Changes in T Lymphocyte Subsets in Different Tumors Before and After Radiotherapy: A Meta-analysis.

Purpose: Radiation therapy (RT) induces an immune response, but the relationship of this response with tumor type is not fully understood. This meta-analysis further elucidated this relationship by analyzing the changes in T lymphocyte subsets in different tumors before and after radiotherapy. Methods: We searched English-language electronic databases including PubMed, EMBASE, and the Cochrane Library to collect studies on the changes in peripheral blood CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes before and after radiotherapy in tumor patients from January 2015 to April 2021. The quality of the included literature was evaluated using the NOS scale provided by the Cochrane Collaboration, and statistical software RevMan 5.4 was used to analyze the included literature. P<0.05 was considered to indicate statistical significance. Results: A total of 19 studies in 16 articles involving 877 tumor patients were included. All data were collected within 1 month before or after radiotherapy. Meta-analysis showed that numbers of CD3+ T lymphocytes (SMD: -0.40; 95% CI [-0.75, -0.04]; p = 0.03) and CD4+ T lymphocytes (SMD: -0.43; 95% CI: [-0.85, -0.02]; p = 0.04) were significantly reduced after radiotherapy compared with before treatment, but there was no statistically significant difference for CD8+ T lymphocytes (SMD: 0.33; 95% CI: [-0.88, 0.74]; p = 0.12). Subgroup analysis showed that peripheral blood T lymphocytes decreased in head and neck cancer. However, in prostate cancer and breast cancer, there was no significant change in peripheral blood. 1 month after radiotherapy, it has a potential proliferation and activation effect on lymphocytes in esophageal cancer and lung cancer. The results showed that CD8+T lymphocytes increased in peripheral blood after SBRT. Radiotherapy alone reduced CD3+ T lymphocyte numbers. Conclusions: Within 1 month of radiotherapy, patients have obvious immunological changes, which can cause apoptosis and reduction of T lymphocytes, and affect the balance of peripheral blood immune cells. The degree of immune response induced by radiotherapy differed between tumor types.

Optical Control of CD8+ T Cell Metabolism and Effector Functions.

Although cancer immunotherapy is effective against hematological malignancies, it is less effective against solid tumors due in part to significant metabolic challenges present in the tumor microenvironment (TME), where infiltrated CD8+ T cells face fierce competition with cancer cells for limited nutrients. Strong metabolic suppression in the TME is often associated with impaired T cell recruitment to the tumor site and hyporesponsive effector function via T cell exhaustion. Increasing evidence suggests that mitochondria play a key role in CD8+ T cell activation, effector function, and persistence in tumors. In this study, we showed that there was an increase in overall mitochondrial function, including mitochondrial mass and membrane potential, during both mouse and human CD8+ T cell activation. CD8+ T cell mitochondrial membrane potential was closely correlated with granzyme B and IFN-γ production, demonstrating the significance of mitochondria in effector T cell function. Additionally, activated CD8+ T cells that migrate on ICAM-1 and CXCL12 consumed significantly more oxygen than stationary CD8+ T cells. Inhibition of mitochondrial respiration decreased the velocity of CD8+ T cell migration, indicating the importance of mitochondrial metabolism in CD8+ T cell migration. Remote optical stimulation of CD8+ T cells that express our newly developed "OptoMito-On" successfully enhanced mitochondrial ATP production and improved overall CD8+ T cell migration and effector function. Our study provides new insight into the effect of the mitochondrial membrane potential on CD8+ T cell effector function and demonstrates the development of a novel optogenetic technique to remotely control T cell metabolism and effector function at the target tumor site with outstanding specificity and temporospatial resolution.

Radiation therapy enhanced therapeutic efficacy of anti-PD1 against gastric cancer.

Radiation therapy is an important method in tumor treatment with distinct responses. This study aimed to investigate the immune effects of radiation therapy on the syngeneic gastric tumor model. Mouse forestomach carcinoma (MFC) cells were irradiated with different X-ray doses. Cell proliferation was determined by clonogenic assay. Gene and protein expression were determined by real-time quantitative PCR and western blot, respectively. The tumor model was established by subcutaneously injecting tumor cells in 615-(H-2 K) mice. Levels of immune-related factors in tumor tissues were determined by immunohistochemistry and flow cytometry. 5 Gy × 3 (three subfractions with 4 h interval) treatment significantly inhibited cell proliferation. Protein expression of stimulator of interferon genes (Sting) and gene expression of IFNB1, TNFα as well as CXCL-9 significantly increased in MFC cells after irradiation. In the MFC mouse model, no obvious tumor regression was observed after irradiation treatment. Further studies showed Sting protein expression, infiltration of dendritic cells and T cells, and significantly increased PD-1/PD-L1 expression in tumor tissues. Moreover, the irradiation treatment activated T cells and enhanced the therapeutic effects of anti-PD1 antibody against MFC tumor. Our data demonstrated that although the MFC tumor was not sensitive to radiation therapy, the tumor microenvironment could be primed after irradiation. Radiation therapy combined with immunotherapy can greatly improve anti-tumor activities in radiation therapy-insensitive tumor models.

Electromagnetic field affects the voltage-dependent potassium channel Kv1.3.

PURPOSE: Theoretical and experimental evidences support the hypothesis that Extremely Low-Frequency Electromagnetic Fields (ELF-EMF) can modulate voltage-gated channels. In this work we investigated the effect of ELF-EMF on Kv1.3, a member of the family of the voltage-gated potassium channels that is thought to be involved in key physiological functions, including the regulation of T-cells activation during the immune response. MATERIALS AND METHODS: Kv1.3 expressing CHO-K1 cells were exposed to a 20 Hz electromagnetic field at two different intensities: 268 µT and 902 µT. Kv1.3 potassium currents were recorded by whole-cell patch-clamp before, during and after field exposure. RESULTS: We found that the Kv1.3 current was increased significantly by the ELF-EMF in a subpopulation of CHO-K1 cells. The increase developed after a few seconds from the start of exposure, reached a steady-state and took several minutes to return to the baseline after field removal. CONCLUSIONS: These findings suggest that Kv1.3 may mediate interactions between ELF-EMF and living cells, disclosing new research opportunities on the molecular mechanisms with which electromagnetic fields affect physiological and pathological processes, including immunomodulation, inflammation and cancer.

Patients with radiation enteritis present regulatory T cell impairment associated with CTLA-4.

Radiation enteritis is one of the most common side effects of ionizing radiation in patients with pelvic cancers. Increasing amounts of evidence indicate that pro-inflammatory responses significantly contribute to the development of radiation enteritis. In this study, we investigated the association between T regulatory (Treg) cells and the risk of developing radiation enteritis in cervical cancer patients. The following observations were made. First, the frequencies of CD25hiFoxp3+ Treg cells were significantly lower in patients with radiation enteritis than in both healthy subjects and cervical cancer patients without radiation enteritis. Also, patients with the more severe grade 3 enteritis presented significantly lower Treg levels than patients with the more common grade 1 enteritis. Second, the expression of several molecules associated with Treg function, including CTLA-4, IL-10, TGF-ß, and perforin, was significantly lower in patients with radiation enteritis than in healthy subjects. In patients without radiation enteritis, however, only CTLA-4, but not other Treg-associated suppressive molecules, was reduced in Treg cells. Third, Treg cells can markedly suppress CD8 T cell proliferation, but in patients with radiation enteritis, this function of Treg cells was significantly impaired, in a manner that was associated with lower CTLA-4 expression. Overall, these data suggest that the frequency and function of Treg cells is negatively associated with the risk of developing enteritis following radiation. In clinical practice, the characteristics of Treg cells may be considered to evaluate the risk of developing enteritis if the cancer patient is receiving ionizing radiation.

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells.

BACKGROUND: The use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human TREG viability, phenotype, and suppressive activity. RESULTS: Both natural and TGF-ß1-induced CD4+ TREG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced TREG cells and the reduction was more robust in induced TREGS. Radiation also modulated the expression of signature iTREG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iTREGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells. CONCLUSIONS: Our findings demonstrate that while human TREG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of TREG signature molecules associated with suppressive activity. Functionally, irradiated TGF-ß1-induced TREGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce TREG activity, particularly when used in combination with cancer immunotherapies.

Engineering light-controllable CAR T cells for cancer immunotherapy.

T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.

B Cells Improve Overall Survival in HPV-Associated Squamous Cell Carcinomas and Are Activated by Radiation and PD-1 Blockade.

PURPOSE: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations. EXPERIMENTAL DESIGN: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV+ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade. RESULTS: RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade. CONCLUSIONS: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

The abscopal effect 67 years later: from a side story to center stage.

For over a century, ionising radiation has been used to treat cancer based on its cytotoxic effects on tumour cells. Technical progress has enabled more precise targeting of the tumour to reduce normal tissue toxicity while delivering higher radiation doses per fraction of treatment.In 1953, unexpected regression in lesions outside of the irradiated field were noted by an observant physician, RH Mole, who named such phenomenon "abscopal effect" from the Latin ab (position away from) and scopus (mark or target), in an article published in this journal. Clinical abscopal responses have been reported over the years but because of their very rare occurrence they could not be methodically studied, remaining akin to a curiosity. Nevertheless, their occurrence has ignited interest in studying the systemic effects of radiotherapy. Progress in dissecting the mechanisms that govern the function of the immune system in cancer has enabled to study the implication of immunity in the abscopal effect of radiation. It has become clear that ionising radiation activates canonical pathways of response to viral infections, and can stimulate antitumour immunity. These immune stimulatory effects of radiation have become clinically relevant in the current era of cancer immunotherapy, rendering abscopal responses in patients an attainable aim. Here, we will briefly review the parallel evolutions of two separate fields of medicine, radiation therapy and cancer immunology, and discuss their therapeutic partnership.

Physiological strength electric fields modulate human T cell activation and polarisation.

The factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.

Ionizing radiation reduces the capacity of activated macrophages to induce T-cell proliferation, but does not trigger dendritic cell-mediated non-targeted effects.

PURPOSE: Previous investigations revealed influences of irradiation up to 2Gy on the cytokine secretion profile of inflammatory and peritoneal mouse macrophages (pMФ). This raised the question if those alterations impact on dendritic cells and consecutive T-cell responses. Further, the impact of irradiation directly on pMФ capacity to induce T-cell responses was analyzed. MATERIALS AND METHODS: pMФ were LPS-activated, irradiated and the expression of activation markers was assessed. Treated pMФ were co-incubated with T-cells to investigate proliferation. To verify modulating properties of pMФ supernatants isolated 24 h after irradiation, bone marrow-derived dendritic cells (BMDC) were co-incubated with supernatants and activation markers as well as the BMDC-induced proliferation of T-cells were measured. RESULTS: pMФ showed a highly significantly decreased major histocompatibility complexII (MHCII) expression within a dose range from 0.7-2Gy. Further, the proliferation rate of cluster of differentiation 4+ (CD4+) T-cells was decreased after co-incubation particularly with 2 Gy irradiated pMФ. The co-incubation of BMDC with supernatants of activated, irradiated pMФ significantly reduced the CD40 expression, but did not impact on the BMDC-derived induction of T-cell proliferation. CONCLUSIONS: Inflammatory macrophages being exposed to irradiation have the potential to modulate consecutive adaptive immune reactions. But supernatants of irradiated macrophages do not influence the dendritic cells (DC)-mediated induction of T cell proliferation.

Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response.

Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.

Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells.

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca2+, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca2+ sensitive K+ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca2+-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.

Polyfunctionality of CD4+ T lymphocytes is increased after chemoradiotherapy of head and neck squamous cell carcinoma.

BACKGROUND: For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients' immune system are not fully understood. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4+ and CD8+ T cells by multicolor flow cytometry, and polyfunctional cytokine expression profiles were determined on a single-cell basis. RESULTS: Expression levels of all measured cytokines in CD4+ T cells were higher in patients after chemoradiotherapy (CRT) as compared to untreated HNSCC patients or normal controls. After CRT, the frequency of polyfunctional CD4+ T cells, which simultaneously expressed multiple cytokines, was significantly increased as compared to untreated patients (p < 0.01). CONCLUSION: CRT increases polyfunctionality of CD4+ T cells in HNSCC patients, suggesting that standard-of-care therapy can promote immune activity in immune cells. These polyfunctional CD4+ T cells in the blood of treated HNSCC patients are expected to be responsive to subsequent immunotherapeutic approaches.

Amotosalen/UVA treatment inactivates T cells more effectively than the recommended gamma dose for prevention of transfusion-associated graft-versus-host disease.

INTRODUCTION: Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication after transfusion of components containing viable donor T cells. Gamma irradiation with doses that stop T-cell proliferation is the predominant method to prevent TA-GVHD. Treatment with pathogen inactivation methodologies has been found to also be effective against proliferating white blood cells, including T cells. In this study, T-cell inactivation was compared, between amotosalen/ultraviolet A (UVA) treatment and gamma-irradiation (2500 cGy), using a sensitive limiting dilution assay (LDA) with an enhanced dynamic range. METHODS AND MATERIALS: Matched plasma units (N = 8), contaminated with 1 × 106 peripheral blood mononuclear cells (PBMCs) per mL, were either treated with amotosalen/UVA or gamma irradiation, or retained as untreated control. Posttreatment, cells were cultured under standardized conditions. T-cell proliferation was determined by the incorporation of 3 H-thymidine and correlated with microscopic detection. RESULTS: Range-finding experiments showed that after gamma irradiation (2500 cGy), significant T-cell proliferation could be observed at a 1 × 107 cell culture density, some proliferation at 1 × 106 , and none at 1 × 105 cells/well. Based on these facts, a quantitative comparison was carried out between amotosalen/UVA at the highest challenge of 1 × 107 PBMCs/well, and gamma irradiation at 1 × 106 and 1 × 105 PBMCs/well. Complete inactivation of the T cells after amotosalen/UVA treatment was observed, equivalent to greater than 6.2 log inactivation. Complete inactivation of the T cells was also observed after gamma irradiation when 1 × 105 PBMCs/well were cultured (>4.2 log inactivation). Proliferation was observed when 1 × 106 PBMCs/well were cultured (≤5.2 log inactivation) after gamma irradiation. CONCLUSION: Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA-GVHD.

Abscopal Effects With Hypofractionated Schedules Extending Into the Effector Phase of the Tumor-Specific T-Cell Response.

PURPOSE: Hypofractionated radiation therapy (hRT) combined with immune checkpoint blockade can induce T-cell-mediated local and abscopal antitumor effects. We had previously observed peak levels of tumor-infiltrating lymphocytes (TILs) between days 5 and 8 after hRT. Because TILs are regarded as radiosensitive, hRT schedules extending into this period might be less immunogenic, prompting us to compare clinically relevant, short and extended schedules with equivalent biologically effective doses combined with anti-programmed cell death 1 (PD1) antibody treatment. METHODS AND MATERIALS: In mice bearing 2 B16-CD133 melanoma tumors, the primary tumor was irradiated with 3 × 9.18 Gy in 3 or 5 days or with 5 × 6.43 Gy in 10 days; an anti-PD1 antibody was given weekly. The mice were monitored for tumor growth and survival. T-cell responses were determined on days 8 and 15 of treatment. The role of regional lymph nodes was studied by administering FTY720, which blocks lymph node egress of activated T cells. Tumor growth measurements after combination treatment using short or extended hRT and control treatment were also performed in the wild-type B16 melanoma and 4T1 breast carcinoma models. RESULTS: In the B16-CD133 model, growth inhibition of irradiated primary and nonirradiated secondary tumors and overall survival were similar with all 3 hRT/anti-PD1 combinations, superior to hRT and anti-PD1 monotherapy, and was strongly dependent on CD8+ T cells. TIL infiltration and local and systemic tumor-specific CD8+ T-cell responses were also similar, regardless of whether short or extended hRT was used. Administration of FTY720 accelerated growth of both primary and secondary tumors, strongly reduced their TIL infiltration, and increased tumor-specific CD8+ T cells in the lymph nodes draining the irradiated tumor. In the 4T1 model, local and abscopal tumor control was also similar, regardless of whether short or extended hRT was used, although the synergy between hRT and anti-PD1 was weaker. No synergies were found in the B16 wild-type model lacking an exogenous antigen. CONCLUSIONS: Our data suggest that combination therapy with hRT schedules extending into the period during which treatment-induced T cells infiltrate the irradiated tumor can provoke local and systemic antitumor effects similar to those with therapy using shorter schedules, if the regional lymph nodes supply sufficient tumor-specific T cells. This has implications for planning clinical RT/immune checkpoint blockade trials.

Resident T Cells in Resolved Psoriasis Steer Tissue Responses that Stratify Clinical Outcome.

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

Enhanced activated T cell subsets in prostate cancer patients receiving iodine-125 low-dose-rate prostate brachytherapy.

Radiotherapy (RT) is one of the most important treatments for prostate cancer. Although RT can kill cancer cells through direct and indirect effects of radiation, it occasionally induces an abscopal effect whereby localized radiation treatment is associated with elimination of metastatic cancer at a distance from the irradiated area. Thus, RT may induce an effective antitumor immune response, although the mechanism involved has remained unclear. The present was designed to evaluate this effect of RT in 36 patients with prostate cancer who provided informed consent prior to enrollment in this clinical trial. Peripheral blood samples were collected periodically after low-dose-rate (LDR) prostate brachytherapy, and lymphocyte subsets were analyzed by flow cytometry. The proportion of activated T cells (CD3+HLA-DR+, CD4+HLA-DR+ and CD8+HLA-DR+) in peripheral blood revealed a gradual and bimodal increase after LDR brachytherapy, whereas memory CD8+ T cells bimodally decreased after treatment. The ratios of activated T cells and regulatory T cells gradually increased after the treatment. Thus, LDR brachytherapy was demonstrated to induce effective immune responses in patients. This increase of activated T cells may contribute to maintenance of remission and reduction of relapse rates.

Expansion of cytotoxic natural killer cells using irradiated autologous peripheral blood mononuclear cells and anti-CD16 antibody.

Natural killer (NK) cells are considered a promising strategy for cancer treatment. Various methods for large-scale NK cell expansion have been developed, but they should guarantee that no viable cells are mixed with the expanded NK cells because most methods involve cancer cells or genetically modified cells as feeder cells. We used an anti-CD16 monoclonal antibody (mAb) and irradiated autologous peripheral blood mononuclear cells (PBMCs) (IrAPs) to provide a suitable environment (activating receptor-ligand interactions) for the NK cell expansion. This method more potently expanded NK cells, and the final product was composed of highly purified NK cells with lesser T-cell contamination. The expanded NK cells showed greater upregulation of various activation receptors, CD107a, and secreted larger amounts of interferon gamma. IrAPs expressed NKG2D ligands and CD48, and coengagement of CD16 with NKG2D and 2B4 caused potent NK cell activation and proliferation. The expanded NK cells were cytotoxic toward various cancer cells in vitro and in vivo. Moreover, irradiation or a chemotherapeutic drug further enhanced this antitumor effect. Therefore, we developed an effective in vitro culture method for large-scale expansion of highly purified cytotoxic NK cells with potent antitumor activity using IrAPs instead of cancer cell-based feeder cells.

Ultraviolet B Exposure Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice by Expanding CD4+Foxp3+ Regulatory T Cells.

BACKGROUND: Pathogenic immune responses are known to play an important role in abdominal aortic aneurysm (AAA) development. Ultraviolet B (UVB) irradiation has been demonstrated to have therapeutic potential not only for cutaneous diseases but also for systemic inflammatory diseases in mice by suppressing immunoinflammatory responses. We investigated the effect of UVB irradiation on experimental AAA. METHODS AND RESULTS: We used an angiotensin II-induced AAA model in apolipoprotein E-deficient mice fed a high-cholesterol diet. Mice aged 10 weeks were irradiated with 5 kJ/m2 UVB once weekly for 6 weeks (UVB-irradiated, n=38; nonirradiated, n=42) and were euthanized for evaluation of AAA formation at 16 weeks. Overall, 93% of angiotensin II-infused mice developed AAA, with 60% mortality possibly because of aneurysm rupture. UVB irradiation significantly decreased the incidence (66%) and mortality (29%) of AAA (P=0.004 and P=0.006, respectively). UVB-irradiated mice had significantly smaller diameter AAA (P=0.008) and fewer inflammatory cells in the aortic aneurysm tissue than nonirradiated mice, along with systemic expansion of CD4+Foxp3+ regulatory T cells and decreased effector CD4+CD44highCD62Llow T cells in para-aortic lymph nodes. Genetic depletion of regulatory T cells abrogated these beneficial effects of UVB treatment, demonstrating a critical role of regulatory T cells. CONCLUSIONS: Our data suggest that UVB-dependent expansion of regulatory T cells has beneficial effects on experimental AAA and may provide a novel strategy for the treatment of AAA.