UNASSIGNED: Tuberculosis (TB) persists as a global health challenge, with its treatment hampered by the side effects of long-term combination drug therapies and the growing issue of drug resistance. Therefore, the development of novel therapeutic strategies is critical. This study focuses on the role of immune checkpoint molecules (ICs) and functions of CD8+ T cells in the search for new potential targets against TB.
UNASSIGNED: We conducted differential expression genes analysis and CD8+ T cell functional gene analysis on 92 TB samples and 61 healthy individual (HI) samples from TB database GSE83456, which contains data on 34,603 genes. The GSE54992 dataset was used to validated the findings. Additionally, a cluster analysis on single-cell data from primates infected with mycobacterium tuberculosis and those vaccinated with BCG was performed.
UNASSIGNED: The overexpression of LAG-3 gene was found as a potentially important characteristic of both pulmonary TB (PTB) and extrapulmonary TB (EPTB). Further correlation analysis showed that LAG-3 gene was correlated with GZMB, perforin, IL-2 and IL-12. A significant temporal and spatial variation in LAG-3 expression was observed in T cells and macrophages during TB infection and after BCG vaccination.
UNASSIGNED: LAG-3 was overexpressed in TB samples. Targeting LAG-3 may represent a potential therapeutic target for tuberculosis.

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis.
METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A.
RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05).
CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.
［摘要］ 目的 探讨淋巴细胞活化基因3 (lymphocyte activation gene 3, LAG3) 缺陷 (LAG3-/-) 对多房棘球蚴感染小鼠自然 杀伤 (natural killer, NK) 细胞功能及肝纤维化的影响。方法 取体质量为 (20 ± 2) g的C57BL/6小鼠, 分为LAG3-/-组和野 生型 (wild type, WT) 组, 分别经肝门静脉接种3 000个多房棘球蚴原头节。感染后12周, 取小鼠肝脏制备肝脏组织切片, 天狼星红染色观察肝脏病灶和纤维化程度。分离小鼠肝脏和脾脏淋巴细胞, 流式细胞术检测小鼠肝脏和脾脏NK细胞比 例, NK细胞表面CD25、CD44和CD69分子表达水平, 以及γ干扰素 (interferon γ, IFN-γ)、肿瘤坏死因子-α (tumor necrosis factor α, TNF-α)、白细胞介素 (interleukin, IL)-4、IL-10、IL-17A 等相关细胞因子分泌水平。结果 天狼星红染色结果显 示, 与WT组相比, LAG3-/-组小鼠肝脏病灶周围炎性细胞带增宽、纤维化结缔组织增生, 且呈现较多胶原纤维沉积。流式 细胞术检测结果显示, LAG3-/-组小鼠肝脏 (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) 和脾脏NK细胞比例 (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) 均低于WT组; LAG3-/-组小鼠肝脏NK细胞表面CD44分子平均荧光强度显著高于WT 组 (t = −3.234, P < 0.01), 而LAG3-/-组和WT组小鼠肝脏和脾脏NK细胞表面CD25和CD69分子平均荧光强度差异均无统 计学意义 (P 均> 0.05)。LAG3-/-组和WT组小鼠肝脏NK细胞分泌IFN-γ、TNF-α、IL-4、IL-10、IL-17A的比例差异均无统计 学意义 (t = −0.723、−0.659、−0.263、−0.455、0.091, P 均> 0.05); LAG3-/-组小鼠脾脏NK细胞分泌IFN-γ的比例显著高于WT 组 (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = −4.042, P < 0.01), 但分泌TNF-α、IL-4、IL-10、IL-17A的比例差异均无统计学 意义 (t = −1.902、−1.333、−1.356、0.529, P均> 0.05)。结论 在多房棘球蚴感染小鼠过程中, LAG3-/-促进脾脏NK细胞高 分泌IFN-γ, 可能参与逆转NK细胞免疫功能, 从而导致肝脏纤维化加重。.

The key role of tissue-resident memory T (TRM) cells in the immune regulation of hepatocellular carcinoma (HCC) has been investigated and reported, but the regulatory mechanism of tumor microenvironment on TRM cells is still unclear. Lymphocyte activating gene 3 (LAG-3) is a promising next-generation immune checkpoint that is continuously expressed due to persistent antigen exposure in the tumor microenvironment. Fibrinogen-like protein 1 (FGL1) is a classical ligand of LAG-3 and can promote T cell exhaustion in tumors. Here, we excavated the effect of FGL1-LAG3 regulatory axis on TRM cells in HCC.
The function and phenotype of intrahepatic CD8+ TRM cells in 35 HCC patients were analyzed using multicolor flow cytometry. Using a tissue microarray of 80 HCC patients, we performed the prognosis analysis. Moreover, we investigated the suppressive effect of FGL1 on CD8+ TRM cells both in in vitro induction model and in vivo orthotopic HCC mouse model.
There was an increase in LAG3 expression in CD8+ TRM cells in end-stage HCC; moreover, FGL1 levels were negatively correlated with CD103 expression and related to poor outcomes in HCC. Patients with high CD8+ TRM cell proportions have better outcomes, and FGL1-LAG3 binding could lead to the exhaustion of CD8+ TRM cells in tumors, indicating its potential as a target for immune checkpoint therapy of HCC. Increased FGL1 expression in HCC may result in CD8+ TRM cell exhaustion, causing tumor immune escape.
We identified CD8+TRM cells as a potential immunotherapeutic target and reported the effect of FGL1-LAG3 binding on CD8+ TRM cell function in HCC.

Objective: To investigate the expression of Lymphocyte activation gene 3 (LAG3) in myelodysplastic syndromes (MDS) patients. Methods: A total of 16 MDS patients newly diagnosed in Hematology Department of Tianjin Medical University were enrolled from January to November 2019. The healthy control (HC) group includes 16 cases of healthy adults. The expression levels of LAG3 on CD8(+)T cells, CD4(+)T cells and regulatory T cells (Treg) in MDS patients and healthy controls were detected by flow cytometry. Results: A total of 16 patients with MDS were included, including 5 males and 11 females, with a median age of 56 (18-80) years old. HC group includes 16 healthy adults, 8 men and 8 women, with a median age of 40 (17-69) years. There was no statistically significant difference in gender and age composition between the two groups (both P>0.05). The expression of LAG3 on CD8(+)T cells in MDS patients (74.45%±22.31%) was significantly higher than that in HC group (58.78%±14.82%, P<0.05). The LAG3 expression on Treg in MDS patients (64.91%±10.32%) were significantly higher than that of HC group (49.09%±13.58%, P<0.05). There was no statistical difference in LAG3 expression on CD4(+)T cells between the two groups. Conclusion: The expression of LAG3 on CD8(+)T cells and Treg increases in MDS patients than that of healthy people.
目的： 探讨淋巴细胞活化基因-3（LAG3）在骨髓增生异常综合征（MDS）患者T细胞亚群中的表达。 方法： 选取2019年1至11月期间于天津医科大学总医院血液科新诊断的MDS患者16例为MDS组，另纳入16名健康成人为健康对照组，采用流式细胞术检测CD8(+)T细胞、CD4(+)T细胞和调节性T细胞在MDS组和健康对照组中的表达水平。 结果： 共纳入MDS患者16例；男5例，女11例；中位年龄56（18~80）岁。健康对照组16名，男女各8名，中位年龄40（17~69）岁，两组性别和年龄组成差异无统计学意义（均P>0.05）。LAG3在MDS组CD8(+)T细胞中的表达显著高于健康对照组（74.45%±22.31%比58.78%±14.82%，P<0.05）。LAG3在MDS组调节性T细胞中的表达显著高于健康对照组（64.91%±10.32%比49.09%±13.58%，P<0.05）。LAG3在CD4(+)T细胞中的表达MDS组与健康对照组间差异无统计学意义（P>0.05）。 结论： LAG3在MDS患者CD8(+)T和调节性T细胞上的表达高于健康人。.

Introduction: Therapeutic targeting of inhibitors of the immune response has reached the clinical setting. Inhibitors of the novel receptor LAG3, which negatively regulates T-cell activation, are under investigation. Here we explore the presence and prognostic role of LAG3 in cancer. Methods: A systematic search of electronic databases identified publications exploring the effect of LAG3 on overall survival (OS) and (for early-stage cancers) disease-free survival (DFS). Hazard ratios (HR) were pooled in a meta-analysis using generic inverse-variance and random effect modeling. Subgroup analyses were conducted based on disease site and tumor type. Results: Fifteen studies met the inclusion criteria. LAG3 was associated with better overall survival [HR 0.81, 95% confidence interval (CI) 0.66-0.99; P = 0.04], with subgroup analysis showing no significant differences between disease-site subgroups. The beneficial effect of LAG3 on OS was of greater magnitude in early-stage malignancies (HR 0.73, 95% CI 0.60-0.88) than in the metastatic setting (HR 1.20, 95% CI 0.70-2.05), but this difference was not statistically significant (subgroup difference p = 0.18). LAG3 did not have a significant association with DFS [HR 1.02, 95% confidence interval (CI) 0.77-1.37; P = 0.87], with subgroup analysis showing worse DFS in patients with lymphoma and improved DFS in those with breast cancer. Conclusions: High expression of LAG3 is associated with favorable overall survival in several solid tumors. A trend toward an association in early-stage disease suggests the importance of immune surveillance in this setting.

Regulatory T (Treg) cells are critical suppressors of inflammation and are thought to exert mainly deleterious effects in cancers. In colorectal cancer (CRC), Foxp3+ Treg accumulation in tumors was associated with poor prognosis. Hence, we examined the circulating Treg cells in CRC patients. Compared to controls, CRC patients presented mild upregulations in CD4+ CD25+/hi T cells and in the more canonical CD4+ CD25+/hi Foxp3+ Treg cells in peripheral blood mononuclear cells. Both of these Treg populations could be roughly divided into lymphocyte activation gene 3 negative T cell immunoglobulin and mucin-domain containing-3 negative (LAG3- TIM3- ) and LAG3+ TIM3+ subsets. In CRC patients, the LAG3+ TIM3+ subset represented approximately half of CD4+ CD25+/hi T cells and greater than 60% of CD4+ CD25+/hi Foxp3+ Treg cells, which was significantly more frequent than in healthy controls. Compared to the LAG3- TIM3- CD4+ CD25+/hi T cells, the LAG3+ TIM3+ CD4+ CD25+/hi T cells presented considerably higher transforming growth factor-β and slightly higher interleukin (IL)-10 secretion, together with higher cytotoxic T-lymphocyte associated protein 4 and Foxp3 expression levels. Notably, macrophages following incubation with LAG3- TIM3- CD4+ CD25+/hi T cells and LAG3+ TIM3+ CD4+ CD25+/hi T cells displayed different characteristics. Macrophages incubated with LAG3+ TIM3+ CD4+ CD25+/hi T cells presented lower expression of major histocompatibility complex class II, CD80, CD86, and tumor necrosis factor-α but higher expression of IL-10, than macrophages incubated with LAG3- TIM3- CD4+ CD25+/hi T cells. Together, our investigations demonstrated that CRC patients presented an enrichment of circulating Treg cells, in which the LAG3+ TIM3+ subset exhibited more potent expression of inhibitory molecules, and furthermore, the LAG3+ TIM3+ Treg cells could suppress the proinflammatory activation of macrophages more potently than the LAG3- TIM3- Treg cells.

Regulatory T cells (Tregs) are necessary for the maintenance of immune tolerance. Tregs are divided into two major populations: one is thymus derived and the other develops in the periphery. Among these Tregs, CD4+CD25+ Tregs, which mainly originate in the thymus, have been extensively studied. Transcription factor Foxp3 is well known as a master regulatory gene for the development and function of CD4+CD25+ Tregs. On the other hand, peripheral Tregs consist of distinct cell subsets including Foxp3-dependent extrathymically developed Tregs and interleukin (IL)-10-producing type I regulatory T (Tr1) cells. Lymphocyte activation gene 3 (LAG3) and CD49b are reliable cell surface markers for Tr1 cells. CD4+CD25-LAG3+ Tregs (LAG3+ Tregs) develop in the periphery and produce a large amount of IL-10. LAG3+ Tregs characteristically express the early growth response gene 2 (Egr2), a zinc-finger transcription factor, and exhibit its suppressive activity in a Foxp3-independent manner. Although Egr2 was known to be essential for hindbrain development and myelination of the peripheral nervous system, recent studies revealed that Egr2 plays vital roles in the induction of T cell anergy and also the suppressive activities of LAG3+ Tregs. Intriguingly, forced expression of Egr2 converts naive CD4+ T cells into IL-10-producing Tregs that highly express LAG3. Among the four Egr gene family members, Egr3 is thought to compensate for the function of Egr2. Recently, we reported that LAG3+ Tregs suppress humoral immune responses via transforming growth factor β3 production in an Egr2- and Egr3-dependent manner. In this review, we focus on the role of Egr2 in Tregs and also discuss its therapeutic potential for the treatment of autoimmune diseases.

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T-cell exhaustion. T-cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co-stimulatory molecules to T-cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia-derived co-stimulatory signals on CD4+ T helper (Th) cell exhaustion, which may limit anti-tumour immunity. Here, CD86 and inducible T-cell co-stimulator ligand (ICOS-LG) co-stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co-cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro-generated exhausted Th cells were defined by up-regulation of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) inhibitory receptors. They were reluctant to proliferate upon re-stimulation and produced reduced amounts of interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Nonetheless, IL-2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co-stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD-1, TIM-3 and/or LAG3 was identified together with CD86+ and/or ICOS-LG+ myeloid blasts. Collectively, co-stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.

BACKGROUND: Surgery has been reported to suppress cell-mediated immunity; however, the detailed mechanisms responsible for this remain unclear. This study determined the expression of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in lymphocytes following surgery for gastric cancer.
METHODS: LAG-3 and PD-1 expression on both CD4+ and CD8+ T cells obtained pre- and post-operatively from gastric cancer patients were evaluated by multicolor flow cytometry.
RESULTS: The total lymphocyte count decreased rapidly from preoperative levels, reaching a minimum on postoperative day 1 and remaining significantly decreased on days 3 and 7. PD-1(+)CD4(+) T cells significantly increased, reaching a maximum on postoperative day 1 and remaining significantly elevated on day 3. PD-1(+)CD8(+) T cells significantly increased and reached a maximum on day 7 before returning to the preoperative level on day 30. There were no statistically significant differences in the frequency of LAG-3(+)CD4(+) or LAG-3(+)CD8(+) T cells after surgery. There were significant positive correlations between PD-1 and LAG-3 expression on both CD4(+) andCD8(+) T cells.
CONCLUSIONS: PD-1 and LAG-3 expression on both CD4(+) and CD8(+) T cells was up-regulated and might be related to impaired cell-mediated immunity after surgery for gastric cancer.

BACKGROUND: The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance.
OBJECTIVE: Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy.
METHODS: In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13).
RESULTS: Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells.
CONCLUSIONS: In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.