Subset Of CD4 Research Articles

Overestimation of clinical N-staging in microsatellite instable gastric cancers is associated with VEGF-C signaling and CD8+ T-cell dynamics

Abstract Background Microsatellite instable (MSI) gastric cancers exhibit reduced lymph node (LN) metastasis and improved survival compared to microsatellite stable (MSS) counterparts. However, to our longstanding observation, clinical N-staging (cN) is frequently overestimated in MSI cases. The clinical implications and underlying mechanisms of this discrepancy warrant further investigation. Materials and methods We conducted a comprehensive review of clinicopathological data from a 141 MSI and 1119 MSS gastric cancer patients. Expression of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 were assessed using qPCR and immunohistochemistry. High-parameter flow cytometry was employed to analyze subsets of CD8+ T cells within the tumors. Results Multivariate analysis revealed that MSI status was an independent prognostic factor, alongside the LN ratio and AJCC8 pathology staging. MSI gastric cancers exhibited a reduced LN ratio, particularly at advanced T-staging, compared to MSS counterparts, while maintaining an equivalent LN yield. Overestimation of cN by computed tomography preoperatively was frequent in MSI gastric cancers but was more commonly underestimated in MSS counterparts. VEGF-C and VEGFR-3 expression were lower in MSI tumors. MSI gastric cancers showed an increased total number of CD8+ T cells, albeit with a lower proportion of effector memory cells expressing CD45RA (EMRA) and CD8+ CXCR4+ T cells, compared to MSS counterparts. Conclusion Frequent overestimation of clinical N-staging in MSI gastric cancers is associated with VEGF-C signaling and CD8+ T-cell dynamics and should be cautiously interpreted, as it might misguide therapeutic options.

Abstract C021: Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung cancer

Abstract Type 1 conventional dendritic cells (cDC1s) are critical for initiating and sustaining tumor-reactive CD8 T cells; lack of functional cDC1s is a key driver for failed tumor immunosurveillance and immunotherapy. However, what factors cause the cDC1 insufficiency in the tumor microenvironment (TME) remain poorly understood. Commensal microbiota has emerged as a key regulator of TME and the efficacy of cancer immunotherapies. Nonetheless, most studies have focused on the intestinal microbiome; it remains underexplored whether and how the local microbiota within tumors influences the anti-cancer immunity. Using the autochthonous Kras LSL-G12D/+ ; p53 flox/flox (KP) mouse model of lung cancer, we found an increased local bacterial burden and altered bacterial composition associated with lung tumors. Importantly, this local microbiota suppresses the anti-tumor CD8 T cell response both at baseline and in response to ICB therapies. Specifically, single-cell RNA-seq analysis revealed that the microbiota shapes the functional subsets of tumor-reactive CD8 T cells. Bacterial depletion promotes the accumulation of stem-like Tpex cells (precursor of exhausted T cells) in the TME, which serve as a reservoir of effector CD8 T cells and mediate an effective response upon ICB treatment. To distinguish the function of the local microbiota within TME from the distal gut microbiota, we developed a new method of aerosolized antibiotic treatment, which selectively ablates the intra-tumoral lung microbiota while preserving the intact gut microbiota. Using this approach, we demonstrated that removal of the intra-tumoral lung microbiota markedly improves the tumor-reactive T cell response and sensitizing “cold” lung tumors to ICB therapies. Mechanistically, we identified cDC1s as a central player in microbiota-mediated suppression of anti-tumor immunity. Local microbiota diminishes cDC1 quality and quantity in the lung TME, resulting in impaired T cell priming and Tpex maintenance. At the molecular level, our data revealed that bacterial products derived from the local microbiota induce type I interferon expression in tumor-associated myeloid cells and enhance GM-CSF production from cancer cells: chronic type I interferon and GM-CSF signaling acts cooperatively to reduce the functional cDC1 population. Combining in vitro cDC1 assays, competitive bone marrow chimera experiments and cDC1-specific knockout models, we establish the causal relationships between the local microbiota, IFN-I and GM-CSF signaling in cDC1s, and the tumor response to ICB. Altogether, our study uncovered a dominant role of tumor-associated microbiota in inhibiting cDC1s and suppressing anti-tumor immunity through type I IFN and GM-CSF dependent pathways in lung cancer. Our findings not only identify the intra-tumoral microbiota as a critical therapeutic target for lung cancer treatment, but also provide new insights into the tissue-specific, context-dependent regulatory mechanisms of cDC1s in the TME. Citation Format: Qiang Dong, Chengcheng Jin. Tumor-associated microbiota suppresses anti-tumor immunity by driving cDC1 dysfunction in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C021.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Augmenting antitumor efficacy of Th17-derived Th1 cells through IFN-γ-induced type I interferon response network via IRF7

The importance of CD4+ T cells in cancer immunotherapy has gained increasing recognition. Particularly, a specific subset of CD4+ T cells coexpressing the T helper type 1 (Th1) and Th17 markers has demonstrated remarkable antitumor potential. However, the underlying mechanisms governing the differentiation of these cells and their subsequent antitumor responses remain incompletely understood. Single-cell RNA sequencing (scRNA-seq) data reanalysis demonstrated the presence of Th171 cells within tumors. Subsequent trajectory analysis found that these Th171 cells are initially primed under Th17 conditions and then converted into IFN-γ-producing cells. Following the in vivo differentiation trajectory of Th171 cells, we successfully established in vitro Th171 cell culture. Transcriptomic profiling has unveiled a substantial resemblance between in vitro-generated Th171 cells and their tumor-infiltrating counterparts. Th171 cells exhibit more potent antitumor responses than Th1 or Th17 cells. Additionally, Th171chimeric antigen receptor T (CAR-T) cells eradicate solid tumors more efficiently. Importantly, Th171 cells display an early exhaustion phenotype while retaining stemness. Mechanistically, Th171 cells migrate faster and accumulate more in tumors in an extracellular matrix protein 1 (ECM1)-dependent manner. Furthermore, we show that IFN-γ up-regulated IRF7 to promote the type I interferon response network and ECM1 expression but decreased the exhaustion status in Th171 cells. Taken together, our findings position Th171 cells as a great candidate for improving targeted immunotherapies in solid malignancies.

Type I Interferon Drives a Cellular State Inert to TCR-Stimulation and Could Impede Effective T-Cell Differentiation in Cancer.

Head and neck squamous cell carcinoma (HNSCC) is linked to human papillomavirus (HPV) infection. HPV-positive and HPV-negative HNSCC exhibit distinct molecular and clinical characteristics. Although checkpoint inhibitors have shown efficiency in recurrent/metastatic HNSCC, response variability persists regardless of HPV status. This study aimed to explore the CD8+ T-cell landscape in HPV-negative HNSCC. We performed simultaneous single-cell RNA and TCR sequencing of CD8+ tumor-infiltrating lymphocytes (TILs) from treatment-naïve HPV-negative HNSCC patients. Additionally, cells were stimulated ex vivo, which allowed for the tracking of clonal transcriptomic responses. Our analysis identified a subset of CD8+ TILs highly enriched for interferon-stimulated genes (ISG). TCR analysis revealed ISG cells are clonally related to a population of granzyme K (GZMK)-expressing cells. However, unlike GZMK cells, which exhibited rapid effector-like phenotypes following stimulation, ISG cells were transcriptionally inert. Additionally, ISG cells showed specific enrichment within tumor and were found across multiple tumor entities. ISG-enriched CD8+ TILs are a consistent feature of various tumor entities. These cells are poorly understood but possess characteristics that may impact antitumor immunity. Understanding the unique properties and functionality of ISG cells could offer innovative treatment approaches to improve patient outcomes in HPV-negative HNSCC and other cancer types.

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype

BackgroundNearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, virus detection and characterization were not at the earliest stages of acute infection. MethodsWe identified an X4-tropic T/F HIV-1 in a participant (40700) in the RV217 acute infection cohort. Coreceptor usage was determined in TZM-bl cell line, NP-2 cell lines, and primary CD4+ T cells using pseudovirus and infectious molecular clones. CD4 subset dynamics were analyzed using flow cytometry. Viral load in each CD4 subset was quantified using cell-associated HIV RNA assay and total and integrated HIV DNA assay. FindingsParticipant 40700 was infected by an X4 tropic HIV-1 without CCR5 using ability. This participant experienced significantly faster CD4 depletion compared to R5 virus infected individuals in the same cohort. Naïve and central memory (CM) CD4 subsets declined faster than effector memory (EM) and transitional memory (TM) subsets. All CD4 subsets, including the naïve, were productively infected. Increased CD4+ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions, while most of the R5 T/F viruses in the same cohort are sensitive to the same panel of bNAbs. InterpretationsX4-tropic HIV-1 is transmissible through mucosal route in people with wild-type CCR5 genotype. The CD4 subset tropism of HIV-1 may be an important determinant for transmissibility and virulence. FundingInstitute of Human Virology, National Institutes of Health, Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.

Single-cell analysis of bone marrow CD8+ T cells in Myeloid Neoplasms reveals pathways associated with disease progression and response to treatment with Azacitidine.

CD8+ T cells are crucial for antitumor immunity. In higher-risk myelodysplastic neoplasms (HR-MDS) and acute myeloid leukemia (AML), CD8+ T cells exhibit altered functionality. To address their role in the course of the disease, we performed in-depth immunophenotypic analysis of 104 pre-treatment bone marrow (BM) samples using mass and flow cytometry and observed an increased frequency of the CD57+CXCR3+ subset of CD8+ T cells in patients who failed azacitidine (AZA) therapy. Furthermore, an increased baseline frequency (>29%) of the CD57+CXCR3+CD8+ T cell subset was correlated with poor overall survival. We performed scRNA-seq to assess the transcriptional profile of BM CD8+ T cells from treatment-naive patients. The response to AZA was positively associated with the enrichment of IFN-mediated pathways, whereas an enhanced TGF-β signaling signature was observed in non-responders. Our results suggest that targeting CD8+ T cells with inhibitors of TGF-β signaling in combination with AZA is a potential therapeutic strategy for HR-MDS and AML.

Age-dependent immune profile in healthy individuals: an original study, systematic review and meta-analysis

BackgroundThe circulatory peripheral immune system is the most convenient approach for determining an individual’s immune status. Due to various reasons, while previous studies have addressed the critical impact of age, most individual studies did not analyze immunosenescence in a systemic manner, which complicates the possibility of building a reference range for age-dependent immune profiles for effective immune monitoring. To address this gap, this study analyzed a group of healthy individuals to establish age-specific reference ranges of the healthy circulatory immune profile, and a systematic review and meta-analysis were conducted to validate the findings and create generalizable immune cell reference ranges.ResultsOur study recruited a total of 363 healthy Taiwanese adults (median age 42 years [IQR 30, 62], age range 21 to 87 years, 43.3% male), including 158 under 40 years old, 127 between 40–64 years old, and 78 over 64 years old. Significant age-related alterations were observed in both adaptive and innate immune cell subsets. CD8 + T cells decreased and CD4/CD8 ratio increased, with notable increases in NK cells. CD4 + T cells were less impacted by aging, while CD8 + T cells significantly lost CD28 and increased CD31 expression with age. A clear reverse trend in naïve and memory subsets of CD4 + and CD8 + T cells was observed. Detailed reference ranges for immune cell subsets in healthy Taiwanese adults were established. A systematic review included 7,425 adults and a meta-analysis of 12 eligible studies confirmed our findings in Taiwan, enhancing generalizability.ConclusionsCombined with previous studies and original data through a systematic review and meta-analysis, we highlighted and quantified significant immune profile differences between older and younger individuals. The sex and age-specific reference ranges for peripheral immune cell subsets can serve as a basis for effective immune monitoring of various aging-related illnesses.

Selenopolysaccharide Isolated from Lentinula edodes Mycelium Affects Human T-Cell Function.

Lentinula edodes polysaccharides are natural immunomodulators. SeLe30, analyzed in this study, is a new mixture of selenium-enriched linear 1,4-α-glucans and 1,3-β- and 1,6-β-glucans isolated from L. edodes mycelium. In the present study, we evaluated its immunomodulatory properties in human T cells. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors' buffy coats. The effects of SeLe30 on CD25, CD366, and CD279 expression, the subsets of CD8+ T cells, and IFN-γ, IL-6, and TNF-α production were analyzed. SeLe30 downregulated CD25, CD279, and CD366 expression on T cells stimulated by the anti-CD3 antibody (Ab) and upregulated in unstimulated and anti-CD3/CD28-Abs-stimulated T cells. It increased the percentage of central memory CD8+ T cells in unstimulated PBMCs and naïve and central memory T cells in anti-CD3-Ab-stimulated PBMCs. SeLe30 decreased the number of central memory and naïve CD8+ T cells in anti-CD3/CD28-stimulated T cells, whereas, in PBMCs, it reduced the percentage of effector memory CD8+ T cells. Moreover, SeLe30 upregulated cytokine production. SeLe30 exhibits context-dependent effects on T cells. It acts on unstimulated T cells, affecting their activation while increasing the expression of immune checkpoints, which sensitizes them to inhibitory signals that can silence this activation. In the case of a lack of costimulation, SeLe30 exhibits an inhibitory effect, reducing T-cell activation. In cells stimulated by dual signals, its effect is further enhanced, again increasing the "safety brake" of CD366 and CD279. However, the final SeLe30 effect is mediated by its indirect impacts by altering interactions with other immune cells.

Single-cell sequencing reveals immune features of treatment response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma

Neoadjuvant immunochemotherapy (nICT) has dramatically changed the treatment landscape of operable esophageal squamous cell carcinoma (ESCC), but factors influencing tumor response to nICT are not well understood. Here, using single-cell RNA sequencing paired with T cell receptor sequencing, we profile tissues from ESCC patients accepting nICT treatment and characterize the tumor microenvironment context. CXCL13+CD8+ Tex cells, a subset of exhausted CD8+ T cells, are revealed to highly infiltrate in pre-treatment tumors and show prominent progenitor exhaustion phenotype in post-treatment samples from responders. We validate CXCL13+CD8+ Tex cells as a predictor of improved response to nICT and reveal CXCL13 to potentiate anti-PD-1 efficacy in vivo. Post-treatment tumors from non-responders are enriched for CXCL13+CD8+ Tex cells with notably remarkable exhaustion phenotype and TNFRSF4+CD4+ Tregs with activated immunosuppressive function and a significant clone expansion. Several critical markers for therapeutic resistance are also identified, including LRRC15+ fibroblasts and SPP1+ macrophages, which may recruit Tregs to form an immunosuppressive landscape. Overall, our findings unravel immune features of distinct therapeutic response to nICT treatment, providing a rationale for optimizing individualized neoadjuvant strategy in ESCC.

Effects of Bacterial Lysates on TLRs/NF-κB Pathway, Immune Function, and Prognosis in Children with Pulmonary Infection Caused by Streptococcus pneumoniae

Background: Bacterial lysates, as immunomodulators, may play a role in regulating the host immune response. Objectives: This study aimed to investigate the effects of bacterial lysates OM85-BV in treating pulmonary infections caused by Streptococcus pneumoniae in children. Methods: A total of 120 children with S. pneumoniae pulmonary infection were divided into two groups: The control group and the OM85-BV group, with a 1: 1 ratio. The control group received intravenous mezlocillin at 100 mL/kg/d, while the OM85-BV group, in addition to mezlocillin, received OM85-BV orally at 3.5 mg per dose. The improvement of clinical symptoms and pulmonary function indexes—including respiratory rate, tidal volume, volume to peak expiratory flow as a proportion of exhaled volume, and time to peak tidal expiratory flow as a proportion of expiratory time—were compared. Additionally, levels of C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), soluble interleukin-2 receptor (sIL-2R), T lymphocyte subsets, and immunoglobulins in serum were analyzed. Results: Compared to the control group, the OM85-BV group showed a significant decrease in respiratory rate and an increase in tidal volume, volume to peak expiratory flow, and time to peak tidal expiratory flow (P < 0.05 for all). The OM85-BV group also had significantly lower levels of serum inflammatory factors such as CRP, TNF-α, IL-6, and sIL-2R. In terms of immune function, the OM85-BV group showed increased levels of T lymphocyte subsets CD3+, CD4+, and CD4+/CD8+ ratios, with a decrease in CD8+, as well as elevated serum IgA and IgG levels. The total effective rate of treatment in the OM85-BV group was significantly higher than in the control group (P < 0.05). Conclusions: The addition of OM85-BV to the treatment of S. pneumoniae pulmonary infection in children significantly improves clinical symptoms, lung function, and immune function, while reducing the inflammatory response. OM85-BV appears to inhibit the activation of the toll-like receptors (TLRs) and nuclear factor kappa-B (NF-κB) pathway.

An abnormal increase in CD26(-)CD28(-) cytotoxic effector CD4 and CD8 T cell populations in patients with systemic lupus erythematosus.

CD26 is a human T cell costimulatory molecule as well as a T cell subset marker, and increase of CD26+ T cells in inflamed tissues and peripheral blood has been reported in diverse autoimmune diseases. In contrast, our group has previously shown that levels of circulating CD26+ T cells are decreased in patients with systemic lupus erythematosus (SLE), although the role of reduced CD26 T cell surface expression in SLE pathology remains to be elucidated. In the present study, we conducted CD26-based T cell subset analyses utilizing peripheral blood mononuclear cells from 57 SLE patients and 31 healthy adult volunteers. We show that the increase in CD26(-) T cell population reflects the abnormal expansion of CD26(-)CD28(-) cytotoxic subsets of both CD8 T cells and CD4 T cells in SLE patients. Single cell RNA sequencing analysis of the CD26(-)CD28(-) CD4 and CD8 T cell populations reveals unique characteristics with similarities to natural killer T cells. In addition, the level of CD26(-)CD28(-) T cells is increased in some active stage SLE patients with renal manifestation. Meanwhile, effect of prednisolone treatment on these populations varies from patient to patient, with levels of these cytotoxic effector populations still being elevated in some inactive stage SLE patients. Taken together, our data suggest that analysis of these populations in SLE may be a useful tool to classify this markedly heterogeneous condition.

Rheumatoid Arthritis: What Inflammation Do We Face?

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by symmetrical joint inflammation, cartilage degradation, and bone erosion. This review explores the multifaceted aspects of RA pathogenesis, focusing on the dynamic interplay between innate and adaptive immune responses, genetic predisposition, and environmental triggers. The development of RA involves genetic susceptibility and trigger events such as infections, trauma, smoking, obesity, and microbiome alterations, fostering autoimmune reactions and tissue/organ destruction. The innate immune response, including toll-like receptor activation and synovial fibroblasts’ roles, contributes to the acceleration of inflammatory processes in joint tissues. Monocytes and macrophages organize and sustain chronic joint inflammation, leading to tissue damage and bone resorption, while highlighting the significance of CD14 and CD16 subsets in RA pathogenesis. In the adaptive immune response, aberrant activation and proliferation of CD4+ T cells and the role of regulatory T cells in maintaining immune tolerance are discussed. Target cytokines like TNF-α, IL-6, IL-1, IL-17, and BAFF, as well as chemokines such as CCL2, CXCL10, CCL5, and CXCL12, have emerged as critical components in managing chronic inflammation and joint damage in RA. This comprehensive overview provides insights into the pathophysiology of RA and potential therapeutic avenues, emphasizing the importance of understanding these complex immunological and genetic mechanisms for developing more effective treatment strategies.

Inhibitory Fcγ receptor deletion enhances CD8 T cell stemness increasing anti-PD-1 therapy responsiveness against glioblastoma

BackgroundCertain cancers present challenges for treatment because they are resistant to immune checkpoint blockade (ICB), attributed to low tumor mutational burden and the absence of T cell-inflamed features. Among these,...

Analysis of Frequencies and Subsets of Peripheral Helper T Cells in Patients with Immune Thrombocytopenia

To investigate the frequencies and subset distribution of peripheral helper (Tph) T cells in patients with immune thrombocytopenia (ITP), and explore the pathogenesis of ITP. A total of 25 newly diagnosed ITP patients treated in The Second Affiliated Hospital of Dalian Medical University from January to December 2022 were selected, and 25 healthy volunteers (age- and sex-matched) were recruited as the control group. Flow cytometry was used to detect the subsets of CD4+ T cells and Tph cells. The frequency of effector memory (CCR7- CD45RO+CD4+) T cells in ITP patients was significantly higher than that in healthy controls(P < 0.05). The frequency of Tph cells in ITP patients was also significantly higher than that in healthy controls (P < 0.001), and most of the Tph cells in ITP patients were effector memory T cells. Furthermore, the expressions of T-cell costimulatory molecules in Tph cells, including ICOS and CD84, were similar to those in follicular helper T(Tfh) cells. CXCR3 - CCR6 -Tph (Tph2) subgroup was dominant in Tph cells, but the frequency of CXCR3+CCR6- Tph (Tph1) cells in ITP patients was much higher than that in healthy controls (P < 0.05). Tph cells, especially Tph1 cells, were abnormally expanded in ITP patients, which may be a potential etiology of ITP.

Tofacitinib alleviated salivary gland inflammation and reduced the percentages of effector T cells in murine Sjögren's disease.

The Janus kinases-signal transducer and activator of transcription (JAK-STAT) signalling pathway plays a crucial role in autoimmunity and the signalling pathways of many cytokines in Sjögren's disease (SjD). Therefore, the aim of this study was to investigate both the therapeutic and immunomodulatory effects of the oral JAK3/JAK2/JAK1 inhibitor tofacitinib in a murine model of SjD. Tofacitinib or vehicle was administered orally to the mice with SjD for 6 weeks. Salivary flow rate was measured every three weeks. Pathological changes of salivary gland were detected by haematoxylin-eosin staining, and the percentages of subsets of CD4+ T cells and B cells in the cervical lymph nodes (cLNs) and spleen was determined by flow cytometry. Tofacitinib significantly ameliorated submandibular gland inflammation compared to the control group, as evidenced by reduced lymphocytic infiltration. Salivary flow rates improved significantly in tofacitinib treated mice compared to controls, indicating restored salivary gland function. The treatment also led to a substantial decrease in follicular helper T (Tfh) cells and the Tfh/Treg ratio in both the spleen and cLNs. Additionally, the frequencies of T helper 1 (Th1) and T helper 17 (Th17) cells were reduced in the spleen and cLNs. Our data indicated that tofacitinib reduced percentages of effector T cells in an animal model of SjD. In addition, tofacitinib alleviated salivary gland inflammation and hypofunction, offering new insights into the clinical management of SjD.

Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain’s innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.

C-CBL/LCK/c-JUN/ETS1/CD28 axis restrains childhood asthma by suppressing Th2 differentiation

BackgroundAsthma is a common immune disease with high morbidity in children. Type 2 inflammation is the center of asthma development, and mainly mediated by a subset of CD4 + T cells, T helper 2 (Th2) cells. Excess Th2 differentiation was generally associated with asthmatic attack. Casitas B-lineage lymphoma (c-CBL) was reported to involved in T cell development and databank showed its decreased expression in CD4 + T cells from peripheral blood of asthmatic children. This study aims to investigate the role of c-CBL in childhood asthma and Th2 differentiation, and explore the underlying mechanism.MethodsWe collected peripheral blood samples from clinical childhood asthma cases and healthy controls, and determined c-CBL expression in CD4 + T cells. Asthma was induced in neonatal mice by ovalbumin (OVA) intraperitoneal injection and aerosol inhalation, and c-CBL expression in CD4 + T cells from peripheral blood and spleen was measured. Gain-of-function experiments was performed to confirm the effects of c-CBL on Th2 differentiation in vitro. Finally, c-CBL was delivered into asthmatic mice via lentivirus infection to verify its effects on experimental asthma.Resultsc-CBL was lowly expressed in CD4 + T cells from asthmatic children than those of healthy controls. Similarly, it was downregulated in CD4 + T cells from peripheral blood and spleen of asthma mice. Overexpression of c-CBL restrained lung pathological injury and type 2 inflammation in experimental asthmatic mice. Gain-of-function experiments demonstrated that c-CBL inhibited Th2 differentiation of CD4 + T cells from healthy children, and mediated the ubiquitination of lymphocyte cell-specific protein-tyrosine kinase (LCK). LCK acted as a kinase to phosphorylate and activate c-JUN, which was predicted to bind promoter sequence of CD28 by bioinformatic analysis. Dual-luciferase reporter assay verified that c-JUN and ETS1 synergically enhanced transcription of CD28, and this transcription activation was aggravated by LCK overexpression.Conclusionc-CBL alleviated asthma and suppressed Th2 differentiation by facilitating LCK ubiquitination, interrupting c-JUN activation and CD28 expression in vivo and in vitro. c-CBL/LCK/c-JUN/ETS1/CD28 axis was partially involved in childhood asthma, and may provide novel insights for clinical treatment for asthma.

Differential expression of HIV target cells CCR5 and α4β7 in tissue resident memory CD4 T cells in endocervix during the menstrual cycle of HIV seronegative women.

Ovarian hormones are known to modulate the immune system in the female genital tract (FGT). We sought to define the impact of the menstrual cycle on the mucosal HIV target cell levels, and tissue-resident CD4 T cells. Here, we characterized the distribution, phenotype, and function of CD4 T cells with special emphasis on HIV target cells (CCR5+ and α4β7+) as well as tissue-resident memory (TRM; CD69+ and CD103+) CD4 T cells in FGT of cycling women. Peripheral blood and Endocervical cells (EC-collected from cytobrush) were collected from 105 healthy women and performed multicolor flow cytometry to characterize the various subsets of CD4 T cells. Cervicovaginal lavage (CVL) were collected for cytokine analysis and plasma were collected for hormonal analysis. All parameters were compared between follicular and luteal phase of menstrual cycle. Our findings revealed no significant difference in the blood CD4 T cell subsets between the follicular and luteal phase. However, in EC, the proportion of several cell types was higher in the follicular phase compared to the luteal phase of menstrual cycle, including CCR5+α4β7-cells (p=0.01), CD69+CD103+ TRM (p=0.02), CCR5+CD69+CD103+ TRM (p=0.001) and FoxP3+ CD4 T cells (p=0.0005). In contrast, α4β7+ CCR5- cells were higher in the luteal phase (p=0.0004) compared to the follicular phase. In addition, we also found that hormonal levels (P4/E2 ratio) and cytokines (IL-5 and IL-6) were correlated with CCR5+ CD4 T cells subsets during the follicular phase of the menstrual cycle. Overall, these findings suggest the difference in the expression of CCR5 and α4β7 in TRM CD4 T cell subsets in endocervix of HIV seronegative women between the follicular and luteal phase. Increase in the CCR5+ expression on TRM subsets could increase susceptibility to HIV infection during follicular phase of the menstrual cycle.

BAFF promotes follicular helper T cell development and germinal center formation through BR3 signal.

T follicular helper (Tfh) cells represent an important subset of CD4+ T cells that is crucial to the maturation and differentiation of B cells and the production of high-affinity antibodies. Since BAFF, a vital B cell survival factor, is also crucial to B cell maturation and differentiation, we assessed the effects of BAFF on Tfh cell development and function. We demonstrate that deficiency of BAFF, but not of APRIL, markedly inhibits Tfh cell development, germinal center (GC) formation, and antigen-specific antibody production. The promoting effect of BAFF on Tfh cell development is dependent on expression of BR3 on T cells, and its promoting effect on GC formation is dependent on expression of BR3 on both T cells and B cells. BAFF directly promotes expression of the Tfh cell-characteristic genes via NF-κB signaling. This effect does need BR3 expression. Thus, BAFF not only has direct effects on B cells, but it also has direct effects on Tfh cell differentiation via engagement of BR3 which collectively promote GC formation and production of high-affinity antibodies. This dual effect of BAFF on B cells and Tfh cells may help explain the clinical utility of BAFF antagonists in the management of certain autoimmune diseases.