Synergistic effects of anti-PDL-1 with ablative radiation comparing to other regimens with same biological effect dose based on different immunogenic response.

INTRODUCTION: Irradiation can induce multiple inhibitory and stimulatory effects on the immune system. In recent studies, it has been noted that administration of radiation with various doses and fractionation plans may influence on immune responses in microenvironment of tumor. But in radiobiology, the Biologically Effective Dose (BED) formula has been designed for calculating isoeffect doses in different regimens of daily clinical practice. In other words, BED has also been used to predict the effects of fractionation schedules on tumor cells.
METHODS: In our study, three different regimens with BEDs of 40 gray (Gy) were analyzed in BALB/c mice. These included conventional fractionated radiotherapy (RT) (3Gyx10), high-dose hypofractionated RT (10Gyx2), and single ablative high-dose RT (15Gyx1).
RESULTS: As BED predicts, all three similarly decreased tumor volumes and increased survival times relative to controls, but after high dose exposure in ablative group, the expression of IFNγ was increased following high infiltration of CD8 cells into the tumor microenvironment. When anti-PDL-1 was combined with RT, single ablative high-dose radiation enhanced antitumor activity by increasing IFNγ in tumors and CD8+ tumor-infiltrating lymphocytes; as a result, this combining therapy had enhanced antitumor activity and lead to control tumor volume effectively and improve significantly survival rate and finally the recurrence of tumor was not observed.
CONCLUSION: Results show distinct radiation doses and fractionation schemes with same BED have different immunogenic response and these findings can provide data helping to design regimens of radiation combined with immune checkpoint blockers (ICBs).

Introduction

The main purpose of irradiation is to eliminate tumor cells through DNA damage in cancer therapy, resulting in apoptosis, necrosis, and autophagy, to ultimately prevent tumor growth [1]. Studies indicate radiotherapy (RT) can stimulate the immune system by inducing immunogenic cell deaths (ICDs) that characterized by the release of Damage-associated molecular patterns (DAMPS), high mobility group box 1 protein (HMGB1), and ATP, and by modifying the tumor microenvironment (TME) [2]. Irradiation has multiple inhibitory and stimulatory effects on the immune system such as increased CD4+, CD8+ and Treg Cells infiltration into TME [3, 4] Some regimens of radiation like ablative RT was associated with increased infiltration of exhausted CD8+ T cells into the tumor, which induced radiation resistance. [5, 6]. The combination of radiotherapy and Immune checkpoint blockers therapy can reduce these inhibitory effects by stimulating immune cells and enhancing the response to radiation. Preclinical studies showed that RT increased PDL-1 expression on tumor cells [7, 8], and anti-PDL1 (αPDL-1) mAb combined with radiation had a synergistic effect on immune response induction dependent IFN-γ producing CD8 T cells activations. [9, 10].

It has also been noted that radiation doses and fractionation schemes distinctly impact both the host immune system and the tumor cell’s immunogenicity [11, 12]. Many studies are ongoing to characterize their inhibitory and stimulatory effects [13], and to improve their effects by combinations of RT and immunotherapy [14, 15].

In radiobiology, the Biologically Effective Dose (BED) formula is used to calculate isoeffect doses in various regimens of daily clinical practice. BED has also been used to predict the effects of fractionation changes on tumors according to the linear quadratic (LQ) model that describes cell response to irradiation [16].

A recent meta-analysis study shows when the BED increases, the occurrence rate of abscopal effects also increases due to immunological responses in preclinical models [17]. However, studies have not yet determined whether different regimens with the same BED (when the biological effects of the regimens are equal) have different immunogenic effects, whether they affect tumor recurrence associated with immunological responses, In addition, can these regimens respond differently to combination therapy? Because it can improve the quality of immune responses into TME by use of modification in the dose and number of fractions radiation to the extent that the radiation clinic limitations and technology of the radiation devices allow us. Also it can lead to memory responses, which prevents tumor recurrence that is a common problem after treatment.

To address these issues, we aimed to evaluate the synergistic effect of RT combined with immunotherapy using αPDL-1 in three different RT regimens with the help of modeled tumor BALB/c mice.

Materials and methods

Mice and cell lines

CT26 murine colon carcinoma cells (Pasteur Institute of Iran, Tehran, Iran) were cultured in RPMI-1640 media (Cassion, US) enriched with 10% fetal bovine serum (FBS) (Gibco, US), 10,000 IU Penicillin, 10,000 mg/mL Streptomycin, and 1% L-glutamine (Cassion, US) and used at limited passage number. Female BALB/c mice (4 to 6 week old) were obtained from Razi Institute of Iran. The mice, eight weeks old at start time, were randomly selected in 8 groups with at least ten mice in each groups [10]. Mice were purchased with a health report according to Pasteur Institute routine health monitoring program and kept in the animal house under standard controlled conditions. All experiments were approved by the Institutional Ethical Committee and Research Advisory Committee of Shahid Beheshti University of Medical Sciences with code ethic number: IR. SBMU. MSP.REC.1395.457.

Tumor challenge

For tumor induction, 1 × 106 CT26 tumor cells were inoculated subcutaneously into the right flank of anesthetized mice as described [18]. Tumor volumes (mm3) were calculated with formula length × width × height × 0.52 that measured by a digital caliper. For tumor re-challenge experiments, long-term surviving (LTS) mice were inoculated with 1 × 106 CT26 tumor cells in the left flank 90 and 150 days after the initial tumor implantation.

Calculate BED

To deliver a 40 Gy equivalent total BED in a single dose as ablative radiation, two fractions as hypofraction radiation, or ten fractions as conventional radiation, the dose per fraction was calculated with LQ models. The biological effect dose of 3 regimens radiation including single dose received 15 Gy (15 Gy × 1), two fractions received 10 Gy per fraction (10 Gy × 2), and 10 fractions received 3 Gy per fraction (3 Gy × 10, is approximately equal 40 Gy (The a/b ratio was considerate 10 Gy for soft tissue tumor like CT26 Tumor model) [19].

Treatment

All mice were irradiated 18 days’ post-initial inoculation. At that time the tumors were at least 300–400 mm3. The experimental schedule of ablative radiation therapy was only one fraction of 15 Gy in 18 days’ post-initial inoculation. In Hypofraction RT regimens, two fractions of 10 Gy were performed on days 18 and 28 after inoculation. In Conventional RT, ten fractions of 3 Gy were given from day 18 to day 31 for 14 days at a time interval of one day or at most two days. (Fig 1A). Before irradiation the mice were anesthetized by intraperitoneal injection with 100 mg/kg ketamine 10% and 12.5 mg/kg Xylazine 2% (Alfasan, Sofia, Bulgaria). Mice were irradiated using a clinical linear accelerator (6 MV photons, Elekta synergy linear accelerator, Stockholm, SE). Welfare considerations were taken to help mice efforts from minimize suffering and distress. The mice were placed in a modified 50 ml plastic tube, which helped the area of the tumor to be irradiated while keeping the rest of the body outside the RT field. All parts of the body except the irradiation field were protected by a 9-cm-thick lead plate. Radiotherapy was delivered to a 3×3 cm2 field with 5-mm margins at 350 Gy/min with 6 MV X-ray using tangential beam delivery. Super flab Bolus Material of 1.5 cm was placed over the tumor, and the source-to-skin distance was 100 cm. Mice received intraperitoneal 200 μg/ml of PDL1-blocking antibody 10F.9G2 clone (Bio X Cell, NH, USA) on day 18 (the starting radiation day), and on days 21 and 24 in the combined therapy groups.(Fig 2A)

Fig 1

Ablative RT comparing to other regimens with same BED increase numbers and ratios of immune cell in the TME but no differences in survival rates or tumor volumes The experimental schedule of radiation therapy in ablative (n = 9), hypofraction (n = 9), and conventional (n = 9) radiation regimens are shown (A). 1 × 106 CT26 cells were inoculated subcutaneously into the right flanks of mice on starting day and irradiation of each group began 18 days after initial tumor implantation. In day 36, 3 mice of each group were analyzed for the percentage of immune cells (CD8+, CD8+ IFNγ+, CD4+, CD4+ CD25+ FOXP3+) that infiltrated into the tumor are shown in (B). Data are presented as means ± SDs and analyzed by Tukey's Multiple Comparison Test; (*: P <0.05, **: P < 0.01). Tumor volumes and survival rates are shown respectly in (C) and (D). There is significant different (***: P < 0.001) between RT therapy and Control group (n = 14) and no significant differences in survival rates were seen between mice treated with the different RT regimens. Results were analyzed by the log-rank (Mantel-Cox) Test; n/s: not significant.

Fig 2

Combining ablative radiation with αPD-L1 mAb increases CD8+ effector T- and Treg cell infiltration in tumors.

The experimental schedule of Combining therapy of Ablative (n = 10), Hypofraction (n = 9) and Conventional (n = 10) Radiation regimens with αPD-L1 mAb are shown (A). In combined therapy groups, mice received 200 μg/ml of αPDL-1 simultaneously with irradiation on day 18 post-initial inoculation and again on days 21 and 24. The percentage of CD8+ IFNγ+, CD4+, and CD4+ CD25+ FOXP3+ cells that infiltrated to tumor, spleen, and lymph node are shown respectively in (B), (C) and (D). Data are presented as means ± SDs and analyzed by Tukey's Multiple Comparison Test; (ns: non-significant*: P < 0.05, **: P < 0.01).

Measurement of survival factors: In vivo study

The time required for a tumor to reach a final volume of 1500 mm3 is called Time to Endpoint (TTE), which is calculated by the formula TTE = [log (end point)-b]/m. A TTE diagram is derived from the linear regression of the log of tumor growth at times that “b” and “m” are the y-intercept and slope, respectively. The end point criteria were 1) tumor volume became greater than 1500 mm3, 2) body weight decreased 15 percent or more of the initial weight, and 3) health decline or dead. Percent of tumor growth delay (%TGD) was calculated from the ratio of TTE in experimental groups to the control group, or each group as described [20].

Measurement of infiltrating immune cells in tumor, lymph nodes, and spleen by flow cytometry

After the treatment protocol, 36 days’ post-tumor challenge, some mice of groups were first anesthetized with isoflurane and then sacrificed by cervical dislocation. The tumors were minced into small pieces then were incubated with Type I collagenase in RPMI medium 1640 (1:1 ratio) at 37°C for two hours. Lymph nodes near the tumor and the spleen were also cut into small pieces, minced, pelleted, and washed two times for 5 min with RBC lysis buffer. The cells were filtered through a 70 μm cell strainer (BD Falcon, USA) and then centrifuged at 300g for 10 min. So, the pellets of cells were suspended in flow cytometry staining buffer (phosphate-buffered saline containing 5% FBS) and analyzed by flow cytometry using fluorochrome antibodies against CD4 (clone GK1.5), CD8 (clone 53–6.7), CD25 (clone 3C7), Foxp3 (clone 150 D), and IgG1 isotype control (clone MOPC-21) (Biolegend, San Diego, California) [21].

Measurement of IFNγ production in tumor, lymph node, and spleen by flow cytometry

The tumor-infiltrating lymphocytes (TILs) and lymph node and spleen cells were cultured with cell activation cocktail (PMA/Ionomycin with Brefeldin A, Biolegend, San Diego, Californian) for 4 hours, centrifuged at 300g for 10 min, and suspended in flow cytometry staining buffer. Cells were analyzed by flow cytometry for the expression of IFNγ (clone XMG1.2), IgG1 isotype control (clone RTK2071), CD8, and CD4. Flow cytometry was performed with a BD FACS Calibur flow cytometer (Becton Dickinson, USA) and analysis with FlowJo 7.6.1 software.

Statistical analysis

The results are presented as means ± standard deviations (SDs) of the means. Statistics were analyzed using the independent t-test, and the post hoc test for one-way ANOVA by GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). Survival was analyzed with the log-rank Mantel–Cox test. P values < 0.05 were considered significant.

Results

Ablative RT comparing to other regimens with same BED increased numbers and ratios of immune cell in the TME but no differences in survival rates or tumor volumes

The radiation therapy schedules are shown in Fig 1A. The percentage of immune cells that infiltrated to the tumor are shown in Fig 1B. Ablative radiation significantly increased infiltration of CD8+ cells expressing IFNγ (CD8+ effector T-cell) and CD4+ CD25+ FOXP3+ (Treg) cells to the tumor while hypofraction and conventional RT did not. The mean tumor volumes and percent survival of mice treated with the 3 regimens were not significantly different, likely due to them all receiving the same BED radiations (Fig 1C and 1D). These data demonstrate that infiltration of immune cells were differed when tumors were irradiated by different regimens with same BED given in different fractions and doses.

Ablative RT combined with αPD-L1 mAb caused CD8+ T cells and Treg cells to infiltrate into tumors in greater numbers than the other regimens

Ablative RT combined with αPD-L1 mAb led to a significant increase in the number of CD8+ T cells expressing of IFNγ and Foxp3+ CD25+ expressing CD4+ T cells infiltrating into the tumor, but not into spleen or lymph nodes (Fig 2B and 2D). The number of CD4+ T cells did not change significantly in the other combined therapy groups (Fig 2C). These data demonstrate that ablative RT, when delivered in combination with αPD-1, leads to changes in tumor infiltration by CD8+ effector T-cell and Treg populations.

Ablative RT leads to IFNγ expression, and when combined with αPDL-1 mAb, significantly increased IFNγ expression in tumors, even in the long term after radiation

To determine whether infiltrated immune cells caused an adaptive change in tumors, the effector cytokine IFNγ was analyzed (Fig 3). We found that ablative radiation increased IFNγ expression in tumors in the long term after radiation relative to the control, while the other regimens decreased it insignificantly (Fig 3A). Also, ablative RT combined with αPDL-1 resulted in a 3-fold increase in IFNγ expression, while the other combination therapies had no different relative to their radiation monotherapies (Fig 3B). Histograms of IFNγ expression showing a shift to the right on the x-axis represent an increase in IFNγ expression on immune cells in the tumors (Fig 3C).

Fig 3

Tumors from mice treated with ablative RT express significantly more IFNγ than tumors from mice treated with hypofractionated or conventional RT, and when ablative treatment combine with αPDL-1 mAb, tumors express significantly more IFNγ than those from mice treated with other regimens.

The percentage of expressing of IFNγ+ on immune cells in tumor are shown (A) in Ablative, Hypofraction and Conventional radiation and are shown in combining regimens of radiations with αPD-L1 (n = 3) (B). Histograms of IFNγ expression showing a shift to the right on the x-axis represent an increase in IFNγ expression on immune cells in the tumors (C). This chart was obtained using FlowJo 7.6.1 software. Data were analyzed by Tukey's Multiple Comparison Test and are presented as means ± SDs; ns: non-significant (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

Ablative RT is the most effective regimen for combining with αPDL-1 mAb at reducing tumor volume and increasing proportion of mice with complete tumor resolution and survival rates

Thirty days after therapy tumors were significantly smaller in all the irradiated mice than in controls, and this effect was even more pronounced when the irradiation was combined with αPDL-1. (Fig 4A and Table 1). The ratios of mice that experienced complete tumor resolution (Fig 4B) and survival rates (Fig 4C) were the same in these groups (Fig 4B). Tumor volumes were lowest and TTE and survival rates and the proportion of mice that experienced complete tumor resolution (6/7 mice) were greatest in mice that received the combining ablative RT with αPDL-1 (Fig 4B and 4C).

Fig 4

When combined with αPDL-1 mAb, ablative radiation is the most effective regimen for reducing tumor volume, inducing complete tumor resolution, and increasing survival rates.

The average tumor volume in groups is shown at the onset of treatment and 30 days’ post-treatment (at least n = 6) (A). On the day the treatment protocol began, tumor volumes were 300–400 mm3 in all groups with no significant differences between groups. The tumor volume changes from the start of treatment to 150 days’ post-treatment in each group are shown as Kaplan Meier curves (B). Survival rates are shown in (C). Combining Abl RT with αPDL-1 group had significant (P < 0.001) when compared with Control and αPDL-1 mice and had significant (P < 0.01) when compared with other therapies groups. ns: non-significant (*: P < 0.05, **: P < 0.01, ***: P < 0.001; Tukey's Multiple Comparison Test and Log-rank (Mantel-Cox) Test.).

Table 1

The means of time to endpoint (TTE) the studied groups and the comparison of tumor growth delay (%TGD) between the studied and control groups, the studied and the αPD-L1 groups, and each combination therapy group with its corresponding RT group were calculated.

Groups	Average of TTE (Day)	% TGD Compared to Control Group	% TGD Compared to Anti-PDL-1 Group	% TGD of each Combination Therapy Group Compared to Radiotherapy Group
Abl RT + Anti-PDL-1	137.12	433.70	279.96	89.23
Abl RT	72.46	182.035	100.79
Hypo RT + Anti-PDL-1	99.79	288.39	176.51	15.75
Hypo RT	86.21	235.54	138.8
Con RT + Anti-PDL-1	88.17	243.16	144.31	7.57
Con RT	81.96	219.00	127.11
Anti-PDL-1	36.09	40.45	0
Ctrl	25.69	0	-28.80

Abl RT: Ablative, RT: Radiotherapy, Hypo: Hypofraction, Con: Conventional, Ctrl: Control

Although their BEDs were equal, the different irradiation regimens had different synergistic effects when combined with αPDL-1. In addition, tumor growth delay from ablative RT plus αPDL-1 was 89.23% greater than with ablative RT alone, hypofraction plus αAPD-L1 was15.75% greater than hypofraction alone, and conventional RT plus αAPD-L1 7.57% greater than conventional RT alone.

Treatment with ablative irradiation plus αPDL-1 mAb inhibit regrowth of new tumor, maybe due to creating a protective anti-tumor immune memory

We next investigated whether immunologic memory was generated in tumor-resolved-long term surviving (LTS) mice after treatment with each combination therapy at in vivo study. All the LTS mice that were treated with ablative (6/6) or hypofraction (2/2) RT plus αPDL-1 completely rejected tumors following re-challenge on days 90 and 150 post-initial challenge while 50% (1/2) of the LTS mice treated with conventional RT rejected tumors 150 days’ post-initial challenge. The percent survival of LTS mice are shown in Fig 5 for 60 days after tumor challenge again. The LTS mice of Conventional RT had less ratio of survival time in compare of others therapy groups.

Fig 5

Treatment with ablative irradiation plus αPDL-1 mAb inhibit regrowth of new tumor, maybe due to creating a protective anti-tumor immune memory.

The curve shows the survival rate in LTS mice (n = 2) for 60 days after tumor re-challenge again with 1 × 106 CT26 cells into the left flanks (another side) of mice. The survival rate in mice treated with ablative RT plus αPDL-1 was significantly greater than that of naïve mice (n = 5). The LTS mice of Conventional RT had less ratio of survival time in compare of others therapy groups (***: P <0.001).

Discussion

Our findings showed a significantly greater number of CD8+ cells expressing IFNγ+ in the TME after ablative radiation than with hypofraction or conventional RT. This finding was previously observed in high-dose irradiation studies [22, 23], and it was found that the expression of some immunogenic cell death (ICD) markers was increased when the radiation dose was greater than 5–10 Gy [24]. Although the BED was 40 Gy for all three regimens in our study, the different regimens had different effects on immune cell infiltration to the TME. This could lead to different responses between the regimens, either with or without αPDL-1.

The mean IFNγ expression in the conventional and hypofraction groups was less than in the control group; however, this difference was not significant. Murthy and colleagues stated that radiation-induced hypoxia can reduce IFNγ expression; as a result, RT is ineffective on the immune system in the tumor area [25].

It is therefore suggested that in further studies, the rate of hypoxia in the tumor area should be considered after irradiation with different regimens. Our study however, showed that IFNγ expression increased in the ablative group following the increase in infiltration of CD8+ cells into the tumor region after high-dose exposure. Another study found that when IFNγ expression by CD8+ cells increased in the tumor site after irradiation, this also increased PDL-1 expression on tumor cells, ultimately leading to exhausted CD8+ cells [26, 27], and so αPDL-1 could be considered an appropriate treatment [10].

We showed that αPDL-1 had synergistic effects when combined with single high-dose RT regimens, and of the regimens in this study, ablative irradiation stimulated IFNγ expression. It is likely that the expression of PDL-1 is different depending on the regimen in the radiated tumor region, just as IFNγ expression differed between regimens [28].

Also, this synergistic effect of high-dose RT with αPDL-1 was entirely TCD8+ dependent, as was shown in other studies [28, 29].

Our study also showed that the population of CD4+ FOXP3+ cells increased significantly in the high-dose single-radiation group with αPDL-1. Given that, unlike other immune cells, Treg cells were resistant to irradiation, Ratikan et al. compared several irradiation regimens with different BEDs and found that increasing the dose per fraction decreased the tumor volume, concurrent with an accumulation of Treg cells [23].

Another study showed that anti-PDL-1 enhanced the cytotoxic effects of IFNγ-dependent CD8+ cells [30]. However, another study in a CT-26 model found no significantly different effects between ablative (1 × 7Gy) RT, hypofraction (3 × 4Gy) RT, or conventional (5 × 2Gy) RT combination therapies with αPD-L1 [31]. We believe the reason for this discrepancy with our study is the difference BEDs in two studies. The total BEDs of the three regimens in their study were 11.2, 16.8 and 12 Gy, respectively. Subsequent meta-analysis studies indicate increasing the irradiation BED could increase the likelihood of observing an immunological-dependent abscopal effect, and when the BED is 60 Gy, the probability of occurrence of this effect is 50% [17]. Low BED regimens are unlikely to stimulate immunity sufficiently; therefore, combining them with αPDL-1 has no additive effect. In another study, αPDL-1 was reported to have greater synergic effects when combined with ablative RTs (8 × 2 Gy) than with conventional regimens (10 × 2 Gy) [32].

Because the CT26 mouse model, in which the immune system is suppressed in TME, is more responsive to immune checkpoint blockade rather other models, it is appropriate for combined therapy with ICBs in pre-clinical studies, in addition to the T [33]. Recent studies suggested this tumor model is used to evaluate the synergistic effects of high-dose RT and ICBs [34], and other studies have suggested the use of the CT26 model due to its severely suppressed environment for immunological responses. The average tumor volume at the beginning of our study was 300–400 mm3. This was largest from other studies [27], and could indicate the combination of ablative RT plus αPDL-1 is more effective than the other combination therapies in large tumor.

Different irradiation regimens with the same BED have equal effects on cell death, as the LQ model predicts [35]. In our study the three radiation regimens with the same BED resulted also in no differences in tumor size or survival rates, but attention should be paid to the immunological changes after irradiation from each of these regimens. For designing of combining immunotherapy with irradiation regimens, clinical researchers should consider how RT affects immunity, leading to effective planning for dose adjustment and number of fractions. Such clinical studies can ultimately accelerate the clinical development of RT regimens and their combination with immunotherapy, which ultimately leads to a strong and sustained immune response that eliminates the tumor and prevents recurrence.

The ARRIVE guidelines checklist.

(PDF)

Click here for additional data file.

(TIF)

Click here for additional data file.

21 Jan 2020

PONE-D-19-33249

Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response

PLOS ONE

Dear Dr Jalali,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it is not fully meet PLOS ONE’s publication criteria. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In addition, during our internal assessments, we noted that 6 animals died due to lowering temperature during the irradiation procedures.Some editors raised the great concerns about the animal welfare. We expect your response about this matter.

We would appreciate receiving your revised manuscript by Mar 06 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jianxin Xue

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. We note that Figures in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a)    You may seek permission from the original copyright holder of the Figures to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b)    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer’s comments

The current study has evaluated the synergistic effect of RT combined with

immunotherapy using the αPDL-1 antibody in three different RT regimens in a xenograft tumor model in BALB/c mice. The synergistic effect of the αPDL-1 antibody in RT regimens is interesting. The findings would be helpful to develop the new immunomodulator and to design the more efficient radiation regimens to control the tumor outgrowth.

Comments to the author

1. In the Introduction section, the author should favorably explain the motivation of the research to differentiate the current study from the other applied papers in this field.

2. The author should explain more about the expression of CD8+ and CD4+ cells after radiation in the first paragraph of the introduction.

Methods:

3. The author should mention the fractionation schedules. How many fractions of RT were given per day and for how many days/ weeks?

4. The mostly used hyper fractionation RT regimens in clinics are 2Gy fraction per day. Is there any specific reason to use 3Gy fractions?

5. Radiation with or without causes nephrotoxicity. The author should evaluate the nephrotoxicity after RT ± anti-PD-L1 and could cite the following paper (PMID: 27836988)

Results and discussion

6. The quality of the figures is unsatisfactory, please improve the quality of the figures.

7. The findings have been discussed to some extent to support the claims made in the hypothesis. The manuscript adds some critical information to advance the field. The results presented in the paper are substantial enough for publication in Plosone after minor revision.

Reviewer #2: Excellent paper. Please add a paragraph after the discussion: Conclusion and discuss clinical implications of your findings and how your findings could be translated into the clinical studies. Lung SBRT is a good example where we deliver ablative dose of radiation.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: ANIS AHMAD

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Mar 2020

February 1, 2020

Prof. Jianxin Xue

Academic Editor

PLOS ONE

Dear Professor Jianxin Xue:

Thank you very much for your letter regarding our manuscript (PONE-D-19-33249) entitled" Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response”. The reviewers’ comments were very helpful, and we made revisions to the manuscript according to their criticisms.

I think described point together with the other minor points that we have addressed made the manuscript much improved. Please find attached a detailed reply to the referees’ comments and description of other changes to the manuscript.

We would like to thank the reviewers again for their important comments. Our responses to the comments are as follows:

#Academic editor: In addition, during our internal assessments, we noted that 6 animals died due to lowering temperature during the irradiation procedures. Some editors raised the great concerns about the animal welfare. We expect your response about this matter.

Response: We appreciate you taking the time and the valuable suggestions offered. The mice were adapted to the conditions after two weeks in the animal research room. Then, after tumor induction, eight groups were randomly selected.

At the stage of tumor induction and irradiation, they were anesthetized to reduce pain tolerance. Symptoms of pain were monitored for up to 4 hours after irradiation, and if any symptoms of pain such as paleness, anorexia, weight loss and back flexion were observed, they were given pain relief. After reaching the final stage and finding endpoint criteria, they were anesthetized by isoflurane and sacrificed ten minutes later.

#Reviwer 1: 1. In the Introduction section, the author should favorably explain the motivation of the research to differentiate the current study from the other applied papers in this field.

Response: We thank the reviewer for their close attention. The motivations for the study were written in the form of a few questions at the end of the last paragraph, which we completed by adding a few sentences.

However, studies have not yet determined whether different regimens with the same BED (when the biological effects of the regimens are equal) have different immunogenic effects, whether they affect tumor recurrence associated with immunological responses. In addition, can these regimens respond differently to combination therapy? Because it can improve the quality of immune responses into TME by use of modification in the dose and number of fractions radiation to the extent that the radiation clinic limitations and technology of the radiation devices allow us. Also it can lead to memory responses, which prevents tumor recurrence that is a common problem after treatment. To address these issues, we aimed to evaluate the synergistic effect of RT combined with immunotherapy using αPDL-1 in three different RT regimens with the help of modeled tumor BALB/c mice.

#Reviwer 1: 2. The author should explain more about the expression of CD8+ and CD4+ cells after radiation in the first paragraph of the introduction.

Response: Yes, Sure, in the first paragraph, we noted the effect of radiation on CD4 and CD8 cells.

Irradiation has multiple inhibitory and stimulatory effects on the immune system such as increased CD4+, CD8+ and Treg Cells infiltration into TME (3, 4) Some regimens of radiation like ablative RT was associated with increased infiltration of exhausted CD8+ T cells into the tumor, which induced radiation resistance. (5, 6). The combination of radiotherapy and Immune checkpoint blockers therapy can reduce these inhibitory effects by stimulating immune cells and enhancing the response to radiation. Preclinical studies showed that RT increased PDL-1 expression on tumor cells (7, 8), and anti-PDL1 (αPDL-1) mAb combined with radiation had a synergistic effect on immune response induction dependent IFN-γ producing CD8 T cells activations. (9, 10).

#Reviwer 1: 3. The author should mention the fractionation schedules. How many fractions of RT were given per day and for how many days/ weeks?

Response: Thank you for pointing this out. In Methods, Treatment section adds these sentences:

The experimental schedule of ablative radiation therapy was only one fraction of 15 Gy in 18 days’ post-initial inoculation. In Hypofraction RT regimens, two fractions of 10 Gy were performed on days 18 and 28 after inoculation. In Conventional RT, ten fractions of 3 Gy were given from day 18 to day 31 for 14 days at a time interval of one day or at most two days. (Figure 1A).

#Reviwer 1: 4. The mostly used hyper fractionation RT regimens in clinics are 2Gy fraction per day. Is there any specific reason to use 3Gy fractions?

Response: We thank the reviewer for their close attention. Given that the clinic conventional RT has a range of 1.8 to 2.5 Gy1, we wanted to assay immunological responses when deliver a 40 Gy equivalent total BED in these regimens. But on the other hand, we were going to have to choose the 2g dose for conventional regimen, we would have to give 17 fractions of radiation, due to the limitation of the number of irradiations in the pre-clinical studies, 3Gy fractions were selected for 10 fractions. Of course, further studies are ongoing for lower dose of BED regimens closer to the clinic regimens.

#Reviwer 1: 5. Radiation with or without causes nephrotoxicity. The author should evaluate the nephrotoxicity after RT ± anti-PD-L1 and could cite the following paper (PMID: 27836988)

Response: The main purpose of this study was to evaluate the immunological responses in irradiated regimens with the same biological effects. Further studies on radiation induced nephropathy should be considered after identifying the most appropriate regimen in any body tissue according to the immunological response and combination of radiation therapy with immunotherapy.

#Reviwer 1: 6. The quality of the figures is unsatisfactory, please improve the quality of the figures.

Response: The correction was made in the revised manuscript. The quality of figures are improved.

#Reviwer 1:7. The findings have been discussed to some extent to support the claims made in the hypothesis. The manuscript adds some critical information to advance the field. The results presented in the paper are substantial enough for publication in Plosone after minor revision.

Response: We greatly appreciate the reviewer’s efforts to carefully review the paper.

#Reviwer 2: Excellent paper. Please add a paragraph after the discussion: Conclusion and discuss clinical implications of your findings and how your findings could be translated into the clinical studies. Lung SBRT is a good example where we deliver ablative dose of radiation.

Response: We greatly appreciate the reviewer’s efforts to carefully review the paper.

References:

1. John. L. Meyer. IMRT, IGRT, SBRT: Advances in the Treatment Planning and Delivery of radiotherapy. Vol. 43. Page 396

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Mar 2020

Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response

PONE-D-19-33249R1

Dear Dr. Jalali,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Jianxin Xue

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: ANIS AHMAD

30 Mar 2020

PONE-D-19-33249R1

Synergistic Effects of Anti-PDL-1 with Ablative Radiation Comparing to other Regimens with Same Biological Effect Dose based on Different Immunogenic Response

Dear Dr. Jalali:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jianxin Xue

Academic Editor

PLOS ONE