Novel Compound Heterozygous Variants in the FAS Gene Lead to Fetal Onset of Autoimmune Lymphoproliferative Syndrome (ALPS).

OBJECTIVE: FAS gene defects lead to autoimmune lymphoproliferative syndrome (ALPS), which is often inherited in an autosomal dominant and rarely in an autosomal recessive manner. We report a case of a newborn girl with novel compound heterozygous variants in FAS and reveal the underlying mechanism. METHODS: Whole-exome sequencing (WES) was used to identify pathogenic variants. Multiparametric flow cytometry analysis, phosflow analysis, and FAS-induced apoptosis assays were used to explore the effects of the variants on FAS expression, apoptosis, and immunophenotype. The HEK293T cells were used to assess the impact of the variants on protein expression and FAS-induced apoptosis. RESULTS: The patient was born with hepatosplenomegaly, anemia, and thrombocytopenia. She also experienced COVID-19, rotavirus infection, herpes simplex virus infection, and severe pneumonia. The proportion of double-negative T cells (DNTs) was significantly elevated. Novel FAS compound heterozygous variants c.310T > A (p.C104S) and c.702_704del (p.T235del) were identified. The apoptotic ability of T cells was defective, and FAS expression on the surface of T cells was deficient. The T235del variant decreased FAS expression, and the C104S protein remained in the endoplasmic reticulum (ER) and could not translocate to the cell surface. Both mutations resulted in loss-of-function in terms of FAS-induced apoptosis in HEK293T cells. The DNTs were mainly terminally differentiated T (TEMRA) and CD45RA+HLA-DR+, with high expression of CD85j, PD-1, and CD57. The percentage of Th1, Tfh, and autoreactive B cells were significantly increased in the patient. The abnormal immunophenotyping was partially attenuated by sirolimus treatment. CONCLUSIONS: We identified two variants that significantly affect FAS expression or localization, leading to early disease onset of in the fetus. Abnormalities in the mTOR pathway are associated with a favorable response to sirolimus.

Double-negative T cells with a distinct transcriptomic profile are abundant in the peripheral blood of patients with breast cancer.

BACKGROUND: Double-negative T (DNT) cells comprise a distinct subset of T lymphocytes that have been implicated in immune responses. The aim of this study was to characterize the peripheral DNT population in breast cancer (BC) patients. METHODS: DNT cells were isolated from the peripheral blood samples of BC patients and healthy controls by flow cytometry. The sorted DNT cells were analyzed by the Smart-seq2 for single-cell full-length transcriptome profiling. The differentially expressed genes (DEGs) between the BC and control groups were screened and functionally annotated by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using R. The protein-protein interaction (PPI) network of the DEGs was constructed using the CytoHubba and MCODE plug-in of Cytoscape software to identify the core genes. Survival status, DNA methylation level, immune infiltration and immune checkpoint expression were analyzed using Kaplan-Meier Plotter, UALCAN, MethSeuvr, TIMER, and TISIDB respectively. The sequencing results were verified by RT-qPCR. RESULT: The percentage of DNT cells was higher in the BC patients compared to healthy controls. We identified 289 DEGs between the DNT populations of both groups. GO and KEGG pathway analyses revealed that the DEGs were mainly related to immunoglobulin mediated immune response, complement activation, and B cell receptor signaling. The PPI networks of the common DEGs were constructed using Cytoscape, and 10 core genes were identified, including TMEM176B, C1QB, C1QC, RASD2, and IFIT3. The expression levels of these genes correlated with the prognosis and immune infiltration in BC patients, and were validated by RT-qPCR (P < 0.05). CONCLUSIONS: DNT cells are abundant in patients with BC, and might exert anti-tumor immune responses by regulating genes such as TMEM176B and EGR1.

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.

Diagnostic and prognostic value of double-negative T cells in colorectal cancer.

Objective: To evaluate the T-lymphocyte subset distribution and the diagnostic and prognosis value of double-negative T (DNT) cells in colorectal cancer (CRC). Methods: This retrospective study compared the T-lymphocyte subsets and DNT of 114 patients with CRC with those of 107 healthy controls (HC). The diagnostic potential of DNT and T-lymphocyte subsets was assessed using the receiver operating characteristic (ROC) curve, and prognostic values were evaluated using the Kaplan-Meier curve and the Cox regression model. Results: The percentages of CD8+ T cells and DNT cells, and value of carcinoembryonic antigen (CEA), were remarkably higher in patients with CRC than in those with HC, but the ratio of CD4+/CD8+ was decreased. Using ROC curve analysis, DNT cell percentage, CEA, and CD4+/CD8+ ratio all had good diagnostic efficacy, with areas under the curve (AUCs) of 0.865, 0.786 and 0.624, respectively. The combination of DNT cell percentage and CEA had an AUC of 0.905, which was significantly higher than that of any single biomarker (p < 0.05). In univariate analysis, the Tumor Node Metastasis (TNM) clinical stage, CD4+/CD8+ ratio, and DNT cell percentage were significantly associated with overall survival (OS) (p < 0.05). In multivariate analysis, TNM clinical staging (HR = 2.37, 95 % CI: 1.15-4.90), a decreased CD4+/CD8+ ratio (HR = 0.33, 95 % CI: 0.15-0.74), and an increased DNT cell percentage (HR = 2.29, 95 % CI: 1.11-4.73) were independent prognostic factors for CRC. Conclusion: The percentage of DNT cells may be useful as an evaluation index for CRC diagnosis and prognosis, which was even better when combined with serum CEA.

Distinct characteristics of unique immunoregulatory canine non-conventional TCRαßpos CD4negCD8αneg double-negative T cell subpopulations.

Conventional CD4pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αßpos CD4negCD8αneg double-negative (dn) T cells of dogs, a novel FoxP3pos Treg-like subset was described that, similar to conventional CD4pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαßpos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25pos (enriched for FoxP3pos Treg-like cells) and the dnCD25neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4posCD25pos Treg cells (positive control) and CD4posCD25neg T cells (negative control). Canine dnCD25pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4posCD25pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4posCD25pos populations, even dnCD25neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25neg T cells have a FoxP3neg phenotype comparable to non-suppressive CD4posCD25neg T cells. Both, CD25pos and CD25neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4posCD25neg T cells to secrete inhibitory factors in contrast to CD4posCD25pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25pos and CD4posCD25pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25neg T cells. Taken together, unique canine non-conventional dnCD25pos FoxP3pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo.

Characterization of double-negative T cells in colorectal cancers and their corresponding lymph nodes.

TCRαß+ CD4- CD8- double-negative T (DNT) cells are minor populations in peripheral blood, and their roles have mostly been discussed in inflammation and autoimmunity. However, the functions of DNT cells in tumor microenvironment remain to be elucidated. We investigated their characteristics, possible origins and functions in colorectal cancer tissues as well as their corresponding tumor-draining lymph nodes. We found a significant enrichment of DNT cells in tumor tissues compared with their corresponding lymph nodes, especially in tumors with lower T cell infiltration. T cell receptor (TCR) sequence analysis of CD4+ T, CD8+ T and DNT cells indicated that TCR sequences detected in DNT cells were found in CD8+ T cells, but rarely in CD4+ T cells, suggesting that a part of DNT cells was likely to be originated from CD8+ T cells. Through a single-cell transcriptomic analysis of DNT cells, we found that a DNT cell cluster, which showed similar phenotypes to central memory CD8+ T cells with low expression of effector and exhaustion markers, revealed some specific gene expression patterns, including higher GZMK expression. Moreover, in flow cytometry analysis, we found that DNT cells lost production of cytotoxic mediators. These findings imply that DNT cells might function as negative regulators of anti-tumor immune responses in tumor microenvironment.

DNT cells mediate resistance to CAR-T cells therapy in a pediatric patient with relapsed and refractory B-ALL.

Chimeric antigen receptor T (CAR-T) cells therapy is a milestone achievement in the immunotherapy of relapsed and refractory (R/R) B cell acute lymphoblastic leukemia (B-ALL). However, some patients treated with CAR-T cells do not achieve complete remission, the mechanisms of which have not been elucidated. In the present study, we report a 9-year-old pediatric patient with refractory B-ALL received a triple infusion of autologous CD19 CAR-T cells therapy after the second relapse. CAR-T cells expanded in the peripheral blood and bone marrow. However, the patient did not achieve complete remission, indicating a lack of response to CAR-T cells therapy. Analysis of etiological factors revealed that the number of CD4 and CD8 double-negative T (DNT) cells was significantly upregulated in the peripheral blood, bone marrow, and autologous CAR-T cells products. In conclusiont, these findings indicate that DNT cells mediated resistance to CAR-T cells therapy in this pediatric patient with R/R B-ALL.

Double-negative T cells ameliorate psoriasis by selectively inhibiting IL-17A-producing γδlow T cells.

BACKGROUND: Psoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism. METHODS: We conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated. RESULTS: DNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5-/- DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions. CONCLUSIONS: DNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic.

CD19-CAR-DNT cells (RJMty19) in patients with relapsed or refractory large B-cell lymphoma: a phase 1, first-in-human study.

Background: Current approved chimeric antigen receptor (CAR) T-cell products are autologous cell therapies that are costly and poorly accessible to patients. We aimed to evaluate the safety and antitumor activity of a novel off-the-shelf anti-CD19 CAR-engineered allogeneic double-negative T cells (RJMty19) in patients with relapsed/refractory large B-cell lymphoma. We report the results from a first-in-human, open-label, single-dose, phase 1 study of allogeneic CD19-specific CAR double-negative T (CAR-DNT) cells. Methods: Eligibility criteria included the presence of measurable lesions, at least 2 lines of prior immunochemotherapy, and an ECOG score of 0-1. We evaluated four dose levels (DL) of RJMty19 in a 3 + 3 dose-escalation scheme: 1 × 106, 3 × 106, 9 × 106 and 2 × 107 CAR-DNT cells per kilogram of body weight. All patients received lymphodepleting chemotherapy with fludarabine and cyclophosphamide. The primary endpoints were dose-limiting toxicities (DLTs), incidence of adverse events (AEs), and clinically significant laboratory abnormalities. Secondary endpoints included evaluation of standard cellular pharmacokinetic parameters, immunogenicity, objective response rates (ORR), and disease control rate (DCR) per Lugano 2014 criteria. Findings: A total of 12 patients were enrolled between 22 July 2022 and 27 July 2023. Among these patients, 66% were classified as stage IV, 75% had an IPI score of 3 or higher, representing an intermediate risk or worse. The maximum tolerated dose was not reached because no DLT was observed. Four patient experienced grade 1 or 2 cytokine release syndrome and dizziness. The most common AEs were hematologic toxicities, including neutropenia (N = 12, 100%), leukopenia (N = 12, 100%), lymphopenia (N = 10, 83%), thrombocytopenia (N = 6, 50%), febrile neutropenia (N = 3, 25%), and anemia (N = 3, 25%). Seven subjects died till the cut-off date, five of them died of disease progression and two of them died of COVID 19. In all patients (N = 12), the ORR was 25% and CRR was 8.3%. DL1 and DL2 patients benefited less from the therapy (ORR: 17%, N = 1; DCR: 33%, N = 2). However, all DL3 patients achieved disease control (N = 3, 100%), and all DL4 patients achieved objective response (N = 3, 100%). Interpretation: Our results demonstrate that CD19-CAR-DNT cells appear to be well tolerated with promising antitumor activity in LBCL patients. Further study of this product with a larger sample size is warranted. This phase 1 study is registered on clinicaltrials.gov (NCT05453669). Funding: Wyze Biotech. Co., Ltd.

Double-Negative T (DNT) Cells in Patients with Systemic Lupus Erythematosus.

Double-negative T (DNT) cells are a rare and unconventional T-lymphocyte subpopulation lacking both CD4 and CD8 markers. Their immunopathological roles and clinical relevance have yet to be elucidated. Beyond autoimmune lymphoproliferative syndrome (ALPS), these cells may also play a role in rheumatic disorders, including systemic lupus erythematosus (SLE); indeed, these two diseases share several autoimmune manifestations (including nephritis). Moreover, one of the main experimental murine models used to investigate lupus, namely the MRL/lpr mouse, is characterized by an expansion of DNT cells, which can support the production of pathogenic autoantibodies and/or modulate the immune response in this context. However, lupus murine models are not completely consistent with their human SLE counterpart, of course. In this mini review, we summarize and analyze the most relevant clinical studies investigating the DNT cell population in SLE patients. Overall, based on the present literature review and analysis, DNT cell homeostasis seems to be altered in patients with SLE. Indeed, most of the available clinical studies (which include both adults and children) reported an increased DNT cell percentage in SLE patients, especially during the active phases, even though no clear correlation with disease activity and/or inflammatory parameters has been clearly established. Well-designed, standardized, and longitudinal clinical studies focused on DNT cell population are needed, in order to further elucidate the actual contribution of these cells in SLE pathogenesis and their interactions with other immune cells (also implicated and/or altered in SLE, such as basophils), and clarify whether their expansion and/or immunophenotypic aspects may have any immunopathological relevance (and, then, represent potential disease markers and, in perspective, even therapeutic targets) or are just an unspecific epiphenomenon of autoimmunity.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Corrigendum: Double-negative T cells regulate hepatic stellate cell activation to promote liver fibrosis progression via NLRP3.

[This corrects the article DOI: 10.3389/fimmu.2022.857116.].

Understanding the Spectrum of Immune Dysregulation Manifestations in Autoimmune Lymphoproliferative Syndrome and Autoimmune Lymphoproliferative Syndrome-like Disorders.

As a disorder of immune dysregulation, autoimmune lymphoproliferative syndrome (ALPS) stems from pathogenic variants in the first apoptosis signal-mediated apoptosis (Fas) and Fas-ligand pathway that result in elevations of CD3+ TCRαß+ CD4- CD8- T cells along with chronic lymphoproliferation, a heightened risk for malignancy, and importantly for the rheumatologist, increased risk of autoimmunity. While immune cytopenias are the most encountered autoimmune phenomena, there is increasing appreciation for ocular, musculoskeletal, pulmonary and renal inflammatory manifestations similar to more common rheumatology diseases. Additionally, ALPS-like conditions that share similar clinical features and opportunities for targeted therapy are increasingly recognized via genetic testing, highlighting the need for rheumatologists to be facile in the recognition and diagnosis of this spectrum of disorders. This review will focus on clinical and laboratory features of both ALPS and ALPS-like disorders with the intent to provide a framework for rheumatologists to understand the pathophysiologic drivers and discriminate between diagnoses.

Autoimmune lymphoproliferative syndrome: A disorder of immune dysregulation.

Autoimmune Lymphoproliferative Syndrome (ALPS) is an autoimmune disease that has been reported in over 2200 patients. It is a rare, genetic disease where pathogenic variants occur in the extrinsic pathway of apoptosis. Various mutations in different genes, such as FAS, FASL, and CASP10, can result in ALPS. Most commonly, pathogenic variants occur in the FAS receptor. This malfunctioning pathway allows for the abnormal accumulation of lymphocytes, namely CD3 + TCRαß+CD4 - CD8- (double negative (DN) T) cells, which are a hallmark of the disease. This disease usually presents in childhood with lymphadenopathy and splenomegaly as a result of lymphoproliferation. Over time, these patients may develop cytopenias or lymphomas because of irregularities in the immune system. Current treatments include glucocorticoids, mycophenolate mofetil, sirolimus, immunoglobulin G, and rituximab. These medications serve to manage the symptoms and there are no standardized recommendations for the management of ALPS. The only curative therapy is a bone marrow transplant, but this is rarely done because of the complications. This review serves to broaden the understanding of ALPS by discussing the mechanism of immune dysregulation, how the symptoms manifest, and the mechanisms of treatment. Additionally, we discuss the epidemiology, comorbidities, and medications relating to ALPS patients across the United States using data from Cosmos.

Severe RAS-Associated Lymphoproliferative Disease Case with Increasing αß Double-Negative T Cells with Atypical Features.

Autoimmune lymphoproliferative syndrome (ALPS) is a disease of lymphocyte homeostasis caused by FAS-mediated apoptotic pathway dysfunction and is characterized by non-malignant lymphoproliferation with an increased number of TCRαß+CD4-CD8- double-negative T cells (αßDNTs). Conversely, RAS-associated leukoproliferative disease (RALD), which is caused by gain-of-functional somatic variants in KRAS or NRAS, is considered a group of diseases with a similar course. Herein, we present a 7-year-old Japanese female of RALD harboring NRAS variant that aggressively progressed to juvenile myelomonocytic leukemia (JMML) with increased αßDNTs. She eventually underwent hematopoietic cell transplantation due to acute respiratory distress which was caused by pulmonary infiltration of JMML blasts. In general, αßDNTs have been remarkably increased in ALPS; however, FAS pathway gene abnormalities were not observed in this case. This case with RALD had repeated shock/pre-shock episodes as the condition progressed. This shock was thought to be caused by the presence of a high number of αßDNTs. The αßDNTs observed in this case revealed high CCR4, CCR6, and CD45RO expressions, which were similar to Th17. These increased Th17-like αßDNTs have triggered the inflammation, resulting in the pathogenesis of shock, because Th17 secretes pro-inflammatory cytokines such as interleukin (IL)-17A and granulocyte-macrophage colony-stimulating factor. The presence of IL-17A-secreting αßDNTs has been reported in systemic lupus erythematosus (SLE) and Sjögren's syndrome. The present case is complicated with SLE, suggesting the involvement of Th17-like αßDNTs in the disease pathogenesis. Examining the characteristics of αßDNTs in RALD, JMML, and ALPS may reveal the pathologies in these cases.

Aging modifies endometrial dendritic cell function and unconventional double negative T cells in the human genital mucosa.

BACKGROUND: Immune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαß + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27-77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract. RESULTS: We discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGß dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively. CONCLUSIONS: Our deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age.

Immuno-Contexture and Immune Checkpoint Molecule Expression in Mismatch Repair Proficient Colorectal Carcinoma.

Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4-CD8-CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. METHODS: A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. RESULTS: Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. CONCLUSION: These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

CD4-/CD8- double-negative tumor-infiltrating lymphocytes expanded from solid tumor tissue suppress the proliferation of tumor cells in an MHC-independent way.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4-/CD8- double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4-CD8- TILs (double-negative TIL, DN-TILs) derived from solid tumor. METHODS: DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. RESULTS: Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3+ TILs, which expanded more than 800-folds in 2 weeks, consisting of a mixture of alpha-beta (αß) and gamma-delta (γδ) T-cell receptor (TCR)-expressing cells (with the majority being αß-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. CONCLUSION: Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL.

TFAP4 promotes the progression of liver fibrosis through regulating double-negative T cell differentiation via OX40.

This work aimed to investigate the role of transcription factor TFAP4-OX40 in promoting the differentiation of double-negative T cells (DNTs). Through prediction and experimental analysis, it was discovered that TFAP4 was the transcription factor of OX40. Therefore, OX40 neutralizing antibody and TFAP4 overexpression transfection were adopted to investigate the role of TFAP4-OX40 in DNTs differentiation, and the effect of differentiated DNTs on hepatic stellate cell (HSC) activation. Moreover, the impact of TFAP4 on liver fibrosis and DNTs in liver tissue was explored using mice with myeloid specific TFAP4 knockout by TFAP4 neutralizing antibody treatment. TFAP4 is the transcription regulatory factor for OX40, which promoted OX40 transcription expression to accelerate DNTs differentiation. Treatment with OX40 neutralizing antibody suppressed DNTs differentiation, while TFAP4 overexpression promoted DNTs differentiation. DNTs produced from the TFAP4 induced differentiation promoted HSC activation. Myeloid specific TFAP4 knockout delayed the progression of liver fibrosis and decreased DNTs in tissue, while treatment with TFAP4 neutralizing antibody suppressed liver fibrosis and DNTs in liver tissue. According to our results, TFAP4 is the transcription factor of OX40, which promotes DNTs differentiation via the OX40 signal, thus promoting the progression of liver fibrosis.