Cytotoxicity In Patients Research Articles

Effectiveness and safety of biosilicate-enhanced bleaching gels on enamel with early erosion lesion.

This study evaluated the efficacy and cytotoxicity of 35% hydrogen peroxide (HP) gel incorporated with 10% (w/w) biosilicate (BioS) on sound enamel and early-stage enamel erosion lesions. Discs of enamel/dentin were selected, subjected to erosive cycles (0.3% citric acid, pH 2.6), and treated with (n = 8): HP (35% HP, positive control); HP_BioS [carboxymethyl cellulose (CMC) + HP + BioS]; BioS (CMC + BioS); CMC (negative control). The discs were adapted to artificial pulp chambers with the enamel exposed for bleaching, and the dentin facing toward the culture medium (Dulbecco's modified Eagle's medium [DMEM]). Bleaching was performed in three 30-min sessions at 7-day intervals. After bleaching, the diffusion product (DMEM extract + diffused HP) was pipetted onto MDPC-23 odontoblastic cell line and inoculated. Color parameters (ΔL, Δa, Δb), color change (ΔE00), and changes in whiteness index (ΔWID) were determined before (T0) and after the last bleaching session (T3). Cell viability (MTT, %), H2O2 diffusion (μg/mL), oxidative cell stress (OxS), and cell fluorescence (live/dead assay, in confocal microscopy) were assessed (ANOVA/Tukey; α = 0.05). No difference in ΔL, Δa, Δb, ΔE00, and ΔWID were found between HP and HP_BioS (p > 0.05). The incorporation of BioS decreased the HP diffusion into the substrates and mitigated oxidative stress in early-stage eroded enamel (p < 0.05). HP_BioS presented significantly higher cell viability compared with HP under erosion conditions. Live/dead assay indicated that BioS_HP maintained viability with larger clusters of viable cells. Incorporating BioS into HP maintained bleaching effectiveness, favored cell viability, reduced the oxidative stress, and the cytotoxicity in teeth with early-stage erosion. BioS formulation showed promising results for reducing cytotoxicity in patients seeking tooth bleaching and presenting undetectable early-stage erosion.

Use of expanded non-genetically modified natural killer cells (SNK01) with enhanced cytotoxicity in patients with Alzheimer's disease — Interim report of a phase I trial

Use of expanded non-genetically modified natural killer cells (SNK01) with enhanced cytotoxicity in patients with Alzheimer's disease — Interim report of a phase I trial

Post-Allograft Romidepsin Maintenance Mitigates Relapse Risk and Stimulates the Graft-Versus-Malignancy Effect through Enhanced NK-Cell Cytotoxicity in Patients with T-Cell Malignancies: Final Results of a Phase I/II Trial

Background: For patients with high risk, and relapsed/refractory T-cell malignancies allogeneic stem cell transplant (allo-SCT) is the only available potentially curative therapy. The efficacy of allo-SCT is limited in this population by high rates of relapse, with rates up to 55-60% post-allo-SCT. We designed a phase I/II trial, evaluating the combination of the histone deacetylases inhibitor romidepsin (rom) with busulfan/fludarabine (BuFlu) conditioning, followed by romidepsin maintenance (m-rom) in patients receiving allo-SCT for T-cell malignancies (NCT02512497). Here we present final clinical results of this therapeutic approach, including an evaluation of the stimulatory effects of m-rom on the graft-versus-malignancy effect through NK-cells post-allo-SCT. Methods: This was a phase I/II clinical trial. Eligible patients had: a diagnosis of T-cell leukemia (including T-acute lymphoblastic leukemia) or T-cell lymphoma (TCL, cutaneous or peripheral) in at least a partial remission requiring an allo-SCT, &amp;lt;70 years of age, with a matched sibling/unrelated donor. Patients received myeloablative (20K) or reduced intensity (16K) BuFlu with rom, followed by standard tacrolimus/methotrexate GVHD prophylaxis and anti-thymocyte globulin for unrelated donors (MUD). An expansion cohort of up to 30 patients (total) was included. M-rom was initiated between day +28 and +100 for 1 year (2 nd year optional), with built in dose reductions for toxicity. An efficacy endpoint of 20% reduction in relapse risk at 1-year in the whole cohort or any individual disease cohort was set based upon historical/CIBMTR (Center for Blood and Marrow Transplant Research) controls was pre-specified. The effect of m-rom on NK-cell cytotoxicity was assessed on samples taken pre-transplant, and 1, 3, 6, 12 months post allo-SCT. NK cytotoxicity was assessed by isolating mononuclear cells from patient samples at each timepoint, comparing those on m-rom (n=13) versus those who did BuFlu controls who did not receive m-rom (n=16). Cells were targeted against K562 targets using the calcein-AM assay. K-M and Fine-Gray models were used to estimate PFS, OS, and cumulative incidence, and compare survival across groups. Results: 28 patients were enrolled on this trial (Table). With a median follow-up time of 15 months, the median OS is 3.3 years (95% CI: 0.85-not reached), with a 1 and 3 year OS probability of 68% and 54%. The median PFS is 2.3 years (95% CI: 0.6-not reached), with 1 and 3-year PFS of 61% and 41%. Cumulative incidence (CI) of NRM at day 100 and 1 year were 14.3% and 21.4%. CI of grade II-IV aGHVD and extensive cGVHD were 46.4% and 38%. The CI of relapse (CIR) was 18.1% at 1 year and 33% at 3 -years. There was no difference between PFS among patients with MRD versus those without MRD prior to transplant (p=0.43). The CIR of patients with TCL was 9.3% at 1 and 3 years, whereas the CIR of patients with leukemia was 25% at 1 year and 48% at 3 years. No patients with PTCL relapsed, 1/2 patients with CTCL relapsed, and 4/6 patients with T-PLL are alive, disease free. Pre-specified general CIR was 55% for all patients. With an overall CIR of 18% at 1 year, and 33% at 3-years, this trial met its pre-specified endpoint of decreasing relapse by 20% at 1-year, and performed well compared to published CIBMTR control populations. Matched propensity score analysis across disease subsets with CIBMTR controls will be presented. 18/28 (64%) of patients received m-rom with a median number of 13 cycles (range 1-47). 8 patients experienced grade 3/4 adverse events (AE), and 1 patient discontinued m-rom due to toxicity, unlikely related to romidepsin (loss of CD3 chimerism). When NK-cytotoxicity was assessed between the two groups after starting maintenance, NK-cytotoxicity in the m-rom group was significantly higher than in those without m-rom (p&amp;lt;0.0001) (Figure). Conclusions: BuFluRom with m-rom is effective at decreasing relapse in patients with T-cell malignancies, with 1-year CI relapse below expected relapse rates for this set of diseases, meeting the pre-specified efficacy endpoint. NK-cell cytotoxicity data in a large sample set of trial patients demonstrates that m-rom enhances NK-cell cytotoxicity post allo-SCT, augmenting the GVL effect and likely accounting for at a minimum, some of the decrease in relapse seen on this trial. M-rom should be considered a new option post allo-SCT to mitigate relapse in patients with T-cell malignancies.

Androgen receptor–specific CD8+ T cells with tumor cell cytotoxicity in patients with prostate cancer in Taiwan.

e17069 Background: Classical androgen receptor (AR) target is the new “tumor-specific” antigen for prostate cancer vaccine development to delay progression and castration-resistant disease. HLA-A2-restricted AR ligand-binding domain (LBD) has been demonstrated to stimulate cytotoxic T lymphocytes (CTL) in HLA-A2+ prostate cancer patients. Although the prevalence of HLA-A2 serotype and LBD-specific CD8+ T cells among Caucasians are known, their frequencies in the Taiwan patients have not been established. In this study, we performed HLA-A2 genotyping, HLA-A2+ LNCaP stimulation of autologous T cell, and CTL tumor cell cytotoxicity from 25 castration-sensitive prostate cancer patients using blood samples collected from a single hospital center. Methods: 20ml whole blood samples are collected in EDTA tube from non-metastatic, castration-sensitive prostate cancer patients. PBMCs are subjected to HLA-A2 allele sequencing, separated by CD3 magnetic bead column, and expanded and activated ex vivo using CD3/CD8 polymeric beads. Autologous T cells are then isolated by CD8 magnetic bead column, stimulated by HLA-A2+ lentivirus-transduced LNCaP (AR+), and targeted against transduced (HLA-A2+) and non-transduced (HLA-A2-) LNCaP (AR+) by Chromium-51 Release Assay. We identified and correlated their frequencies with patient’s age at diagnosis, Gleason grades, PSA level, and family history. Results: A total of 25 blood samples were tested and analyzed for HLA-A2 genotype with 44% (11/25) positive for the HLA-A2 [28% (7/25) heterozygotes, 16% (4/25) homozygotes]. Among the 11 HLA-A2+ patients, 72% (8/11) were HLA-A*02:07 allele, 100% (11/11) CD8+ T cells were successfully stimulated with HLA-A2+ transduced LNCaP (AR+) and 45% (5/11) patients demonstrated HLA-A2+ AR-specific CTL tumor cell lysis. No clinical association with patient age (54-86 y.o.), Gleason grades (6-9), PSA level (3.84-62.8 ng/ml), and family history (1/25). Conclusions: In this single hospital center study, we observed a similar prevalence of HLA-A2 alleles in Taiwan prostate cancer patient cohort with a predominant HLA-A2*02:07 allele, and higher frequencies of HLA-A2-restricted CD8+ T cell stimulation and HLA-A2+ AR-specific tumor cell cytotoxicity. Our findings support the application of AR LBD as tumor antigen for prostate cancer vaccine development for Taiwan prostate cancer patients.

Higher proinflammatory responses possibly contributing to suppressed cytotoxicity in patients with COVID-19 associated mucormycosis

IntroductionCOVID-19 Associated Mucormycosis (CAM), an opportunistic fungal infection, surged during the second wave of SARS Cov-2 pandemic. Since immune responses play an important role in controlling this infection in immunocompetent hosts, it is required to understand immune perturbations associated with this condition for devising immunotherapeutic strategies for its control. We conducted a study to determine different immune parameters altered in CAM cases as compared to COVID-19 patients without CAM. MethodologyCytokine levels in serum samples of CAM cases (n = 29) and COVID-19 patients without CAM (n = 20) were determined using luminex assay. Flow cytometric assays were carried out in 20 CAM cases and 10 controls for determination of frequency of NK cells, DCs, phagocytes, T cells and their functionalities. The cytokine levels were analyzed for their association with each other as well as with T cell functionality. The immune parameters were also analyzed with respect to the known risk factors such as diabetes mellitus and steroid treatment. ResultsSignificant reduction in frequencies of total and CD56 + CD16 + NK cells (cytotoxic subset) was noted in CAM cases. Degranulation responses indicative of cytotoxicity of T cell were significantly hampered in CAM cases as compared to the controls. Conversely, phagocytic functions showed no difference in CAM cases versus their controls except for migratory potential which was found to be enhanced in CAM cases. Levels of proinflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-17, IL-1β, IL-18 and MCP-1 were significantly elevated in cases as compared to the control with IFN-γ and IL-18 levels correlating negatively with CD4 T cell cytotoxicity. Steroid administration was associated with higher frequency of CD56 + CD16- NK cells (cytokine producing subset) and higher MCP-1 levels. Whereas diabetic participants had higher phagocytic and chemotactic potential and had higher levels of IL-6, IL-17 and MCP-1. ConclusionCAM cases differed from the controls in terms of higher titers of proinflammatory cytokines, reduced frequency of total and cytotoxic CD56 + CD16 + NK cell. They also had reduced T cell cytotoxicity correlating inversely with IFN-γ and IL-18 levels, possibly indicating induction of negative feedback mechanisms while diabetes mellitus or steroid administration did not affect the responses negatively.

Contribution of Mitochondrial Activity to Doxorubicin-Resistance in Osteosarcoma Cells

Osteosarcoma is considered the most common bone tumor affecting children and young adults. The standard of care is chemotherapy; however, the onset of drug resistance still jeopardizes osteosarcoma patients, thus making it necessary to conduct a thorough investigation of the possible mechanisms behind this phenomenon. In the last decades, metabolic rewiring of cancer cells has been proposed as a cause of chemotherapy resistance. Our aim was to compare the mitochondrial phenotype of sensitive osteosarcoma cells (HOS and MG-63) versus their clones when continuously exposed to doxorubicin (resistant cells) and identify alterations exploitable for pharmacological approaches to overcome chemotherapy resistance. Compared with sensitive cells, doxorubicin-resistant clones showed sustained viability with less oxygen-dependent metabolisms, and significantly reduced mitochondrial membrane potential, mitochondrial mass, and ROS production. In addition, we found reduced expression of TFAM gene generally associated with mitochondrial biogenesis. Finally, combined treatment of resistant osteosarcoma cells with doxorubicin and quercetin, a known inducer of mitochondrial biogenesis, re-sensitizes the doxorubicin effect in resistant cells. Despite further investigations being needed, these results pave the way for the use of mitochondrial inducers as a promising strategy to re-sensitize doxorubicin cytotoxicity in patients who do not respond to therapy or reduce doxorubicin side effects.

Galactosyl- and glucosylsphingosine induce lysosomal membrane permeabilization and cell death in cancer cells.

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.

Post-Allograft Romidepsin Maintenance Mitigates Relapse Risk and Stimulates the Graft-Versus-Malignancy Effect through Enhanced NK-Cell Cytotoxicity in Patients with Aggressive T-Cell Malignancies in a Phase I/II Trial

Background: For patients with high risk, and relapsed/refractory T-cell malignancies allogeneic stem cell transplant (allo-SCT) is the only available potentially curative therapy. The efficacy of allo-SCT is limited in this population by high rates of relapse, with rates up to 55-60% post-allo-SCT. We designed a phase I/II trial, evaluating the combination of the histone deacetylases inhibitor romidepsin (rom) with FluBu conditioning, followed by romidepsin maintenance (m-rom) in patients receiving allo-SCT for T-cell malignancies (NCT02512497). Here we present an evaluation of the stimulatory effects of m-rom on the graft-versus-malignancy effect through NK-cells post-allo-SCT in the context of clinical results. Methods: This is a phase I/II clinical trial of patients with T-leukemia/lymphoma in at least a partial remission requiring an allo-SCT, <70 years of age, with a MRD/MUD donor. Patients received BuFlu (AUC 20000 or 16000) with rom, followed by tacrolimus/methotrexate GVHD prophylaxis with ATG for MUDs. An expansion cohort of up to 30 patients (total) was included. M-rom was initiated between day +28 and +100 for 1 year (2nd year optional), with built in dose reductions for toxicity. The effect of m-rom on NK-cell cytotoxicity was assessed on samples taken pre-transplant, and 1, 3, 6, 12 months post allo-SCT. NK cytotoxicity was assessed by isolating mononuclear cells from patient samples at each timepoint, comparing those on m-rom (n=10) versus those who did not receive m-Rom (n=5). Cells were targeted against K562 and T-cell lymphoma targets using the calcein-AM assay. Fine-Gray models were used to estimate PFS, OS, and cumulative incidence, and compare survival across groups. Results: To date, 28 patients have been enrolled, with a median age at transplant of 55, 50% female, 75% received MUD donor, and 68% utilized PB marrow source with 32% receiving BM. 16/28 (57%) had a diagnosis of T-cell leukemia, while 12/28 (42%) had a diagnosis of lymphoma. These included: T-ALL (18%), ETP-ALL (14%), ATLL (4%), T-PLL (21%), CTCL (7%), and PTCL (36%). 18% of patients entered transplant in a PR, while the remaining entered transplant in CR1 (61%) or CR2+ (21%). With a median follow-up time of 12 months, the median OS is 40 months (95% CI: 7.5-not reached), with a 1 and 3 year OS probability of 67% and 56%. The median PFS is 28.2 months (95% CI: 6.5-not reached), with 1 and 3-year PFS of 58% and 32%. Cumulative incidence (CI) of NRM at day 100 and 1 year were 15.6% and 19.8%. CI of grade II-IV aGHVD and extensive cGVHD were 42.9% and 19.1%. The CI of relapse (CIR) was 22.2% at 1 year (95% CI: 7.7-41.3%). There was no difference between PFS among patients with MRD versus those without MRD prior to transplant (p=0.71), and no difference in 1-year CIR (p=0.83). PFS at 1 year was substantially better in the lymphoma versus leukemia patients (78% vs 44%, p=0.02), though CIR at 1 year was not significantly different (13% vs 29%, p=0.12). No patients with PTCL relapsed, and 4/6 patients with T-PLL are alive, disease free. 16/28 (57%) of patients received m-rom with a median number of 12 cycles (range 1-39). 8 patients experienced grade 3/4 adverse events (AE), and 1 patient discontinued m-rom due to toxicity, unlikely related to romidepsin (loss of CD3 chimerism). NK-cytotoxicity was significantly higher at each time point in patients who received m-rom compared to those who did not (p<0.01). Further, when NK-cytotoxicity was assessed between the two groups after starting maintenance, NK-cytotoxicity in the m-rom group was significantly higher than in those without m-rom (p<0.0001) (Figure). Studies evaluating NK-cytotoxicity against patient T-cells are ongoing and will be presented at the ASH meeting. Conclusions: BuFluRom with m-rom is effective at decreasing relapse in patients with T-cell malignancies, with 1-year CI relapse below expected relapse rates for this set of diseases. NK-cell cytotoxicity data in a large sample set of trial patients demonstrates that m-rom enhances NK-cell cytotoxicity post allo-SCT, augmenting the GVL effect and likely accounting for at a minimum, some of the decrease in relapse seen on this trial. These results suggest long-term romidepsin past one year may decrease late relapse. Long-term follow-up is needed to evaluate these results, but these results suggest the BuFluRom regimen with m-rom should be strongly considered in patients receiving allo-SCT for T-cell malignancies to mitigate relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Decreased CD57 expression of natural killer cells enhanced cytotoxicity in patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory biliary disease for which the immunopathological basis remains an enigma. Natural killer (NK) cells are key components of innate immunity and seemingly play diversified roles in different autoimmune disorders (AIDs). The aim of this study was to determine the role of NK cells in the pathogenesis of PSC. The frequency and phenotype of circulating NK cells in a large cohort of patients with PSC and healthy controls (HCs) were systematically examined. In addition, the functional capacity of NK cells including cytotoxicity and cytokine production was studied. The frequency of CD3-CD56dimCD16+ (defined as CD56dim) NK cells in PSC patients was significantly lower in comparison to HCs. CD56dim NK cells from PSC displayed a more immature phenotype including high expression of the natural killing receptor NKp46 and downregulation of the highly differentiated NK cell marker CD57. Interestingly, the reduction of CD57 expression of NK cells was associated with the disease severity of PSC. In addition, PSC CD56dim NK cells exhibited increased CD107a degranulation and cytolytic activity toward target cells compared with HCs. Further analysis demonstrated that CD57-CD56dim NK cells from PSC had elevated expression of NKp46, NKp30, IL-2 receptor, and KLRG1 and higher cytotoxic capacity as compared to CD57+CD56dim NK cells. Our data demonstrate that the differentiation of PSC NK cells is dysregulated with enhanced cytotoxic activity. This change is likely to be functionally involved in pathogenesis and disease progression, deducing the potential of NK-directed immunotherapy for PSC.

Ketamine Does Not Change Natural Killer Cell Cytotoxicity in Patients Undergoing Cancer Surgery: Basic Experiment and Clinical Trial.

Background The natural killer cell cytotoxicity (NKCC) suppressed by nociceptive stimuli, systemic inflammation, and drugs used during cancer surgery may be associated with poor outcomes. We investigated the potential modulation of ketamine on NKCC in vitro and in a clinical setting during cancer surgery. Subjects and Methods. The NK cell line KHYG1 was cultured for the in vitro experiments. The NK cells were treated with 3 and 10 μM ketamine (the ketamine groups) or without ketamine (the control) for 4, 24, and 48 h. The posttreatment NKCC was measured with a lactate dehydrogenase assay and compared among the treatment groups. For the clinical study, lung cancer patients (n = 38) and prostate cancer patients (n = 60) who underwent radical cancer surgeries at a teaching hospital were recruited. The patients received propofol and remifentanil superposed with or without ketamine (ketamine group, n = 47; control group, n = 51). The primary outcome was the difference in NKCC between these groups. Results In the in vitro experiment, the cytotoxicity of NK cells was similar with or without ketamine at all of the incubation periods. The patients' NKCC was also not significantly different between the patients who received ketamine and those who did not, at the baseline (36.6 ± 16.7% vs. 38.5 ± 15.4%, p = 0.56) and at 24 h (25.6 ± 12.9% vs. 27.7 ± 13.5%, respectively, p = 0.49). Conclusion Ketamine does not change NKCC in vitro or in the clinical setting of patients who undergo cancer surgery. This trial is registered with UMIN000021231.

Romidepsin in Conditioning and Maintenance Mitigates Relapse Risk and Enhances NK-Cell Cytotoxicity in Patients Receiving Allogeneic Stem Cell Transplant for Aggressive T-Cell Malignancies: Results of a Phase I/II Clinical Trial

Romidepsin in Conditioning and Maintenance Mitigates Relapse Risk and Enhances NK-Cell Cytotoxicity in Patients Receiving Allogeneic Stem Cell Transplant for Aggressive T-Cell Malignancies: Results of a Phase I/II Clinical Trial

Activation of Toll-like receptor 2 enhances peripheral and tumor-infiltrating CD8+ T cell cytotoxicity in patients with gastric cancer

BackgroundToll-like receptors (TLRs) play central roles in the initiation of innate immune response, and also control adaptive immunity activation. Thus, the aim of the study was to investigate the regulation of TLR activation to CD8+ T cells has not been fully elucidated in gastric cancer (GC).Materials and methodsThirty-two GC patients and twenty-three healthy controls were enrolled. Expression profile of TLRs in peripheral and tumor-infiltrating CD8+ T cells was investigated. Purified CD8+ T cells were stimulated with Pam3Csk4, an agonist of TLR2, and cytotoxic and co-inhibitory molecules in CD8+ T cells was measured. Direct and indirect contact coculture system between CD8+ T cells and AGS cells was set up. Modulation of TLR2 activation to CD8+ T cells was assessed by measuring lactate dehydrogenase release and cytokine secretion.ResultsTLR2 mRNA and TLR2+ cell percentage was down-regulated in GC derived peripheral and tumor-infiltrating CD8+ T cells. CD8+ T cells from GC patients showed exhausted phenotype, which presented as decreased perforin/granzyme B, increased programmed death-1, and reduced cytotoxicity to AGS cells. TLR2 activation by Pam3Csk4 enhanced perforin and granzyme B expression in CD8+ T cells, however, did not affect either proinflammatory cytokine production or co-inhibitory molecules expression. Pam3Csk4 stimulation enhanced cytolytic activation of peripheral and tumor-infiltrating CD8+ T cells from GC, but not those from healthy individuals.ConclusionThe present data revealed an important immunomodulatory activity of TLR2 to CD8+ T cells in GC patients.

Downregulation of Programmed Death-1 Pathway Promoting CD8 + T Cell Cytotoxicity in Primary Biliary Cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease. CD8 + T cell (CTLs) cytotoxicity played a crucial rule in of PBC with unclear detailed pathogenesis. The role of the programmed death-1 (PD-1) pathway in CD8 + T cell cytotoxicity in patients with PBC was determined. We recruited 69 patients with PBC and 57 healthy controls (HCs). PD-1 pathway in peripheral CD8 + T cells and related cytokines were detected, and gene expression levels were detected. Immunofluorescence staining of PD-1/PD-L1 was performed on liver tissue. PD-1 ± CTLs were cocultured with human intrahepatic biliary epithelial cells (HiBECs) to measure CTL cytotoxicity, proliferation and cytokine levels and HiBEC apoptosis. The upstream signaling pathway of PD-1 was detected. PBC patients exhibited Tbet gene upregulation and PD-1 downregulation in CTLs, with PD-1 expression reduced in CTLs and PD-L1 reduced in the liver portal region relative to HCs. Higher plasma IL-10, interferon-γ and transforming growth factor-β concentrations were observed in the PBC group than the HC group. In CTL and HiBEC coculture experiment, compared with PD-1- CTLs, PD-1 + CTLs exhibited weaker cytotoxicity, less proliferation and lower cytokine production. When the system was blocked by anti-PD-1 antibodies, these effects were antagonized. PD-1 expression in CD8 + T cells decreased, and PD-1 pathway-related cytokines changed in patients with PBC. PD-1/PD-L1 pathway silencing increased CD8 + T cell proliferation, related cytokine production and CTL cytotoxic effects on HiBECs in coculture experiment. The PD-1/PD-L1 pathway might represent an important pathway in the immunological mechanism underlying PBC.

Abnormality in the NK-cell population is prolonged in severe COVID-19 patients

BackgroundOur understanding of adaptive immune responses in patients with coronavirus disease 2019 (COVID-19) is rapidly evolving, but information on the innate immune responses by natural killer (NK) cells is still insufficient.ObjectiveWe aimed to examine the phenotypic and functional status of NK cells and their changes during the course of mild and severe COVID-19.MethodsWe performed RNA sequencing and flow cytometric analysis of NK cells from patients with mild and severe COVID-19 at multiple time points in the course of the disease using cryopreserved PBMCs.ResultsIn RNA-sequencing analysis, the NK cells exhibited distinctive features compared with healthy donors, with significant enrichment of proinflammatory cytokine-mediated signaling pathways. Intriguingly, we found that the unconventional CD56dimCD16neg NK-cell population expanded in cryopreserved PBMCs from patients with COVID-19 regardless of disease severity, accompanied by decreased NK-cell cytotoxicity. The NK-cell population was rapidly normalized alongside the disappearance of unconventional CD56dimCD16neg NK cells and the recovery of NK-cell cytotoxicity in patients with mild COVID-19, but this occurred slowly in patients with severe COVID-19.ConclusionsThe current longitudinal study provides a deep understanding of the NK-cell biology in COVID-19.

The low cytotoxic activity of peripheral blood NK cells may relate to unexplained recurrent miscarriage.

Unexplained recurrent miscarriage (uRM) is defined as two or more spontaneous abortions prior to 20weeks of gestation with unknown etiology. Peripheral blood natural killer (pNK) cells contact with the villus and exert important role in normal pregnancy. However, it is still controversial about the association between pNK cytotoxicity and uRM, and the underlying mechanism remains unknown so far. In this study, we aim to compare the percentage, immunophenotype, and function of pNK cells between patients with uRM and fertile controls. The peripheral blood was collected from 49 patients with uRM and 11 fertile women in their middle luteal phase of the menstrual cycle. pNK cells were co-cultured with K562 cells at different cell ratios to measure the cytotoxicity. The percentage of CD3- CD56+ , CD3- CD56bright , and CD3- CD56dim pNK was analyzed by flow cytometry and quantified to evaluate the expression of cytotoxic granules (granzyme B, granulysin, and perforin), and the cell surface receptors related to pNK cell cytotoxicity (NKG2D, NKp30, NKp46, CD158a, and CD158b) were also detected. The general linear model analysis showed that pNK cell cytotoxicity in patients with uRM was significantly lower than that in fertile controls. In addition, the ratios of NKG2D/CD158a, NKp30/CD158a, and NKp46/CD158a in CD3- CD56bright pNK subsets were significantly lower in uRM group than that in fertile control. The logistical regression analysis showed that the reduced NKp30/CD158a, NKp46/CD158a ratios in CD3- CD56bright pNK subsets were significantly associated with uRM. Our results suggested that a low pNK cytotoxicity, which is mediated by inhibitory signals, might be associated with uRM.

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

Interleukin-9-secreting CD4+ T cells regulate CD8+ T cells cytotoxicity in patients with acute coronary syndromes.

T cells play vital roles in the development and progression of acute coronary syndromes (ACS), including cytotoxicity mediated by CD8+ T cells and immunoregulatory activity mediated by CD4+ T cells. Interleukin (IL)-9-secreting CD4+ T cells (Th9 cells) were recently found to be involved in the onset of ACS. We investigated regulatory role of Th9 cells to CD8+ T cells in patients with stable angina pectoris, unstable angina pectoris, and acute myocardial infarction (AMI). Circulating Th9 cells percentage, plasma IL-9 level, and PU.1 mRNA relative level was up-regulated in AMI patients compared with controls. There was no significant difference of IL-9-secreting CD8+ T cells percentage among groups. CD8+ T cells from AMI patients revealed increased cytotoxicity than those from controls, which presented as enhanced cytotolytic activity to target cells, increased interferon-γ and tumor necrosis factor-α secretion, elevated perforin and granzyme B production, and reduced programmed death-1 and cytotoxic T lymphocyte-associated protein 4. IL-9 stimulation did not affect proliferation, but promoted CD8+ T-cell cytotoxicity from both controls and AMI patients. IL-9-secreting CD4+ T cells were enriched in CD4+ CCR4- CCR6- CXCR3- cells. The enhancement of CD8+ T-cell cytotoxicity induced by CD4+ CCR4- CCR6- CXCR3- cells was dependent on IL-9 secretion. The present results indicated that up-regulation of IL-9-secreting CD4+ T cells may contribute to pathogenesis of AMI through enhancement of CD8+ T-cell cytotoxicity.

A global antiB cell strategy combining obinutuzumab and daratumumab in severe pediatric nephrotic syndrome.

BackgroundSteroid-sensitive nephrotic syndrome (SSNS) is, in most patients, a chronic disease with 80% experiencing at least one relapse after first flare. B cell depletion using rituximab is effective in preventing relapse in steroid-dependent (SDNS) patients but fails to maintain long-term remission following B cell recovery, possibly due to development of autoreactive long-lived plasma cells. We investigated sequential combination of antiCD20 antibody targeting all B cell subsets, and antiCD38 antibody with high plasma cell cytotoxicity in patients with uncontrolled SDNS after failure of one or several attempts at B cell depletion.MethodsFourteen patients with median disease duration 7.8 years received 1000 mg/1.73 m2 obinutuzumab followed by 1000 mg/1.73 m2 daratumumab 2 weeks later. Oral immunosuppression was discontinued within 6 weeks, and biological monitoring performed monthly until B cell recovery.ResultsMedian age at treatment was 11.0 [IQR 10.4–14.4] years. B cell depletion was achieved in all patients, and B cell reconstitution occurred in all at median 9.5 months after obinutuzumab injection. After median follow-up 20.3 months (IQR 11.5–22.6), 5/14 patients relapsed including 4 within 100 days following B cell repletion. Relapse-free survival was 60% at 24 months from obinutuzumab infusion. Mild infusion reactions were reported in 3/14 patients during obinutuzumab and 4/14 during daratumumab infusions. Mild transient neutropenia (500–1000/mm3) occurred in 2/14 patients. Intravenous immunoglobulins were given to 12/14 patients due to hypogammaglobulinemia. Low IgA and IgM levels were noted in 8 and 14 patients, respectively. No severe infection was reported.ConclusionGlobal antiB cell strategy combining obinutuzumab and daratumumab induces prolonged peripheral B cell depletion and remission in children with difficult-to-treat SDNS.Electronic supplementary materialThe online version of this article (10.1007/s00467-020-04811-0) contains supplementary material, which is available to authorized users.

Coronavirus disease (COVID-19) and sirtuins

The NAD+dependent proteins deacetylases are called Sirtuins (SIRT). Objectives: this review is to study the sirtuins involved in cancer, as well as SIRT1 inhibition studies in patients with coronavirus disease COVID-19. For this, a search was made in Medline, Scopus and WOS, where descriptive studies of each of the functions of sirtuins were included, adjusted to recent scientific research. SIRT1 inhibition reduces CD8 T cell cytotoxicity in patients with systemic erythematosus lupus, being susceptible to SARS Cov-2 infections. SIRT2 is regulated by the secretion of IL-4 by eosinophils and the increase in SIRT2 increases hyperplasia, in contrast, SIRT3 promotes angiogenesis, inducing cardiac remodeling. SIRT4 is a tumor suppressor, in contrastto SIRT5 that promotes cell proliferation causing colorectal cancer; SIRT6 attenuates herpes virus associated with Kaposi's Sarcoma (KSHV) in immune compromised patients. Suppression of SIRT7 inhibits the growth of endometrial cancer cells. It is concluded that SIRT1, SIRT2 and SIRT4 are involved in the development of cancer, the suppression of SIRT5 and SIRT7 promotes the apoptosis of cancer cells and SIRT6 attenuates the replication of KSHV, in addition to the molecular pathology pathway of COVID-19 is associated with the inhibition of SIRT1 activity that may be related to inflammatory processes.

The effects of two different anesthesia and analgesia techniques on NK cell cytotoxicity in patients undergoing surgery for colorectal cancer

The effects of two different anesthesia and analgesia techniques on NK cell cytotoxicity in patients undergoing surgery for colorectal cancer