementary Materials and Methods and Supplementary Table.

Selective depletion of intratumoral Tregs by anti-CD25-F(ab′)2 NIR-PIT suppresses tumor growth

To selectively deplete intratumoral Tregs, we employed anti-CD25-F(ab′)2 NIR-PIT (13). CD25 was highly and selectively expressed in CD3+CD4+Foxp3+ Tregs residing in tumor (Supplementary Figs. S1A and S1B), and anti-CD25-F(ab′)2 NIR-PIT selectively depleted intratumoral CD3+CD4+CD25+Foxp3+ Tregs by more than 90% within 30 minutes of NIR-light exposure (Fig. 1A), whereas Tregs in the lymph nodes and spleen and CD3+CD4+Foxp3 non-Tregs in tumor remained intact (Figs. 1A and B). We further confirmed that immune cells other than Tregs were not depleted, through a comprehensive analysis of lymphoid and myeloid cell populations in tumor tissues (Supplementary Figs. S1A–S1F and S2A and S2B). IR-700-conjugated anti-CD25-F(ab′)2 does not bind to tumor cells nor does it induce nonspecific cell death upon NIR-light irradiation (13). The fraction of Tregs among CD4+ T cells began to recover from day 2 and reached near pretreatment levels by day 4 (Fig. 1C). This recovery was much quicker than that after systemic depletion of Tregs in Foxp3DTR mice by intravenous injection of diphtheria toxin (DT; Supplementary Figs. S3A and S3B), indicating that recruitment of circulating Tregs, not conversion from non-Treg CD4 T cells, mainly contributes to the replenishment of intratumoral Tregs after anti-CD25-F(ab′)2 NIR-PIT.

Figure 1.

Selective depletion of intratumoral Tregs suppresses tumor growth. A, Selective depletion of intratumoral Tregs analyzed 30 minutes after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (approximately 100 mm3). Representative flow cytometry data showing frequency of CD25+Foxp3+ Tregs in CD3+CD4+ T cells in indicated tissues (n = 3 each). B, Frequency and cell number of CD3+CD4+CD25+Foxp3+ Tregs and CD3+CD4+Foxp3 non-Treg cells in total cells in MC38 tumor 30 minutes after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT, analyzed by flow cytometry (n = 3 each). C, Changes in the frequency of Foxp3+CD25+ Tregs among CD3+CD4+ T cells in MC38 and EO771 tumors over 4 days after anti-CD25-F(ab′)2 NIR-PIT, analyzed by flow cytometry (n = 3 at each time point). D, Frequency of indicated subsets of lymphocytes in total cells in untreated MC38, EO771, LL/2, and 4T1 tumors, analyzed by flow cytometry (n ≥ 5 each). *, compared with MC38 tumor; †, compared with EO771 tumor. E, Tumor growth after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38, EO771, LL/2, and 4T1 tumors. * and †, P < 0.05; ** and ††, P < 0.01.

Figure 1.

Selective depletion of intratumoral Tregs suppresses tumor growth. A, Selective depletion of intratumoral Tregs analyzed 30 minutes after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (approximately 100 mm3). Representative flow cytometry data showing frequency of CD25+Foxp3+ Tregs in CD3+CD4+ T cells in indicated tissues (n = 3 each). B, Frequency and cell number of CD3+CD4+CD25+Foxp3+ Tregs and CD3+CD4+Foxp3 non-Treg cells in total cells in MC38 tumor 30 minutes after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT, analyzed by flow cytometry (n = 3 each). C, Changes in the frequency of Foxp3+CD25+ Tregs among CD3+CD4+ T cells in MC38 and EO771 tumors over 4 days after anti-CD25-F(ab′)2 NIR-PIT, analyzed by flow cytometry (n = 3 at each time point). D, Frequency of indicated subsets of lymphocytes in total cells in untreated MC38, EO771, LL/2, and 4T1 tumors, analyzed by flow cytometry (n ≥ 5 each). *, compared with MC38 tumor; †, compared with EO771 tumor. E, Tumor growth after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38, EO771, LL/2, and 4T1 tumors. * and †, P < 0.05; ** and ††, P < 0.01.

Close modal

Next, to analyze how CD8 T- and NK-cell infiltration levels affect the antitumor effect of intratumoral Treg depletion, we performed anti-CD25-F(ab′)2 NIR-PIT on different murine tumors that showed similar Treg frequencies (Fig. 1D). Interestingly, the growth of tumors with higher CD8 T- and NK-cell infiltration (MC38 and EO771) was significantly suppressed by intratumoral Treg depletion, whereas minimal effects were observed in tumors with lower T- and NK-cell infiltration (LL/2 and 4T1; Fig. 1E). In responsive tumor models (MC38 and EO771), the tumor shrunk from day 1 to day 3 after anti-CD25-F(ab′)2 NIR-PIT but re-grew after day 5, coinciding with the recovery of Tregs in the tumors (Fig. 1C). When intratumoral Treg depletion was repeated on MC38 tumor, the period of tumor regression/suppression extended but all tumors re-grew after day 8 (Supplementary Figs. S4A and S4B). We used MC38 and EO771 tumors in subsequent experiments to examine the mechanism of the antitumor effect of intratumoral Treg depletion.

Antitumor effect of intratumoral Treg depletion depends on IFNγ signaling in non-bone marrow–derived stromal cells

Our previous study demonstrates that the antitumor effect of intratumoral Treg depletion by anti-CD25-F(ab′)2 NIR-PIT requires IFNγ production by CD8 T and NK cells (13). Similarly, the antitumor effect observed in systemic depletion of Tregs in tumor-bearing Foxp3DTR mice is IFNγ-dependent (7, 14). However, the exact target of IFNγ after intratumoral Treg depletion remains unknown.

We first analyzed kinetics of IFNγ expression in CD8 T and NK cells after anti-CD25-F(ab′)2 NIR-PIT intratumoral Treg depletion. IFNγ was produced within 30 minutes of intratumoral Treg depletion, without exogenous stimulations such PMA and ionomycin, but began to decrease between 2 and 6 hours after treatment on MC38 and EO771 tumors (Fig. 2A and B). This rapid IFNγ production was associated with increased expression of perforin (Fig. 2A) and activation markers including CD69 (13), but expression of TNFα and granzyme B did not change (Fig. 2A). Ex vivo stimulation of tumor-infiltrating CD8 T and NK cells with PMA and ionomycin enabled production of IFNγ and perforin regardless of prior intratumoral Treg depletion (Supplementary Figs. S5A and S5B). These results indicate that although tumor-infiltrating CD8 T and NK cells possessed the ability to express IFNγ and perforin, intratumoral Tregs actively inhibited the expression of these effector molecules, and that the short but synchronized induction of IFNγ up to 6 hours of treatment accounted for the antitumor effect of anti-CD25-F(ab′)2 NIR-PIT. Of note, anti-CD25-F(ab′)2 NIR-PIT did not significantly change the frequencies of tumor-infiltrating CD8 T and NK cells (Supplementary Fig. S1D), contrasting to systemic depletion of Tregs in Foxp3DTR mice model that induces tumor-infiltration of activated CD8 and CD4 T cells (6–9).

Figure 2.

IFNγR expression in non-bone marrow–derived stromal cells is required for the antitumor effect of intratumoral Treg depletion. A, Induction of IFNγ and perforin, but not TNFα and granzyme B, in CD3+CD8+ T and CD45+NK1.1+ NK cells 30 minutes after anti-CD25-F(ab′)2 on MC38 tumor, analyzed by flow cytometry (n ≥ 4 each). Tumor tissues were minced and incubated with brefeldin A (4 hours) without exogenous stimulation before flow cytometry analysis. Refer to Supplementary Fig. S5 for results after PMA/ionomycin stimulation. B, Changes in the frequency of IFNγ or perforin-expressing cells in CD3+CD8+ T cells and CD45+NK1.1+ NK cells, using the method depicted in A, in MC38 and EO771 tumors following anti-CD25-F(ab′)2 NIR-PIT (n ≥ 4 at each time point). C and D, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor was abrogated by intravenous injection of anti-IFNγ antibody (100 μg, 1 day before NIR-PIT; C) and in Ifngr1−/− mice (D). E and F, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor was abrogated in Ifngr1−/− mice reconstituted with WT bone marrow (WTIfngr1−/−; E), whereas that was maintained in WT mice reconstituted with Ifngr1−/− bone marrow (Ifngr1−/−WT; F). *, P < 0.05; **, P < 0.01.

Figure 2.

IFNγR expression in non-bone marrow–derived stromal cells is required for the antitumor effect of intratumoral Treg depletion. A, Induction of IFNγ and perforin, but not TNFα and granzyme B, in CD3+CD8+ T and CD45+NK1.1+ NK cells 30 minutes after anti-CD25-F(ab′)2 on MC38 tumor, analyzed by flow cytometry (n ≥ 4 each). Tumor tissues were minced and incubated with brefeldin A (4 hours) without exogenous stimulation before flow cytometry analysis. Refer to Supplementary Fig. S5 for results after PMA/ionomycin stimulation. B, Changes in the frequency of IFNγ or perforin-expressing cells in CD3+CD8+ T cells and CD45+NK1.1+ NK cells, using the method depicted in A, in MC38 and EO771 tumors following anti-CD25-F(ab′)2 NIR-PIT (n ≥ 4 at each time point). C and D, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor was abrogated by intravenous injection of anti-IFNγ antibody (100 μg, 1 day before NIR-PIT; C) and in Ifngr1−/− mice (D). E and F, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor was abrogated in Ifngr1−/− mice reconstituted with WT bone marrow (WTIfngr1−/−; E), whereas that was maintained in WT mice reconstituted with Ifngr1−/− bone marrow (Ifngr1−/−WT; F). *, P < 0.05; **, P < 0.01.

Close modal

Next, to determine the target and role of IFNγ after intratumoral Treg depletion, we neutralized IFNγ or used various Ifngr1-deficient mouse models. The antitumor effect of intratumoral Treg depletion by anti-CD25-F(ab′)2 NIR-PIT was abrogated in wild-type (WT) mice injected with anti-IFNγ antibody 1 day before NIR-PIT (Fig. 2C), in Ifngr1−/− mice (Fig. 2D), and in Ifngr1−/− mice reconstituted with WT bone marrow cells (WTIfngr1−/−; Fig. 2E), suggesting that both IFNγ production and IFNγR1 expression on non-bone marrow–derived cells were required for the efficacy. In contrast, anti-CD25-F(ab′)2 NIR-PIT remained effective in WT mice reconstituted with Ifngr1−/− bone marrow cells (Ifngr1−/−WT; Fig. 2F), indicating that IFNγR1 expression on bone-marrow-derived cells was dispensable. In both Ifngr1−/−WT and WTIfngr1−/− mice, more than 95% of peripheral blood and intratumoral leukocytes were derived from reconstituted bone marrow cells (Supplementary Figs. S5C and S5D). The frequency of CD8 T and NK cells in tumor and their IFNγ expression was compatible between the tumors in Ifngr1−/− and WT mice (Supplementary Fig. S5E). These results collectively indicate that after selective depletion of intratumoral Tregs by anti-CD25-F(ab′)2 NIR-PIT, IFNγ produced from activated CD8 T and NK cells directly acted on non-bone marrow–derived host stromal cells in tumor tissue.

Selective depletion of intratumoral Tregs causes IFNγ-dependent acute tumor vessel regression

Among the stromal cells in tumor tissue, fibroblasts and pericytes can differentiate from bone marrow-derived cells (15–17), whereas little evidence suggest that endothelial cells are bone marrow derived (18). A recent study demonstrated that forced expression of IFNγ in tumor cells directly acts on endothelial cells to cause rapid vessel regression and intratumoral ischemia (19). However, the magnitude of IFNγ forced to be produced from all the tumor cells is considered to be much higher than that of IFNγ physiologically produced from tumor-infiltrating CD8 T and NK cells that usually consist of small fractions in the tumor (Fig. 1D). Therefore, it is still undetermined whether more physiological, short-lasting induction of IFNγ from CD8 T and NK cells observed upon depletion of intratumoral Treg (Fig. 2B) is sufficient to cause similar vascular changes. Thus, we next analyzed the changes in tumor vasculature after intratumoral Treg depletion.

IHC analysis of tumor vessels using an endothelial cell marker CD31 showed narrowed vessel lumens, occurring within 4 hours of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors (Fig. 3A and B). Blood perfusion was decreased in these vessels (demonstrated by the lack of endothelial cell labeling by intravenously injected-DyLight 594-conjugated Tomato-lectin) in both MC38 and EO771 tumors (Fig. 3A and C; Supplementary Figs. S6A–S6C). Empty structures of collagen IV+ basement membrane sleeves without CD31+ endothelial cell lining remained after vessel regression (Fig. 3A). These changes were clearly observed up to 48 hours after depletion of intratumoral Tregs (Fig. 3B and C), leading to decreased tumor vessel density (Fig. 3D). Anti-CD25-F(ab')2 NIR-PIT registrant 4T1 tumor showed poor vessel perfusion, which did not change by intratumoral Treg depletion (Supplementary Figs. S6B and S6C). These findings suggest a role for Tregs to maintain tumor vessels and blood perfusion. Of note, we did not detect CD25 expression in endothelial cells nor were tumor vessels rapidly depleted by anti-CD25-F(ab′)2 NIR-PIT (Fig. 3D and E). Contrasting to the tumor vessels, blood vessel structure and perfusion remained unchanged in adjacent nontumor tissue, where lymphocytes were scarce (Supplementary Fig. S6D). Importantly, the acute tumor vessel regression upon anti-CD25-F(ab′)2 NIR-PIT was observed in Ifngr1−/−WT mice but not in Ifngr1−/− and WTIfngr1−/− mice bearing MC38 tumors (Fig. 3F; Supplementary Fig. S6E), indicating that this process required the expression of IFNγR1 in non-bone marrow–derived stromal cells in tumor tissue.

Figure 3.

Selective depletion of intratumoral Tregs causes IFNγ-dependent rapid vessel regression. A, Representative IHC of CD31 (top), DyLight 594-conjugated tomato-lectin and CD31 (tomato-lectin perfusion assay, middle), and Collagen IV and CD31 (bottom) 4 hours after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 each). Bar, 100 μm. B and C, Quantitation of regressed vessels (B) and vessel perfusion (C) on IHC, as in A, at indicated time points following control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 at each time point). D, Quantitation of vessel density on CD31 IHC at indicated time points after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 at each time point). E, CD25 was not expressed in endothelial cells (CD45CD31+) in MC38 tumor, analyzed by flow cytometry (n = 3, representative data). F, Vessel perfusion in MC38 tumors, analyzed by tomato-lectin perfusion assay, was maintained in Ifngr1−/− and WTIfngr1−/− mice, but was impaired in Ifngr1−/−WT bone-marrow chimera mice, 4 hours after anti-CD25-F(ab′)2 NIR-PIT (n = 3 each). G and H, Impaired tumor vessel perfusion, analyzed by tomato-lectin perfusion assay, in early phase of Treg depletion in MC38 tumor-bearing Foxp3DTR mice. DT (25 ng/g) was intraperitoneally injected on day −3, −2, and −1 of analysis. Anti-IFNγ antibody injection (100 μg on day −3 and −1) abrogated impaired perfusion. Representative immunohistochemistry (G) and quantitation summary (n = 3 each; H). Bar, 100 μm. I, TEM images show degeneration of endothelial cells (white arrowheads, obscured intracellular organelle with or without cytoplasmic vacuolation; black arrowheads, swollen mitochondria and increased electron densities in cytoplasm) 4 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor. Bar, 2 μm. J, Quantitation of degenerated vessels in I. In each group, 22 vessels from two tumors were analyzed with χ2 test. *, compared with no treatment; †, compared with control-F(ab′)2 NIR-PIT; * and †, P < 0.05; ** and ††, P < 0.01.

Figure 3.

Selective depletion of intratumoral Tregs causes IFNγ-dependent rapid vessel regression. A, Representative IHC of CD31 (top), DyLight 594-conjugated tomato-lectin and CD31 (tomato-lectin perfusion assay, middle), and Collagen IV and CD31 (bottom) 4 hours after control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 each). Bar, 100 μm. B and C, Quantitation of regressed vessels (B) and vessel perfusion (C) on IHC, as in A, at indicated time points following control-F(ab′)2 or anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 at each time point). D, Quantitation of vessel density on CD31 IHC at indicated time points after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor (n ≥ 3 at each time point). E, CD25 was not expressed in endothelial cells (CD45CD31+) in MC38 tumor, analyzed by flow cytometry (n = 3, representative data). F, Vessel perfusion in MC38 tumors, analyzed by tomato-lectin perfusion assay, was maintained in Ifngr1−/− and WTIfngr1−/− mice, but was impaired in Ifngr1−/−WT bone-marrow chimera mice, 4 hours after anti-CD25-F(ab′)2 NIR-PIT (n = 3 each). G and H, Impaired tumor vessel perfusion, analyzed by tomato-lectin perfusion assay, in early phase of Treg depletion in MC38 tumor-bearing Foxp3DTR mice. DT (25 ng/g) was intraperitoneally injected on day −3, −2, and −1 of analysis. Anti-IFNγ antibody injection (100 μg on day −3 and −1) abrogated impaired perfusion. Representative immunohistochemistry (G) and quantitation summary (n = 3 each; H). Bar, 100 μm. I, TEM images show degeneration of endothelial cells (white arrowheads, obscured intracellular organelle with or without cytoplasmic vacuolation; black arrowheads, swollen mitochondria and increased electron densities in cytoplasm) 4 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor. Bar, 2 μm. J, Quantitation of degenerated vessels in I. In each group, 22 vessels from two tumors were analyzed with χ2 test. *, compared with no treatment; †, compared with control-F(ab′)2 NIR-PIT; * and †, P < 0.05; ** and ††, P < 0.01.

Close modal

We next examined the effects of Treg depletion on tumor vessels in another model, Foxp3DTR mice. Previous studies using B16 melanoma-bearing Foxp3DTR mice have shown that Treg depletion by intravenous DT injection causes tumor vessel normalization, characterized by decreased vessel density and improved vessel perfusion, 3 to 4 days after DT treatment (9). We hypothesized that this decreased vessel density was preceded by vessel regression, and thus performed Tomato-lectin perfusion assay 1 day after completion of DT treatment, when more than 95% of intratumoral and systemic Tregs were depleted (Supplementary Figs. S3A and S3B). We found that vessel perfusion in MC38 tumor was significantly impaired in DT-treated mice, but not when combined with an IFNγ-neutralizing antibody injection (Figs. 3G and H). These results indicate that systemic Treg depletion induced the vessel regression in an IFNγ-dependent manner, similar to the findings with intratumoral Treg depletion by anti-CD25-F(ab′)2 NIR-PIT.

To further characterize tumor vessel regression induced by anti-CD25-F(ab′)2 NIR-PIT intratumoral Treg depletion, we performed TEM analysis (Fig. 3I). The most characteristic changes observed were various degrees of shrinkage or swelling of endothelial cells, associated with obscured intracellular organelle with or without cytoplasmic vacuolation (Fig. 3I, white arrowheads) or associated with swollen mitochondria and increased electron densities in cytoplasm (Fig. 3I, black arrowheads). These changes were rarely observed in tumors after control-F(ab′)2 NIR-PIT (Fig. 3I and J).

To this end, we performed in vivo live perfusion imaging of the tumor by injecting IR800-conjugated bovine serum albumin 18 hours after anti-CD25-F(ab′)2 NIR-PIT and observed decreased tumor vessel perfusion (IR800 optical signal) extending whole tumor both in vivo and in harvested tumors (Supplementary Figs. S7A–S7D).

Vessel function is partially normalized after intratumoral Treg depletion

Increased vessel leakiness is an important factor causing uneven and poor blood perfusion in tumor vessels, which can be normalized by IFNγ (20–23). To assess the changes in vessel leakiness after anti-CD25-F(ab′)2 NIR-PIT intratumoral Treg depletion, a dextran leakage assay was performed. MC38 tumor showed area with dextran leakage, which decreased after intratumoral Treg depletion (day 3), even with adjustment for the number of tumor vessels (Supplementary Figs. S8A–S8C), suggesting a partial normalization of vessel function following the acute vessel regression. Tumor vessel coverage by NG2+ pericytes did not change after anti-CD25-F(ab′)2 NIR-PIT (Supplementary Fig. S8D). However, collagen IV+ basement membrane structure, that was maintained in the early phase of vessel regression (4 hours, Fig. 3A), degraded over time (18 hours, Supplementary Fig. S8D). Endothelial cells of tumor vessels are characterized by decreased VCAM-1 expression and increased FasL expression, both of which negatively affect the influx of CD8 T cells to the tumor tissue (24), but the expression levels of these molecules did not change by intratumoral Treg depletion (Supplementary Fig. S8E).

Tumor vessel regression by intratumoral Treg depletion leads to tumor ischemia and necrosis/apoptosis

We next asked if tumor vessel regression induced by intratumoral Treg depletion caused ischemia in the tumor. We first performed pimonidazole-binding assay at various timepoints after anti-CD25-F(ab′)2 NIR-PIT and mice in control groups. Pimonidazole binding to both tumor (MC38 and EO771) and immune cells increased as early as 4 hours after intratumoral Treg depletion and persisted for 48 hours (Figs. 4A–D). Similarly, expression of carbonic anhydrase IX (CAIX), another ischemic/hypoxic marker, increased after intratumoral Treg depletion (Supplementary Figs. S9A and S9B). Systemic Treg depletion in Foxp3DTR mice also demonstrated increased pimonidazole-positive ischemic areas in MC38 tumors on day 1 of DT treatment completion, which was diminished by IFNγ-neutralization (Supplementary Figs. S9C and S9D). Moreover, development of intratumoral ischemia was dependent on IFNγR1 expression in non-bone marrow–derived stromal cells as was seen in Ifngr1−/−WT but not in WTIfngr1−/− bone marrow chimera mice (Figs. 4E and F). The acute intratumoral ischemia resulted in a significant increase in necrosis and apoptosis as demonstrated by propidium iodide injection assay (Fig. 4G and H) and TUNEL assay (Fig. 4I and J), respectively, both of which were abrogated in Ifngr1−/− mice (Fig. 4G–J). Altogether, these data indicate that tumor vessel regression induced by intratumoral Treg depletion led to tumor ischemia, apoptosis, and necrosis in a manner dependent on physiologically produced IFNγ that acted on non-bone marrow–derived host stromal cells.

Figure 4.

IFNγ-dependent rapid vessel regression is followed by tumor ischemia and necrosis/apoptosis of tumor. A and B, Flow cytometry indicates increased pimonidazole positivity in CD45 tumor cells and CD45+ immune cells in pimonidazole-binding assay following anti-CD25-F(ab′)2 NIR-PIT. Representative histograms at 18 hours after anti-CD25-F(ab′)2 NIR-PIT (A) and changes of pimonidazole+ hypoxic tumor and immune cell frequencies in MC38 and EO771 tumors (n = 3 at each time point; B). C and D, Increased pimonidazole+ areas in pimonidazole-binding assay in MC38 tumors 18 hours after anti-CD25-F(ab′)2 NIR-PIT. Representative IHC (C) and quantitation summary (n = 3 each; D). Bar, 250 μm. E and F, Flow cytometry analysis of pimonidazole positivity in CD45 tumor cells and CD45+ immune cells in pimonidazole-binding assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor in Ifngr1−/−, and Ifngr1−/−WT, and WTIfngr1−/− bone-marrow chimera mice. Representative histograms (E) and quantitation summery in CD45 tumor cells (n ≥ 3 each; F). G and H, Increased necrotic areas positive for propidium iodide in propidium iodide injection assay, observed 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor, which was abrogated in Ifngr1−/− mice. Representative histologic images (G) and quantitation summary (n = 3 each; H). Bar, 250 μm. I and J, Increased TUNEL-positive apoptotic cells 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38, which was abrogated in Ifngr1−/− mice. Representative histologic images (I) and quantitation summary (n = 3 each; J). Bar, 250 μm. *, P < 0.05; **, P < 0.01.

Figure 4.

IFNγ-dependent rapid vessel regression is followed by tumor ischemia and necrosis/apoptosis of tumor. A and B, Flow cytometry indicates increased pimonidazole positivity in CD45 tumor cells and CD45+ immune cells in pimonidazole-binding assay following anti-CD25-F(ab′)2 NIR-PIT. Representative histograms at 18 hours after anti-CD25-F(ab′)2 NIR-PIT (A) and changes of pimonidazole+ hypoxic tumor and immune cell frequencies in MC38 and EO771 tumors (n = 3 at each time point; B). C and D, Increased pimonidazole+ areas in pimonidazole-binding assay in MC38 tumors 18 hours after anti-CD25-F(ab′)2 NIR-PIT. Representative IHC (C) and quantitation summary (n = 3 each; D). Bar, 250 μm. E and F, Flow cytometry analysis of pimonidazole positivity in CD45 tumor cells and CD45+ immune cells in pimonidazole-binding assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor in Ifngr1−/−, and Ifngr1−/−WT, and WTIfngr1−/− bone-marrow chimera mice. Representative histograms (E) and quantitation summery in CD45 tumor cells (n ≥ 3 each; F). G and H, Increased necrotic areas positive for propidium iodide in propidium iodide injection assay, observed 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumor, which was abrogated in Ifngr1−/− mice. Representative histologic images (G) and quantitation summary (n = 3 each; H). Bar, 250 μm. I and J, Increased TUNEL-positive apoptotic cells 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38, which was abrogated in Ifngr1−/− mice. Representative histologic images (I) and quantitation summary (n = 3 each; J). Bar, 250 μm. *, P < 0.05; **, P < 0.01.

Close modal

Compatible with ischemic reaction, we observed induction of cytokines and chemokines related to acute inflammation (Supplementary Fig. S10), induction of VEGF expression (Supplementary Fig. S10), influx of neutrophils (Supplementary Fig. S1D), and polarization of intratumoral macrophages and monocytes into regulatory phenotypes, exhibiting CD206highMHCIIlow and Ly6Clow, respectively (Supplementary Figs. S1D–S1F), in the tumor.

IFNγ directly acts on endothelial cells to cause acute tumor vessel regression

To determine if IFNγ directly acted on endothelial cells to cause tumor vessel regression, we generated Tie2CreIfngr1flox/flox mice. In these mice, IFNγR1 was selectively depleted in endothelial cells, among non-bone marrow–derived stromal cells in the tumor tissue (Fig. 5A). Although Tie2Cre is also expressed in hematopoietic-lineage cells (25) and thus these cells in Tie2CreIfngr1flox/flox mice lack IFNγR1, our bone marrow reconstitution experiments using WT and Ifngr−/− mice excluded the contribution of IFNγR1 expressed on bone marrow-derived cells to the antitumor effect of anti-CD25-F(ab')2 NIR-PIT (Figs. 2 and 3). In Tie2CreIfngr1flox/flox mice, the therapeutic effect on MC38 tumors was abrogated, whereas it was maintained in control Ifngr1flox/flox mice (Fig. 5B). We repeated the experiment using Tie2CreIfngr1flox/flox mice reconstituted with WT bone marrow (WTTie2CreIfngr1flox/flox) and control mice (WTIfngr1flox/flox). The therapeutic effect was abrogated in WTTie2CreIfngr1flox/flox mice, but was maintained in control WTIfngr1flox/flox mice (Fig. 5C). Of note, in both WTIfngr1flox/flox and WTTie2CreIfngr1flox/flox mice, more than 95% of intratumoral Tregs and Tie2-expressing macrophages were derived from reconstituted bone marrow (Supplementary Figs. S11A–S11C). Importantly, induction of acute vessel regression and ischemia were also abrogated in Tie2CreIfngr1flox/flox mice, whereas they were observed in control Ifngr1flox/flox mice (Fig. 5D–G). These results indicate that IFNγR1 expression on tumor vascular endothelial cells was necessary for the antitumor effect of intratumoral Treg depletion and that IFNγ produced by CD8 T and NK cells directly acted on endothelial cells to cause acute vessel regression and intratumoral ischemia.

Figure 5.

IFNγ directly targets endothelial cells to cause vessel regression. A, IFNγRI expression in endothelial cells (CD45panCKCD31+), pericytes (CD45panCKCD31NG2+), and fibroblasts (CD45panCKCD31PDFGRα+) in MC38 tumors in WT, Ifngr1−/−, or Tie2CreIfngr1flox/flox mice, analyzed by flow cytometry (n = 2 each). B, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors was abrogated in Tie2CreIfngr1flox/flox mice. Ifngr1flox/flox mice were used as control. C, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors was abrogated in WTTie2CreIfngr1flox/flox mice. WTIfngr1flox/flox mice were used as control. D and E, IHC of DyLight 594-conjugated tomato-lectin and CD31 (endothelial cells) in tomato-lectin perfusion assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors in Ifngr1flox/flox or Tie2CreIfngr1flox/flox mice. Representative images (bar, 250 μm; D) and quantitation summary (n = 3 each; E). F and G, IHC of pimonidazole and CD31 in pimonidazole-binding assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors in Ifngr1flox/flox or Tie2CreIfngr1flox/flox mice. Representative images (bar, 500 μm; F) and quantitation summary (n = 3 each; G). **, P < 0.01.

Figure 5.

IFNγ directly targets endothelial cells to cause vessel regression. A, IFNγRI expression in endothelial cells (CD45panCKCD31+), pericytes (CD45panCKCD31NG2+), and fibroblasts (CD45panCKCD31PDFGRα+) in MC38 tumors in WT, Ifngr1−/−, or Tie2CreIfngr1flox/flox mice, analyzed by flow cytometry (n = 2 each). B, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors was abrogated in Tie2CreIfngr1flox/flox mice. Ifngr1flox/flox mice were used as control. C, Antitumor effect of anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors was abrogated in WTTie2CreIfngr1flox/flox mice. WTIfngr1flox/flox mice were used as control. D and E, IHC of DyLight 594-conjugated tomato-lectin and CD31 (endothelial cells) in tomato-lectin perfusion assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors in Ifngr1flox/flox or Tie2CreIfngr1flox/flox mice. Representative images (bar, 250 μm; D) and quantitation summary (n = 3 each; E). F and G, IHC of pimonidazole and CD31 in pimonidazole-binding assay 18 hours after anti-CD25-F(ab′)2 NIR-PIT on MC38 tumors in Ifngr1flox/flox or Tie2CreIfngr1flox/flox mice. Representative images (bar, 500 μm; F) and quantitation summary (n = 3 each; G). **, P < 0.01.

Close modal

CD8 T and NK cells induce IFNγ/STAT1 signaling around tumor vessels and in endothelial cells

It is intriguing how a small number of intratumoral CD8 T and NK cells can induce tumor vessel regression in such an effective manner (Figs. 3A–C). Interestingly, CD8 T and NK cells and Tregs were found, histologically, near tumor vessels in both MC38 and EO771 tumors (Fig. 6A and B). Thus, we next hypothesized that intratumoral Treg depletion could induce IFNγ-mediated STAT1 signaling in cells around tumor vessels. Without treatment, only weak phosphorylation of STAT1 Tyr701, the main residue phosphorylated by IFNγ (26), was observed in scattered areas in MC38 tumors (Fig. 6C). Upon intratumoral Treg depletion, cells nearby vessels and endothelial cells readily showed STAT1 Tyr701 phosphorylation (Fig. 6C and D). These findings suggest that IFNγ produced by CD8 T and NK cells around tumor vessels effectively targeted endothelial cells.

Figure 6.

Depletion of intratumoral Tregs causes IFNγ/phospho-STAT1 signaling around tumor vessels. A and B, Perivascular localization of CD8+ T cells, NKp46+ NK cells, and CD4+Foxp3+ Tregs analyzed by IHC in untreated MC38 tumors. Representative images (bar, 100 μm; A) and frequency of indicated lymphocytes located in indicated distances from tumor vessels (n = 3 each; B). C, Increased phospho-STAT1 (Tyr701) positivity around tumor vessels analyzed by IHC 60 minutes after anti-CD25-F(ab′)2 NIR-PIT (n = 3 each, representative images). For negative control, frozen sections were treated with lambda protein phosphatase (λ-PP) before the phospho-STAT1 staining. Inset, phospho-STAT1 (Tyr701) positivity CD31+ endothelial cells. Bar, 500 μm. D, Percentage of phospho-STAT1-positive areas analyzed by distance from tumor vessels (top) and phospho-STAT1–positive tumor vessels (bottom) in C (n = 3 each). **, P < 0.01.

Figure 6.

Depletion of intratumoral Tregs causes IFNγ/phospho-STAT1 signaling around tumor vessels. A and B, Perivascular localization of CD8+ T cells, NKp46+ NK cells, and CD4+Foxp3+ Tregs analyzed by IHC in untreated MC38 tumors. Representative images (bar, 100 μm; A) and frequency of indicated lymphocytes located in indicated distances from tumor vessels (n = 3 each; B). C, Increased phospho-STAT1 (Tyr701) positivity around tumor vessels analyzed by IHC 60 minutes after anti-CD25-F(ab′)2 NIR-PIT (n = 3 each, representative images). For negative control, frozen sections were treated with lambda protein phosphatase (λ-PP) before the phospho-STAT1 staining. Inset, phospho-STAT1 (Tyr701) positivity CD31+ endothelial cells. Bar, 500 μm. D, Percentage of phospho-STAT1-positive areas analyzed by distance from tumor vessels (top) and phospho-STAT1–positive tumor vessels (bottom) in C (n = 3 each). **, P < 0.01.

Close modal

Increased complete eradication is achieved by anti-CD25-F(ab′)2 NIR-PIT combined with rhIL-15 therapy, not PD-1 blockade

Finally, we aimed to boost the antitumor effect of CD8 T and NK cells to eradicate established tumors by combining anti-CD25-F(ab′)2 NIR-PIT with another treatment. A combination with anti-PD-1 antibody therapy failed to enhance effector activity of CD8 T and NK cells after intratumoral Treg depletion, showing only marginal additional tumor control (Supplementary Fig. S12A and S12B). PD-L1 expression levels on tumor cells, monocytes, macrophages, and dendritic cells in MC38 tumors did not change after intratumoral Treg depletion (Supplementary Fig. S12C).

We then evaluated another combination with recombinant human IL15 (rhIL15) treatment (Fig. 7A). This combination therapy significantly decreased tumor volume, leading to complete regression of some MC38 and EO771 tumors (Fig. 7B) and significantly improved survival (Supplementary Fig. S13A). Anti-CD25-F(ab′)2 NIR-PIT and rhIL15 treatment synergistically increased the frequency of tumor-infiltrating CD8 T and NK cells, and their granzyme B expression was increased by rhIL15 (Figs. 7C–E). However, expression of IFNγ and perforin did not increase. Histologic analysis revealed that rhIL15 treatment alone increased CD8 T-cell infiltration to the periphery of the tumor but not to the center, which did not change by the combination therapy (Fig. 7F and G). In contrast, anti-CD25-F(ab′)2 NIR-PIT preferentially targeted more centrally located tumor cells through induction of ischemic necrosis/apoptosis (Fig. 5). The combination of anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 therapy, therefore, targeted both the center and periphery of the tumor, while increasing the cytotoxic activity of lymphocytes by rhIL15. This likely explains the increased tumor control and complete tumor eradication.

Figure 7.

Combination of rhIL-15 treatment with anti-CD25-F(ab')2 NIR-PIT synergize to increase complete eradication of tumor. A, Regimen of anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 combination therapy. B, Anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 synergistically impaired growth of MC38 and EO771 tumors. Sample size and complete regression (CR) rate in each group are indicated. χ2 test was used to calculate P values of CR ratio. *, compared twith no treatment group; †, compared with other three groups. See Supplementary Fig. S13A for the survival curves. C, Frequency of indicated lymphocyte subsets 5 days after indicated treatments on MC38 tumor, analyzed by flow cytometry (n ≥ 5 each). *, compared with no treatment group and anti-CD25-F(ab′)2 NIR-PIT group; †, compared with rhIL-15 group. D, Positivity of IFNγ, granzyme B, and perforin in CD3+CD8+ T cells or CD45+NK1.1+ NK cells, analyzed by flow cytometry 3 days after indicated treatments on MC38 or EO771 tumor (day 3, n ≥ 5 each). *, compared with no treatment group and anti-CD25-F(ab′)2 NIR-PIT group; †, compared with rhIL-15 group. E, Granzyme B expression in CD3+CD8+ T cells or CD45+CD3NK1.1+ NK cells analyzed by flow cytometry 3 days after indicated treatments on MC38 tumor (n ≥ 5). F and G, rhIL-15 treatment induced infiltration of CD8 T cells to the periphery of EO771 tumors (day 3). Representative IHC (bar, 250 μm; F) and quantitation summary (n = 3 each; G). H, Prevention of lung metastasis formation after rechallenge by intravenous injection of 1 × 106 MC38 or EO771 tumor cells at 1 or 3 months of complete regression by anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 combination therapy (n ≥ 3 each). * and †, P < 0.05; ** and ††, P < 0.01.

Figure 7.

Combination of rhIL-15 treatment with anti-CD25-F(ab')2 NIR-PIT synergize to increase complete eradication of tumor. A, Regimen of anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 combination therapy. B, Anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 synergistically impaired growth of MC38 and EO771 tumors. Sample size and complete regression (CR) rate in each group are indicated. χ2 test was used to calculate P values of CR ratio. *, compared twith no treatment group; †, compared with other three groups. See Supplementary Fig. S13A for the survival curves. C, Frequency of indicated lymphocyte subsets 5 days after indicated treatments on MC38 tumor, analyzed by flow cytometry (n ≥ 5 each). *, compared with no treatment group and anti-CD25-F(ab′)2 NIR-PIT group; †, compared with rhIL-15 group. D, Positivity of IFNγ, granzyme B, and perforin in CD3+CD8+ T cells or CD45+NK1.1+ NK cells, analyzed by flow cytometry 3 days after indicated treatments on MC38 or EO771 tumor (day 3, n ≥ 5 each). *, compared with no treatment group and anti-CD25-F(ab′)2 NIR-PIT group; †, compared with rhIL-15 group. E, Granzyme B expression in CD3+CD8+ T cells or CD45+CD3NK1.1+ NK cells analyzed by flow cytometry 3 days after indicated treatments on MC38 tumor (n ≥ 5). F and G, rhIL-15 treatment induced infiltration of CD8 T cells to the periphery of EO771 tumors (day 3). Representative IHC (bar, 250 μm; F) and quantitation summary (n = 3 each; G). H, Prevention of lung metastasis formation after rechallenge by intravenous injection of 1 × 106 MC38 or EO771 tumor cells at 1 or 3 months of complete regression by anti-CD25-F(ab′)2 NIR-PIT and rhIL-15 combination therapy (n ≥ 3 each). * and †, P < 0.05; ** and ††, P < 0.01.

Close modal

Trans-presentation of IL15/IL15Rα complex by myeloid and epithelial cells to lymphocytes is important for IL15 efficacy (27, 28). We analyzed changes in IL15Rα expression in tumor and immune cells and found increased IL15Rα expression in Ly6Chigh inflammatory-monocytes after anti-CD25-F(ab′)2 NIR-PIT (Supplementary Figs. S13B and S13C). However, because the frequency of Ly6Chigh inflammatory-monocytes decreased after the NIR-PIT (Supplementary Fig. S1F), the contribution of these cells in trans-presenting IL15 may be limited.

After complete remission of local tumor, systemic antitumor immunity lasted for at least several months and prevented the formation of lung metastasis when the mice were re-challenged with intravenous administration of the same tumors (Fig. 7H).

Although the pivotal roles of Tregs to inhibit antitumor immunity is well-established (6–9), the specific roles that intra- and extratumor Tregs play remain unclear due to technical difficulties in selectively depleting or manipulating Tregs in a spatially specific manner. In this study, we took advantage of a highly selective method of depleting cells from the tumor microenvironment, NIR-PIT (11–13), which enabled us to observe the effects of selective removal of intratumoral Tregs without employing systemic depletion strategies.

Our previous study of anti-CD25-F(ab′)2 NIR-PIT demonstrated that depletion of intratumoral Tregs caused tumor regression in a manner dependent on IFNγ production from CD8 T and NK cells (13). IFNγ can exhibit antitumor effects directly on tumor cells and also indirectly through T cells, macrophages, and stromal cells, including tumor vessels, depending on the context of IFNγ production (26). Previous investigations have shown that doxycycline-mediated induction of IFNγ in tumor cells directly acts on endothelial cells to cause rapid tumor vessel regression and intratumoral ischemia (19, 29), however, magnitude of IFNγ induced from all tumor cells would be significantly higher than that induced upon intratumoral Treg depletion. Therefore, the mechanism by which IFNγ causes tumor regression after intratumoral Treg depletion remained to be elucidated.

In this study, we utilized anti-CD25-F(ab′)2 NIR-PIT (13) and various Ifngr1 knockout models and identified that IFNγ produced by tumor-infiltrating CD8 T and NK cells upon intratumoral Treg depletion directly acted on endothelial cells through IFNγR1, inducing acute vascular regression and intratumoral ischemia, leading to impaired tumor growth. It was surprising how effectively the physiological, short-lasting (up to 6 hours) production of IFNγ from limited numbers of tumor-infiltrating CD8 T and NK cells after intratumoral Treg depletion induced these changes. Because of the perivascular distribution of CD8 T and NK cells, IFNγ/phospho-STAT1 signaling was preferentially induced around tumor vessels and in the endothelial cells, suggesting effective IFNγ targeting of vascular endothelial cells. As a source for IFNγ, CD8 T and NK cells may equally contribute, because they show similar IFNγ production kinetics upon intratumoral Treg depletion and similar perivascular distribution patterns. This seems consistent with our previous finding showing similar contributions by CD8 T and NK cells to the antitumor effect of anti-CD25-F(ab′)2 NIR-PIT (13). In addition to IFN-γ induction, simultaneous deprivation of Treg-derived VEGF (30) upon intratumoral Treg depletion may also have additional effects on the regression of tumor vasculature and following vessel recovery.

Interestingly, intratumoral Treg depletion alone did not increase the number and effector activity of tumor-infiltrating CD8 T and NK cells, unlike systemic Treg depletion in Foxp3DTR mouse model and by administration Treg-depleting antibodies (6–9). IFNγ can also modulate tumor vasculature by polarizing vasculature-supporting M2 macrophages into pro-inflammatory M1 macrophages, and by inducing CXCL9/10 in tumors (9, 26). However, we neither observed macrophage polarization into M1 phenotype nor induction of CXCL9/10 after intratumoral Treg depletion, which may be due to the limited amount and duration of IFNγ production and developing ischemia upon intratumoral Treg depletion.

There is an interesting relationship between IFNγ-dependent vessel regression observed in our study and tumor vessel normalization, which is also reported to be IFNγ-dependent (20, 22, 23) but was only partially observed in this study. Tumor vessel normalization is observed during pruning and reorganization after some therapies and is associated with improved tumor oxygenation and lymphocyte infiltration into tumor (20). Studies have shown that IFNγ expressed from CD4 and CD8 T cells by immune checkpoint inhibitors (22, 23) induces vessel normalization. On the other hand, doxycycline-mediated IFN-γ induction in tumor cells causes acute regression of tumor vessels as discussed above (19, 29). The question of whether IFNγ can cause both tumor vessel regression and normalization may be reconciled by considering differences in the amount and duration of IFNγ expressed in tumors (31). High levels of IFNγ expression (19, 29) or short-lasting but synchronized expression of IFNγ, as observed in this study, may preferentially cause rapid vessel regression, whereas low level but long-lasting expression of IFNγ from tumor-infiltrating CD4 and CD8 T cells induced by immune checkpoint inhibitors (22, 23) may shift towards tumor vessel normalization.

Although development of intratumoral ischemia after intratumoral Treg depletion could control tumor growth, it failed to achieve complete regression. Re-growth of the tumors observed is likely due to the relative resistance to ischemia at the periphery of tumors and to the limited increase in number and cytotoxic activity of CD8 T and NK cells after intratumoral Treg depletion. To augment therapeutic efficacy, we added rhIL15 administrations (1 μg × 5 doses) to anti-CD25-F(ab′)2 NIR-PIT (28, 32). IL15 was chosen over IL2 because, whereas IL15 preferentially expand and activate cytotoxic lymphocytes, IL2 also expand Tregs systemically (27), which can accelerate recovery of intratumoral Tregs after their depletion. Consequently, rhIL15 combination achieved increased complete eradication of MC38 and EO771 tumors. Although depletion of intratumoral Tregs preferentially targets the center of tumors by inducing tumor vessel regression and intratumoral ischemia, rhIL15 treatment increased infiltration of cytotoxic lymphocytes at the periphery of tumors and their cytotoxic action, eradicating residual tumor cells. It is also intriguing that the combination of IL15 may enhance and prolong antitumor memory T-cell development and survival (27). We observed strong anti-tumor memory at 3 months after achieving complete tumor regression by the combination of IL15 therapy, as shown by the rejection of a tumor rechallenge.

There are some limitations in this study. First, anti-CD25-F(ab′)2 used in this study is derived from clone PC-61.5.3 that blocks IL2 binding to CD25 and can potentially inhibit the function of IL2 produced in initial activation of lymphocytes after intratumoral Treg depletion (33). Thus, we plan to use an anti-CD25-F(ab′)2 derived from a clone that has minimal effects on IL2 activity (34). It would also be prudent to use a non-IL2 blocking antibody when we clinically translate anti-CD25-F(ab′)2 NIR-PIT intertumoral Treg depletion therapy in the future, possibly combined with tumor cell-targeting NIR-PIT.

Another limitation is that we could not perform experiments using human tumor samples because of technical difficulties in using human tumors for selective intratumoral Treg depletion, requiring further studies in the future. The structure of tumor vessels vary more significantly in human tumors compared with mouse experimental models (35) and these vessels may exhibit different sensitivities to IFNγ. Also intriguingly, some types of human tumors are more heavily infiltrated by cytotoxic lymphocytes compared with murine tumors (36–39). The results of this study suggest that these lymphocyte-rich human tumors would be more sensitive to intratumoral Treg depletion by anti-CD25-F(ab′)2 NIR-PIT than murine tumors.

In summary, we demonstrated that IFNγ induced in CD8 T and NK cells upon selective depletion of intratumoral Tregs directly targeted endothelial cells to cause rapid vessel regression, intratumoral ischemia, and necrosis/apoptosis of tumor cells. Combination with rhIL15 treatment further increased complete tumor eradication. These data shed light on the role that Tregs play in the tumor microenvironment and help advance clinical translation of anti-Treg cancer therapies such as anti-CD25-F(ab′)2 NIR-PIT.

P.L. Choyke reports a patent for photoimmunotherapy method issued, licensed, and with royalties paid from Rakuten Medical. N. Sato reports a patent for photoimmunotherapy of suppressor cells pending. No disclosures were reported by the other authors.

Y. Kurebayashi: Conceptualization, data curation, investigation, methodology, writing–original draft, writing–review and editing. C.P. Olkowski: Resources. K.C. Lane: Resources. O.V. Vasalatiy: Resources. B.C. Xu: Resources. R. Okada: Resources. A. Furusawa: Resources. P.L. Choyke: Supervision, funding acquisition, writing–review and editing. H. Kobayashi: Resources, supervision, funding acquisition. N. Sato: Conceptualization, data curation, supervision, investigation, methodology, project administration, writing–review and editing.

We thank Mr. Kunio Nagashima for great assistance for electron microscopy studies and Ms. Erina He for her assistance in the creation of the graphical abstract.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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