Human CD8+CD28- T suppressor cells expanded by common gamma chain (γc) cytokines retain steady allospecific suppressive capacity in vivo.

BACKGROUND: CD8+CD28- T suppressor (Ts) cells play critical role in transplant tolerance. Our previous study has generated CD8+CD28- Ts cells in vitro which exert robust allospecific suppressive capacity in vitro. RESULTS: CD8+CD28- Ts cells were expanded by stimulating human CD8+ T cells with allogeneic antigen presenting cells in the presence of the common gamma chain cytokines IL-2, IL-7 and IL-15 in vitro, and were further verified in vitro through day 7 to 11 for their persistency of the allospecific suppressive capacity. When CD8+CD28- Ts cells were adoptively transferred into NOG mice, their capacity to inhibit CD4+ T cell proliferation in allospecific manner remained potent on 11 days after their injection. The mechanisms for expansion of CD8+CD28- Ts cells by the common gamma chain cytokines were investigated. These included promoting CD8+CD28- T cells proliferation, converting CD8+CD28+ T cells to CD8+CD28- T cells and decreasing CD8+CD28- T cell death. Furthermore, the expanded CD8+CD28- Ts cells showed upregulation of the co-inhibitory molecule Tim-3 and down-regulation of the cytotoxic molecule granzyme B. CONCLUSIONS: In summary, these results demonstrated that the in vitro-expanded human CD8+CD28- T cells retained potent allospecific suppressive capacity in vivo and depicted multiple mechanisms for the expansion of Ts cells, which might promote further bench to clinic research.

Maturation of induced pluripotent stem cell-derived cardiomyocytes and its therapeutic effect on myocardial infarction in mouse.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have an irreplaceable role in the treatment of myocardial infarction (MI), which can be injected into the transplanted area with new cardiomyocytes (Cardiomyocytes, CMs), and improve myocardial function. However, the immaturity of the structure and function of iPSC-CMs is the main bottleneck at present. Since collagen participates in the formation of extracellular matrix (ECM), we synthesized nano colloidal gelatin (Gel) with collagen as the main component, and confirmed that the biomaterial has good biocompatibility and is suitable for cellular in vitro growth. Subsequently, we combined the PI3K/AKT/mTOR pathway inhibitor BEZ-235 with Gel and found that the two combined increased the sarcomere length and action potential amplitude (APA) of iPSC-CMs, and improved the Ca2+ processing ability, the maturation of mitochondrial morphological structure and metabolic function. Not only that, Gel can also prolong the retention rate of iPSC-CMs in the myocardium and increase the expression of Cx43 and angiogenesis in the transplanted area of mature iPSC-CMs, which also provides a reliable basis for the subsequent treatment of mature iPSC-CMs.

Hydrochemical analysis and groundwater suitability for drinking and irrigation in an arid agricultural area of the Northwest China.

Groundwater is the foremost water source in the arid and semiarid regions of Northwest China. Assessing groundwater's drinking and irrigation quality is essential for protecting these valuable groundwater resources. In this study, a total of 24 confined groundwater samples and 54 phreatic groundwater samples were collected in the southern and central Ningxia area for hydrochemical analysis and quality assessment. The hydrochemical results revealed that hydrochemical types of phreatic and confined groundwater consistently belonged to Na-SO4-Cl and Na-Mg-SO4-Cl types. The driving forces of groundwater chemistry were determined by gypsum dissolution, silicate dissolution, and positive cation exchange for phreatic and confined aquifers. The entropy-weighted water quality index (EWQI) and irrigation water quality index (IWQI) showed that the drinking water quality and irrigation quality were better in phreatic groundwater than in confined groundwater due to the Neogene-Paleogene groundwater system recharge and strong evaporation. Measures such as controlling groundwater extraction and optimizing well placement need to be implemented. The achievements would be helpful for groundwater management and protection in agricultural areas under semi-arid and arid climates.

In vitro and In vivo CD8+ T Cell Suppression Assays.

CD8+CD28- T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells. 3H-thymidine is a classical immunological technique for assaying T cell proliferation but this approach has drawbacks such as the inconvenience of working with radioactive materials. Labeling T cells with CFSE allows relatively easy tracking of generations of proliferated cells. In this report, we utilized antigen presenting cells (APCs) and T cells matched for human leukocyte antigen (HLA) class I or class II to study CD8+CD28- T cell suppression generated in vitro by this novel approach of combining allogeneic APCs and γc cytokines. The expanded CD8+CD28- T cells were isolated (purity 95%) and evaluated for their suppressive capacity in mixed lymphocyte reactions using CD4+ T cells as responders. Here, we present our adapted protocol for assaying the Ts allospecific suppression of CFSE-labeled responder T cells.

WITHDRAWN: Knocking out c-Jun promotes cardiomyocyte differentiation from embryonic stem cells.

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Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming.

BACKGROUND: Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. METHODS: The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. RESULTS: ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. CONCLUSIONS: In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease.

Advances in 3D bioprinting technology for cardiac tissue engineering and regeneration.

Cardiovascular disease is still one of the leading causes of death in the world, and heart transplantation is the current major treatment for end-stage cardiovascular diseases. However, because of the shortage of heart donors, new sources of cardiac regenerative medicine are greatly needed. The prominent development of tissue engineering using bioactive materials has creatively laid a direct promising foundation. Whereas, how to precisely pattern a cardiac structure with complete biological function still requires technological breakthroughs. Recently, the emerging three-dimensional (3D) bioprinting technology for tissue engineering has shown great advantages in generating micro-scale cardiac tissues, which has established its impressive potential as a novel foundation for cardiovascular regeneration. Whether 3D bioprinted hearts can replace traditional heart transplantation as a novel strategy for treating cardiovascular diseases in the future is a frontier issue. In this review article, we emphasize the current knowledge and future perspectives regarding available bioinks, bioprinting strategies and the latest outcome progress in cardiac 3D bioprinting to move this promising medical approach towards potential clinical implementation.

A Promising Candidate: Heparin-Binding Protein Steps onto the Stage of Sepsis Prediction.

Sepsis is a systemic inflammatory response syndrome caused by infection. With high morbidity and mortality of this disease, there is a need to find early effective diagnosis and assessment methods to improve the prognosis of patients. Heparin-binding protein (HBP) is a granular protein derived from polynuclear neutrophils. The biosynthetic HBP in neutrophils is rapidly released under the stimulation of bacteria, resulting in increased vascular permeability and edema. It is reasonable to speculate that the HBP in plasma may serve as a novel diagnostic marker for sepsis, bacterial skin infection, acute bacterial meningitis, leptospirosis, protozoan parasites, and even some noncommunicable diseases. It implies that in the detection and diagnosis of sepsis, it will be possible to make relevant diagnosis through this new indicator in the future. In this review, we summarize the typical biological function of HBP and its latest research progress to provide theoretical basis for clinical prediction and diagnosis of sepsis.

Different Effects of Interleukin 21 and Interleukin 15 on In Vitro Expanded CD8+ T Cells Stimulated by Alloantigen.

OBJECTIVE: To investigate the effects of IL (interleukin) 21 on CD8+ T cells stimulated by alloantigen in the presence of IL-15 in vitro. METHODS: CD8+ T cells sorted with MicroBeads from fresh human peripheral blood mononuclear cells were cocultured with antigen-presenting cells derived from HLA-A, -B, and -DR full-mismatched individuals for 9 days without any cytokines, in the presence of IL-15, IL-21, and IL-15 combined with IL-21, respectively. The proliferation and phenotypic characteristics of CD28+ and CD28- subsets were measured after 9 days of culture. RESULTS: The proliferation of CD8+ T cells can be promoted either by IL-15 alone or in combination with IL-21 compared with IL-21. Cells expanded in the presence of IL-15 are mainly CD8+CD28- T cells, while those expanded in the presence of IL-15 combined with IL-21 are mostly CD8+CD28+ T cells. In the presence of IL-15, most CD8+CD28+ T cells shifted to CD8+CD28- T cells during the process of proliferation, but In the presence of IL-15 combined with IL-21, CD8+CD28+ T cells didn't shift to CD8+CD28- T cells during proliferation, moreover, CD8+CD28- T cells cannot transform in reverse to CD8+CD28+ T cells. IL-21 combined with IL-15 can promote the expression of granzyme B and perforin in CD8+CD28+ and/or CD8+CD28- T cells compared with IL-15 alone. CONCLUSION: IL-21 cannot promote the proliferation of CD8+ T cells under allogeneic stimulation unless combined with IL-15. IL-21 prevents the loss of CD28 molecules caused by IL-15 but cannot promote its re-expression in CD28- T cells. CD8+ T cells expanded by IL-21 combined with IL-15 is characterized by cytotoxic phenotype.

Human CD8+CD28- T Suppressor Cells Expanded by IL-15 In Vitro Suppress in an Allospecific and Programmed Cell Death Protein 1-Dependent Manner.

CD8+CD28- T suppressor cells (Ts) have been recently documented to play an important role in alloimmunity. Therefore, understanding and optimizing the conditions under which these cells are generated and/or expanded would greatly facilitate further research and potential clinical use. In this study, we describe rapid expansion of human allospecific CD8+CD28- Ts cells through coculture of CD8+ T cells with human leukocyte antigen-mismatched donor antigen-presenting cells plus IL-15 in a relative short period of time in vitro. Interestingly, IL-15 promotes the expansion of CD8+CD28- Ts cells through several parallel mechanisms. The expanded CD8+CD28- Ts cells upregulate expression of CD132, CD25, and programmed cell death protein 1 (PD-1), but downregulate expression of CD122, GZM-B, and perforin, while exhibiting no cytotoxicity. Most importantly, the expanded CD8+CD28- Ts cells vigorously inhibit CD4+ T cells proliferation in a contact-dependent and donor-specific manner both in vitro and in vivo. Interestingly, the co-inhibitory molecules PD-1 and programmed death-ligand 1 play an obligatory role in the mechanisms of CD8+CD28- Ts cells suppression. Taken together, our study report novel methodology for IL-15-induced expansion of human CD8+CD28- Ts cells and possible mechanisms. These findings may facilitate understanding of transplant rejection and promote clinical application of CD8+CD28- Ts cell-based strategies for inducing and monitoring transplant tolerance in the future.