Search Results (21)

Immune checkpoint inhibitors (ICIs) show limited efficacy in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma due to its non-inflamed tumor microenvironment (TME). While quadruple therapy combining chemotherapy, anti-programmed death-ligand 1 (PD-L1), and anti-vascular endothelial growth factor (VEGF) antibodies has shown inconsistent results in EGFR-tyrosine kinase inhibitor (TKI)-pretreated patients, whether anti-VEGF therapy can modulate the intrinsic non-inflamed TME remains unknown. We employed an EGFR-TKI-naïve syngeneic Egfr-mutant mouse model and evaluated effects of anti-VEGF, anti-PD-L1, carboplatin, and paclitaxel as monotherapies and combinations, with TME analysis via immunohistochemistry (IHC), flow cytometry, and RNA sequencing. Anti-PD-L1 showed no antitumor effect, and adding anti-VEGF failed to convert the TME to an inflamed status. Although paclitaxel—but not carboplatin—combined with low-dose anti-VEGF inhibited tumor growth, adding anti-PD-L1 provided no benefit, indicating the anti-VEGF-A antibody evaluated here has a limited role in sensitizing tumors to anti-PD-L1 regardless of chemotherapy. CD8⁺ T-cell depletion did not attenuate the effect of paclitaxel plus low-dose anti-VEGF, and IHC and RNA sequencing revealed increased natural killer cell infiltration, suggesting a CD8⁺ T-cell-independent, innate immune mechanism. These findings provide preclinical evidence that the evaluated anti-VEGF has limited immunomodulatory activity in EGFR-TKI-naïve Egfr-mutant tumors with a non-inflamed TME, and suggest immunotherapeutic strategies beyond CD8⁺ T-cell-mediated immunity warrant investigation. Full article

Background: Immunological monitoring in multiple sclerosis (MS) patients treated with disease-modifying drugs may help predict infectious complications and guide treatment. The main objective of this study was to evaluate whether anti-CD20 treatments in MS patients induce immunodeficiency and whether certain immunological parameters can predict the risk of infection and response to treatment. Methods: This retrospective, observational, single-centre study included MS patients who started treatment with ocrelizumab or rituximab and received follow-up in the Neuroimmunology Unit of our centre between January 2017 and January 2023. The study was conducted in collaboration with the Immunology Department of this hospital. Results: Fifty-five patients were included, with a mean age of 47 years and a follow-up period of 24 months. Analyses of lymphocyte subpopulations (T, B, NK) and immunoglobulin levels (IgG, IgA, IgM) were performed before treatment and at 6-, 12- and 24-month follow-ups. In addition, we carried out an exhaustive study of B cells in the baseline analysis. Sixty-four percent of patients presented infections, mostly due to COVID-19. Three patients developed cryptogenic organising pneumonia. IgG hypogammaglobulinemia was the main risk factor for developing infections. Patients with infections had fewer mature memory B cells and a lower percentage of NK cells. Furthermore, a lower proportion of naïve and mature memory B cells was associated with inflammatory activity and disease progression, respectively. The absence of CD20 depletion during follow-up was associated with clinical worsening. Conclusions: Baseline immunophenotype and immunological monitoring can help predict the risk of infections and the efficacy of anti-CD20 therapy in MS patients. Full article

Porcine circovirus 2 (PCV2) is one of the most widespread immunosuppressive viruses, impairing the protective efficacy of vaccines in pig herds. Previous studies have shown that PCV2 infection reduces the generation of immune memory and antibody secretion induced by vaccination in hosts. In this study, we used single-cell mRNA sequencing of mice splenic cells to show that PCV2 infection decelerates the differentiation of light zone germinal center (GC) B cells into memory B cells and plasma cells. We found that, although PCV2 infection led to lymphocyte depletion in the spleens of mice, the remaining splenic B cells were activated by the infection. The percentage of naïve B cells in PCV2-infected mice decreased mainly due to differentiation rather than death. Meanwhile, the percentages of memory B cells and plasma cells increased without significant enhancement of functional gene expression. Focusing on the GC B cells, we found that PCV2 infection activated the proliferation of dark zone GC B cells, but not the differentiation of light zone GC B cells. Furthermore, the transcriptional level of Prdm1 was not significantly altered by PCV2 infection, and the level of Bach2 was dramatically reduced. Further analysis showed that the interactions between light zone GC B cells and dendritic cells, macrophages, and follicular helper T cells were weakened in the spleens of PCV2-infected mice. In conclusion, this study found that PCV2 infection impairs the differentiation of B cells into functional memory B cells and plasma cells. This may be an important and previously unrecognized reason why PCV2 infection impairs vaccine efficiency. Full article

Hepatic viruses, such as hepatitis B and C (HBV and HCV), evade immune defenses and drive liver cirrhosis and cancer. They remain a major global health burden, requiring deeper research into immune responses; specifically, adaptive immunity. This study aims to analyze T cellular subsets in chronic HBV and HCV infection and investigate their potential role in the immunopathogenesis of these conditions. Methods: For our study, we collected 123 blood samples taken from patients infected with HCV (n = 36) and HBV (n = 34) and healthy volunteers (n = 53). With the use of flow cytometry, we assessed levels of CD4+ and CD8+ minor T cell subpopulations (naïve, central, and effector memory cells (CM and EM), terminally differentiated EM (TEMRA), Th1, Th2, Th17, Tfh, Tc1, Tc2, Tc17, Tc17.1). Results: Despite similar total CD4+ T cell frequencies across chronic HCV, HBV, and healthy groups, patients with hepatitis showed elevated TEMRA, EM, and CM subsets alongside depleted naïve Th cells and specific CM subpopulations compared to controls. Patients with chronic HCV and HBV showed elevated CD8+ T cell frequencies versus controls, with disease-specific shifts: reduced EM CTLs but increased TEMRA CTLs, Tc1/Tc17.1 depletion (notably Tc17.1 in HCV), and higher Tc2 levels. Conclusions: Viral clearance in HBV and HCV requires a delicate balance between immunity and viral activity. Despite similar T cell frequencies (CD3+/CD4+/CD8+), minor subsets revealed distinct patterns differentiating HCV, HBV, and healthy controls. Full article

Introduction: In mouse models of chronic lymphocytic leukemia (CLL), an effective anti-leukemia immune response was obtained by depleting a specific regulatory T-cell (Treg) subset. While STAT5 signaling could alter the homeostasis of naïve (nTreg) and activated (aTreg) subsets, which are capable of suppressing also CLL patients’ responses to microbial antigens, perturbed STAT3 signaling could drive CXCR5 expression in circulating T-follicular regulatory cells (Tfr) and their entry into the lymph node/tumor microenvironment. Materials and Methods: By using phospho-specific flow cytometry, we monitored STAT signaling/phosphorylation (pSTAT), in vitro responses to Sars-Cov2-antigen-specific stimulation, and circulating Treg subsets in relation to Binet stage and total tumor mass/tumor distribution (TTM/TD) scoring in 62 patients with CLL. Results: The percentage of CXCR5⁺ Treg significantly increased in patients with Binet stage B disease, and Tfr-like subsets were associated with higher TTM and lower TD. The pSTAT3 levels in CD4⁺ T-cells were only significantly increased in patients undergoing therapy. Lower nTreg percentages correlated with increased disease duration, and an increased aTreg/nTreg ratio correlated with SARS-CoV-2-antigen-induced STAT5 signaling responses. Conclusions: The results show increased amounts of circulating CXCR5⁺ Tfr-like subsets in patients with extensive lymph node involvement and augmented STAT3 signaling in patients on therapy. While STAT5 responses may drive nTreg differentiation into aTreg, nTreg decline is associated with increased disease duration. Full article

Introduction: The efficacy of anti-CD20 antibodies has significantly contributed to advancing our understanding of disease pathogenesis and improved treatment outcomes in relapsing-remitting multiple sclerosis (RRMS). A comprehensive analysis of the peripheral immune cell profile, combined with prospective clinical characterization, of RRMS patients treated with ocrelizumab (OCR) or ofatumumab (OFA) was performed to further understand immune reconstitution following B-cell depletion. Methods: REBELLION-MS is a longitudinal analysis of RRMS patients treated with either OCR (n = 34) or OFA (n = 25). Analysis of B, T, natural killer (NK) and natural killer T (NKT) cells at baseline, month 1, and 12 was performed by multidimensional flow cytometry. Data were analyzed by conventional gating and unsupervised computational approaches. In parallel, different clinical parameters were longitudinally assessed. Twenty treatment-naïve age/sex-matched RRMS patients were included as the control cohort. Results: B-cell depletion by OCR and OFA resulted in significant reductions in CD20⁺ T and B cells as well as B-cell subsets, alongside an expansion of CD5⁺CD19⁺CD20⁻ B cells, while also elevating exhaustion markers (CTLA-4, PD-1, TIGIT, TIM-3) across T, B, NK, and NKT cells. Additionally, regulatory T-cell (T_REG) numbers increased, especially in OCR-treated patients, and reductions in double-negative (CD3⁺CD4⁻CD8⁻) T cells (DN T cells) were observed, with these DN T cells having higher CD20 expression compared to CD4 or CD8 positive T cells. These immune profile changes correlated with clinical parameters, suggesting pathophysiological relevance in RRMS. Conclusions: Our interim data add weight to the argumentation that the exhaustion/activation markers, notably TIGIT, may be relevant to the pathogenesis of MS. In addition, we identify a potentially interesting increase in the expression of CD5+ on B cells. Finally, we identified a population of double-negative T cells (KLRG1+HLADR+, in particular) that is associated with MS activity and decreased with CD20 depletion. Full article

The amount of sphingosine 1-phosphate (S1P) found in the synovial tissue of individuals with rheumatoid arthritis is five times greater than that in those with osteoarthritis. Our study aims to determine whether inhibiting S1P₂ can mitigate collagen-induced rheumatoid arthritis (CIA) by using an S1P₂ antagonist, JTE-013, alongside DBA-1J S1pr2 wild-type (WT) and knock-out (KO) mice. CIA causes increases in arthritis scores, foot swelling, synovial hyperplasia, pannus formation, proteoglycan depletion, cartilage damage, and bone erosion, but these effects are markedly reduced when JTE-013 is administered to S1pr2 WT mice. CIA also elevates mRNA expression levels of pro-inflammatory Th1/Th17 cytokines in the foot and spleen, which are significantly decreased by JTE-013 in S1pr2 WT mice. Additionally, CIA raises Th1/Th17 and Treg cell counts, while JTE-013 reduces these elevations in the spleens of S1pr2 WT mice. Treatment with JTE-013 or the absence of S1pr2 curtails the differentiation of naïve T cells into Th1 and Th17 cells in a dose-dependent manner. In SW982 human synovial cells, JTE-013 lowers LPS-induced increases in pro-inflammatory cytokine levels. Overall, these findings propose that blocking S1P₂ in immune and synovial cells may alleviate rheumatoid arthritis symptoms and offer a potential therapeutic approach. Full article

Background/Objectives: Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) with a relapsing nature and complex etiology. Bioinformatics analysis has been widely applied to investigate various diseases. This study aimed to identify crucial differentially expressed genes (DEGs) and explore potential therapeutic agents for UC. Methods: The GSE47908 and GSE55306 colon tissue transcriptome gene datasets were downloaded from the Gene Expression Omnibus-NCBI (GEO) database. GEO2R and Gene Set Enrichment Analysis (GSEA) were used to screen for DEGs in patients with UC compared to the normal population based on weighted gene co-expression network analysis (WGCNA). GO-BP analysis and KEGG enrichment analysis were performed on the intersecting differential genes via the Metascape website, while hub genes were analyzed by STRING11.0 and Cytoscape3.7.1. The expression of hub genes was verified in the dataset GSE38713 colon tissue specimens. Finally, the gene expression profiles of the validation set were analyzed by immuno-infiltration through the ImmuCellAI online tool, and the CMap database was used to screen for negatively correlated small molecule compounds. Results: A total of 595 and 926 genes were screened by analysis of GSE47908 and GSE55306 datasets, respectively. Combined WGCNA hub module intersection yielded 12 hub genes (CXCL8, IL1β, CXCL1, CCL20, CXCL2, CXCR2, LCN2, SELL, AGT, LILRB3, MMP3, IDO1) associated with the pathogenesis of UC. GSEA analysis yielded intersecting pathways for both datasets (colorectal cancer pathway, base excision repair, cell cycle, apoptosis). GO-BP and KEGG enrichment analyses were performed to obtain key biological processes (inflammatory response, response to bacteria, leukocyte activation involved in the immune response, leukocyte–cell adhesion, apoptosis, positive regulation of immune effector processes) and key signaling pathways (cytokine–cytokine receptor interactions, IBD, NOD-like receptor signaling pathways). The immune cell infiltration analysis suggested that the incidence of UC was mainly related to the increase in CD4+T cells, depletion of T cells, T follicular helper cells, natural killer cells, γδ T cells and the decrease in CD8 naive T cells, helper T cells 17 and effector T cells. The CMap database results showed that small molecule compounds such as vorinostat, roxarsone, and wortmannin may be therapeutic candidates for UC. Conclusions: This study not only aids in early prediction and prevention but also provides novel insights into the pathogenesis and treatment of UC. Full article

Background: Gastrointestinal mucosal damage due to human immunodeficiency virus (HIV) infection leads to microbial translocation and immune activation, contributing to the development of non-infectious comorbidities (NICM) in people living with HIV (PLWH). Additionally, persistent proviral HIV-1 in the gut-associated lymphatic tissue (GALT) can trigger immunological changes in the epithelial environment, impacting the mucosal barrier. However, the role of zonulin, a modulator of epithelial tight junctions in GALT during HIV infection, remains poorly understood. Methods: We measured zonulin in serum and intestinal tissue sections from five treatment-naive (HIV⁺_NAIVE) and 10 cART-treated (HIV⁺_cART) HIV⁺ individuals, along with 11 controls (CTRL). We compared zonulin levels with clinical characteristics, inflammatory markers (IFN-α, CXCR3, and PD-1), and the viral reservoir in peripheral blood (PB) and terminal ileum (TI). Results: Upon HIV infection, TI was found to harbor more HIV DNA than PB. Circulating zonulin levels were highest in HIV⁺_NAIVE compared to HIV⁺_cART or CTRL. Surprisingly, in the gut tissue sections, zonulin levels were higher in CTRL than in HIV⁺ individuals. Elevated circulating zonulin levels were found to be correlated with CD4⁺T-cell depletion in PB and TI, and with intestinal IFN-α. Conclusions: The findings of this study indicate a shift in zonulin levels from the gut to the bloodstream in response to HIV infection. Furthermore, elevated systemic zonulin levels are associated with the depletion of intestinal CD4⁺ T cells and increased gut inflammation, suggesting a potential link between systemic zonulin and intestinal damage. Gaining insight into the regulation of gut tight junctions during HIV infection could offer valuable understanding for preventing NICM in PLWH. Full article

The proportion of patients with type I allergy in the world population has been increasing and with it the number of people suffering from allergic symptoms. Recently we showed that prophylactic cell therapy employing allergen-expressing bone marrow (BM) cells or splenic B cells induced allergen-specific tolerance in naïve mice. Here we investigated if cell therapy can modulate an established secondary allergen-specific immune response in pre-immunized mice. We sensitized mice against the grass pollen allergen Phl p 5 and an unrelated control allergen, Bet v 1, from birch pollen before the transfer of Phl p 5-expressing BM cells. Mice were conditioned with several combinations of low-dose irradiation, costimulation blockade, rapamycin and T cell-depleting anti-thymocyte globulin (ATG). Levels of allergen-specific IgE and IgG1 in serum after cell transfer were measured via ELISA and alterations in cellular responses were measured via an in vitro proliferation assay and transplantation of Phl p 5⁺ skin grafts. None of the tested treatment protocols impacted Phl p 5-specific antibody levels. Transient low-level chimerism of Phl p 5⁺ leukocytes as well as a markedly prolonged skin graft survival were observed in mice conditioned with high numbers of Phl p 5⁺ BMC or no sensitization events between the day of cell therapy and skin grafting. The data presented herein demonstrate that a pre-existing secondary allergen-specific immune response poses a substantial hurdle opposing tolerization through cell therapy and underscore the importance of prophylactic approaches for the prevention of IgE-mediated allergy. Full article

Aging attenuates the overall responsiveness of the immune system to eradicate pathogens. The increased production of pro-inflammatory cytokines by innate immune cells under basal conditions, termed inflammaging, contributes to impaired innate immune responsiveness towards pathogen-mediated stimulation and limits antigen-presenting activity. Adaptive immune responses are attenuated as well due to lowered numbers of naïve lymphocytes and their impaired responsiveness towards antigen-specific stimulation. Additionally, the numbers of immunoregulatory cell types, comprising regulatory T cells and myeloid-derived suppressor cells, that inhibit the activity of innate and adaptive immune cells are elevated. This review aims to summarize our knowledge on the cellular and molecular causes of immunosenescence while also taking into account senescence effects that constitute immune evasion mechanisms in the case of chronic viral infections and cancer. For tumor therapy numerous nanoformulated drugs have been developed to overcome poor solubility of compounds and to enable cell-directed delivery in order to restore immune functions, e.g., by addressing dysregulated signaling pathways. Further, nanovaccines which efficiently address antigen-presenting cells to mount sustained anti-tumor immune responses have been clinically evaluated. Further, senolytics that selectively deplete senescent cells are being tested in a number of clinical trials. Here we discuss the potential use of such drugs to improve anti-aging therapy. Full article

The breakdown of the blood–brain barrier (BBB) and the trans-endothelial migration of lymphocytes are central events in the development of multiple sclerosis (MS). Autoreactive T cells are major players in MS pathogenesis, which are rapidly depleted following alemtuzumab treatment. This modulation, in turn, inhibits CNS inflammation, but alemtuzumab’s effect on T cell migration into the CNS has been less studied. Human brain endothelial cells were stimulated with pro-inflammatory cytokines to mimic an inflamed BBB in vitro. Peripheral blood mononuclear cells from healthy controls, untreated or alemtuzumab-treated patients with relapsing-remitting MS (RRMS) were added to the BBB model to assess their transmigratory capacity. Here, the migration of CD4⁺ effector memory T (T^EM) and CD8⁺ central memory T (T^CM) cells across the BBB was impaired in alemtuzumab-treated patients. Naïve T (T^naïve) cells were unable to migrate across all groups. CD38 was lowly expressed on CD8⁺ T^CM cells, particularly for RRMS patients, compared to CD8⁺ T^naïve cells. CD62L expression was lower on CD4⁺ T^EM cells than CD4⁺ T^naïve cells and decreased further in alemtuzumab-treated patients. These data suggest that repopulated memory T cells are phenotypically different from naïve T cells, which may affect their transmigration across the BBB in vitro. Full article

Sickle cell disease (SCD) is a condition of functional hypo-/a-splenism in which predisposition to bacterial infections is only a facet of a wide spectrum of immune-dysregulation disorders forming the clinical expression of a peculiar immunophenotype. The objective of this study was to perform an in-depth immunophenotypical characterization of SCD pediatric patients, looking for plausible correlations between immunological biomarkers, the impact of hydroxyurea (HU) treatment and clinical course. This was an observational case–control study including 43 patients. The cohort was divided into two main groups, SCD subjects (19/43) and controls (24/43), differing in the presence/absence of an SCD diagnosis. The SCD group was split up into HU+ (12/19) and HU− (7/19) subgroups, respectively receiving or not a concomitant HU treatment. The principal outcomes measured were differences in the immunophenotyping between SCD patients and controls through chi-squared tests, t-tests, and Pearson’s correlation analysis between clinical and immunological parameters. Leukocyte and neutrophil increase, T-cell depletion with prevalence of memory T-cell compartment, NK and B-naïve subset elevation with memory and CD21low B subset reduction, and IgG expansion, significantly distinguished the SCD HU− subgroup from controls, with naïve T cells, switched-memory B cells and IgG maintaining differences between the SCD HU+ group and controls (p-value of <0.05). The mean CD4+ central-memory T-cell% count was the single independent variable showing a positive correlation with vaso-occlusive crisis score in the SCD group (Pearson’s R = 0.039). We report preliminary data assessing plausible clinical implications of baseline and HU-related SCD immunophenotypical alterations, which need to be validated in larger samples, but potentially affecting hypo-/a-splenism immuno-chemoprophylactic recommendations. Full article

Lymphodepletion, reconstitution and active-specific tumor cell vaccination (LRAST) enhances the induction of tumor-specific T cells in a murine melanoma model. Myeloid-derived suppressor cells (MDSC) may counteract the induction of tumor-reactive T cells and their therapeutic efficacy. Thus, the aim of the study was to evaluate a possible benefit of MDSC depletion using anti-Gr-1 antibodies (Ab) in combination with LRAST. Female C57BL/6 mice with 3 days established subcutaneous (s.c.) D5 melanoma were lymphodepleted with cyclophosphamide and reconstituted with naive splenocytes. Vaccination was performed with irradiated syngeneic mGM-CSF-secreting D5G6 melanoma cells. MDSC depletion was performed using anti-Gr-1 Ab (clone RB6-8C5). Induction of tumor-specific T cells derived from tumor vaccine draining lymph nodes (TVDLN) was evaluated by the amount of tumor-specific interferon (IFN)-γ release. LRAST combined with anti-Gr-1 mAb administration enhanced the induction of tumor-specific T cells in TVDLN capable of releasing IFN-γ in a tumor-specific manner. Additional anti-Gr-1 mAb administration in LRAST-treated mice delayed growth of D5 melanomas by two weeks. Furthermore, we elucidate the impact of anti-Gr-1-depleting antibodies on the memory T cell compartment. Our data indicate that standard of care treatment regimens against cancer can be improved by implementing agents, e.g., depleting antibodies, which target and eliminate MDSC. Full article

Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3⁺ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19⁺ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19⁻ CD27⁻ CD38^low/+ CD138⁻ cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels. Full article