% No. % Any AE 8 80.00 1 10.00 9 50.00 1 5.55Fever 4 40.00 0 0 2 11.11 0 0Fatigue 7 70.00 0 0 6 33.33 0 0Chill 2 20.00 0 0 0 0 0 0 Emesis 1 10.00 0 0 1 5.55 0 0 Muscle soreness 4 40.00 0 0 1 5.55 0 0Injection site reaction 0000 1 5.55 0 0 Dizziness 0000 1 5.55 0 0 Nausea 1 10.00 0 0 0 0 0 0Upper gastrointestinal haemorrhage 1 10.00 0 0 0 0 0 0 Weight loss 0000 1 5.55 0 0 Acute allergy 0 0 1 10.00 0 0 1 5.55 BA C FIGURE 1 Better clinical response in RFA+Vac patients upon receiving iNeo-Vac. (A)Swimmer plots of RFA+Vac patients (with yellow background) and Vac patients (with blue background); (B)Waterfall plots of RFA+Vac patients (shown in yellow) and Vac patients (shown in blue). Dashed line above 20% or below -30% indicate 20% increase ( ≥20% is considered as progressive disease) or 30% reduction ( ≤-30% is considered as partial response) of the sum of the longest diameters of the tumors, respectively. # suggested that new lesion(s) was found; (C)Spider plots of RFA+Vac patients and Vac patients. The “best clinical response ”in panel b was de ﬁned as the largest reduction of target lesion in size during treatment.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 06 trial (RFA+Vac colorectal group) (n=5) were compared to that of colorectal cancer patients receiving RFA treatment alone (RFAcolorectal group) (n=8) during the same period. By comparing the patients ’baseline characteristics, includi ng age, sex distribution, tumor metastases (excluding lung metastases), prior systemic treatment as well as RFA treatment, no signi ﬁcant differences were found between RFA+Vac colorectal and RFA colorectal groups ( Figure 2C ). The mOS of RFA+Vac colorectal patients was 21.27 months (11.93-42.03 months), while RFA colorectal patients 11.55 months (2.53-18.03 months) ( Figure 2D ), indicating the ef ﬁcacy of iNeo-Vac-P01 vaccination after RFA in improving colorectal patients ’OS. Neoantigen vaccination elicited stronger neoantigen-speci ﬁc immune response in RFA+Vac patients Previously, the potentials of iNeo-Vac-P01 vaccination in eliciting T-cell-mediated im mune response targeting tumorneoantigens have been demonstrated in patients with advanced solid tumors ( 10). In this study, T cell activation post neoantigen vaccination was con ﬁrmed in 88.89% patients (24/27) by IFN- gELISpot assay ( Figure 3A ). All evaluated patients (n=27) showed an increase of spot counts after vaccination ( p< 0.05), except for patients P006, which might be attributed to the noisy background at baseline. Overall, 77.97% (92/118) individual long peptides and 84.48% (49/58) long peptide pools elicited measurable neoantigen peptide- speci ﬁc immune response (positive results in ELISpot assay after vaccination) in 21 patients, respectively ( Supplementary Table S6 ). TCR sequencing of peripheral T cells at baseline and after vaccination revealed that abundant novel peripheral T cell clones were detected in 60.00% (12/20) patients ( Supplementary Table S7 ). These data suggested the effectiveness of iNeo-Vac- P01 in inducing neoantigen-speci ﬁc T cell activation. Although RFA+Vac patients showed higher baseline response for IFN- gELISpot assay compared to Vac patients, B CDA FIGURE 2 RFA+Vac patients showed better progression-f ree survival (PFS) and overall survival (OS). (A)Progression-free survival of Va cp a t i e n t s( s h o w ni nb l u e )a n d RFA+Vac patients (shown in yellow). (B)Overall survival of Vac patients (blue) and RFA+Vac patients (yellow). (C)Characteristics of patients in RFA+Vac colorectal group, in comparison to that of patients in RFA colorectal group. (D)Overall survival of RFA-pretreated colorectal cancer patients (RFA+Vac colorectal) (yellow) in this study, in comparison to RFA-treated colorect al cancer patients (RFA colorectal) (green) during the same period at Sir R un Run Shaw Hospital. Longer OS was observed for patients receiving additional neoantigen vaccination ( p= 0.037). Survival data were compared by logrank test.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 07 BC DEA FIGURE 3 RFA+Vac patients showed better immune responses upon receiving iNeo-Vac-P01. (A)Ex vivo ELISpot assay results of each patient. Green triangle and red diamond represent the relative response rate, calculated per the ratios of peptides with positive ELISpot reads to all peptidesimmunized before and after vaccination, respectively. The bar chart shows the spot numbers of the peptide or peptide pool with best reads per10 5PBMCs. # Indicated that peptide pools were used for ELISpot assay for this patient; + indicated that relatively high abundant novel clones of peripheral T cells were detected for this patient after vaccination. (B,C)Data shown in Box and Whiskers (with minimum to maximum, showing all points and median). (B)Baseline and post-vaccination ELISpot spot counts per 105PBMCs of RFA+Vac (yellow) and Vac (blue) patients. c) ELISpot spot counts per 105PBMCs at baseline and post vaccination of the peak response for RFA+Vac patients with t int< 3 months, or 3 ≤tint< 6 months. No signi ﬁcant difference was found between the two groups at baseline (blue and yellow); however, the difference was intensi ﬁed post vaccination, as patients with t int< 3 months showed stronger peak response (red). Baseline response and peak response of all RFA+Vac patients showed statistically signi ﬁcant difference ( p< 0.0051). (D)Flow cytometric analyses of RFA+Vac and Vac patients on T cell subsets. RFA +Vac and Vac patients were further divided into two subgroups: patients with good response (OS ≥12 months) and poor response (OS < 12 months); number of active CD4+T cells (# per mL), and proportion of CD4+or CD8+T cells that expressed CTLA4 (%) at baseline, prime phase and boost phase were analyzed, respectively. (E)Flow cytometric analyses of RFA+Vac and Vac patients on cytokines including IFN- g, IL-6 and TNF- aat baseline, prime phase, and boost phase. A t-test analysis was applied to indicate the signi ﬁcance in data difference with a two-tailed pvalue. ns, not signi ﬁcant (p > 0.05); *p ≤0.05; **p ≤0.01.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 08 the difference was not signi ﬁcant ( p= 0.0969). A signi ﬁcant change in spot counts was observed in both RFA+Vac and Vac patients post neoantigen vaccination, indicating the feasibility of personalized neoantigen peptide pools in eliciting robust cellular response. To be noted, RFA+Vac patients exhibited a higher immune response in relative to Vac patients after vaccination with a p-value less than 0.05 ( Figure 3B ), showing that RFA prior to vaccination could help enhance antitumor responsesdrastically. Although not signi ﬁcantly, the time required for RFA+Vac group to witness a peak response was 55.2 days, longer than that of Vac group (51.8 days) ( Supplementary Figure S1 ). This phenomenon indicated that the time to obtain an optimal immune response post neoantigen vaccination was probably unrelated to RFA treatment. To determine the optimal timing of the ﬁrst vaccination after RFA treatment, RFA+Vac patients were categorized into two subgroups: patients with an RFA-vaccination interval (t int) less than 3 months (t int≤3 months) (n=5), and patients with an RFA-vaccination interval between 3 months and 6 months (3 months < t int< 6 months) (n=5). No signi ﬁcant difference in IFN-g-speci ﬁc cellular response at baseline was found between these two groups. Differently, patients in RFA-Vac t int≤3 months group showed insigni ﬁcantly higher peak response upon neoantigen vaccination ( Figure 3C ). A study with larger sample size would be needed to validate whether shorter RFA- vaccination interval [i.e., (t int≤3 months)] could contribute to a better antitumor immune response. Good clinical response was associated with T cell activation and pro-in ﬂammatory cytokine secretion To further explore the potential reasons for the discrepancy in clinical bene ﬁts among patients in this study, patients were further divided into two subgroups according to their clinical response, that are, patients with good response (OS ≥12 months) group and patients with poor response (OS < 12 months) group ( Figure 3D ;Supplementary Table S9 ). Although not statistically signi ﬁcant, it was observed in RFA +Vac group that good response patients showed more active CD4+T cells, as well as higher proportions of CD4+CTLA4+and CD8+CTLA4+T cells than poor response patients at all time points. Differently in Vac group, poor response patients exhibited insigni ﬁcantly larger active CD4+T cell numbers than good response patients at both baseline and after prime vaccination It is also noteworthy that in All group (including all patients in both RFA+Vac and Vac groups), good response patients displayed larger number of active CD4+T cells, as well as higher proportion of CD4+CTLA4+or CD8+CTLA4+T cells at each time point. However, only the proportion of CD8+ CTLA4+T cells after boost immunization showed signi ﬁcant difference between good and poor response patients (All group)(p= 0.0337). These results demonstrated a stronger T cell activation in good response patients than that in poor response patients. In other words, good clinical response was likely to be resulted from ef ﬁcacious T cell activation by neoantigen vaccination. Potential biomarkers for cl inical response were also investigated in this study, including IFN- g, TNF- aand IL-6 (Figure 3E ;Supplementary Table S10 ). Similar tendency of the change in IFN- gsecretion post-vaccination was observed compared to our previous study ( 10). In brief, low IFN- g secretion was observed at baseline, except for RFA+Vac poor response patients. After boost immunization, IFN- gsecretion in good response patients was upregulated, showing a higher level compared to poor response patients in All group ( p= 0.0037), Vac group ( p= 0.0189) and RFA+Vac group ( p= 0.085), indicating that the ef ﬁcacy of neoantigen vaccination in upregulating IFN- gsecretion was probably unbiased for Vac and RFA+Vac patients. Overall, the difference in IFN- gsecretion between good response and poor response patients was intensi ﬁed after vaccination. No disparity in TNF- aexpression at baseline was found between good and poor response patients in Vac group, while in RFA+Vac group, good response patients showed a higher bassline TNF- aexpression, demonstrating RFA ’se fﬁcacy in the activation of immune system. To be noted, the difference in TNF- aexpression between good response and poor response patients was ﬁrst intensi ﬁed after prime immunization, and then lowered after boost immunization ( Figure 3E ). As a potential tumor growth factor for different cancer types ( 18), the secretion of IL-6 was also evaluated. Higher level of circulating IL-6 was found in poor response patients compared to good response patients at all time points in all groups, which was in line with previously published studies ( 10,19,20). Also, lower IL-6 expression was found in poor response patients of RFA+Vac group after vaccine administration, indicating that combination therapy may still beneﬁt poor response patients by lowering their circulating IL- 6 level. Case report of an intrahepatic cholangiocarcinoma patient P015 Good clinical response and immune response were observed in several patients in RFA+Vac group. Here, a case report of a patient who received two batches of customized vaccines, was presented to highlight the importance of applying adjustable and timely personalized treatment for patients to improve their survival. In the case of patient P015, a 56-year-old male diagnosed with adenocarcinoma by right lobe liver lump puncture in August 2017, had multiple liver RFA treatments prior to iNeo-Vac-P01 vaccination. He went through the combinational treatment of Tegafur and Gemcitabine 7 times post-diagnosis, before his ﬁrst liver RFA treatment in lateShou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 09 November 2017. After another liver RFA under the guidance of ultrasound in January 2018, he had Tegafur orally for 2 cycles. Followed by his third liver RFA in late February 2018, he took Apatinib orally for targeted therapy. His most recent RFA prior to neoantigen vaccination was in May 2018, around 2 months before his ﬁrst dose of iNeo-Vac-P01. For patient P015, the ﬁrst batch of immunization comprising 5 prime doses and 4 boost doses of iNeo-Vac-P01 started from11 thJuly 2018 ( Figure 4A ). During the ﬁrstﬁve months post ﬁrst vaccination, continuous reduction in size of the liver lump was observed, the sum of longest diameters (SLDs) decreasing from 79.6 mm at baseline, to 71.7 mm at the second month and then 67.8 mm at the ﬁfth month, respectively ( Figure 4B ). However, multiple new lesions were observed 11.5 months after ﬁrst vaccination. Sequencing of his recent biopsy sample enabled the preparation of a new batch of vaccine. The second batch of iNeo-Vac-P01 was delivered to him since 7thAugust 2019 (Figure 4A ). It is noted that each peptide pool managed to elicit robust neoantigen-speci ﬁc T cell responses, and the peak response for each pool was observed 7 weeks post ﬁrst immunization (Figures 4C ,Dand Supplementary Table S6 ). The gradual decline of T cell responses in PHA group at week 3, 7 and 11 in Figure 4D was probably resulted from the differences of cell status at these time points. TCR bchain of peripheral T cells was sequenced and the results revealed that the abundance of seven TCR clones considerably increased after vaccination (Figure 4E ). These data suggested that multiple subsets of T cells with speci ﬁcities induced by iNeo-Vac-P01 could be successfully activated to kill tumor cells. Both the density and proportion of CD4+Granzyme B+and CD8+Granzyme B+T cells exhibited an increase post-vaccination, indicating the enhancement of the inﬁltration of T cells into tumor tissues ( Figure 4F ). Compared to other patients, patient P015 had a relatively long survival time ( Figure 1A ). This could be correlated with the good antitumor immune response, as evaluated by ex vivo ELISpot assay. Multiple RFA treatments prior to neoantigen vaccination might contribute to the release of tumor-speci ﬁc antigens, thereby leading to the immune priming of several neoantigens. Moreover, the timely preparation and delivery of the second batch of iNeo-Vac-P01 after disease progression was also considered bene ﬁcial to prolong his survival. Combination treatment of RFA and neoantigen vaccination inhibited tumor growth in mice Inspired by the good clinical and immune response of neoantigen peptide vaccination after RFA treatment amongst patients with different cancer types, mouse models were employed to validate whether a combinational treatment modality incorporating RFA and subsequent individualized neoantigen-based vaccinatio n could generate strongerantitumor effects than either of the single modality alone. As it has been reported that the ablation of a tumor could lead to the shrinkage of another distant tumor ( 21–23). Thus, to study the synergetic antitumor ef ﬁcacy of combination treatment and the abscopal effects of RFA, we constructed a murine unilateral CT26 tumor model (unilateral tumor), as well as bilateral tumor models with CT26 or 4T1 tumor implanted on the left ﬂank and CT26 implanted on the right ﬂank (bilateral tumor) ( Figure 5A ). Compared to the CT26 tumor growth of mice bearing unilateral CT26 tumor that received only PBS (PBS group), mice bearing bilateral heterogeneous tumors (4T1 on the left ﬂank and CT26 on the right ﬂank) showed similar CT26 tumor growth on the right ﬂank after RFA was given on the left ﬂank (Figure 5B ), indicating no evident antitumor effects. This phenomenon demonstrated that the ablation of an irrelevant tumor 4T1 did not slow down the growth of target tumor CT26. In contrast, slower CT26 tumor growth on the right ﬂank was observed in mice bearing bilateral homogenous CT26 tumors (RFA group) after RFA treatment on the left ﬂank, indicating the abscopal effects induced by RFA were probably resulted from the activated immune cells that could recognize and kill the tumor cells harboring the same antigens as those released from the eliminated tumor cells through ablation. Similarly, when neoantigen vaccines were administered to mice bearing unilateral CT26 tumor (Vac group), evident inhibition of CT26 tumor growth was reported. Compared to mice in both PBS and Vac groups, mice bearing bilateral CT26 tumors receiving combination treatment of RFA and neoantigen vaccination (RFA+Vac group) displayed further inhibition of CT26 tumor growth on the right ﬂank, proving the stronger antitumor ef ﬁcacy of RFA+Vac combination treatment ( Figure 5B ). ICIs, especially anti-PD-1 antibodies, have previously shown synergistic effects with RFA or neoantigen vaccination in pre- clinical and clinical studies. Also, CT26 colon cancer cell line has been reported to be sensitive to checkpoint blockade ( 5,24). Taken these together, it was hypothesized that in the above mice models, the introduction of PD-1 blockades to RFA+Vac combination treatment (RFA+V ac+anti-PD-1 group) could further inhibit the growth of CT26 tumor on the right ﬂank. As expected, a more signi ﬁcant reduction of CT26 tumor growth on the right ﬂank was observed in the mice of RFA+Vac+anti- PD-1 group, compared with that of mice in RFA, Vac, RFA +anti-PD-1, and RFA+Vac groups ( Figure 5C ). It was most likely that RFA+Vac combination treatment might have upregulated the expression of PD-1 in tumor tissues, turning the “cold ”tumor into “hot”tumor, thereby making ICI more efﬁcacious. This hypothesis was validated by ﬂow cytometry to some extent, as higher PD-1 expression in tumor tissues was found in RFA+Vac group (11.50%), compared to RFA+anti-PD- 1 group (9.66%), RFA group (7.54%) and PBS group (5.37%). To be noted, the highest PD-1 expression was observed in RFA+Vac +anti-PD-1 group (25.20%) ( Figure 5D ). This ﬁnding was also in line with several published studies, where subsets of PD-1+TShou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 10 B CD E FA FIGURE 4 Case report of patient P015, who had multiple RFA treatments prior to vaccination. (A)The treatment scheme of patient P015. Two batches of iNeo-Vac-P01 were scheduled for this patient. (B)CT scan images of the tumor at different time points during the treatment. The red arrows indicated the corresponding tumor lesion. SLDs: sum of longest diameters. SLDs: sum of the longest diameters. (C, D) Ex vivo ELISpot assay of IFN- g+PBMCs during vaccination. (E)Increased abundance of peripheral neoantigen-speci ﬁc T cell clones post vaccination as detected by TCR sequencing. (F)Multiplexed IF images of FFPE samples obtained from patient P015 pre- and post-vaccination. Signals of CD8, CD4 and Granzyme B are shown in fuchsias, green and yellow. Double stranded DNA is shown in blue. Shown on the right are the proportion (%) anddensity (counts per mm 2) of CD4+Granzyme B+and CD8+Granzyme B+cells.Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 11 B C D E FA G FIGURE 5 Combination treatment of RFA and neoantigen vaccination inhibited tumor growth and elicited robust local and systemic antitumor immune responses in mice. (A)Schematic illustration of the treatment mice received. Mouse models with unilateral or bilateral tumor(s) were established; mice with bilateral tumors received RFA treatment on the left ﬂank tumor. (B, C) Tumor growth of mice receiving different treatments. Ablation of an irrelevant tumor (4T1) did not help inhibit the growth of CT26 tumor. Combinational use of RFA and iNeo-Vac-P01 (RFA+Vac) signi ﬁcantly slowed down the growth of CT26 tumor, and the inclusion of anti-PD-1 further enhanced this antitumor effect. (D)Flow cytometric analyses of PD-1 expression in tumor tissues (n=3). (E)Flow cytometric analyses of multifunctional T cells, gating on CD8+perforin+ cells (n=3). (F)Flow cytometric analyses of cytotoxic T cells, gating on CD8+IFN- g+cells (n=3). (G)Ex vivo ELISpot assay. Spleen cells were collected and re-stimulated by neoantigen peptides. Data shown in Mean ± SD; two-tailed pvalue was obtained through unpaired t test. ns, not signiﬁcant (p > 0.05); **p ≤0.01; ***p ≤0.001; ****p ≤0.0001Shou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 12 cells elicited robust neoantigen-speci ﬁc responses in melanoma (25,26), suggesting that a stronger TCR signaling was correlated with the addition of anti-PD-1 antibody in the combinational modality. To conclude, the favorable antitumor outcome of RFA plus neoantigen vaccination was probably associated with the enhanced in ﬁltration of activated PD-1+T cells, which could be reinvigorated upon PD-1 blockade, as reported in a previously published study ( 27). Combinational modality elicited stronger local and systemic immune responses in mice The antitumor functions of cytotoxic CD8+T cells include releasing granzymes to induce the apoptosis of target cells, and/ or perforin to generate large transmembrane pores on the membrane of target cells for the diffusion of granzymes ( 28). Here, the antitumor ability of CD8+T cells was evaluated by ﬂow cytometry. Mice in RFA+Vac+anti-PD-1 group showed the highest proportions of CD8+perforin+T cells (25.50%) and CD8+Granzyme B+T cells (3.36%), followed by RFA+Vac (20.10% and 2.89%), RFA+anti-PD-1 (11.10% and 1.55%), RFA alone (9.33% and 1.11%) and PBS (5.36% and 0.36%) (Figure 5E andSupplementary Figure S2 ). To further explore whether the combination treatment of RFA, iNeo-Vac-P01 and/or anti-PD-1 could mount systemic antitumor responses, mice spleen cells were collected for IFN- g secretion analyses through ﬂow cytometry. Not surprisingly, combination treatment consisting of all three modalities showed the highest CD8+IFN-g+T cell proportion (23.00%), followed by RFA+Vac (14.80%), RFA+anti-PD-1 (7.66%), RFA (4.03%) and PBS (1.90%) ( Figure 5F ). Consistent with the ﬂow cytometry results, ELISpot assays for IFN- gsecretion upon ex vivo re-stimulation of T cells with selected neoantigen peptides also demonstrated that the combined three modalities had most spot counts amongst all groups, with RFA+Vac taking the second place ( Figure 5G ). Altogether, RFA in combination with iNeo-Vac-P01 could enhance both local and systemic antitumor immune responses, with anti-PD-1 further promoted the antitumor effects. Discussion Considered as a standard local treatment, RFA is widely used for different cancer types. However, RFA alone cannot prevent cancer progression suf ﬁciently, as new remote lesions might appear ( 29). To date, different modalities including ICIs, TLR9 agonists and c-MET inhibitors displayed their synergies with RFA ( 5,30,31), while no study had been reported to validate the synergies between neoantigen vaccination and RFA. During the past ﬁve years, an increasing number of researchers havedemonstrated that neoantigen is crucial to the success of cancer immunotherapy; more than 130 neoantigen-targeting clinical trials are currently active and many of these neoantigen-based therapies are being studied in combination with checkpoint modulator. It is likely that personalized neoantigen vaccines could become the turning point in improving the survival of cancer patients in the near future. Although vaccination of neoantigens has been proven toaugment local radiotherapy ’s antitumor activity in a few studies ( 32); to our knowledge, our study was the ﬁrst to investigate the synergy between RFA and neoantigen vaccination. As reported previously, some patients who received RFA before neoantigen vaccination seemed to have relatively long OS, implying that RFA could induce tumor necrosis and thereby releasing tumor neoantigens that acted as pro-in ﬂammatory signals ( 10). Herein, we sought to explore the rationale of combining peptide neoantigen vaccination with RFA to improve their antitumor effects. All patients enrolled in our study were late-staged patients with relatively bad prognosis. We selected PFS and OS as the markers to indicate the ef ﬁcacy of neoantigen vaccine, considering that the overall response rate (ORR) and duration of response (DoR) might not be good indicators for the ef ﬁcacy of the treatment for late-staged patients. Besides, both PFS and OS have been reported to be good therapeutic ef ﬁcacy markers for late-stage patients who received immunotherapy or chemotherapy ( 33–39). Patients in our study displayed comparable or longer mPFS and mOS in relative to patients with the same cancer types in these abovementioned studies. Herein, our patients were retrospectively divided into two groups: one received RFA treatment within 6 months before neoantigen vaccination, the other did not. As expected, patients who received pre-vaccination RFA treatment showed longer mPFS and mOS, which correlated with their stronger immune response at baseline and post vaccination. Based on the clinical response, patients were further divided into two subgroups: patients with good clinical response (OS ≥12 months) and patients with poor clinical response (OS < 12 months) to better understand the discrepancy in their clinical response in terms of the differences in T cell subsets as well as cytokine pro ﬁles. Similar tendency in IFN- g, TNF- aand IL-6 expression (pg/mL) was observed, compared to that of our previous published data (16). IFN- gplays an important role in modulating immune response against cancer. No signi ﬁcant difference in IFN- g expression was found between RFA+Vac and Vac patients after vaccination, suggesting that the vaccination of personalized neoantigen peptides contributed to most of IFN- g expression ( Figure 3C ). On the contrary, a larger discrepancy in TNF- aexpression at baseline between patients with good response and patients with poor response in RFA+Vac group was observed, in comparison to that of Vac group, suggesting that RFA might have contributed to higher TNF- aexpression at baseline. Moreover, after vaccination, IL-6 expression was lowerShou et al. 10.3389/ ﬁmmu.2022.1000681 Frontiers in Immunology frontiersin.org 13 in patients with good response compared to patients with poor response, which was in line with a published study concluding that the elevation of IL-6 correlated with poor survival in pancreatic cancer patients ( 40). Our result here demonstrated that neoantigen vaccination could downregulate the expression of tumor-promoting cytokines such as IL-6 to help achieve better clinical response. In addition, a larger proportion of activated T cells in patients with good response was foundcompared to that of patients with poor response upon vaccination ( Figure 3D ). As expected, the differences in the proportions of activated CD4 +,C D 4+CTLA4+and CD8+ CTLA4+T cells between patients with good response and patients with poor response in RFA+Vac group were larger than that in Vac group, although not signi ﬁcantly. This not only explained that patients with good response might have undergone strong T cell activation upon vaccination, thereby upregulating CTLA4, the negative regulator of T cell activation, as a halting mechanism; but also suggested that the inclusion of anti-CLTA-4 immunotherapy might further bene ﬁtt h e s e patients ’clinical response ( 41). Moreover, patient P015 who received multiple RFA treatments, and two batches of vaccines showed more granzyme B in post-vaccination tumor tissue (Figure 4F ), indicating the better in ﬁltration activity of T cells upon vaccination. In addition, CD4+T cells amongst all enrolled patients exhibited a signi ﬁcant increase in PD-1 expression after vaccination ( Supplementary Figure S3 andSupplementary Table S8), suggesting that the addition of anti-PD-1 may further beneﬁt these patients. Combination treatment of RFA and neoantigen vaccination was performed in mi ce to further validate their synergies ( Figure 5 ). Mice receiving RFA and neoantigen vaccination showed slower tu mor growth than mice receiving single modality alone ( Figure 5B ). Triple therapy consisting of RFA, Vac and anti-PD-1 further inhibited tumor growth and showed stronger immune responses compared to double therapy ( Figures 5C –G). Highest IFN- gand perforin expression was found in CD8+cells of the mice receiving triple therapy, respectively ( Figures 5E ,F), suggesting the effective activation of multifunctional T cells by triple therapy. In line with our clinical data ( Supplementary Figures S3 ;Supplementary Figures S4 ;Supplementary Table S8), PD-1 expression in mice receiving RFA+Vac treatment was also found higher compar ed to mice receiving single modality. To be noted, with the inclusion of anti-PD-1, the expression of PD-1 in triple therapy was the highest. This could be explained by the reinvigoration of a T cell subset with high tumor in ﬁltration ability upon the blockade of PD-1, as discussed earlier in the Results Section. Taken these together, our results indicated that cold tumors could potentially turn into hot tumors when neoantigen vaccination is coupled with RFA. More importantly, the antitumor effects of RFA together with neoantigen vaccination co uld be further promoted witht h eb l o c k a g eo fP D - 1 .F u r t h e rs tudies should be conducted to investigate the ef ﬁcacy of triple therapy combining RFA, neoantigen vaccination and anti-PD-1 in patients with different cancer types. Conclusions It was demonstrated in this study that RFA and peptide neoantigen vaccination could h ave synergies in combating existing tumors. Patients who received pre-vaccination RFA treatment displayed better clin ical response upon vaccination of personalized cancer vaccine. Also, the timing for neoantigen vaccination could be of vital importance to control tumor progression, as patients showing better antitumor responses in this study turned out to be those who received more timely vaccination. With the addition of checkpoint inhibition, the synergies of RFA and neoantigen vaccination were further improved in mice model. Taken these together, we conclude that the combinational use of neoantigen vaccination and RFA may bring about longer OS for patients with a variety of cancer types, with immune checkpoint blockade further enhancing the clinical response. Data availability statement The original contributions presented in the study are included in the article/ Supplementary Material .