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Patients with chronic hepatitis B (CHB) undergoing interferon (IFN)-α-based therapies often exhibit a poor HBeAg serological response. Thus, there is an unmet need for new therapies aimed at CHB. This study comprised two clinical trials, including 130 CHB patients, who were treatment-naïve; in the first, 92 patients were systematically analyzed ex vivo for interleukin-2 receptor (IL-2R) expression and inhibitory molecules expression after receiving Peg-IFN-α-2b therapy. In our second clinical trial, 38 non-responder patients, in whom IFN-α therapy had failed, were treated with or without low-dose IL-2 for 24 weeks. We then examined the hepatitis B virus (HBV)-specific CD8+ T-cell response and the clinical outcome, in these patients. Although the majority of the participants undergoing Peg-IFN-α-2b therapy were non-responders, we observed a decrease in CD25 expression on their CD4+ T cells, suggesting that IFN-α therapy may provide a rationale for sequential IL-2 treatment without increasing regulatory T cells (Tregs). Following sequential therapy with IL-2, we demonstrated that the non-responders experienced a decrease in the numbers of Tregs and programmed cell death protein 1 (PD-1) expression. In addition, sequential IL-2 administration rescued effective immune function, involving signal transducer and activator of transcription 1 (STAT1) activation. Importantly, IL-2 therapy significantly increased the frequency and function of HBV-specific CD8+ T cells, which translated into improved clinical outcomes, including HBeAg seroconversion, among the non-responder CHB patients. Our findings suggest that sequential IL-2 therapy shows efficacy in rescuing immune function in non-responder patients with refractory CHB.

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0–21 years of age) admitted to Children’s Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention's case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.

IL‐2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti‐immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL‐2, Mutakine‐6 (MK‐6), with reduced IL‐2Rα–binding capability. MK‐6 induced comparable cell growth potential toward IL‐2Rβγ–positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL‐2, in vivo administration of MK‐6 produced minimal adverse effects. Using CT26 and B16F10‐syngeneic tumor models, we found MK‐6 was highly efficacious on tumor regression. Serum albumin conjugation to MK‐6 prolonged in vivo half‐life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor‐infiltrating leukocytes analysis revealed that albumin‐fused MK‐6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK‐6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation., Albumin‐conjugated IL‐2 mutein, MK‐6, exerts a potent antitumor effect in the CT26 tumor model.

ObjectivesGiant cell arteritis (GCA) is the most common primary vasculitis, preferentially affecting the aorta and its large-calibre branches. An imbalance between proinflammatory CD4+ T helper cell subsets and regulatory T cells (Tregs) is thought to be involved in the pathogenesis of GCA and Treg dysfunction has been associated with active disease. Our work aims to explore the aetiology of Treg dysfunction and the way it is affected by remission-inducing immunomodulatory regimens.MethodsA total of 41 GCA patients were classified into active disease (n=14) and disease in remission (n=27). GCA patients’ and healthy blood donors’ (HD) Tregs were sorted and subjected to transcriptome and phenotypic analysis.ResultsTranscriptome analysis revealed 27 genes, which were differentially regulated between GCA-derived and HD-derived Tregs. Among those, we identified transcription factors, glycolytic enzymes and IL-2 signalling mediators. We confirmed the downregulation of forkhead box P3 (FOXP3) and interferon regulatory factor 4 (IRF4) at protein level and identified the ineffective induction of glycoprotein A repetitions predominant (GARP) and CD25 as well as the reduced T cell receptor (TCR)-induced calcium influx as correlates of Treg dysfunction in GCA. Inhibition of glycolysis in HD-derived Tregs recapitulated most identified dysfunctions of GCA Tregs, suggesting the central pathogenic role of the downregulation of the glycolytic enzymes. Separate analysis of the subgroup of tocilizumab-treated patients identified the recovery of the TCR-induced calcium influx and the Treg suppressive function to associate with disease remission.ConclusionsOur findings suggest that low glycolysis and calcium signalling account for Treg dysfunction and inflammation in GCA.

Osteoporosis is one of the chronic and often neglected bone diseases in aging postmenopausal women that affect the quality of life. Studies on ovariectomized mice models indicated the reciprocal role of Th17 cells and Treg cells in the aetiology of osteoporosis. While Th17 cells promote osteoclastogenesis, Treg cells exhibit anti-osteoclastogenic activity. This exploratory study aimed to determine the difference in the frequency of these T-cell subtypes in pre-and postmenopausal women and to examine their association with BMD. In our study, the frequency of Treg cells, analyzed by flow cytometry, did not differ between pre-and postmenopausal women. However, plasma levels of IL-10 along with IL-10+CD4+T cells were higher in post- compared to premenopausal women. The frequency of Th17 cells was higher in postmenopausal women irrespective of their BMD, however, only postmenopausal women with low BMD had elevated IL-17 levels and their T-scores were associated with Th17 frequency. Collectively, the results suggest that estrogen insufficiency in postmenopausal women may lead to increased Th17 cell frequency and elevated IL-17 levels which are associated with low BMD. This study highlights, Th17 cells and IL-17 as key players in the pathogenesis of osteoporosis and they can be the potential targets for immunotherapy in the treatment of osteoporosis.

Background Prognostic markers for disease severity and identification of therapeutic targets in COVID‐19 are urgently needed. We have studied innate and adaptive immunity on protein and transcriptomic level in COVID‐19 patients with different disease severity at admission and longitudinally during hospitalization. Methods Peripheral blood mononuclear cells (PBMCs) were collected at three time points from 31 patients included in the Norwegian SARS‐CoV‐2 cohort study and analysed by flow cytometry and RNA sequencing. Patients were grouped as either mild/moderate (n = 14), severe (n = 11) or critical (n = 6) disease in accordance with WHO guidelines and compared with patients with SARS‐CoV‐2‐negative bacterial sepsis (n = 5) and healthy controls (n = 10). Results COVID‐19 severity was characterized by decreased interleukin 7 receptor alpha chain (CD127) expression in naïve CD4 and CD8 T cells. Activation (CD25 and HLA‐DR) and exhaustion (PD‐1) markers on T cells were increased compared with controls, but comparable between COVID‐19 severity groups. Non‐classical monocytes and monocytic HLA‐DR expression decreased whereas monocytic PD‐L1 and CD142 expression increased with COVID‐19 severity. RNA sequencing exhibited increased plasma B‐cell activity in critical COVID‐19 and yet predominantly reduced transcripts related to immune response pathways compared with milder disease. Conclusion Critical COVID‐19 seems to be characterized by an immune profile of activated and exhausted T cells and monocytes. This immune phenotype may influence the capacity to mount an efficient T‐cell immune response. Plasma B‐cell activity and calprotectin were higher in critical COVID‐19 while most transcripts related to immune functions were reduced, in particular affecting B cells. The potential of these cells as therapeutic targets in COVID‐19 should be further explored.

Background: novel approaches are required to improve outcomes in relapsed or refractory classical Hodgkin lymphoma and non-Hodgkin lymphoma. We aimed to evaluate camidanlumab tesirine, an anti-CD25 antibody-drug conjugate, in this patient population.Methods: this was a phase 1, dose-escalation (part 1), dose-expansion (part 2), multicentre trial done in 12 hospital sites (seven in the USA and five in the UK). Adults (≥18 years old) with pathologically confirmed relapsed or refractory classical Hodgkin lymphoma or non-Hodgkin lymphoma, an Eastern Cooperative Oncology Group performance status 0-2, who had no therapies available to them with established clinical benefit for their disease stage were enrolled. Camidanlumab tesirine was administered intravenously (3-150 μg/kg) once every 3 weeks. Primary objectives were to assess dose-limiting toxicity, determine maximum tolerated dose and recommended expansion dose(s), and assess safety of camidanlumab tesirine. Safety was assessed in all treated patients; antitumour activity was assessed in patients with one or more valid baseline and post-baseline disease assessment and in those who had disease progression or died after first study-drug dose. This trial was registered with ClinicalTrials.gov, NCT02432235.Findings: between Oct 5, 2015, and Jun 30, 2019, 133 patients were enrolled (77 [58%] had classical Hodgkin lymphoma and 56 (42%) had non-Hodgkin lymphoma). Median follow-up was 9·2 months (IQR 4·2-14·3). Eight dose-limiting toxicities were reported in five (6%) of 86 patients who were evaluable; the maximum tolerated dose was not reached. The recommended doses for expansion were 30 μg/kg and 45 μg/kg for patients with classical Hodgkin lymphoma and 80 μg/kg for patients with T-cell non-Hodgkin lymphomas. No recommended doses for expansion were defined for B-cell non-Hodgkin lymphomas. Grade 3 or worse treatment-emergent adverse events (reported by ≥10% of the 133 patients) included increased γ-glutamyltransferase (20 [15%] patients), maculopapular rash (16 [12%]), and anaemia (15 [11%]); 74 (56%) patients had serious treatment-emergent adverse events, most commonly pyrexia (16 [12%]). One (1%) fatal treatment-emergent adverse event and two (2%) deaths outside the reporting period were considered at least possibly study-drug related. Antitumoural activity was seen in classical Hodgkin and non-Hodgkin lymphomas; notably in all patients with classical Hodgkin lymphoma, the overall response was 71% (95% CI 60-81).Interpretation: these results warrant evaluation of camidanlumab tesirine as a potential treatment option for relapsed or refractory lymphoma, particularly in patients with classical Hodgkin lymphoma.Funding: ADC Therapeutics.

Background The Identification of B cell subsets with regulatory functions might open the way to new therapeutic strategies in the field of transplantation, which aim to reduce the dose of immunosuppressive drugs and prolong the graft survival. CD25 was proposed as a marker of a B-cell subset with an immunosuppressive action termed Bregs. The effect of CD19 + CD25 + Bregs on graft function in renal transplant recipients has not yet been elucidated. We investigated a potential impact of CD19 + CD25 + Bregs on renal graft function as well as a possible interaction of CD19 + CD25 + Bregs with peripheral Tregs in healthy controls, end-stage kidney disease patients (ESKD), and renal transplant recipients. Moreover, we aimed to investigate the association of CD19 + CD25 + Bregs with serum IL-10, TGF-ß1, and IFN-γ in the same study groups. Method Thirty-one healthy controls, ninety renal transplant recipients, and eighteen ESKD patients were enrolled. We evaluated the CD19 + CD25 + Bregs and Treg absolute counts. Next, we investigated CD19 + CD25 + Bregs as predictors of good graft function in multiple regression and ROC analyses. Finally, we evaluated the association between CD19 + CD25+ Bregs and serum IL-10, TGF-ß, and IFN-γ. Results ESKD patients and renal transplant recipients showed lower counts of CD19 + CD25+ Bregs compared to healthy controls (p p = 0.02). In these patients, higher CD19 + CD25+ Bregs were independently associated with higher Treg counts (unstandardized B = 2.8, p = 0.004). In ROC analysis, cut-offs for CD19 + CD25 + Breg counts and serum TGF-ß1 of 0.12 cell/μl and 19,635.4 pg/ml, respectively, were shown to provide a good sensitivity and specificity in identifying GFR ≥ 30 ml/min (AUC = 0.67, sensitivity 77%, specificity 43%; AUC = 0.65, sensitivity 81%, specificity 50%, respectively). Finally, a significant positive association between CD19 + CD25+ Bregs and TGF-ß1 was shown in renal transplant recipients (r = 0.255, p = 0.015). Conclusions Our findings indicate that higher counts of CD19 + CD25+ Bregs are independently associated with better renal function and higher absolute Treg counts in renal transplant recipients.

Summary Follicular T helper (Tfh) and regulatory (Tfr) cells are distinct subsets of CD4+ T lymphocytes, regulating humoral immune responses in the germinal center. It is widely accepted that dysregulated Tfh and Tfr cells are associated with autoimmunity. In this study, we evaluated the frequencies of circulating chemokine receptor (CXCR)5+ programmed cell death 1 (PD-1+) Tfh (cTfh) and CXCR5+PD-1+forkhead box protein 3 (FoxP3+) CD25+ Tfr (cTfr) cells, and their corresponding cytokines from the peripheral blood mononuclear cells of 28 patients with relapsing–remitting multiple sclerosis (MS) and 16 age- and sex-matched healthy controls (HC). Subsets of cTfh cells by Th1- and Th17-related surface markers (CXCR3 and CCR6) were also evaluated. We found that the frequency of cTfh cells was significantly higher in MS patients compared to that of HC. Conversely, the frequency of cTfr cells was lower in MS patients than that of HC. Interleukin (IL)-21-producing cTfh cells were significantly increased in MS patients, while IL-10-secreting cTfr cells were lower in MS patients compared to levels in HC. Among cTfh cells, cTfh17.1 cells were the major subtypes that were significantly increased in MS patients compared to HC, with the frequency of IL-21-secreting cells being the highest. These results suggest that an imbalanced distribution of cTfh and cTfr exist in MS patients, which contributes to the reciprocally altered IL-21 and IL-10 production.

SLE is a disease of the adaptive immune system where T lymphocyte dysfunction has an important role as well. We assessed the plasma levels of checkpoint receptors expressed on T cells, along with Galectin-9 to reflect type-1 IFN activity and IL-2Rα in childhood SLE patients. Forty-nine children with SLE and15 healthy controls were included. SLEDAI scores were evaluated at the time of sampling. CD25 (IL-2Rα), 4-1BB, B7.2 (CD86), TGF-β1, CTLA-4, PD-L1, PD-1, Tim-3, LAG- 3, Galectin-9 levels were studied by cytometric bead-based multiplex assay panel. Galectin-9 and PD-L1 were significantly higher in SLE patients. Other checkpoint proteins and IL-2Rα were also higher but did not reach statistical significance. There were significant correlations between SLEDAI and IL-2Rα, Galectin-9 and PDL1. There were three clinical clusters: Cluster 1 included patients with no major organ involvement, cluster 2 had predominantly haematological involvement(n=16) and cluster 3 (n=11) had predominantly renal involvement. Checkpoint proteins were not different among these three clusters. Our data supports that Galectin 9 and IL-2Rα are good markers for disease activity in childhood SLE. We need larger series to evaluate differences between disease clusters in SLE. We failed to show a significant correlation with checkpoint proteins and SLEDAI except for PDL1.

This study is to investigate the pathogenesis of vulvar lichen sclerosus (VLS) by analyzing the level of Foxp3, DNMTs, methylation of Foxp3 promoter region, and CD4 + CD25 + CD127low Regulatory T cells (Tregs). This study enrolled 15 VLS patients and 25 controls. Lesional and extralesional vulvar skin tissues, normal vulvar skin tissues and peripheral blood were collected. Compared with the control group, Foxp3 protein in the lesional and extralesional skin of VLS group was significantly reduced. The levels of DNMT1 and DNMT3b proteins in lesional skin of VLS group were significantly increased. There was no difference in the total methylation rates of the promoter region of the Foxp3 gene. The methylation rates of CpG1, CpG4, CpG9, and CpG10 were significantly higher in lesional skin of VLS group than in control group. There was no correlation between the total methylation rates of 10 CpG sites and the level of Foxp3 and DNMT1 proteins; there was a positive correlation between Foxp3 and DNMT1 protein in lesional skin of VLS group (r = 0.675, p

CD25, the alpha chain of the IL-2 receptor, is expressed on activated effector T cells that mediate immune graft damage. Induction immunosuppression is commonly used in solid organ transplantation and can include antibodies blocking CD25. However, regulatory T cells (Tregs) also rely on CD25 for their proliferation, survival, and regulatory function. Therefore, CD25-blockade may compromise Treg protective role against rejection. We analysed in vitro the effect of basiliximab (BXM) on the viability, phenotype, proliferation and cytokine production of Treg cells. We also evaluated in vivo the effect of BXM on Treg in thymectomized heart transplant children receiving BXM in comparison to patients not receiving induction therapy. Our results show that BXM reduces Treg counts and function in vitro by affecting their proliferation, Foxp3 expression, and IL-10 secretion capacity. In pediatric heart-transplant patients, we observed decreased Treg counts and a diminished Treg/Teff ratio in BXM-treated patients up to 6-month after treatment, recovering baseline values at the end of the 12-month follow up period. These results reveal that the use of BXM could produce detrimental effects on Tregs, and support the evidence suggesting that BXM induction could impair the protective role of Tregs in the period of highest incidence of acute graft rejection.

The aim of this study was to investigate the relationship between pre-pregnancy blood immune status and unexplained recurrent pregnancy loss (URPL), and to evaluate the predictive value of pre-pregnancy blood Treg levels for subsequent miscarriage in patients with URPL. We retrospectively analyzed 76 women who had experienced two or more miscarriages before 24 weeks of gestation for no obvious reason, and 74 women who had achieved live births as controls. Flow-cytometric analysis of peripheral blood CD4 + T cells, CD8 + T cells, NK cells, NKT cells, B cells, NK cell subpopulations (including CD56

IntroductionMany patients are referred to multiple sclerosis (MS) tertiary centers to manage brain white matter hyperintensities (WMH). Multiple diagnoses can match in such situations, and we lack proper tools to diagnose complex cases.ObjectiveThis study aimed to prospectively analyze and correlate with the final diagnosis, cerebrospinal fluid (CSF) interleukin (IL)-1β, soluble IL-2 receptor (CD25), IL-6, IL-10, and kappa free light chains (KFLC) concentrations in patients presenting with brain WMH.MethodsAll patients over 18 years addressed to our MS tertiary center for the diagnostic workup of brain WMH were included from June 1, 2020, to June 1, 2021. Patients were separated into three groups—MS and related disorder (MSARD), other inflammatory neurological disorder (OIND), and non-inflammatory neurological disorder (NIND) groups—according to clinical presentation, MRI characteristics, and biological workup.ResultsA total of 176 patients (129 women, mean age 45.8 ± 14.7 years) were included. The diagnosis was MSARD (n = 88), OIND (n = 35), and NIND (n = 53). Median CSF KFLC index and KFLC intrathecal fraction (IF) were higher in MSARD than in the OIND and NIND groups; p < 0.001 for all comparisons. CSF CD25 and IL-6 concentrations were higher in the OIND group than in both the MSARD and NIND groups; p < 0.001 for all comparisons. KFLC index could rule in MSARD when compared to NIND (sensitivity, 0.76; specificity, 0.91) or OIND (sensitivity, 0.73; specificity, 0.76). These results were similar to those with oligoclonal bands (sensitivity, 0.59; specificity, 0.98 compared to NIND; sensitivity, 0.59; specificity, 0.88 compared to OIND). In contrast, elevated CSF CD25 and IL-6 could rule out MSARD when compared to OIND (sensitivity, 0.58 and 0.88; specificity, 0.95 and 0.74, respectively).DiscussionOur results show that, as OCBs, KFLC biomarkers are helpful tools to rule in MSARD, whereas elevated CSF CD25 and IL-6 rule out MSARD. Interestingly, CSF IL-6 concentration could help identify neuromyelitis optica spectrum disorder, myelin oligodendrocyte glycoprotein antibody-associated disease, and central nervous system (CNS) vasculitis. These results need to be confirmed within more extensive and multicentric studies. Still, they sustain that KFLC, CSF CD25, and CSF IL-6 could be reliable biomarkers in brain WMH diagnostic workup for differentiating MSARD from other brain inflammatory MS mimickers.

Progesterone supplementation is recommended to prevent spontaneous preterm birth (sPTB) in clinical practice. However, the exact mechanism is still unclear. This study aims to better understand the mechanisms that progesterone can prevent PTB.Late pregnant mice were given various doses of progesterone receptor antagonist mifepristone, and pregnancy outcomes were observed. Then, non-pregnant and pregnant mice were given a subcutaneous injection of 40 mg/kg progesterone and 5 mg/kg mifepristone, respectively. CD4Mifepristone induced preterm birth, and an obvious dose-response was found. Proportions of CD4Progesterone promotes pregnancy immune homeostasis by up-regulating Treg cells and TGF-β1 expression in the maternal-fetal interface. It may be one of the mechanisms of progesterone in preventing sPTB.

Regulatory T cells (Treg) deficits, both quantitative and qualitative, are known to be possible triggers for the development of autoimmune disorders by causing T and B cells dysfunction. The contribution of Treg deficiency in the etiology of systemic lupus erythematosus (SLE) is still being debated. The aim of the present study is to evaluate the percentage of circulating CD450 female patients with SLE together with an equal number of age- and sex-matched healthy controls were enrolled in the study. Flow cytometric determination of peripheral Treg cells was carried out for all participants by detecting the percentage of CD4CD4Our study showed impaired production of CD4

Dendritic cells (DCs) are first in line to sense invading microbes and to deliver signals to other immune cells. Plasmacytoid DCs (pDC) produce high amounts of type I interferons (IFNs) but also regulate immune responses. Using the Clec4C (BDCA2)-diphtheria toxin receptor mouse model allowing conditional pDC depletion, we identified an essential role for pDCs in regulating intestinal inflammation locally in the gut. In pDC-depleted mice, Citrobacter rodentium infection led to enhanced activation of conventional DCs and induction of IFN-γ-producing Th1-cells in colon-draining lymph nodes, while induction of Foxp3+ /CD25+ Treg and IL-17-producing Th17 cells was impaired. Concomitantly, F4/80+ macrophages accumulated into the colon lamina propria in excess, and levels of Il-1β and Tnf transcripts increased and Foxp3+ Treg were fewer. Our results indicate that pDCs control inflammation in the gut during C. rodentium infection and that they have an important immune regulatory role in colon-draining lymph nodes.

It is estimated that about 10-20 million peoples are infected with human T-cell leukemia virus type 1 (HTLV-1) around the world and suffered from HTLV-related diseases. The present study was aimed to evaluate the cellular immunity, T-cell activation, humoral immunity, and inflammatory response hallmarks which affect HTLV-1-associated disease progression. A total of 78 participants were included in the study, comprising 39 HTLV-1 asymptomatic careers (ACs) and 39 healthy controls. The HTLV-proviral load (PVL) was determined via real-time PCR technique, and anti-HTLV antibody, sIL2R, sCD30, Neoptrin, hs-CRP, IgE, anti-VCA, anti-EBNA, and anti-EA were assessed by ELISA method. Mean PVL in ACs was 352.7 ± 418.7 copies/104 PBMCs. A significant higher level of sIL-2R was observed in ACs (P < 0.0001). Anti-VCA antibody titer in ACs and healthy controls was 80.72 ± 105.95 and 156.05 ± 130.71, respectively (P = 0.007). Intriguingly, suppression in ACs immune response was not observed. Resultantly, HTLV-1 infection has no effect on the humoral immune response in ACs but greater T-cell activation and function cellular responses were detected. Finally, more studies on various immune markers in adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients are greatly needed to illuminate the association of ACs' immune status with the development of the related diseases.

This single-center, retrospective study aimed to explore the immune characteristics of COVID-19 and biomarkers to predict the severity of this disease. Patients infected with SARS-CoV-2 (n = 215) treated at the First Affiliated Hospital of Nanchang University from January 24 to March 12, 2020, were included in the study and classified into severe and non-severe groups. Peripheral immunocyte count and cytokine statuses were compared. The correlation between immune status, cytokine levels, and disease severity was analyzed. Leukocyte numbers were normal in both groups; however, they were relatively high (7.19 × 109/L) in patients of the severe group. Leukocyte distributions differed between the two groups; the severe group had a higher percentage of neutrophils and lower percentage of lymphocytes compared with the non-severe group, and absolute lymphocyte numbers were below normal in both groups, and particularly deficient in patients in the severe group. Lymphocyte counts have negative correlation with duration of hospital period whereas neutrophil count has no significant correlation with it. Of tested cytokines, IL-6 levels were significantly higher in the severe group (P = 0.0418). Low level of lymphocyte predicts severity of COVID-19. IL-6 levels were significantly higher in the severe group, especially in some extremely severe patients. But we did not detect the significant correlation between severity of COVID-19 with IL-6 level which may be due to limited case numbers. Our observations encourage future research to understand the underlying molecular mechanisms and to improve treatment outcome of COVID-19. Electronic supplementary material The online version of this article (10.1007/s12026-020-09156-2) contains supplementary material, which is available to authorized users.

BACKGROUND : This study aimed to assess the significance of combined expression of interleukin-2 receptor (CD25) and the interleukin-3 receptor (CD123) in acute myeloid leukemia (AML) patients. METHODS : The expression of CD25 and CD123 on blast cells in bone marrow samples were identified by flowcytometry in 94 patients (⩽ 60 years old) with de novo acute myeloid leukemia (AML) treated at the Mansoura University Oncology Center (MUOC). RESULTS : Of the 94 samples at diagnosis there were 17 (18.1%) CD25+/CD123+ (double positive) cases; 25 (26.6%) CD25+/CD123- (single positive); 32 (34.0%) CD25-/CD123+ (single positive) cases; 20 (21.3%). CD25-/CD123- (double negative). Most of the AML patients have double CD25+/CD123+ were significantly associated with poor and intermediate risk as compared to those associated with those in the good risk group (P= 0.005). The lowest induction of remission was recorded in AML patients have double CD25+/CD123+ expression as compared to the remaining AML patient group. Study the effect of these biomarkers on the overall survival reveal that AML patients exhibited double CD25+/CD123+ expression had significantly shorter overall survival as compared to negative ones. CONCLUSION: Double CD25+/CD123+ co-expression in AML patients is a dismal prognostic marker and could be used as novel biomarker for risk stratification for AML patients.

Sepsis occurs when an infection induces a dysregulated immune response, and is most commonly bacterial in origin. This condition requires rapid treatment for successful patient outcomes. However, the current method to confirm infection (blood culture) requires up to 48 h for a positive result and many true cases remain culture-negative. Therefore, new diagnostic tests are urgently needed. Recent clinical studies suggest that CD69, CD64 and CD25 may serve as useful biomarkers of sepsis. In this study, we evaluated the cecal ligation and puncture (CLP) and cecal slurry (CS) mouse models as tools to study these biomarkers in young and aged mice, and elucidate the timeliness and specificity of sepsis diagnosis. Fluorescence-activated cell sorting (FACS) analysis revealed that all three biomarkers were elevated on blood leukocytes during sepsis. CD69 was specifically upregulated during sepsis, while CD64 and CD25 were also transiently upregulated in response to sham surgery. The optimal biomarker, or combination of biomarkers, depended on the timing of detection, mouse age and presence of surgery. CD69 demonstrated an excellent capacity to distinguish sepsis, and in some scenarios the diagnostic performance was enhanced by combining CD69 with CD64. We also analyzed biomarker expression levels on specific cell populations (lymphocytes, monocytes and neutrophils) and determined the cell types that upregulate each biomarker. Elevations in blood biomarkers were also detected via microfluidic analyses; in this case CD64 distinguished septic mice from naive controls. Our results suggest that CD69 and CD64 are valuable biomarkers to rapidly detect sepsis, and that mouse models are useful to study and validate sepsis biomarkers.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2–based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

Based on CD25 expression, T follicular helper cells (Tfh) could be divided into T follicular regulatory (Tfr)-like subset (CD25+CD4+CXCR5+) and CD25- Tfh subset (CD25-CD4+CXCR5+). Patients with diffuse large B cell lymphoma (DLBCL) display high level of Tfr-like cells in blood and tumor. This Tfr-like subset could suppress CD8 T cell response while promote tumor cell proliferation. In this study, we investigated the transcription factors and regulatory elements associated with Tfr-like cells in DLBCL patients. Both circulating and tumor-infiltrating Tfr-like cells presented slightly higher Blimp-1 expression and significantly higher Foxp3 expression than the CD25- Tfh subset. As the IL-2 receptor, CD25 could be moderately upregulated in stimulated CD25- Tfh cells. However, stimulated CD25- Tfh cells could not upregulate Foxp3, indicating that the distinction between Foxp3-low CD25-CXCR5+CD4+ T cells and Foxp3-high CD25+CXCR5+CD4+ T cells was not due to differences in stimulation status. Regarding cytokine production, while both Tfr-like and CD25- Tfh cells upregulated IL-21 and IL-10 during stimulation, the CD25- Tfh cells presented significantly higher IL-21 and lower IL-10 expression than the Tfr-like cells, and the TGF-β expression was only increased in Tfr-like cells. Interestingly, IL-21 secreted from CD25- Tfh cells negatively regulated the expression of Foxp3 and IL-10 of autologous Tfr-like cells. Together, these results demonstrated that the Tfr-like and CD25- Tfh subsets of circulating Tfh cells presented different functions and should be investigated separately.

Background: TGF-β has long been considered as the main inducer of Tregs in tumor microenvironment and is the reason for the aberrant number of Tregs in tumor-bearing individuals. Recently, it has been suggested that the enzyme arginase I is able to mediate the induction of Tregs in a TGF-β-independent fashion. The recombinant WW2/WW3 domains from smad ubiquitination regulatory factor 2 molecule was demonstrated to increase TGF-β signaling while reducing arginase I gene expression. In this study, we aimed to examine the effects of this recombinant protein on CD4+CD25+/CD4+ proportion in the spleen of 4T1 mammary carcinoma-bearing BALB/c mice. Methods: Flow cytometry was used to evaluate CD4+CD25+ spleen cell populations of the tumor-bearing mice that received WW2/WW3 protein treatment and those of the control group. Results: The results indicated a significant rise in CD4+CD25+/CD4+ ratio, along with an average increase in tumor mass of the subjects that underwent protein treatment. Conclusion: It can be inferred that the heightened CD4+CD25+/CD4+ proportion in the spleen of protein-treated tumor-bearing mice can be the result of the increased TGF-β signaling despite the reduced arginase I expression.

Background: Although most patients with COVID-19 pneumonia have a good prognosis, some patients develop to severe or critical cases, and the mortality of critical cases was up to 61.5%. However, specific information and molecular mechanism about critical patients with COVID-19 was poorly understood. Methods: A total of 54 patients were enrolled and divided into three groups, among which 34 were common type, 14 were severe type, and 6 were critical type. The constitution of peripheral blood mononuclear cells (PBMC) in patients was analyzed by CyTOF. The profile of cytokines was performed in plasma of patients using Luminex. The IL-2 signaling pathway was investigated in the PBMC of patients by qRT-PCR. Results: The lymphocytes count and percentage were significantly decreased in critical patients compared to common and severe patients with COVID-19 pneumonia. CD8+T cells were remarkably decreased in critical patients compared to common, severe patients and normal controls, which suggested the decrease of lymphocytes may be attributed to the reduction of T cells in critical patients. The expression of IL-2R, JAK1, and STAT5 decreased in immune cells of common, severe, and critical patients, but IL-2 level was elevated in severe patients and decreased in critical patients with COVID-19 pneumonia. Conclusion: The decrease of CD8+T cells in critical patients with COVID-19 pneumonia may be related to the IL-2 signaling pathway. The inhibition of IL-2/IL-2R gives rise to CD8+T cell and lymphocyte decrease through JAK1-STAT5 in critical patients with COVID-19 pneumonia. Funding Statement: This study was financially supported by COVID-19 Key Technology Research and Development Funding of Beijing Hospital Authority; The National Natural Science Foundation of China (81672026); National Science and Technology Major Project of China (2018ZX10302205-005). Declaration of Interests: The authors confirm that there are no conflicts of interest. Ethics Approval Statement: The study was approved by the Ethics Committee of Beijing Youan Hospital, Capital Medical University, China.

Summary Effective laboratory markers for the estimation of disease severity and predicting the clinical progression of coronavirus disease‐2019 (COVID‐19) is urgently needed. Laboratory tests, including blood routine, cytokine profiles and infection markers, were collected from 389 confirmed COVID‐19 patients. The included patients were classified into mild (n = 168), severe (n = 169) and critical groups (n = 52). The leukocytes, neutrophils, infection biomarkers [such as C‐reactive protein (CRP), procalcitonin (PCT) and ferritin] and the concentrations of cytokines [interleukin (IL)‐2R, IL‐6, IL‐8, IL‐10 and tumor necrosis factor (TNF)‐α] were significantly increased, while lymphocytes were significantly decreased with increased severity of illness. The amount of IL‐2R was positively correlated with the other cytokines and negatively correlated with lymphocyte number. The ratio of IL‐2R to lymphocytes was found to be remarkably increased in severe and critical patients. IL‐2R/lymphocytes were superior compared with other markers for the identification of COVID‐19 with critical illness, not only from mild but also from severe illness. Moreover, the cytokine profiles and IL‐2R/lymphocytes were significantly decreased in recovered patients, but further increased in disease‐deteriorated patients, which might be correlated with the outcome of COVID‐19. Lymphopenia and increased levels of cytokines were closely associated with disease severity. The IL‐2R/lymphocyte was a prominent biomarker for early identification of severe COVID‐19 and predicting the clinical progression of the disease., IL‐2R/lymphocyte was a superior marker for the identification of severe COVID‐19 patients.

We previously demonstrated that a deficiency of natural antibodies against CD25, Mucin 1 (MUC1), and vascular endothelial growth factor receptor 1 (VEGFR1) could contribute to high risk of non‐small cell lung cancer (NSCLC). This study was designed to investigate whether natural IgG antibodies against POU domain class 5 transcription factor 1 (POU5F1), tumor necrosis factor‐α (TNF‐α), and the combination of CD25, VEGFR1, and MUC1 could play an anti‐tumorigenic role against developing NSCLC. An ELISA was developed in‐house to examine plasma IgG against peptide antigens derived from POU5F1, TNF‐α, and a combination of peptide antigens derived from CD25, MUC1, and VEGFR1 in 211 patients with NSCLC and 200 healthy controls. Mann–Whitney U test demonstrated that plasma IgG levels for the combination of peptide antigens derived from CD25, MUC1, and VEGFR1 were significantly lower in NSCLC patients than control subjects (Z = −12.978, P, Group I as an early stage (stages 1 and 2A, n = 121), group II as stage 2B (n = 41), and group III as stages 3 and 4 (n = 49). ROC curve analysis showed that the CD25‐MUC1‐VEGFR1 combination‐based ELISA showed the largest AUC of 0.883 (95% CI 0.844–0.923) with a sensitivity of 49.6% against the specificity of 95.0% in patients with early‐stage NSCLC. Natural antibodies against the combination of CD25, VEGFR1, and MUC1 may be an effective biomarker for early diagnosis of NSCLC.

Aims Soluble tumour necrosis factor‐α receptor 1 (sTNF‐αR1) and interleukin‐2 receptor α (sIL‐2Rα) predict incident heart failure (HF) in the elderly population. However, the association of these biomarkers with HF in a multi‐ethnic asymptomatic population is unclear. We aimed to investigate the association of sTNF‐αR1 and sIL‐2Rα with incident HF in a multi‐ethnic population of middle age and older participants. Methods and results The multi‐ethnic study of atherosclerosis is a prospective population‐based study of 6814 participants aged 45–84 years who were free of clinical cardiovascular disease at enrolment. We included 2869 participants with available sTNF‐αR1 or sIL‐2Rα level measurement at baseline multi‐ethnic study of atherosclerosis exam (2000–2002). We used Cox proportional‐hazards model to investigate the association between sTNF‐αR1 and sIL‐2Rα with incident HF after adjusting for traditional cardiovascular risk factors and coronary artery calcium score measured by cardiac computed tomography. Among the included participants, the mean (standard deviation) age was 61.6 (10.2) years and 46.7% were men. The median (interquartile range) sTNF‐αR1 and sIL‐2Rα were 1293 (1107–1547) and 901 (727–1154) pg/mL. During a median follow‐up of 14.2 (interquartile range: 11.7–14.8) years, 130 participants developed HF. In multivariable analysis, the hazard ratio (95% confidence interval, P value) of incident HF for each standard deviation increment of log‐transformed sTNF‐αR1 and sIL‐2Rα was 1.43 (1.21–1.7, P ≤ 0.001) and 1.26 (1.04–1.53, P = 0.02), respectively. Excluding participants with interim coronary heart disease, we found a statistically significant association between sTNF‐αR1 and HF with hazard ratio of 1.39 (95% confidence interval: 1.11 to 1.74, P = 0.005) and sIL‐2Rα and HF showing a hazard ratio of 1.39 (95% confidence interval: 1.09 to 1.76, P = 0.007). Conclusions sTNF‐αR1 and sIL‐2Rα are associated with a higher risk of incident HF in a multi‐ethnic cohort without a previous history of cardiovascular disease.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700–targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3(+)CD25(+)CD4(+) regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3(+)CD25(+)CD4(+) Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

Objective: To assess the clinical utility of the ratio of CD4+CD25+CD127low regulatory T cells (Tregs) in subjects at high risk of HCC, investigate the relationship between the percentage of Tregs and the expression of transforming growth factor (TGF)-β1 and interleukin (IL)-10 in patients with hepatocellular carcinoma before and after treatment. Methods: Peripheral venous blood was collected from patients with liver cancer before and after treatment. The proportion of CD4+CD25+CD127low Tregs was detected by flow cytometry. The levels of TGF-β1 and IL-10 in serum were detected by enzyme-linked immunosorbent assay, and were compared with healthy subjects as a control group. Results: The proportion of CD4+CD25+CD127low to CD4+T lymphocytes in patients with hepatocellular carcinoma was significantly higher than that in healthy controls (P

Low Foxp3+ regulatory T-cell (Treg) presence in the tumor-infiltrating lymphocytes (TILs) is considered favorable in breast cancer, and numerous CD25-targeting agents have been applied in the attempt to remove Foxp3+ Treg cells, which typically present CD4+ CD25+/hi surface phenotype. However, CD25 is not Treg-exclusive and can be upregulated by effector T cells. Hence, CD25 depletion may cause the elimination of activated T cells that are responding to tumor-specific antigens. In this study, the composition and function of CD4+ CD25+ cells inside the microenvironment of triple-negative breast carcinoma (TNBC) were investigated. Directly ex vivo, the Foxp3+ Treg cells represented a minor subset in total CD4+ CD25+ TILs. Significant differences were observed in the expression of Treg-associated molecules between CD4+ CD25+ Foxp3+ TILs and CD4+ CD25+ Foxp3- TILs. While both the CD4+ CD25+ Foxp3+ and the CD4+ CD25+ Foxp3- TILs could express CTLA-4 and LAG-3, the expression levels were significantly higher in CD4+ CD25+ Foxp3+ TILs than in CD4+ CD25+ Foxp3- TILs. Upon TCR stimulation, the expression of TGF-beta was significantly higher in CD4+ CD25+ Foxp3+ TILs, while the expression of IL-10 was significantly higher in CD4+ CD25+ Foxp3- TILs. These differences were conserved in the blood counterparts of these cells. Interestingly, the level of CD25+ Foxp3+ cells in circulating CD4+ T cells was positively correlated with the level of CD25+ Foxp3+ cells in CD4+ TILs, but the level of CD25+ Foxp3- cells in circulating CD4+ T cells was not associated with the level of CD25+ Foxp3- cells in CD4+ TILs. Th17-polarizing medium could readily remodel CD4+ CD25+ Foxp3- , but not CD4+ CD25+ Foxp3+ , T cells into RORgammat and IL-17-expressing T cells, demonstrating stronger plasticity of the former subset. Together, these data demonstrated that the CD4+ CD25+ TILs were composed of distinctive Foxp3- and Foxp3+ cells, with the former representing the major subset. The antigen specificity and effector molecule expression of the CD4+ CD25+ Foxp3- thus require further analyses.

Thymoglobulin, or antithymocyte globulin (ATG), and anti-interleukin 2α (IL-2α) chain receptor antibody (IL-2αRAb) achieve comparable good results in kidney transplantation notwithstanding different actions on immune cells. Previously, we reported the usefulness of flow cytometry (FC) analysis of lymphocyte subsets present in peripheral blood sample (PBL) and fine-needle aspiration biopsies (FNABs) for clinical surveillance, as, FC reaches very high predictive positive values for acute rejection diagnosis. Now we report an FC study on 2 kidney transplantation (KT) groups under ATG (n = 19) and IL-2αRAb (n = 24) treatment. Both groups were further treated with calcineurin inhibitors mycophenolate mofetil (MMF) and prednisone. PBL and FNAB samples were collected on day 7 post-KT, stained for several T- and B-lymphocyte subsets, and acquired using FACScan. Statistical analysis were done by Mann-Whitney U test. FNAB results showed a significant downregulation by ATG of CD3 (P .001), CD4 (P = .009), CD4CD29 (P = .003), and CD2 (P ≤ .001) and significant upregulation of death receptor (DR) (P = .03), CD3CD69 (P .001), and CD3CD25 (P .0001) as compared to groups treated with IL-2αRAb. For PBL, the same trend was seen for CD3, CD4, CD2, CD3CD25, CD3CD69, CD4CD29, and DR plus a downregulation of CD45RO (P = .001) and an upregulation of CD4CD45RA (P .0001) in IL-2αRAb. This study shows that among stable KTs, ATG as compared to IL-2αRAb induces a significant downregulation of a subset of T-memory (CD4CD29) cells but an upregulation of antigen-experienced cells (CD45RO). Further, ATG decreases CD2, CD3, CD4, and naïve (CD45RA) and stimulates T cells as translated by CD3CD69 and DR. As it should be expected from an IL-2αRAb agent, CD25 cells were virtually eliminated.

IntroductionFor end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells.MethodsPeripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry.ResultsWithin the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (pvia CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells.ConclusionHigher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.

Human γδ T cells expressing Vγ9Vδ2 T cell receptors exert a robust response to pathogens and malignant cells. These cells are activated by BTN3A1, which is expressed by pathogen-derived phosphoantigens (pAgs) or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Activated Vδ2 (+) T cells exert multiple effector functions; therefore, they are a promising candidate for immunotherapy. However, not all donors have γδ T cells with adequate proliferative activity. Here, we performed ex vivo culture of γδ T cells from 20 healthy donors and explored factors that may affect their expansion efficiency. Consistent with previous studies, we found that amplification of γδ T cells requires CD14

BackgroundImmunization with tumor neoantigens is a promising vaccine approach to promote antitumor immunity due to their high immunogenicity, lack of expression in normal tissue, and preferential induction of tumor neoantigen-specific T cells, which are central mediators of the anti-cancer response. A drawback to targeting tumor neoantigen-specific T cells is that these cells are found at a low frequency in patients with cancer, limiting their therapeutic benefit. Interleukin-2 (IL-2) promotes expansion and persistence of tumor-reactive T cells. However, its clinical use has been hampered by toxicities arising from its multiple cellular targets. Thus, new engineered IL-2 receptor (IL-2R) agonists with distinctive cell type selectivity have been designed to harness the potential of IL-2 for tumor immunotherapy.MethodsWe investigated the potential to amplify neoantigen-specific CD4+and CD8+T cell immune responses to promote antitumor immunity through vaccination with tumor neoantigens. Following T cell receptor (TCR)-mediated induction of the high-affinity IL-2R on these T cells, amplification of the neoantigen-specific T cell response was achieved using a high dose of the mouse IL-2/CD25 (mIL-2/CD25) fusion protein, an IL-2R agonist with more favorable pharmacokinetics and pharmacodynamics than IL-2 and selectivity toward the high-affinity IL-2R.ResultsAdministration of a high dose of mIL-2/CD25 shortly after antigen-dependent induction of the high-affinity IL-2R amplified the numbers and function of TCR transgenic tumor-reactive tyrosinase-related protein-1 (TRP-1) CD4+T cells, leading to antitumor immunity to B16-F10 melanoma. This approach was adapted to amplify endogenous polyclonal B16-F10 neoantigen-specific T cells. Maximal expansion of these cells required prime/boost neoantigen vaccinations, where mIL-2/CD25 was optimal when administered only after the boosting steps. The ensuing mIL-2/CD25-driven immune response supported antitumor immunity to B16-F10 and was more effective than treatment with a similar amount of IL-2. Optimal antitumor effects required amplification of CD4+and CD8+neoantigen-specific T cells. High-dose mIL-2/CD25 supported a tumor microenvironment with higher numbers of CD4+and CD8+T effectors cells with increased granzyme B expression and importantly a more robust expansion of neoantigen-specific T cells.ConclusionThese results indicate that neoantigen-based vaccines are optimized by potentiating IL-2R signaling in CD4+and CD8+neoantigen-reactive T cells by using high-dose mIL-2/CD25, leading to more effective tumor clearance.

Inflammation after acute CNS injury plays a dual role. The interplay between immune cells and inflammatory mediators is critical to the outcome of injured neurons. Microglia/macrophages are the first sensors and regulators of the immune response. We previously found that the enhancement of macrophages on neuron survival does not persist in thymectomized rats. How T lymphocytes and macrophages interact and benefit neuron survival is not fully elucidated. To this point, we introduce and characterize a cell-retina co-culture model that mimics the recruitment of peripheral lymphocytes at the injury site. Three-day post-optic nerve transection (ONT) in Fischer 344 rats, transected retinas were co-cultured with either peripheral lymph node-derived lymphocytes (injury-activated) or from intact rats as the control. The injury-activated lymphocytes preserved retinal ganglion cells (RGCs) and caused extensive retina microglial/macrophage infiltration. CD4+CD25+ T cells were upregulated in the injury-activated lymphocytes and increased RGC survival, suggesting that CD4+CD25+ T cells suppressed the cytotoxicity of control lymphocytes. When microglia/macrophages were depleted by clodronate, neuron loss was more extensive, the cytotoxicity of control lymphocytes on RGCs was alleviated, and the neuroprotective effect of injury-activated lymphocytes remain unchanged Cytokine detection showed an increase in IL-6 and TNF-α levels that were reduced with microglia/macrophage depletion. Our results suggest that microglial/macrophage infiltration into axotomized retinas promotes RGC survival by secreting cytokines to induce CD4+CD25+ T cells and suppress T cell-mediated RGC toxicity. These findings reveal a specific role for microglia/macrophage and CD4+CD25+ T cells in inflammation after CNS injury, thereby adding to the mechanistic basis for the development of microglial/macrophage modulation therapy for traumatic CNS injury.

Aims/hypothesis Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes expressing an αβ T cell antigen receptor that recognises the MHC-related 1 molecule. MAIT cells are altered in children at risk for and with type 1 diabetes, and mouse model studies have shown MAIT cell involvement in type 1 diabetes development. Since several studies support heterogeneity in type 1 diabetes physiopathology according to the age of individuals, we investigated whether MAIT cells were altered in adults with type 1 diabetes. Methods MAIT cell frequency, phenotype and function were analysed by flow cytometry, using fresh peripheral blood from 21 adults with recent-onset type 1 diabetes (2–14 days after disease onset) and 47 adults with long-term disease (>2 years after diagnosis) compared with 55 healthy blood donors. We also separately analysed 17 women with long-term type 1 diabetes and an associated autoimmune disease, compared with 30 healthy women and 27 women with long-term type 1 diabetes. Results MAIT cells from adults with recent-onset type 1 diabetes, compared with healthy adult donors, harboured a strongly activated phenotype indicated by an elevated CD25+ MAIT cell frequency. In adults with long-term type 1 diabetes, MAIT cells displayed an activated and exhausted phenotype characterised by high CD25 and programmed cell death 1 (PD1) expression and a decreased production of proinflammatory cytokines, IL-2, IFN-γ and TNF-α. Even though MAIT cells from these patients showed upregulated IL-17 and IL-4 production, the polyfunctionality of MAIT cells was decreased (median 4.8 vs 13.14% of MAIT cells, p

Human regulatory T (Treg) cells play a central role in controlling allergic inflammation in the airways. A reduced number of peripheral Treg cells and decreased suppressive function have been previously reported in the pathogenesis of allergic asthma. However, the characteristic role of specific Treg cell subsets and their mechanisms in the pathogenesis of allergic asthma remain unclear. In this study, we examined the proportion of different Treg cell subsets in both healthy subjects and patients with allergic asthma using flow cytometry and single-cell RNA sequencing. The migration function of the cells was compared using cell sorting and Transwell experiments. Furthermore, two allergen-challenged mouse models and a cell transfer experiment were used to examine the role of these Treg subsets. We found that the proportion of CD25+Foxp3+CD127- Treg cells in the peripheral blood of patients with allergic asthma was lower than in those of healthy subjects. Furthermore, the circulating Treg cells expressed lower levels of CCR6 and IL-17 compared with healthy subjects. The chemokine from the airway mucosa, CCL20, was abundantly expressed, and Transwell experiments further proved that this chemokine promoted CCR6+ Treg cell migration in vitro. A mouse model induced by house dust mite (HDM) revealed that the number of CCR6+ Treg cells in the lung tissue increased remarkably. The incidence of allergic asthma may be related to an increase in Treg cells secreting IL-17 in the lung tissue. Recruited CCR6+ Treg cells are likely to differentiate into Th17-like cells under the Th17 environment present in the lungs. IL-17 derived from Th17-like cells could be associated with the pathology of allergic asthma by promoting Th17 responses, thereby favoring HDM-induced asthma exacerbations.

Background and Purpose: T lymphocytes contribute to secondary brain damage after stroke. It has not been fully investigated whether this contribution is caused by antigen-specific or antigen-nonspecific activation of T lymphocytes. Lymphocytes from Nur77 GFP transgenic mice express a fluorescent protein upon activation via the TCR (T-cell receptor), allowing the differentiation of activation mode in a natural repertoire of immune cells and antigens. Methods: Middle cerebral artery occlusion or sham surgery was performed, and T-lymphocyte activation was analyzed by flow cytometry in the brain, spleen, and blood 16 hours, 2 days, 3 days, 4 days, and 7 days after surgery. Results: Ipsilateral hemispheric T-lymphocyte invasion peaked on day 4 poststroke. Here, we observed PD-1 (programmed cell death protein 1) expression on almost all invading T lymphocytes, while CD25 expression was low. CD25+, CD69+, or PD-1+ T lymphocytes predominantly displayed antigen-specific activation; the opposite was observed for T lymphocytes isolated from the blood. A mixed activation that favored antigen-specific activation was observed in the spleen. PD-1 was upregulated within the brain, whereas CD25 was not. Antigen-specific T lymphocytes home to the brain, while antigen-nonspecifically activated cells remain within the blood. Conclusions: Our data clearly demonstrate antigen-specific activation of T lymphocytes infiltrating ischemic brain lesions in stroke. The high expression of inhibitory PD-1 and low expression of CD25 on activated T lymphocytes in the brain most likely reflect immunosuppressive mechanisms.

Immune-mediated, drug-induced hepatitis is a rare complication of halogenated volatile anesthetic administration. IL-4-regulated Th2-polarized reactions initiate this type and other types of hepatitis, while the mechanisms that regulate the severity remain elusive. IL-33 is an innate, IL-4-inducing, Th2-polarizing cytokine that has been detected in patients with liver failure and has been associated with upregulated ST2+Foxp3+CD4+CD25+ T cells; however, roles for IL-33 in drug-induced hepatitis are unclear. We investigated IL-33 in an anesthetic, immune-mediated hepatitis modeled in BALB/c, IL-33-/- and ST2-/- mice, as well as in patients with anesthetic hepatitis. The hepatic IL-33 and ST2 levels were elevated in BALB/c mice (p 0.05) with hepatitis, and anti-IL-33 diminished hepatitis (p 0.05) without reducing IL-33 levels. The complete absence of IL-33 reduced IL-10 (p 0.05) and ST2+Foxp3+CD4+CD25+ T cells (p 0.05), as well as reduced the overall survival (p 0.05), suggesting suppressive roles for IL-33 in anesthetic, immune-mediated hepatitis. All of the mice demonstrated similar levels of CD4+ T-cell proliferation following direct T-cell receptor stimulation, but we detected splenic IL-33 and ST2-negative Foxp3+CD4+CD25+ T cells in ST2-/- mice that developed less hepatitis than BALB/c mice (p 0.05), suggesting that ST2-negative Foxp3+CD4+CD25+ T cells reduced hepatitis. In patients, serum IL-33 and IPEX levels were correlated in controls (r