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The crucial role of CD4 + and CD8 + T cells in shaping and controlling immune responses during immune disease and cancer development has been well established and used to achieve marked clinical benefits. CD3 + CD4 - CD8 - double-negative (DN) T cells, although constituting a rare subset of peripheral T cells, are gaining interest for their roles in inflammation, immune disease and cancer. Herein, we comprehensively review the origin, distribution and functions of this unique T cell subgroup. First, we focused on characterizing multifunctional DN T cells in various immune responses. DN regulatory T cells have the capacity to prevent graft-versus-host disease and have therapeutic value for autoimmune disease. T helper-like DN T cells protect against or promote inflammation and virus infection depending on the specific settings and promote certain autoimmune disease. Notably, we clarified the role of DN tumor-infiltrating lymphocytes and outlined the potential for malignant proliferation of DN T cells. Finally, we reviewed the recent advances in the applications of DN T cell-based therapy for cancer. In conclusion, a better understanding of the heterogeneity and functions of DN T cells may help to develop DN T cells as a potential therapeutic tool for inflammation, immune disorders and cancer.

Background Alzheimer's disease (AD) is a debilitating neurodegenerative disorder that is difficult to predict and is typically diagnosed only after symptoms manifest. Recently, CD4+ T cell‐derived double‐negative T (DNT) cells have shown strong immuno‐regulatory properties in both in vitro and in vivo neuronal inflammation studies. However, the effectiveness of DNT cells in treating on AD are not yet fully understood. Objective This study's aims were three‐fold, to (1) evaluate the efficacy of CD4+ T cell‐derived DNT cells treatment on AD mice, (2) understand how DNT treatment make changes in different cell types of 5FAD mice, (3) identify the side effects of DNT treatment. Methods We performed tail vein injection of transformed and amplified CD4+ T cell‐derived DNT cells into 5 × FAD mice, while using WT mice and saline injection 5FAD mice as controls. DNT suspensions or NaCl alone were administered to 5 × FAD mice at the 6 months of age. For intravenous injection (n = 10 for both DNT and control injections), 5 × FAD mice were injected with a total of 5 × 106 DNT cells suspended in 200 μL of 0.9% NaCl or 0.9% NaCl alone via the lateral tail vein. Behavioral tests and pathology tests were carried out 30 days after cell transplantation. Results Through qualitative analysis, we identified 6 main themes. DNT from young wild‐type mice enhance the capability of spatial learning and memory in AD mice. DNT cell treatment rejuvenates the microglial function. DNT cell treatment improves the state of oligodendrocytes. DNT cell treatment finetunes the activation of the immune system. DNT cell treatment improves the synaptic plasticity and increases the complexity of neurons. DNT cell treatment reduces the density of amyloid Beta plaques deposition in the cortex and hippocampus of 5 × FAD mice. Discussion The findings from this study reveal that DNT treatment improved spatial memory and learning abilities, reduced Aβ deposition, and enhanced synaptic plasticity, contrasting with previous reports on thymus‐derived DNT cells. Additionally, CD4+ T cell‐derived DNT therapy exhibited anti‐inflammatory effects and modulated microglial function, promoting a neuroprotective environment. Notably, DNT treatment also reduced tau pathology by decreasing levels of abnormally phosphorylated tau. These findings suggest that CD4+ T cell‐derived DNT cells hold therapeutic potential for AD, effectively targeting both Aβ and tau pathologies. We focused on a subset of CD4‐derived double‐negative T (DNT) cells called TCRαβ+NK1.1−CD4−CD8− for treating Alzheimer's disease, and we found their injection can improve cognition of the 5×FAD mouse model. We thus identify DNT cell transplantation as a potential therapeutic approach for future AD treatments.

Background/Aim: Double-negative T (DNT) cells are phenotypically CD3+CD4−CD8−T cells. This study aimed to investigate the anti-cancer activity of DNT cells against pancreatic cancer cells. Materials and Methods: DNT cells were isolated from human peripheral blood. The effect of DNT cells on proliferation and invasion of the human pancreatic cell line Panc-1 was assessed. Expression of Nrf2 and Fas in Panc-1 cells co-cultured with DNT cells was analyzed with RT-PCR. The supernatants of Panc-1 and DNT co-cultures were analyzed with ELISA for IFN-r and FasL levels. Results: The isolated DNT cell phenotype was CD4−CD8−CD56− CD3+TCR (T cell receptor) α/β+ T cells with more than 90% purity. Panc-1 cell proliferation was significantly inhibited by co-culture with DNT cells. Panc-1 cells co-cultured with DNT cells showed significantly reduced cell invasion. Panc-1 cells co-cultured with DNT cells showed increased Nrf2 and Fas mRNA expression. Increased INF-r and FasL levels were detected in the supernatants of co-cultures of DNT and pancreatic cells. Conclusion: DNT cells inhibited proliferation and invasion of human pancreatic cancer cells. The INF-r, Fas/FasL pathway and Nrf2 may be involved in the anti-cancer effect of DNT cells against human pancreatic cancer.

Background While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. Methods Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. Results Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. Conclusions Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

Background Double-Negative T (DNT) cells, lacking both CD4 and CD8 expression, play critical roles in cancer immunology, and have garnered increasing attention in cancer research. However, their heterogeneity and functional diversity within the tumor microenvironment (TME) remain underexplored. Methods In-house and publicly available single-cell RNA sequencing (scRNA-seq) data for different cancer types were integrated after quality control and batch effect correction, followed by DNT cells separation from CD3 + T cells subtypes. Functional characteristics, intercellular communication, differentiation trajectories, regulatory networks, and clinical relevance were analyzed among different DNT subsets. Key findings were validated using multiplex immunofluorescence and spatial transcriptomics to investigate the spatial localization of DNT subsets and their interactions within the TME. Impact of γδ T cells on immunotherapy response was also assessed using MC38-based murine tumor model. Results By integrating scRNA-seq data from 2,369 samples across 23 cancer types, we established a comprehensive single-cell atlas of 157,025 high-quality DNT cells. Fourteen distinct DNT subsets (6 αβ DNT and 8 γδ T cell subsets) were identified, demonstrating tumor both type-specific and shared distribution patterns, as well as unique cell-cell interaction network within the TME. These subsets displayed specialized functional profiles, including cytotoxicity, antigen presentation, and immune modulation, indicating that the functional diversity of DNT cells is largely subset-specific rather than a manifestation of multifunctionality within a single population. We also delineated divergent trajectories for αβ DNT and γδ T cell subsets, including the functional plasticity of gut-resident γδ T cells transitioning between cytotoxic and immunosuppressive states. Notably, several DNT subsets were significantly associated with favorable clinical treatment outcomes, including improved responses to cancer immunotherapy. Consistently, depletion of γδ T cells in the murine tumor model significantly decreased the efficacy of PD-1 blockade, underscoring their critical role in therapeutic response. Conclusions Our study uncovers the previously underappreciated heterogeneity and functional diversity of DNT cells in the TME and demonstrates their profound impact on tumor progression and immunotherapy outcomes.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a second-line treatment with curative potential for leukemia patients. However, the prognosis of allo-HSCT patients with disease relapse or graft-versus-host disease (GvHD) is poor. CD4 + or CD8 + conventional T (Tconv) cells are critically involved in mediating anti-leukemic immune responses to prevent relapse and detrimental GvHD. Hence, treatment for one increases the risk of the other. Thus, therapeutic strategies that can address relapse and GvHD are considered the Holy Grail of allo-HSCT. CD3 + CD4 − CD8 − double-negative T cells (DNTs) are unconventional mature T cells with potent anti-leukemia effects with “off-the-shelf” potential. A phase I clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy for patients with relapsing acute myeloid leukemia (AML) post-allo-HSCT. Here, we studied the impact of DNTs on the anti-leukemic and GvHD-inducing activities of Tconv cells. DNTs synergized with Tconv cells to mediate superior anti-leukemic activity. Mechanistically, DNTs released soluble factors which activated and evoked potent anti-leukemic activities of Tconv cells. In contrast, DNTs suppressed GvHD-inducing activities of Tconv cells in a CD18-dependent manner by mediating cytotoxicity against proliferative Tconv cells. The seemingly opposite immunological activities of DNTs were dictated by the presence or absence of AML cells. Collectively, these results support the potential of DNTs as an adjuvant to allo-HSCT to address both disease relapse and GvHD. Key points DNTs enhance T cells’ anti-leukemic activity in the presence of AML cells, while suppressing T cells to inhibit GVHD in the absence of AML cells.

Background CD3 + CD20 + T cells (T B cells) are a subset of lymphocytes in the human body that are associated with inflammation. They originate from T cells interacting with B cells, and their levels are abnormally elevated in individuals with immune disorders, as well as in some cancer patients. The interplay between tumor immunity and inflammation is intricate, yet the specific involvement of T B cells in local tumor immunity remains uncertain, with limited research on their subtypes. Methods Lung cancer surgical samples were stained using multi-color immunofluorescence to study the subtypes and distribution patterns of T B cells. Results T B cells were confirmed to exist in a scattered pattern within tertiary lymphoid structures (TLS) in lung cancer tissues, with higher abundance in mature TLS. In subtype analysis, the CD4-CD8- double-negative T B cell subtype was predominant, comprising over 90% in samples with abundant TLS infiltration and over 60% in samples with poor infiltration. This was followed by the CD4 + CD8- and CD4-CD8 + single-positive T B cell subtypes, while the CD4 + CD8 + double-positive T B cell subtype was nearly absent. During the maturation of TLS, the proportion of B cells gradually increased, while the proportion of CD4-CD8- T cell subtype decreased. Conclusions T B cells extensively infiltrate the TLS regions in tumor tissues, with the double-negative subtype being predominant, potentially playing a crucial regulatory role in the local tumor immune microenvironment. This finding could facilitate the advancement of novel cancer treatment strategies.

Pediatric primary immune thrombocytopenia (ITP) is an acquired autoimmune disease that can be partially restored by glucocorticoids. TCRαβ CD4 CD8 double negative T cells (TCRαβ DNT) has been linked to the pathophysiology of ITP; however, the role of TCRαβ DNT in response to high-dose dexamethasone (HD-DXM) is unclear. In this study, we aimed to explore the alteration in TCRαβ DNT in ITP and the effect of HD-DXM on this subset. Pediatric patients (aged <18 years) newly diagnosed with ITP were recruited for this retrospective study. Th1, Th17, Treg, and TCRαβ DNT levels were measured by flow cytometry using specific antibodies. All patients received HD-DXM treatment and underwent periodic outpatient follow-up for 2-6 months. Patients were divided into the overall response (OR) and no response (NR) groups according to their responses to HD-DXM treatment. We enrolled 130 pediatric patients with ITP (OR, 95 cases; NR, 35 cases) and 50 age- and sex-matched healthy controls. Compared with Th17-to Treg, Th17, and Th1, univariate analysis identified that the proportion of TCRαβ DNT at baseline was more effective in predicting the response to HD-DXM ( <0.05). A significantly increased frequency of TCRαβ DNT was found in patients with ITP compared to healthy controls (percentage of T cells: 1.31% vs. 1.00%, <0.0001; percentage of lymphocytes: 0.76% vs. 0.68%, =0.010). Patients in the NR group had a higher percentage of TCRαβ DNT than the OR at the initial diagnosis (TCRαβ DNT/T: 1.52% vs. 1.30%, <0.01; TCRαβ DNT/Lym: 0.84% vs. 0.72%, <0.01). After treatment with HD-DXM, the elevated TCRαβ DNT was effectively reduced in the OR group, but not in the NR group (TCRαβ DNT/T: <0.05; TCRαβ DNT/Lym: =0.001; TCRαβ DNT counts: <0.01). TCRαβ DNT appears to play a significant role in the pathogenesis of pediatric ITP and may be involved in the immune response to HD-DXM. The correction of elevated TCRαβ DNT in patients who respond to HD-DXM may provide a novel insight for immune therapy in pediatric ITP.

Tumor recurrence is one of the major life-threatening complications after liver transplantation for liver cancer. In addition to the common mechanisms underlying tumor recurrence, another unavoidable problem is that the immunosuppressive therapeutic regimen after transplantation could promote tumor recurrence and metastasis. Transplant oncology is an emerging field that addresses oncological challenges in transplantation. In this context, a comprehensive therapeutic management approach is required to balance the anti-tumor treatment and immunosuppressive status of recipients. Double-negative T cells (DNTs) are a cluster of heterogeneous cells mainly consisting of two subsets stratified by T cell receptor (TCR) type. Among them, TCRαβ + DNTs are considered to induce immune suppression in immune-mediated diseases, while TCRγδ + DNTs are widely recognized as tumor killers. As a composite cell therapy, healthy donor-derived DNTs can be propagated to therapeutic numbers in vitro and applied for the treatment of several malignancies without impairing normal tissues or being rejected by the host. In this work, we summarized the biological characteristics and functions of DNTs in oncology, immunology, and transplantation. Based on the multiple roles of DNTs, we propose that a new balance could be achieved in liver transplant oncology using them as an off-the-shelf adoptive cell therapy (ACT).

Previous studies have reported that deletion of the transcription factor, early growth response protein 2 (EGR2), in normal C57BL/6 (B6) resulted in the development of lupus-like autoimmune disease. However, increased EGR2 expression has been noted in human and murine lupus, which challenges the notion of the autoimmune suppressive role of EGR2 in B6 mice. In this study, we derived both conditional EGR2 -/- B6/ lpr and EGR2 -/- B6 mice to elucidate the immune and autoimmune regulatory roles of EGR2 in autoinflammation (B6/lpr) versus physiologically normal (B6) conditions. We found that conditional EGR2 deletion increased spleen weight, enhanced T cell activation and IFNγ production, and promoted germinal center B cells and LAG3 + regulatory T cells development in both B6/lpr and B6 mice. Nevertheless, EGR2 deletion also showed strikingly differential effects in these two strains on T lymphocyte subsets profile, Foxp3 + Tregs and plasma cell differentiation, anti-dsDNA autoantibodies and immunoglobulins production, and on the induction of IL-17 in in vitro activated splenocytes. Specifically, EGR2 deletion in B6/lpr mice significantly decreased serum levels of anti-dsDNA autoantibodies, total IgG, IgM, IgG1, and IgG2a with reduced plasma cells differentiation. Furthermore, EGR2 deletion in B6/lpr mice had no obvious effect on IgG immunocomplex deposition, medium caliber vessel, and glomeruli inflammation but increased complement C3 immunocomplex deposition and large caliber vessel inflammation in the kidneys. Importantly, we demonstrated that EGR2 deletion in B6/lpr mice significantly reduced pathogenic CD4 - CD8 - CD3 + B220 + double negative T cells, which correlated with the reduced anti-dsDNA autoantibodies in serum and decreased IL-17 production in splenocytes of EGR2 -/- B6/lpr mice. Together, our data strongly suggest that the role of EGR2 is complex. The immunoregulatory role of EGR2 varies at normal or autoinflammation conditions and should not be generalized in differential experimental settings.