The transcription factor IRF4 determines the anti-tumor immunity of CD8+ T cells.

Understanding the factors that regulate T cell infiltration and functional states in solid tumors is crucial for advancing cancer immunotherapies. Here, we discovered that the expression of interferon regulatory factor 4 (IRF4) was a critical T cell intrinsic requirement for effective anti-tumor immunity. Mice with T-cell-specific ablation of IRF4 showed significantly reduced T cell tumor infiltration and function, resulting in accelerated growth of subcutaneous syngeneic tumors and allowing the growth of allogeneic tumors. Additionally, engineered overexpression of IRF4 in anti-tumor CD8+ T cells that were adoptively transferred significantly promoted their tumor infiltration and transition from a naive/memory-like cell state into effector T cell states. As a result, IRF4-engineered anti-tumor T cells exhibited significantly improved anti-tumor efficacy, and inhibited tumor growth either alone or in combination with PD-L1 blockade. These findings identify IRF4 as a crucial cell-intrinsic driver of T cell infiltration and function in tumors, emphasizing the potential of IRF4-engineering as an immunotherapeutic approach.

A human pan-cancer system analysis of regulator of chromatin condensation 2.

Regulation of chromosome condensation 2 (RCC2) is associated with the cell cycle and is a crucial regulator of the chromatin condensation 1 (RCC1) family. The members of this family were normally regulators in the process of DNA replication and nucleocytoplasmic transport. RCC2 overexpression may lead to tumor formation and poor prognosis in some tumors including breast cancer and lung adenocarcinoma. However, the possible role of RCC2 in tumor formation and its prognostic function remains unclear. In this study, expression analysis from databases including The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) were combined to perform the first integrative and comprehensive analysis of RCC2 in human pan-cancer. RCC2 was highly expressed in most tumors which may lead to a poor prognosis. RCC2 expression was associated with immune/stromal infiltration, immune checkpoints, tumor mutational burden, and microsatellite instability. Thus, RCC2 could be a novel biomarker for prognosis and a promising cancer therapy target.

PLODs: Novel prognostic biomarkers and potential immunotherapy targets for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) comprise a group of malignant tumors arising from the squamous epithelium of the oral cavity, pharynx, and larynx. HNSCC is the 6th most common cancer in the world, with approximately 650,000 new cases and 400,000 deaths annually. Although survival rates have improved, HNSCC therapy may result in short - or long-term morbidity in approximately 50% of cases. Previous studies have also indicated that the overexpression of procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenases (PLOD) family proteins could lead to certain diseases or even tumors. However, there has been no dedicated evaluation of the relationship between PLOD family members and HNSCC. Here we used data from the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) databases to explore the potential role of PLOD family proteins in HNSCC. Our evaluations suggest that increased expression of PLOD family proteins may be associated with poorer prognosis and increased immune infiltration in HNSCC, making these proteins a potential biomarker for personalized treatment of HNSCC.

Continuous Expression of Interferon Regulatory Factor 4 Sustains CD8+ T Cell Immunity against Tumor.

T-cell-based immunotherapy is gaining momentum in cancer treatment; however, our comprehension of the transcriptional regulation governing T cell antitumor activity remains constrained. The objective of this study was to explore the function of interferon regulatory factor 4 (IRF4) in antitumor CD8+ T cells using the TRAMP-C1 prostate cancer and B16F10 melanoma model. To achieve this, we generated an Irf4GFP-DTR mouse strain and discovered that CD8+ tumor-infiltrating lymphocytes (TILs) expressing high levels of IRF4.GFP exhibited a more differentiated PD-1high cell phenotype. By administering diphtheria toxin to tumor-bearing Irf4GFP-DTR mice, we partially depleted IRF4.GFP+ TILs and observed an accelerated tumor growth. To specifically explore the function of IRF4 in antitumor CD8+ T cells, we conducted 3 adoptive cell therapy (ACT) models. Firstly, depleting IRF4.GFP+ CD8+ TILs derived from ACT significantly accelerated tumor growth, emphasizing their crucial role in controlling tumor progression. Secondly, deleting the Irf4 gene in antitumor CD8+ T cells used for ACT led to a reduction in the frequency and effector differentiation of CD8+ TILs, completely abolishing the antitumor effects of ACT. Lastly, we performed a temporal deletion of the Irf4 gene in antitumor CD8+ T cells during ACT, starting from 20 days after tumor implantation, which significantly compromised tumor control. Therefore, sustained expression of IRF4 is essential for maintaining CD8+ T cell immunity in the melanoma model, and these findings carry noteworthy implications for the advancement of more potent immunotherapies for solid tumors.

Melatonin Improved the Survival of Multi-Territory Perforator Flaps by Promoting Angiogenesis and Inhibiting Apoptosis via the NRF2/FUNDC1 Axis.

Background: Multi-territory perforator flaps are a reconstructive measure for repairing large soft tissue defects caused by tumors or trauma. However, the use of these flaps in clinical practice has been restricted due to the uncertain blood supply. Therefore, promoting the survival of the multi-territory perforator flap is critical for clinical repair and reconstruction. In our study, we explored the effects of melatonin (MLT) on multi-territory perforator flaps and the possible molecular mechanisms. Materials and Methods: Seventy-two Sprague-Dawley rats (250-300 g) were randomly divided into 3 groups (n = 24): Control, MLT and MLT + ML385 groups. First, we assessed the survival area of the flap, followed by the micro-vessel density and CD31-positive vessel expression. Apoptosis of the skin flap under immunofluorescence and expression of the apoptosis-related proteins Bcl-2, Bax and Caspase3 were measured. Additionally, angiogenesis of the skin flaps was shown by angiography, and NRF2 and FUNDC1 mRNA and protein expression was detected by real-time PCR and western blotting. Results: The results showed that MLT increased the survival area of the multi-territory perforator flap, which was related to increased angiogenesis and decreased apoptosis. We also found that mRNA and protein of NRF2 and FUNDC1 levels were significantly increased after MLT treatment, and an NRF2 inhibitor reversed the ability of MLT to enhance multi-territory perforator flap survival, promote angiogenesis and inhibit apoptosis and reduced FUNDC1 protein expression. Conclusion: MLT promoted angiogenesis and inhibited apoptosis to promote the survival of multi-territory perforator flaps, which may be regulated via the NRF2/FUNDC1 axis.

Exosomes from LPS-preconditioned bone marrow MSCs accelerated peripheral nerve regeneration via M2 macrophage polarization: Involvement of TSG-6/NF-κB/NLRP3 signaling pathway.

Lipopolysaccharide (LPS)-preconditioned mesenchymal stem cells (MSCs) possessed strong immunomodulatory and anti-inflammatory functions by secreting exosomes as major paracrine effectors. However, the specific effect of exosomes from LPS pre-MSCs (LPS pre-Exos) on peripheral nerve regeneration has yet to be documented. Here, we established a sciatic nerve injury model in rats and an inflammatory model in RAW264.7 cells to explore the potential mechanism between LPS pre-Exos and peripheral nerve repair. The local injection of LPS pre-Exos into the nerve injury site resulted in an accelerated functional recovery, axon regeneration and remyelination, and an enhanced M2 Macrophage polarization. Consistent with the data in vivo, LPS pre-Exos were able to shift the pro-inflammation macrophage into a pro-regeneration macrophage. Notably, TNF stimulated gene-6 (TSG-6) was found to be highly enriched in LPS pre-Exos. We obtained si TSG-6 Exo by the knockdown of TSG-6 in LPS pre-Exos to demonstrate the role of TSG-6 in macrophage polarization, and found that TSG-6 served as a critical mediator in LPS pre-Exos-induced regulatory effects through the inhibition of NF-ΚΒ and NOD-like receptor protein 3 (NLRP3). In conclusion, our findings suggested that LPS pre-Exos promoted macrophage polarization toward an M2 phenotype by shuttling TSG-6 to inactivate the NF-ΚΒ/NLRP3 signaling axis, and could provide a potential therapeutic avenue for peripheral nerve repair.

Customized reconstruction of a complex soft-tissue defect around the knee with a free perforator flap.

Reconstruction of a complex defect around the knee, particularly involving a large soft-tissue defect or disruption of the extensor mechanism, is always a challenging problem. The purpose of this study was to introduce the use of a customized free perforator flap for complex soft-tissue reconstruction around the knee. Between June 2010 and March 2017, 16 patients underwent this procedure. The choice of flap design is based on the location of the wound, the required pedicle length, the missing tissue components and their volumes, and the risk of donor-site morbidity. The reconstruction was performed using anterolateral thigh perforator (ALTP) flaps in five cases, modified ALTP flaps in two cases, chimeric ALTP flaps in four cases, dual-skin paddle ALTP flaps in two cases, and chimeric thoracodorsal artery perforator flaps in two cases. Multiple perforator flaps and vascularized fascia lata were used in one case. All flaps survived postoperatively. No vascular congestion was observed, and partial necrosis was observed in only one case. Primary closure of the donor site was performed for all patients. At a mean follow-up time of 16.5 months, most cases showed satisfactory flap contours and acceptable functional outcomes. A free perforator flap is a reliable option for repairing complex soft-tissue defects in the knee region, especially when local and pedicled flaps are unavailable. Various flap designs allow for more individualized treatment approaches and can achieve better results.

Adoptive CD8+ T cell therapy generates immunological memory to inhibit melanoma metastasis.

Adoptive T cell therapy has emerged as a promising treatment for cancer. However, it is unknown whether adoptively transferred anti-tumor T cells can form immunological memory and provide continuous protection against cancer metastasis. Herein, we used TCR transgenic Pmel-1 CD8+ T cells as a model to investigate whether early transferred Pmel-1 CD8+ T cells can generate immunological memory to prevent later melanoma metastasis. Upon stimulation with the cognate melanoma-associated hgp100 antigen, in vitro cultured Pmel-1 CD8+ T cells developed into effector T (Teff) cells that exhibited potent cytotoxic activity against B16F10 melanoma cells. Next, B16F10 melanoma cells were intravenously injected into C57BL/6 (B6) mice to establish experimental lung metastasis. In vitro generated Pmel-1 Teff cells were adoptively transferred into the mice on the same day of or three weeks prior to B16F10 cell inoculation. We found that adoptive Pmel-1 Teff cell therapy significantly inhibited the B16F10 lung metastasis and prolonged the animal survival. Importantly, Pmel-1 Teff cells transferred three weeks prior to tumor inoculation were as potent as the Pmel-1 Teff cells transferred on the same day in inhibiting melanoma metastasis. Hence, our results suggest that adoptive CD8+ Teff cell therapy generates immunological memory that continuously protect against melanoma metastasis.

Interferon regulatory factor 4 deficiency in CD8+ T cells abrogates terminal effector differentiation and promotes transplant acceptance.

Allogeneic CD8+ cytotoxic T cells play an essential role in rejecting transplanted allografts, but how their effector function is regulated on a transcriptional level remains unclear. Herein, we investigate the role of interferon regulatory factor 4 (IRF4) in controlling CD8+ T-cell function in response to transplant. B6.Rag1-/- mice were adoptively transferred with CD8+ T cells isolated from either Irf4fl/fl Cd4-Cre (T-cell-specific Irf4-deficient) or Irf4fl/fl control mice, followed by BALB/c skin transplantation. Recipients that received Irf4-deficient CD8+ T cells permanently accepted the skin allografts, whereas recipients that received control CD8+ T cells acutely rejected the transplanted skins. Mechanistically, compared with the transferred control CD8+ T cells in B6.Rag1-/- recipients, the transferred Irf4-deficient CD8+ T cells lost the capacity to differentiate into CD127- KLRG1+ terminal effector cells, barely produced effector cytokines and cytotoxic molecules (e.g. IL-2, IFN-γ, TNF-α, granzyme A and granzyme B), and displayed defect in proliferative capacity, evident by their decreased Ki67 expression and lower frequencies. Moreover, the transferred Irf4-deficient CD8+ T cells displayed low expression of transcription factors ID2 and T-bet that govern the terminal effector T-cell programmes, and high expression of transcription factor TCF1 that maintains the naïve-memory T-cell programmes. Hence, IRF4 deficiency in CD8+ T cells abrogates their terminal effector differentiation and promotes transplant acceptance. These findings suggest that targeting IRF4 expression represents an attractive and promising therapeutic approach for inducing transplant acceptance.

T follicular helper and memory cell responses and the mTOR pathway in murine heart transplantation.

BACKGROUND: The mammalian target of rapamycin (mTOR) inhibitors are valuable immunosuppressants in clinical transplantation; however, the mTOR regulation of allogeneic T-cell responses is not fully understood yet. Therefore, the objective of this study is to investigate the effects of T-cell-specific mTOR deletion on the allogeneic T-cell responses and heart transplant survival. METHODS: BALB/c heart allografts, with or without BALB/c skin sensitization, were transplanted in the wild-type C57BL/6, Mtorfl/flCd4-Cre, Stat3fl/flCd4-Cre, and Mtorfl/flStat3fl/flCd4-Cre mice. Graft survival and histology, as well as T-cell frequencies and phenotypes, were evaluated after transplantation. RESULTS: In the absence of donor skin sensitization, long-term heart allograft survival was achieved in the Mtorfl/flCd4-Cre recipients, which was associated with significantly decreased frequencies of CD62L-CD44+ effector T cells and BCL-6+CXCR5+ T follicular helper (Tfh) cells in the periphery. Long-term heart allograft survival was also achieved in the donor skin-sensitized Mtorfl/flStat3fl/flCd4-Cre mice, whereas the heart allograft survival was prolonged in the donor skin-sensitized Mtorfl/flCd4-Cre and Stat3fl/flCd4-Cre mice. CONCLUSIONS: mTOR is required for Tfh cell response in murine heart transplantation. T-cell-specific deletion of both mTOR and Stat3 abrogates the memory response to heart transplants. These findings help us to better understand the molecular mechanisms underlying the T cell immunity to transplanted organs.

CD4+ T cell exhaustion leads to adoptive transfer therapy failure which can be prevented by immune checkpoint blockade.

Cytotoxic CD8+ T cell exhaustion is one of the mechanisms underlying the tumor immune escape. The paradigm-shifting immune checkpoint therapy can mitigate CD8+ T lymphocyte exhaustion, reinvigorate the anticancer immunity, and achieve durable tumor regression for some patients. Emerging evidence indicates that CD4+ T lymphocytes also have a critical role in anticancer immunity, either by directly applying cytotoxicity toward cancer cells or as a helper to augment CD8+ T cell cytotoxicity. Whether anticancer CD4+ T lymphocytes undergo exhaustion during immunotherapy of solid tumors remains unknown. Here we report that melanoma antigen TRP-1/gp75-specific CD4+ T lymphocytes exhibit an exhaustion phenotype after being adoptively transferred into mice bearing large subcutaneous melanoma. Exhaustion of these CD4+ T lymphocytes is accompanied with reduced cytokine release and increased expression of inhibitory receptors, resulting in loss of tumor control. Importantly, we demonstrate that PD-L1 immune checkpoint blockade can prevent exhaustion, induce proliferation of the CD4+ T lymphocytes, and consequently prevent tumor recurrence. Therefore, when encountering an excessive amount of tumor antigens, tumor-reactive CD4+ T lymphocytes also enter the exhaustion state, which can be prevented by immune checkpoint blockade. Our results highlight the importance of tumor-specific CD4+ T lymphocytes in antitumor immunity and suggest that the current immune checkpoint blockade therapy may achieve durable anticancer efficacy by rejuvenating both tumor antigen-specific CD8+ T lymphocytes and CD4+ T lymphocytes.

Metformin induces the M2 macrophage polarization to accelerate the wound healing via regulating AMPK/mTOR/NLRP3 inflammasome singling pathway.

BACKGROUND: Skin wound healing is a challenging problem, especially in aging or diabetic patients, which becomes more difficult to heal, and easily results in considerable public health burden. The purpose of this study was to investigate the effects of metformin on wound healing and explore its underlying mechanism. METHODS: Metformin was local topical application in rat skin defect models. Alterations in the wounded skin were observed, and angiogenesis in the wound also was analyzed by immunohistochemical staining. The markers associated with differentiation macrophage were analyzed by immunofluorescence staining. The roles of AMPK singling pathway and the relative protein of NLRP3 inflammasome in wound were also analyzed by western blotting. In addition, AMPK/mTOR/NLRP3 inflammasome signaling axis was investigated to further analyze the molecular mechanism of metformin treatment on inducing M2 macrophage polarization in vitro. RESULTS: Out results showed that metformin improved wound healing and angiogenesis which was paralleled by M2 macrophage polarization. We also found that the level of relative proteins of NLRP3 inflammasome was markedly decreased after metformin treatment. Furthermore, blockage of AMPK or activation of mTOR abolished the effects of metformin treatment on depressing NLRP3 inflammasome activation, M2 polarization and improving wound healing. It suggested that the treatment effects of metformin on wound healing were through regulating AMPK/mTOR/NLRP3 inflammasome signaling axis. CONCLUSION: Metformin regulated AMPK/mTOR singling pathway to inhibit NLRP3 inflammasome activation, which boosted M2 macrophage polarization to accelerate the wound healing. These findings provided new insights into the molecular mechanism of metformin therapy and its therapeutic potential in wound healing.