CXCR5^+ monocyte emigration impairs the radiation-induced antitumor immune response

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
A substantial portion of patients experience radioresistance, which impedes clinical benefit. The radiation-induced ‘protumor’ immune response is previously demonstrated to limit antitumor efficacy. However, the detailed mechanism remains to be explored. In this study, we observe CXCR5+ monocytes are enriched in tumor upon radiation. CXCR5 expression on monocytes in host is induced by tumor-derived VEGF through PI3K/mTOR/HIF-1α axis. Local radiation enhances CXCL13 expression from tumor cells, a specific ligand of CXCR5, which leads to the recruitment of CXCR5+ monocytes. Tumor-infiltrating CXCR5+ monocytes induce radioresistance by inhibiting CD8+ T cells through PD-1/PD-L1 interaction. Moreover, radiation-induced GM-CSF promotes the differentiation of CXCR5+ monocytes toward M2-like macrophages. In contract, inhibiting VEGFR signaling, neutralizing CXCL13 and GM-CSF, or blocking PD-L1 facilitates radiation-induced tumor control by abrogating CXCR5+ monocyte-mediated immunosupression. Furthermore, the CXCR5+ and CD14+ populations are increased in patients with cancer following radiotherapy. Monocyte is increased in the peripheral blood of patients with progressive disease following radiotherapy. These findings suggest potential strategies for blocking the CXCR5/CXCL13 axis to improve radiotherapy efficacy.

Introduction
Radiotherapy (RT) is widely applied as an effective cytotoxic therapy for cancer treatment. More than 50% of patients with tumors require radiation therapy 1-4. Despite its extensive use in oncotherapy, the weakened effectiveness of RT, such as locoregional recurrence following treatment, occurs in some patients and leads to a worse prognosis 5,6. Thus, radioresistance-induced therapeutic failure has become the main concern in cancer management. The immune system is recognized for its crucial role in the efficacy of RT 7. Nevertheless, the immunological mechanism of radioresistance is unclear.

Neoantigens, cytokines, and chemokines are released in tumor tissue following ionizing radiation (IR) exposure. These signals aroused immune response, typically the activation of cytotoxic T cells, which is required for the full antitumor effect of RT 8. In addition to “antitumor” immune responses, radiotherapy elicits “protumor” effects and profoundly affects the prognosis of cancer treatment. IR upregulates type I IFN expression, thereby inducing programmed death ligand 1 (PD-L1) expression on myeloid cells, both dampen the antitumor response 9-11. Moreover, some subsets of neutrophils, monocytes, macrophages, and Tregs are enhanced following local tumor radiation 12. These cells mediate tumor radioresistance by promoting immunosuppression and interfering with T-cell activation 13,14. Exploring an efficient strategy that could overcome IR-induced “protumor” effects is necessary to amplify the antitumor immune response during RT and enhance curability.

Monocytes and macrophages are major components of the tumor microenvironment and account for up to 50% of solid tumors 15,16. Previous studies have shown that CD11b+Ly6Chigh myeloid cells are increased in irradiated tumors and that some subsets are protumorigenic due to their immunosuppressive effects on CD8+ T cells 12,14,17. Considering that CD8+ T cells play a critical role in the radiation-induced antitumor response, reducing protumorigenic cells and attenuating their immunosuppression specifically are essential goals. Combining immune checkpoint blockade with RT has been confirmed to result in better control of tumors in mouse models and patients 18,19. This finding illustrates that strengthening CD8+ T-cell function by thwarting the immunosuppressive response can potentially prevent radioresistance.

In the present study, we report that radiation enhances CXCL13 expression, which promotes CXCR5+ monocytes infiltration. CXCR5+ monocytes, as an immunosuppressive factor, are induced by tumor-derived VEGF and have the potential to differentiate into CXCR5+ macrophages with M2 features. Inhibiting the infiltration, function, or differentiation of CXCR5+ monocytes restores T-cell function and enhances tumor control during radiation. These results reveal the mechanism of radiotherapy-induced protumor effects and provide a novel direction for therapeutic strategies to overcome radioresistance.

Results
1. CXCL13 and CXCR5+ monocytes increased in the tumor microenvironment after IR
Emerging evidence has shown that tumor infiltration of monocytes may account for therapy resistance. To determine the impact of radiation on the tumor immune microenvironment, we examined tumor immune cells at the early phase, revealing a significant increase of monocytes (Ly6ChighLy6Glow) (Supplementary Fig. 1A). We next confirmed these findings in MC38, PanC02, and 4T1 tumor models on the 3rd day and 7th day of IR. Similarly, a striking increase in the number of tumor-infiltrated monocytes was observed 3 days after irradiation (Figures 1A–C), whereas neutrophils (Ly6GhighLy6Clow) were not induced by radiation (Supplementary Fig. 1B). Furthermore, no remarkable differences in tumor volume or necrosis (rarely observed) between the untreated and irradiated groups at the indicated time points (Supplementary Fig. 1C and 1D). Bulk tumor RNA-seq revealed that Ly6c2 was highly expressed post-IR, whereas other leukocyte antigen-6 genes did not significantly increase after radiation (Supplementary Fig. 1E and 1F).

Since an abundance of chemokines can be released from tumor tissues and myeloid cells can be recruited at the initial stage after IR exposure, we next scanned chemokines in tumor tissues subjected to irradiation or not. We noted that CXCL13 was increased post-IR (Figures 1D and 1E), which has been reported to be an immunosuppressive mediator in chemotherapy and immune checkpoint blockade treatment. The CXCL13–CXCR5 axis facilitates tumor metastasis and is associated with poor prognosis in colorectal cancer and breast cancer patients 20-23. However, the function of the CXCL13–CXCR5 axis in the IR-induced immune response has not been characterized in detail. Thus, we sought to determine whether CXCL13 induced monocyte infiltration following irradiation. We analyzed CD45+CXCR5+ subsets of tumor 3 days and 7 days after IR. CD45+CXCR5+ cells were significantly increased on the day 3 of IR; notably, the increased CXCR5+ population was mainly monocytes. We also noted that on the day 7 post IR, CXCR5+ monocytes receded, whereas the proportions of CXCR5+ mono-macrophages and CXCR5+ macrophages increased (Figures 1F and 1G, Supplementary Fig. 1G). These results are consistent with those of RNA-seq, which revealed that the expression of myeloid cell differentiation-associated genes Lyz2 and Csfr1 increased after IR (Supplementary Fig. 1F).

To further characterize CXCR5+ monocytes, we compared them with CXCR5⁻ monocytes and tumor infiltrated monocytes (CD45+CD11b+Ly6ChighLy6Glow in untreated tumor). Result showed that: 1) both with and without radiation, the proportion of the CX3CR1+CCR2⁻ subset was similar across tumor infiltrated monocytes, CXCR5⁻ and CXCR5+ monocytes; 2) the CX3CR1⁻CCR2+ subset accounted for a small proportion of these three cell types; and 3) CCR2⁻CX3CR1⁻CXCR5+ monocytes were expanded in irradiated tumors (Figure 1H). Furthermore, the proportion of CXCR5+ monocytes significantly increased in myeloid cells and tumor tissues on the 3rd day of IR in MC38 tumor-bearing mice (Figure 1I and Supplementary Fig. 2A); nevertheless, local tumor irradiation did not affect the CXCR5− monocyte population or CXCR5 expression on monocytes (Supplementary Fig. 2B–E). A similar effect was observed in the PanC02, 4T1, and LLC tumor models (Figures 1J, 1K, and Supplementary Fig. 2F–H). Thus, the increased CXCL13 in irradiated tumors may recruit CXCR5+ monocytes specifically, resulting in monocyte expansion.

2. Tumor-derived VEGF induced CXCR5 expression on monocytes via the PI3K/Akt/mTOR/HIF-1α pathway
These findings prompted us to further explore the source of CXCR5+ monocytes. We analyzed monocytes in the PB and BM of naïve mice and tumor-bearing mice. In tumor-bearing mice, monocytes in the PB and BM expanded at 15 days post tumor inoculation (Supplementary Fig. 3A). Moreover, there was a significant expansion of CXCR5+ monocytes but not of CXCR5- monocytes in the PB and BM of tumor-bearing mice (Supplementary Fig. 3B). Compared with that in naïve mice, CXCR5 expression on monocytes from PBs (Figure 2A) and BMs (Figure 2B) was increased in tumor-bearing mice regardless of ionizing radiation. We next validated our findings via the use of BM-derived monocytes in vitro. CXCR5 expression increased in monocytes cultured with MC38 cells (Figure 2C). Given that CXCR5+ monocytes expanded in the PB and BM of tumor-bearing mice and that tumor cells can induce CXCR5 expression on monocytes, we postulated that tumors may upregulate CXCR5 expression on monocytes remotely. To address this hypothesis, we separated the supernatants of the tumor cells and filtered the cell components with a 0.44 µM filter. The monocytes supplemented with tumor supernatant (TS) highly expressed CXCR5 (Figure 2C). The same results were observed for monocytes treated with TS from various tumor cell lines (Supplementary Fig. 3C and 3D) and for monocytes from PB and BM (Supplementary Fig. 3E). In addition, TS-induced CXCR5 expression occurred in a dose- (Supplementary Fig. 3F) and time-dependent (Supplementary Fig. 3G) manner. In the MC38 tumor model, CXCR5 expression increased in PBMCs (Supplementary Fig. 3H) and tumor-infiltrated monocytes (Supplementary Fig. 3I) with increasing inoculation time. These results suggest that tumors are necessary for the induction of CXCR5+ monocytes.

We next sought to uncover how tumors induce CXCR5 expression on monocytes. Wnt signaling and NF-𝜅B signaling were reported to be responsible for CXCR5 expression on immune cell 24 25 26 and tumor cells 27. However, an NF-𝜅B inhibitor (BAY 11-7082) or a Wnt/β-catenin inhibitor (salinomycin) did not reduce TS-induced CXCR5 expression on monocytes (Supplementary Fig. 4A). Notably, differentially expressed genes in of CXCR5+ and CXCR5- monocytes were enriched in PI3K-Akt signaling (Figure 2D). Furthermore, inhibiting PI3K with LY294002 rescued highly expressed CXCR5 in TS-treated monocytes (Figure 2E). Our results are consistent with those of a previous report indicating that PI3K activation mediates CXCR5 expression on Tfh cells 28. The PI3K/Akt/mTOR (PAM) signaling pathway is a highly conserved signal transduction network which involved in M1/M2 polarization, anti-inflammatory effects, and polymorphonuclear neutrophil chemotaxis 29-33. Here, we used rapamycin, an mTORC1 inhibitor, to confirm whether the PAM regulates CXCR5 expression in monocytes. We observed a decrease in the CXCR5+ population in rapamycin-pretreated monocytes after TS treatment (Figure 2F). mTOR has been reported to regulate various transcription factors 34-36, among which Eif4g2, Hif1a, and Maf are highly expressed in CXCR5+ monocytes (Supplementary Fig. 4B). We constructed eIF4G2-, HIF1a-, and Maf-knockdown monocytes with siRNA (Supplementary Fig. 4C‒E). With TS treatment, HIF1a-knockdown monocytes expressed less CXCR5 (Figure 2G), while WT monocytes highly expressed HIF-1α (Figure 2H). Taken together, these findings indicate that tumors can release certain soluble substances to induce monocyte CXCR5 expression, which is PI3K/Akt/mTOR/HIF-1α dependent.

To further explore which soluble substances in tumors induce CXCR5 expression on monocytes via PAM, we cultured monocytes with TS filtered through membranes with different pore sizes or with inhibited tumor exosomes, and the results revealed that CXCR5 expression was not affected by insoluble factors, including extracellular vesicles (Supplementary Fig. 4F and 4G). Previous studies reported that tumor cells can generate prostaglandin E2 (PGE2) 37 and induces CXCR5 expression on THP-1 cells 26. Therefore, we applied PGE2 to monocytes as described in previous studies 26. Although 20 ng/ml PGE2 enhanced CXCR5 expression (Supplementary Fig. 4H), inhibiting cyclooxygenase-2 (a key synthetase of PGE2) in MC38 or monocytes (Supplementary Fig. 4I) or knocking out Ptgs2 in the MC38 tumor cell line (Supplementary Fig. 4J) did not rescue the upregulation of CXCR5 in TS-treated monocytes. These results suggest that PGE2 is not the main mediator of CXCR5 expression in monocytes under tumor conditions.

Tumor-derived small molecules or soluble proteins have been verified to mediate myelopoiesis and enhance tumor-promoting properties 38, we next focused on the tumor-derived soluble immunosuppressive factors thromboxane A2 (TXA2), indoleamine 2,3-dioxigenase (IDO), adenosine, TGF-𝛽, EGF, and VEGF 39-42. We treated monocytes with receptor antagonists or inhibitors of these factors before culture with TS. The results showed that inhibiting TGF-𝛽, EGF, and VEGF rescued TS-induced CXCR5 expression (Figure 2I). Moreover, recombinant murine (rm) VEGF and EGF enhanced CXCR5 expression on monocytes (Figure 2J). Compared with WT-TS-induced CXCR5, Vegfa knock out (KO)-TS and Egf KO-TS weakened CXCR5 expression on monocytes (Figure 2K). In addition, anti-VEGF-A reversed TS-induced CXCR5 expression (Figure 2L). We also found that rmVEGF was activated in monocytes (Figure 2M) and that a VEGFR inhibitor impaired TS-induced PAM signaling activation (Figure 2N). In naïve mice, rmVEGF enhanced CXCR5 expression on monocytes in BM and PB (Figure 2O). Anti-VEGF-A reduced CXCR5 expression on monocytes in tumor-bearing mice (Figure 2O). Vegfa-KO MC38 inoculation and regorafenib treatment inhibited CXCR5 expression on monocytes in the tumor, PB, and BM of tumor-bearing mice (Figures 2P and 2Q). Furthermore, regorafenib reduced the proportion of monocyte in PBs and tumors (Figure 2R). These findings establish that tumors release VEGF and induce CXCR5 expression on monocytes. The expanded CXCR5+ monocytes served as a “reserve pool” and were prepared for radiation-induced monocyte infiltration.

3. IR promoted the tumoral infiltration of CXCR5+ monocytes through the CXCR5–CXCL13 axis
Since numerous chemokines are upregulated in tumor tissues after IR (Figure 1D), we investigated which of these contribute to monocyte recruitment using selective antagonists and neutralizing antibodies. Blockade of CXCL13 signaling, but not CCL4, CCL5, CXCL1, and CXCL10 signaling, abrogated radiation-induced monocyte infiltration (Figure 3A and Supplementary Fig. 5A), which suggested that CXCL13-CXCR5 axis is more important for monocytes recruitment in irradiated tumor.

In the IR condition, we observed a significant increase of the percentage and number of monocytes (Figure 3B), especially CXCR5+ monocytes, in the PB (Figure 3C), which is consistent with the findings in tumor tissues. To further investigate whether increased CXCL13 levels accelerated CXCR5+ monocyte migration and tumor infiltration, we performed an ex vivo transwell assay. Homogenates of tumor tissues from untreated or IR-treated mice were added to the lower chambers. TS-treated WT or CXCR5−/− monocytes were added to the upper chambers. After 4 h of culture, the cells in the lower chamber were collected and analyzed via flow cytometry (Figure 3D). We found that compared with untreated tumors, IR-treated tumors recruited more WT monocytes but fewer CXCR5−/− monocytes in irradiated tumors (Figure 3E and Supplementary Fig. 5B). In accordance with the results ex vivo, monocytes increased in the tumors of WT mice post-IR but decreased in those of CXCR5−/− (Figure 3F), anti-VEGF-A treated (Figure 3G), and Vegfa-KO tumor-bearing mice (Figure 3H). These data suggested that IR recruited monocytes via CXCR5 on monocytes. To further verify whether irradiated tumors recruit CXCR5+ monocytes via enhanced CXCL13, we next added a CXCL13-neutralizing antibody to the homogenate of IR-treated tumors (Figure 3I). The results showed that neutralizing CXCL13 reduced radiation-induced CXCR5+ monocyte migration (Figure 3J). In tumor-bearing mice, CXCL13 neutralization significantly weakened the infiltration of CXCR5+ monocytes and monocytes post-IR (Figure 3K). However, CXCL13 neutralization did not affect CXCR5− monocyte infiltration (Supplementary Fig. 5C). These findings demonstrated that by inducing CXCL13 production, local tumor radiation recruited monocytes, most of which were CXCR5+ cells.

To determine the source of CXCL13 after IR, we analyzed IR-treated tumors and found that the proportion of CXCL13+ cells increased (Figures 3L and Supplementary Fig. 5D), among which CD45- cells strikingly expanded post-IR (Figure 3M). Hence, we hypothesized that tumor cells are the main source of CXCL13 during radiation. We then delivered radiation to the tumor cells directly. Interestingly, the level of CXCL13 increased slightly in tumor cells after IR. Tumor cells cultured with macrophages also showed mild upregulation of CXCL13. Notably, IR-treated tumor cells cultured with macrophages (with or without IR) highly expressed CXCL13 (Figure 3N). We concluded that macrophages were necessary for tumor cells to produce CXCL13 following IR. We also found that, in Cxcl13 KO MC38 tumor-bearing mice and macrophage-deficient mice, IR did not significantly enhance CXCL13 in tumor tissues (Supplementary Fig. 5E). Moreover, monocytes, especially CXCR5+ monocytes, were reduced in the PB and tumor tissue after local irradiation (Figures 3O and Supplementary Fig. 5F). Blocking the CXCL13–CXCR5 axis (CXCR5 deficiency in monocytes or CXCL13 reduction in tumors) decreased monocyte and CXCR5+ monocyte infiltration after IR. Taken together, our data indicate that radiation, as a stimulus, increases CXCL13 in tumor cells, thereby prompting CXCR5+ monocytes to migrate from the “reserve pool” to the tumor tissue.

4. Reducing tumor-infiltrating CXCR5+ monocytes enhanced CD8+ T-cell-dependent antitumor immunity
We next sought to determine whether and how CXCR5+ monocytes affect the efficacy of RT. Tumor-bearing mice were adoptively transferred with CXCR5− and CXCR5+ monocytes from the day of irradiation. The tumor growth revealed that CXCR5+ monocytes had an impaired therapeutic effect, whereas CXCR5- monocytes did not (Figure 4A). We then employed CXCR5−/− tumor-bearing mice and administered IR. Compared with WT mice, CXCR5−/− mice presented pronounced inhibition of tumor growth (Figure 4B). Vegfa-KO tumors presented better control following IR than did WT tumors (Figure 4C). Furthermore, CXCL13 deficiency in tumor cells (Figure 4D) and CXCL13 neutralization in irradiated tumors (Figure 4E) enhanced tumor control and prolonged the survival of tumor-bearing mice after radiation (Figure 4F). These data suggested that CXCR5+ monocytes weakened tumor control after IR treatment.

To understand how CXCR5+ monocytes impair IR efficiency, we conducted rechallenge models using tumor-rejected mice in the IR and anti-CXCL13 combination groups (Figure 4G). In naïve mice, the tumor volume of MC38 cells increased progressively. Conversely, MC38 was completely inhibited in tumor-rejected mice (Figure 4H). The LLC tumors grew normally in these two groups (Figure 4I). These results suggested that CXCR5+ monocytes weakened adaptive immunity. We then depleted CD8+ T cells in mice and observed that neutralizing CXCL13 after IR did not enhance antitumor efficiency (Figure 4J). Thus, we hypothesized that CXCR5+ monocytes impaired the CD8+ T-cell response after radiation. In accordance with the enhanced efficiency, IR induced greater expression of TNF and IFN𝛾 in tumor-infiltrating CD8+ T cells from CXCR5-/- mice (Figure 4K) and Cxcl13 KO tumors (Figure 4L) than in those from control mice. Furthermore, host CXCR5 deficiency or neutralization CXCL13 after radiation augmented total and activated CD8+ T cells in tumor tissues (Supplementary Fig. 6A‒C). To determine whether CXCR5+ monocytes have direct effects on T cells, we conducted a T-cell activation assay and observed that, compared with CXCR5− monocytes, CXCR5+ monocytes more strongly inhibited the proliferation and expression of TNF and IFN𝛾 in CD8+ T cells (Figures 4M–O). Consistent effects were observed in CD4+ T cells (Supplementary Fig. 6D–F). These results demonstrated that CXCR5+ monocytes have increased immunosuppressive activity and could impair the efficacy of radiation by inhibiting CD8+ T-cell function.

Next, we identified the genes expressed in CXCR5+, CXCR5− monocytes from irradiated tumors and monocytes from untreated tumors to investigate how CXCR5+ monocytes attenuate CD8+ T-cell function. The results revealed that CXCR5⁺ monocytes demonstrate an enhanced expression of protumor genes, including Arg1, Hif1a, and Spp1, when compared with either tumor inflitrated monocytes or CXCR5− monocytes. In contrast, CXCR5+ monocytes exhibited an attenuated antitumor gene profile, including IL12a, CD86, and ICOSL (Supplementary Fig. 6G). In accordance with the gene expression profiles of CXCR5−/+ monocytes, PD-L1 highly expressed in tumor-infiltrated CXCR5+ monocytes in MC38, PanC02, and 4T1 tumor-bearing mice (Figure 4P, Supplementary Fig. 6H and 6I). In TS-treated monocytes, PD-L1 expression increased in CXCR5+ monocytes (Supplementary Fig. 6J). We next investigated whether PD-L1/PD-1 signaling mediated CXCR5+ monocyte-induced CD8+ T-cell inhibition. CXCR5+ monocytes pretreated with or without anti-PD-L1 were cultured with activated T cells. The result showed that anti-PD-L1 reversed the CXCR5+ monocyte-induced inhibition of CD8+ and CD4+ T-cell proliferation (Figure 4Q and Supplementary Fig. 6K). Furthermore, RT combined with anti-PD-L1 and anti-CXCL13 significantly improved tumor control (Figure 4R). On the basis of this observation, we surmised that reducing CXCR5+ monocyte infiltration and blocking immune regulatory signals enhanced the effect of RT.

5. CXCR5+ monocytes differentiated into CXCR5+ macrophages
The results revealed that CXCR5+ monocytes did not increase consistently after IR; nevertheless, the proportion of CXCR5+ macrophages increased (Figures 1G and 5A). Considering that monocytes can migrate to tumors and differentiate into macrophages, we hypothesized that CXCR5+ monocytes may convert to CXCR5+ macrophages in irradiated tumors. To test this hypothesis, we inhibited CXCR5 expression on monocytes and neutralized CXCL13 in tumors after IR, revealing that the proportion of CXCR5+ macrophages decreased after radiation (Figure 5B, Supplementary Fig. 7A and 7B). We next investigated how CXCR5+ monocytes convert to CXCR5+ macrophages after irradiation and revealing that that granulocyte‒macrophage colony-stimulating factor (GM-CSF), which can promote hemopoietic stem cell differentiation, increased after radiation (Figure 5C). In addition, the possibility that tumors induce macrophages expressing CXCR5 directly cannot be excluded. Hence, we performed two processes in vitro. In process 1, macrophages were induced with GM-CSF and treated with TS. In process 2, monocytes were treated with TS and differentiated into macrophages under GM-CSF conditions. The results revealed that TS cannot increase CXCR5 expression on macrophages and that CXCR5+ macrophages are derived from CXCR5+ monocytes (Figure 5D). Furthermore, iPI3K inhibited CXCR5 expression in monocytes but did not affect macrophages (Supplementary Fig. 7C). By transferring CXCR5+ monocytes (CD45.1.2) into CD45.2 mice and delivering radiation to their tumors, we verified the differentiation of CXCR5+ monocytes in vivo (Figure 5E). The results revealed that most adoptive CXCR5+ monocytes polarized into macrophages in irradiated tumors, which persistently expressed CXCR5 and highly expressed CD206 (Figure 5F). These data indicate that the increased proportion of CXCR5+ macrophages following irradiation is derived mainly from CXCR5+ monocytes.

M2 macrophages exhibit immunosuppressive features in the tumor 43. We suspected that the CXCR5+ macrophages derive from CXCR5+ monocytes were M2-like macrophages with immunosuppressive effects. To test this, TS-induced CXCR5+ monocytes were labeled with APC-Cy7 and then cultured with GM-CSF for 72 h. The results revealed that the APC-Cy7+ population contained more CD206+MHC II- macrophages than the APC-Cy7- population (Figure 5G). We also found that CXCR5+ macrophages expressed higher levels of CD206 after irradiation, consistent with the in vitro results (Figure 5H). Moreover, the proportion of M2 macrophages increased in the tumor 7 days after irradiation (Figure 5I), the same trend as that observed with CXCR5+ macrophages (Figure 5A). Blockade of the CXCR5‒CXCL13 axis reversed the increase in M2-like macrophages after IR (Figures 5J, 5K, and Supplementary Fig. 7D). Moreover, neutralizing IR-induced GM-CSF decreased the M2-like macrophage population but enhanced M1-like macrophages (Figures 5L and Supplementary Fig. 7E). We analyzed the gene expression of CXCR5+/- monocytes and observed that CXCR5+ monocytes expressed higher Hif1a and Arg1 but lower Citta (Supplementary Fig. 7F). DEGs in CXCR5-/+ monocytes were enriched in the PPAR signaling pathway, which regulates M2 polarization (Supplementary Fig. 7G). These results suggested that IR induced CXCR5+ M2-like macrophages and that their characteristics were determined at the monocyte stage.

To further characterize CXCR5+ macrophages in detail, we analyzed gene expression differences between CXCR5− and CXCR5+ macrophages across functional modules. Antigen presentation- and cell cycle-associated genes were expressed at lower levels in CXCR5+ macrophages than in CXCR5− cells (Supplementary Fig. 8A). Conversely, the expression of immunosuppressive genes, such as Cd274, Arg1, Ctla2b, Mrc1, and Ms4a4a, was increased in CXCR5+ macrophages (Supplementary Fig. 8A). Enrichment analyses revealed that CXCR5+ macrophages were less enriched in myeloid leukocyte activation and the inflammatory response (Supplementary Fig. 8B). We next confirmed whether CXCR5+ M2-like macrophages play an immunosuppressive role and found that CXCR5+ macrophages strongly inhibited CD8+ T-cell proliferation (Figure 5M), TNF, and IFN𝛾 (Figure 5N). Consistent with the findings in CXCR5+ monocytes, PD-L1 expression was greater in CXCR5+ macrophages (Figure 5O). A transfer assay demonstrated that CXCR5+ monocytes differentiated into macrophages that highly expressed CD206 and PD-L1 (Supplementary Fig. 8C). These results demonstrate that CXCR5+ monocytes differentiate into CXCR5+ macrophages with an M2-like phenotype, which inhibiting CD8+ T-cell immunity. In tumor-bearing mice, combination treatment with IR and anti-GM-CSF enhanced tumor control (Figure 5P). This finding suggests that neutralizing GM-CSF is a potential strategy for promoting the efficacy of RT by interrupting CXCR5+ M2-like macrophage differentiation after irradiation.

6. Radiation induced human CXCR5+ monocyte infiltration in tumors
To verify the relevance of our findings to cancer patients, we integrated datasets from the Gene Expression Omnibus. In pancreatic cancer (PDAC) patients, the tumor of patients who received stereotactic radiosurgery (SBRT) or conventional radiotherapy (XRT) presented high expression of Cd14, Cxcr5, and Cd274 (Figure 6A). We performed a systematic analysis of 31 solid tumors from the TCGA database. The results demonstrated that the expression of Cxcr5, Cd14, and immunosuppressive factors was positively correlated with Cxcl13 (Supplementary Fig. 9A). Given these observations, we sought to verify whether irradiated tumors recruit CXCR5+ monocytes. Human PBMCs were treated with TS of human pancreatic and breast cancer cell lines (Figure 6B). After 48 h of treatment, CXCR5 highly expressed in the CD14+ population (Figures 6C and Supplementary Fig. 9B). The Transwell assay showed that the supernatant from irradiated cocultured cells recruited more TS-induced PBMCs, which expressed higher levels of CXCR5 than did the supernatant from the untreated group (Figures 6B and 6D). In vivo, we established a human colonic tumor model in nude mice with HCT116 cells and transferred CTV-labeled THP-1 cells, a human monocyte-like cell line that expresses high levels of CXCR5 (Supplementary Fig. 9C). The results revealed that local tumor irradiation enhanced the infiltration of CTV+CXCR5+ THP-1 cells (Figure 6E).

We next evaluated monocytes in the PB and tumor responses (according to the Response Evaluation Criteria in Solid Tumors, RECIST) of cancer patients and found that monocytes significantly increased in progressive disease (PD) patients after IR (Figures 6F and 6G). However, it did not change in partial response (PR) or stable disease (SD) patients (Figures 6F and 6G). In addition, there was no significant change in the number of blood platelets (PLTs) in the PR, SD, or PD patients before and after irradiation (Supplementary Fig. 9D). Immunohistochemistry showed CXCL13 expression and CXCR5+ monocyte infiltration in the tumors of rectal cancer patients who received radiotherapy (Figures 6H and 6I). High expression of CXCR5 was associated with poor overall survival in TCGA rectum, colon, pancreatic adenocarcinoma, and breast invasive carcinoma cohorts of (Supplementary Fig. 9E).

In consistent with the mouse model data, VEGFR, PI3K, or mTOR inhibitors reversed TS-induced CXCR5 expression on human monocytes (Figure 6J). Correlation analysis of whole blood from the Genotype-Tissue Expression database revealed that CXCR5 expression was positively correlated with Pik3r1, Akt1, Mtor, and Hif1a (Supplementary Fig. 9F). PBMCs from healthy donors showed a baseline level of CXCR5+CD14+, which consistent with the findings of a previous report 44. The proportion of CXCR5+CD14+ cells is increased with human recombinant VEGF-AA treatment (Figure 6K). In whole blood, CXCR5 expression was positively correlated with VEGFA (Supplementary Fig. 9G). PD-L1 was highly expressed on the CXCR5+CD14+ population (Figure 6L) and was positively correlated with whole blood CXCR5 expression (Supplementary Fig. 9H). In cancer patients, PD-L1+ monocytes infiltrated into tumors following radiation treatment (Figure 6M). We next examined the quantity and immunosuppressive function of CXCR5⁺ monocytes in tumors treated with different radiation doses, and fractionation regimens at various time points following IR. Radiation-induced CXCR5⁺ monocyte infiltration was found to be dose-dependent (Supplementary Fig. 9I); however, the radiation dose did not affect CXCR5⁺ monocyte function (Supplementary Fig. 9J). Given that regorafenib reduced the proportion of CXCR5+ monocytes in a mouse model (Figure 2Q), we investigated the therapeutic effect of combination treatment with regorafenib and RT. The results showed that regorafenib enhanced radiation-induced tumor control in the colonic, pancreatic, and breast tumor models (Figures 6N–P). Taken together, these findings indicate that RT induces monocyte infiltration through the CXCR5‒CXCL13 axis, which is correlated with a poor prognosis and is a potential candidate for enhancing the efficacy of RT.

Methods
Human study
All uses of human material and the clinical data of patients and PBMCs obtaining from healthy donors have been approved by the Research Ethics Committee of The First Affiliated Hospital of Xi’an Jiaotong University and complied with all relevant ethical regulations. All recruited volunteers provided written informed consent.

Mice
All animal experiments were performed in accordance with protocols approved by the Xi’an Jiantong University Institutional Animal Care and Use Committee (IACUC). All animals were maintained in pathogen-free conditions and cared for in accordance with the International Association for Assessment and Accreditation of Laboratory Animal Care policies and certification. Mice were euthanized by carbon dioxide (CO₂) asphyxiation at the end point. Wild-type C57BL/6 (N000013), BALB/c (N000020) and BALB/c-nu (D000521) were purchased from GemPharmatech and Laboratory Animal Center of Xi’an Jiaotong University Health Science Center. CD45.1.2 strain was kindly provided by Zhang’s lab71. C57BL/6 CXCR5-/- strain (S-KO-01232) was purchased from Cyagen. The mice aged six to eight weeks were used in the experiments. Experimental and control animals were bred separately. All WT mice were randomly grouped. Given the nature of the transgenic mice, grouping was not random but rather based on their genotypes. However, the age and the weight of the mice was equally distributed.

Tumor growth and treatment
To establish subcutaneous tumor model, 1.5× 106 tumor cells were subcutaneously (s.c.) injected in the right flank of mice. Tumors were measured twice weekly for 3–4 weeks (length × width × depth/2). Mice were pooled and randomly divided into different groups at 10 days after tumor inoculation (tumor volume reached approximately 100 mm3). In accordance with the approved protocol of IACUC, the maximum tumor volume was set at 1,500 mm3. No tumors in this study exceeded this limit, and animals were euthanized upon reaching this endpoint or if any signs of distress were observed. For IR treatment, X-ray radiation (225 keV, dose rate 3.072 Gy/min) was delivered to the tumors using a RS2000pro irradiation system (American). Tumors were irradiated at 10 days post tumor inoculation with one dose of 12 Gy. All irradiated mice were shielded under anesthesia.

For rmVEGF treatment, 175 𝜇 g/kg of rmVEGF (450-32, PeproTech) was injected intraperitoneally to naïve mice every other day for 4 times in total. For anti-VEGF-A treatment, 5 mg/kg InVivoMAb anti-mouse VEGF-A (BE0399, BioXcell) was injected intraperitoneally on the 7 days after tumor inoculation every other day for 4 times in total. For regorafenib treatment experiment, 10 mg/kg regorafenib (HY-10331, MCE) was given by gavage daily starting 3 days before IR for a total of 14 times. For CCR5 and CXCR3 antagonist, 5 mg/kg TAK-220 (HY-19974, MCE) or 15 mg/kg AMG 487 (HY-15319, MCE) was given by gavage on the day of IR and every other day thereafter till endpoint. For CXCL13 neutralization experiment, 2 𝜇 g antibody (MAB470-100, R&D) was injected intratumorally every other day starting on the day of IR for a total of 5 times. For mono-mac depletion, 25 mg/kg Invivo anti-mouse CSF1R Recombinant mAb (S0B1139, STARTER) was injected intratumorally on the day of IR and every other day thereafter till endpoint. For CD8+ T cell depletion experiment, 7.5 mg/kg of antibody (BE0004-1, BioXcell) was injected intraperitoneally every three days starting 1 day before IR for a total of 4 times. For anti-PD-L1 treatment experiment, 7.5 mg/kg antibody (BE0101, BioXcell) was injected intraperitoneally every three days starting on the day of IR for a total of three times. For GM-CSF neutralization experiment, 2 𝜇 g/tumor antibody (A2148, Selleck) was injected intratumorally every other day starting on the day of IR for a total of 6 times.

For rechallenge model, more than 4 weeks after tumor regression, 5 × 105 cancer cells were injected into the left flank. For monocytes transfer experiment, 5 × 106 CXCR5+ or CXCR5- monocytes were injected intravenously every three days starting on the day of IR treatment for a total of 4 times. For THP-1 transfer assay, BALB/c-nu mice were inoculated with HCT116. Ten days after tumor inoculation, tumor received 10 Gy radiation and 5×106 CTV-labeled THP-1 intravenous injection. Tumor were separated 3 days after radiation and analyzed by flow cytometry.

Cell lines
Murine tumor cell lines, HCT116 (SCSP-5076), RKO (SCSP-5236), Mia (SCSP-568), 1806 (SCSP-5019), and SW1990 (SCSP-5302) were purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College. 231 was gift of Jiao’s lab72. All cell lines were grown in Dulbecco’s modified Eagle’s high glucose medium (C11995500BT, Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, 12B108, EXcell), 1% Penicillin/Streptomycin (P/S, G4003, Servicebio), and were maintained in a humidified incubator with 5% CO2 at 37°C. Tumor supernatant was collected after 24 h culture.

Flow cytometry analysis of cell surface and intracellular protein expression
For flow cytometry analysis, tumors, blood, or bone marrow were collected from mice. Tumor tissues were cut into small pieces and incubated with 1 mg/ml collagenase IV (11088866001, Sigma) and 0.2 mg/ml deoxyribonuclease I (D8070, Sigma) for 1 h at 37°C. Samples were then filtered through a 50 μm nylon strainer and washed twice with staining buffer (phosphate-buffered saline supplemented with 2% FBS). Peripheral blood was collected using heparin as the anti-coagulant and lysed by double distilled H2O. Bone marrow from femur was flushed with 6 ml of phosphate-buffered saline (PBS). After centrifugation, the cell pellet was incubated in Red Blood Cell Lysis buffer (r90003, Biosharp) and neutralized by staining buffer. The cells were re-suspended in staining buffer and blocked with anti-FcR (2.4G2, BioXcell). For cell surface staining, the cells were incubated with 200-fold diluted fluorescence-labeled antibodies for 45 min at 4°C in the dark and then detected by flow cytometry with a BD Celesta. For intracellular staining, 3 × 105 cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium (C11875500BT, Gibco) with Cell Stimulation Cocktail (00-4970-93, eBioscience) and Protein Transport Inhibitor Cocktail (00-4980-93, eBioscience) in 96-well plates. Five hours later, cells were harvested and fixed with Fixation Buffer (554722, BD) for 60 min on ice, and then washed twice with diluted Permeabilization Buffer (554723, BD). The appropriate antibodies were added to cells and incubated at 4°C. The antibodies involved in this study are summarized in Supplementary Table 1. Data were analyzed by FlowJo software.

Bone marrow derived monocytes/macrophages culture and treatment
Single-cell suspensions of bone marrow cells were obtained from wild-type or Cxcr5-KO C57BL/6 mice. The cells were cultured in induce medium, RPMI 1640 medium containing 10% FBS (C04002-500, VivaCell) and 20 ng/ml murine recombinant GM-CSF (96-315-03, PeproTech), at day 0. Fresh induce medium was added at day 3 and day 5. The bone marrow (BM)-derived monocytes and macrophages were obtained at day 4 and day 7.

To detect CXCR5 expression on monocytes and macrophages, cells were cultured with 25% tumor supernatant (TS) or treated with murine recombinant protein for 24 h. Monocytes pretreated with inhibitors for 30 min, and then cultured with 25% TS and the inhibitors.

To obtain CXCR5- and CXCR5+ monocytes, BM-derived monocytes were cultured with 50% TS for 48 h. To obtain CXCR5+ macrophages, CXCR5+ monocytes were cultured in induce medium for 3 days. These two subpopulations were separated by fluorescence-activated cell sorting. To track the differentiation of CXCR5+ monocytes, TS-induced monocytes were stained with APC-Cy7 labeled CXCR5. After that, all of monocytes kept cultured in induce media for 72 h. Finally, all cells were collected, stained, and then analyzed by flow cytometry.

Human PBMCs separation and treatment
Peripheral blood was freshly collected in heparin coated tube and then separated by Human Lymphocyte Separation Medium (7111011, Dake we) in accordance with the manufacturer’s instructions. To detect CXCR5 expression on monocytes, PBMCs were cultured with 25% TS or treated with human recombinant protein for 24 h. In transwell migration assay, PBMC and tumor cells were co-cultured for 2 h and then received 10 Gy radiation or not. 48 h later, supernatant was collected. After filtering with 0.22 µM filter, 500 µl supernatant was added into lower chamber. 3×105 TS-treated PBMC in complete RPMI medium were added to the upper chamber. After 1.5 h and 4 h incubating, migrated cells in bottom chamber were collected and counted.

RNA interference and sgRNA-mediated gene deletion
Nonspecific control siRNA and siRNAs for HIF1α, Maf, and eIF4G2 were purchased from Sangon Biotech (China). siRNA transfection of cells was performed with Lipofectamine RNAiMAX Transfection Reagent following the manufacturer's instructions. Single-guide RNAs (sgRNAs) were designed using the Benching online tool (https://benchling.com/) and cloned into the lentiCRISPRv2 (52961). Lentivirus production involved the co-transfection of 293T cells using the lentiCRISPRv2 alongside psPAX2 and pMD2.G packaging plasmids. For effective viral infection, MC38 cell line was treated with the lentivirus amid the presence of 8 μg/mL polybrene, followed by a stringent selection process using 1 μg/ml puromycin for a duration of 4–6 days. Sequences of siRNA and sgRNA oligonucleotides used in this study are summarized in Supplementary Table 2.

T-cell activation assay
Bone marrow derived monocytes and macrophages were performed for the suppression assay. T cells were harvested from the lymph nodes of naïve mice and then stained with CellTrace Violet (C34557, Invitrogen). The CTV-labeled T cells (2 × 105 per well) were cultured in plate-bound 5 µg/ml of anti-CD3 (clone 145-2C11; Thermo Fisher Scientific) and 2 µg/ml of anti-CD28 (Clone 37.51; Thermo Fisher Scientific) with complete RPMI medium containing 5 µM β-mercaptoethano. CXCR5- and CXCR5+ monocytes or macrophages were then added into T cell cultures at a ratio of 1:8 (myeloid cells:T cells) for 72 h. Anti-PD-L1 were added into CXCR5+ monocytes for the concentration of 1 𝜇 g/ml and incubated for 30 min. Incubated CXCR5+ monocytes were then cultured with T cell at a ratio of 1:8 (monocytes:T cells) with 1 𝜇g/ml of anti-PD-L1, 5 µg/ml of anti-CD3 and 2 µg/ml of anti-CD28 as previously mentioned for 72 h. Cells were then harvested and CTV-negative signal in the CD8+ and CD4+ gate was measured by flow cytometry.

Transwell migration assay
We used 24-well transwell plates with 6 mm inserts in polyethylene terephthalate track-etched membranes (Corning). Tumor was precisely weighed (100 ± 0.5 mg), then grinded and centrifuged at 13000G for 15 min. The supernatant was collected and added to bottom chamber. The tumor homogenate culture medium was comprised by 40% supernatant and 60% complete medium of RPMI for 500 μl in total. In ex vivo CXCL13 neutralization assay, the supernatant of homogenate from the same irradiated tumor was divided into two parts, one of which was pretreated with 150 ng/ml anti-CXCL13 for 30 min. After that, the supernatant was added to bottom chamber. The tumor homogenate culture medium was comprised as previously mentioned and 150 ng/ml anti-CXCL13. 3× 105 TS-treated monocytes in complete RPMI medium were added to the upper chamber. After 4 h incubating, migrated cells in bottom chamber were collected, counted, and then analyzed by flow cytometry.

Cytometric Bead Array
For tumor chemokines and cytokines quantifications, tumor tissues were homogenized in PBS with protease inhibitor followed by addition of Triton X-100. The LEGENDplex MU Proinflam Chemokine Panel (Biolegend) and the LEGENDplex Mouse Inflammation Panel (Biolegend) were used for quantitative analysis of CXCL1, CCL5, CCL20, CCL11, CCL17, CCL2, CXCL9, CXCL10, CCL3, CCL4, CXCL13, CXCL5, CCL22, IL-23, IL-1α, IFN-γ, TNF, MCP-1, IL-12p70, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and GM-CSF in accordance with the manufacturer’s instructions.

Western Blot Analysis
Whole-cell protein was extracted with Triton X-100 buffer (150 mM sodium chloride, 50 mM Tris, 1% Triton X-100; pH 8.0) with proteinase inhibitors and phosphatase inhibitors (HY-K0010, HY-K0021, MCE). Immuno-blotting analyses were performed as previously described9. Primary antibodies against the following proteins were utilized in this study: PI3K (A4992, ABclonal), p-PI3K (AP0854, ABclonal), AKT (A18675, ABclonal), p-AKT (AP0637, ABclonal), mTOR (2983T, CST), p-mTOR (5536T, CST), HIF1α (36169T, CST), Maf (A3361, ABclonal), and eIF4G (A2897, ABclonal). Horseradish peroxidase-linked anti-rabbit (78430, Thermofisher) secondary antibody was used. GAPDH mAb (A19056, ABclonal) was used to normalize for the amount of loaded protein.

Immunofluorescence and confocal imaging
Tumor tissues were separated and embedded in Optimal Cutting Temperature Compound. The cyoblock was cut at a thickness of 5 µm and washed with PBS for 10 min. The cryosection was permeabilized with 0.5% Triton X-100 for 20 min and blocked with 10% normal goat serum for 1 h at room temperature. The samples were incubated with PE anti-mouse CD45 (Clone 30-F11; Biolegend), FITC anti-mouse Ly6C (Clone HK1.4; BioLegend), APC/Fire 750 anti-mouse CD185 (Clone L138D7; BioLegend), APC anti-mouse CXCL13 (Clone DS8CX1; Ebioscience). The cryosections were then thoroughly washed with PBS and counterstained with DAPI (D8417, Sigma). Regarding formalin-fixed paraffin-embedded tissue section, the section was deparaffinized and rehydrated before antigen retrieval. After serum blocking (as previously mentioned), the samples were incubated with anti-human CD14 (Clone HCD14; BioLegend), CXCR5 (Clone J252D4; BioLegend), CXCL13 (orb13426, biorbyt), and PD-L1 (66248, proteintech) overnight at 4 °C. The slides were then thoroughly washed with PBS and incubated with secondary antibody at room temperature for 50 min in the dark. DAPI counterstain is same as previously mentioned. Images were acquired on an Olympus FV3000 confocal microscope. Image analysis was performed using Image J.

Hematoxylin-eosin (HE) staining
Tumor tissue sections embedded in paraffin were stained with haematoxylin and eosin (HE) in accordance with the manufacturer's protocol. Following deparaffinisation, the sections were stained with Mayer's haematoxylin. After differentiation using a hydrochloric acid and alcohol solution, followed by ammonia treatment, the sections were stained with eosin.

RT–qPCR analysis
RNA was extracted from the cells using TRIzol (P118-05, GenStar) or RNeasy Mini Kit (74804, QIAGEN), treated with DNase using a TURBO DNA-free Kit (AM1907, Thermofisher) or an RNase-Free DNase Set (11119915001, Sigma), and transcribed into cDNA using the HighCapacity cDNA Reverse Transcription Kit (205311, Qiagen). PCR was performed using SYBR green (RK21203, Yeasen) with specific primers (Supplementary Table 3). Reactions were run on an ABI QuantStudio 5 Real-time PCR System (Thermo Fisher Scientific). The cycling conditions were as follows: (a) 95°C 3 min, 1 cycle; (b) 95°C 5 secs, followed by 60°C 30 secs, 40 cycles. Following the amplification, the Ct values for target genes and the reference gene, GAPDH, were recorded. Fold induction was calculated using the ΔΔCt method.

Bulk RNA-seq analysis
The total RNA was extracted according to the instruction manual of the TRlzol Reagent (Life technologies). RNA concentration and purity was measured using NanoDrop 2000 (Thermo Fisher Scientific). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality. The StringTie Reference Annotation Based Transcript assembly method was used to construct and identify both known and novel transcripts from Hisat2 alignment results. Gene function was annotated based on the KEGG Ortholog database and Gene Ontology. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis of two conditions/groups was performed using the DESeq2. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.01 & Fold Change≥2 found by DESeq2 were assigned as differentially expressed. Differential expression analysis of two samples was performed using the edgeR. The FDR < 0.01 & Fold Change ≥2 was set as the threshold for significantly differential expression.

TCGA data analysis
TCGA data was acquired and analyzed by the Gene Expression Profiling Interactive Analysis Platform. The mRNA expression of tumors was analyzed by the Multiple Gene Comparison module. The overall survival of rectum adenocarcinoma, colon adenocarcinoma, pancreatic adenocarcinoma, and breast invasive carcinoma was analyzed by the Kaplan-Meier estimator and tested by the log rank using the survival module.

Statistics and Reproducibility
Analyses were performed using GraphPad Prism Version 9.0.0. Data were analyzed by one-way ANOVA with Tukey’s Multiple Comparison Test or Student’s t test. All statistical tests were two-sided. P values < 0.05 were considered statistically significant (ns > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). The in vitro experiments have been repeated at least twice. The in vivo experiments have been performed in at least two to three independent batches or have at a minimum 5-10 independent biological replicates for reproducibility. For Figure 6H, 6I, and 6M, the experiments have been performed in 3 individuals and repeated twice.

Data availability
The mRNA-seq data generated in this study have been deposited in the SRA database under the [PRJNA1420777] [https://www.ncbi.nlm.nih.gov/sra/PRJNA1420777]. All data are included in the Supplementary Information or available from the authors, as are unique reagents used in this Article. The raw numbers for charts and graphs are available in the Source Data file whenever possible.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors demonstrated that Ly6Chigh monocytes/macrophages infiltrate tumors and abrogate anti-cancer immune responses following radiation therapy. Radiation induced CXCL13 expression and the accumulation of CXCR5-positive myeloid cells in mouse subcutaneous tumor models. VEGF was suggested to induce CXCR5 expression. Anti-CXCL13 treatment inhibited the recruitment of CXCR5-positive cells after irradiation.\nComments:\n#1:\nMonocyte/macrophage counts typically vary depending on tumor size and the extent of necrosis. Therefore, the authors should include control data for Figure 1, comparing untreated tumor nodules of similar size to those in the irradiated group.\n#2:\nThe CXCL13-positive signal in Figure 1E appears to resemble background staining. Have you checked the specificity of anti-CXCL13 antibody by yourselves? In situ hybridization is recommended to confirm CXCL13 expression more reliably.\n#3:\nCCL3, CCL4, and CCL5 are known ligands for CCR5 (not CXCR5) and are significantly upregulated following radiation, as shown in Figure 1D. CCR5 seems to be more important for protumor mechanisms in radiation therapy more than CXCR5. Please describe this point in the discussion.\n#4:\nLines 132–134:\nThe authors state that CXCL13 is associated with poor prognosis in cancer. However, a recent study (PMID: 39696312) demonstrated that CXCL13 production by T cells promotes TLS formation and correlates with better prognosis in colorectal cancer. Moreover, many studies in the PubMed database report that increased CXCL13 expression is associated with favorable outcomes in several cancer types. This contradiction should be discussed.\n#5:\nThe percentage of Ly6Chigh cells among CD11b-positive cells is less than 5% in Figure 1B. This suggests that the majority of CD11b-positive, Ly6Clow/negative cells may consist of myeloid-derived suppressor cells (MDSCs). The authors describe an increase in CXCR5-positive cells after irradiation; however, the proportion appears quite low in Figures 1H–1J. The authors should evaluate the presence of MDSCs in Figure 1 to strengthen their analysis.\n#6:\nRegorafenib is a multikinase inhibitor and is not specific to VEGFR. Therefore, the authors should additionally utilize recombinant VEGF protein and anti-VEGF antibodies to more specifically assess the role of VEGF in inducing CXCR5 expression. Several soluble factors could contribute to CXCR5 induction. Furthermore, VEGF knockout tumors likely have significantly reduced tumor size compared to wild-type tumors. The authors should clarify whether tumor sizes were matched in the analyses shown in Figures 2O and 2P.\n#7:\nIn Figure 3, the authors added tumor tissue homogenates to the culture medium. Given that homogenates contain abundant DAMPs (damage-associated molecular patterns), it remains unclear whether this method is standard in the field. The authors should justify and discuss the validity of this experimental approach.\n#8:\nAs shown in Figure 5G, it is difficult to clearly distinguish M1 and M2 macrophage populations. The M1/M2 classification has become less favored in recent literature. Since multiple markers have been proposed to assess macrophage polarization, the authors should incorporate additional markers to provide a more comprehensive evaluation of the M1/M2 balance.\n#9:\nDirect evidence from human tumor tissues treated with radiation should be included in Figure 6. Immunohistochemistry or in situ hybridization assays are recommended to strengthen the clinical relevance of the findings.", "reviewer_2": "CXCR5⁺ monocyte emigration impairs the radiation-induced antitumor immune response\nby Yutiantian Lei et al.\nSummary\nThis manuscript presents an original and comprehensive investigation into how a subset of CXCR5⁺ monocytes, mobilized following radiotherapy (RT), impairs the antitumor immune response. The study identifies a novel VEGF–PI3K–HIF1α–CXCR5 axis in monocytes and a tumor-derived CXCL13–CXCR5 chemotactic loop that facilitates the recruitment of immunosuppressive monocytes post-RT. These monocytes express PD-L1 and differentiate into macrophages with a suppressive M2-like phenotype, ultimately dampening CD8⁺ T-cell responses. The authors further explore combinatorial strategies to block this axis and enhance RT efficacy.\nStrengths\nOriginality: The identification of CXCR5⁺ monocytes as regulators of post-RT immune suppression is novel and timely.\nScientific depth: The manuscript combines transcriptomic analysis, mouse models, clinical samples, and ex vivo validation in a robust and multidisciplinary framework.\nClinical relevance: The study’s findings have clear translational implications, suggesting new avenues for improving RT efficacy through combinatorial immunotherapy.\nMajor Comments\nConsideration of Other Cytokines and Chemokines:\nWhile the focus on the CXCL13–CXCR5 axis is well justified, the tumor microenvironment produces a variety of cytokines and chemokines known to affect myelopoiesis and monocyte trafficking. In particular, CCL2, a key monocyte chemoattractant, should be evaluated.\nThe authors should experimentally assess or at least discuss the role of CCL2 and other cytokines such as G-CSF, IL-6, or CXCL12, in shaping the monocyte response following RT.\nThis would clarify whether CXCR5⁺ monocytes represent a unique immunosuppressive pathway or part of a broader myeloid reprogramming post-irradiation.\nImpact of Radiation Dose, Fractionation, and Kinetics:\nThe current study appears to use a single RT dose and schedule. However, in clinical practice, RT regimens vary widely.\nIt would be important to investigate how different radiation doses, fractionation schemes, and time kinetics (early vs. late effects post-RT) affect the induction and function of CXCR5⁺ monocytes.\nThis could help determine whether this suppressive pathway is dose-dependent or universally activated, thereby influencing its clinical relevance and therapeutic targeting.\nHuman Relevance and Clinical Data:\nThe translational value of the study would be enhanced by a more detailed characterization of the clinical cohorts. The methodology for PBMC isolation, timing post-RT, and correlation with clinical outcomes (e.g., RECIST responses) should be clarified.\nThe authors should also acknowledge the limitations of extrapolating murine immune responses to human biology in the discussion.\nFigure Clarity and Model Schematic:\nA comprehensive model summarizing the proposed mechanism should be placed more prominently, ideally in the main figures or at the end of the discussion.\nSome figures are dense; simplification or rearrangement could improve clarity, particularly for multiparametric flow cytometry data.\nMinor Comments\nThe introduction could be streamlined to better emphasize the novelty of the CXCL13–CXCR5 axis in the context of radiation response.\nSome figure legends would benefit from more methodological detail, particularly for non-standard assays.\nMinor typographical issues are present but do not hinder comprehension.\nThis is a strong, well-executed study that advances our understanding of radiation-induced immune modulation. Addressing the potential roles of other cytokines (notably CCL2), exploring variable RT parameters (dose, fractionation, timing), and clarifying human data will enhance the translational robustness of the work. Last, the focuss on regorafenib, a commercial anti antiangiogenic agent could be questionned and I would rather refer to and VEGF agents (and validate also this in vitro).", "reviewer_3": "This manuscript presents a series of experiments investigating the effects of radiation therapy (RT) in subcutaneous tumor models of colon carcinoma, pancreatic cancer, and mammary tumors. Three days post-irradiation, the authors report an accumulation of monocytes at the tumor site. Notably, they observe elevated levels of CXCL13 following irradiation, which led them to explore CXCR5 expression on monocytes. They find that these CXCR5⁺ monocytes impair CD8⁺ T-cell-mediated antitumor immunity via the PD-1/PD-L1 axis. Mechanistically, the VEGF–PI3K/Akt/mTOR–HIF1α signaling pathway is implicated in the upregulation of CXCR5 on monocytes. The study further demonstrates that these monocytes differentiate into immunosuppressive CXCR5⁺ M2-like macrophages, and that their depletion or functional inhibition (e.g., via CXCL13 neutralization or VEGFR blockade) significantly enhances tumor control.\nThis work addresses an important issue in oncology—the immunosuppressive effects of RT and the cellular mechanisms contributing to radioresistance. However, several key concerns need to be addressed to support the author conclusions:\nThe focus on CXCL13 appears somewhat narrow, especially given the upregulation of multiple chemokines post-irradiation (as shown in Figure 1B). The authors should justify their selection and provide a broader analysis of immune cell dynamics following RT, including the involvement of cell types beyond monocytes.\nThe phenotypic and functional profile of CXCR5⁺ monocytes is insufficiently detailed. It is not clear how this population differs from other monocyte subsets or from M-MDSCs. Additionally, previous reports have shown CXCR5 expression on intermediate human monocytes; the authors should reconcile these findings with their own and discuss the expression of other proteins in the CXCR5⁺ population.\nAlthough RNA sequencing was performed, only a limited number of genes are shown (Figure 1). A more comprehensive analysis of differentially expressed genes would enhance the mechanistic depth of the study. Furthermore, there is no mention of public deposition of RNA-seq data, which is necessary for reproducibility.\nThe manuscript lacks critical methodological details regarding tumor implantation and treatment protocols, including: number of cells injected per tumor model, timing and size of tumors at treatment initiation, specific timepoints for control and treated groups, concentrations and durations of inhibitor and antibody treatments. Without this information, reproducibility is compromised.\nImportantly, the potential systemic or off-target immune consequences of targeting the CXCL13–CXCR5 axis—particularly in the context of chronic infections or autoimmune disease—are not discussed. Arethe treatments affecting B cells that are the main CXCR5 expressing cells?"}, "author_response": "Q1:\nMonocyte/macrophage counts typically vary depending on tumor size and the extent of necrosis. Therefore, the\nauthors should include control data for Figure 1, comparing untreated tumor nodules of similar size to those in the\nirradiated group.\nWe appreciate the comments and helpful suggestions. We have added the tumor sizes of each sample for the\ndifferent groups. There is no remarkable difference in tumor volume between the untreated and irradiated groups at\nthe indicated time (see below, current Figure S1D), while an increase in monocytes was observed. In addition,\nnecrosis is rarely observed at this time point (see below, current Figure S1C). The finding that neutralizing CXCL13\ndecreases radiation-induced monocytes and macrophage tumor infiltration (current Figures 3H) suggests that the\nchanges in monocyte and macrophage counts post-irradiation mainly result from enhanced CXCL13.\nUpper: Tumor volume of mice with MC38, PanC02, and 4T1 tumor inoculation at the indicated time. B6 mice were\ninoculated s.c. with 1 106 MC38 cells or PanC02 cells. BALB/c mice were inoculated s.c. with 1 106 4T1 cells.\nTen days post inoculation, the tumors received one dose of 12 Gy ironizing radiation (IR). Three and seven days\nafter IR, the tumors w ×er e measured, and the tumor volume was calculated from the results (lengt h× width\ndepth/2).\n× ×\nBottom: Hematoxylin–eosin (HE) staining of tumor tissue. Mice were inoculated with MC38 and received IR as\ndescribed above, and the tumors were separated at 3 days post-IR.\nQ2:\nThe CXCL13-positive signal in Figure 1E appears to resemble background staining. Have you checked the specificity\nof anti-CXCL13 antibody by yourselves? In situ hybridization is recommended to confirm CXCL13 expression more\nreliably.\nThank you for these questions. We stained irradiated WT and Cxcl13 KO tumors with the anti-CXCL13 antibody to\nassess its specificity.The IR-treated tumor showedenhanced CXCL13 expression, whereas the Cxcl13 KO tumor\nshowed an absence of the CXCL13 signal (current Figure S3G). We agree that in situ hybridizationwould be a more\nreliable method for confirming CXCL13 expression; however, the reagents and kits required for this experiment have\na delivery time of 3–4 months. Therefore, it is challenging for us to complete this experiment within the given time\nframe. We refined the experimental procedure to prevent background staining by implementing more stringent\nblocking strategies and reducing the concentration of direct fluorescent antibody. The results of the replicated\nexperiment using the optimized protocol are presented in Figure 1E (also see below).\nRepresentative immunofluorescence (IF) staining of CXCL13 in tumors with or without radiation. CXCL13 (magenta);\nnuclear (blue). Mice were inoculated with MC38 and received IR as in Q1, and tumors were removed at 3 days post-\nIR.\nQ3:\nCCL3, CCL4, and CCL5 are known ligands for CCR5 (not CXCR5) and are significantly upregulated following\nradiation, as shown in Figure 1D. CCR5 seems to be more important for protumor mechanisms in radiation therapy\nmore than CXCR5. Please describe this point in the discussion.\nWe appreciate this comment. To determine whether radiation-induced monocyte tumor infiltration occurs through the\nCCL3/CCL4/CCL5–CCR5 axis, we treated mice with TAK-220 (a selective CCR5 antagonist that blocks CCR5-\nmediated migration) from the day of IR treatment and analyzed tumor-infiltrating monocytes/macrophages post-IR.\nTAK-220 did not decrease radiation-induced monocyte or macrophage infiltration (see below). In contrast,\nneutralizing CXCL13 reversed the increase in monocytes and macrophages post-IR, consistent with\nmonocyte/macrophage depletion via anti-mouse CSF1R. Collectively, these data suggested that the CXCL13–\nCXCR5 axis is more important for monocyte/macrophage tumor infiltration in radiation therapy. As suggested, we\nhave added these data to the results section (Figure S3A).\nThe proportion of monocytes (Ly6ChighLy6Glow) and macrophages (F4/80+) in the tumors. Mice were inoculated with\nMC38 and received IR as in Q1. Other treatments (CCR5 antagonist, i.g., 5 mg/kg; anti-CXCL13, i.t., 2 g/tumor;\nanti-CSF1R, i.p., 350 g/20 mg) were administered on the day of IR and every other day. Tumors were removed and\nanalyzed 3 days post-IR. 𝜇\n𝜇\nQ4:\nLines 132–134: The authors state that CXCL13 is associated with poor prognosis in cancer. However, a recent study\n(PMID: 39696312) demonstrated that CXCL13 production by T cells promotes TLS formation and correlates with\nbetter prognosis in colorectal cancer. Moreover, many studies in the PubMed database report that increased\nCXCL13 expression is associated with favorable outcomes in several cancer types. This contradiction should be\ndiscussed.\nWe agree with the comments that CXCL13+ T cells show antitumor effects. The cited study (PMID: 39696312)\ndemonstrated that CD8+ tissue-resident memory T (TRM) cells secrete higher levels of CXCL13 and recruit B cells\ninto tumor tissues through the CXCL13/CXCR5 signaling axis, thereby forming tertiary lymphoid structures and\nparticipating in antitumor immune responses. In addition, studies have demonstrated that CXCL13+ T cells exhibit\nenhanced antitumor properties within liver metastases (PMID: 37327339). However, irradiated tumors may differ.\nRadiation can induce the reduction or even depletion of tertiary lymphoid structures in tumors (PMID: 30038899,\nPMID: 34862242). The role of CXCL13 is more complex in the IR condition. By acting on different cells, CXCL13\nexhibits dual functional activity, including both anti- and protumor effects. Our findings indicate that IR promotes the\ninfiltration of immunosuppressive CXCR5+ monocytes via CXCL13 and induces a protumor immune response, thus\nelucidating a mechanism underlying the protumor effect of CXCL13 in radiotherapy. We cite the references\nmentioned above in the discussion section (line 447).\nThe association between CXCL13 expression and patient prognosis varies across different tumors. This study\nfocuses specifically on the impact of CXCL13 on radioresistance. Given the wide application of radiotherapy in\ncolorectal, breast, and pancreatic cancers, our survival analysis incorporated cohorts with these malignancies from\nthe TCGA and mechanistic investigations using corresponding animal tumor models.\nQ5:\nThe percentage of Ly6Chigh cells among CD11b-positive cells is less than 5% in Figure 1B. This suggests that the\nmajority of CD11b-positive, Ly6Clow/negative cells may consist of myeloid-derived suppressor cells (MDSCs). The\nauthors describe an increase in CXCR5-positive cells after irradiation; however, the proportion appears quite low in\nFigures 1H–1J. The authors should evaluate the presence of MDSCs in Figure 1 to strengthen their analysis.\nThe main population of MDSCs varies across different tumor types. In MC38, Ly6Chigh cells and PMN-MDSC\n(Ly6ClowLy6Ghigh cells) increase post-IR. In PanC02 and 4T1, PMN-MDSC predominates in the tumor immune\nmicroenvironment; however, this population was not induced by IR. Ly6Chigh cells are consistently increased in\nirradiated MC38, PanC02, and 4T1 tumors (see below, current Figure S1B). Therefore, we focused on Ly6Chigh cells\nand conducted further investigations into their role in the radiation-induced immune response.\nThe proportion of monocytes (Ly6Chigh) and neutrophils (Ly6GhighLy6Clow). Mice were treated as in Q1. Tumors were\nremoved and analyzed at 3 and 7 days post-IR.\nQ6:\nRegorafenib is a multikinase inhibitor and is not specific to VEGFR. Therefore, the authors should additionally utilize\nrecombinant VEGF protein and anti-VEGF antibodies to more specifically assess the role of VEGF in inducing\nCXCR5 expression. Several soluble factors could contribute to CXCR5 induction. Furthermore, VEGF knockout\ntumors likely have significantly reduced tumor size compared to wild-type tumors. The authors should clarify whether\ntumor sizes were matched in the analyses shown in Figures 2O and 2P.\nThank you for your comment and suggestion, which improves the rigor of our study. We employed recombinant\nmouse VEGF (rmVEGF), which induced CXCR5 expression on monocytes in vitro (Figure 2J).In addition,\nneutralizing VEGF with anti-VEGF-A reduced tumor supernatant-induced CXCR5 expression on monocytes\n(see below, current Figure 2L).\nCXCR5 expression on monocytes. Monocytes were cultured with 25% tumor supernatant (TS) with or without 2\ng/ml anti-VEGF-A for 24 h. TS was collected 24 h after radiation and filtered with 0.44 filter.\n𝜇Next, we verified these results in vivo. Anti-VEGF-A reduced CXCR5 expression on monocytes in the tumor,\nperipheral blood, and bone marrow. rmVEGFA induced CXCR5 expression on monocytes from the peripheral blood\nand bone marrow of naïve mice (see below, current Figure 2O).\nCXCR5 expression on monocytes. Mice were inoculated with MC38 as in Q1. At 7 days post inoculation, anti-VEGFA\nwas administered via i.p. injection, 100 g/20 mg, and then every other day until 15 days post inoculation. Naïve\nmice were treated with 3.5 μg/20mg rmVEGFA via i.p. injection every other day from 8 days before the endpoint.\n𝜇\nIn the IR condition, anti-VEGF-A reduced tumor-infiltrating CXCR5+ monocytes (see below, current Figure 3F).\nThe proportion of CXCR5+ monocytes in tumor tissue. Mice were inoculated with MC38 as in Q1. Anti-VEGFA was\nadministrated same as above. Ten days post inoculation, tumors were treated with 12 Gy radiation. Three days post\nIR, tumors were separated and analyzed.\nWe analyzed the tumor size at the time point when we determined monocyte CXCR5 expression (current Figures\nS2T) and found that the WT and Vegfa KO tumor sizes were comparable (see below, left). In Figure 4C (also see\nbelow, right), it is shown that the WT and Vegfa KO tumor sizes were comparable from day 10 to day 21 post-tumor\ninoculation, and that the Vegfa KO tumor showed a reduced size only at the endpoint (day 24 post-tumor\ninoculation). These data suggest that tumor-induced CXCR5 expression is attributable to VEGF signaling, not tumor\nsize.\nTumor volume of mice with WT and Vegfa KO MC38 at the endpoint.\nTumor growth of mice with WT and Vegfa KO MC38 tumor with or without IR.\nQ7:\nIn Figure 3, the authors added tumor tissue homogenates to the culture medium. Given that homogenates contain\nabundant DAMPs (damage-associated molecular patterns), it remains unclear whether this method is standard in the\nfield. The authors should justify and discuss the validity of this experimental approach.\nWe did not exclude the function of DAMPs. In this study, we focus on CXCL13 and intervene it. We demonstrate that\nCXCL13 plays an important role in radiation-induced CXCR5+ monocyte recruitment. As validated in current Figures\nS3C and S3D, homogenate from the same irradiated tumor were divided into two parts, one of which was\nsupplemented with anti-CXCL13. The paired comparisons confirmed that CXCL13 neutralization reduced monocyte\nmigration to homogenates of IR-treated tumors. This result is consistent with CXCL13 neutralization in vivo. In\naddition, a commonly used method for homogenate preparation was applied to tumors across all groups.\nHomogenate supernatant was separated and employed as described in previous studies (PMID: 28967875, PMID:\n39773263, PMID: 23169301). We have detailed this process in the methods section (line 618).\nQ8:\nAs shown in Figure 5G, it is difficult to clearly distinguish M1 and M2 macrophage populations. The M1/M2\nclassification has become less favored in recent literature. Since multiple markers have been proposed to assess\nmacrophage polarization, the authors should incorporate additional markers to provide a more comprehensive\nevaluation of the M1/M2 balance.\nWe agree with the reviewer that in earlier studies, macrophages were identified as M1 and M2 subtypes, while\nadvances in sequencing technologies enabled more elaborate characterization of macrophage. To further\ncharacterize the polarization of CXCR5+ monocytes derived macrophage in detail, we analyzed gene expression\nbetween CXCR5- and CXCR5+ macrophages across functional modules based on recent reports (PMID: 40215981,\n38057662). The result revealed that antigen presentation-associated genes and cell cycle-associated genes were\nexpressed at lower levels in CXCR5+ macrophages. Conversely, the expression of immunosuppressive genes such\nas Cd274, Arg1, and Ctla2b were increased in CXCR5+ macrophages (see below, current Figure S5H). Moreover,\nwe prefer to characterize CXCR5+ macrophages as M2-like macrophages due to its high expression of CD206, a\ncanonical M2 markers, and its immunosuppressive function. We have revised the relevant descriptions in the\nmanuscript.\nGene expression of CXCR5- and CXCR5+ macrophages. These two populations were separated by fluorescence-\nactivated cell sorting.\nQ9:\nDirect evidence from human tumor tissues treated with radiation should be included in Figure 6.\nImmunohistochemistry or in situ hybridization assays are recommended to strengthen the clinical relevance of the\nfindings.\nWe appreciate this comment. We collected tissue sections from rectal cancer patients who received radiotherapy.\nRegrettably, tissue sections before treatment were not available due to institutional policy restrictions. To validate our\nfindings in patients, we examined CXCL13 expression, CXCR5+ monocyte infiltration, and PD-L1 expression on\nmonocytes in tumor tissues from patients who received radiotherapy. We have added these results to current\nFigures 6H, 6I and 6M.\nIF staining of tumor tissuefrom rectal cancer patients who received radiotherapy. Upper: CXCL13 (red), nuclear\n(blue); middle: CD14 (green), CXCR5 (red), nuclear (blue); bottom: CD14 (green), PD-L1 (red), nuclear (blue).\nReviewer #2:\nMajor Comments\nConsideration of Other Cytokines and Chemokines:\nWhile the focus on the CXCL13–CXCR5 axis is well justified, the tumor microenvironment produces a variety of\ncytokines and chemokines known to affect myelopoiesis and monocyte trafficking. In particular, CCL2, a key\nmonocyte chemoattractant, should be evaluated.\nThe authors should experimentally assess or at least discuss the role of CCL2 and other cytokines such as G-CSF,\nIL-6, or CXCL12, in shaping the monocyte response following RT.\nThis would clarify whether CXCR5 monocytes represent a unique immunosuppressive pathway or part of a broader\n+\nmyeloid reprogramming post-irradiation.\nWe appreciate these comments. In our previous study (PMID: 29170400), we reported that the anti-CCR2\nmonoclonal antibody decreased the accumulation of the CCR2+Ly6Chi population and that CCR2 blockade in\nestablished tumors reversed radiation resistance, achieving greater antitumor efficacy. The distinctions between the\npresent and previous study are as follows:\n1. Different receptor expression patterns: CCR2 is broadly expressed on monocytes in both naïve and tumor-bearing\nmice, whereas CXCR5 expression on monocytes is tumor-mediated.\n2. Divergent regulating mechanisms of ligand expression: CCL2 is up-regulated by STING–IFN I signaling and\nproduced mainly in both tumor cells and myeloid cells. Meanwhile, CXCL13 is primarily derived from tumor cells\nrather than myeloid cells and is induced by radiation-mediated crosstalk between tumor cells and macrophages.\n3. Functional difference in immune modulation: Our previous study demonstrated that the STING–IFN I pathway\nenhanced immunosuppressive response in tumors by recruiting myeloid cells, in part via the CCR2 pathway. This\nfinding emphasized that monocytic (M-)MDSCs (CD11b+Ly6ChiLy6G-) mediate the immunosuppressive response as\na unit, and the CCL2–CCR2 axis induces their mobilization. In current study, we conducted a more precise\nidentification of a group of monocytes characterized by the expression of CXCR5. We found that CXCR5+\nmonocytes, which highly express PD-L1, exhibited stronger immunosuppressive capacity than CXCR5-\nmonocytes. In addition, our current data demonstrate that CXCR5+ monocytes can further differentiate into\nCXCR5+ macrophages (M2-like), subsequently perpetuating immune suppression.\nWe add this information in discussion section (line 431).\nRegarding CXCL12, we examined the expression of CXCR2, the CXCL12 receptor, on monocytes. Radiation did not\nchange CXCR2+ monocyte tumor infiltration (see below, current Figure S3B). Therefore, we consider that IR may not\npromote CXCR2+ monocyte mobilization and that CXCL12 is not the key mediator in monocyte migration post-IR.\nThe proportion of CXCR2+ monocytes in tumor tissue. B6 mice were inoculated s.c. with 1 106 MC38 cells. Ten days\npost inoculation, tumors received one dose of 12 Gy IR. Three days post IR, tumors were separated and analyzed.\n×\nIL-6 is thought to exhibit dual functions in antitumor immunity. To clarify the role of IL-6 in monocyte migration after\nradiation, we treated tumor-bearing mice with an IL-6 neutralizing antibody post-IR. The results showed that anti-IL-6\ndid not reverse the increase in monocytes or macrophages, whereas CXCL13 neutralization significantly reduced the\nnumber of tumor-infiltrating monocytes and macrophages (see below).\nThe proportions of monocytes (Ly6ChighLy6Glow) and macrophages (F4/80+) in tumors. Mice were inoculated with\nMC38 and received IR as mentioned before. Other treatments (anti-CSF1R, i.p., 350 g/20 mg; anti-CXCL13, i.t., 2\ng/tumor) were administered on the day of IR and every other day thereafter. Tumors were removed and analyzed\n3 days post-IR. 𝜇\n𝜇\nDue to the long delivery time required for the in vivo administration of G-CSF-neutralizing antibodies, it is challenging\nfor us to conduct IL-6 neutralization experiments within the limited time frame. It is generally accepted that G-CSF\nregulates neutrophil production within the bone marrow and affects neutrophil progenitor proliferation. In our study,\nwe observed an elevated proportion of neutrophils following radiation (see below). Thus, we hypothesized that the\nenhanced G-CSF might contribute to an expanded population of neutrophils. Studies have demonstrated that G-CSF\ncan increase the population of monocytes in peripheral blood (PMID: 16735597), explaining the mechanism by which\nG-CSF treats clinical myelosuppression. Furthermore, G-CSF may partially contribute to monocyte differentiation and\nmacrophage polarization (PMID: 30683906).\nThe proportion of neutrophils (Ly6ChighLy6Glow) in tumor. Mice were inoculated with MC38 and received IR same as\nmentioned before. Tumors were separated and analyzed at 3 days and 7 days post IR.\nCollectively, our findings indicate that it is CXCR5+monocytes and other myeloid cells, including CCR2+monocytes,\nthat collaboratively induce protumor immunity in radiation and the radioresistance of solid tumors, as opposed to a\nunique immunosuppressive pathway.\nImpact of Radiation Dose, Fractionation, and Kinetics:\nThe current study appears to use a single RT dose and schedule. However, in clinical practice, RT regimens vary\nwidely.\nIt would be important to investigate how different radiation doses, fractionation schemes, and time kinetics (early vs.\nlate effects post-RT) affect the induction and function of CXCR5 monocytes.\n+\nThis could help determine whether this suppressive pathway is dose-dependent or universally activated, thereby\ninfluencing its clinical relevance and therapeutic targeting.\nWe appreciate this comment, which improves the clinical relevance of this study. We treated tumors with different\ndoses and fractionations, then examined CXCR5+ monocytes at different time points post-IR.\nDuring the early phase (day 3 post-IR), single low-dose IR had a minimal impact on monocyte infiltration, whereas\nsingle high-dose radiation led to a dose-dependent increase in CXCR5+ monocyte infiltration. Fractionated radiation\nresulted in increased CXCR5+monocyte infiltration compared with that observed with single low-dose irradiation;\nhowever, this infiltration remained lower than that observed with high-dose irradiation (see below, current Figure\nS6I).\nBy the intermediate phase (day 7 post-radiation), CXCR5+ monocytes did not change in the single-dose IR group.\nThe data shown in Figure 5 indicate that the CXCR5+ monocytes differentiated into CXCR5+macrophages. In\naddition, fractionated radiation decreased the percentage of CXCR5+ monocytes in the tumor (see below, current\nFigure S6I).\nIn the late phase (day 14 post-radiation), all groups exhibited reduced CXCR5+ monocyte infiltration compared with\nthat observed in the early and intermediate phases, although infiltration was increased in all irradiated groups\ncompared with that in the untreated group. At this stage, the tumor volume and microenvironment had undergone\nsignificant changes, which could have impacted the presence of CXCR5+ monocytes (see below, current\nFigure S6I).\nWe administered a single dose of radiation at 4Gy, 8Gy, or 12Gy or fractionated radiation (4Gy × 3 fractions) to\ntumor-bearing mice at 10 days post tumor inoculation.\nTo investigate how different radiation doses affect the induction and function of CXCR5 monocytes, we analyzed the\n+\nimmunosuppressive function of CXCR5 monocytes in tumors treated with different radiation doses. We found that\n+\nCXCR5 monocytes exhibited a stronger suppressive effect on T cells than did CXCR5− monocytes, regardless of the\n+\nradiation dose administered to the tumor (see below, current Figure S6J).\nT cell activation assay. T cells were cultured with monoclonal anti-CD3/anti-CD28 antibodies and were co-cultured\nwith CXCR5- or CXCR5+ monocytes at a ratio of 8:1. The acquisition of these two populations were separated by\nfluorescence-activated cell sorting from irradiated tumor 3 days post 8 Gy and 12 Gy radiation.\nThus, radiation-induced CXCR5 monocyte infiltration is dose-dependent, whereas the radiation dose did not affect\n+\nCXCR5+monocyte immunosuppressive function. In addition, fractionated IR induced CXCR5 monocyte infiltration,\n+\nalthough the induction was not strong as that observed with high-dose IR. These data suggest that the induction of\nimmunosuppressive CXCR5 monocytes post radiotherapy is a universal effect.\n+\nHuman Relevance and Clinical Data:\nThe translational value of the study would be enhanced by a more detailed characterization of the clinical cohorts.\nThe methodology for PBMC isolation, timing post-RT, and correlation with clinical outcomes (e.g., RECIST\nresponses) should be clarified.\nThe authors should also acknowledge the limitations of extrapolating murine immune responses to human biology in\nthe discussion.\nWehave modified the methods for PBMC isolation. Peripheral blood of patients was tested within a 7-day window\nbefore and after the radiotherapy initiation. All patients were assessed according to RECIST. We added this\ninformation in the figure legend and supplement detailed patients’ information in Supplementary Table 4.\nWe agreed with reviewer that mouse models and humans have considerable differences in regulation such as the\nproportion of leukocyte subsets, T cell costimulation and responses to antibody. Efficient interventions in mouse\nmodels require further validation in humanized animal models and clinical studies to confirm their efficacy in cancer\npatients. We added the limitations to the discussion.\nFigure Clarity and Model Schematic:\nA comprehensive model summarizing the proposed mechanism should be placed more prominently, ideally in the\nmain figures or at the end of the discussion.\nSome figures are dense; simplification or rearrangement could improve clarity, particularly for multiparametric flow\ncytometry data.\nWe modified the manuscript, including simplifying the flow cytometry data in both main and supplementary figures to\nimprove layout clarity and moving the graphical abstract to Figure 7.\nMinor Comments\nThe introduction could be streamlined to better emphasize the novelty of the CXCL13–CXCR5 axis in the context of\nradiation response.\nWe have modified the manuscript accordingly.\nSome figure legends would benefit from more methodological detail, particularly for non-standard assays.\nWe appreciate the comment and detailed methods.\nMinor typographical issues are present but do not hinder comprehension.\nWe appreciate this comment and have made the necessary corrections.\nReviewer #3:\nThe focus on CXCL13 appears somewhat narrow, especially given the upregulation of multiple chemokines post-\nirradiation (as shown in Figure 1B). The authors should justify their selection and provide a broader analysis of\nimmune cell dynamics following RT, including the involvement of cell types beyond monocytes.\nWe appreciate the comment and suggestion, which improves the quality of our study. We observed that, as shown in\nthe heatmap in Figure 1D, CCL2, CCL4, CCL5, CXCL1, CXCL10, and CXCL13 were enhanced post-IR. CCL4 and\nCCL5 are the ligands of CCR5. CXCL10 is the ligand of CXCR3. TAK-220 is a selective CCR5 antagonist that\ninhibits the binding of chemokines to CCR5. AMG 487 is a selective antagonist of CXCR3 that inhibits the binding of\nCXCL10 to CXCR3. To verify whether radiation-induced monocyte tumor infiltration occurs through the CCL4/CCL5–\nCCR5 axis or the CXCL10–CXCR3 axis, we treated mice with these two antagonists or neutralized CXCL13 on the\nday of IR treatment. Subsequently, we analyzed tumor-infiltrated monocytes 3 days after radiation. The results\nshowed that antagonizing ligands of CCR5 or the CXCL10–CXCR3 axis did not reduce monocyte migration to the\nirradiated tumor. However, neutralizing CXCL13 in the tumor reversed the increase in monocytes and macrophages\npost-IR (sew below, current Figure S3A). Moreover, the efficiency of this neutralization is comparable to whole body\nmonocyte/macrophage depletion (in vivo anti-mouse CSF1R). Collectively, we propose that the CXCL13–CXCR5\naxis is more important for protumor mechanisms in radiation therapy.\nThe proportion of monocytes (Ly6ChighLy6Glow) and macrophages (F4/80+) in tumor. B6 mice were inoculated s.c.\nwith 1 106 MC38 cells. Ten days post inoculation, tumors received one dose of 12 Gy IR. Three days post IR,\ntumors were separated and analyzed. Other treatments (CCR5 antagonist, i.g., 5 mg/kg; CXCR3 antagonist, i.g., 5\nmg/kg×; anti-CXCL13, i.t., 2 g/tumor; anti-CSF1R, i.p., 350 g/20 mg) were administered at the day of IR, every\nother day. Tumors were separated and analyzed 3 days post IR.\n𝜇 𝜇\nRegarding CXCL12, we examined monocytes expressing its receptor, CXCR2. Radiation did not enhance CXCR2+\nmonocyte tumor infiltration. Therefore, we consider that IR may not promote CXCR2+ monocyte mobilization and that\nCXCL12 is not the key mediator in monocyte migration post-IR.\nThe proportion of CXCR2+ monocytes in tumor tissue. Mice were inoculated with MC38 and received IR same as\nmentioned before. Tumors were separated and analyzed 3 days post IR.\nOur previous study demonstrated that the anti-CCR2 monoclonal antibody clone decreases the accumulation of the\nCCR2+Ly6Chi population and that CCR2 blockade in established tumors reverses radiation resistance, achieving\ngreater antitumor efficacy (PMID:29170400). The distinctions between these studies are as follows:\n1. Differential receptor expression patterns: CCR2 is broadly expressed in both naïve and tumor-bearing mice,\nwhereas CXCR5 expression on monocytes is tumor-mediated.\n2. Divergent regulating mechanisms of ligand expression: CCL2 is up-regulated by STING–IFN I signaling and\nproduced mainly in both tumor cells and myeloid cells. Meanwhile, CXCL13 is primarily derived from tumor cells\nrather than myeloid cells and is induced by radiation-mediated crosstalk between tumor cells and macrophages.\n3. Functional heterogeneity in immune modulation: In our previous study, wedemonstrated that the STING/type I\ninterferon pathway enhances suppressive inflammation in tumors by recruiting myeloid cells, in part via the CCR2\npathway. This finding emphasized that monocytic (M-)MDSCs (CD11b+Ly6ChiLy6G-) mediate the immunosuppressive\nresponse as a unit and that the CCL2–CCR2 axis induces their mobilization. In this study, weaimed to identify the\nmore specific subsets that weaken radiotherapy efficacy and elucidate their mechanisms in suppressing antitumor\nimmunity. We found that CXCR5+ monocytes exhibited stronger immunosuppressive capacity than CXCR5-\nmonocytes, and this effect was PD1/PD-L1-dependent. Additionally, CXCR5+ monocytes can further differentiate into\nCXCR5+ macrophages, perpetuating immune suppression.\nWe add this information in discussion section (line 431).\nMoreover, we systematically analyzed immune cells in irradiated tumors (3 days post-IR). Monocytes were enhanced\npost-IR, whereas macrophages and CD4+ T cells decreased (see below, current Figure S1A).\nThe immune cell populations in irradiated tumor. Mice were inoculated with MC38 and received IR same as\nmentioned before. Tumors were separated and analyzed 3 days post IR.\nThe phenotypic and functional profile of CXCR5 monocytes is insufficiently detailed. It is not clear how this\n+\npopulation differs from other monocyte subsets or from M-MDSCs. Additionally, previous reports have shown CXCR5\nexpression on intermediate human monocytes; the authors should reconcile these findings with their own and\ndiscuss the expression of other proteins in the CXCR5 population.\n+\nWe conducted mRNA-seq and KEGG pathway analysis to investigate the detailed differences between CXCR5+\nmonocytes and classical M-MDSCs. The differentially expressed genes (DEGs) of CXCR5+ monocytes and classical\nM-MDSCs were enriched in immune system and signal transduction pathway, similar to the DEGs of CXCR5+\nmonocytes versus CXCR5- monocytes. The heatmap revealed that CXCR5 monocytes demonstrate an enhanced\n+\nexpression profile in terms of genes with protumor characteristics when compared with either classical M-MDSCs or\nCXCR5− monocytes. In contrast, CXCR5+ monocytes exhibited an attenuated antitumor gene profile when compared\nwith classical M-MDSCs.\nUpper: KEGG pathway analysis of differentially expressed genes of CXCR5+ monocytes versus classical M-MDSCs\nor CXCR5- monocytes.\nBottom: Heatmap showing the mRNA expression of anti- and pro- tumor associated genes in M-MDSCs. M-MDSCs\n(Ly6ChighLy6Glow) were sorted from untreated tumors and CXCR5− and CXCR5+monocytes from irradiated tumors.\nWe agree withthe reviewer’s comment that CXCR5 is expressed on some monocytes in human peripheral blood. In\nour study, we analyzed peripheral blood mononuclear cells from healthy donors and found a baseline level of\nCXCR5+CD14+ (current Figure 6C and 6K), consistent with the findings of a previous report (PMID: 38742110).\nMoreover, PD-L1 showed higher expression in CXCR5+CD14+ than CXCR5-CD14+ at this stage, consistent with the\nfindings in mice. We have added this information to the Result 6 section (line 397).\nAlthough RNA sequencing was performed, only a limited number of genes are shown (Figure 1). A more\ncomprehensive analysis of differentially expressed genes would enhance the mechanistic depth of the study.\nFurthermore, there is no mention of public deposition of RNA-seq data, which is necessary for reproducibility.\nIn Figure 1D, we quantified various chemokines in tumor with or without radiation by employed cytometric bead array\n(LEGENDplex™ MU Proinflam Chemokine Panel, Biolegend) and we showed all quantified chemokines in Figure 1D.\nIn addition, we added volcano plot in Figure S1E to comprehensively exhibit differentially expressed genes before\nand after radiation in tumor tissue. All relevant data had been deposited to the public repository when resubmit.\nThe manuscript lacks critical methodological details regarding tumor implantation and treatment protocols, including:\nnumber of cells injected per tumor model, timing and size of tumors at treatment initiation, specific timepoints for\ncontrol and treated groups, concentrations and durations of inhibitor and antibody treatments. Without this\ninformation, reproducibility is compromised.\nWe appreciate the critique and have added methodological details in method section and figure legends accordingly.\nImportantly, the potential systemic or off-target immune consequences of targeting the CXCL13–CXCR5 axis—\nparticularly in the context of chronic infections or autoimmune disease—are not discussed. Are the treatments\naffecting B cells that are the main CXCR5 expressing cells?\nWe treated tumor-bearing mice with anti-CXCL13 post-IR and found that CXCL13 neutralization did not change the\npercentage of B cells in the irradiated tumor (see below). Consider that CXCL13 shows protective effects against\nHBV infection and is upregulated in various autoimmune diseases. Therefore, clinical applications of anti-CXCL13 for\ncancer treatment should consider potential risks of increased susceptibility to infections or the interference of\nautoimmune diseases diagnostics. We have added this information to the discussion section (line 443).\nThe proportion of B cells in tumor tissue. Mice were inoculated with MC38 and received IR same as mentioned\nbefore. Anti-CXCL13 were administered same as Q1. Tumors were separated and analyzed 3 days post IR."}, {"round": 2, "reviewer_comments": {"reviewer_1": "No further comments in this article.", "reviewer_2": "Substantial changes to the manuscript have been made and the queries have been answered appropriately, in my opinion the manuscript can be published as is, there is only a need to integrate into the discussion the recent Nature paper about the »badscopal » effect of radiotherapy from the Chicago group (Wechselbaum’s team) which is also in line with the current PI3K/VEGF/chemokines regulatory track described In the current manuscript.", "reviewer_3": "In the revised manuscript, the authors provide additional data to support their hypothesis that CXCR5⁺ monocytes represent the main immunosuppressive population following irradiation. I have appreciated the increased experimental details provided in the figure legends, which improve the overall clarity of the presentation.\nHowever, the experimental approach remains incomplete and presents several conceptual and methodological issues.\nSpecifically, the use of inhibitors to exclude the involvement of other chemokines is only partial and does not convincingly rule out alternative mechanisms. Moreover, there are factual inaccuracies in the interpretation of chemokine–receptor interactions — for instance, CXCL12 is not a ligand of CXCR2.\nIn addition, my previous question regarding the characterization of the CXCR5⁺ monocyte population remains unanswered. It is still unclear whether CXCR5 expression is upregulated after irradiation within a specific monocyte subpopulation. A FACS analysis showing LY6C, CCR2, CX3CR1 and CXCR5 expression is required.\nOverall, the manuscript requires substantial revision and clarification of these key aspects before it can be considered suitable for publication."}, "author_response": "Reviewer #1:\nNo further comments in this article.\nWe appreciate the reviewer for the previous comments, which strengthened the clarity and quality of our manuscript.\nReviewer #2:\nSubstantial changes to the manuscript have been made and the queries have been answered appropriately, in my\nopinion the manuscript can be published as is, there is only a need to integrate into the discussion the recent Nature\npaper about the »badscopal » effect of radiotherapy from the Chicago group (Wechselbaum’s team) which is also in\nline with the current PI3K/VEGF/chemokines regulatory track described In the current manuscript.\nWe appreciate the reviewer’s affirmation of our revision work. We carefully reviewed Nature paper (Piffko, A. et al.\n2025. Nature) and found the proposed concept of \"badscopal\" highly intriguing. It reports that radiation induces\ntumor-derived amphiregulin which reprograms EGFR+ myeloid cells towards immunosuppressive phenotype and\npromotes metastasis. This funding provides an insight for re-evaluating radiotherapy's systemic impacts. In our study,\nwe find that radiation promotes immunosuppressive CXCR5+ monocytes infiltrating in local tumor and these cells are\ninduced by tumor derived VEGF. Nature paper and our study both focus on the tumor-derived factors that induce\nimmunosuppressive myeloid cells and reveal the systemic and local protumor effects of radiation respectively. We\nhave added these to the discussion (line 437–442).\nReviewer #3:\nIn the revised manuscript, the authors provide additional data to support their hypothesis that CXCR5 monocytes\nrepresent the main immunosuppressive population following irradiation. I have appreciated the increas⁺ed\nexperimental details provided in the figure legends, which improve the overall clarity of the presentation.\nHowever, the experimental approach remains incomplete and presents several conceptual and methodological\nissues. Specifically, the use of inhibitors to exclude the involvement of other chemokines is only partial and does not\nconvincingly rule out alternative mechanisms. Moreover, there are factual inaccuracies in the interpretation of\nchemokine–receptor interactions — for instance, CXCL12 is not a ligand of CXCR2.\nWe thank the reviewer for the positive feedback of the revised work and for further comments. To investigate the\npotential effects of other chemokines and their corresponding receptors on monocyte recruitment, we examined the\ntumor infiltrated monocytes with or without radiation treatment and analyzed the receptors of other radiation-\nenhanced chemokines (including CCL2, CCL4, CCL5, CXCL1 and CXCL10 as shown in the heatmap in Figure 1D).\nSingle-cell RNA sequencing shows that monocytes enhanced in irradiated tumor (see below, A) and CCR2+, CCR5+\nsubset expands post-radiation (see below, D) which is consist with flow cytometry analysis (see below, E–F).\nOur pervious study (Liang, H. et al. 2017. Nat Commun) has reported the role of CCL2-CCR2 axis in monocytes\nrecruitment following radiation. The differences of these two studies (CCL2-CCR2 axis versus CXCL13-CXCR5 axis)\nhave been disscussed in last response and the current Disscussion (line 443–448). Despite the increase of CCR5+\nmonocytes in irradiated tumor, CCR5 antagonist did not reverse radiation-induced monocytes infiltration or other\nmyeloid cells (see below, G), but reduce CD8+ T cells in irradiated tumor (see below, H). Recent study reports that\nCCL5 promote M1-like macrophage polarization and correlated with better patient overall survival rates in multiple\nhuman tumors (Yu, Z. et al. 2025. Cell). Similarly, Seo, In-Ho. et al. found that CCR5 expression on memory CD8+\nT cell mediated its migration (Seo, In-Ho et al. 2025. Cell Reports). Together with our findings, these studies suggested\nthat the CCL5-CCR5 axis mediates antitumor immunity, which is distinct from the radiation-induced protumor effect\nrevealed by our current study. We intend to investigate this issue in future research.\nA CD45+ immune cells in tumor with or without IR.\nB–D The expression of enhanced chemokines associated receptors on monocytes in tumor.\nE The proportion of monocytes in tumor infiltrated immune cells.\nF and G The proportion of CCR2+ (F) and CCR5+ (G) monocytes in tumor infiltrated immune cells.\nH CD45+ immune cells in tumor with or without IR.\nB6 mice were inoculated s.c. with 1 106 MC38 cells. Ten days post inoculation, the tumors received one dose of 12\nGy ironizing radiation (IR). Three days post IR, tumors were separated and analyzed. CCR5 antagonist (TAK-220)\nwere administered i.g., 5 mg/kg at th ×e day of IR, every other day.\nIn the last response, we summarized the enhanced chemokines we detected “as shown in the heatmap in Figure 1D,\nCCL2, CCL4, CCL5, CXCL1, CXCL10, and CXCL13 were enhanced post-IR”. CXCR2 is reported as the receptor of\nCXCL1, we analyzed tumor infiltrated CXCR2+ monocytes before and after radiation and did not find difference in\nthese two groups (see below, current Figure S3B). Besides, CXCR2+CD45+ population decreased post IR.\nTherefore, we propose that CXCL1-CXCR2 does not mediate monocyte migration following IR treatment. We would\nlike to apologize sincerely for the typographical error of writing \"CXCL12\" instead of \"CXCL1\" in our initial response.\nWe have corrected the typographical error in current manuscript (line 223). Actually, we did not observe CXCL12\nenhanced in irradiated tumor in our study.\nThe proportion of CXCR2+ monocytes and CXCR2+ immune cells in tumor tissue. Mice were inoculated with MC38\nand received IR as previous. Three days post IR, tumors were separated and analyzed.\nIn addition, my previous question regarding the characterization of the CXCR5 monocyte population remains\nunanswered. It is still unclear whether CXCR5 expression is upregulated after ⁺irradiation within a specific monocyte\nsubpopulation. A FACS analysis showing LY6C, CCR2, CX3CR1 and CXCR5 expression is required.\nWe appreciate the reviewer's additional explanation, which helps us characterize the CXCR5 monocyte population\nmore precisely. In pervious response, by conducting mRNA-seq and KEGG pathway analysis⁺, we found that CXCR5+\nmonocytes exhibited an enhanced protumor gene profile when compared with classicalM-MDSCs. In this revision,\nas suggested, we further detected the expression of CCR2 and CX3CR1 on M-MDSCs, CXCR5- and CXCR5+\nmonocytes. The results showed that, although a certain proportion of both CXCR5+ monocytes and M-MDSCs\nexpressed CCR2 and CX3CR1, CXCR5+ monocytes had higher expression of these receptors than M-MDSCs. By\ncontrast, some CXCR5- monocytes expressed CX3CR1 without expressing CCR2 (see below, A, current Figure 1H).\nWe also noted that: 1) the proportion of the CX3CR1+CCR2- subset was similar across M-MDSCs, CXCR5- and\nCXCR5+ monocytes, both with and without radiation; 2) the CX3CR1-CCR2+ subset accounted for a small proportion\nof these three cell types. 3) CCR2-CX3CR1-CXCR5+ monocytes expanded in irradiated tumor (see below, A, current\nFigure 1H). These results suggested that CXCR5+ monocytes were recruited by radiation regardless of the\nexpression of CCR2 and CX3CR1.\nCollectively, the results demonstrated that radiotherapy-induced CXCR5+ monocyte is a specific monocyte\nsubpopulation, which is different from M-MDSC，CCR2+, or CX3CR1+ monocyte subpopulation (see below, B).\nTo investigate whether radiation induced CXCR5 expression on a specific monocyte subpopulation, we analyzed\nCCR2+ and CX3CR1+ monocytes and found that CXCR5 was not upregulated in these monocytes following radiation\ntreatment (see below, C and D, current Figures S1K and S1L).\nA The expression of CCR2 and CX3CR1 on tumor infiltrated monocytes.\nB A schematic diagram of the expression of CXCR5, CCR2 and CX3CR1 on tumor infiltrated monocytes.\nC CXCR5 expression on CCR2- or CCR2+ monocytes in tumor with or without radiation.\nD CXCR5 expression on CX3CR1- or CX3CR1+ monocytes in tumor with or without radiation.\nMice were inoculated with MC38 and received IR as previous. Three days post IR, tumors were separated and\nanalyzed.\nOverall, the manuscript requires substantial revision and clarification of these key aspects before it can be\nconsidered suitable for publication.\nThank you again for your careful review and constructive feedback, which have further improved the quality and rigor\nof our work."}, {"round": 3, "reviewer_comments": {"reviewer_2": "This manuscript investigates the role of CXCR5⁺ monocytes in mediating radiotherapy-induced immunosuppression and radioresistance. The authors propose a mechanistic model in which tumor-derived VEGF induces CXCR5 expression on monocytes via the PI3K/mTOR/HIF-1α pathway, while radiation-induced CXCL13 recruits these CXCR5⁺ monocytes into tumors, where they suppress CD8⁺ T-cell function through PD-1/PD-L1 signaling and differentiate into M2-like macrophages. Targeting components of this axis improves tumor control after irradiation.\nOverall, the study addresses an important and timely question in radiation oncology and tumor immunology, namely how radiotherapy can paradoxically induce protumor immune responses. The work is extensive, technically sophisticated, and supported by multiple tumor models and patient correlative data.\nMajor Strengths\n1. Conceptual relevance: The identification of CXCR5⁺ monocytes as mediators of radiation-induced immunosuppression is novel and fits well with emerging concepts such as the “badscopal” effects of radiotherapy.\n2. Mechanistic depth: The VEGF–PI3K/mTOR/HIF-1α–CXCR5 pathway is systematically explored using pharmacologic inhibition, gene knockdown, and in vivo validation.\n3. Breadth of models: Findings are reproduced across several murine tumor models and supported by human patient data, strengthening translational relevance.\n4. Therapeutic implications: The study provides multiple actionable strategies (VEGFR inhibition, CXCL13 neutralization, PD-L1 blockade) that could be integrated with radiotherapy.\nMajor Concerns\n1. Incomplete exclusion of alternative chemokine pathways\nAlthough the authors analyze several chemokines induced by radiation, the inhibitor-based approach does not fully rule out contributions from parallel recruitment pathways (e.g., CCR2- or CCR5-dependent mechanisms). The dominance of the CXCL13–CXCR5 axis, while plausible, is not conclusively demonstrated as exclusive.\n2. Characterization of CXCR5⁺ monocyte subsets\nWhile additional flow cytometry and scRNA-seq data have been provided, it remains somewhat unclear whether CXCR5 expression defines a stable monocyte lineage or reflects a radiation-induced activation state within existing subsets. A clearer phenotypic definition (Ly6C, CCR2, CX3CR1, CXCR5 co-expression) in a single integrated analysis would strengthen the conclusions.\n3. Interpretational overreach\nSome statements imply that CXCR5⁺ monocytes are the primary drivers of radioresistance. Given the complexity of the irradiated tumor microenvironment, this conclusion should be more carefully qualified in the Discussion.\nMinor Comments\n-The Discussion could better integrate recent high-impact literature on systemic protumor effects of radiotherapy.\n- Minor factual inaccuracies in chemokine–receptor pairing identified in earlier versions appear corrected but should be carefully rechecked throughout the text.\n- Some figure legends remain dense and could be streamlined for clarity.", "reviewer_3": "I acknowledge that the authors have made an effort to address most of the points raised. However, I note that in the revised version of the manuscript the term MDSC is introduced without being spelled out or formally defined at its first occurrence.\nIn addition, by definition, MDSCs represent a functional category, and in the absence of functional validation, the use of the term MDSC should be avoided. The myeloid populations analyzed should instead be referred to based on their phenotypic markers (for example, CD11b⁺ Ly6C^high Ly6G+ cells), with the gating strategy clearly specified. This would improve conceptual clarity and prevent overinterpretation of the functional identity of these cells."}, "author_response": "Reviewer #2 (Remarks to the Author):\nThis manuscript investigates the role of CXCR5 monocytes in mediating radiotherapy-\ninduced immunosuppression and radioresistance. The authors propose a mechanistic\n⁺\nmodel in which tumor-derived VEGF induces CXCR5 expression on monocytes via the\nPI3K/mTOR/HIF-1α pathway, while radiation-induced CXCL13 recruits these CXCR5\nmonocytes into tumors, where they suppress CD8 T-cell function through PD-1/PD-L1\n⁺\nsignaling and differentiate into M2-like macrophages. Targeting components of this axis\n⁺\nimproves tumor control after irradiation.\nOverall, the study addresses an important and timely question in radiation oncology and\ntumor immunology, namely how radiotherapy can paradoxically induce protumor immune\nresponses. The work is extensive, technically sophisticated, and supported by multiple\ntumor models and patient correlative data.\nMajor Strengths\n1. Conceptual relevance: The identification of CXCR5 monocytes as mediators of\nradiation-induced immunosuppression is novel and fits well with emerging concepts such\n⁺\nas the “badscopal” effects of radiotherapy.\n2. Mechanistic depth: The VEGF–PI3K/mTOR/HIF-1α–CXCR5 pathway is systematically\nexplored using pharmacologic inhibition, gene knockdown, and in vivo validation.\n3. Breadth of models: Findings are reproduced across several murine tumor models and\nsupported by human patient data, strengthening translational relevance.\n4. Therapeutic implications: The study provides multiple actionable strategies (VEGFR\ninhibition, CXCL13 neutralization, PD-L1 blockade) that could be integrated with\nradiotherapy.\nWe are sincerely grateful for the positive feedback of the reviewer, and we appreciate the\ntime and effort dedicated to evaluating our work.\nMajor Concerns\n1. Incomplete exclusion of alternative chemokine pathways\nAlthough the authors analyze several chemokines induced by radiation, the inhibitor-based\napproach does not fully rule out contributions from parallel recruitment pathways (e.g.,\nCCR2- or CCR5-dependent mechanisms). The dominance of the CXCL13–CXCR5 axis,\nwhile plausible, is not conclusively demonstrated as exclusive.\nWe agree with reviewer that our current work did not fully rule out the contributions of all\nother chemokines on radiation-induced CXCR5 monocytes recruitment. We utilized\ninhibitors, neutralizing antibodies, and genetic knocking-out mice to assess the chemokine\n⁺\nwhich is most likely to influence CXCR5 monocyte infiltration in irradiated tumor.\nWe found\nthat CXCL13 promoted the infiltration of CXCR5 monocytes into irradiated tumors and\n⁺\nthese cells suppressed CD8 T cells induced antitumor immunity, thereby impacting\n⁺\nradiotherapy efficacy. The limitation of our investigation is that it has not excluded the\n⁺\ncontribution of all other chemokines to CXCR5 monocyte recruitment during radiation.\nThe role of other chemokines and their receptors in radiation-induced immune responses\n⁺\nand the underlying mechanism requires further investigation, which were add into the\ndiscussion (Line 450).\n2. Characterization of CXCR5 monocyte subsets\nWhile additional flow cytometry and scRNA-seq data have been provided, it remains\n⁺\nsomewhat unclear whether CXCR5 expression defines a stable monocyte lineage or\nreflects a radiation-induced activation state within existing subsets. A clearer phenotypic\ndefinition (Ly6C, CCR2, CX3CR1, CXCR5 co-expression) in a single integrated analysis\nwould strengthen the conclusions.\nWe sincerely thank the reviewer for the constructive comment. As suggested, we analyzed\nthe co-expression of Ly6C, CCR2, CX3CR1, and CXCR5 and found that, among\nmonocytes, Ly6C and CXCR5 double-positive, Ly6C and CCR2 double-positive, Ly6C\nsingle-positive, and quadruple-positive subpopulations significant increased post radiation\n(see below, A). This result is consisting with: 1) Figure 1H which suggested that part of\nCXCR5+ monocytes were recruited by radiation regardless of the expression of CCR2 and\nCX3CR1; and 2) our pervious study of CCL2-CCR2 axis induced monocytes infiltration in\nirradiated tumor (PMID: 29170400). To investigate whether radiation induced CXCR5\nexpression on a specific monocyte subpopulation, we analyzed CCR2+ and CX3CR1+\nmonocytes and found that CXCR5 expression was not upregulated in these monocytes\nfollowing radiation treatment (Supplementary Fig. 2D and 2E).\nThese results suggested that CXCR5 expression defines a stable monocyte lineage which\nincreases following radiation and is different from CCR2+ or CX3CR1+ monocyte\nsubpopulation (see below, B).\nA The percentage of indicated cell subsets in tumor infiltrated monocytes.\nB A schematic diagram of the expression of CXCR5, CCR2 and CX3CR1 on monocytes in irradiated tumor.\nB6 mice were inoculated s.c. with 1 106 MC38 cells. Ten days post inoculation, the tumors received one dose of 12\nGy ironizing radiation (IR). Three days post IR, tumors were separated and analyzed.\n×\n3. Interpretational overreach\nSome statements imply that CXCR5 monocytes are the primary drivers of radioresistance.\nGiven the complexity of the irradiated tumor microenvironment, this conclusion should be\n⁺\nmore carefully qualified in the Discussion.\nWe revised the inappropriate statements in the Discussion section. Specifically, we deleted\nthe statement that emphasized the priority of CXCR5 monocytes in radioresistance.\n⁺\nMinor Comments\n-The Discussion could better integrate recent high-impact literature on systemic protumor\neffects of radiotherapy.\nAs suggested, we integrate recent high-impact literature on radiation inducedsystemic\nprotumor effects (PMID: 39325547 and PMID: 38427437) and investigated the\nradiotherapy-induced immunosuppressive response (Line 441-448).\n- Minor factual inaccuracies in chemokine–receptor pairing identified in earlier versions\nappear corrected but should be carefully rechecked throughout the text.\nWe thank the reviewer for the kindly remind. The manuscript has been carefully proofread.\n- Some figure legends remain dense and could be streamlined for clarity.\nWe simplified the figure legends to improve the readability of the manuscript.\nReviewer #3 (Remarks to the Author):\nI acknowledge that the authors have made an effort to address most of the points raised.\nHowever, I note that in the revised version of the manuscript the term MDSC is introduced\nwithout being spelled out or formally defined at its first occurrence.\nWe thank the reviewer for recognizing our revisions and for providing valuable comments\non our manuscript. In response to the suggestion, we have ensured that the abbreviation\n\"MDSC\" (myeloid-derived suppressor cells) is now fully defined at its first occurrence in\nthe main text (Line 446).\nIn addition, by definition, MDSCs represent a functional category, and in the absence of\nfunctional validation, the use of the term MDSC should be avoided. The myeloid\npopulations analyzed should instead be referred to based on their phenotypic markers (for\nexample, CD11b Ly6C^high Ly6G+ cells), with the gating strategy clearly specified. This\nwould improve conceptual clarity and prevent overinterpretation of the functional identity\n⁺\nof these cells.\nWe agree with the reviewer that MDSCs represent a functional category. Therefore, we\nrefer to these CD45+CD11b+Ly6ChighLy6Glow cells as tumor-infiltrating monocytes in our\nresults. The gating strategy is shown in Supplementary Fig. 10B."}]

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