TCR-T cell therapy: current development approaches, preclinical evaluation, and perspectives on regulatory challenges.

TCR-T cell therapy represents a promising advancement in adoptive immunotherapy for cancer treatment. Despite its potential, the development and preclinical testing of TCR-T cells face significant challenges. This review provides a structured overview of the key stages in preclinical testing, including in silico, in vitro, and in vivo methods, within the context of the sequential development of novel therapies. This review aimed to systematically outline the processes for evaluating TCR-T cells at each stage: from in silico approaches used to predict target antigens, assess cross-reactivity, and minimize off-target effects, to in vitro assays designed to measure cell functionality, cytotoxicity, and activation. Additionally, the review discusses the limitations of in vivo testing in animal models, particularly in accurately reflecting the human tumor microenvironment and immune responses. Performed analysis emphasizes the importance of these preclinical stages in the safe and effective development of TCR-T cell therapies. While current models provide valuable insights, we identify critical gaps, particularly in in vivo biodistribution and toxicity assessments, and propose the need for enhanced standardization and the development of more representative models. This structured approach aims to improve the predictability and safety of TCR-T cell therapy as it advances towards clinical application.

NPRL2 promotes TRIM16-mediated ubiquitination degradation of Galectin-3 to prevent CD8+T lymphocyte cuproptosis in glioma.

BACKGROUND: Our previous study found that tumor suppressor nitrogen permease regulator like-2(NPRL2) is frequently downregulated in glioma, leading to malignant growth. However, NPRL2-mediated crosstalk between tumor cells and immune cells remains unclear. METHODS: The regulatory effects of NPRL2 on tripartite motif-containing protein 16(TRIM16) dependent ubiquitination degradation of Galectin-3(Gal-3) were explored. The effects of Gal-3 on copper uptake, immunocompetence and cuproptosis were investigated in CD8+T lymphocytes(CD8+T cells). The ability of NPRL2 to protect CD8+T cells from Gal-3 damage was evaluated. Furthermore, the correlations among NPRL2, TRIM16, Gal-3 and CD8+T cell accumulation were analyzed in glioma clinical specimens. RESULTS: NPRL2 increased the TRIM16 expression via inactivation of ERK1/2, which in turn promoted the ubiquitination-mediated degradation of Gal-3 and diminished Gal-3 release from glioma cells. Moreover, Gal-3 accelerated copper uptake and triggered cuproptosis in CD8+T cells, whereas NPRL2 increased CD8+T cell recruitment and prevented impairment of CD8+T cells by Gal-3. Clinical samples revealed that NPRL2 expression was positively associated with TRIM16 expression and negatively correlated with Gal-3, but Gal-3 expression was negatively associated with CD8+T cell accumulation. CONCLUSION: Glioma-derived NPRL2/TRIM16/Gal-3 axis participates in the regulation of CD8+T cell cuproptosis, which provides a promising strategy to rescue the immune activity of CD8+T cells and reverse immunosuppression in glioma.

Impact of oxaliplatin and trastuzumab combination therapy on tumor markers and T lymphocyte subsets for advanced gastric cancer.

BACKGROUND: Advanced gastric cancer (AGC) remains a challenging malignancy with poor prognosis. The combination of oxaliplatin and trastuzumab has shown promising results in AGC treatment. This study aimed to investigate the effects of oxaliplatin and trastuzumab combination therapy on serum tumor markers and T lymphocyte subsets in patients with AGC and to explore their potential as predictive biomarkers for treatment response. AIM: To investigate the impact of oxaliplatin and trastuzumab combination therapy on serum markers and T cell subsets in patients with AGC. METHODS: This prospective study enrolled 60 patients with AGC. All patients received oxaliplatin (130 mg/m2, every 3 weeks) and trastuzumab (8 mg/kg loading dose, followed by 6 mg/kg every 3 weeks) for six cycles. Serum carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), and cancer antigen 72-4 (CA72-4) were measured before and after treatment. T-lymphocyte subsets, including CD3+, CD4+, CD8+, and CD4+ /CD8+ ratios, were also evaluated. The clinical response was assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: After six cycles of treatment, the CEA, CA19-9, and CA72-4 serum levels significantly decreased compared to baseline levels (P < 0.001). The percentages of CD3+ and CD4+ T lymphocytes increased significantly (P < 0.05), whereas the percentage of CD8+ T lymphocytes decreased (P < 0.05). The CD4+/CD8+ ratio also significantly increased after treatment (P < 0.05). Patients with a higher decrease in serum tumor markers (≥ 50% reduction) and a higher increase in CD4+/CD8+ ratio (≥ 1.5-fold) showed better clinical response rates (P < 0.05). CONCLUSION: Oxaliplatin and trastuzumab combination therapy effectively reduced serum tumor marker levels and modulated T lymphocyte subsets in patients with AGC. Combination therapy not only has a direct antitumor effect, but also enhances the immune response in patients with AGC. Serum tumor markers and T lymphocyte subsets may serve as potential predictive biomarkers for treatment response in patients with AGC receiving combination therapy.

Reconceptualization of immune checkpoint inhibitor-associated gastritis.

In recent years, with the extensive application of immunotherapy in clinical practice, it has achieved encouraging therapeutic effects. While enhancing clinical efficacy, however, it can also cause autoimmune damage, triggering immune-related adverse events (irAEs). Reports of immunotherapy-induced gastritis have been increasing annually, but due to its atypical clinical symptoms, early diag-nosis poses a certain challenge. Furthermore, it can lead to severe complications such as gastric bleeding, elevating the risk of adverse outcomes for solid tumor patients if immunotherapy is interrupted. Therefore, gaining a thorough under-standing of the pathogenesis, clinical manifestations, diagnostic criteria, and treatment of immune-related gastritis is of utmost importance for early identification, diagnosis, and treatment. Additionally, the treatment of immune-related gastritis should be personalized according to the specific condition of each patient. For patients with grade 2-3 irAEs, restarting immune checkpoint inhibitors (ICIs) therapy may be considered when symptoms subside to grade 0-1. When restarting ICIs therapy, it is often recommended to use different types of ICIs. For grade 4 irAEs, permanent discontinuation of the medication is necessary.

[Development of off-the-shelf universal T cell therapies from ES/iPS cells: applications in cancer and viral infection].

Methods in which patient-derived T cells are genetically modified in vitro and administered to patients have been demonstrated effective in the area of cancer immunotherapy. However, these methods have some unresolved issues such as cost, time, and unstable quality. Several groups have developed strategies to overcome these barriers by regenerating T cells from iPSCs. We have been developing a method in which specific TCR genes are introduced into iPSCs and T cells are regenerated from these iPSCs (TCR-iPSC method). We are now using starting iPSCs from the iPSC stock lines provided by CiRA-F, as the iPSC stock cells are less likely to be rejected. A study aimed at application to solid tumors demonstrated the therapeutic effect of regenerated T cells in a patient tissue xenograft model of WT1 antigen-positive renal cell carcinoma. This article will also discuss strategies by other groups to regenerate various types of T cells from iPSCs.

Soluble cytotoxic T-lymphocyte antigen-4 (sCTLA-4) in children with immune cytopenia: relation to disease activity.

Cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) is a costimulatory receptor exhibiting a potent inhibitory signal on antigen-activated immune responses. A soluble form, sCTLA-4, has been identified and was found to be increased in several autoimmune diseases. We aimed to evaluate serum levels of sCTLA-4 in different immune cytopenias, and to determine its possible relation to the disease activity. We measured serum levels of sCTLA-4 in 47 patients with immune cytopenias and compared them to 47 age- and sex-matched healthy controls. sCTLA-4 levels were significantly higher in patients with immune cytopenias compared to healthy controls (p < 0.001), however, levels were comparable between different groups of immune cytopenias (p = 0.084). Serum sCTLA-4 inversely correlated with age at diagnosis and hemoglobin level (p = 0.048, and p = 0.039 respectively), while it directly correlated with disease duration (p = 0.023) as well as markers of hemolysis including reticulocyte count, serum LDH and indirect bilirubin (p = 0.025; p = 0.019; p = 0.004 respectively). In the AIHA group, serum sCTLA-4 levels were significantly lower in patients in remission compared to patients with active disease (p = 0.026). Children with immune cytopenia exhibit significantly higher levels of circulating sCTLA-4 which correlated with disease activity, yet the prognostic significance and its use to tailor treatment regimen require additional studies.

Apoptosis Regulation in Dental Pulp Cells and PD-1/PD-L1 Expression Dynamics Under Ozone Exposure - A Pilot Approach.

This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context.

Hemoglobin alpha is a redox-sensitive mitochondrial-related protein in T-lymphocytes.

Hemoglobin subunits, which form the well-characterized, tetrameric, oxygen-carrying protein, have recently been described to be expressed in various non-canonical cell types. However, the exact function of hemoglobin subunits within these cells remains to be fully elucidated. Herein, we report for the first time, the expression of hemoglobin alpha-a1 (Hba-a1) in T-lymphocytes and describe its role as a mitochondrial-associated antioxidant. Within naïve T-lymphocytes, Hba-a1 mRNA and HBA protein are present and highly induced by redox perturbations, particularly those arising from the mitochondria. Additionally, preliminary data using a T-lymphocyte specific Hba-a1 knock-out mouse model indicated that the loss of Hba-a1 led to an exacerbated production of mitochondrial reactive oxygen species and inflammatory cytokines after a stress challenge, further supporting the role of HBA acting to buffer the mitochondrial redox environment. Interestingly, we observed Hba-a1 expression to be significantly upregulated or downregulated depending on T-lymphocyte polarization and metabolic state, which appeared to be controlled by both transcriptional regulation and chromatin remodeling. Altogether, these data suggest Hba-a1 may function as a crucial mitochondrial-associated antioxidant and appears to possess critical and complex functions related to T-lymphocyte activation and differentiation.

Rewiring the T cell-suppressive cytokine landscape of the tumor microenvironment: a new frontier for precision anti-cancer therapy.

T lymphocytes that infiltrate the tumor microenvironment (TME) often fail to function as effective anti-cancer agents. Within the TME, cell-to-cell inhibitory interactions play significant roles in dampening their anti-tumor activities. Recent studies have revealed that soluble factors released in the TME by immune and non-immune cells, as well as by tumor cells themselves, contribute to the exacerbation of T cell exhaustion. Our understanding of the cytokine landscape of the TME, their interrelationships, and their impact on cancer development is still at its early stages. In this review, we aim to shed light on Interleukin (IL) -6, IL-9, and IL-10, a small group of JAK/STAT signaling-dependent cytokines harboring T cell-suppressive effects in the TME and summarize their mechanisms of action. Additionally, we will explore how advancements in scientific research can help us overcoming the obstacles posed by cytokines that suppress T cells in tumors, with the ultimate objective of stimulating further investigations for the development of novel therapeutic strategies to counteract their tumor-promoting activities.

T lymphocyte proportion in Alzheimer's disease prognosis.

Bai et al investigate the predictive value of T lymphocyte proportion in Alzheimer's disease (AD) prognosis. Through a retrospective study involving 62 AD patients, they found that a decrease in T lymphocyte proportion correlated with a poorer prognosis, as indicated by higher modified Rankin scale scores. While the study highlights the potential of T lymphocyte proportion as a prognostic marker, it suggests the need for larger, multicenter studies to enhance generalizability and validity. Additionally, future research could use cognitive exams when evaluating prognosis and delve into immune mechanisms underlying AD progression. Despite limitations inherent in retrospective designs, Bai et al's work contributes to understanding the immune system's role in AD prognosis, paving the way for further exploration in this under-researched area.

Evaluation of CTLA-4 and PD-L1 Expression in Thyroid Carcinoma and Its Prognostic Significance.

Introduction Immune checkpoint inhibitors (ICIs) targeting cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-ligand 1 (PD-L1) have revolutionised treatment and improved outcomes in various malignancies. We aimed to evaluate CTLA-4 and PD-L1 immunoexpression in thyroid tumours and correlated them with clinicopathological parameters. Methods The study included 90 cases of thyroid malignancies comprising papillary thyroid carcinoma (PTC) (n = 64, 54.2%), follicular thyroid carcinoma (FTC) (n = 19, 16.1%), anaplastic thyroid carcinoma (ATC) (n = 3, 2.5%), and poorly differentiated carcinoma (n = 4, 3.4%), two cases (1.69%) of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) along with 26 cases (22%) of benign thyroid lesions. CTLA-4 (UMAB249) and PD-L1 (SP263) expression were assessed in all the cases of thyroid tumours. Results were compared with clinicopathologic parameters and overall survival. Results PD-L1 was positive in all three cases of anaplastic thyroid carcinoma (ATC), 33% (n = 21) cases of PTC, and 16% (n = 3) cases of FTC. PD-L1 positivity was significantly associated at tumour proportion score (TPS) ≥1% with lymphovascular invasion and age ≤40 years and at TPS ≥50% with tumour necrosis and N-stage. Immune proportion score (IPS) did not correlate with any clinicopathological parameters except for the N-stage. CTLA-4 was positive in six cases of PTC (1-5%); five showed lymph node involvement (p = 0.032). IPS was positive in 14 cases, and a significant association was seen with lymph node metastasis, lymphocytic infiltration, and lymphovascular invasion. Three cases of PTC showed co-expression for PD-L1 and CTLA-4 in tumour cells. No significant association was seen between PD-L1 expression and survival. Conclusion The current data suggest that PD-L1 is expressed in differentiated thyroid carcinoma, mainly PTC and ATC, indicating higher responsiveness to immunotherapy. A subset of PTC showed co-expression of PD-L1 and CTLA-4. These findings suggest the need for further investigation to utilise combinational immunotherapy, including anti-PD-L1 and anti-CTLA-4.

Interleukin-12p40 deficiency attenuates myocardial ferroptosis in doxorubicin-induced chronic cardiomyopathy by inhibiting Th17 differentiation and interleukin-17A production.

AIMS: Interleukin (IL)-12p40 is a common subunit of the bioactive cytokines IL-12 and IL-23, and it also has its own intrinsic functional activity. However, its role in doxorubicin-induced chronic cardiomyopathy (DICCM) as well as the underlying mechanisms are still unknown. METHODS AND RESULTS: In this study, we used IL-12p40-knockout mice, IL-23p19-knockout mice, Rag1-knockout mice, a ferroptosis inhibitor, recombinant IL-12 (rIL-12), rIL-23, rIL-12p40, rIL-12p80, and anti-IL17A to investigate the effects of IL-12p40 on DICCM and elucidate the underlying mechanisms. We found that myocardial ferroptosis were increased in DICCM and that the inhibition of ferroptosis protected against DICCM. The expression of IL-12p40 was upregulated, and IL-12p40 was predominantly expressed by CD4+ T cells in the hearts of mice with DICCM. IL-12p40 knockout attenuated cardiac dysfunction, fibrosis and ferroptosis in DICCM, and similar results were observed in the context of CD4+ T cell IL-12p40 deficiency in Rag1-/- mice. Treatment with rIL-23, but not rIL-12, rIL-12p40 monomer or rIL-12p80, abolished the protective effects of IL-12p40 knockout. Moreover, rIL-23 treatment and IL-23p19 knockout exacerbated and ameliorated DICCM, respectively. IL-12p40 knockout might protect against DICCM by inhibiting Th17 differentiation and IL-17A production but not Th1, Th2 and Treg differentiation. Neutralizing IL-17A with an antibody also attenuated cardiac dysfunction, fibrosis and ferroptosis. The IL-12p40/Th17/IL-17A axis might promote cardiomyocyte ferroptosis by activating TNF receptor-associated factor 6 (TRAF6)/mitogen-activated protein kinase (MAPK)/P53 signaling in DICCM. CONCLUSIONS: Interleukin-12p40 deficiency protects against DICCM by inhibiting Th17 differentiation and the production of IL-17A, which plays critical roles in cardiomyocyte ferroptosis in DICCM via activating TRAF6/MAPK/P53 signaling. Our study may provide novel insights for the identification of therapeutic targets for treating DICCM in the clinic.

Luteolin exerts anti-tumour immunity in hepatocellular carcinoma by accelerating CD8+ T lymphocyte infiltration.

Luteolin, a commonly used traditional Chinese medicine, has been utilized for several decades in the treatment of hepatocellular carcinoma (HCC). Previous research has demonstrated its anti-tumour efficacy, but its underlying mechanism remains unclear. This study aimed to assess the therapeutic effects of luteolin in H22 tumour-bearing mice. luteolin effectively inhibited the growth of solid tumours in a well-established mouse model of HCC. High-throughput sequencing revealed that luteolin treatment could enhance T-cell activation, cell chemotaxis and cytokine production. In addition, luteolin helped sustain a high ratio of CD8+ T lymphocytes in the spleen, peripheral blood and tumour tissues. The effects of luteolin on the phenotypic and functional changes in tumour-infiltrating CD8+ T lymphocytes were also investigated. Luteolin restored the cytotoxicity of tumour-infiltrating CD8+ T lymphocytes in H22 tumour-bearing mice. The CD8+ T lymphocytes exhibited intensified phenotype activation and increased production of granzyme B, IFN-γ and TNF-α in serum. The combined administration of luteolin and the PD-1 inhibitor enhanced the anti-tumour effects in H22 tumour-bearing mice. Luteolin could exert an anti-tumour immune response by inducing CD8+ T lymphocyte infiltration and enhance the anti-tumour effects of the PD-1 inhibitor on H22 tumour-bearing mice.

Replacement Therapy with Blood Products in People Living with HIV.

Cytopenias or coagulation deficiencies can occur in people living with HIV (PLWH). The severity of these disorders is influenced by the low levels of CD4+ lymphocytes, viral load, and the stage of viral infection. The aim of our retrospective observational study was to determine the frequency of cytopenias and coagulation deficiencies in PLWH as well as the need for replacement therapy with blood products. We sought to determine whether there is an association between severe anemia or thrombocytopenia (requiring replacement therapy) and CD4+T lymphocyte levels. All 29 patients were critically ill, with 27 out of 29 (93%) in advanced stages of HIV disease and 23 out of 29 (79%) having CD4+ lymphocyte counts below 200 cells/microL. Most patients were either late presenters (45%) or had been lost to follow-up (41%). In addition to HIV infection, various conditions that could alter hematologic parameters were associated, including co-infections with hepatitis viruses, tuberculosis at various sites, malignant diseases, sepsis, SARS-CoV-2 infection, or other opportunistic infections. No significant correlation was found between severe anemia or severe thrombocytopenia or coagulation deficiencies and the CD4+T lymphocyte count. Our data suggest that these hematological disorders in patients with advanced HIV infection are more likely to be associated comorbidities rather than the HIV infection per se.

Ophthalmic Immune-Related Adverse Events and Association with Survival: Results from a Real-World Database.

PURPOSE: Assessing immune-related ocular toxicities from immune checkpoint inhibitors (ICIs) is crucial, though rare. This study, utilizing real-world data, examines the occurrence of ophthalmic immune-related adverse events (irAEs) after ICI treatment and their impact on overall survival. DESIGN: A retrospective cohort study METHODS: Data were obtained from TriNetX, an aggregated electronic health records database. Patients who developed ophthalmic irAEs within 1 year after the first instance of ICI therapy were included. Participants with defined ocular toxicities 6 months before ICI treatment were excluded. Subjects were paired with controls using propensity scores derived from demographics and cancer type. A Cox proportional hazard model was used to determine hazard ratios. A Kaplan-Meier survival function was evaluated with the log-rank test based on the development of ophthalmic irAEs in a 12-month landmark analysis. RESULTS: A cohort of 41,020 patients comprising 57.4% males with a mean age of 65.2±11.9 years was included. The five most prevalent ophthalmic irAEs in this cohort were dry eye syndrome (2%), conjunctivitis (0.87%), blepharitis (0.51%), anterior uveitis (0.39%), and keratitis (0.38%). Dry eye syndrome was the most common irAE among all ICI classes. Subjects taking CTLA-4 inhibitor plus PD-1 inhibitor and CTLA-4 inhibitors had higher rates of anterior uveitis (1.39% and 1.29%, respectively) than PD-1 inhibitors (0.27%) and PD-L1 inhibitors (0.14%) within 1 year after taking ICI. After a 12-month landmark analysis, there was a significant decreased chance of survival for the following categories: any ophthalmic irAE (HR, 1.37; 95% CI, 1.20-1.56; P < 0.0001), neuro-ophthalmic irAE (HR, 1.53; 95% CI, 1.09-2.14; P = 0.0124), and cornea and ocular surface irAE (HR, 1.34; 95% CI, 1.15-1.56; P < 0.0001). CONCLUSIONS: Ophthalmic irAEs involving the anterior segment are more frequent than the posterior segment, regardless of ICI class. Ophthalmic irAEs may also portend decreased survival. This insight could help guide clinicians aggressively manage irAEs and allow patients to continue ICI therapy despite having ocular issues.

Agent-Based Modeling of Virtual Tumors Reveals the Critical Influence of Microenvironmental Complexity on Immunotherapy Efficacy.

Since the introduction of the first immune checkpoint inhibitor (ICI), immunotherapy has changed the landscape of molecular therapeutics for cancers. However, ICIs do not work equally well on all cancers and for all patients. There has been a growing interest in using mathematical and computational models to optimize clinical responses. Ordinary differential equations (ODEs) have been widely used for mechanistic modeling in immuno-oncology and immunotherapy. They allow rapid simulations of temporal changes in the cellular and molecular populations involved. Nonetheless, ODEs cannot describe the spatial structure in the tumor microenvironment or quantify the influence of spatially-dependent characteristics of tumor-immune dynamics. For these reasons, agent-based models (ABMs) have gained popularity because they can model more detailed phenotypic and spatial heterogeneity that better reflect the complexity seen in vivo. In the context of anti-PD-1 ICIs, we compare treatment outcomes simulated from an ODE model and an ABM to show the importance of including spatial components in computational models of cancer immunotherapy. We consider tumor cells of high and low antigenicity and two distinct cytotoxic T lymphocyte (CTL) killing mechanisms. The preferred mechanism differs based on the antigenicity of tumor cells. Our ABM reveals varied phenotypic shifts within the tumor and spatial organization of tumor and CTLs despite similarities in key immune parameters, initial simulation conditions, and early temporal trajectories of the cell populations.

Effects of combined cytotoxic T-lymphocyte antigen 4 and programed death 1 ligand-receptor blockade on interferon-gamma production in bovine leukemia virus-infected cattle.

Background and Aim: In chronic viral infections, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) significantly suppress immune responses. The CTLA-4 receptor abundance in regulatory T cells showed a positive association with viral load and a negative association with interferon-gamma (IFN-γ) production in bovine leukemia virus (BLV)-infected cattle. Blocking this receptor boosted IFN-γ production, recovering immune response against this illness. In human cancer patients, not everyone responded positively to non-immunotherapy using CTLA-4 receptor antibodies. The present study analyzed the synergistic effects of CTLA-4 and PD-L1 receptor blockade on IFN-γ production in BLV+ cattle in vitro. Materials and Methods: The genes for bovine CTLA-4 and PD-L1 were artificially produced. The amino acid sequences of the extracellular receptor domains were sourced from the National Center for Biotechnology Information PubMed database. The western blotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were employed for the characterization of recombinant CTLA-4 (rCTLA-4) and recombinant PD-L1 (rPD-L1) proteins. The immunoinhibitory effects of recombinant proteins in Staphylococcus enterotoxin B (SEB)-stimulated cattle peripheral blood mononuclear cells (PBMCs) were investigated. Enzyme-linked immunosorbent assay (ELISA) was used to analyze monoclonal antibodies against rCTLA-4 and rPD-L1. Antibodies generated from peripheral blood mononuclear cells of healthy and BLV-seropositive cows were employed to evaluate their blocking capabilities. Results: The resulting recombinant proteins specifically reacted with commercial homogeneous monoclonal antibodies (mAbs) using ELISA and anti-His-tag mAbs using western blotting. Analysis of the proteins using LC-MS/MS revealed correspondence with the sequences of rCTLA-4 and rPD-L1 located in the Mascot database. rCTLA-4 and rPD-L1 proteins inhibited IFN-γ production in bovine PBMCs of activated SEB. When PBMCs from cows were cultured with activated SEB containing rCTLA-4 and rPD-L1, the mAbs increased IFN-γ production in PBMCs. The combined cultivation of mAbs and PBMCs from BLV+ cattle enhanced IFN-γ production in the cells. Conclusion: These findings suggest that the combined blockade of bovine CTLA-4 and PD-L1 receptors can be used as a therapy for bovine leukemia. However, it was shown that a single PBMC sample from a BLV-positive donor did not amplify the synergistic effect. Therefore, it is necessary to perform further studies on a larger population and assessing a wider range of cytokines.

Differential proteins from EVs identification based on tandem mass tags analysis and effect of Treg-derived EVs on T-lymphocytes in COPD patients.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a widespread respiratory disease. This study examines extracellular vesicles (EVs) and proteins contained in EVs in COPD. METHODS: Blood samples were collected from 40 COPD patients and 10 health controls. Cytokines including IFN-γ, TNF-α, IL-1ß, IL-6, IL-8, and IL-17, were measured by ELISA. Small EVs samples were extracted from plasma and identified by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), and Western blot. Protein components contained in EVs were analyzed by Tandem Mass Tags (TMT) to identify differential proteins. Treg-derived EV was extracted and added to isolated CD8+, Treg, and Th17 subsets to assess its effect on T-lymphocytes. RESULTS: ELISA revealed higher levels of all cytokines and flow cytometry suggested a higher proportion of Treg and Th17 cells in COPD patients. After identification, TMT analysis identified 207 unique protein components, including five potential COPD biomarkers: BTRC, TRIM28, CD209, NCOA3, and SSR3. Flow cytometry revealed that Treg-derived EVs inhibited differentiation into CD8+, CD4+, and Th17 cells. CONCLUSION: The study shows that cytokines, T-lymphocyte subsets differences in COPD and Treg-derived EVs influence T-lymphocyte differentiation. Identified biomarkers may assist in understanding COPD pathogenesis, prognosis, and therapy. The study contributes to COPD biomarker research.

Single-cell profiling of intrahepatic immune cells reveals an expansion of tissue-resident cytotoxic CD4+ T lymphocyte subset associated with pathogenesis of alcoholic-associated liver diseases.

BACKGROUND & AIMS: The immunological mechanisms underpinning the pathogenesis of alcoholic-associated liver disease (ALD) remain incompletely elucidated. This study aims to explore the transcriptomic profiles of hepatic immune cells in ALD compared to healthy individuals and those with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: We utilized single-cell RNA sequencing to analyze liver samples from healthy subjects, MASLD, and ALD patients, focusing on the immune cell landscapes within the liver. Key alterations in immune cell subsets were further validated using liver biopsy samples from additional patient cohorts. RESULTS: We observed a significant accumulation of CD4+ T cells in ALD patients' livers, surpassing the prevalence of CD8+ T cells, in contrast to MASLD and healthy counterparts, while natural killer (NK) cells and γδT cells exhibited reduced intrahepatic infiltration. In-depth transcriptional and developmental trajectory analyses unveiled that a distinct CD4+ subset characterized by granzyme K (GZMK) expression, displaying a tissue-resident signature and terminal effector state, prominently enriched among CD4+ T cells infiltrating the livers of ALD patient. Subsequent examination of an independent ALD patient cohort corroborated the substantial enrichment of GZMK+CD4+ T lymphocytes, primarily within liver fibrotic zones, suggesting their potential involvement in disease progression. Additionally, we noted shifts in myeloid populations, with expanded APOE+ macrophage and FCGR3B+ monocyte subsets in ALD samples relative to MASLD and healthy tissues. CONCLUSIONS: In summary, this study unravels the intricate cellular diversity within hepatic immune cell populations, highlighting the pivotal immune pathogenic role of the GZMK+CD4+ T lymphocyte subset in ALD pathogenesis.

Correlations of PSGL-1 VNTR polymorphism with the susceptibility to severe HFMD associated with EV-71 and the immune status after infection.

Enterovirus 71 (EV-71) has strong neurotropism, and it is the main pathogen causing severe hand, foot, and mouth disease (HFMD). In clinical observations, significant differences were observed in the severity and prognosis of HFMD among children who were also infected with EV-71. Genetic differences among individuals could be one of the important causes of differences in susceptibility to EV-71-induced HFMD. As P-selectin glycoprotein ligand-1 (PSGL-1) is an important receptor of EV-71, the correlation between single-nucleotide polymorphisms (SNPs) in PSGL-1 and the susceptibility to severe HFMD following EV-71 infection is worth studying. Given the role of PSGL-1 in immunity, the correlations between PSGL-1 SNPs and the immune status after EV-71 infection are also worth studying. Meanwhile, PSGL-1 variable number of tandem repeats (VNTR) represents a research hotspot in cardiovascular and cerebrovascular diseases, but PSGL-1 VNTR polymorphism has not been investigated in HFMD caused by EV-71 infection. In this study, specific gene fragments were amplified by polymerase chain reaction, and PSGL-1 VNTR sequences were genotyped using an automatic nucleic acid analyzer. The correlations of PSGL-1 VNTR polymorphism with the susceptibility to EV-71-associated severe HFMD and the post-infection immune status were analyzed. The PSGL-1 VNTR A allele was identified as a susceptible SNP for severe HFMD. The risk of severe HFMD was higher for AA + AB genotype carriers than for BB genotype carriers. The counts of peripheral blood lymphocyte subsets were lower in AA + AB genotype carries than in BB genotype carries. In conclusion, PSGL-1 VNTR polymorphism is associated with the susceptibility to EV-71-induced severe HFMD and the immune status after infection. PSGL-1 VNTR might play a certain role in the pathogenesis of severe cases.