Low Percentage of Perforin-Expressing NK Cells during Severe SARS-CoV-2 Infection: Consumption Rather than Primary Deficiency.

Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.

NFIL3 contributes to cytotoxic T lymphocyte-mediated killing.

Cytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs.

Involvement of M1-Activated Macrophages and Perforin/Granulysin Expressing Lymphocytes in IgA Vasculitis Nephritis.

We investigated the polarisation of CD68+ macrophages and perforin and granulysin distributions in kidney lymphocyte subsets of children with IgA vasculitis nephritis (IgAVN). Pro-inflammatory macrophage (M)1 (CD68/iNOS) or regulatory M2 (CD68/arginase-1) polarisation; spatial arrangement of macrophages and lymphocytes; and perforin and granulysin distribution in CD3+ and CD56+ cells were visulaised using double-labelled immunofluorescence. In contrast to the tubules, iNOS+ cells were more abundant than the arginase-1+ cells in the glomeruli. CD68+ macrophage numbers fluctuated in the glomeruli and were mostly labelled with iNOS. CD68+/arginase-1+ cells are abundant in the tubules. CD56+ cells, enclosed by CD68+ cells, were more abundant in the glomeruli than in the tubuli, and co-expressed NKp44. The glomerular and interstitial/intratubular CD56+ cells express perforin and granulysin, respectively. The CD3+ cells did not express perforin, while a minority expressed granulysin. Innate immunity, represented by M1 macrophages and CD56+ cells rich in perforin and granulysin, plays a pivotal role in the acute phase of IgAVN.

Cell softness renders cytotoxic T lymphocytes and T leukemic cells resistant to perforin-mediated killing.

Mechanical force contributes to perforin pore formation at immune synapses, thus facilitating the cytotoxic T lymphocytes (CTL)-mediated killing of tumor cells in a unidirectional fashion. How such mechanical cues affect CTL evasion of perforin-mediated autolysis remains unclear. Here we show that activated CTLs use their softness to evade perforin-mediated autolysis, which, however, is shared by T leukemic cells to evade CTL killing. Downregulation of filamin A is identified to induce softness via ZAP70-mediated YAP Y357 phosphorylation and activation. Despite the requirements of YAP in both cell types for softness induction, CTLs are more resistant to YAP inhibitors than malignant T cells, potentially due to the higher expression of the drug-resistant transporter, MDR1, in CTLs. As a result, moderate inhibition of YAP stiffens malignant T cells but spares CTLs, thus allowing CTLs to cytolyze malignant cells without autolysis. Our findings thus hint a mechanical force-based immunotherapeutic strategy against T cell leukemia.

ERG mediates the inhibition of NK cell cytotoxicity through the HLX/STAT4/Perforin signaling pathway, thereby promoting the progression of myocardial infarction.

This study aimed to investigate the role of ERG in the HLX/STAT4/Perforin signaling axis, impacting natural killer (NK) cell cytotoxicity and myocardial infarction (MI) progression. NK cell cytotoxicity was assessed via co-culture and 51Cr release assays. Datasets GSE34198 and GSE97320 identified common differentially expressed genes in MI. NK cell gene expression was analyzed in MI patients and healthy individuals using qRT-PCR and Western blotting. ERG's regulation of HLX and STAT4's regulation of perforin were studied through computational tools (MEM) and ChIP experiments. HLX's influence on STAT4 was explored with the MG132 proteasome inhibitor. Findings were validated in a mouse MI model.ERG, a commonly upregulated gene, was identified in NK cells from MI patients and mice. ERG upregulated HLX, leading to STAT4 proteasomal degradation and reduced Perforin expression. Consequently, NK cell cytotoxicity decreased, promoting MI progression. ERG mediates the HLX/STAT4/Perforin axis to inhibit NK cell cytotoxicity, fostering MI progression. These results provide vital insights into MI's molecular mechanisms.

Upregulation of CX3CR1 expression in circulating T cells of systemic lupus erythematosus patients as a reflection of autoimmune status through characterization of cytotoxic capacity.

OBJECTIVE: This study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. METHODS: Peripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1's diagnostic potential for SLE. RESULTS: CX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. CONCLUSION: CX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.

CCL5 mediated astrocyte-T cell interaction disrupts blood-brain barrier in mice after hemorrhagic stroke.

The crosstalk between reactive astrocytes and infiltrated immune cells plays a critical role in maintaining blood-brain barrier (BBB) integrity. However, how astrocytes interact with immune cells and the effect of their interaction on BBB integrity after hemorrhagic stroke are still unclear. By performing RNA sequencing in astrocytes that were activated by interleukin-1α (IL-1α), tumor necrosis factor α (TNFα), and complement component 1q (C1q) treatment, we found CCL5 was among the top upregulated genes. Immunostaining and western blot results demonstrated that CCL5 was increased in mice brain after hemorrhagic stroke. Flow cytometry showed that knockout of astrocytic CCL5 reduced the infiltration of CD8+ but not CD4+ T and myeloid cells into the brain (p < 0.05). In addition, knockout CCL5 in astrocytes increased tight junction-related proteins ZO-1 and Occludin expression; reduced Evans blue leakage, perforin and granzyme B expression; improved neurobehavioral outcomes in hemorrhagic stroke mice (p < 0.05), while transplantation of CD8+ T cells reversed these protective effects. Moreover, co-culture of CD8+ T cells with bEnd.3 cells induced the apoptosis of bEnd.3 cells, which was rescued by inhibiting perforin. In conclusion, our study suggests that CCL5 mediated crosstalk between astrocytes and CD8+ T cells represents an important therapeutic target for protecting BBB in stroke.

Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

Harnessing the Power of CAR-NK Cells: A Promising Off-the-Shelf Therapeutic Strategy for CD38-Positive Malignancies.

Background: CD38 is highly expressed on multiple myeloma (MM) cells and has been successfully targeted by different target therapy methods. This molecule is a critical prognostic marker in both diffuse large B-cell lymphoma and chronic lymphocytic leukemia. Objective: We have designed and generated an anti-CD38 CAR-NK cell applying NK 92 cell line. The approach has potential application as an off-the-shelf strategy for treatment of CD38 positive malignancies. Methods: A second generation of anti-CD38 CAR-NK cell was designed and generated, and their efficacy against CD38-positive cell lines was assessed in vitro. The PE-Annexin V and 7-AAD methods were used to determine the percentage of apoptotic target cells. Flow cytometry was used to measure IFN-γ, Perforin, and Granzyme-B production following intracellular staining. Using in silico analyses, the binding capacity and interaction interface were evaluated. Results: Using Lentivirus, cells were transduced with anti-CD38 construct and were expanded. The expression of anti-CD38 CAR on the surface of NK 92 cells was approximately 25%. As we expected from in silico analysis, our designed CD38-chimeric antigen receptor was bound appropriately to the CD38 protein. NK 92 cells that transduced with the CD38 chimeric antigen receptor, generated significantly more IFN-γ, perforin, and granzyme than Mock cells, and successfully lysed Daudi and Jurkat malignant cells in a CD38-dependent manner. Conclusion: The in vitro findings indicated that the anti-CD38 CAR-NK cells have the potential to be used as an off-the-shelf therapeutic strategy against CD38-positive malignancies. It is recommended that the present engineered NK cells undergo additional preclinical investigations before they can be considered for subsequent clinical trial studies.

High-free Fatty Acid Treatment Induced Anti-inflammatory Changes in a Natural Killer (NK) Cell Line.

Background: Natural killer (NK) cells play a role in the pathogenesis of various metabolic diseases related to obesity. While our initial findings have indicated a potential involvement of NK cells in the pathogenesis of type 2 diabetes mellitus, the precise mechanism underlying NK cell-mediated development of this form of diabetes remains inadequately comprehended. Objective: To investigate the impact and the underlying mechanism of high glucose and elevated levels of free fatty acids (FFAs) on immune and inflammatory responses and oxidative stress in NK92 cells. Methods: In this experiment, the CCK8 cytotoxicity assay was used to select the 44.4 mM and 1.5 mM concentrations of high glucose and high FFAs, respectively, to treat NK92 cells for 4 days. The concentrations of superoxide dismutase (SOD) and glutathione (GSH) were determined using a biochemical analyzer. Intracellular reactive oxygen species (ROS) levels, cytokines concentrations (TNF-α, IFN-γ, IL-6, and IL-10), and the expression levels of intracellular molecules (perforin and granzyme B) were assessed by flow cytometry. Results: The number of NK92 cell clumps was significantly reduced in the high-FFA (HF) group. In addition, the production of ROS and levels of cytokines (TNF-α, IFN-γ, IL-6, and IL-10) significantly decreased in the HF group but showed no significant change in the high-glucose (HG) group. This observation was consistent with the expression levels of perforin and granzyme B that decreased in the HF group. Conclusion: High FFAs induced morphological changes and serious damage to oxidative stress and inflammatory response in NK92 cells.

Expression of Tim-3/Galectin-9 pathway and CD8+T cells and related factors in patients with cystic echinococcosis.

OBJECTIVE: One of the primary reasons for the successful patriotization of Echinococcus multilocularis in patients is its ability to induce host immune tolerance. This study examined the expression of the immunosuppressive Tim-3/Galectin-9 pathway, CD8+T cells, and related factors in AE patients. The aim was to analyze the relationship between the Tim-3/Galectin-9 pathway and CD8+T cells in this disease and further understand the mechanism of immune tolerance induced by cystic echinococcosis. METHODS: Using flow cytometry, we evaluated the expression of CTL, CD8+CD28-T cells, CD8+CD28 + IFN-γ + T cells, CD8+CD28+perforin + T cells, CD8+CD28+granzyme B + T cells, CD8+CD28-IL-10 + T cells, CD8+CD28-TGF-ß+T cells, and Tim-3 expression on CD8+T cells in the peripheral blood of control (n = 30) and AE patients (n = 33). qRT-PCR was used to measure CD107a and Tim-3/Galectin-9 mRNA levels in PBMCs from the control and AE groups. Immunohistochemistry was employed to detect IL-10, TGF-ß, and Tim-3/Galectin-9 expressions in the infected livers of AE patients. RESULTS: AE patients exhibited a significant decrease in peripheral blood CTL ratio (P < 0.001) and an increase in CD8+CD28+IFN-γ+T cell ratio (P < 0.001). No significant changes were observed in the ratios of CD8+CD28+perforin + T cells (P = 0.720) and CD8+CD28+granzyme B + T cells (P = 0.051). The proportions of CD8+CD28-T cells (P < 0.001), CD8+CD28-IL-10 + T cells (P < 0.001), and CD8+CD28-TGF-ß+T cells (P < 0.001) were notably higher than in the control group. The expression of Tim-3 on CTL and CD8+CD28-T cells in AE patients was significantly upregulated (P < 0.001, P < 0.001). AE patients displayed a substantial decrease in peripheral blood PBMC CD107a mRNA levels (P < 0.001) and significant elevations in Tim-3/Galectin-9 mRNA levels (P < 0.001, P < 0.001). A negative correlation was observed between CD107a mRNA levels and both Tim-3 (r^2 = 0.411, P < 0.001) and Galectin-9 (r2 = 0.180, P = 0.019) mRNA levels. Expressions of IL-10 (P < 0.001), TGF-ß (P < 0.001), and Tim-3/Galectin-9 (P < 0.001, P < 0.001) in AE patient-infected livers were significantly higher than in uninfected regions. IL-10 and TGF-ß expressions showed a positive correlation with Tim-3/Galectin-9. CONCLUSION: This study suggests that the high expression of Tim-3 on CD8+T cell surfaces in AE patients might promote an increase in CD8+CD28-T cells and related factors, while suppressing CTL and related factor expressions. This potentially induces the onset of immune tolerance, which is unfavorable for the clearance of Echinococcus multilocularis in patients, leading to the exacerbation of persistent infections.

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18). Though individually tolerated, mice with dual susceptibility (Prf1⁻/⁻Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T cells drove MAS after a viral (lymphocytic choriomeningitis virus), but not innate (toll like receptor 9), trigger. In vitro, CD8 T cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced but perforin deficiency impaired immunoregulatory restimulation-induced cell death (RICD). Paralleling hyperinflammation, DS mice displayed massive postthymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and interferon gamma production, which contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and antiretrovirals failed to ameliorate the disease. Attempting to genetically "fix" TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation, chiefly on T cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state, combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.

Fragment-based and structure-guided discovery of perforin inhibitors.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.

Surface plasmon resonance and microscale thermophoresis approaches for determining the affinity of perforin for calcium ions.

Perforin is a pore-forming protein that plays a crucial role in the immune system by clearing virus-infected or tumor cells. It is released from cytotoxic granules of immune cells and forms pores in targeted lipid membranes to deliver apoptosis-inducing granzymes. It is a very cytotoxic protein and is therefore adapted not to act in producing cells. Its activity is regulated by the requirement for calcium ions for optimal activity. However, the exact affinity of perforin for calcium ions has not yet been determined. We conducted a molecular dynamics simulation in the absence or presence of calcium ions that showed that binding of at least three calcium ions is required for stable perforin binding to the lipid membrane. Biophysical studies using surface plasmon resonance and microscale thermophoresis were then performed to estimate the binding affinities of native human and recombinant mouse perforin for calcium ions. Both approaches showed that mouse perforin has a several fold higher affinity for calcium ions than that of human perforin. This was attributed to a particular residue, tryptophan at position 488 in mouse perforin, which is replaced by arginine in human perforin. This represents an additional mechanism to control the activity of human perforin.

Limocitrin increases cytotoxicity of KHYG-1 cells against K562 cells by modulating MAPK pathway.

Natural killer (NK) cells are gaining popularity in the field of cancer immunotherapy. The present study was designed to investigate the effect of a natural flavonol compound limocitrin in increasing cytotoxicity of a permanent NK leukemia cell line KHYG-1 against an aggressive leukemia cell line K562. The findings revealed that limocitrin increased the expressions of cytolytic molecules perforin, granzymes A and B, and granulysin in KHYG-1 cells by inducing phosphorylation of transcription factor CREB, leading to increased lysis of K562 cells. Mechanistically, limocitrin was found to increase the expressions of t-Bid, cleaved caspase 3, and cleaved PARP to induce K562 cell apoptosis. Moreover, limocitrin reduced the expressions of SET and Ape1 to inhibit DNA repair mechanism, leading to caspase-independent K562 cell death. At the molecular level, limocitrin was found to increase the phosphorylation of ERK, p38, and JNK to increase granzyme B expression in KHYG-1 cells. Taken together, the study indicates that limocitrin increases cytotoxicity of NK cells against a range of cancer cells.

Analyzing the activity of the proteases granzyme B and caspase-8 inside living cells using fluorescence localization reporters.

Natural Killer (NK) cells are innate lymphocytes that are important for early immune reactions against viral infections and cancer. Their cytotoxic activity is mediated by the release of perforin and granzymes or by engaging death receptors on the surface of their target cells. Here we provide a protocol for the use of fluorescence localization reporters to measure the activity of granzyme B or caspase-8 activity inside living target cells. This method can be used to investigate how these two killing pathways are used by NK cells. By modifying the modular structure of the reporters, they can be adapted to study other cytotoxic effector cells or signaling pathways, where proteases play an important role.

Dynamic changes of phenotype and function of natural killer cells in peripheral blood before and after thermal ablation of hepatitis B associated hepatocellular carcinoma and their correlation with tumor recurrence.

BACKGROUND: Thermal therapy induces an immune response in patients with hepatocellular carcinoma (HCC), but the dynamic characteristics of the natural killer (NK) cell immune response post-thermal ablation remain unclear. We conducted a prospective longitudinal cohort study to observe the dynamic changes of phenotype and function of NK cells in peripheral blood before and after thermal ablation of hepatitis B-associated HCC and their correlation with tumor recurrence. METHODS: Fifty-six patients clinically and pathologically confirmed with hepatitis B-associated HCC were selected for thermal ablation. Peripheral blood was collected on day 0, day 7, and month 1. NK cell subsets, receptors, and killing function were detected by flow cytometry, and the LDH levels were examined. Overall recurrence and associated variables were estimated using Kaplan-Meier, log-rank, and Cox proportional-hazards analyses. RESULTS: The frequency of CD3-CD56+ cells was increased on day 7 (P < 0.01) without significant differences between D0 and M1. NKG2D, NKp44, NKp30, CD159a, and CD158a expression was increased on M1 (all P < 0.05). The granzyme B and IFN-γ expression in NK cells were higher on M1 vs. D0 (P < 0.05). On day 7, the NK cell lysis activity of the target K562 cells was increased (P < 0.01) but decreased on M1 (P < 0.05). Survival analysis showed that CD158a expression and IFN-γ and perforin release on day 0 were associated with the risk of HCC recurrence. Cox regression analysis showed that the expression changes in CD56, NKp46, granzyme B, and perforin (D7-D0) induced by thermal ablation were associated with recurrence-free survival (RFS) of patients with HCC. CONCLUSION: Thermal ablation increased the frequency and function of CD3-CD56+ NK cells in the peripheral blood of patients with HCC. These cells tended to be more differentiated and activated. Notably, expression levels of NK cell receptors NKp46, perforin, and granzyme B were associated with RFS.

Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs.

For a long time, ex vivo-expanded peripheral-blood-derived γδT cell (PBγδT)-based immunotherapy has been attractive, and clinical trials have been undertaken. However, the difficulty in expanding cytotoxic γδT cells to an adequate number has been a major limitation to the efficacy of treatment in most cases. We successfully re-generated γδT cells from γδT cell-derived human induced pluripotent stem cells (iPSCs). The iPSC-derived γδT cells (iγδTs) killed several cancer types in a major histocompatibility complex (MHC)-unrestricted manner. Single-cell RNA sequencing (scRNA-seq) revealed that the iγδTs were identical to a minor subset of PBγδTs. Compared with a major subset of PBγδTs, the iγδTs showed a distinctive gene expression pattern: lower CD2, CD5, and antigen-presenting genes; higher CD7, KIT, and natural killer (NK) cell markers. The iγδTs expressed granzyme B and perforin but not interferon gamma (IFNγ). Our data provide a new source for γδT cell-based immunotherapy without quantitative limitation.

[Experimental Study on the Characteristic Changes of the Immunological Microenvironment at the Maternal-Fetal Interface in IVF-ET Pregnancy].

Objective: To investigate the characteristic functional changes of the decidual natural killer (NK) cells and γδ T cells, two immunocytes in the decidua, at the maternal-fetal interface in in vitro fertilization-embryo transfer (IVF-ET) pregnancy. Methods: Decidual samples were collected from 12 women of natural pregnancy (NP) and 32 women of IVF-ET pregnancy, who were enrolled in the NP group and the IVF-ET group, respectively. Then part of the decidual samples were paraffin-embedded for HE staining and immunofluorescence staining, while the rest of the samples were digested and Percoll was used for isolating decidual immunocytes (DICs) by gradient centrifugation. Flow cytometry was used to determine the cell counts of decidual NK cells and γδ T cells and the expression levels of their surface activation markers, CD69 and NKG2D in the NP and the IVF-ET groups. In addition, the expression levels of IFN-γ, TNF-α, IL-17A, and IL-10, the intracellular cytokines, and granzyme B, perforin, and granulysin, the cytolytic granules, were measured. The characteristic changes in the relevant immunological indicators were compared and analyzed. Results: HE staining of the tissue specimens showed that the typical structure of decidua was observed, and that lymphocytes were enriched in the decidua. Immunofluorescence staining showed that the percentage of decidual NK (dNK) cells in nucleated cells of the IVF-ET group was significantly lower than that of the NP group ( P<0.05). Flow cytometry analysis of DICs showed that, compared with those of the NP group, the percentage of dNK cells of the IVF-ET group was decreased ( P<0.05) and the expression levels of IL-10 and perforin were significantly decreased in the IVF-ET group ( P<0.05). However, there was no significant difference in the decidual γδ T (dγδT) cell count between the two groups. The expression of IL-10, IL-17A, and perforin was downregulated in the IVF-ET group ( P<0.05). There was no significant difference in the expression of IFN-γ, TNF-α, granzyme B, and granulysin, the cellular function indicators ( P>0.05). Conclusion: The dNK cell count and the secretion of some intracellular cytokines of dNK and dγδT cells of women of IVF-ET pregnancy decreased to some degree, which suggests that certain changes may have taken place in the immunological microenvironment at the maternal-fetal interface. The specific effect of these changes on pregnancy outcomes needs further investigation.

Drop-off-reinitiation at the amino termini of nascent peptides and its regulation by IF3, EF-G, and RRF.

In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the anticodon of fMet-tRNAini and then relocates the fMet-tRNAini to an active position. Here, we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of the codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson-Crick, are more preferred, but other types of base pairs, for example, G/U wobble, are also tolerated; weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNAini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the amino-terminal fMet. We refer to this event as the amino-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNAini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the amino-terminal drop-off-reinitiation.