Intracellular Staining Research Articles

Age-specific dynamics of neutralizing antibodies, cytokines, and chemokines in response to La Crosse virus infection in mice.

La Crosse virus (LACV) is a primary cause of pediatric arboviral encephalitis in the United States, particularly affecting children aged 16 years or younger. This age-related susceptibility extends to murine models, where weanling mice (3 weeks old) succumb to LACV infection, while adults (≥6 weeks old) demonstrate resistance. Despite its clinical relevance, the host immune response to LACV is not fully understood. In this study, we investigated the roles of neutralizing antibodies (nAbs), cytokines, and chemokines in weanling and adult mice following infection with 5 × 105 plaque-forming units (PFU) of LACV. Weanling mice demonstrated early disease onset with elevated peripheral viremia, but passive transfer of adult serum, confirmed to have nAbs, to naïve weanlings prior to infection completely rescued them from death. Moreover, adult mice had increased Th1 cytokines, Th9/Th17/Th22/Treg cytokines, and many chemokines. In contrast, weanlings had higher Th2 cytokines, correlating with symptoms onset. Flow cytometry and intracellular cytokine staining further demonstrated that weanling mice produced higher levels of IL-4 by CD4+ and CD8+ T cells compared to adults, regardless of infection status. Conversely, LACV-infected adult mice had increased IFN-γ production by CD8+ T cells compared to uninfected controls. Finally, the adoptive transfer of splenocytes from immune adult mice to naïve weanlings delayed neurological symptoms and improved survival. In conclusion, this study links nAbs and cytokine and chemokine responses to protective immunity in adult mice, contrasting with the pathogenesis seen in weanlings. These findings underscore the importance of further research into innate and adaptive immune mechanisms during LACV infection.IMPORTANCELa Crosse virus (LACV) is a primary cause of pediatric encephalitis in the United States, with an impact on children aged 16 years or younger. This age-related susceptibility is recapitulated in mouse models, where young mice succumb to LACV-induced disease, while adults demonstrate resistance. Our understanding of host responses to LACV remains underexplored. This study sheds light on the dynamics of neutralizing antibodies (nAbs), cytokines, and chemokines following LACV infection in both adult and weanling mice. Our study reveals age-specific variations in viremia, neutralizing antibody titers, survivability, and levels of cytokines and chemokines. Adult mice exhibit significantly elevated levels of Th1 cytokines, contrasting with elevated levels of Th2 cytokines observed in weanling mice, often coinciding with the onset of symptoms. These data reveal age-specific dynamics in cytokines and chemokines associated with protective versus pathogenic immunity, emphasizing the need for further studies on innate and adaptive immunity.

Fibrocytes in tumor microenvironment: Identification of their fraction and novel therapeutic strategy.

Fibrocytes were identified as bone marrow-derived myeloid cells that also have fibroblast-like phenotypes, such as ECM production and differentiation to myofibroblasts. Although fibrocytes are known to contribute to various types of tissue fibrosis, their functions in the tumor microenvironment are unclear. We focused on fibrocytes as pivotal regulators of tumor progression. Our previous studies have indicated that fibrocytes induce angiogenesis and cancer stem cell-like phenotypes by secreting various growth factors. In contrast, immune checkpoint inhibitor (ICI)-treated fibrocytes demonstrated antigen-presenting capacity and enhanced antitumor T cell proliferation. Taken together, these findings indicate that fibrocytes have multiple effects on tumor progression. However, the detailed phenotypes of fibrocytes have not been fully elucidated because the isolation of distinct fibrocyte clusters has not been achieved without culturing in ECM-coated conditions or intracellular staining of ECM. The development of single-cell analyses partially resolves these problems. Single-cell RNA sequences in CD45+ immune cells from tumor tissue identified ECM-expressing myeloid-like cells as distinct fibrocyte clusters. In addition, these findings enabled the isolation of tumor-infiltrating fibrocytes as CD45+CD34+ cells. These tumor-infiltrating fibrocytes demonstrated both antigen-presenting ability and differentiation into myofibroblast-like cancer-associated fibroblasts. Considering these functions of fibrocytes in tumor progression, molecular-targeting agents for the migration, activity, and differentiation of fibrocytes are promising therapeutic strategies. Furthermore, identification of specific cell surface markers and master regulators of fibrocytes will advance novel fibrocyte-targeting therapies. In this review, we discuss the multiple roles of tumor-infiltrating fibrocytes and novel cancer therapeutic strategies.

γδ T17 Cells Regulate the Acute Antiviral Response of NK Cells in HSV-1-Infected Corneas.

To determine whether γδ T cells regulate natural killer (NK) cells in the herpes simplex virus 1 (HSV-1)-infected cornea. CD57Bl/6 (wild-type [WT]), TCRδ-/-, and IFN-γ-/- mice were infected intracorneally with HSV-1. TCR-/- mice were treated with IL-17A at 24 hours post-infection (PI), and the WT mice received treatments of fingolimod (FTY720) and anti-IL-17A. At 48hours PI, corneas were excised, and intracellular staining flow cytometry was performed, as well as multiplex analysis. Additionally, single-cell RNA sequencing (scRNAseq) was done to analyze the transcriptome of NK cells from WT and TCRδ-/- mice. In mice lacking γδ T cells, there were significantly fewer NK cells following ocular HSV-1 infection. This reduction of NK cells corresponded with lower levels of cytokines and chemokines associated with the antiviral response. Furthermore, NK cells from WT mice had enriched IL-17A signaling compared to those from TCRδ-/- mice. The NK cell response was partially rescued in TCRδ-/- mice by administration of IL-17A. Correspondingly, the NK cell response could be blunted in WT mice by administration of anti-IL-17A. Finally, IFN-γ-/- mice had significantly less IL-17A production compared to WT mice. γδ T17 cells promote NK cell accumulation in HSV-1-infected corneas. In turn, NK cells secrete IFN-γ, which negatively regulates further IL-17A production by γδ T cells.

Hyperlipidemia interferes with the behavior of variant NKT cells

Abstract Background NKT cells significantly influence atherosclerosis development. However, inconsistent findings on the function of the NKT cells and the invariant subset in atherosclerosis remains a challenge. Moreover, there is no clarity on the role and phenotype of the variant (vNKT) subset in atherosclerosis. Purpose To understand the role of the vNKT subsets in HFD-induced atherosclerosis. Methods We extracted the data for meta-analysis to understand the role of the whole NKT and iNKT cells in atherosclerosis. PRISMA guidelines were followed for the meta-analysis. In addition, C57BL/6 mice (WT) were fed with HFD for 20 weeks from 6-8 weeks of age. vNKTs were identified as CD3+NK1.1+CD1d-tetramer- within the CD3+NK1.1+ NKT cell population in both liver and spleen by flow cytometry. Intracellular cyotkines staining was performed. Statistical differences inthe means of two groups are calculated by two-tailed Student’s t-test with nonparametric Mann-Whitney tests. P-value<0.05 was considered significant. All data represented as mean±SEM. Results The NKT cell numbers increased significantly in the spleen, lymph nodes, blood, and liver in the HFD-fed mice fed from 1.5 to 24 weeks in Apoe-/-, Ldlr-/- in comparison to wild-type mice (SMD, 2.51 [95% CI, 1.42, 3.61]). Contrary to this, the iNKT cell numbers and the iNKT TCR gene expression levels, decreased significantly in the HFD-fed mice (SMD, -2.04 [95% CI, -3.34, -0.75]). The reduction in iNKT cell numbers is also supported by the clinical data from the AMI patients when compared to healthy controls [SMD = -1.81, 95% CI = -2.89, -0.74] (Figure 1). In HFD-fed C57BL/6 hyperlipidemic mice, significantly increased levels of total cholesterol (HFD vs Chow; 226.5±29.66 vs 118.5±10.56 mg/dL, n=4) and LDLc (44.56±11.96 vs 16.69±1.13 mg%, n=4) was observed. The number of vNKTs in the liver was significantly increased by 46% (HFD vs Chow 28.35±2.28 vs 19.41±1.36, n=6-7) (Figure 2A). However, the CD8+ vNKTs were significantly decreased by 68.6% in the liver (4.72±0.99 vs 15.06±0.96) and the CD4+ vNKTs decreased more than two times in the HFD-fed WT liver (7.39±0.76 vs 18.28±0.36) (Figure 2B). Also, the intracellular IFN-γ+ vNKT cells doubled in their numbers in the HFD-fed spleen (14.31±1.39 vs 6.367±0.87) compared to the chow-fed mice (Figure 2C). Conclusion(s) Meta-analysis revealed dual nature of the NKT cells and its invariant subsets in atherosclerosis, since the whole NKTs increase whereas the iNKTs decrease in number. The vNKT subset actively participates in immune modulation in the HFD-induced hyperlipidemia by increasing their number, as well as by differentially regulating CD8 and CD4 co-receptors between chow and HFD. HFD induced a pro-inflammatory nature in the splenic vNKTs but not in liver vNKTs, that in turn may aggravate other immune cells. This data confirms the dual nature of the NKT subsets and reveals a crucial systemic involvement of vNKT cells in the progression of dyslipidemia.

Long term T cell response and safety of a tetravalent dengue vaccine in healthy children

As robust cellular responses are important for protection against dengue, this phase 2 study evaluated the kinetics and phenotype of T cell responses induced by TAK-003, a live-attenuated tetravalent dengue vaccine, in 4–16-year-old living in dengue-endemic countries (NCT02948829). Two hundred participants received TAK-003 on Days 1 and 90. Interferon-gamma (IFN-γ) enzyme-linked immunospot assay [ELISPOT] and intracellular cytokine staining were used to analyze T cell response and functionality, using peptide pools representing non-structural (NS) proteins NS3 and NS5 matching DENV-1, -2, -3, and -4 and DENV-2 NS1. One month after the second TAK-003 dose (Day 120), IFN-γ ELISPOT T cell response rates against any peptide pool were 97.1% (95% CI: 93.4% to 99.1%), and similar for baseline dengue seropositive (96.0%) and seronegative (98.6%) participants. IFN-γ ELISPOT T cell response rates at Day 120 were 79.8%, 90.2%, 77.3%, and 74.0%, against DENV-1, -2, -3, and -4, respectively, and remained elevated through 3 years post-vaccination. Multifunctional CD4 and CD8 T cell responses against DENV-2 NS peptides were observed, independent of baseline serostatus: CD8 T cells typically secreted IFN-γ and TNF-α whereas CD4 T cells secreted ≥ 2 of IFN-γ, IL-2 and TNF-α cytokines. NAb titers and seropositivity rates remained substantially elevated through 3 years post-vaccination. Overall, TAK-003 was well tolerated and elicited durable T cell responses against all four DENV serotypes irrespective of baseline serostatus in children and adolescents aged 4–16 years living in dengue-endemic countries. TAK-003-elicited CD4 and CD8 T cells were multifunctional and persisted up to 3 years post-vaccination.

The impact of cryopreservation on cytokine secretion and polyfunctionality in human PBMCs: a comparative study.

Human peripheral blood mononuclear cells (hPBMCs) are widely used in fundamental research and clinical applications as studying their responses to in vitro activation is an effective way to uncover functional alterations and disease associated phenotypes. However, the availability of samples in large numbers at a specific time and location remains challenging, hence they often might preferably be collected and cryopreserved for later analysis. While the effect of cryopreservation on viability and cell surface expression is well established, changes in activity and cytokine secretion still lead to conflicting results as it is often measured in bulk or within the cells. Here, we used our platform for dynamic single-cell multiplexed cytokine secretion measurement and compared it to a traditional intracellular cytokine staining to quantify the effect of cryopreservation on cytokine secretion and expression of individual hPBMCs. Following stimulation with LPS or anti-CD3/CD28 antibodies for up to 36 or 72 h incubation, we observed distinct alterations in cytokine responses due to cryopreservation when comparing to fresh samples, but also remarkable consistencies for some cytokines and parameters. In short, the frequencies of cytokine-secreting cells in cryopreserved samples were lower for IL-6 (LPS), IL1-β (CD3/CD28) and IFN-γ (CD3/CD28), while the frequency and dynamics of IL-8 secretion were strongly impacted in all cases. We observed a large disconnect between cytokine expression and secretion for TNF-α, where the expression dramatically increased after cryopreservation, but actual secretion was, in comparison, remarkably stable. The polyfunctionality of single cells was altered by cryopreservation in specific co-secreting populations led by the effects on IL-6 or IL-8 secretion. Among immune cells, cryopreservation seemed to affect lymphocytes and monocytes differently as effects appeared early on in lymphocytes while generally observed in later time points in monocytes. Together, this study offers an in-depth quantitative insight into the biological behavior of immune cells in response to cryopreservation and stimulation, further providing some insights into conflicting results in theliterature as well as guidelines for researchers planning to assess cytokine-secreting from frozen hPBMCs in immunological research or clinical applications.

Hyperfunctional T cell responses unchecked by regulatory T cells are unable to resolve hepaciviral infection without humoral contribution

Background & AimsThe most recent T cell-based vaccine against hepatitis C virus (HCV) in human subjects failed to swing the pendulum from chronicity to resolution despite eliciting cellular responses in the majority of individuals. These results naturally evoke the question of whether hyperactivated responses of a single adaptive immune arm are capable of inducing HCV clearance or if coordinated efforts between antibodies and T cells are indeed necessary. Here, we sought to address this point in determining whether the suppression of antiviral T cell and IgG responses by regulatory T cells (Tregs) is a critical prerequisite of delayed viral clearance or overt chronicity. MethodsUsing a surrogate model of HCV infection, rodent hepacivirus (RHV) infection in mice, we utilized Foxp3-DTR mice to assess how Tregs modulate the generation of acute antiviral adaptive immune responses and indirectly dictate infection fate via intracellular flow cytometry staining, ELISA, RNA sequencing, and qPCR. ResultsTransient depletion of Tregs prior to infection decreased viral-specific CD4+ T cell function, IgG production, and delayed viral clearance. In contrast, transient Treg depletion after infection increased both T cell functionality and IgG production, thereby facilitating accelerated viral clearance. Hyperactivated T cells, achieved via transient Treg depletion, were unable to clear the virus as an isolated effector arm without the help of viral-specific IgG production. ConclusionsTregs control the outcome of RHV infection via direct modulation of CD4+ T cells and IgG production. Hyperactivated T cell responses are incapable of compensating for experimentally induced lack of antibodies, further reinforcing the notion of cooperative interplay between adaptive immune arms in facilitating hepaciviral clearance. Impact and implicationsWe demonstrate herein how timing of Treg depletion determines the fate of effector T cells, humoral responses, and the kinetics of viral clearance. Our observations provide direct evidence that functional T cell responses are incapable of compensating for suboptimal humoral responses in facilitating viral resolution. Our results imply that future HCV vaccine regimens should not solely rely on eliciting focused responses of a single effector arm, but rather incorporate immunogens capable of inducing durable features of both humoral and cellular memory.

Novel H-2Db-restricted CD8 epitope derived from mouse MAGE-type antigen P1A mediates antitumor immunity in C57BL/6 mice

BackgroundMelanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse...

Rapamycin Prevents Expansion of Costimulation Blockade-resistant CD8+ Alloreactive Memory Cells following Depletional Induction in Renal Transplant Recipients.

Alemtuzumab induction with belatacept/rapamycin-based maintenance immunotherapy (ABR) prevents kidney allograft rejection and specifically limits early costimulation blockade-resistant rejection (CoBRR). To evaluate the mechanisms by which this regimen alters CoBRR, we characterized the phenotype and functional response of preexisting memory cells to allogeneic endothelial cells using intracellular cytokine staining and flow cytometry. IL-7-induced lymphocyte proliferation in the presence or absence of rapamycin was assessed to characterize the phenotype of proliferating cells. Lymphocytes from 40 recipients who underwent transplant using the ABR regimen were studied longitudinally. The rapid immunoresponses of preexisting alloreactive cells to allogeneic endothelial cells were predominantly CD8+TNF-α+/IFN-γ+ cells. These cells were effector memory (TEM) and terminally differentiated effector memory cells lacking CD28 expression, and most were CD57+PD1-. Neither rapamycin nor belatacept directly inhibited these cells. IL-7, a cytokine induced during lymphopenia postdepletion, provoked dramatic CD8+ TEM cell proliferation and a low level of CD8+CD57+PD1- cell expansion invitro. The IL-7 stimulation induced CD8+ cell mTOR phosphorylation, and rapamycin treatment markedly inhibited IL-7-induced TEM and CD57+PD1- cell expansion. This effect was evident in patients receiving the ABR in that the repopulation of CD8+CD57+PD1- TEM cells was substantially suppressed for at least 36 mo after transplant. These findings help define one mechanism by which a costimulation blockade/rapamycin-based therapy following alemtuzumab induction minimizes CoBRR, namely that in the presence of rapamycin, costimulation-resistant alloreactive cells are disproportionately ineffective at repopulating following post-transplant T cell depletion.

The role of CD24hiCD27+ regulatory B cells in human chronic rhinosinusitis with/without nasal polyps

BackgroundRegulatory B cells (Bregs) reduce allergic and autoimmune inflammation. However, their role in chronic rhinosinusitis (CRS) remains unknown. This study investigated the frequency and function of Breg subsets in the peripheral blood of patients with CRS. MethodsThe demographic and clinical characteristics were compared among control, CRSsNP, neCRSwNP, and eCRSwNP groups. The expression of various Breg subtypes was evaluated in peripheral blood mononuclear cells (PBMCs) of patients with eosinophilic CRS with nasal polyps (eCRSwNP), non-eosinophilic CRS with nasal polyps (neCRSwNP), CRS without nasal polyps (CRSsNP). CD19+CD24hiCD27+ B cells (B10 cells) were isolated by flow cytometry, followed by RNA sequencing (RNA-seq). Finally, IL-10 secreted by B10 cells were evaluated through the intracellular stain. ResultsA higher number of eosinophils in peripheral blood and nasal polyps were found in eCRSwNP compared with neCRSwNP, CRSsNP, and control groups. The frequency of B10 in the peripheral blood B cells (B10%) of patients with eCRSwNP was significantly lower than that in the neCRSwNP and control groups. B10% was negatively correlated with the quantity of tissue eosinophils, and the percentage and absolute value of peripheral blood eosinophils. The eCRSwNP, neCRSwNP and control groups had 1403 differentially expressed genes, 35 of which were identified in four highly enriched pathways. Additionally, the frequency of IL-10+B10 cells in peripheral blood was lower in patients with eCRSwNP than in the neCRSwNP and control groups. ConclusionThis study is the first to reveal differences in both the quantity and IL-10 secretion of B10 cells in patients with eCRSwNP and neCRSwNP. These variations were strongly negatively associated with eosinophils in nasal polyps and peripheral blood. IL-10+B10 cells may play a key role in the pathological mechanisms of CRS, particularly the recurrence of eCRSwNP.

Different Antigen-Specific CD4+ and CD8+ T-Cell Response against HCMV Proteins in Pregnant Women with Primary Infection and in Control Subjects with Remote Infection.

Background/Objectives: Human cytomegalovirus (HCMV) is the most frequent cause of congenital infections. The HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection in pregnancy. In this study, the antigen-specific T-cell response against different HCMV proteins (IE-1, pp65, gB, gHgLpUL128L) was investigated in pregnant women with primary infection and in control subjects with remote infection to identify possible components of a vaccine. Methods: Blood samples from 35 pregnant women with HCMV primary infection and 30 HCMV-seropositive healthy adult subjects with remote infection were tested. The antigen-specific T-cell response was measured using cytokine intracellular staining after stimulation with IE-1, pp65, gB and gHgLpUL128L peptides pool. Results: The pp65-specific CD4+ T-cell response was higher in pregnant women with HCMV primary infection at the late time point and in control subjects with remote infection, while the pregnant women at the early time point showed a higher gB-specific CD8+ T-cell response. Regarding the CD4+ and CD8+ T-cell phenotypes, we observed that HCMV-specific CD4+ and CD8+ T cells expressing CD45RA+ remained constant in pregnant women with primary infection at the early and late time points and in subjects with remote infection, while HCMV-specific CD4+ and CD8+ T cells expressing IL-7R+ or producing IL-2 were higher in control subjects with remote infection than in pregnant women with HCMV primary infection. Conclusions: The T-cell response was higher against gB in the early phase of infection and against pp65 in the late phase. Therefore, these proteins should be taken into consideration as candidates for a vaccine.

Abstract P176: Elevated Circulating T Cell-Monocyte Complexes in Long COVID-19 Tachycardia Syndrome: Implications of Immune Dysregulation, Inflammation, and Disease Progression

Background: Postural orthostatic tachycardia syndrome (POTS) is a common complication in Long-COVID. Long-COVID POTS (LCP) is characterized by abnormal orthostatic tachycardia, symptoms of orthostatic intolerance, and persistent inflammation. Hypothesis: Novel parameters of immune activation exist in and likely contribute to the pathogenesis of LCP. Methods: We enrolled 15 LCP patients and 5 subjects who recovered from COVID-19 infection without lingering effects as controls. Hemodynamic parameters were measured at baseline and during a 10-minute 75° head-up tilt. Flow cytometry was used to detect circulating T cells, monocyte subsets, and their cytokines. Immune synapse formation between T cells and monocytes was detected using Forster Resonance Energy Transfer (FRET) between T cell receptors (TCR) and human leukocyte antigens (HLAs). Intracellular staining was used to detect the cytokines IL-17A, IFN-γ and IL-6. Results: Subjects with LCP exhibited a greater increase in heart rate (HR) during 10-min upright tilt compared with controls (50.23± 4.6 vs. 20.33 ± 3.17 bpm, p<0.01, Figure). Circulating monocyte/T cell complexes were increased in LCP vs controls and these exhibited high TCR/HLA FRET ( p<0.005 ), suggesting immune synapse formation. Importantly, complexed T cells demonstrated higher levels of IL-17A and IFN-γ, compared to non-complexed T cells and a profound increase in intracellular IL-6 compared to non-complexed monocytes ( p<0.005 ). Notably, the percent of IFN-γ + and IL-17A + T cells in the monocyte/T cell complexes of LCP subjects positively correlated with their increase in HR (ΔHR) in 10 min active standing test (Figure). Conclusion: Circulating T cell-monocyte complexes are markedly increased in humans with LPC, and exhibit evidence of functional and dynamic cellular interactions. We propose that these contribute to persistent inflammation and the pathogenesis of autonomic dysfunction in these subjects.

Attenuation of HIV-Specific T Cell Responses Among People with HIV on art Following Dipyridamole Treatment.

Twelve weeks of dipyridamole increased extracellular adenosine levels and decreased T cell activation in people with HIV. In this analysis, we investigated the effect of dipyridamole on HIV-specific T cell responses. We compared changes in Gag- and Env-specific T cell responses using intracellular cytokine staining, following 12 weeks of dipyridamole treatment vs placebo. We evaluated whether frequencies of polyfunctional HIV-specific T cells were associated with purines in the adenosine pathway and with measures of HIV persistence and chronic inflammation. There was a significant decrease in CD4+ polyfunctional T cell responses to Gag (-62.6% vs -23.0%; p<0.001) and Env (-56.1% vs -6.0%; p<0.001) in the dipyridamole arm. In the dipyridamole group, lower frequencies of polyfunctional Env-specific CD4+ T cells were associated with higher plasma levels of adenosine (r= -0.85; p<0.01) and inosine (r= -0.70; p=0.04). Higher adenosine levels induced by dipyridamole treatment is associated with decreased HIV-specific CD4+ T cell polyfunctional responses in people with HIV on antiretroviral therapy.

SARS-CoV-2-Specific T Lymphocytes Analysis in mRNA-Vaccinated Patients with B-Cell Lymphoid Malignancies on Active Treatment.

Patients with B-lymphocyte malignancies (BCMs) receiving B-lymphocyte-targeted therapies have increased risk of severe COVID-19 outcomes and impaired antibody response to SARS-CoV-2 mRNA vaccination in comparison to non-hematologic oncologic patients or general population. Consequently, it is vital to explore vaccine-induced T-lymphocyte responses in patients referred for the understanding of immune protection against SARS-CoV2 infections. The objective of the present study was to analyze the recall immune responses carried out by T lymphocytes after two COVID-19 mRNA vaccine doses. We enrolled 40 patients with BCMs and 10 healthy controls (HCs) after 4 weeks from the second mRNA vaccine dose. Spike (S)-specific T-lymphocyte responses were assessed in peripheral blood mononuclear lymphocytes (PBMCs) by intracellular IFN-γ staining combined with flow cytometry. Furthermore, the humoral response was assessed with the measurement of anti-spike antibodies. From March to July 2021, 40 patients (median age 68) received mRNA vaccines. The overall antibody response for BCMs was 52.5% versus 100% for the healthy controls (p = 0.008). The antibody response was different across BCMs: 18.75% for non-Hodgkin lymphoma, 54.5% for chronic lymphocytic leukemia, and 92.3% for multiple myeloma. Responses varied by malignancy type and treatment, with anti-CD20 therapies showing the lowest response (6.7%). T-lymphocyte analysis revealed reduced numbers and altered differentiation stages in patients compared to the controls. However, the vaccine-induced T response was generally robust, with variations in specific T subpopulations. mRNA vaccines induced significant humoral and cellular immune responses in B-cell lymphoid malignancy patients, although responses varied by treatment type and malignancy. Further research is needed to optimize vaccination strategies in this population.

Antifibrotic and Pro-regenerative Effects of SMAD3 siRNA and Collagen I mRNA-Loaded Lipid Nanoparticles in Human Tenocytes.

Tendinopathy involves the inflammation and degeneration of the tendon due to repetitive strain injury. Current treatments primarily target inflammation resolution, yet they do not aim at tissue regeneration. In this study, a microfluidics approach is harnessed to develop a platform of lipid nanoparticles (LNPs) loaded simultaneously with SMAD3 siRNA and collagen I mRNA, aiming to explore its potential dual antifibrotic and regenerative effects in human tenocytes. The developed LNPs displayed size homogeneity and colloidal stability and exhibited high cytocompatibility in human tenocytes. Moreover, LNPs allowed for efficient uptake and transfection efficiency of the RNAs. In the in vitro efficacy studies, the gene expression and production of SMAD3 and collagen I were tested by real-time quantitative chain polymerase reaction and immuno- and intracellular staining, revealing collagen I production enhancement, SMAD3 inhibition, and modulation of other tendon repair factors by the LNPs. Overall, the potential of this platform of RNA-loaded LNPs to be used as a dual therapeutic approach to prevent fibrosis and promote tissue remodeling in late stages of tendon diseases was confirmed.

The effects of combustible cigarettes and electronic nicotine delivery systems on immune cell-driven inflammation and mucosal healing in ulcerative colitis.

The effects of combustible cigarettes (CCs) and electronic nicotine delivery systems (ENDS) on immune cell-driven colon inflammation and intestinal healing of patients with ulcerative colitis (UC) are still unknown and, therefore, were examined in this study. Intracellular staining and flow cytometry analysis of immune cells isolated from UC patients who used ENDS (UCENDS), CCs (UCCC) and who were non-smokers (UCAIR) were performed to elucidate cellular mechanisms which were responsible for CCs and ENDS-dependent modulation of immune response during UC progression. Additionally, dextran sulfate sodium (DSS)-colitis was induced in ENDS/CC/air-exposed mice (DSSENDS/ DSSCC/DSSAIR groups) to support clinical findings. Significantly increased number of immunosuppressive, IL-10, TGF-β and IL-35-producing, FoxP3-expressing CD3+CD4+T regulatory cells (Tregs) was observed in the blood of UCENDS patients while reduced presence of inflammatory, TNF-α and IFN-γ-producing, Tbx21-expressing CD3+CD4+ Th1, IL-4-producing Gata3-expresing Th2 and IL-17, IL-22-producing, RORγT, IL-23R-expressing Th17 cells were noticed in the blood of UCCC patients. Exposure to either CCs or ENDS was associated with enhanced mucosal healing, ameliorated spontaneous recovery and improved survival of DSS-treated mice. An expansion of immunosuppressive cells (IL-10-producing tolerogenic CD11c+ dendritic cells, alternatively activated CD206, Arginase 1-expressing, IL-10-producing F4/80+macrophages, IL-10-producing FoxP3-expressing Tregs) was noticed in the colons of DSSENDS-treated mice, while reduced number of inflammatory, IL-17- and IL-4-producing T lymphocytes was observed in the colons of DSSCC-compared to DSSAIR-treated mice. Despite different mechanisms of action, both ENDS and CCs attenuated on-going colon inflammation, enhanced healing and ameliorated recovery of injured intestines of DSS-treated mice and UC patients. This is the first study that compared the effects of CCs and ENDS on immune cells of patients suffering from ulcerative colitis, providing new information about molecular and cellular mechanisms which were responsible for ENDS and CCs-dependent modulation of immune cell-driven colon injury and inflammation. Obtained results showed that both ENDS and CCs had capacity to attenuate detrimental immune response, enhanced healing and ameliorated recovery of injured intestines.

Active Heme Metabolism Suppresses Macrophage Phagocytosis via the TLR4/Type I IFN Signaling/CD36 in Uterine Endometrial Cancer.

Uterine endometrial cancer (UEC) is a common gynecological estrogen-dependent carcinoma, usually accompanied by intermenstrual bleeding. Active heme metabolism frequently plays an increasingly important role in many diseases, especially in cancers. Tumor-associated macrophages (TAMs) are the major population in the immune microenvironment of UEC. However, the roles of heme metabolisms in the crosstalk between UEC cells (UECCs) and macrophages are unclear. In our study, by using TCGA database analysis, integration analysis of the protein-protein interaction (PPI) network and sample RNA transcriptome sequencing were done. The expression level of both heme-associated molecules and iron metabolism-related molecules were measured by quantitative real-time polymerase chain reaction. Heme level detection was done through dehydrohorseradish peroxidase assay. In addition to immunohistochemistry, phagocytosis assay of macrophages, immunofluorescence staining, intracellular ferrous iron staining, as well as enzyme-linked immune sorbent assay were performed. In the study, we verified that heme accumulation in UECCs is apparently higher than in endometrial epithelium cells. Low expression of succinate dehydrogenase B under the regulation of estrogen contributes to over-production of succinate and heme accumulation in UECC. More importantly, excessive heme in UECCs impaired macrophage phagocytosis by regulation of CD36. Mechanistically, this process is dependent on toll-like receptor (TLR4)/type I interferons alpha (IFN Iα) regulatory axis in macrophage. Collectively, these findings elucidate that active heme metabolism of UECCs directly decreases phagocytosis by controlling the secretion of TLR4-mediated IFN Iα and the expression of CD36, and further contributing to the immune escape of UEC.

The course of tuberculosis infection in hyper-susceptible mice carrying the &lt;i&gt;H2&lt;/i&gt;ᵛ haplotype

Genetic regulation of the host susceptibility to Mycobacterium tuberculosis (Mtb) and severity of tuberculosis (TB) infection remain incompletely investigated. Identification of particular genes and involved in TB control and immune reactions regulated by these genes is essential for our understanding of pathogenesis of the disease, discovery of drug targets and rational vaccine development. We have shown that mice of the B10.SM (H2ᵛ) strain are extremely TB susceptible; meanwhile, the general genome structure of this mouse strain and the H2ᵛ haplotype itself are poorly characterized. We selected a pool of Mit genetic markers differentiating B10.SM mice from Chr. 17-congenic mice of the B10 strain by the PCR products motility in the electrophoresis setting. TB susceptibility of B10 mice is much lower than that of B10.SM. In the model of infection triggered by two different dosed of Mtb (100 and 600 CFU per mouse) administered via respiratory tract we demonstrated that B10.SM mice have significantly shorter survival time and significantly higher lung mycobacterial multiplication compared to B10 mice. We demonstrated (intracellular staining and ELISA) that IFNγ production in the lungs of infected mice of the two strains corresponds well to their disease phenotypes. Thus, more resistant B10 mice possess significantly more lung IFNγ-positive CD4⁺ T cells and a higher level of IFNγ secretion. We have established (B10х B10.SM) F1 hybrids and demonstrated that the post-infection phenotypes of survival time, lung mycobacterial multiplication and IFNγ production in these mice are intermediate compared to parental mice. Thus, we deal with the genetic trait with incomplete dominance expression. These data were confirmed in F2 hybrids by segregation genetic analysis. To characterize the phenotype of B10.SM mice in more detail, we vaccinated these mice with the BCG vaccine before TB challenge. Vaccination significantly prolonged survival time, diminished mycobacterial multiplication in the lungs and the degree of lung tissue pathology. Thus, a high level of susceptibility to primary infection did not interfere with BCG vaccination efficacy. We intend to continue genetic and immunologic analyses of TB-hyper-susceptible B10.SM mice. Experimental data regarding the cause of extreme disturbances in protection against infection are prerequisite for our better understanding causality of the wide spectrum of TB manifestations in human populations, as well as for rational search for novel vaccines and medications against TB infection.

Circulating CD8 + LGALS9 + T Cell Population Exhibiting Low Cytotoxic Characteristics are Decreased in Patients with Systemic Lupus Erythematosus.

LGALS9, also known as Galectin-9 and a member of the β-galactosidase family, plays a crucial role in immune regulation. However, its expression and function in CD8 T cells, as well as its association with cytotoxic T lymphocytes (CTL), remain unclear. This study aims to investigate LGALS9 expression patterns in human circulating CD8 T lymphocytes and elucidate its clinical significance in Systemic Lupus Erythematosus (SLE). Blood samples from 56 healthy controls and 50 new-onset SLE patients were collected. Flow cytometry was utilized to analyze LGALS9 expression in circulating CD8 T lymphocytes via intracellular staining. Compared to LGALS9 + CD8 + T cells, LGALS9-CD8 + T cells showed increased secretion of Granzyme B (GZMB) and Perforin, along with elevated expression levels of GPR56, CX3CR1, KLRD1, KLRF1, PD1, and CD29. A higher proportion of Tn (naive T cells) and TCM (central memory T cells) showed LGALS9 positivity, compared to TEM (effector memory T cells) and TEMRA (terminally differentiated effector memory T cells re-expressing CD45RA). Clinically, the downregulation of LGALS9 expression was significant in SLE patients. LGALS9 + CD8 + T cells exhibited an Area Under the Curve (AUC) of 0.6916, while CX3CR1 + in LGALS9 + CD8 + T cells had an AUC of 0.6478, and KLRF1 + had an AUC of 0.6419, for distinguishing SLE from healthy individuals. In conclusion, CD8 + LGALS9 + T cells display characteristics of low cytotoxicity, and their reduction is evident in SLE patients, potentially implicating them in SLE pathogenesis and providing diagnostic assistance.

Effects of Combustible Cigarettes and Heated Tobacco Products on Systemic Inflammatory Response in Patients with Chronic Inflammatory Diseases.

Smoke derived from combustible cigarettes (CCs) contains numerous harmful chemicals that can impair the viability, proliferation, and activation of immune cells, affecting the progression of chronic inflammatory diseases. In order to avoid the detrimental effects of cigarette smoking, many CC users have replaced CCs with heated tobacco products (HTPs). Due to different methods of tobacco processing, CC-sourced smoke and HTP-derived aerosols contain different chemical constituents. With the exception of nicotine, HTP-sourced aerosols contain significantly lower amounts of harmful constituents than CC-derived smoke. Since HTP-dependent effects on immune-cell-driven inflammation are still unknown, herein we used flow cytometry analysis, intracellular staining, and an enzyme-linked immunosorbent assay to determine the impact of CCs and HTPs on systemic inflammatory response in patients suffering from ulcerative colitis (UC), diabetes mellitus (DM), and chronic obstructive pulmonary disease (COPD). Both CCs and HTPs significantly modulated cytokine production in circulating immune cells, affecting the systemic inflammatory response in COPD, DM, and UC patients. Compared to CCs, HTPs had weaker capacity to induce the synthesis of inflammatory cytokines (IFN-γ, IL-1β, IL-5, IL-6, IL-12, IL-23, IL-17, TNF-α), but more efficiently induced the production of immunosuppressive IL-10 and IL-35. Additionally, HTPs significantly enhanced the synthesis of pro-fibrotic TGF-β. The continuous use of CCs and HTPs aggravated immune-cell-driven systemic inflammation in COPD and DM patients, but not in UC patients, suggesting that the immunomodulatory effects of CC-derived smoke and HTP-sourced aerosols are disease-specific, and need to be determined for specific immune-cell-driven inflammatory diseases.