T-cell immunophenotype correlations with cortical thickness and white matter microstructure in bipolar disorder.

BACKGROUND: Inflammation and immunological alterations, such as T-cell and cytokine changes, are implicated in bipolar disorder (BD), with some evidence linking them to brain structural changes (e.g., cortical thickness (CT), gray matter (GM) volume and white matter (WM) microstructure). However, the connection between specific peripheral cell types, such as T-cells, and neuroimaging in BD remains scarcely investigated. AIMS OF THE STUDY: This study aims to explore the link between T-cell immunophenotype and neuroradiological findings in BD. METHODS: Our study investigated 43 type I BD subjects (22 depressive, 21 manic) and 26 healthy controls (HC), analyzing T lymphocyte immunophenotype and employing neuroimaging to assess CT for GM and fractional anisotropy (FA) for WM. RESULTS: In lymphocyte populations, BD patients exhibited elevated CD4+ and CD4+ central memory (TCM) cells frequencies, but lower CD8+ effector memory (TEM) and terminal effector memory (TTEM) cells. Neuroimaging analysis revealed reduced CT in multiple brain regions in BD patients; and significant negative correlations between CD4 + TCM levels and CT of precuneus and fusiform gyrus. Tract-based spatial statistics (TBSS) analysis showed widespread alteration in WM microstructure in BD patients, with negative and positive correlations respectively between FA and radial diffusivity (RD) and CD4 + TCM. Additionally, positive and negative correlations were found respectively between FA and RD and the CD8 + TEM and CD8 + TTEM subsets. CONCLUSIONS: Our research revealed distinct T lymphocyte changes and brain structure alterations in BD, underscoring possible immune-brain interactions, warranting further study and therapeutic exploration.

Guadecitabine increases response to combined anti-CTLA-4 and anti-PD-1 treatment in mouse melanoma in vivo by controlling T-cells, myeloid derived suppressor and NK cells.

BACKGROUND: The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). METHODS: We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. RESULTS: In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. CONCLUSIONS: These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.

An optimized flow cytometry protocol for simultaneous detection of T cell activation induced markers and intracellular cytokines: Application to SARS-CoV-2 immune individuals.

Antigen (ag)-specific T cell analysis is an important step for investigation of cellular immunity in many settings, such as infectious diseases, cancer and vaccines. Multiparameter flow cytometry has advantages in studying both the rarity and heterogeneity of these cells. In the cellular immunologist's toolbox, the expression of activation-induced markers (AIM) following antigen exposure has made possible the study and sorting of ag-specific T cells without using human leukocyte antigen (HLA)-multimers. In parallel, assessing the cytokine profile of responding T cells would support a more comprehensive description of the ongoing immune response by providing information related to cell function, such as polarization and effector activity. Here, a method and flow cytometry panel were optimized to combine the detection of activated CD4+ and CD8+ T cells in a TCR-dependent manner with the evaluation of cytokine production by intracellular staining, without affecting the positivity of activation markers. In particular, the expression of CD134 (OX40) and CD69 have been tested in conjunction with intracellular (ic) CD137 (4-1BB) to detect SARS-CoV-2 Spike protein-specific activated T cells. In our setting, CD134 provided minimal contribution to detect the pool of AIM+ T cells, whereas a key role was described for ic-CD69 which was co-expressed with ic-CD137 in both CD4+ and CD8+ lymphocytes. Moreover, the analysis of TCR-triggered cytokine-producing T cells (IFNγ, TNFα and IL-2 were assessed) further confirmed the capacity of ic-CD69 to identify functionally responsive antigen-specific T cells which were often largely negative or weakly positive for CD134 expression. In parallel, the use of CD45RA, CCR7 and CXCR5 allowed us to describe the T cell matuarion curve and detect T follicular helper (Tfh) CD4+ cells, including the antigen specific activated subsets. In conclusion, we optimized a method and flow cytometry panel combining assessment of activation induced markers and intracellular cytokines that will be useful for measuring TCR stimulation-dependent activation of CD4+ and CD8+ T cells.

Trojan-horse silk fibroin nanocarriers loaded with a re-call antigen to redirect immunity against cancer.

BACKGROUND: The current challenge for immunotherapies is to generate effective antitumor immunity. Since tumor immune escape mechanisms do not impact pre-existing and consolidated immune responses, we tested the hypothesis of redirecting a pregenerated immunity to cancer: to recall a non-tumor antigen response against the tumor, silk fibroin nanoparticles (SFNs) have been selected as 'Trojan-horse' carriers, promoting the antigen uptake by the tumor cells. METHODS: SFNs have been loaded with either ovalbumin (OVA) or CpG oligonucleotide (CpG) as antigen or adjuvant, respectively. In vitro uptake of SFNs by tumor (B16/F10 melanoma and MB49 bladder cancer) or dendritic cells, as well as the presence of OVA-specific T cells in splenic and tumor-infiltrating lymphocytes, were assessed by cytometric analyses. Proof-of-concept of in vivo efficacy was achieved in an OVA-hyperimmune B16/F10 murine melanoma model: SFNs-OVA or SFNs-CpG were injected, separately or in association, into the subcutaneous peritumoral area. Cancer dimensions/survival time were monitored, while, at the molecular level, system biology approaches based on graph theory and experimental proteomic data were performed. RESULTS: SFNs were efficiently in vitro uptaken by cancer and dendritic cells. In vivo peritumor administration of SFNs-OVA redirected OVA-specific cytotoxic T cells intratumorally. Proteomics and systems biology showed that peritumoral treatment with either SFNs-OVA or SFNs-CpG dramatically modified tumor microenvironment with respect to the control (CTR), mainly involving functional modules and hubs related to angiogenesis, inflammatory mediators, immune function, T complex and serpins expression, redox homeostasis, and energetic metabolism. Both SFNs-OVA and SFNs-CpG significantly delayed melanoma growth/survival time, and their effect was additive. CONCLUSIONS: Both SFNs-OVA and SFNs-CpG induce effective anticancer response through complementary mechanisms and show the efficacy of an innovative active immunotherapy approach based on the redirection of pre-existing immunity against cancer cells. This approach could be universally applied for solid cancer treatments if translated into the clinic using re-call antigens of childhood vaccination.

Intermediate monocytes expansion and homing markers expression in COVID-19 patients associate with kidney dysfunction.

Patients with severe SARS-CoV-2 infection have an overwhelming inflammatory response characterized by remarkable organs monocyte infiltration. We performed an immunophenotypic analysis on circulating monocytes in 19 COVID-19 patients in comparison with 11 naïve HIV-1 patients and 10 healthy subjects. Reduced frequency of classical monocytes and increased frequency of intermediate monocytes characterized COVID-19 patients with respect to both HIV naïve patients and healthy subjects. Intensity of C-C motif chemokine receptor 2 (CCR2) monocyte expression highly correlated with parameters of kidney dysfunction. Our data indicate that highly activated monocytes of COVID-19 patients may be pathogenically associated with the development of renal disease.

Expression of type 1 cannabinoid receptor gene in bipolar disorder.

BACKGROUND: The Endocannabinoid System (ECBs) may have a crucial role in bipolar disorder (BD). Previous reports have not detected abnormalities in the expression of the cannabinoid receptor gene CNR1, encoding for CB1. However, we hypothesized that differentiating between mania and depression may uncover differences in CNR1 expression levels. METHODS: We recruited 44 subjects with BD type I (BD-I), in mania (n = 22) and depression (n = 22) and 25 Healthy Controls (HC). CNR1 gene expression was analyzed using a quantitative real-time polymerase chain reaction from peripheral blood mononuclear cells. Data were analyzed using frequentist non-parametric and Bayesian approaches (generalized location-scale model based on lognormal and gamma distributions). RESULTS: Using the frequentist non-parametric approach, the depression group had lower CNR1 expression compared to the mania group (p = 0.004). In addition, there was a negative correlation between CNR1 expression and Hamilton Depression Scale score (rho = -0.37; p = 0.007). Bayesian analyses further revealed that CNR1 expression in the mania group was higher and less variable than among HC (>95% probability), while CNR1 expression in the depression group was lower and more variable than among HC (100% probability). LIMITATIONS: Lack of participants with bipolar disorder in the euthymic phase, lack of toxicology screening and evaluation of CNR1 variants. CONCLUSION: CNR1 expression is higher and less variable in mania than in depression. It is highly probable that these differences also distinguish individuals in different illness phases from healthy controls. Future studies are needed to clarify the role of the endocannabinoid system in bipolar disorder.

Longitudinal Cluster Analysis of Hemodialysis Patients with COVID-19 in the Pre-Vaccination Era.

Coronavirus disease 2019 (COVID-19) in hemodialysis patients (HD) is characterized by heterogeneity of clinical presentation and outcomes. To stratify patients, we collected clinical and laboratory data in two cohorts of HD patients at COVID-19 diagnosis and during the following 4 weeks. Baseline and longitudinal values were used to build a linear mixed effect model (LME) and define different clusters. The development of the LME model in the derivation cohort of 17 HD patients (66.7 ± 12.3 years, eight males) allowed the characterization of two clusters (cl1 and cl2). Patients in cl1 presented a prevalence of females, higher lymphocyte count, and lower levels of lactate dehydrogenase, C-reactive protein, and CD8 + T memory stem cells as a possible result of a milder inflammation. Then, this model was tested in an independent validation cohort of 30 HD patients (73.3 ± 16.3 years, 16 males) assigned to cl1 or cl2 (16 and 14 patients, respectively). The cluster comparison confirmed that cl1 presented a milder form of COVID-19 associated with reduced disease activity, hospitalization, mortality rate, and oxygen requirement. Clustering analysis on longitudinal data allowed patient stratification and identification of the patients at high risk of complications. This strategy could be suitable in different clinical settings.

Healthcare Worker Study Cohort to Determine the Level and Durability of Cellular and Humoral Immune Responses after Two Doses of SARS-CoV-2 Vaccination.

We prospectively studied immunological response against SARS-CoV-2 after vaccination among healthcare workers without (group A) and with previous infection (group B). The analyses were collected at T0 (before the BNT162b2), T1 (before the second dose), T2 and T6 (1 and 6 months after the second dose). For cellular immune response, the activation-induced cell marker assay was performed with CD4 and CD8 Spike peptide megapools expressed as Stimulation Index. For humoral immune response, we determined antibodies to Spike-1 and nucleocapsid protein. The linear mixed model compared specific times to T0. The CD4+ Spike response overall rate of change was significant at T1 (p = 0.038) and at T2 (p < 0.001), while decreasing at T6. For CD8+ Spike reactivity, the interaction between the time and group was significant (p = 0.0265), and the p value for group comparison was significant at the baseline (p = 0.0030) with higher SI in previously infected subjects. Overall, the anti-S Abs significantly increased from T1 to T6 compared to T0. The group B at T6 retained high anti-S titer (p < 0.001). At T6, in both groups we found a persistent humoral response and a high CD4+ T cell response able to cross recognize SARS-COV-2 variants including epsilon, even if not a circulating virus at that time.

Evidence of alterations of Beta-endorphin levels and Mu-opioid receptor gene expression in bipolar disorder.

Despite the well-recognized effects of endogenous opioids on mood and behavior, research on its role in bipolar disorder (BD) is still limited to small or anecdotal reports. Considering that Beta-endorphins (ß-END) and Mu-opioid receptors (MOR), in particular, have a crucial activity in affective modulation, we hypothesized their alteration in BD. A cross-sectional study was conducted. We compared: (1) BD type I (BD-I) patients (n = 50) vs healthy controls (n = 27), (2) two BD-I subject subgroups: manic (MAN; n = 25) vs depressed (DEP; n = 25) subjects. Plasma levels of ß-END and MOR gene expression in peripheral blood mononuclear cells were analyzed using ELISA Immunoassay qRT-PCR. We found that subjects with BD exhibited a significant upregulation of MOR gene expression and a decrease of ß-END (p<0.0001 for both). MAN display higher MOR levels than DEP (p<0.001) and HC (p<0.0001). Plasma levels of ß-END were lower in DEP compared to MAN (p<0.05) and HC (p<0.0001). The main limitations are the cross-sectional design and the lack of a group of euthymic subjects. Although preliminary, our results suggest a dysregulation of the endogenous opioid systems in BD. In particular, both MAN and DEP showed a reduction of ß-END levels, whereas MAN was associated with MOR gene overexpression.

Case report: Sotrovimab, remdesivir and nirmatrelvir/ritonavir combination as salvage treatment option in two immunocompromised patients hospitalized for COVID-19.

COVID-19 in immunocompromised patients is difficult to treat. SARS-CoV-2 interaction with the host immune system and the role of therapy still remains only partly understood. There are no data regarding the use of monoclonal antibodies and the combination of two antivirals in fighting viral replication and disease progression. We report the cases of two patients, both treated with rituximab for non-Hodgkin lymphoma and granulomatosis with polyangiitis, respectively, and both hospitalized for COVID-19 with positive SARS-CoV-2 RNAemia, who were successfully treated with a salvage combination therapy with sotrovimab, remdesivir and nirmatrelvir/ritonavir.

The Longest Persistence of Viable SARS-CoV-2 With Recurrence of Viremia and Relapsing Symptomatic COVID-19 in an Immunocompromised Patient-A Case Study.

BACKGROUND: Immunocompromised patients show prolonged shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs. We report a case of prolonged persistence of viable SARS-CoV-2 associated with clinical relapses of coronavirus disease 2019 (COVID-19) in a patient with mantle cell lymphoma who underwent treatment with rituximab, bendamustine, cytarabine with consequent lymphopenia and hypogammaglobulinemia. METHODS: Nasopharyngeal swabs and blood samples were tested for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR). On 5 positive nasopharyngeal swabs, we performed viral culture and next-generation sequencing. We analyzed the patient's adaptive and innate immunity to characterize T- and NK-cell subsets. RESULTS: SARS-CoV-2 RT-PCR on nasopharyngeal swabs samples remained positive for 268 days. All 5 performed viral cultures were positive, and genomic analysis confirmed a persistent infection with the same strain. Viremia resulted positive in 3 out of 4 COVID-19 clinical relapses and cleared each time after remdesivir treatment. The T- and NK-cell dynamic was different in aviremic and viremic samples, and no SARS-CoV-2-specific antibodies were detected throughout the disease course. CONCLUSIONS: In our patient, SARS-CoV-2 persisted with proven infectivity for >8 months. Viremia was associated with COVID-19 relapses, and remdesivir treatment was effective in viremia clearance and symptom remission, although it was unable to clear the virus from the upper respiratory airways. During the viremic phase, we observed a low frequency of terminal effector CD8+ T lymphocytes in peripheral blood; these are probably recruited in inflammatory tissue for viral eradication. In addition, we found a high level of NK-cell repertoire perturbation with relevant involvement during SARS-CoV-2 viremia.

Telomerase-based GX301 cancer vaccine in patients with metastatic castration-resistant prostate cancer: a randomized phase II trial.

Debate is around the optimal immunization regimen for cancer vaccines since too intense vaccination schedules may exhaust reactive lymphocytes. GX301 is a telomerase-based cancer vaccine whose safety and immunological effects were tested in a phase I trial applying an eight administrations schedule. Main objective of this study was to comparatively analyse safety and immunological response to three GX301 regimens in metastatic castration-resistant prostate cancer patients with response/disease stability after docetaxel chemotherapy. This was a multicentre, randomized, parallel-group, open-label trial registered with EudraCT (2014-000095-26) and ClinicalTrials.gov (NCT02293707, 2014). Ninety-eight patients were randomized to receive either eight (regimen 1), four (regimen 2) or two (regimen 3) vaccine administrations. Sixty-three patients were assessable for the primary immunological end-point. Vaccine-specific immune responses were evaluated by intracellular staining for IFN, elispot and cytotoxic assay at 90 and 180 days from baseline. No major side effects were recorded. A 54% overall immune responder rate was observed with 95% of patients showing at least one vaccine-specific immune response. Rate of immunological responders and number of immunizations were proportionally related, suggesting superiority of regimens 1 and 2 over regimen 3. Overall survival did not differ among regimens in both immunological responders and non-responders and was inversely associated (P = 0.002) with increase in the number of circulating CD8 + T regulatory cells at 180 days. These data indicate that GX301 cancer vaccine is safe and immunogenic in metastatic castration-resistant prostate cancer patients. Schedules with high number of administrations should be preferred in future studies due to their better immunological outcome.

Development of Exhaustion and Acquisition of Regulatory Function by Infiltrating CD8+CD28- T Lymphocytes Dictate Clinical Outcome in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) has a poor clinical outcome despite the presence of a rich CD8+ T cell tumor infiltrate in the majority of patients. This may be due to alterations of tumor infiltrating CD8+ T cells. Here, we performed a characterization of HNSCC infiltrating CD8+ T cells in a cohort of 30 patients. The results showed that differential intratumoral frequency of CD8+CD28+ T cells, CD8+CD28- T cells, and CD8+CD28-CD127-CD39+ Treg distinguished between HNSCC patients who did or did not respond to treatment. Moreover, high PD1 expression identified a CD8+CD28- T cell subpopulation, phenotypically/functionally corresponding to CD8+CD28-CD127-CD39+ Treg, which showed a high expression of markers of exhaustion. This observation suggests that development of exhaustion and acquisition of regulatory properties may configure the late differentiation stage for intratumoral effector T cells, a phenomenon we define as effector-to-regulatory T cell transition.

Transcription Factor Evaluation by Flow Cytometry.

The development of T helper (Th) cell subsets requires activated T cells that respond to a polarizing cytokine environment, resulting in the activation and expression of specific transcription factors. The subset-specific transcription factors are located either in the cytoplasm or in the nucleus, which determine the functional profile of Th populations, inducing the production of specific effector cytokines and functions. Flow cytometry analysis of transcription factors has become very common not only in research but also in immunologic follow-up protocols of patients recruited in clinical trials (as evaluation of CD4+CD25+ FOXP3+ T regulatory cells). Here, we propose and describe one-step protocols to evaluate the expression of transcription factors in mouse and human CD4+ lymphocytes, focusing the critical points of this cytometric approach.

Characterization of T lymphocytes in severe COVID-19 patients.

In this observational study, 13 patients with severe COVID-19 and 10 healthy controls were enrolled. The data concerning the analysis of circulating T cells show that, in severe COVID-19 patients, the expansion of these cell compartments is prone to induce antibody response, inflammation (CCR4+ and CCR6+ TFH) and regulation (CD8+ Treg). This pathogenic mechanism could lead us to envision a possible new form of biological target therapy.

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847.

: Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes.

White matter microstructure alterations correlate with terminally differentiated CD8+ effector T cell depletion in the peripheral blood in mania: Combined DTI and immunological investigation in the different phases of bipolar disorder.

BACKGROUND: White matter (WM) microstructural abnormalities and, independently, signs of immunological activation were consistently demonstrated in bipolar disorder (BD). However, the relationship between WM and immunological alterations as well as their occurrence in the various phases of BD remain unclear. METHOD: In 60 type I BD patients - 20 in manic, 20 in depressive, 20 in euthymic phases - and 20 controls we investigated: (i) diffusion tensor imaging (DTI)-derived fractional anisotropy (FA), radial diffusivity (RD) and axial diffusivity (AD) using a tract-based spatial statistics (TBSS) approach; (ii) circulating T cell subpopulations frequencies, as well as plasma levels of different cytokines; (iii) potential relationships between WM and immunological data. RESULTS: We found: (i) a significant widespread combined FA-RD alteration mainly in mania, with involvement of the body of corpus callosum (BCC) and superior corona radiata (SCR); (ii) significant increase in CD4+ T cells as well as significant decrease in CD8+ T cells and their subpopulations effector memory (CD8+ CD28-CD45RA-), terminal effector memory (CD8+ CD28-CD45RA+) and CD8+ IFNγ+ in mania; (iii) a significant relationship between WM and immunological alterations in the whole cohort, and a significant correlation of FA-RD abnormalities in the BCC and SCR with reduced frequencies of CD8+ terminal effector memory and CD8+ IFNγ+ T cells in mania only. CONCLUSIONS: Our data show a combined occurrence of WM and immunological alterations in mania. WM abnormalities highly correlated with reduction in circulating CD8+ T cell subpopulations that are terminally differentiated effector cells prone to tissue migration, suggesting that these T cells could play a role in WM alteration in BD.