DNT Cells Research Articles

Double-negative T cells in pediatric rheumatic diseases.

Double-negative (CD4-CD8-) T (DNT) cells have been implicated in Autoimmune Lymphoproliferative Syndrome (ALPS), where their expansion inside the circulating pool of T cells represents a diagnostic criterion. Recent experimental evidence has supported the immunomodulatory roles of DNT cells, and studies in adult patients have suggested that they may be altered in some immune-mediated conditions. This study aimed to retrieve available data on circulating DNT cells in pediatric rheumatic disorders that do not arise in the context of ALPS through a systematic literature review of three scientific databases (PubMed, Scopus, and Web of Science). The final output of the systematic literature search consisted of eight manuscripts, including cross-sectional (n=6) and longitudinal (n=2) studies. Overall, the pooled population of patients includes children affected with pediatric Systemic Lupus Erythematosus (n=104), Juvenile Idiopathic Arthritis (n=92), Behçet's disease (n=15), mixed connective tissue disease (n=8), Juvenile Dermatomyositis (n=6), and Kawasaki disease/multisystem inflammatory disease in children (n=1 and n=14, respectively); moreover, one study also included 11 children with a high titer of antinuclear antibody but no diagnosis of rheumatic disease. All studies except one included a control group. The number of DNT cells were increased in most studies of children with rheumatic diseases. Even if such a limited number of studies and their great heterogeneity in several methodological aspects do not allow for reliable conclusions about the relevance of DNT cells in specific rheumatic conditions in children, this cell population deserves further investigation in this pathological setting through well-designed clinical studies.

P-390 The percentage of regulatory T cells is associated with T follicular helper cell quantity in the peripheral blood of pregnant women

Abstract Study question Is there a relationship between the percentage of peripheral regulatory T cells (Treg) and other T cell subtypes in the first trimester of pregnancy? Summary answer The percentage of Treg cells showed a significant positive correlation with the quantity of T follicular helper cells (TFH). What is known already Regulatory T cells (Treg) play an essential role in the process of embryo implantation and maintenance of normal pregnancy. The quantity of these cells in the peripheral blood expands during the first trimester of pregnancy. Several studies report the importance of Treg in the control of the inflammatory process and in the pathophysiology of common adverse pregnancy outcomes. However, there is a scarce data about the quantitative relation between Treg and other important T sub-types during pregnancy. Study design, size, duration This is an observational study of 93 women, 28 to 53 years of age (mean 38 years), who had a blood sample during the first trimester (at 8-10 weeks of pregnancy). The study was conducted between November 2022 and October 2023. The exclusion criteria were history of recent inflammatory or infection disorders, recent antibiotic treatment, autoimmune diseases, oncological diseases, moderate or severe endometriosis, and endometrial polyps. Written informed consent was obtained from each participant. Participants/materials, setting, methods SIx peripheral T cell subtypes were measured by flow cytometry: T helpers % (CD3+CD4+CD8-/CD3+), T killers % (CD3+CD4-CD8+/CD3+), double-positive T cells % (CD3+CD4+CD8+/CD3+), double-negative T cells % (CD3+CD4-CD8-/ CD3+), T follicular helper cells % (CD3+CD4+CD8-CD185+/CD4+), T reg cells (CD4+CD8-CD25+CD127−/CD4+). Spearman’s correlation test was performed with all studied T cell subtypes in SPSS 21 package program. Main results and the role of chance The median percentage (range) of the studied T cell subtypes was: T helpers (51.7%, 12.1%÷91.45%), T killers (39.0%, 4.1%÷72.5%), DPT cells (1.2%, 0÷8.75), DNT cells (8.4%, 1.9%÷46.2%), Treg cells (4.6%, 1.9%÷12.7%), TFH cells (7.6%, 1.4%÷42.6%). To examine the association between the percentage of Treg cells and the other T cell subtypes, the Spearman’s Rho Correlation coefficient for non-paramentric data was used. No correlation was found between the quantity of Treg and T helpers, T killers, Total T cells, double-positive T cells (p > 0.05). In contrast, the percentage of Treg showed a significant positive correlation with the percentage of TFH (R = 0.36; p = 0.01), and a significant negative correlation with double-negative T cells (R= - 0.29; p = 0.04). Limitations, reasons for caution The main limitation of the study is that it was carried out only in a limited period of time during the pregnancy. Thus, it would be beneficial to compare the percentage of Treg cells with other T cell sub-types in the second and third trimester of pregnancy. Wider implications of the findings Our study demonstrated that Treg cells have stronger association with TFH cells than with other T cell subtypes. The strength of this quantitative relationship might be important for the normal progress of pregnancy, and it remains subject of further studies. Trial registration number not applicable

P-306 GnRH agonists exert a positive influence on regulatory immune cells in the endometrium of women with adenomyosis

Abstract Study question How does treatment with GnRH-agonist (GnRH-a) prior to frozen embryo transfer (FET) in women with adenomyosis affect immune cell populations in endometrium and peripheral blood? Summary answer GnRH-a induces a decrease in monocytes and an increase in CD4+T-cells in peripheral blood and a decrease classical monocytes and T-regulatory cells in the endometrium What is known already Adenomyosis is associated with lower implantation and higher miscarriage rates. Observational studies have reported improved pregnancy outcomes in women with adenomyosis after assisted reproductive technologies (ART) and pre-treatment with GnRH-a prior to FET. However, the specific mechanisms of GnRHa action beyond central hormonal suppression remain unclear, but may be mediated through local and/or systemic immunomodulation. Study design, size, duration Prospective cohort study including infertile women with adenomyosis and GnRH-a treatment ≥3 months prior to FET. Pre- and post-treatment analysis of blood and endometrium samples were compared. Recruitment is ongoing with n = 30 women included thus far, 16 of whom have completed the study, while the remaining are still undergoing GnRH-a therapy. Participants/materials, setting, methods Adenomyosis was diagnosed sonographically if ≥ 2 criteria published by the MUSA group were met. Peripheral blood and endometrial samples were obtained before and after treatment with GnRH-a. Plasma cells and uterine Natural Killer (uNK) cells in endometrial biopsies were quantified with immunhistochemistry (IHC), and leukocyte populations in all samples were analysed using flow cytometry (FC). Pre- and post-treatment levels of immune cells and percentage of leukocyte populations were compared using Wilcoxon rank-sum tests (p < 0.05 significant) Main results and the role of chance Interim analysis of immune cell populations in peripheral blood and endometrium of the 16 women who already completed the study was performed. At baseline, IHC showed elevated plasma cell concentrations indicating chronic endometritis in 3/30 patients (10%), while uNK cells were elevated (>300/mm2) in 13/30 patients (43.3%). FC analysis of peripheral blood showed a significant decrease in percentage of monocytes (p = 0.01) and increase in percentage of T-cells (p = 0.03), specifically concentration of CD4+T-cells (p = 0.03), after treatment with GnRH-a as compared to pre-treatment levels. In the endometrium, following GnRH-a therapy, we observed a decrease in both the percentage of classical monocytes (p = 0.03) and T-regulatory cells (p = 0.03). All 16 participants who completed the study already underwent FET, resulting in ten pregnancies (62.5%), although four resulted in miscarriage, while the outcome of the remaining pregnancies is still pending. Limitations, reasons for caution These are preliminary results whose generalizability needs to be confirmed in a larger study population. No correction for multiple comparisons was performed and significant findings have to be interpreted with caution. Wider implications of the findings Elevated uNK on IHC cells seen at baseline may be associated with implantation failure, miscarriage. CD4+ and DN T-cells are important mediators of reproductive immune tolerance. In women with adenomyosis, GnRH-a may have a positive impact on these regulatory immune cells in peripheral blood as well as in the endometrium. Trial registration number Not applicable

P-593 Inflammaging in the ovary: focus on foamy cells generation

Abstract Study question Could the dysregulation of the inflammatory response in the aged ovary be associated with foamy cells generation and be ameliorated using an alternative therapeutic treatment? Summary answer Aged ovarian macrophages show an inflammatory profile associated with incorporation of lipid droplets that can be reduced after culture with platelet-rich plasma from fertile women. What is known already The concept of ‘inflammaging’ represents the inflammation associated to aging, which is systemic, chronic and linked to the increase of pro-inflammatory mediators. This microenvironment is associated with the presence of ovarian foamy macrophages, a unique subset with intracytoplasmic lipids accumulation. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging, focusig on the generation of foamy macrophages and their impact in oocyte quality. Since, oocytes develop in the context of ovarian follicles, we obtained follicular fluid (FF) from women with different ages, to investigate ovarian immune microenvironment and their modulation after in vitro platelet-rich plasma (PRP) treatment. Study design, size, duration Mononuclear cells were recovered from FF from women of different ages and IL-1β production and macrophages and lymphocytes profiles tested by immune-staining and FACS analysis. To test foamy cells generation, we measured surface and intracellular CD68 expression (scavenger receptor involved in lipid accumulation). Immune cells profiles were correlated with clinic parameters as number of oocytes, M2 and atretic follicles. Finally, isolated ovarian macrophages were treated with PRP and modulation of macrophage profile was tested. Participants/materials, setting, methods We studied FF (n = 70) from women from ovodonation (21-29y), with cryopreservation desire (30-35y) and ART indication (36-42y). The San Isidro Ethics Committee, Buenos Aires, Argentina, approved this study. Mononuclear cells were isolated using Ficoll-Hypaque and macrophages profile (IL-1β+, CD68+) and the novel lymphocyte regulatory subpopulation (CD3+CD4-CD8-) tested by flow cytometry. Lipid droplets were measured using BODIPY 493/503 probe. Ovarian macrophages were isolated by adherence and treated with PRP (obtained from fertile women) for 5 days. Main results and the role of chance FF macrophages from aged women (35-42y) showed a significant increase in IL-1β production in comparison with the youngest group (21-29y), that also correlates negatively with the number of M2 oocytes recovered. Focusing on macrophage characterization, FF from aged women displayed an increased frequency of CD68+ macrophages in comparison with young women. Since CD68 shuttles between the cytoplasm and the plasmatic membrane to incorporate lipids, we evaluated lipid accumulation in FF macrophages to generate foamy cells. Interestingly, we found an increase of lipid droplets in FF macrophages from aged women in comparison with the younger group. Therefore, we discriminated surface and intracellular CD68 detection and found higher intracellular CD68 expression in aged macrophages, correlating with lipid droplets accumulation. Then, we isolated ovarian macrophages from aged women and treated with fertile PRP to evaluate its modulatory effects and observed a reduction in the frequency of CD68+IL-1β cells in comparison with those cultures without treatment. Finally, we focused on a novel T lymphocyte regulatory subpopulation, CD3+CD4-CD8- (DNT), that is emerging as effector cells capable of mediating immune tolerance in the female reproductive system. We found that FF from aged women displayed a reduced frequency of DNT, in comparison with FF from younger women. Limitations, reasons for caution The present results were obtained using FF samples from women from different ages. Even though the samples represent the ovarian microenvironment, it is difficult to study the human ovary per se and further studies are necessary to elucidate whether these mechanisms operate similarly in vivo. Wider implications of the findings We demonstrated that dysregulation of the inflammatory response during aging leads to the generation of foamy cells and reduction of DNT cells associated to the regulation of the immune ovarian microenvironment impacting in oocyte quality. In vitro PRP treatment displayed immunomodulatory effects, suggesting its relevance as a potential therapeutic treatment. Trial registration number not applicable

Abstract 1520: A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for hepatocellular carcinoma

Abstract Tumor immunotherapies represent promising strategies for treating various cancers, including hepatocellular carcinoma. In this context, immune cells, including the adoptive T cell transfer treatment, become the focus of interests. Notably, alpha/beta TCR+CD4−CD8−T (DNT) cells is a subgroup of T cells that is ignored for a long time but was re-focused recently. These DNT cells are a group of cells with opposite functions in different condition, including pro-inflammatory or suppressive functions and possibly originated from over-stimulation of peripheral CD4+T cells/CD8+T cells or a special group of T cells formed in thymus. In our research results, we have found these DNT cells increased in murine hepatoma model, especially in the tumor microenvironment. In addition, these DNT cells from tumor microenvironment of murine hepatoma possessed potent tumor-killing ability. Furthermore, we analyzed the transcriptome profile of T cells in tumor microenvironment by single cell RNAseq approach (CITE-seq). We found DNT cells could be categorized into two main clusters, one is cytolytic DNT cells, resemble the potent CD8+T cells, and another is the CD4+T cell-like DNT cells. These cytolytic DNT cells are not exhausted but with high expression of cytolytic molecule and low hypoxia-inducible factor-1alpha (HIF-1a) expression. By trajectory analysis, cytolytic DNT cells come from the conventional CD8+T cells but following a unique developmental pathway that is different from the exhausted CD8+T cells. Interesting, these cytolytic DNT cells also expressed high level of Fcer1g molecule, a marker of innate CD8+T cells. In addition, these DNT cells could be generated and expanded in-vitro by IL-15 without TCR activation. In conclusion, we identified a cluster of cytolytic DNT cells, originated from CD8+T cells, exhibits innate T cells behavior and potent killing ability. In addition, these cytolytic DNT cells could be generated and expanded in-vitro for the adoptive T cell transfer treatment of hepatoma. Citation Format: Cheng-Heng Wu, Te-Wei Tseng, Chen-Yang Huang, Ping-Hsun Ou, Yi-Chen Lin, Pin-Jung Chen, Po-Ting Lin, Chan-Keng Yang, Wei Teng, Tsung-Han Wu, Yung-Chang Lin, Chun-Yen Lin. A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1520.

Increase of Dnts Correlated with Clinic Outcome after Allogeneic DNT Cells Infusion in r/r AML Patients

Background: The treatment of relapsed/refractory AML (r/r AML) patients are still an unmet medical need in clinic. In recent years, cellular immunotherapy especially CAR-T cell therapy played more important roles in hematological malignancies. Unfortunately, the safety and efficacy of existing cell therapies to r/r AML patients still facing many challenges and needs to be further improved. Our previous studies have demonstrated that Double Negative T cells (DNTs) showed strong cytotoxicity to AML blasts and can improve the prognosis of AML patients relapsed after allogeneic stem cell transplantation. Here, we reported our ongoing phase I clinical trial of RC1012 injection (allogeneic DNT cells) in patients with r/r AML (NCT05471323). This study protocol was approved by Chinese NMPA (National Medical Products Administration). Informed consent was obtained in accordance with the Declaration of Helsinki. Objective: Explore the efficacy indicator in r/r AML patients after allogeneic DNT cells therapy. Methods: 7 patients with r/r AML were enrolled in the trial according to the inclusion and exclusion criteria. DNTs from peripheral blood of healthy donors were manufactured in GMP facility. After lymphodepleting chemotherapy with fludarabine and cyclophosphamide, each patient received three infusions of HLA-mismatched DNT cells at 1.5×10^8 cells/kg with one week interval. The PK data were collected and analyses as well as the checkpoint inhibitors on T cells was monitored. Results: As reported before, DNT cells had shown good safety profile in the treatment of r/r AML. Multiple infusions of allogeneic DNT cells can benefit some patients with r/r AML. Allogeneic DNT cell therapy can stimulate the patient's DNT cells to proliferate, and the DNT cells level in peripheral blood corelated to the disease outcomes. Allogeneic DNT cells can also stimulate the patient's CD4 and CD8 cells to proliferate and is favorite the CD8 cells to grow, indicating its potential to enhance the anti-tumor activities after DNT cells therapy. Allogeneic DNT cells have no toxicity to normal bone marrow cells, and the quick recovery of platelet counts in peripheral blood corelated to the efficacy of the DNT cells therapy. TIGIT, PD-1, CD57 are increased on patients' T cells which may hinder the effect of immunotherapy in r/r AML patients. Conclusions: Allogeneic DNTs therapy are safe in clinical treatment and can reduce tumor burden after infusion with the increase of patient's DNT cells in complete remission and partial remission patients. These findings warrant the allogeneic DNTs as a novel adjuvant therapy with allo-HSCT to enhance the treatment efficacy in r/r AML. The finding of TIGIT, PD-1, CD57 molecules were increased on patients' T cells indicated that the combination of DNTs with immune checkpoint inhibitors may further improve the DNTs efficacy in r/r AML patients. Key words: Double negative T cells (DNTs), AML, Immunotherapy

A Phase 1 Study of RJMty19: Anti-CD19 Humanized CAR-Engineered Allogeneic Double Negative T Cells in Adults with B-Cell Non-Hodgkin's Lymphoma

Introduction: Autologous CAR-T cell therapies has been shown to benefit patients with relapsed/refractory B-cell non-Hodgkin's lymphoma (B-NHL). However, the intrinsic characteristics of autologous CAR-T cell products such as preparation failure, high cost, long production cycle, and bridging therapy required for some patients make it less accessible for a wide range of patients. RJMty19 is a first-in-class humanized anti-CD19 CAR-engineered allogeneic double negative T cells (DNTs). RJMty19 exhibits the advantages of both adaptive and natural immunity and has a high proportion of stem cell-like memory T cells (Tscm). Furthermore, RJMty19 fulfills the requirements of an off-the-shelf cellular immunotherapy, including not causing graft-versus-host disease (GvHD), resistance to host-versus-graft (HvG) rejection, scalability, and storability (WYZE 2022). Preclinical studies had shown that CD19-CAR-DNT has a better safety profile compared to conventional CD19-CAR-T (Zhang 2022). Hence, the first-in-human clinical evaluation of RJMty19 is being undertaken in this Phase 1 study in patients with B-NHL. Methods: This is a first-in-class, open-label, single-dose, phase 1 study of CD19-CAR-DNT cells. The study is designed to evaluate five dose levels (DL) of RJMty19 in a 3+3 dose-escalation scheme: 1×10 6, 3×10 6, 9×10 6, 2×10 7 and 3×10 7 viable CAR + DNT cells per kilogram of body weight. Eligibility criteria included the presence of measurable lesions, at least 2 lines of prior therapy, ECOG score of 0 to 1. All patients received lymphodepleting chemotherapy with fludarabine and cyclophosphamide. The primary endpoint is dose-limiting toxicities, incidence of adverse events and clinically significant laboratory abnormalities. Secondary endpoints include evaluation of standard cellular PK parameters, PD, immunogenicity, objective response rates (ORR) and disease control rate (DCR) per Lugano 2014 Criteria. This phase 1 study is registered on clinicaltrials.gov (NCT05453669). Results ： As of July 31 2023, 15 patients with B-NHL were enrolled and 12 patients were evaluable (including one subject finished infusion for just 10 days). Of the 11 patients who had completed DLT evaluations at Day 28, five (45.5%) were male, and the average age was 59.7 years (range 45-74). At baseline, 73% of patients with IPI score ≥ 3 (range 0-4); the median volume of target lesion was 169,000 (4520-513,688) mm 3. The median number of prior therapies was three (range2-9). Furthermore, five patients had prior BTKi therapy and four patients had prior lenalidomide/venetoclax therapy before recruiting into the trial. No patients had been treated with autologous CD19-CART products. Following single RJMty19 infusion, there was no cases of GvHD, ICANS and SAEs. Only one patient developed a grade 2 CRS which was resolved within 24 hours. Except for fever, all of ≥grade 3 adverse events were hematologic disorders including leukopenia, lymphocytopenia, neutropenia, reduced platelet count and anemia (Table 1). Among patients who received ≥ 9×10 6 CAR-DNT cells/kg (DL ≥3; N=5), best DCR and ORR were 100% (5/5) and 40% (2/5) respectively at Day 28 with B-ultrasound imaging. One of patients in DL4 achieved 87% reduction of tumor lesions (SPD) at Day 31 and Day 61. CD19-CAR-DNT cell kinetics improved in a dose-dependent manner with peak cell expansion occurring between Day 5 and 7 at DL4 based on flow cytometry. Additionally, the mean of C max in DL4 is 384 CAR + DNT cells per microliter (μl) blood (N=3), and the highest peak value of 936 CAR + DNT cells/μl was detected in the recent enrolled patient.

Mass cytometry immune profiling to detect signatures of clinical activity of bintrafusp alfa (BA) in patients with advanced non-small cell lung cancer (NSCLC).

e21016 Background: BA, a bifunctional fusion protein targeting programed death-ligand 1 and transforming growth factor-β, is clinically active in a subset of patients (pts) with advanced NSCLC. In this study, mass cytometry was used to identify immune features associated with response and survival in pts with NSCLC treated with BA. Methods: Peripheral blood mononuclear cells were collected at baseline and after 1 and 3 doses of BA from 12 NSCLC pts treated every 2 weeks in a dose expansion biomarker cohort of a phase I clinical trial (NCT02517398). Samples were analyzed using a 44-marker mass cytometry panel designed to detect immune cell subsets and functional states of the innate and adaptive immune system. Manually gated immune cell subpopulations and unsupervised clusters were analyzed with Kruskal Wallis and Wilcoxon Rank Sum testing for multigroup comparison; and with linear correlation, univariate Cox regression and Kaplan Meier methods for progression free survival (PFS) analysis. Results: Among 12 pts, 3 had an objective response (OR), 6 had stable disease (SD) and 3 developed progressive disease (PD). Immune cell subsets associated with clinical activity by multigroup analyses included memory B cells, CD16+ natural killer (NK) cells, and double negative T cells (DNT). Pts with OR had a 2-fold higher frequency of memory B cells at baseline compared to pts who developed SD or PD. After 1 dose of BA, pts who subsequently developed an OR or SD had a 5-fold increase of CD16+ NK cells compared to pts with PD. After 3 doses of BA, pts who subsequently developed an OR had 4-fold more memory B cells and 3-fold more T-bet+ DNTs compared to pts with PD or SD. Immune cell subpopulations associated with survival included 3 unsupervised clusters annotated by the clustering algorithm as CD16+ NK cells, DNTs and classical monocytes. Fewer CD16+ NK cells at baseline were associated with longer PFS (p = 0.0053). Longer PFS was also associated with higher CD161+ CD127+ DNT cells after 1 dose of BA relative to baseline (p = 0.029), while an expansion of IgG4+ PD-1+ classical monocytes after 3 doses of BA was associated with shorter PFS (p = 0.015). Linear correlation analysis did not find strong associations between any one immune state and survival. Conclusions: Our study demonstrates the ability of mass cytometry immune profiling to detect potential immunological markers of response and survival in NSCLC pts treated with BA. The association between CD16+ NK cell frequencies and clinical benefit could reflect the ability of BA to facilitate antibody-dependent cellular cytotoxicity by NK cells in NSCLC. A high fraction of memory B cells at baseline and after 3 doses identifies pts likely to respond to BA. If validated in larger studies, these novel immune signatures can be used to improve clinical outcomes in pts with NSCLC considering immunotherapy.

ALPS Diagnosis: Different Phenotype in Child and Mother

Autoimmune lymphoproliferative syndrome (ALPS) is a group of genetic disorders characterized by early-onset lymphoproliferation, autoimmune cytopenias, and susceptibility to malignancy. We present two cases: mother and son with different phenotype at onset.A 7-year-old child presented with anemia, thrombocytopenia and splenomegaly. In his family history: born from non-consanguineous parents, his mother had a splenectomy due to hemolytic anemia of unknown cause. In the child, initial differential diagnosis ruled out infection or malignancy. Bone marrow aspirate had erythroid hyperplasia, giant metamyelocytes and bands and low thrombopoyesis. His immunological evaluation had hypergammaglobulinemia, elevated TCRαβ+-DNT cells (10%). ALPS was suspected and he started Prednisolone and Mycophenolate.A mutation in FAS gene was identified (exon 9, c.785T > A, p.Ile262Asn). His mother, a 45-year-old female presented with a history of lymphadenopathy. In her past medical history, she had hemolytic anemia and splenomegaly, a splenectomy was performed and the anemia resolved. Initial evaluation was started because breast cancer was suspected. Lymph node biopsy was performed where malignancy was ruled out, but unspecific inflammatory infiltrate was found. An immunological evaluation was performed. The first that was identified was the history of his son with ALPS (not specified by any other physician). Due to her history, the lymphoproliferation and anemia Sanger sequencing of her sońs mutation was performed. It revealed the same mutation and treatment was started. FAS gene with 85% frequency is the main etiological cause of genetically diagnosed patients with ALPS phenotype. The factors that determine the penetrance of clinical ALPS have not been entirely determined. Penetrance appears to be relatively associated with the location and type of the mutation and even varies in individuals carrying the same genetic mutation.

Frequency and functional profile of circulating TCRαβ+ double negative T cells in HIV/TB co-infection

BackgroundIncreased frequency of circulating double negative T (DNT, CD4−CD8−CD3+) cells with protective immune function has been observed in human immunodeficiency virus (HIV) infection and tuberculosis (TB). Here the role of circulating TCRαβ+ DNT cells was further investigated in HIV/TB co-infection.MethodsA cross-sectional study was conducted to investigate the frequency and functional profiles of peripheral TCRαβ+ DNT cells including apoptosis, chemokine and cytokine expression among healthy individuals and patients with TB, HIV infection and HIV/TB co-infection by cell surface staining and intracellular cytokine staining combined with flow cytometry.ResultsSignificantly increased frequency of TCRαβ+ DNT cells was observed in HIV/TB co-infection than that in TB (p < 0.001), HIV infection (p = 0.039) and healthy controls (p < 0.001). Compared with TB, HIV/TB co-infection had higher frequency of Fas expression (p = 0.007) and lower frequency of Annexin V expression on TCRαβ+ DNT cells (p = 0.049), and the frequency of Annexin V expression on Fas+TCRαβ+ DNT cells had no significant difference. TCRαβ+ DNT cells expressed less CCR5 in HIV/TB co-infection than that in TB (p = 0.014), and more CXCR4 in HIV/TB co-infection than that in HIV infection (p = 0.043). Compared with healthy controls, TB and HIV/TB co-infection had higher frequency of TCRαβ+ DNT cells secreting Granzyme A (p = 0.046; p = 0.005). In TB and HIV/TB co-infection, TCRαβ+ DNT cells secreted more granzyme A (p = 0.002; p = 0.002) and perforin (p < 0.001; p = 0.017) than CD4+ T cells but similar to CD8+ T cells.ConclusionsReduced apoptosis may take part in the mechanism of increased frequency of peripheral TCRαβ+ DNT cells in HIV/TB co-infection. TCRαβ+ DNT cells may play a cytotoxic T cells-like function in HIV/TB co-infection.

Elevated Foxp3+ double-negative T cells are associated with disease progression during HIV infection.

Persistent immune activation, which occurs during the whole course of HIV infection, plays a pivotal role in CD4+ T cells depletion and AIDS progression. Furthermore, immune activation is a key factor that leads to impaired immune reconstitution after long-term effective antiretroviral therapy (ART), and is even responsible for the increased risk of developing non-AIDS co-morbidities. Therefore, it’s imperative to identify an effective intervention targeting HIV-associated immune activation to improve disease management. Double negative T cells (DNT) were reported to provide immunosuppression during HIV infection, but the related mechanisms remained puzzled. Foxp3 endows Tregs with potent suppressive function to maintain immune homeostasis. However, whether DNT cells expressed Foxp3 and the accurate function of these cells urgently needed to be investigated. Here, we found that Foxp3+ DNT cells accumulated in untreated people living with HIV (PLWH) with CD4+ T cell count less than 200 cells/µl. Moreover, the frequency of Foxp3+ DNT cells was negatively correlated with CD4+ T cell count and CD4/CD8 ratio, and positively correlated with immune activation and systemic inflammation in PLWH. Of note, Foxp3+ DNT cells might exert suppressive regulation by increased expression of CD39, CD25, or vigorous proliferation (high levels of GITR and ki67) in ART-naive PLWH. Our study underlined the importance of Foxp3+ DNT cells in the HIV disease progression, and suggest that Foxp3+ DNT may be a potential target for clinical intervention for the control of immune activation during HIV infection.

Targeting breast cancer with a combination of DNT and LAG3 checkpoint blockage and its mechanism.

IntroductionThe characteristics of the tumor immune microenvironment (TIME) are closely related to immunotherapy. Breast cancer can benefit from immunotherapy, and its TIME is still unclear.MethodsWe utilized mass cytometry to explore the immune cell heterogeneity in breast cancer. Double‐negative T cells (DNTs) from healthy volunteers (HBs) were enriched in vitro. Flow cytometry was used to detect the cell surface receptors of cancer cells and DNT cells. The correlation between immune checkpoints and the abundance of immune cells or prognosis of breast cancer was analyzed by the TCGA database. The messenger RNA (mRNA) expression of functional genes was performed by quantitative real‐time PCR.ResultsWe found that the frequencies of Granzyme B (GZMB)+CD8+T and GZMB+DNT cells in cancer tissues (CA) of breast cancer were lower than those in blood samples of patients (PB), and the frequencies of programmed cell death protein 1 (PD1)+CD8+T and PD1+DNT cells in CA were higher than those in PB. DNTs from HBs had a cytotoxic effect on MDA‐MB‐231. LAG3Ab could upregulate the mRNA expression of interferon gamma and perforin by increasing T‐BET transcription to enhance the cytotoxicity of DNT cells in vitro.ConclusionOur study revealed the suppressive status of TIME in breast cancer and supported DNT cells had the potential to be applied as a novel adoptive cell therapy for TNBC either alone or combined with LAG3Ab.

Monogenic Disorder Associated with Autoimmune Lymphoproliferative Syndrome -Like Phenotype: A Systematic Review

Background: Autoimmune lymphoproliferative syndrome (ALPS) is a congenital disorder that results in an apoptosis impairment of lymphocytes, leading to chronic lymphoproliferation and autoimmunity, mainly autoimmune cytopenia. Some of autoreactive T cells cannot become apoptosis in activation-induced cell death (AICD) pathway; therefore, they have accumulation of autoreactive TCRαβ+CD4−CD8− doublenegative T (αβ- DNT) cells, leading to cytopenia, splenomegaly, lymphadenopathy, autoimmune disorders and a greatly increased lifetime risk of lymphoma. FAS, FASL, CASP8 and CASP10 gene defects are often responsible for the disease, the phenotype of which can vary from asymptomatic/mild forms to severe disease. More rarely, defects are associated to other genes involved in ALPS-like phenotype.&#x0D; Methods: A systematic literature search was performed in Web of Science, PubMed and Scopus from the earliest available date to march 2021 with standard keywords to find patients with ALPS-like phenotypes. Demographic, clinical, immunological and molecular data were extracted.&#x0D; Results: In this systematic review we reported 61 patients with genetically determined ALPS-like. Most of ALPS-like cases carry mutations in the STAT3 (n=15), LRBA (n=11) and CARD11 (n=8) genes. The most common presentation was splenomegaly and lymphadenopathy followed by hepatomegaly. The most common autoimmunity was autoimmune hemolytic anemia and immune thrombocytopenic purpura followed by auto immune neutropenia. Elevated serum immunoglobulin was reported especially in IgG, IgM and IgA.&#x0D; Conclusion: In the present study, 61 patients with genetically determined ALPS-like were examined. Our results showed that most of ALPS-like cases carry mutations in the STAT3. We reported that the most common presentations were splenomegaly and lymphadenopathy. Elevated serum immunoglobulin, IL-10, vitamin B12 and increased proportion of DNT cells were reported.

High-Dimensional Immune Profiling by Mass Cytometry Revealed the Circulating Immune Cell Landscape in Patients With Intracranial Aneurysm.

BackgroundIncreasing evidence supports a critical role of chronic inflammation in intracranial aneurysm (IA). Understanding how the immunological alterations in IA provides opportunities for targeted treatment. However, there is a lack of comprehensive and detailed characterization of the changes in circulating immune cells in IA.ObjectiveTo perform a comprehensive and detailed characterization of the changes in circulating immune cells in patients with IA.MethodsPeripheral blood mononuclear cell samples from IA patients (n = 26) and age-and sex-matched healthy controls (HCs, n = 20) were analyzed using high dimensional mass cytometry, and the frequency and phenotype of immune cell subtypes were assessed.ResultsWe identified 28 cell clusters and found that the immune signature of IA consists of cluster changes. IA patients exhibited dysfunction of immunity, with dysregulation of CD4+ T-cell clusters, increased B cells and monocytes, and decreased CD8+ T cells, DNT cells, and DPT cells. Moreover, compared with findings in HC, IA was associated with enhanced lymphocyte and monocyte immune activation, with a higher expression of HLA-DR, CXCR3, and CX3CR1. In addition, the expression of TLR4, p-STAT3, and the exhaustion marker PD1 was increased in T cells, B cells, and NK cells in IA patients.ConclusionsOur data provide an overview of the circulating immune cell landscape of IA patients, and reveal that the dysfunction of circulating immunity may play a potential role in the development of IA.

Abstract 5510: Preclinical study of allogeneic CD19-CAR-DNT cells as an off-the-shelf immunotherapy drug for NHL

Abstract Introduction Allogeneic Double Negative T (DNT, CD3+CD4-CD8-) cells have shown great potential as a novel off-the-shelf immunotherapy drug candidate in clinic trials for r/r/AML patients. This is our first report to demonstrate that CD19-CAR-DNT cells with high percentage of Tscm+Tcm phenotypes have been successfully developed with strong immunotherapeutic effects on Raji-luciferase tumor cells from non-Hodgkin’s lymphoma both in vitro and in vivo. Methods DNT cells were expanded ex vivo from healthy donors and then were transduced with CD19-CAR lentivirus in the presence of IL-2. The CD19-CAR-DNT cell expansions were performed under GMP condition and then harvested and cryo-preserved by CryStor®CS5 in liquid nitrogen for future use. Summary The ex vivo expanded CD19-CAR-DNT cells has more than 50% of Tscm and Tcm phenotypes. The viability and CAR+ percentage of CD19-CAR-DNT cells were 84.40% and 34.98% respectively. The killing of CD19-CAR-DNT cells on Raji-luciferase cells was 77.92% (n=6) at the effector to target ratio of 4:1. Elevated IFN-γ release from CD19-CAR-DNT cells after overnight co-culture with Raji cells was observed. The antitumor efficacy was evaluated in the xenografted NHL mice model. After the first infusion of the cells, the tumor growth inhibitory rates (TGI %) of CD19-CAR-DNT cells at 1×106, 5×106 and 1×107 per mouse on day14 were 80.54%, 98.31% and 99.05% respectively, the killing effects were in a dose-dependent manner. After the second infusion of the CD19-CAR-DNT cells at day 14, median survival times (MST) of the treated three groups mice were elongated till Day60, that is twice longer than that of the xenografted vehicle group. In the subsequent PKPD study, infusion CD19-CAR-DNT cells into Raji-luciferase cell xenografted mice were primarily distributed in lungs, peripheral blood, spleen, and bone marrow within 72h, homing back to liver and spleen on Day36. All animal body weights in both fresh and frozen reconstituted CD19-CAR-DNT cells treated mice were increased with no observed toxicities. Conclusions ex vivo expansion of CD19-CAR-DNT cells were successfully developed to explore its potential as a novel off-the-shelf universal CART drug for malignant B cells leukemia. The cell subsets consist of more than 50% of Tscm and Tcm cells whereas most immunological check point inhibitors were not elevated after priming by target tumor cells except a slight increase of PD-1 molecule. Fresh or frozen CD19-CAR-DNT cells can kill Raji cells in vitro and in vivo studies in a dose-dependent manner although the frozen one showed a delayed killing in vivo. The tumor cells were almost depleted and the MST was significantly increased with NOAEL at 1x107 cells/mouse. The amounts of CD19-CAR-DNT cells are re-distributed from early lungs, peripheral blood to live, spleen later. Citation Format: Liming Yang, Dan Wang, Shangzhi Xu, Liuyang Wang, Jianjun Tong, Honglin Zhu, Man Xu, Xiancai Li, Letu Ji, Chuntao Zheng, Zhiqiang Xiang, Qinghua Sun, Hengcai Wang. Preclinical study of allogeneic CD19-CAR-DNT cells as an off-the-shelf immunotherapy drug for NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5510.

Thyroid Cancer Screening Using Tumor-Associated DN T Cells as Immunogenomic Markers.

BackgroundThyroid nodules are an extremely common entity, and surgery is considered the ultimate diagnostic strategy in those with unclear malignant potential. Unfortunately, strategies aiming to predict the risk of malignancy have inadequate specificity. Our group recently found that the microenvironment of thyroid cancer is characterized by an enhanced immune invasion and activated immune response mediated by double-negative T lymphocytes (DN T) (CD3+CD4-CD8-), which are believed to enable or promote tumorigenesis. In the present work, we try to use the DN T cells’ proportion in thyroid fine-needle aspiration (FNA) material as a predictor of the risk of malignancy.MethodsWe recruited 127 patients and obtained ultrasound-guided FNA samples from subjects with cytology-positive or suspicious for malignancy and from those with benign nodular goiter associated with compressive symptoms (such as dysphagia, shortness of breath, or hoarseness), Hashimoto thyroiditis, and Graves’ disease. Out of 127, we investigated 46 FNA samples of patients who underwent total thyroidectomy and for which postoperative histological diagnosis by the academic pathologists was available. We specifically measured the number of cells expressing CD3+CD4-CD8- (DN T) as a function of total CD3+ cells in FNA samples using flow cytometry. We correlated their FNA DN T-cell proportions with the pathological findings.ResultsThe DN T cells were significantly more abundant in lymphocytic infiltrates of thyroid cancer cases compared to benign nodule controls (p < 0.0001). When the DN T-cell population exceeded a threshold of 9.14%, of total CD3+ cells, the negative likelihood ratio of being cancer-free was 0.034 (96.6% sensitivity, 95% CI, 0.915–1.000, p < 0.0001). DN T cells at <9.14% were not found in any subject with benign disease (specificity 100%). The high specificity of the test is promising, since it abolishes a false-positive diagnosis and in turn unnecessary surgical procedures.ConclusionThe present study proposes DN T cells’ proportion as a preoperative diagnostic signature for thyroid cancer that with integration of RNA transcriptomics can provide a simplified technology based on the PCR assay for the ease of operation.

Decreased CD73+ Double-Negative T Cells and Elevated Level of Soluble CD73 Correlated With and Predicted Poor Immune Reconstitution in HIV-Infected Patients After Antiretroviral Therapy.

Although extensive use of antiretroviral therapy (ART) has made great progress in controlling HIV replication and improving CD4+ T cell recovery, the immune reconstitution remained insufficient in some patients, who were defined as poor immunological responders (PIRs). These PIRs were at a high risk of AIDS-related and non-AIDS complications, resulting in higher morbidity and mortality rate. Thus, it is a major challenge and urgently needed to distinguish PIRs early and improve their immune function in time. Immune activation is a key factor that leads to impaired immune reconstitution in people living with HIV (PLWH) who are receiving effective ART. Double negative T cells (DNT) were reported to associate with the control of immune activation during HIV infection. However, the precise mechanisms by which DNT cells exerted their suppressive capacity during HIV infection remained puzzled. CD73, both a soluble and a membrane-bound form, display immunosuppressive effects through producing adenosine (ADO). Thus, whether DNT cells expressed CD73 and mediated immune suppression through CD73-ADO pathway needs to be investigated. Here, we found a significant downregulation of CD73 expression on DNT cells in treatment-naïve PLWH (TNs) compared to healthy controls, accompanied with increased concentration of sCD73 in plasma. Both the frequency of CD73+ DNT cells and the level of plasma sCD73 recovered after ART treatment. However, PIRs showed decreased percentage of CD73+ DNT cells compared to immunological responders (IRs). The frequency of CD73+ DNT cells was positively correlated with CD4+ T cell count and CD4/CD8 ratio, and negatively correlated with immune activation in PLWH. The level of sCD73 also showed a negative correlation to CD4+ T cell count and CD4/CD8 ratio. More importantly, in the present cohort, a higher level of sCD73 at the time of initiating ART could predict poor immune reconstitution in PLWH after long-term ART. Our findings highlighted the importance of CD73+ DNT cells and sCD73 in the disease progression and immune reconstitution of PLWH, and provided evidences for sCD73 as a potential biomarker of predicting immune recovery.

Correlation between Peripheral T Cell Subsets and the Activity of Thyroid-Associated Ophthalmopathy.

Introduction Thyroid-associated ophthalmopathy (TAO) is the most common orbital immunological disease in adults. T cells play an important role in the pathogenesis of TAO. However, our knowledge of the circulating T cell subsets in TAO is limited. Objective To investigate the circulating T cell subsets in TAO and the correlations between them and the activity of TAO. Methods Thirty-eight TAO patients (19 active and 19 nonactive) were enrolled. The absolute number and percentage of total lymphocytes, CD3+T cells, CD4+T cells, CD8+ T cells, CD3+CD4-CD8-T cells (DNT cells), and CD3+CD4+CD8+ T cells (DPT cells) in peripheral blood were measured by flow cytometer. Results TAO patients were divided into the active group and the nonactive group by the clinical activity score (CAS). The mean CAS was 4 ± 1.11 in the active group and 1.47 ± 0.61 in the nonactive group. No statistical differences were found in gender, age, and the levels of FT3, FT4, TSH, and TRAb between the two groups. The percentage of DNT cells was lower in the active group than in the non-active group, and it was negatively correlated with CAS (r = −0.349, P=0.032), but not the absolute number. The CD4/CD8 ratio, the absolute number and percentage of CD3+ T cells, CD4+ T cells, CD8+ T cells, and DPT cells did not differ between the two groups. ConclusionIn the present study, we found the percentage of DNT cells was significantly lesser in the active TAO than in the nonactive TAO, and it was negatively correlated with the activity of the TAO. It suggests that DNT cells may involve in the immunopathogenesis of TAO and can serve as a clinical biomarker of the disease activity.

Differential blood leukocyte populations based on individual variances and age.

Blood was collected from the New York State Department of Health (NYSDOH) employees to assess variances in leukocyte numbers in January, May, and September throughout a year and over many years. Women and men of ages 20 to 80 volunteered to donate for this program. Most of the blood came from healthy individuals, and many remained healthy throughout the years of their blood donations. The major objective was to determine the extent that blood leukocyte numbers change so that transient vs more lingering changes may be helpful in assessing health status. Since some donors remained in the program for 14 years, age influences over time could be determined. Within a short period of 2–3 years, the flow cytometric immunophenotypic profile of blood lymphocyte is relatively stable with a CV% of < 20%. However, as humans age, the blood CD3+ T cell, CD8+ T cell, B cell, NKT cell, and CD4−/CD8− double-negative T cell (DN-T cell) subsets declined in cell numbers/μL, but the double-positive CD4+/CD8+ T cells (DP-T cells) increased in numbers. The extent and chronology of a variance, e.g., a subset exceeding its 75th or 90th percentile, might be indicative of a transient or chronic physiological or psychosocial stress affecting health or a developing pathology; however, because of the wide ranges of cell numbers/μL for each subset among individuals reported as healthy, everyone’s immunity and health must be carefully evaluated. A CD4 to CD8 ratio (4/8R) of < 1 has been used to define an immunodeficiency such as HIV-induced AIDS, but a high 4/8R is less well associated with health status. A high 4/8R or granulocyte to lymphocyte ratio (GLR) might be an indicator of a stress, infection, or immune-related pathology. Sporadic and longitudinal increases of GLRs are reported. The results suggest that there are some age and sex differences in leukocyte numbers; stress influences on the blood profile of leukocytes likely exist. However, some values exceeding 2 standard deviations from means do not necessarily predict a health concern, whereas a longitudinal increase or decline might be indicative of a need for further evaluations.

Predictors for symptomatic intracranial hemorrhage after intravenous thrombolysis with acute ischemic stroke within 6\u2009h in northern China: a multicenter, retrospective study

Background and purposeThis study assessed the predictive factors for symptomatic intracranial hemorrhage (sICH) in patients with acute ischemic stroke (AIS) after receiving intravenous thrombolysis (IVT) within 6 h in northern China.MethodsWe retrospectively analyzed ischemic stroke patients who were treated with IVT between November 2016 and December 2018 in 19 hospitals in Shandong Province, China. Potential predictors of sICH were investigated using univariate and multivariate analyses.ResultsOf the 1293 enrolled patients (845 men, aged 62 ± 11 years), 33 (2.6%) developed sICH. The patients with sICH had increased coronary heart disease (36.4% vs. 13.7%, P = 0.001), more severe stroke (mean National Institutes of Health Stroke Scale [NIHSS] score on admission of 14 vs.7, P < 0.001), longer door-to-needle time [DNT] (66 min vs. 50 min, P < 0.001), higher blood glucose on admission, higher white blood cell counts (9000/mm3 vs. 7950/mm3, P = 0.004) and higher neutrophils ratios (73.4% vs. 67.2%, P = 0.006) et al. According to the results of multivariate analysis, the frequency of sICH was independently associated with the NIHSS score (OR = 3.38; 95%CI [1.50–7.63]; P = 0.003), DNT (OR = 4.52; 95%CI [1.69–12.12]; P = 0.003), and white blood cell count (OR = 3.59; 95%CI [1.50–8.61]; P = 0.004).When these three predictive factors were aggregated, compared with participants without any factors, the multi-adjusted odds ratios (95% confidence intervals) of sICH for persons concurrently having one, two or three of these factors were 2.28 (0.25–20.74), 15.37 (1.96–120.90) and 29.05 (3.13–270.11), respectively (P for linear trend < 0.001), compared with participants without any factors.ConclusionNIHSS scores higher than 10 on admission, a DNT > 50 min, and a white blood cell count ≥9000/mm3 were independent risk factors for sICH in Chinese patients within 6 h after IVT for AIS.