The Transcription Factor Zfp335 Promotes Differentiation and Survival of Effector Th1 Cells by Directly Regulating Lmna Expression.

Ag-specific effector CD4+ T cells play a crucial role in defending against exogenous pathogens. However, the mechanisms governing the differentiation and function of IFN-γ-producing effector CD4+ Th1 cells in immune responses remain largely unknown. In this study, we elucidated the pivotal role of zinc finger protein 335 (Zfp335) in regulating effector Th1 cell differentiation and survival during acute bacterial infection. Mice with Zfp335 knockout in OT-II cells exhibited impaired Ag-specific CD4+ T cell expansion accompanied by a significant reduction in resistance to Listeria infection. Furthermore, Zfp335 deficiency restricted the effector CD4+ Th1 cell population and compromised their survival upon Listeria challenge. The expression of T-bet and IFN-γ was accordingly decreased in Zfp335-deficient Th1 cells. Mechanistically, Zfp335 directly bound to the promoter region of the Lmna gene and regulated its expression. Overexpression of Lmna was able to rescue the survival and function of Zfp335-deficient effector Th1 cells. Therefore, our study provides novel insights into the mechanisms governing effector Th1 cell differentiation and survival during acute infection.

Effect of Med1 on T cell development and CD4+ T cell differentiation in immune response. / Med1对T细胞发育及CD4+ Tç»†èƒžåœ¨å ç–«åº”ç­”ä¸­åˆ†åŒ–çš„å½±å“.

OBJECTIVES: The differentiation of CD4+ T cells is regulated by a complex and fine signaling pathway composed of many molecules during immune response, and the molecular mechanism for regulating T-bet expression is unclear. Mediator complex subunit 1 (Med1) can combine with a variety of co-factors to regulate gene transcription, promote cell proliferation and survival, and affect invariant natural killer T cell (iNKT) development. This study aims to investigate the effect of Med1 on T cell development and CD4+ T cell differentiation in immune response. METHODS: Mice with T cell-specific knockout of Med1 gene (Med1F/FCD4cre+, KO) were constructed and verified. The percentage and number of CD4+ and CD8+ T cells in thymus, spleen, and lymph nodes of KO mice and control (Con) mice (Med1F/FCD4cre-) were detected by flow cytometry. After 8 days of infection with lymphocytic choriomeningitis virus (LCMV), the percentage and number of CD4+ T cells or antigen-specific (GP66+) CD4+ T cells, the percentage and number of Th1 cells (Ly6c+PSGL1+) in CD4+ T cells or antigen-specific CD4+ T cells were examined in the spleen of mice. Moreover, the fluorescence intensity of T-bet in CD4+ T cells or antigen-specific CD4+ T cells was analyzed. RESULTS: Compared with the Con group, the percentage and number of CD4+ T cells and CD8+ T cells in the thymus, CD4+ T cells in the spleen and lymph nodes of the KO group showed no significant differences (all P>0.05), but the percentage and number of CD8+ T cells in the spleen and lymph nodes of the KO group were diminished significantly (all P<0.05). After 8 days of infection with LCMV, there was no significant difference in the percentage and number of CD4+ T cells or antigen-specific CD4+ T cells in the spleen between the KO group and the Con group (all P>0.05), while in comparison with the Con group, the percentage and number of Th1 cells in CD4+ T cells or antigen-specific CD4+ T cells, and the expression of T-bet in CD4+ T cells or antigen-specific CD4+ T cells were significantly reduced in the spleen of the KO group (all P<0.05). CONCLUSIONS: Specific knockout of Med1 in T cells does not affect the development of CD4+ and CD8+ T cells in the thymus, but does affect the maintenance of peripheral CD8+ T cells. In the immune response, Med1 gene deletion affects the expression of transcription factor T-bet, which in turn to reduce Th1 cell differentiation.

The application value of GeneXpert MTB/RIF Ultra in the detection of special specimens of tuberculosis / 中国热带医学

@#Abstract: Objective To compare the diagnostic efficacy of the upgraded version of the GeneXpert automated fluorescent quantitative PCR system (GeneXpert MTB/RIF Ultra, GeneXpert Ultra) and the original version of the GeneXpert system (GeneXpert MTB/RIF, Xpert), real-time fluorescent quantitative nucleic acid detection (FQ-PCR), real-time fluorescent thermostatic amplification of Mycobacterium tuberculosis RNA (SAT-RNA), real-time fluorescent thermostatic amplification detection of DNA (thermostatic amplification method) and traditional BACTEC MGIT 960 liquid culture (culture method) for special specimens of tuberculosis, in order to analyze its application value in clinical detection. Methods Using prospective research methods, a total of 170 special specimens (including 47 pleural and ascites effusion samples, and 34 24-hour urinary sediment specimens, 49 tissue specimens and 40 fester specimens) were collected i'an Chest Hospital from January to September 2021. GeneXpert Ultra, Xpert, FQ-PCR, SAT-RNA, isothermal amplification, and traditional culture were used for detection. Clinical diagnosis was used as the standard, and sensitivity, specificity, positive predictive value, negative predictive value, coincidence rate, and Kappa value were compared among the methods. Results The sensitivities of GeneXpert Ultra, Xpert, FQ-PCR, SAT-RNA, isothermal amplification, and traditional culture were 65.18% (73/112), 49.11% (55/112), 37.50% (42/112), 19.64% (22/112), 8.04% (9/112), and 22.32% (25/112), respectively. The sensitivity of GeneXpert Ultra was higher than that of the other five methods, and the differences were statistically significant (χ2=66.25, 42.10, 28.89, 13.09, 4.92, 15.18, all P<0.05). GeneXpert Ultra result analysis showed that: 5.48%(4/73) cases had trace, that is, trace Mycobacterium tuberculosis load, 79.45% (58/73) cases were extremely low, 10.96% (8/73) cases were low, 2.74% (2/73) were medium, , and 1.36% (1/73) were high load. In 4 trace samples, the Xpert detection was negative for all. Of the 73 GeneXpert Ultra positive reports, 63 were rifampicin-sensitive, 6 were rifampicin-resistant, and 4 were rifampicin-resistant but of unclear resistance. Of the 55 Xpert positive reports, 45 were rifampicin-sensitive, 2 were rifampicin-resistant, and 8 were rifampicinresistant but of unclear resistance.. Conclusions The new generation of GeneXpert MTB/RIF Ultra has high sensitivity, specificity and drug resistance detection rate, and its advantage is even more apparent in the pathogenic diagnosis of special specimens of tuberculosis. It can be used as one of the preferred methods in samples with low bacterial load.

Med1 Controls Effector CD8+ T Cell Differentiation and Survival through C/EBPß-Mediated Transcriptional Control of T-bet.

Effector CD8+ T cells are crucial players in adaptive immunity for effective protection against invading pathogens. The regulatory mechanisms underlying CD8+ T cell effector differentiation are incompletely understood. In this study, we defined a critical role of mediator complex subunit 1 (Med1) in controlling effector CD8+ T cell differentiation and survival during acute bacterial infection. Mice with Med1-deficient CD8+ T cells exhibited significantly impaired expansion with evidently reduced killer cell lectin-like receptor G1+ terminally differentiated and Ly6c+ effector cell populations. Moreover, Med1 deficiency led to enhanced cell apoptosis and expression of multiple inhibitory receptors (programmed cell death 1, T cell Ig and mucin domain-containing-3, and T cell immunoreceptor with Ig and ITIM domains). RNA-sequencing analysis revealed that T-bet- and Zeb2-mediated transcriptional programs were impaired in Med1-deficient CD8+ T cells. Overexpression of T-bet could rescue the differentiation and survival of Med1-deficient CD8+ effector T cells. Mechanistically, the transcription factor C/EBPß promoted T-bet expression through interacting with Med1 in effector T cells. Collectively, our findings revealed a novel role of Med1 in regulating effector CD8+ T cell differentiation and survival in response to bacterial infection.

The Transcription Factor Zfp335 Promotes Differentiation and Persistence of Memory CD8+ T Cells by Regulating TCF-1.

Memory CD8+ T cells play an essential role in providing effective and lifelong protection against pathogens. Comprehensive transcriptional and epigenetic networks are involved in modulating memory T cell development, but the molecular regulations of CD8+ memory T cell formation and long-term persistence remain largely unknown. In this study, we show that zinc finger protein 335 (Zfp335) is indispensable for CD8+ T cell memory establishment and maintenance during acute infections. Mice with Zfp335 deletion in CD8+ T cells exhibit a significant reduction of memory T cells and memory precursor cells in the contraction phase. Zfp335 deficiency in CD8+ T cells resulted in decreased expression of memory featured genes Eomes and IL-2Rß, leading to a loss of memory identity and an increase of apoptosis in response to IL-7 and IL-15. Mechanistically, Zfp335 directly binds to and regulates TCF-1, known to be critical for memory T cell development. Importantly, overexpression TCF-1 could rescue the defects in the survival of both CD8+ memory precursors and memory T cells caused by Zfp335 deficiency. Collectively, our findings reveal that Zfp335 serves as a novel transcriptional factor upstream of TCF-1 in regulating CD8+ T cell memory.

DExD/H-box helicase 9 intrinsically controls CD8+ T cell-mediated antiviral response through noncanonical mechanisms.

Upon virus infection, CD8+ T cell accumulation is tightly controlled by simultaneous proliferation and apoptosis. However, it remains unclear how TCR signal coordinates these events to achieve expansion and effector cell differentiation. We found that T cell-specific deletion of nuclear helicase Dhx9 led to impaired CD8+ T cell survival, effector differentiation, and viral clearance. Mechanistically, Dhx9 acts as the key regulator to ensure LCK- and CD3ε-mediated ZAP70 phosphorylation and ERK activation to protect CD8+ T cells from apoptosis before proliferative burst. Dhx9 directly regulates Id2 transcription to control effector CD8+ T cell differentiation. The DSRM and OB_Fold domains are required for LCK binding and Id2 transcription, respectively. Dhx9 expression is predominantly increased in effector CD8+ T cells of COVID-19 patients. Therefore, we revealed a previously unknown regulatory mechanism that Dhx9 protects activated CD8+ T cells from apoptosis and ensures effector differentiation to promote antiviral immunity independent of nuclear sensor function.

Single-cell sequencing reveals antitumor characteristics of intratumoral immune cells in old mice.

BACKGROUND: Aging has long been thought to be a major risk factor for various types of cancers. However, accumulating evidence indicates increased resistance of old animals to tumor growth. An in-depth understanding of how old individuals defend against tumor invasion requires further investigations. METHODS: We revealed age-associated alterations in tumor-infiltrating immune cells between young and old mice using single-cell RNA and coupled T cell receptor (TCR) sequencing analysis. Multiple bioinformatics methods were adopted to analyze the characteristics of the transcriptome between two groups. To explore the impacts of young and old CD8+ T cells on tumor growth, mice were treated with anti-CD8 antibody every 3 days starting 7 days after tumor inoculation. Flow cytometry was used to validate the differences indicated by sequencing analysis between young and old mice. RESULTS: We found a higher proportion of cytotoxic CD8+ T cells, naturally occurring Tregs, conventional dendritic cell (DC), and M1-like macrophages in tumors of old mice compared with a higher percentage of exhausted CD8+ T cells, induced Tregs, plasmacytoid DC, and M2-like macrophages in young mice. Importantly, TCR diversity analysis showed that top 10 TCR clones consisted primarily of exhausted CD8+ T cells in young mice whereas top clones were predominantly cytotoxic CD8+ T cells in old mice. Old mice had more CD8+ T cells with a 'progenitor' and less 'terminally' exhausted phenotypes than young mice. Consistently, trajectory inference demonstrated that CD8+ T cells preferentially differentiated into cytotoxic cells in old mice in contrast to exhausted cells in young mice. Importantly, elimination of CD8+ T cells in old mice during tumor growth significantly accelerated tumor development. Moreover, senescent features were demonstrated in exhausted but not cytotoxic CD8+ T cells regardless of young and old mice. CONCLUSIONS: Our data revealed that a significantly higher proportion of effector immune cells in old mice defends against tumor progression, providing insights into understanding the altered kinetics of cancer development and the differential response to immunotherapeutic modulation in elderly patients.

A Genetic Model Reveals Biological Features of Neonatal CD4 Helper Cells Undergone Homeostasis in Mice.

CD4+ T cells are essential for regulating effective immune response to pathogens and immune balance. Recent studies have demonstrated the unique features of T cells in neonate mice, such as more sensitive to antigen response and preference toward T helper 2 (Th2) response and regulatory T cells (Tregs) differentiation. However, the biological characteristics of neonatal age-derived CD4+ T cells following homeostasis remain unclear. Here we utilized a lineage tracing model of TCRδ CreER R26 ZsGreen to mark neonatal- and adult-derived CD4+ T cells followed by a combination analysis of activation, proliferation, survival, and differentiation. Our results showed that neonatal CD4+ T cells had higher capacity of activation, proliferation, apoptosis, and differentiation toward Th2 and T helper 17 (Th17) lineages, accompanied by a reduced potential for T helper 1 (Th1), T helper 9 (Th9), and Treg lineages. In contrast, tracked neonatal CD4+ T cells exhibited similar characters of above-mentioned of tracked adult cells in adult mice. Therefore, our data support a natural requirement for CD4+ T cells to acquire fully-equipped functional potentials of adult cells.

Med1 controls CD8 T cell maintenance through IL-7R-mediated cell survival signalling.

Under steady-state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8+ T cells compared with WT counterparts. Finally, Med1-deficient CD8+ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.

Age-Related Gene Alteration in Naïve and Memory T cells Using Precise Age-Tracking Model.

In aged individuals, age-related changes in immune cells, especially T cell deficiency, are associated with an increased incidence of infection, tumor, and autoimmune disease, as well as an impaired response to vaccination. However, the features of gene expression levels in aged T cells are still unknown. Our previous study successfully tracked aged T cells generated from one wave of developing thymocytes of young age by a lineage-specific and inducible Cre-controlled reporter (TCRδ CreER R26 ZsGreen mouse strain). In this study, we utilized this model and genome-wide transcriptomic analysis to examine changes in gene expression in aged naïve and memory T cell populations during the aging process. We identified profound gene alterations in aged CD4 and CD8 T cells. Both aged CD4+ and CD8+ naïve T cells showed significantly decreased organelle function. Importantly, genes associated with lymphocyte activation and function demonstrated a significant increase in aged memory T cells, accompanied by upregulation of immunosuppressive markers and immune checkpoints, revealing an abnormal T cell function in aged cells. Furthermore, aging significantly affects T cell survival and death signaling. While aged CD4 memory T cells exhibited pro-apoptotic gene signatures, aged CD8 memory T cells expressed anti-apoptotic genes. Thus, the transcriptional analysis of gene expression and signaling pathways in aged T cell subsets shed light on our understanding of altered immune function with aging, which will have great potential for clinical interventions for older adults.

Lineage Tracking the Generation of T Regulatory Cells From Microbial Activated T Effector Cells in Naïve Mice.

Regulatory T cells (Tregs) are essential for the maintenance of gut homeostasis by suppressing conventional CD4+ helper T cells (Tconvs) that are activated by microbial antigens. Although thymus is the major source of the peripheral Tregs, peripheral conversion from Tconvs to Tregs have also been shown to occur under various experimental conditions. It remains less clear about the frequency of lineage conversion from Tconvs to Tregs in naïve animals. Here we used a newly established reporter system to track a group of post expansion Tregs (eTregs), which exhibited a stronger suppressive ability than the non-lineage marked Tregs. Notably, microbial antigens are the primary driver for the formation of eTregs. TCR repertoire analysis of Peyer's patch T cells revealed that eTregs are clonally related to Tconvs, but not to the non-lineage tracked Tregs. Adoptive transfer of Tconvs into lymphopenic hosts demonstrated a conversion from Tconvs to eTregs. Thus, our lineage tracking method was able to capture the lineage conversion from microbial activated effector T cells to Tregs in naïve animals. This study suggests that a fraction of clonally activated T cells from the natural T cell repertoire exhibits lineage conversion to Tregs in response to commensal microbes under homeostatic conditions.