It has become apparent that innate immune cells are involved in the regulation of adaptive T cell immunity. Our previous studies revealed that cryo-thermal therapy induced M1 macrophage polarization and DCs maturation were required for the shaping of systemic long-lived T cell mediated anti-tumour memory immunity. Eosinophils are multifunctional innate effector cells and there is lack of knowledge on the role of eosinophils in cryo-thermal-induced anti-tumour immunity. This study revealed that cryo-thermal therapy activated eosinophils in spleen at early stage following the treatment. Furthermore, cryo-thermal-activated eosinophils exerted versatile immunologic regulation from innate immunity to anti-tumour adaptive immunity, such as M1 macrophage polarization, DCs maturation, differentiation of CD4-CTL subtypes and enhanced cytotoxicity of CD8+ T cells. Our study indicated that the cryo-thermal-activated eosinophils was essential for the shaping of durable anti-tumour memory immunity. Thus, our results present a new concept for eosinophils mediated anti-tumour immunity after cryo-thermal therapy.Previous, we established a novel therapeutic approach to tumour of cryo-thermal therapy, which can induce durable anti-tumour memory immunity mediated by CD4 Either RFA or cryosurgery alone was reported to elicit immunological responses. RFA modulates immune system and induces anti-tumour immunity5. RFA can induce tumour cell necrosis and subsequent release of tumour antigen4. The physical barriers of tumours are ablated after the treatment, which results in increasing infiltration of immune cells, including NK cells, APCs and T cells5. In addition, cryoablation can also induce potent anti-tumour immune response through recovering tumour-educated dendritic cells (DCs) or activating of effector T cells7. However, there is no lasting and enough response to establish a sustained anti-tumour immunity for clinical applications9.Accumulating evidences show that inducing durable anti-tumour immunity are essential in cancer therapy. Local thermal ablation, such as conventional RFA (using temperature >60 °C), or cryosurgery (using temperature −20 °C to −40 °C) is generally accepted for the treatment of metastatic tumour in clinic11. Cryo-thermal therapy significantly causes tumour cell necrosis and markedly improves cancer survival on 4T1 murine mammary carcinoma and B16F10 murine melanoma model13. Cryo-thermal therapy induces a specific and durable anti-tumour memory immunity which dependents on antigen-specific CD4+ T cells12. The modulation and sustain of T cell-mediated adaptive immunity is critical for efficacious tumour cell killing and generating effective anti-tumour memory immunity13. The innate immune cells are capable of bridging innate and adaptive anti-tumour immune responses14. A large body of data demonstrated that activating the innate immune response could stimulate both local and systemic antitumour T-cell responses. The innate immune cells, such as DCs and macrophages, are capable of cross-prime T cells leading to the activation of adaptive immune response15. Infiltration of mature, immunogenic DCs into tumours confers immune activation16. Fully mature DCs can present antigen to CD4+ and CD8+ T cells leading to trigger antigen-specific cytotoxic T lymphocytes (CTLs)17. Tumour-associated macrophages are divided into alternative M2 and classical M1 macrophages. M2 macrophages have inherent immune suppressive activity and promote tumour growth18. In contrast, M1 macrophages are characterized by high expression level of pro-inflammatory factors and presentation of tumour-specific antigens to T cells19. Previously, cryo-thermal-induced M1 macrophage polarization was exclusively responsible for the subsequent DC maturation, differentiation of CD4-CTL and Th1 subsets, which was crucial for mediating long-term anti-tumour memory immunity after cryo-thermal therapy20. But whether other innate immune cells also mediated cryo-thermal-induced anti-tumour memory immunity should be addressed.To enhance the curative power of thermal ablation, we establish a new kind of therapeutic approach to tumour of cryo-thermal therapy through the alternative cooling and heating of tumour tissues21. Growing evidence supported eosinophils contributing to anti-tumour immunity. Clinically, the infiltration of tumour-associate tissue eosinophils (TATE) are usually indicated a good prognosis, such as head and neck cancer, colorectal cancer, and prostate cancer24. Eosinophils can exert cytotoxic properties for tumour cell upon activating via the release of several cytotoxic granules, including eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN), major basic protein (MBP) and eosinophils peroxidase (EPO). Meanwhile, eosinophils are also a major source of numerous immunologically active factors, for example, Th1 and Th2 cytokines and chemokines25. Moreover, eosinophils can also intact with other immune cells such as DCs, macrophages and T cells to modulate anti-tumour immunity28. However, if eosinophils contribute to anti-tumour immunity, especially, the role of eosinophils in cryo-thermal-induced long-lasting anti-tumour memory immunity remained unclear.Eosinophils are multifunctional granulocytes linked to the modulation of diverse inflammatory response including allergic diseases, helminth infections, tissue injury and tumour immunityin vivo after cryo-thermal therapy was constructed evaluate the role of cryo-thermal-activated eosinophils in shaping of long–term anti-tumour immunity. We discovered that cryo-thermal therapy induced the activation of eosinophils at early stage following the treatment. Cryo-thermal-activated eosinophils play a crucial role in M1 macrophage polarization, DCs maturation, functional differentiation of CD4+ T cells, generation of cytotoxic CD8+ T cells, and finally triggering long-lasting anti-tumour memory immunity. Thus, our study presented a new concept of eosinophils mediated anti-tumour immunity after cryo-thermal therapy that would lead to novel therapeutic strategies.In this study, we explored whether eosinophils would serve as key cells that contributed to tumour-specific T cell responses mediated by cryo-thermal therapy. We investigated the phenotype and function of eosinophils after local cryo-thermal therapy using a murine B16F10 melanoma model. And eosinophils depletion 29. In this study, we also repeated to study the therapeutic effect of this therapy, and survival rates in murine B16F10 melanoma was over 80%  was evaluated by CCK-8. On day 3 following the treatment, there was no significant difference in cytolysis of eosinophils from cryo-thermal or tumour-bearing mice co-cultured with B16F10 cells  on day 3, 5, 14 after cryo-thermal therapy. For further verification, the cytotoxicity of splenic eosinophils against B16F10 melanoma cells was tested lls Fig. . But on lls Fig. . The res27. The above results revealed that eosinophils were activated from day 3 after cryo-thermal therapy  and M1 macrophage (CD11b+F4/80+CD86+MHCII+) on day 3 following cryo-thermal therapy  in spleen and blood were up-regulated after cryo-thermal therapy  in spleen and blood, but resulted in less M1 macrophage population (CD11b+F4/80+CD86+MHCII+) as compared to cryo-thermal therapy + isotype group . The splenic eosinophils were isolated from tumour-bearing mice on day 15 following tumour inoculation (termed as tumour-bearing eosinophils), and cryo-thermal treated mice on day 3 . The tumour-bearing DCs alone was as the control. The tumour-bearing eosinophils were co-cultured with tumour-bearing DCs at a ratio of 1:1 for 24 h (tumour-bearing eosinophils + tumour-bearing DCs). Meanwhile, cryo-thermal eosinophils were co-cultured with tumour-bearing DCs, adding isotype IgG1 , IFN-γ neutralizing antibody , IL-4 neutralizing antibody , respectively. We analyzed the phenotype and function of DCs by using flow cytometry and RT-qPCR, respectively. The proportion of total CD11c+ cells was not significant difference in all the groups  was significantly down-regulated when DCs were co-cultured with tumour-bearing eosinophils as compared to DCs cultured alone. Interestingly, higher level of matured DCs was induced when tumour-bearing DCs were co-cultured with cryo-thermal eosinophils as compared to tumour-bearing DCs co-cultured with tumour-bearing eosinophils. The percentage of matured DCs in IL-4 neutralizing group was significantly increased as compared to cryo-thermal eosinophils + tumour-bearing DCs group, but blockade of IFN-γ had no difference . Siglec-F+ eosinophils were isolated from tumour-bearing mice on day 15 following tumour inoculation (termed as tumour-bearing eosinophils), and cryo-thermal treated mice on day 3 . The groups of in vitro assays were listed as follows: tumour-bearing macrophage alone was considered as the control group; tumour-bearing eosinophils + tumour-bearing macrophage group; cryo-thermal eosinophils + tumour-bearing macrophage group; cryo-thermal eosinophils + tumour-bearing macrophage + anti-IFN-γ antibody, termed as IFN-γ neutralizing group; cryo-thermal eosinophils + tumour-bearing macrophage + anti-IL-4 antibody, termed as IL-4 neutralizing group. The phenotype and function of co-cultured macrophages were analyzed by using flow cytometry and RT-qPCR, respectively. The percentage of total F4/80+ macrophages was higher in IL-4 neutralizing group as compared to cryo-thermal eosinophils + tumour-bearing macrophage group  population as compared to tumour-bearing macrophages alone. Blockade of IFN-γ with neutralizing antibodies resulted in slightly high proportion of M1 macrophage compared with that in cryo-thermal eosinophils + tumour-bearing macrophage group, and IL-4 neutralizing reduced it  was determined. There was no significant difference in cytolysis of splenic CD8+ T cells co-cultured with B16F10 cells at effect-to-target cell ratio of 1:2 in three groups. However, when CD8+ T cells from cryo-thermal + Siglec-F mAb treated mice were co-cultured with B16F10 cells at ratio of 1:1, the cell viability of B16F10 cells was significantly increased as compared to cryo-thermal + isotype groups. More interestingly, when CD8+ T cells from cryo-thermal + Siglec-F mAb treated mice were co-cultured with B16F10 cells at ratios of 4:1 and 8:1, CD8+ T cells promoted the proliferation of B16F10 cells . Eosinophils are typical innate immune cells which can release several cytokines and interact with various innate and adaptive immune cells upon activation37. Moreover, eosinophils are associated with a favorable prognosis, particularly in solid tumours38. However, the role of eosinophils in the process of cryo-thermal-induced anti-tumour immunity is unclear. In our study, cryo-thermal therapy activated eosinophils in the early stage led to the induction of strong innate and adaptive anti-tumour immune response. Considering that cryo-thermal therapy triggered strong anti-tumour memory immunity to improve the long-term survival in murine B16F10 melanoma13, we suggested that the activated eosinophils in early stage following cryo-thermal therapy was essential to mediate effective and durable anti-tumour immunity.It is generally accepted that an effective innate immunity is a prerequisite for inspiring a persistent adaptive immunity25. Moreover, cryo-thermal therapy improved the cytolytic activity of eosinophils, as demonstrated by the up-regulation of cytotoxic molecules (e.g. granzyme-B and perforin), and enhanced tumour-killing capacity on tumour cells. More importantly, cryo-thermal-activated eosinophils enhanced M1 macrophage polarization, DCs maturation, differentiation of CD4+ T cells into CD4-CTL subset, and generation of cytotoxic of CD8+ T cells. We believed that eosinophils were very important in the process of cryo-thermal-induced anti-tumour immunity from innate immunity to adaptive immunity.In our study, cryo-thermal therapy induced the accumulation of eosinophils into spleen and elicited fully activation of eosinophils from day 3 after the treatment. The splenic eosinophils in cryo-thermal-treated mice expressed high level of chemokines and pro-inflammatory cytokines , which could create immunostimulatory microenvironment contributing to anti-tumour immunity16. But only mature DCs can stimulate T cells and activate a strong adaptive immune response39. Therefore, DCs maturation was required for subsequently T cells activation and shaping of effective anti-tumour immune response. DCs maturation relies on the local microenvironment and was regulated by other immune cells40. CpG activated eosinophils induce DCs maturation during chronic inflammatory disease26. In this study, cryo-thermal-activated eosinophils played a regulatory role in induction of maturation DCs. Eosinophils depletion down-regulated the expression of pro-inflammatory cytokines (IL-7 and IL-6) and up-regulation the production of immunosuppressive molecules (IDO2 and PD-L1) in DCs, while the inflammatory cytokines were required for T cell activation and immunosuppressive molecules could induce T-cell tolerance42. Hence, our findings indicated that cryo-thermal-activated eosinophils could induce the activation and maturation of DCs.DCs present antigens to T cells and provide a series of crucial signals (co-stimulatory molecules and cytokines) for T cell activation and differentiation, thereby modulate the adaptive immune response43. But M2 macrophages promote tumour invasion and metastasis18. Only M1 macrophages serve as a pivotal role in the activation of T cells and formation of durable anti-tumour immunity18. Macrophages can change their functional phenotype in response to specific environmental stimuli44. Hence, reversing macrophages from immunosuppressive M2 phenotype to immunostimulatory M1 phenotype is useful in promoting T cells activation and differentiation. In this study, cryo-thermal-activated eosinophils modulated M1 macrophages polarization, along with the up-regulated expression of co-stimulatory surface molecules (CD86 and MHC II), M1-associated cytokines  and down-regulation of immunoregulatory molecules (Arg-1 and CD206).Among these effector cells of innate immunity, macrophages represent the most important cell of innate immunity. First, macrophages could kill neoplastic cells by phagocytosis. Second, antigen processing by macrophages can convert the antigen into several antigenic epitopes, which can be recognized by T lymphocytes. Moreover, macrophages also secrete cytokines  that play a variety of roles in promotion of inflammation, enhancement of phagocytosis, activation of resting T lymphocytesIn this study, the percentage of matured DCs in spleen and peripheral blood was not changed after cryo-thermal therapy with eosinophils depletion, while M1 macrophage was decreased. Meanwhile, eosinophils depletion resulted in a partial reduction of pro-inflammatory cytokines  in DCs, but a comprehensive down-regulation of M1-associated cytokines in macrophage, which indicated that cryo-thermal-activated eosinophils had a stronger effect on M1 macrophage polarization than DCs maturation. In our previous study, cryo-thermal therapy triggered M1 macrophage polarization on day 5 following the treatment, while fully maturation of DCs was emerged on day 14. Therefore, we suggested that M1 macrophage polarization occurred prior to DCs maturation after cryo-thermal therapy, and cryo-thermal-activated eosinophils firstly induced M1 macrophage polarization. However, the interactions of cryo-thermal-activated eosinophils, macrophages and DCs should be furtherly studied.31. IL-4 produced by eosinophils is always associated with anti-inflammatory activity and maintains the balance of Th1/Th245. IL-4-expressing eosinophils play an important role in macrophage development45. In this study, blockade of IFN-γ produced by eosinophils inhibited DCs maturation, but IL-4 neutralization promoted DCs maturation, which suggested that cryo-thermal-activated eosinophils modulated DCs maturation via the antagonistic interaction between IFN-γ and IL-4. In addition, blockade of IFN-γ produced by eosinophils inhibited M1 macrophage polarization and promoted the activation of M2 macrophage. However, IL-4 neutralization down-regulated the expression of M1-associated cytokines and pleiotropic regulated the expression of immunoregulatory molecules. We speculated that, unlike the antagonistic effects of IFN-γ and IL-4 on DCs maturation, IFN-γ and IL-4 derived from cryo-thermal-activated eosinophils would synergistically promote M1 macrophage polarization. But the role of IFN-γ and IL-4 derived from cryo-thermal-activated eosinophils in M1 macrophage polarization and DCs maturation should be further investigated.Cryo-thermal therapy induced the expression of IFN-γ and IL-4 in eosinophils from day 3 after the treatment. IFN-γ can enhance macrophage and DCs activation through TLR ligation+ T cells have direct cytotoxic activity against tumour cells via the expression of cytotoxic perforin/granzyme-B and IFN-γ46. The optimal polarization of CD4+ T cells is essential to obtain durable systemic anti-tumour immunity47. CD4+ CTL and Th1 subsets mainly exert anti-tumour activity, while the regulatory T cells (Tregs) have potent tumour-promoting activity49. The function of other CD4+ T cell subsets, Th2 and Th17 depend on the certain circumstances47. Eosinophils have the ability to physically interact with CD4+ T cells, and provide costimulatory signals for the activation of CD4+ T cells38. In this study, Eosinophils depletion inhibited the differentiation of CD4+ T cells into CD4-CTL, and inhibited the differentiation of Tregs and Th17 cells. And low level of chronic exposure to Th17-associated cytokines may facilitate cancer progression50. The Th2-assoicated cytokine IL-4 was decreased, and IL-5 was up-regulated after eosinophils depletion. IL-4 produced by CD4+ T cells may inhibit angiogenesis, promote the infiltration of eosinophils and macrophages into tumour and exert anti-tumour effects, while IL-5 mainly has immunosuppressive and tumour-promoting function51. Generally, cryo-thermal-activated eosinophils could significantly promote the differentiation of CD4+ T cells into CD4-CTL subset and inhibit the differentiation of Tregs, Th17 and Th2 cells. Meanwhile, cryo-thermal-activated eosinophils could enhance the cytotoxic activity of CD8+ T cells. The result showed that cryo-thermal-activated eosinophils have the capacity to directly activate the anti-tumour CD4+ T cells and cytotoxic CD8+ T cells.Effector T cells are the major players in adaptive immunity for tumour destruction. Activated CD852. The proper balance between IL-12 and IL-10 controls the appearance of immune response53. After eosinophils depletion, the decreased expression of IL-12 in macrophage would limit the expression of IL-10 to maintain the balance of IL-12/IL-10. iNOS is a critical pro-inflammatory cytokine which can mediate the production of nitric oxide (NO) and inhibit the production of macrophage-derived TGF-β54. NO is an essential mediator of macrophage mediated cytotoxicity in anti-tumour immunity55. CCL22 can contribute to the infiltration and inflammatory function of macrophage56. Hence, we speculated that the down-regulated expression of iNOS and CCL22 in macrophage after eosinophils depletion would hamper cryo-thermal-induced anti-tumour immunity.In this study, the expression of IL-10, inducible nitric oxide synthase (iNOS) and CCL22 in macrophages was decreased after eosinophils depletion. IL-10 acts as an anti-inflammatory cytokine which plays a key role in limiting the extent of immune activation57. The high expression of IDO1 would play an important role in limiting exaggerated inflammatory reactions after cryo-thermal therapy. In addition, Arg-1 was expressed at a much higher level after tumour-bearing macrophage co-cultured with cryo-thermal-activated eosinophils as compared to tumour-bearing macrophage co-cultured with tumour-bearing eosinophils. The role of macrophage-derived Arg-1 in tumour immunity remains controversial58. Macrophage could induce anti-tumour activity through the depletion of L-arginine by Arg-1 within the tumour microenvironment59. So, the high level of Arg-1 in macrophage could be helpful for anti-tumour immunity after cryo-thermal therapy.In our study, the expression level of IDO1 was up-regulated after tumour-bearing DCs co-cultured with cryo-thermal-activated eosinophils as compared to tumour-bearing DCs co-cultured with tumour-bearing eosinophils. IFN-γ can induce the expression of IDO in DCs and IFN-γ-IDO axis is responsible for avoiding harmful hyperinflammatory response29. Since DAMPs can act as a danger signal to activate eosinophils and prolong their lifespan61, we speculated that cryo-thermal therapy induced up-regulated DAMPs release would play a vital role in the activation of eosinophils. In the near future study, we will investigate the mechanism of cryo-thermal therapy induced activation of eosinophils based on the above hypothesis.In our previous study, cryo-thermal therapy caused extensive tumor necrosis and elevated extracellular release of DAMPs (damage associated molecular patterns), such as HSP70, CRT and HMGB1 (unpublished data) from necrotic tumor cells+ T cells differentiation into CD4+ CTL and generation of cytotoxic CD8+ T cells shaping strong systematic anti-tumour immune response, which enhanced the therapeutic efficacy for metastatic tumour.In summary, our study demonstrated that cryo-thermal therapy induced eosinophils activation at early stage after the treatment, which was a pivotal cell of the innate immune system involved in the regulation of cryo-thermal-induced adaptive immunity. Cryo-thermal-activated eosinophils could promote M1 macrophage polarization, and DCs maturation, meanwhile, cryo-thermal-activated eosinophils could directly promote CD45 B16F10 murine melanoma cells.All animal experiments have been approved by the Animal Welfare Committee of Shanghai Jiao Tong University. Experimental methods were conducted according to the guidelines of Shanghai Jiao Tong University Animal Care (approved by Shanghai Jiao Tong University Scientific Ethics Committee). The female C57BL/6 mice were purchased directly from Shanghai Slaccas Experimental Animal Co., Ltd (China). Mice were maintained in isolated cages. At 6–8 weeks of age, the mice were injected subcutaneously in the right femoral region with 5 × 1029. Briefly, the mice were divided into two groups: tumour-bearing group without any treatment and cryo-thermal group on day 12 after tumour inoculation. The mice in cryo-thermal group were injected intraperitoneally (i.p.) with 1.6% pentobarbital sodium  for anesthesia. The subcutaneous tumour was freezed with liquid nitrogen at −20 °C for 5 min, then rewarming to 6–8 °C at room temperature and heated with radiofrequency (RF) at 50 °C for 10 min. The therapy system was developed by our laboratory. All the procedures were conducted at aseptic condition.Cryo-thermal therapy was constructed as previously described in detailThe spleen and peripheral blood were collected after therapy. Single-cell suspension of peripheral blood cells and splenocytes were prepared after treatment with erythrocyte-lysing reagent to remove red blood cells. For cell surface staining, the cells were stained with fluorescence conjugated antibodies which could bind cell specific surface marker for at 4 °C 30 min. For intercellular staining, the cells were stimulated with Cell Activation Cocktail  for 4 hours, cells were incubated with anti-FcγR antibody, followed by surface staining with corresponding surface marker antibodies then fixed, permeabilized and incubated with antibodies binding specific intercellular marker. Data were acquired using BD FACS Aria II cytometer (BD Biosciences) and analyzed using FlowJo software. Fixation Buffer, Intracellular Staining Permeabilization Wash Buffer and Cell Activation Cocktail (with Brefeldin A) were purchased from Biolegend . Fluorochrome-conjugated monoclonal antibodies: CD3-FITC (clone 145-2C11), CD11b-FITC (clone M1/70), CD11c-FITC (clone N418), CD86-PE (clone GL-1), I-A/I-E-PerCP/Cy5.5 (clone M5/114.15.2), Gr-1-PE (clone R86-8C5), IL-4 (clone 11B11) . Siglec-F-BV421 (clone E50-2440), CD4-APC (clone RM4–5), IFN-γ (clone XMG1.2) . CD8-APC (clone 53.6.7), F4/80-APC (clone BM8) .+ and CD8+ T cells. DCs were isolated by DC isolation micro-bread kit (StemCell) in line with manufacturer’s instructions. DCs with a purity of >90% were used for experiments. For macrophages isolation, splenocytes were plated in DMEM supplemented with 10% FBS at 37 °C in a humidified 5% CO2 incubator for 1 h, then the supernatant fraction was poured off and the adherent splenocyte fraction was processed for CD68+ macrophages isolation by using EasySepTM PE positive selection kit (StemCell) in line with manufacturer’s instructions. Macrophages with a purity of >90% were used for experiments. Siglec-F+ eosinophils were isolated by using EasySepTM PE positive selection kit (StemCell) in line with manufacturer’s instructions. Eosinophils with a purity of >90% were used for experiments. CD4+ and CD8+ T cells were isolated by EasysepTM CD4+ T cell negative selection kit and EasysepTM CD8+ T cell negative selection kit (StemCell), respectively. CD4+ and CD8+ T cells with a purity of >90% were used for experiments.Single-cell suspension of splenocytes were prepared to isolate DCs, macrophages, eosinophils, CD4+ and CD8+ T cells. The RNA purity of all samples was measured using the absorbance at 260/280 nm, which was above 1.9. PrimeScript RT reagent kit (TaKaRa) was used to made cDNA. Reaction system for quantitative real-time qPCR was made using SYBR Premix Ex Taq (TaKaRa) in 284-well plates, and performed on ABI 7900HT sequence detection system (Applied Biosystems). Data was analyzed using SDS software. The primer sequence of each gene used in our study was submitted in Supplementary Table TRIzol Reagent (TaKaRa) was used to isolated total freshly RNA from purified splenic DCs, macrophages, eosinophils, CD4For the depletion of eosinophils, 15 μg anti Siglec-F  was injected intraperitoneally on day 1 before cryo-thermal therapy and day 3, 7, 11 after cryo-thermal therapy. Control groups consisted of mice were injected intraperitoneally with isotype-matched Ab (R&D Systems).+ eosinophils were isolated from tumour-bearing mice on day 15 after the tumour inoculation and cryo-thermal treated mice on day 3 after therapy as previously described. The splenic CD11c+ DCs, CD68+ macrophages, CD4+ and CD8+ were isolated from tumour-bearing mice on day 15 following tumour inoculation. The tumour-bearing DCs or macrophages were co-cultured with eosinophils from tumour-bearing mice at a ratio of 1:1, respectively. The co-cultured cells were plated in DMEM supplemented with 20% serum from tumor-bearing mice at 37 °C in a humidified 5% CO2 incubator for 24 h. Meanwhile, the tumour-bearing DCs or macrophages were also co-cultured with eosinophils from cryo-thermal mice at a ratio of 1:1, respectively. The co-cultured cells were plated in DMEM supplemented with 20% serum from tumor-bearing mice at 37 °C in a humidified 5% CO2 incubator for 24 h. Furthermore, the tumour-bearing T cells were co-cultured with eosinophils from tumour-bearing or cryo-thermal mice at a ratio of 5:1, respectively. The co-cultured cells were plated in DMEM supplemented with 20% serum from tumor-bearing mice or cryo-thermal mice at 37 °C in a humidified 5% CO2 incubator for 24 h. The tumour-bearing DCs, macrophages and T cells were considered as control. The co-cultured cells were analyzed by flow cytometry, and purified for RT-qPCR assay.The splenic Siglec-Fin vitro, we used neutralizing antibodies to IL-4 or IFN-γ (anti-mouse IL-4 (clone 11B11), anti-mouse IFN-γ (clone XMG1.2), 10ug/ml, and all from Sungene Biotech) and the isotype control antibody Rat IgG1 .For evaluated the effect of IL-4 and IFN-γ derived from eosinophils on DCs and macrophages 2 . After incubation for 24 hours, the supernatants were poured off, and the adherent cells were survival B16F10 cells. Then 90ul fresh DMEM and 10ul CCK-8 solution were added to each well and additionally incubated for 2 hours. And a microplate reader was used to measure the absorbance at 450 nm. The B16F10 cells were used as control, and cell culture media alone was used as blank, respectively. The survival rate of B16F10 cells was calculated as /[(control-blank)] × 100%.For evaluating the eosinophils mediated cytotoxicity against tumour cells after cryo-thermal therapy, a colorimetric cell-counting kit  assay was used according to the manufacturer’s instructions. Briefly, splenic eosinophils were isolated from tumour-bearing or cryo-thermal-treated mice. Eosinophils were co-cultured with B16F10 cells at the ratio of eosinophils: B16F10 cells = 1:1, 2:1, 4:1, 8:1 and 16:1 at 37 °C in 5% CO+ or CD8+ T cells after cryo-thermal therapy with eosinophils depletion. Briefly, splenic CD4+ or CD8+ T cells were isolated from tumour-bearing mice, cryo-thermal with isotype treated mice and cryo-thermal with Siglec-F mAb treated mice. CD4+ or CD8+ T cells were co-cultured with B16F10 cells at the ratio of CD4+/CD8+ T cells: B16F10 cells = 1:2, 1:1, 4:1 and 8:1 at 37 °C in 5% CO2 . After incubation for 24 hours, the supernatants were poured off, and the adherent cells were survival B16F10 cells. Then 90ul fresh DMEM and 10ul CCK-8 solution were added to each well and additionally incubated for 2 hours. And a microplate reader was used to measure the absorbance at 450 nm. The B16F10 cells were used as control, and cell culture media alone was used as blank, respectively. The survival rate of B16F10 cells was calculated as /[(control-blank)] × 100%.Similar to the above, a CCK-8 assay was used to evaluate the cytotoxicity against tumour cells mediated by CD45 B16F10 melanoma cells (i.v.) 45 days after the therapy. Two weeks later the mice were sacrificed for lung tumor nodules counting.To investigate the effect of eosinophils on systemic immunologic memory response after cryo-thermal therapy, primary B16F10 melanoma tumor in C57BL/6 mice were treated with cryo-thermal therapy, and eosinophils were depleted according to previous description(n = 6 per group). The living mice were rechallenged with 1 × 10*p < 0.05, **p < 0.01, ***p < 0.001. P-values < 0.05 was considered to be statistically significant.onThe student’s t-test and two-way ANOVA were used for statistical comparisons using Graph Pad Prism 6. Figures denoted statistical significance of Supplementary_information


Glioma, which accounts for more than 30% of primary central nervous system tumours, is characterised by symptoms such as headaches, epilepsy, and blurred vision. Glioblastoma multiforme is the most aggressive, malignant, and lethal brain tumour in adults. Even with progressive combination treatment with surgery, radiotherapy, and chemotherapy, the prognosis for glioma patients is still extremely poor. Compared with the poor outcome and slowly developing technologies for surgery and radiotherapy, the application of targeted chemotherapy with a new mechanism has become a research focus in this field.Moreover, targeted therapy is promising for most solid tumours. The tumour-tropic ability of stem cells, including neural stem cells and mesenchymal stem cells, provides an alternative therapeutic approach. Thus, mesenchymal stem cell-based therapy is based on a tumour-selective capacity and has been thought to be an effective anti-tumour option over the past decades. An increasing number of basic studies on mesenchymal stem cell-based therapy for gliomas has yielded complex outcomes.In this review, we summarise the biological characteristics of human mesenchymal stem cells, and the current status and potential challenges of mesenchymal stem cell-based therapy in patients with malignant gliomas. Gliomas are the most common aggressive primary central nervous system tumours in adults , and theDifferent types of stem and progenitor cells, including neural stem cells (NSCs), mesenchymal stem cells (MSCs), haematopoietic progenitor cells (HPCs), and embryonic stem cells (ESCs), have been shown to have the capacity for tumour-tropic homing and migration both in vitro and in vivo –11. In tIn this current review, we summarise the biological characteristics of human MSCs and present the principle of MSC-based therapy. Then, we analyse the current status and potential challenges in basic and preclinical studies for MSC-based therapy in patients with gliomas.Friendenstein et al. reported the existence of MSCs in bone marrow in 1966 . SubsequThe regulation of the biological features of MSCs is complex and multi-layered. The microenvironment conditions in different sources is thought to be the main reason for the slight differences in biological characteristics and genome patterns of MSCs. In vitro, TGF-β, bone morphogenetic proteins, insulin, dexamethasone, and other differentiation factors could induce MSCs into multiple cell lines –35. SeveRecently, tumour-associated MSCs (TA-MSCs) have been regarded as integral components involved in several hallmarks of different tumours, such as initiation, promotion, progression, and metastasis. Some of these TA-MSCs already exist in many tissues, but some of them are recruited into tumour microenvironments to take part in the progression of tumours via the production of various growth factors, chemokines, and cytokines and to cross-talk with tumour cells , 44.MSCs are rarely found in the brains of normal mice and humans, and these MSCs with classical features are located in a vascular niche , 43. An Based on their tumour-tropism and facile ability to cross the blood-brain barrier, MSCs have been utilised as carrying vectors for glioma therapy.MSC-based therapy for gliomas is a promising novel approach to transport different genes, proteins, and viruses. Various therapy approaches could suppress the growth of gliomas through different mechanisms, such as immunotherapy, suicide protein therapy, virus-based therapy, cytotoxic factor-based therapy, and anti-angiogenesis therapy, which are discussed more in the following sections.Over the past decade, the ability of tumours to evade the immune system has always been a significant research direction for the rapid development of tumour immunotherapy. Malignant gliomas could escape the host immune system by secretion of immunosuppressive agents, inhibition of T-cell proliferation, and reduction in immune responses , 47. ThuIn 2004, Nakamura et al. found that intra-cranial administration of gene-modified MSCs expressing IL-2 could migrate towards a glioma site  and then+ and CD8+ T-cell infiltration and then stimulating subsequent cascade immune networks; these modified MSCs can be exploited to a therapeutic advantage against gliomas.This review provides a summary of currently open and recruiting MSC-based immune-therapy studies for gliomas, as shown in Table Suicide protein-based therapy is a widely applied form of gene therapy in the cancer field. This approach entails mRNA encoding a pro-drug-activating enzyme (suicide protein) transduced into MSCs, the injection of these MSCs into the tumour sites, and the subsequent conversion of non-toxic pro-drugs into toxic pro-drugs, leading to regression of tumour cells in vivo , 53. To The HSV-TK/GCV system has been most reported in glioma treatment. This system is based on the ability of HSV-TK to efficiently phosphorylate the pro-drug ganciclovir to its monophosphate state, which is further phosphorylated by cellular enzymes to GCV-triphosphate (GCV-TP) . MSCs exLater, De Melo et al. designed a strategy using adipose-derived MSCs (AT-MSCs) expressing HSV-TK combined with GCV, which was able to exert a cytotoxic effect on U87 cells in vitro and diminish tumour size , 59. SimCytosine deaminase (CD) is another pro-drug-activating enzyme that can convert the non-toxic pro-drug 5-fluorocytosine (5-FC) to toxic 5-fluorouracil (5-FU), which effectively inhibits tumour growth. Early in 2012 a related study reported the use of CD-expressing MSCs combined with 5-FC for the treatment of intra-cranial rat gliomas and protected normal brain tissue from damage . The CD/Oncolytic virotherapy is also a novel approach in which viruses are genetically modified to selectively replicate in tumour cells. The virus is released from its carrier at tumour sites and then selectively kills tumour cells and protects normal tissues from injury. However, the viral particles could be attacked and eliminated by the host immune system. In addition, the low-efficiency virus is far away from the tumour site. To overcome the barriers, tumour-tropic migratory cells may be used to deliver viral particles to the distant sites of tumours and shield a therapeutic virus from the host immune system .Many studies using MSCs loaded with oncolytic adenovirus CRADs and Delta-24-RGD have demonstrated extended delivery of oncolytic viruses and prolonged survival of glioma-bearing animals treated with stem cell-mediated oncolytic virotherapy. In 2008, Sonabend et al. reported that MSCs provide CRAD delivery to distant glioma cells and that this delivery significantly enhanced the infection and apoptosis of tumour cells compared with injection of distant CRADs alone, showing a therapeutic advantage. Later, MSCs carrying Δ24-RGD (hMSC-Δ24) were injected into the carotid artery of mice and then migrated to tumour sites, resulting in inhibited growth and improved survival of mice. Taken together, previous results indicated that MSCs migrate and deliver CRADs and Delta-24-RGD to distant glioma cells and improved the outcome of oncolytic virotherapy for glioma –69. ThesIn the future, there is a possibility that tumour-specific antigens will replace the use of viruses in which the anti-viral immune response is caused by carrier proteins, and thus the virus could combine the benefits of virotherapy with immune-therapy to achieve the aim of treating cancer.One novel strategy of tumour treatment is to induce apoptosis of tumour cells. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor (TNF) superfamily and could induce apoptosis of tumour cells through activation of the TNF/CD95L axis and spare the majority of non-malignant cells . StudiesAngiogenesis is an important characteristic event in the development and progression of gliomas. The use of factors that inhibit angiogenesis is thought to be one of the strategies for glioma treatment.Pigment epithelial-derived factor (PEDF) is a 50-kDa secreted glycoprotein that can activate the Fas/FasL pathway to induce endothelial cell death and regulate the balance between inducers and inhibitors of angiogenesis , 74. PreThus, engineered MSCs act as an inhibitory molecular vehicle to promote apoptosis and attenuate angiogenesis in gliomas, and they may have the potential as a therapeutic agent in the clinical application of stem cell therapy against gliomas.Although MSC-based therapy for malignant gliomas has become increasingly popular, all MSC-based therapies, similar to other stem cell-based therapies, appear to be limited in their effectiveness. Some recent studies on cross-talk between MSCs and glioma cells reveal the potential challenges to MSC-based therapy for gliomas.There is no clear conclusion on whether MSCs themselves support or suppress the progression of glioma. AT-MSCs can increase the size of glioma tissue by a reduction in apoptosis and secretion of VEGF . UC-MSCsTherefore, MSCs from different sources showed different abilities to promote or suppress the growth of glioma cells under different conditions. If these MSCs were investigated as delivery vectors, the effects of MSCs supporting or suppressing the growth of gliomas should also be considered.− gbMSCs produced more VEGF and prostaglandin E2 (PGE2) than did CD90+ gbMSCs, and these two subpopulations had 211 differentially expressed genes but showed no mRNA differences [− gbMSCs could differentiate into pericytes and contribute to neovascularization in the glioma microenvironment [+ gbMSCs could increase the growth and invasion of glioma cells under a serum deprivation condition (data not shown). Figueroa et al. also found that gbMSCs could increase the tumourigenicity of glioma stem cells by microRNA-1587 exosome transfer [gbMSCs were first isolated from fresh glioma tissues and identified by our team in 2014. We found that gbMSCs showed classical features of morphology, surface markers, and differentiation. The gbMSCs secrete different factors dependent on the inter-cellular cross-talk and hypoxia conditions . Moreoveferences . Our teaironment . CD90+ gtransfer  and mainAs well as an assistant role of MSCs in tumour microenvironments, some studies have reported that MSCs can directly transform to malignant cells and have revealed a new challenge to MSC-based therapy for cancer. In 2004, Houghton et al. suggested that BMSCs were recruited to the sites of pre-cancerous lesions by inflammatory cytokines and then transformed into gastric cancer cells to promote cancer progression through a series of transformations . Serakin− gbMSCs showed stronger ability for tumour immunosuppression than their CD90+ counterparts [In tumour microenvironments, MSCs could support tumour growth by suppression of host immune responses. Once MSCs are recruited into a tumour microenvironment, they can secrete several ligands to help tumour cells build the immunosuppressive environment by recruiting monocytes, macrophages, and myeloid-derived suppressor cells. These immune cells then inhibit the anti-tumour immune responses of T cells . In glioterparts . HoweverCurrently, MSC-based therapy for gliomas has become an increasing focus of research. MSCs are regarded as effective vectors to deliver therapeutic agents to tumour sites. Despite MSC-related studies making great strides in the field of glioma treatment, there remain some powerful challenges. Moreover, there are no on-going clinical trials on MSC-based therapy for malignant gliomas. In conclusion, MSC-based therapy for glioma is still in its infancy, and we need a better understanding of the biological consequences of MSC-based therapy before it is widely used in the treatment of patients with malignant gliomas.


Halyomorpha halys (Stål), or brown marmorated stink bug (BMSB), has become a major pest and nuisance for both agricultural growers and homeowners since its arrival in North America and Europe. The nutritional ecology of BMSB is important for understanding its life history and rearing requirements. However, little is known about the nutritional status of wild populations, especially in the U.S. This research monitored the nutrient status of nymphal and adult BMSB collected from English holly in western Oregon. We measured their weight, nutrient index (weight/(prothorax × width)3), lipid, glycogen and sugar levels and egg load from May–September/October. First, glycogen and sugar levels of adults were often lowest sometime in June-August with a general increase by September. Meanwhile, their lipid levels varied without a discernible trend. Second, adult females had few eggs in May, with the highest egg load in June and July, and no eggs by September. Lastly, first and second nymphal instars were found in June, and fourth and fifth instars in September. Because nothing is known about the nutrient levels of nymphs, the reported values from this survey can assist future research on physiological responses of BMSB to treatments or environmental impacts in the field. Halyomorpha halys (Stål), was first detected in the United States in 1996 and Europe in 2004 [The brown marmorated stink bug (BMSB),  in 2004 . It has  in 2004 . In Oreg in 2004 .3) [Ilex aquifolium L., over two years.Understanding the nutritional ecology of BMSB improves our knowledge of this pest’s life history. Prior work on the development of BMSB on different diets has enabled researchers to optimize rearing BMSB for research use ,9,10,11.3) , lipid, This first objective on nutrient profiles of BMSB was based on a preliminary study in 2012–2014 . In thatUnderstanding the reproductive phenology of BMSB is essential to develop predictive models that assist growers to predict pest occurrence. While reproductive development on BMSB has been recorded in multiple states and crops , this inBicyrtis quadrifasciatus Say (Hymenoptera: Crabronidae) and Astata unicolor Say (Hymenoptera: Crabronidae), prefer to raise their young on live nymphal BMSB. Bicyrtis quadrifasciatus nests contained on average over 50 BMSB nymphs. Currently, the nutritional value of BMSB nymphs have not been measured, whereas BMSB egg masses are known to provide ~25.5 μg of lipid, 3 µg glycogen and 3 µg of sugar per egg [Monitoring the activity of nymphal instars of BMSB is important for pest control. Insecticide applications against nymphs can be more efficacious because soft-bodied nymphs are more susceptible than adults . Further per egg . TherefoThis monitoring paper highlights potential seasonal variation among the nutrient status of adult BMSB, describes the nutritional content of nymphal BMSB to predators and provides baseline information for future studies examining BMSB nutritional ecology in various habitats, management or environmental conditions.BMSB adults and nymphs were collected from holly trees at five sites in the Willamette Valley of Oregon: Corvallis (44°31′49″ N 123°16′09″ W), Albany (44°37′53″ N 123°07′09″ W), Aurora (45°13′42″ N 122°47′09″ W), Molalla (45°11′42″ N 122°34′54″ W) and Monmouth (44°50′55″ N 123°14′20″ W). Sites had ornamental or commercial plantings of holly where BMSB were collected by beat sheet at least twice a month per site from May–September or October when adults were present. Nymphal instars were only collected in 2015. BMSB were transported live in well-ventilated mesh containers with a water wick from the field and quickly frozen at −80 °C in the lab.3 (mg/mm3)), which is a body mass index for BMSB used previously to evaluate pre- and post-overwintering physiological status [After removal from the −80 °C freezer, all BMSB were weighed, and the prothorax width was measured to calculate the nutrient index (body weight/(prothorax × width)l status . Adult fl status  based onl status , this wag to collect the glycogen precipitate. For adults, the supernatant (~1000 µL) was transferred into a glass test tube, vortexed and aliquoted further: 50 µL for the lipid assay and 50 µL for the sugar assay. For nymphs, the final aliquots of the supernatant used to react for sugar and lipid assays was based on nymphal instar . Lipid content was estimated from the absorbance values of lipid standards made for each vanillin reagent. To calibrate the standard, 0, 1, 5, 10, 35 and 50 µg of canola oil were reacted with vanillin as described above, and the relationship between the absorbance value and lipid content was calculated by a linear equation. Similar calibrations were done for glycogen and sugar standards. For the glycogen assay, 975 µL of anthrone reagent were added and vortexed until the precipitate dissolved. For adults, an aliquot of 50 µL of this mixture was transferred to a new tube, to which an additional 950 µL anthrone was added. For nymphs, the aliquot of supernatant was taken based on nymphal instar , and a cTo simplify the models for adult BMSB, each sex and year were run independently. Females were expected to have higher nutrient levels and weight than males . The weiThe relationships between the egg load and nutrient reserves were tested by regression in PROC REG . A regreNo statistics were run for the nutrient levels of nymphal instars because this information was intended to provide baseline measurements for the five instars. Furthermore, different nymphal instars were collected each month, so observed differences between instars could also be influenced by varying conditions at the time of collection.Results are presented per individual  and per Glycogen and sugar levels dipped in June–August in seven out of eight trend lines g–j. Gener2-values of <0.05 (r2-values of 0.227–0.478.Lipid levels did not significantly vary with glycogen or sugar levels, or only weakly with low of <0.05 . GlycogeThe egg load for the females peaked in June and July k,l. The r2-values of 0.25–0.34. Egg load of females had no or minimal relationships with total lipid, glycogen or sugar levels measured from the entire female, which included eggs. The regressions were not significant or had very low r2-values of <0.01. There was a weak negative association in 2015; as egg load increased, total lipid or glycogen levels decreased.Female egg load significantly correlated with individual weight and nutrient index , with moA total of 605 nymphs were collected over the 2015 summer. The percentage of nymphs per month was tracked over the summer months of June–September . In JuneAs expected, there was an increase in total lipids, glycogen and sugar level, weight and prothorax width with the progression of nymphal instars . Not surThe nutrient levels of BMSB adults collected from holly fluctuated from May–September/October. Generally, sugar and glycogen levels dipped around July. Notably, the dip occurred with repeated sampling from five sites and one host type over two summers and is consistent with observations from more random sampling across 12 sites in Northwestern Oregon from multiple hosts in 2012–2014 . First, Lipid levels showed substantial variation, which may reflect the numerous metabolic pathways associated with lipids . FemalesThe egg load, ovarial and spermathecal ranks of female BMSB collected from holly consistently showed that females were beginning reproduction in May, peaked in June–July and were in reproductive diapause by September in 2015–2016. This trend is consistent with previous BMSB collections from the Willamette Valley of Oregon, from 2012–2014 from a range of hosts [Females with greater egg loads often weighed more, or had higher nutrient index values, which may reflect the added weight of eggs. The relationship between female egg load and total lipid, glycogen or sugar content was not as clear. Females with more eggs tended to have lower lipid levels, although this trend was weak. In another pentatomid species, ovarian development was observed to coincide with decreased body fat . It is pPaulownia tomentosa also suggested a univoltine population in the Northeastern U.S. [1 generation on holly may not be reproductively mature before fall weather sets in and they start moving to overwintering sites.The progression of nymphal instars from June–September 2015 suggests that there was just one generation per year on holly hosts. Previous trends observed on ern U.S. . Nymphs As expected, lipid, glycogen and sugar levels progressively rose with the nymphal instars. Average nutrient levels among the fourth and fifth nymphal instars sometimes overlapped. Smaller instars were observed to have high lipid content per mg of body mass compared to larger instars. This suggests that smaller BMSB have a greater density of lipids in their body.BMSB is an important pest across North America and Europe, and exploring the nutrient status of wild populations provides insight on its life history and nutritional quality as prey. Adult BMSB nutrient levels fluctuate through spring to fall with glycogen and sugar levels often dipping in summer. BMSB may be univoltine on holly; first and second instars were present in June, and fourth and fifth instars in September. Among adults and all nymphal instars, lipids were the predominant energy reserve, followed by sugar, and glycogen the lowest. Future studies should examine how different hosts or management tactics affect the energetic reserves of BMSB in the landscape.


Participants (35–52 years) were primary caregivers for ≥ one child, the primary household food shoppers/preparers, enrolled in the Supplemental Nutrition Assistance Program (SNAP) and able to speak/read English. Participants reported having “go-to” family recipes and regularly searching online for new recipes, especially those using ingredients available/preferred by family members. Recipe websites with highest appeal were polished and engaging to mothers/children, offered user-ratings/comments and were reachable from search engines. Results identified key recommendations: (1) understand the target audience; (2) aim to add healthy/customizable recipes to family “go-to’ recipe rotations and understand the impact of generational influences (e.g. how mothers/grandmothers cooked) on family meals; and (3) create websites that meet target audience criteria. Seeking the target audience’s input about the content/delivery of recipes is an important formative step for obesity-prevention projects that include healthy recipes.We describe a 5-year (2011–2015) qualitative evaluation to refine the content/delivery of the Food Hero social marketing campaign recipes to low-income mothers. Objectives were to: (1) identify characteristics looked for in recipes; (2) determine recipe sources; (3) understand motivation for seeking new recipes and recipe adaptations; and (4) identify recipe website characteristics users valued. Nine focus groups ( Lower0 kg/m2) . Mills ense diet . Tiwari n = 139,219) in 2003 and 2016, reported that 67–70% of women cooked at home for 47–50 min/day and 35–46% of men cook for 15–20 min/day . Or, I go back to things that my mom made, which are a lot of creative-type recipes that you didn’t have to measure. You just kind of put it together. It wasn’t a recipe as far as measurements. You take a little bit of this and a little of that.” Although those who spoke of this generational connection largely shared positive memories associated with learning to cook with family members, they disagreed about the importance of passing on family cooking traditions—including recipes—to their own children. About half of this group spoke of deliberately adopting healthier eating practices than those they were exposed to growing up: “I thought [my weight] was just hereditary, because I have always been heavier. But it’s just that my family culture was so centered around food and celebration, so it was always something.”Although the FG guides did not direct participants to discuss family generational influences on their recipe choices, these influences on participants’ home meal preparation behaviors and recipe selection emerged repeatedly in responses. Participants were especially likely to bring up a mother’s or grandmother’s influence when describing how they decided what to cook for their families. One participant explained how her mother’s “go-to” meal repertoire had become folded into her own HPM decision-making process: “Participants identified their sources for new recipes, their reasons for seeking them out and the methods they used to adapt these recipes to fit their family’s needs. Each of these is described below.Food Network; and friends and family  reported regularly using the Internet for meal ideas and recipe searches. In order of significance, the primary sources of new recipes were: websites, social media ; cookbooks; magazines, newspapers and television channels such as the d family .“Every so often [we’ll use a new recipe], like my daughter and one of her friends, just this weekend, wanted to make sugar cookies. They just looked it up online, just pulled it up and put in ‘sugar cookies.’”Motivation for Seeking New Recipes. There were five main reasons participants sought new recipes: (1) to add variety to their meal routine while meeting family food preferences; (2) to engage in a creative activity involving food ; (3) to find new ways to use up surplus ingredients or leftovers ; (4) to prepare foods for special occasions such as birthdays or holidays; and, (5) to prepare foods for a special diet such as gluten-free, vegetarian, low sugar or vegan . Discuss“Just that I can put my own flavor and spin on it, like if I don’t stick to a recipe and kind of can change it up and do different things with it to suit the taste of the people that I’m feeding it to.” Others echoed this sentiment across groups: “There’s a lot of meat [in these recipes] but a lot of that I’d just change. I’d make it different. I’d probably use tofu. We use a lot of tofu.” “I have something in a recipe that I never heard of before and I couldn’t find it anywhere. So I just eliminated it and didn’t use it and it seemed fine. You can substitute concentrated apple juice [for] oil, which is a cheaper, healthier way to do a lot of things.”Methods of Adapting Recipes. As previously described, participants typically reported adapting new recipes by substituting ingredients family members preferred, healthier ingredients, and/or ingredients they had available. In describing how she commonly adapted recipes, one mother commented, For the majority of participants, the wide variety of options available on the Internet made online searching a highly salient method for finding recipes. When describing what they looked for in recipe websites, their responses fell into the four categories described below . The whole process is telling you whatever you’re looking at, straight and to the point—click and you go to the next one, reading a little bit, no more than five lines—three, four or five lines.” Bright, colorful, eye-catching pictures were also an important feature for participants and their children. One participant described a website she used often: “When you get on it, it’s very bright and visual and you’re interested in looking at the pictures and seeing what they’re doing.” In contrast, reasons not to revisit a website included overly complex recipe instructions and cooking tips or the perception they were not the intended audience. For example, one woman gave this evaluation of a popular website: “Chef.com isn’t one of my favorites. It really was written for high-level home cooks, like the ones that have every kind of tool. Foo-foo. I think that just doesn’t work for a family.”Simple and Visually Interesting. This was the top characteristic of preferred recipe websites. Participants stressed the importance of balancing a simply formatted recipe website with visually interesting content: “Say I have got a bushel of broccoli or something. I could click on broccoli and I’d see something with broccoli.” Another explained, “I like to garden and I have this big garden. It’s like, ‘okay, I’ve got all this squash. What am I going to do?’” Being able to search by recipe name was also mentioned: “If I do a 7-UP cake, I put in ‘7-UP’ and it will give you the different recipes. I’ll go through them and try to find the simplest.” Some reported using multiple key words to increase the number of recipes in the search results: “If you type in, ‘ways to make quinoa,’ then you have five or six websites that will pop up.” Still others reported narrowing the search field by making the query more specific; “I put ‘simple’ or ‘easy’ so they give you the most simple thing that comes up first.”Efficient Search Mechanism. Participants generally searched for recipes from search engines like Google using a specific key word(s) or ingredient(s) versus by recipe name, yet both methods were used. As one participant elaborated, ““A database that has the fruits and vegetables or that would match colors or have things that would be appropriate for kids as well as adults.” However, participants reported that they did not typically find this characteristic in the recipe websites they viewed/used. Participants often stressed the importance of having content that could appeal to a range of children’s ages and abilities. As one participant explained, “I have [a child] who is ready to cut the tomatoes herself and then one that has no idea what he’s doing, so something simpler that is for younger kids and then something as they get older in stages—kind of like in school.” Child-friendly ideas mentioned across groups included the use of bright, eye-catching content and interactive animated characters to guide children step-by-step through recipe preparation.Engages Children. Participants preferred recipe websites with child-centered content and a design that allowed adults to use recipes side-by-side with their children. One mother, in explaining her idea of a child-centered website, said, Allrecipes.com, a popular website among FG participants, one woman said: “They’ve got the top comments. I looked up jerked chicken and they said, ‘Well, this recipe is good but all you need to do is just subtract this or this...’” The majority of participants talked about reading online recipe comments but not choosing to post comments themselves: “But I don’t want to post it, because I guess the constructive criticism, like if somebody was like, ‘Ugh, that looks nasty’…well, my son liked it, so it’s okay.” “I would probably read them but I wouldn’t post them.”User Ratings and Reviewer Comments. Favorite recipe websites also included a user-rating system. Participants reported looking for a rating system—either star-ratings or text-based reviewer comments—before making a recipe. They relied on these systems to decide whether a recipe had high user approval or if users had given tips or provided ideas for substitutions to adapt or improve a recipe. For example, explaining why she liked using Findings from our FGs were used to refine the content and delivery of recipes through the Food Hero SM campaign, specifically to SNAP-enrolled mothers. Our research yielded three key recommendations for disseminating recipes to low-income audiences through a SM campaign: (1) understand the target audience to better define and support the recipe characteristics they may be looking for; (2) aim to add healthy, customizable recipes to family “go-to’ recipe rotations and understand generational influences on family meals; and (3) when delivering recipes online, create website content and design that meet the criteria the target audience seeks. n = 9,569) found that low-income participants  reported cooking either more frequently (24% cooked dinner 6–7 times/week) or less frequently (22% cooked dinner 0–1 times/week) as compared to those with higher incomes , who cooked a moderate amount (86% cooked dinner 2–5 times/week) [When asked to describe cooking habits, participants reported enjoying cooking, making HPM daily and rarely eating out with their families because of budgetary constraints. Research consistently shows that US women of all backgrounds and income levels do the majority of HPM cooking; however, while most of the participants in our study reported cooking daily, the cooking frequency reported nationally among low-income US adults varies widely ,28. For ts n = 2,68 reportes/week) ,30,31,32n = 40) women (mean 25 years) living in southwest Georgia regarding their food sources at the grocery store and when eating out. They found that mothers talked about using low-cost ingredients they were sure family members would eat and in doing so, sticking closely to planned meal rotations [The majority of our FG participants reported consulting recipes for HPM. Participants all had an existing set of “go-to” recipes, many of which they cooked from memory but most also sought out new recipes and were confident about customizing both new and existing “go-to” recipes to their family’s liking. Our finding that mothers rely on “go-to” recipes is similar to Raskind et al., who interviewed  and a web survey  to determine where participants learned to cook [Research about a generational flow of recipes is emerging. Wolfson et al. examined US adults (parenthood status unreported) using FGs  gave higher aesthetic ratings to bright and colorful websites with high symmetry and low complexity [n = 101) relied heavily on Google searches to find relevant online nutritional information [Although our recipe website research is novel, our results are similar to those found for general use of websites. First, our FG participants wanted a simple yet polished and eye-catching appearance in a recipe website. This is comparable to research showing that people form value judgments of online content in microseconds and their initial impressions—based largely on appearance—carry over into other evaluations of the website’s attributes and content . This fimplexity . Second,ormation . Egri anormation . Third, ormation ,49. To oThis research has four key strengths. First, to our knowledge this is the first study to focus on describing the characteristics low-income mothers look for in recipes, including characteristics valued by mothers when selecting recipes online. Second, we used five years (2011–2015) of FG data to examine how low-income mothers select and use recipes for HPM. Third, results from the FGs provided insights that have been used to continually refine the content and delivery of recipes through the ongoing Food Hero SM campaign. Fourth, only SNAP-enrolled participants were recruited through a strong partnership with Oregon’s DHS.These findings used subjective narratives to provide unique insights about recipe use among English-speaking, low-income mothers; thus, they cannot be generalized to all households. It is possible those who attended the FGs are a sub-set of the population who rely more heavily on HPM compared to the general SNAP population. Findings related to a distinct subset, however, may still have useful implications for a broader audience when viewed through a social cognitive lens of sharing and modeling HPM strategies and observational learning through successful outliers . FG dynaDespite routine promotion of recipes within nutrition and public health programs as a way to encourage HPM and healthy eating, there is no consensus regarding best practices for types of healthy recipes to offer, nor how or to whom to deliver them. Our research identified criteria used by low-income Oregon SNAP eligible mothers who generally cook HPM often and enjoy cooking, when searching for and choosing new and “go to” recipes. This study underscores that these mothers customize recipes to fit within their family’s unique constellation of preferences and circumstances and thus are not looking for one-size-fits-all prescriptive recipes. Rather, to Oregon SNAP eligible mothers who regularly cook HPM, health educators should consider promoting low-cost, easily customizable, healthy recipes that use common ingredients and can be delivered on interactive platforms to encourage self-efficacy and child involvement with planning/cooking. Strategies should recognize that children and adults are key influencers of current and future family food environments. Our findings also point to the potential need for search engine optimization of healthy online recipes to make them easier and more convenient to find. Since the research described in this paper was conducted, results have been used to develop two adult intercept surveys that aim to empirically test related research questions with a larger sample size. Future research in this area should focus on input from diverse population segments about desired recipe traits, readiness to try new healthy recipes and adapt existing recipes to be more healthful and best ways to access new recipes and recipe healthy adaptation tips. Additionally, nutrition educators can encourage online promotion of healthy recipes by using bright and polished photographs, facilitating high name recognition in Google searches and incorporating interactive engagement opportunities like user comments and ratings. More research, however, is needed on what combination of qualities and delivery mechanisms is ideal. Gathering a target audience’s input about desired recipe traits and how best to deliver recipes is an important formative step of obesity prevention projects that include recipes as a tool to promote healthy eating habits.


Pancreatic cancer exhibits a poor prognosis and often presents with metastasis at diagnosis. Immunotherapeutic approaches targeting private cancer mutations (neoantigens) are a clinically viable option to improve clinical outcomes.3/40 TIL lines  were more closely examined for neoantigen recognition. Whole-exome sequencing was performed to identify non-synonymous somatic mutations. Mutant peptides were synthesised and assessed for antigen-specific IFN-γ production and specific tumour killing in a standard Cr51 assay. TIL phenotype was tested by flow cytometry. Lymphocytes and HLA molecules in tumour tissue were visualised by immunohistochemistry.G12v mutation, while a PanTT39 CD4+ TIL clone recognised the neoepitope (GLLRYWRTERLF) from an aquaporin 1-like protein (gene: K7N7A8). Repeated stimulation of TILs with the autologous tumour cells line lead to focused recognition of several mutated targets, based on IFN-γ production. TILs and corresponding PBMCs from PanTT77 showed shared as well as mutually exclusively tumour epitope recognition (TIL-responsive or PBMC-responsive).PanTT26 and PanTT39 TILs recognised and killed the autologous tumour cells. PanTT26 TIL recognised the KRASThis study provides methods to robustly screen T-cell targets for pancreatic cancer. Pancreatic cancer is immunogenic and immunotherapeutic approaches can be used to develop improved, targeted therapies. These statistics commensurate with limited treatment options for patients with pancreatic cancer, which include surgery  or chemotherapy with gemcitabine, sometimes in combination with the tyrosine kinase inhibitor erlotinib.2 A regimen of gemcitabine plus nab-paclitaxel (Abraxane®) improves overall and progression-free survival in patients with locally advanced or metastatic pancreatic cancer compared to gemcitabine alone, albeit with treatment-related toxicities.4 The combination regimen of oxaliplatin, irinotecan, fluorouracil and leucovorin (FOLFIRINOX) lead to improved progression-free survival of patients compared to gemcitabine alone (median of 6.4 months versus 3.3 months).6 Due to toxicity (grades 3–4) related to the use of FOLFIRINOX, this regimen is recommended for younger patients.7The World Health Organization (WHO) reported that pancreatic cancer claimed >330,000 lives in 2012, with 68% of deaths occurring in countries with a high to very high human development index (HDI).11 Monoclonal antibody-based blockade of the immune checkpoint inhibitors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has further revolutionised the field. The clinical efficacy of PD-1 blockade in patients with metastatic cancer appears to depend on activation of T-cell populations with T-cell receptors (TCRs), which specifically recognise mutated host molecules, ie, neoepitopes.12 These mutant proteins harbouring point mutations, termed neoepitopes, represent a clinically significant step towards refining T-cell-based immunotherapies and cancer vaccines. Neoepitopes differ from one patient to another , giving rise to a highly personalised antigen signature. In line with this, specialised T cell-based therapies targeting private mutations in patients with metastatic cancers have resulted in remarkable clinical responses.15Cancer immunotherapy is a progressively expanding discipline within modern oncology. Immune-based treatment of metastatic melanoma using interleukin (IL) 2-conditioned autologous lymphocytic cells from patients’ blood has existed since the 1980s.16 In the present study, we characterised the neoepitope recognition profile of TILs as well as peripheral blood T cells from patients with pancreatic cancer.We have previously reported that tumour-infiltrating lymphocytes (TILs) can be reliably and successfully isolated from pancreatic cancer lesions and expanded in vitro using a cocktail of IL-2, IL-15 and IL-21.Pancreatic tumour samples from three patients with pancreatic cancer were obtained as part of a clinical research project approved by the Regional Ethical Review Board  at Karolinska Institutet, Sweden (EPN: 2013/1332-31/3 and 2013/977-31/1). The clinical characteristics of the patients are presented in Table 17 Tumour cells required for the cytotoxicity experiments were obtained during passage 15–20.Pancreatic cancer TILs and autologous tumour cell lines were generated as previously described.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood (PanTT77) over a Ficoll-Hypaque gradient  and washed twice in sterile PBS prior to use in experiments.18. Briefly, genomic DNA from patient samples (tumour tissue and TILs) was fragmented for constructing an Illumina DNA library . Regions of DNA corresponding to exons were captured in solution using the Agilent SureSelect 50 Mb kit Version 3 as per manufacturer’s instructions . Paired-end sequencing resulting in 100 bases from each end of every fragment was performed using a HiSeq 2000 Genome Analyser (Illumina). Results of the sequencing data were mapped to the reference human genome sequence. Alterations within the sequencing data were determined by comparing over 50 million bases of tumour DNA from non-malignant lesions. A high fraction of the sequences obtained for each sample was found to occur within the captured coding regions. More than 43 million bases of target DNA were analysed in the tumour and normal samples; an average of 42–51 reads per base was obtained for both sample types. The tags were aligned to the human genome reference sequence (hg18) using the Eland algorithm of CASAVA 1.6 software (Illumina). The chastity filter of the BaseCall software of Illumina was used to select sequence reads for subsequent analysis. The ELANDv2 algorithm of CASAVA 1.6 software (Illumina) was applied for identifying point mutations, small insertions, deletions or stop codons in the sequences obtained. Mutation polymorphisms recorded in the Single Nucleotide Polymorphism Database (dbSNP) were excluded from analysis. Potential somatic mutations were filtered out as previously described,18 while only non-synonymous single and dinucleotide substitutions, respectively, were listed in an Excel spreadsheet for downstream work. The filter criterion for selecting candidate peptides is that the expression level of mutated genes in tumour tissue surpasses 5%. Alternatively spliced products or mutated sequences with stop codons may result in epitopes that are shorter than the standard 15-mer peptides that are used for screening immunogenicity. The length of the resulting peptide sequences was set at 15-mer to include all possible epitopes presented by HLA class I (8–10 amino acids) as well as HLA class II (11–20 amino acids) molecules.Isolation and purification of genomic DNA, library construction, exome capture of all coding genes as well as next-generation sequencing of tumour tissue and control patient samples (TILs) were performed as previously described by Jones et al.After identification of mutations through whole-exome sequencing followed by in silico analysis, the 15-mer peptides were constructed by placing the mutation at the centre position of the 15-amino acid sequence . The corresponding wild-type epitopes were also synthesised to compare the matched mutant and wild-type sequences (peptide pairs) in immunological assays.5 cells) were cultured in 200 μl of T-cell medium with 1 μg of the individual wild-type or mutated peptide in round-bottom 96-well microtiter plates. Negative controls contained assay medium alone while the positive control contained 30 ng/mL of the anti-human CD3 antibody clone OKT3  for maximal TCR stimulation. Cells were incubated for 3 days at 37 °C with 5% CO2, after which supernatants were harvested for interferon (IFN)-γ production using a standard sandwich enzyme-linked immunosorbent assay (ELISA) kit . Values from the negative control (medium) were subtracted from epitope (peptide)-specific responses and the data reported to reflect the IFN-γ production (in pg/3 days/1.0 × 105 TILs representing the net IFN-γ production from T-cell populations). The mAbs w6/32  and L243 (anti-HLA-DR) were used as blocking antibodies to asses MHC class I or -class I restriction.TILs or PBMCs (1.0 × 106 TILs: 1 × 106 tumour cells) containing T-cell medium for 7 days, after which TILs were stimulated with (autologous) tumour cells two more times.TILs were stimulated with the autologous tumour cell line in six-well tissue culture plates  by flow cytometry. A total of 2 × 105 T cells were co-cultured with 4 × 104 autologous tumour cells for 5 h at 37 °C (and 5% CO2) in a 96-well tissue culture plate containing 200 μl assay medium/well . During the incubation period, 1.3 μg/ml of monensin , and 4 μl of the anti-human CD107a-Alexa Fluor 700 antibody  were added. PMA = phorbol 12-myristate 13-acetate (PMA) was used as the positive control and assay medium alone without tumour cells was used as negative control. After 5 h of incubation, the cells were stained with anti-human CD3-PE/Cy7 , anti-human CD4-V450 (Clone RPA-T4) and anti-human CD8-APC/Cy7 (Clone SK1) , and analysed by flow cytometry.The CD417TILs were stained with anti-CD3 Brilliant violet 570, anti-CD4 Brilliant violet 510, anti-CXCR3 FITC  and anti-CD8a APC-Cy7 . After 15 min, cells were washed in PBS-0.1% FBS, and analysed by flow cytometry. Differentiation and maturation marker analysis based on CD45RA and CCR7 expression was performed as described previously.All flow cytometry experiments were performed on a BD FACS Aria flow cytometer, while data analysis was performed using FlowJo software version 7 (both from BD Biosciences).Resected pancreatic specimens were fixed in 4% formalin and processed for routine histopathological diagnosis. The fixed tissue blocks were then embedded in paraffin, sectioned (thickness of 4 μm) and stained with haematoxylin-eosin for light microscopy. A specialist pancreatic pathologist (Dr. C. F. Moro) chose from each specimen a tissue block in which the tumour centre, tumour border and surrounding non-tumour pancreatic tissue was represented. The selected blocks were subsequently sectioned and processed for immunohistochemistry. To preserve the relative spatial distribution of the lymphocyte populations, chromophore-conjugated antibodies were combined into the following 2-plex immunostainings: CD3-CD20 and CD4-CD8. Details of the individual antibodies are as follows: anti-CD3 , anti-CD20 , anti-CD4 , anti-CD8 . Staining was performed using a Leica BOND III automated immunostainer (Leica Biosystems). The primary antibodies were detected using 3,3′-diaminobenzidine (DAB—brown) as chromogen and the secondary antibodies with alkaline phosphatase (AP—red). HLA class I and class II immunohistochemistry staining was performed separately, according to the above-mentioned protocol. Technical details of the reagents used are as follows: anti-human HLA-class I ABC antibody (clone EMR8-5) and anti-human HLA-class II DR, DP and DQ antibody (clone CR3/43) . The antibodies were detected using 3,3′-diaminobenzidine (DAB—brown).+ and CD8+ TILs from pancreatic cancer tissue using IL-2, IL-15 and IL-21, particularly within the central and effector memory compartments, is shown in Supplementary Figure The reliable expansion of CD4+ and double-negative  T cells among TILs derived from patient PanTT26 (annotated as Panc17 in our previous publication16) are able to produce IL-2 and IL-17, while CD8+ T cells do not produce cytokines after 6 h of stimulation with autologous tumour cells.16 In addition, we also observed that PanTT26 TILs could kill autologous tumour cells within 4 h of culture initiation, using a standard Cr51-release assay. We, therefore, intended to further explore the anti-tumour characteristics of PanTT26 in the present study.TILs and the corresponding tumour cell line was established from patient PanTT26. We previously showed that CD4+ T cells and 22% CD4+ T cells , while CD4+ T cells were entirely absent (from 22% pre-stimulation to 0% post-stimulation with tumour cells) based on flow cytometric data. Using the W6/32 (anti-HLA-I) and L243 (anti-HLA-DR) antibodies in Cr51-release assays, we found that TILs, prior to 3× stimulation with the autologous tumour cell line, displayed a dampened cytotoxic effect with HLA class II inhibition, while the W6/32 (anti-MHC class I) antibody abrogated tumour recognition completely  was produced by young TIL or tumour cell-stimulated TIL in response to subsequent exposure to wild-type or mutated peptides (Table Whole-exome sequencing of the pancreatic tumour tissue from patient PanTT26 was performed to identify cancer-related mutations that may give rise to mutated antigens (neoantigens). Mutated peptide sequences (containing neoepitopes) were predicted by placing the mutation at the centre of the sequence and flanking it with seven amino acids on either side. The resulting peptide sequences were synthesised along with the corresponding wild-type sequences and tested for T-cell reactivity with TILs by measuring antigen-specific IFN-γ production in the culture supernatants by sandwich ELISA. In total, 298 peptides  were tested for both ‘young’ TILs and TILs that were stimulated three times with the autologous tumour cells. We found that >150 pg IFN-γ (per 10es Table . A more VGVGKSAL (representing the well-described KRASG12V mutation) was observed after 3× stimulation with the autologous tumour cell-stimulated TILs compared to young TILs (Table 19 Garcia-Silva and Aranda have shown that thyroid hormone nuclear receptors (TRs) can repress RAS-dependent cellular transformation and tumour growth.20 A subsequent study from the same group showed that cellular nuclear receptor co-repressor 1 (NCOR1) levels could increase upon TR(s) expression.21 Intriguingly, the strongest IFN-γ response by both PanTT26 young TILs and tumour cell-stimulated TILs was observed after exposure to a mutated peptide from NCOR1 . Young TILs produced 327 pg IFN-γ/10e5 TILs in response to mutated NCOR1, while tumour cell-stimulated TILs showed 710 pg IFN-γ/10e5 TIL (Table NCOR1 gene product in the PanTT39 tumour harboured a mutation at position 120 (R120L).Increased IFN-γ production to the mutated KRAS peptide KLVVVGALs Table . The cliIL Table . We also23 However, the significance of WDFY4 in cancer is yet to be explored. PanTT26 TILs also showed strong IFN-γ responses to a mutated peptide derived from WDFY4 (RKFISLHKKALESDF). We noticed that 17% of mutations (25/149 mutations) in PanTT26 are associated with zinc-finger proteins (ZNF), which display diverse biological functions.24 The recognition of a ZNF730-derived peptide was pronounced following stimulation of PanTT26 TILs with autologous tumour cells, although four other wild-type ZNF peptides were recognised (Table WD repeat- and FYVE domain-containing protein 4 (WDFY4) is highly expressed in lymph nodes and the spleen; previous studies have shown that aberrations in this gene are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.ed Table . It is p+ T cells (>99%)  was also identified. This is of note, since BRCA1 mutations are implicated as a key contributing factor related to the burden of somatic mutations in pancreatic cancer.25 We also found seven-point mutations in the HLA-A alleles, two-point mutations in the HLA-B alleles and eight-point mutations in the HLA-C alleles, which ultimately gave rise to amino acid changes in the resulting protein products associated with the HLA class I antigen processing and presentation pathway , which derives from an uncharacterised protein product of 449 amino acids encoded by the K7N7A8 gene. The CD4+ TCR Vβ9+ TIL clone that recognises the K7N7A8 mutated peptide GLLRYWRTERLF produced a cytotoxic T-cell response against the autologous tumour cell line, which was assessed in a standard CD107a induction assay  in a dose-dependent manner  revealed that the peptide belongs to a putative water channel transporter like aquaporin 1 (AQP1)  surrounding B cells.Akin to PanTT26, immunohistology of PanTT39 tumour showed weak T-cell infiltration into the centre of the tumour mass Fig.  comparedtre Fig. . Interes+ T cells and 14% CD8+ T cells , a protein that is expressed in the brain, testes and kidneys but overexpressed in gliomas;28 Ankyrin Repeat And Sterile Alpha Motif Domain Containing 1B (ANKS1B), which is expressed in normal brain tissue and is required for development, but also implicated in the pathogenesis of Alzheimer’s Disease and downregulated in smoking-related clear-cell renal cell carcinoma;30 Ciliogenesis Associated TTC17 Interacting Protein (CATIP/C2orf62), a protein involved in cilium biogenesis by inducing actin polymerisation;31 Calcium Voltage-Gated Channel Subunit Alpha1 S (CACNA1S), a subunit of a voltage-gated calcium channel with an important role in interacting with the ryanodine receptor in muscle cells for excitation–contraction coupling.32 Interestingly, some of the mutated peptides were recognised by TILs and PBMCs (six mutated peptides) and triggered stronger IFN-γ production in PBMCs (up to IFN-γ 350 pg/10e5 PBMCs) compared to TILs (up to 141 pg IFN-γ/10e5 TIL). A single mutated peptide, derived from the Proline Rich Transmembrane Protein 1 , induced strong IFN-γ by PBMCs and TILs. PRRT1 is part of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complex, which is involved in glutamate transport in the central nervous system and is important for synaptic transmission.34 No mutations were found in the HLA class I and class II pathways in this patient’s tumour.PanTT77 TILs comprised ~84% CD4lls Fig. . Immunorlls Fig. , showingWe have been able to reliably grow TILs from patients with pancreatic cancer that could be used for the adoptive cellular therapy. Using a combination of co-culture techniques, we gauged the capacity, breadth and specificity of an individual patient’s T cells to react to private neoepitopes. To the best of our knowledge, this is the first report to describe individual neoepitope recognition patterns in patients with pancreatic cancer and argues that pancreatic cancer exhibits a certain degree of ‘immunogenicity’, making this malignancy potentially amenable to immunotherapeutic approaches. An integral component of the present study is the use of autologous tumour cell lines to profile TIL neoepitope recognition. The availability of samples for this purpose can be scarce, since surgery is mostly performed for patients who present without metastasis at diagnosis. Thus, the use of PBMCs to screen for neoepitope recognition is also a viable approach for developing personalised cellular therapies.G12D mutation in a patient with metastatic colorectal cancer,15 and we observed that repetitive exposure to autologous tumour cells can result in enrichment of KRAS-specific T cells that was non-detectable in the ‘young’ TILs population.Repeated exposure to particular antigenic targets is likely to enrich for certain T-cell populations capable of durable anti-tumour responses. This is based on the differential recognition of mutated peptides (as opposed to the wild-type/native form), arising from important driver mutations. This has been shown for the KRAS+ T cells. The IFN-γ-producing capacity of CD4+ TILs directed against cancer neoepitopes may confer productive clinical responses, as shown in a patient with cholangiocarcinoma (directed against mutated ERBB2IP).13 Several other CD4+ T-cell neoantigens have been described and concisely reviewed elsewhere,35 a majority of which are restricted by the HLA-DR alleles. Of note, mutations within the HLA class I alleles, mostly in HLA-A and HLA-C , professional and non-professional alike, including CD8+ and gamma-delta (γδ) T cells that express HLA-DR39 may present tumour-associated antigens to CD4+ TILs in the tumour microenvironment, even when the tumour itself may harbour mutations in the HLA class II pathway. Expanded TILs from patient PanTT77 were also enriched for CD4+ T cells coupled with broad neoepitope recognition despite the absence of mutations in either HLA pathway, thus underlining the importance of CD4+ T cells in targeted immunotherapy.TILs derived from patient PanTT39 comprised mainly CD4+ TIL response. Interestingly, the immunostaining results showed a dominant composition of CD4+ TILs in the lymphocytic infiltrate in tumour sections from patient PanTT39 (including the TLS). Considering that this patient harboured mutations in the HLA-I pathway, the CD4+ TILs were likely to be the ‘fitter’ T-cell subpopulation that managed to expand in vitro in response to IL-2, IL-15 and IL-21 stimulation. In contrast, patient PanTT26 did not have any mutations in the MHC-I pathway (like patient PanTT77) and displayed a prominent T-cell presence especially in the TLS. IL-2, IL-15 and IL-21 conditioning of total TILs from the resected PanTT26 tumour piece (which includes the TLS) was enriched for CD8+ T cells, as confirmed in the cytotoxicity assay performed with the autologous tumour cell line. The same TILs, following 3× stimulation with the autologous tumour cell line, yielded a CD8+ T-cell population, which included a KRASG12V reactive subset. Previous study showed large lymphocytic infiltration in the tumour-associated TLS  may reflect survival benefit in patients with solid tumours including pancreatic cancer.41 The survival period post-tissue sampling recorded for patient PanTT26 (547 days) was much longer than for patient PanTT39 (411 days) . However, the antigen processing and presentation machinery was not compromised, as reflected by the strong  CD4+ TIL clone recognised a mutant peptide derived from the aquaporin (AQP)-like putative transmembrane ion/water channel K7N7A8 protein product, which also induced potent cytotoxicity against the autologous tumour cell line. At least 40 different mutations in the AQP2 gene (resulting in protein product variants) have been linked to the onset of autosomal nephrogenic diabetes insipidus, where loss of responsiveness to vasopressin disrupts the kidneys’ ability to concentrate urine, resulting in an excessive excretory volume.42 Although site-specific mutations in the AQP4 molecule have been associated with reduced binding of potentially pathogenic IgG (in conjunction with the neurodegenerative disease neuromyelitis optica),43 clinically relevant immune responses to mutant AQPs remain unknown. To the best of our knowledge, the present study is the first to describe CD4+ TIL anti-tumour responses in pancreatic cancer directed against a mutant epitope derived from an AQP-like molecule.The PanTT39 CD444 Vagus nerve stimulation also reduces tumour necrosis factor-α production by gut macrophages.45 Intratumoural TGF-β, associated with disease progression, is a known suppressor of effective anti-tumour immune responses in pancreatic cancer.46 Strong TGF-β production in the tumour microenvironment impedes TIL activity and reduces the chances of tumour regression.48 Low-dose gemcitabine as well as cyclophosphamide can lead to reduction in regulatory T-cell (Treg) numbers, a major source of TGF-β and represents an effective strategy to improve local anti-tumour immune responses.51 Cyclophosphamide is already used as a pre-emptive drug in adoptive cell therapy clinical protocols.52 Interestingly, pharmacological depletion of Tregs using cyclophosphamide results in the reduction of intracellular adenosine triphosphate (ATP) turnover53 and their impaired ability to repair DNA double-strand breaks (DSBs).51 Surface-bound CD73 is an ecto-5′-nucleotidase that dephosphorylates AMP to produce adenosine, which goes on to trigger several different pathways in the tumour microenvironment including but not limited to aberrant neovascularisation, conversion of inflammatory M1 macrophages to anti-inflammatory M2 macrophages and apoptosis of tumour-infiltrating T cells.54 Hypoxia and an overt inflammatory milieu can lead to CD73 upregulation on stromal and tumour cells, which would then create a rather inhospitable environment  for tumour-directed immune cells. Anti-CD73 strategies are now in clinical assessment as an adjunctive immunotherapeutic measure to complement anti-PD-1/PD-L1 therapy for cancer, qualifying CD73 as a novel immune checkpoint . Increased potassium channel activity and its dysregulation is yet another characteristic of solid tumours linked to invasiveness and uncontrolled cancer cell proliferation.55 At least three different potassium channel genes are known to be overexpressed in pancreatic cancer. Immunologically, dying tumour cells release an excess of potassium into the tumour microenvironment, which has a deleterious effect on infiltrating T cells by impairing the TCR-Akt-mTOR signalling cascade for effector functions, including IFN-γ production.57 Thus, the effect of induction of cell death as well as specific immune-suppressive activity in the tumour microenvironment and how they influence adoptive T-cell therapies warrant consideration in developing cellular therapies.Of note, specific immunomodulatory processes linked to the pancreas would inevitably affect the outcome of TIL therapy. For instance, stimulation of the vagus nerve serving the pancreas is likely to induce insulin production while inhibiting hepatic glucose release.69 Exposure to antigens, either by direct contact with tumour cells or associated products presented by professional APCs or exosomes70, may activate T cells bearing TCRs that are either silenced or tolerised by the tumour.Satellite studies performed alongside clinical trials of novel cancer immunotherapies, ie, vaccines, immune checkpoint blockade or TIL therapy show that immune correlates of protection ) observed in responding patients are partly concomitant with the emergence of specific populations of neoantigen-directed T cells in peripheral blood.Needle biopsies (such as for patient PanTT77) can also be used to cultivate TILs. Alternatively, biopsy material may also be used for constructing the patient’s mutanome to help screen for neoepitope-reactive circulating T cells, particularly if a limited number of tumour mutations are targeted by a specific TIL product. Future studies will also focus on whether gamma-delta (γδ) TCRs can recognise mutated targets relevant to cancer immunotherapy, although we did not observe TCR γδ+populations in the three TIL lines examined in greater detail in this report.71Several T-cell-based approaches to treat pancreatic cancer are currently pursued. The NCI is currently carrying out a clinical study of IL-2-stimulated TIL infusion in patients with metastatic pancreatic cancer . Another study is evaluating the safety and efficacy of EGFR-directed bispecific antibody-expressing T cells (BATs) in patients with locally advanced or metastatic pancreatic cancer who have already undergone one to two rounds of chemotherapy , although the T cells themselves will be harvested from blood. IL-2, IL-15 and IL-21-stimulated TIL therapy is actively pursued at the Krankenhaus Nordwest  for patients with resectable locally advanced or metastatic pancreatic cancer. Future clinical studies are likely to benefit from translational data linking anti-tumour T cells in pancreatic cancer to recognition of specific private mutations to improve survival as it was recently shown for a patient with metastatic breast cancer.+ TILs with TCRs directed against neoepitopes may kill autologous tumour cells and (ii) repeated exposure of TILs to the tumour cells (expressing neoantigens) can refine the TCR repertoire to produce a more focused immune response; (iii) PBMCs may be used as a viable source for T cells directed against tumour mutations and (iv) microdissection of tumour tissue may help achieve a more precise extraction and biologically relevant expansion of TILs.The results presented in this study have clinically relevant implications: (i) CD4Supplementary Tables S1-S3Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3


The authors of “A Cardiopulmonary Monitoring System for Patient Transport Within HospitalsUsing Mobile Internet of Things Technology: Observational Validation Study” :e12048) wish to add their grant number to the Acknowledgments section.Thus, the sentence “This study was supported by a grant from Korea Health IndustryDevelopment Institute” has been changed to “This study was supported by a grant from KoreaHealth Industry Development Institute (grant number HI17C0178).”The correction will appear in the online version of the paper on the JMIR website on December20, 2018, together with the publication of this correction notice. Because this was made aftersubmission to PubMed, PubMed Central, and other full-text repositories, the corrected articlealso has been resubmitted to those repositories.