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CD8 downregulation is an overlooked component of self-reactive CD8+ T cell tolerance. CD8 downregulation is accompanied by T cell inhibition at the signaling and transcriptional levels. Loss of CD8 expression impairs the capacity of T cells to engage peptide-major histocompatibility complex complexes. CD8 downregulation may be a temporary event, as cells may regain CD8 expression and effector function. CD8 downregulation in mammals may be a part of a multilayered mechanism that inactivates self-reactive CD8+ T cells. Loss of CD8 may impair their capacity to engage target cells, while other simultaneous changes curb their activation and effector functions. Understanding this process represents a central element in our interpretation of CD8+ T cell physiology and its role in autoimmune diseases and cancer immunology. Activated CD8+ T cells directly kill target cells. Therefore, the regulation of their function is central to avoiding immunopathology. Mechanisms that curb effector functions in CD4+ and CD8+ T cells are mostly shared, yet important differences occur. Here, we focus on the control of CD8+ T cell activity and discuss the importance of a poorly understood aspect of tolerance that directly impairs engagement of target cells: the downregulation of CD8. We contextualize this process and propose that it represents a key element during CD8+ T cell modulation. [ABSTRACT FROM AUTHOR]

Our previous study found that double negative T cells (DNTs) could promote the NLRP3 activation through high expression of TNF‐α, thereby leading to hepatic fibrosis progression. We focused on investigating the role and mechanism of DNTs in regulating the Th9 cells differentiation in liver fibrosis. In our results, among patients with liver fibrosis, the proportions of peripheral blood DNTs and Th9 cells were up‐regulated and positively correlated. While promoting the progression of liver fibrosis in mice, DNTs could elevate the proportion of Th9 cells and activate the TNFR2‐STAT5‐NF‐κB pathway. The use of IL‐9 and TNF‐α monoclonal antibodies (mAbs) inhibited the effect of DNTs and lowered the proportion of Th9 cells in tissues. In vitro experiments showed that DNTs could promote the Th9 cells differentiation of Naive T cells, while TNF‐α mAbs could inhibit such effect of DNTs to lower the proportion of Th9 cells. We found that DNTs can activate TNFR2‐STAT5‐NF‐κB pathway by secreting TNF‐α, thereby promoting the Th9 Cells differentiation to facilitate the progression of liver fibrosis. There is interaction between DNTs and Th9 cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

This study was intended to delineate the profile of double‐negative T cells (DNTs) in NOD.Cg‐Prkdcscid Il2rgtm1wj/SzJ mice and cytokines released from DNTs in vivo and in vitro. Total 4 × 107 cells of RC1012 injection per mice were intravenously infused. IFN‐γ, TNF‐α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10 were measured in vivo and in vitro. A quantitative polymerase chain reaction (PCR) was employed to determine the gene copies of Notch2‐NLA per DNT cell from collected organs. Cytokines were significantly increased in vitro (4 h) and in vivo (3 h). DNT cells were distributed into the lung, liver, heart, and kidney earlier, and redistributed to lymphocyte homing spleen and bone marrow, which seemed to frame a two‐compartment pharmacokinetics (PK) model but more data are needed to confirm this, and the clearance of DNT cells fell into first‐order kinetics. [ABSTRACT FROM AUTHOR]

Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αβ+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance.

Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αβ+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance. [ABSTRACT FROM AUTHOR]

• The proportion of TCR-αβ+ DNTs was elevated in patients with aAA than HD. • SAA/VSAA patient with CR after IST had higher proportion of TCR-αβ+ DNTs at diagnosis. • Higher proportion of TCR-αβ+ DNTs at diagnosis implied a good response to IST. The pathophysiology of acquired aplastic anemia (aAA) is most associated with T cell mediated immune dysfunction, but the role of CD4- CD8- double negative T cells (DNTs) in pediatric patients with aAA is unclear. In this study, we aimed to investigate the proportion of TCR-αβ+ DNTs in pediatric patients with aAA and correlation with the response to immunosuppressive therapy (IST). Assessment of DNTs from peripheral blood was done by sensitive multi-color flow cytometry. The potential clinical value of TCR-αβ+ DNTs was then assessed by the receiver operating characteristic (ROC) curves. The retrospective study evaluated 164 pediatric patients with aAA and 105 healthy donors (HD). Our data showed higher proportion of TCR-αβ+ DNTs in total lymphocytes [1.04% (0.79%-1.40%) vs 0.69% (0.47%-0.87%), p < 0.001] and CD3+ T cells [1.52% (1.10%-1.96%) vs 1.10% (0.70%-1.40%), p < 0.001] in aAA compared to HD. Patients with SAA/VSAA achieving complete response (CR) after IST had a higher proportion of TCR-αβ+ DNTs at initial diagnosis, than those not achieving CR for total (1.21%±0.39 vs 0.78%±0.38, p < 0.05) and CD3+ T cells (1.74%±0.53 vs 1.15%±0.59, p < 0.05). The ROC analysis showed areas under the curves (AUCs) for TCR-αβ+ DNT proportion in lymphocytes and CD3+ T cells were 0.756 (cutoff value 1.33, p < 0.05) and 0.758 (cutoff value 1.38, p < 0.05), respectively. And the complete response rate was higher in TCR-αβ+ DNT proportion high group than in TCR-αβ+ DNT proportion low group at baseline (p < 0.001). Our observations suggest that elevated TCR-αβ+ DNTs seems to play a role in the pathogenesis of aAA, and it was involve in immune response to IST. [ABSTRACT FROM AUTHOR]

Abstract: In this present investigation, flow cytometry was utilized to evaluate 13 healthy controls and 31 HIV-1 infected patients who had advanced to the AIDS stage of infection (CD4 count below 200cells/mm3), for the expression of CD161 on CD3+ double negative (DN) (CD3+CD4-CD8-) T cells, CD4+ T cells, CD8+ T cells and γδ T cells. The observed depletion of CD161+ T cells from peripheral circulation was due primarily to the loss of CD4+CD161+ T cells; as these cells represented 8.67±0.74% of the total healthy control peripheral T cell population, while the CD4+CD161+ T cells of the AIDS group represented only 3.35±0.41% (p=<0.0001) of the total peripheral T cell population. We have also shown here that the DN T cell population was more than doubled in the AIDS group, with the DN T cell population expanding from 3.29±0.45% of the healthy control peripheral T cell population to 8.64±1.16% (p=0.0001) of the AIDS group peripheral T cell population. By evaluating the expression of CD161 on the surface of the DN T cells we showed that within the healthy control group, 47.4±4.99% of the DN T cells were positive for the expression of CD161, while only 26.4±3.54% (p=0.002) of the AIDS group''s DN T cells expressed CD161. Despite CD161 expression being halved on the DN T cells of the AIDS group, when we compared the total peripheral T cell percentage of CD161+ DN T cells between the healthy control group and the AIDS group, there was no statistical difference. Even though only 26.4% DN T cells within the AIDS group were positive for CD161+, the overall DN T cell population had expanded to such an extent that there was no statistical difference between the groups with regard to CD161+ DN T cells as a percentage of the total peripheral T cell population. Furthermore, we showed that within the DN T cell population, there was an approximate 2:1 ratio of γδ to αβ T cells, and this ratio was maintained in both the healthy control group and the AIDS group. While evaluating γδ T cells we also discovered that CD8+ γδ T cells were expanded from 0.62±.09% of the healthy control peripheral T cell population to 5.01±.88% (p=<0.0001) of the peripheral T cell population of the AIDS group; and that this population of CD8+ γδ T cells underwent the same reduction in percentage of cells expressing CD161+, further demonstrated that the phenomenon of CD161+ percentage reduction and compensatory increase in total cell population was affecting the entire circulating γδ T cell population. [Copyright &y& Elsevier]

Simple Summary: Persistence of infused cells is an important factor that dictates the outcome of adoptive cellular therapy (ACT). DNT therapy is a novel form of ACT with promising result in treating relapsed or refractory AML in preclinical and early clinical studies. However, in vivo kinetics of human DNTs in cancer-bearing host have not been previously investigated. This study was the first to investigate the persistence of DNTs and ways to improve it in patient-derived xenograft models. DNTs persistence was observed up to 50 days in various organs of leukemia-bearing hosts. However, the detected DNT level was low while significant level of persisting AMLs was observed. To improve the in vivo persistence and therapeutic efficacy of DNTs, we expanded DNTs in the presence of an PI3Kδ inhibitor, idelalisib (Ide). Ide treatment of healthy donor-derived DNTs promoted early memory subsets and improved overall fitness, reducing exhaustion while improving viability. These Ide-induced attributes led to prolonged persistence of DNTs, resulting in superior anti-leukemic activity in vivo. Further, Ide-treated DNTs improved the durability of the treatment response. Collectively, our study highlights the importance of DNT persistence and Ide-mediated improvements in the overall fitness of DNTs, which promote longer persistence in vivo and better treatment outcome. The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome. [ABSTRACT FROM AUTHOR]

CD4 and CD8 coreceptor double negative TCRαβ+ T (DNT) cells are increasingly being recognized for their critical and diverse roles in the immune system. However, their molecular and functional signatures remain poorly understood and controversial. Moreover, the majority of studies are descriptive because of the relative low frequency of cells and non-standardized definition of this lineage. In this study, we performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of male C57BL/6 mice using strict fluorescence-activated cell sorting. The data was replicated in a subsequent study. Our analysis revealed five transcriptionally distinct naïve DNT cell clusters, which expressed unique sets of genes and primarily performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, in comparison with CD4+, CD8+ T cells and NK cells, Ikzf2 was highly expressed by both naïve and activated cytotoxic DNT cells. In conclusion, we provide a map of the heterogeneity in naïve and active DNT cells, addresses the controversy about DNT cells, and provides potential transcription signatures of DNT cells. The landscape approach herein will eventually become more feasible through newer high throughput methods and will enable clustering data to be fed into a systems analysis approach. Thus the approach should become the "backdrop" of similar studies in the myriad murine models of autoimmunity, potentially highlighting the importance of DNT cells and other minor lineage of cells in immune homeostasis. The clear characterization of functional DNT subsets into helper DNT, cytotoxic DNT and innate DNT will help to better understand the intrinsic roles of different functional DNT subsets in the development and progression of autoimmune diseases and transplant rejection, and thereby may facilitate diagnosis and therapy. • We delineated the heterogeneity of naïve and activated DNT cells in mice. • nDNT subgroups primarily performed T helper, cytotoxic and innate immune functions. • Anti-CD3/CD28 activation enhanced DNT cell T helper and cytotoxic functions. • Ikzf2 is an important transcriptional factor for cytotoxic DNT cells. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease of uncertain etiology that affects multiple tissues and organs. Tetra‐arsenic tetra‐sulfide (As4S4), a traditional Chinese medicine, is effective on acute promyelocytic leukemia with mild side effects. In our previous study, BXSB lupus‐prone mice treated with As4S4 has showed improved monocytosis, decreased serum interleukin (IL)‐6 and suppressed skin, liver and renal lesions with well‐tolerance. In this study, we explored the effect and mechanism of As4S4 on the MRL/lpr mice. MRL/lpr and wild MRL/MpJ mice were divided into control and As4S4 treatment groups and dosed with As4S4 or placebo for 8 weeks. We found that As4S4 prevented the skin, renal and lung lesions of MRL/lpr mice. As4S4 significantly decreased the double negative T (DN T) cells and reduced the serum levels of IL‐17, IL‐10, and antinuclear antibodies titer. Further results revealed that the FasL was decreased, and activated caspases elevated in DN T cells in As4S4 treated MRL/lpr mice. Taken together, As4S4 could selectively suppresses DN T cells by inducing apoptosis. It also reduced inflammatory cytokines IL‐17, which may be produced by DN T cells. As4S4 may represent a new therapy for SLE. [ABSTRACT FROM AUTHOR]