Pericoronary Fat Attenuation Index Is Associated With Vulnerable Plaque Components and Local Immune‐Inflammatory Activation in Patients With Non‐ST Elevation Acute Coronary Syndrome

Background The pericoronary fat attenuation index (FAI) is assessed using standard coronary computed tomography angiography, and it has emerged as a novel imaging biomarker of coronary inflammation. The present study assessed whether increased pericoronary FAI values on coronary computed tomography angiography were associated with vulnerable plaque components and their intracellular cytokine levels in patients with non‐ST elevation acute coronary syndrome. Methods and Results A total of 195 lesions in 130 patients with non‐ST elevation acute coronary syndrome were prospectively included. Lesion‐specific pericoronary FAI, plaque components and other plaque features were evaluated by coronary computed tomography angiography. Local T cell subsets and their intracellular cytokine levels were detected by flow cytometry. Lesions with pericoronary FAI values >−70.1 Hounsfield units exhibited spotty calcification (43.1% versus 25.0%, P =0.015) and low‐attenuation plaques (17.6% versus 4.2%, P =0.016) more frequently than lesions with lower pericoronary FAI values. Further quantitative plaque compositional analysis showed that increased necrotic core volume (Pearson’s r=0.324, P <0.001) and fibrofatty volume (Pearson’s r=0.270, P <0.001) were positively associated with the pericoronary FAI, and fibrous volume (Pearson’s r=−0.333, P <0.001) showed a negative association. An increasing proinflammatory intracellular cytokine profile was found in lesions with higher pericoronary FAI values. Conclusions The pericoronary FAI may be a reliable indicator of local immune‐inflammatory response activation, which is closely related to plaque vulnerability. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04792047.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

Characterization of Conserved and Promiscuous Human Rhinovirus CD4 T Cell Epitopes

Human rhinovirus (RV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from RV A and 8 from RV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in RV A and C serotypes and predicted to bind to multiple human leukocyte antigen class II (HLA II) molecules. We found positive T cell recall responses by interferon gamma (IFNγ)-ELISPOT assays to eight peptides, validating seven of them (three from RV A and four from RV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these seven epitopes could be recognized by >95% of the world population. We actually determined IFNγ responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor RV infections and to develop peptide-based vaccines against most RV A and C serotypes.

Characterization of Conserved and Promiscuous Human Rhinovirus CD4 T Cell Epitopes

Human Rhinovirus (HRV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from HRV A and 8 from HRV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in HRV A and C serotypes and predicted to bind to multiple HLA II molecules. We found positive T cell recall responses by IFN&gamma;-ELISPOT assays to 8 peptides, validating 7 of them (3 from HRV A and 4 from HRV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these 7 epitopes could be presented and recognized by &gt; 95 % of the world population. We actually determined IFN&gamma; responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor HRV infections and to develop peptide-based vaccines against most HRV A and C serotypes.

Influence of immunomodulation on intracellular cytokine expression by spleen T-helpers of mice immunized by Yersinia pestis EV NIIEG

Aim. To characterize the intracellular expression of cytokines by spleen T-helpers and the spontaneous production of cytokines in the blood of BALB/c mice immunized with Yersinia pestis EV NIIEG against the background of immunomodulation.Materials and methods. Intracellular expression of CD4+IFN-γ+, CD4+IL-4+, CD4+IL-17+ was determined in mice spleen cell suspensions by flow cytometry, IFN-γ and IL-10 were measured in ELISA in blood supernatants on day 3 and day 21 after the immunization with Y. pestis EV against the background of immunomodulation. On day 21 after the immunization animals were infected by Y. pestis 231 at a dose of 400 LD50.Results. Differences in cytokine response to studied drugs, correlated with CD4+IFN-γ+ levels in animals, were identified. On day 3, a significant decrease in CD4+IFN-γ+ was observed in response to Y. pestis EV and to recombinant gamma interferon (Ingaron). A significant increase in CD4+IFN-γ+ was detected in response to vaccine strain administered with azoximer bromide (Polyoxidonium). Intracellular expression of IFN-γ, IL-4 and IL-17 increased on day 21by an average of 2,3 times when immunomodulators were used in the immunization schedule. In addition, on day 21 a significant (p ˂ 0.05) increase in the proportion of T-helpers expressing IFN-γ, as well as in level of spontaneous IFN-γ production in blood supernatants was observed only in animals immunized by schedules that included immunomodulators. After the challenge with Y. pestis 231 of animals previously immunized by schedules that included Polyoxidonium, the correlation analysis confirmed the association (r = 0,94; p = 0,0004) of mice survival with intensity of CD4+IFN-γ+ expression.Conclusion. The data obtained confirm the effectiveness of Polyoxidonium application in experimental animal Y. pestis EV immunization schedule and the usefulness of intracellular cytokine expression measurement for assessment of the level of protection following the immunization.

Identification of HLA-A*02:01-restricted candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 that may be natural targets of CD8+ T cell recognition in vivo

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three response patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. IMPORTANCE For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three reaction patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

Cytokine Secretion and Proliferative Capacity of CD4+ T Cells in Delayed Type Hypersensitivity Reactions due to Ciprofloxacin

Introduction: Ciprofloxacin (CPFX), a frequently prescribed quinolone, may induce cutaneous adverse drug reactions. Delayed type hypersensitivity reactions (DTHR) are often difficult to deal with, therefore, in vitro testing for DTHR is the long-anticipated method for their management. This study aimed to evaluate potential value of lymphocyte transformation test (LTT) and intracellular cytokine secretion of drug stimulated CD4+ T cells in patients with DTHR against ciprofloxacin. Material and Methods: Patients experienced DTHR with CPFX (n=8) and healthy subjects (n=10) were enrolled. CPFX skin prick, patch and intradermal tests were performed. LTT by flow cytometry aimed to determine CPFXspecific CD4+ T cell proliferation. Intracellular IL-4, IL-10, IL-2 & IFN-γ levels were analysed by flow cytometry in CPFX-specific CD4+ T cells. Cytokine contents of cell culture supernatants were evaluated by ELISA. Results: In patients with DTHR, 5 and 10 μg/mL CPFX induced significant CD4+ T cell proliferation (p=0.014 and p=0.05, respectively). IL-2 (p=0.02, p=0.001 and p=0.001, respectively) and IL-4 (p=0.001) secreting CD4+ T cell percentages were increased, while IFN-γ+ (p=0.001, p=0.011 and p=0.012, respectively) and IL-10+ (p=0.001, p=0.001 and p=0.002, respectively) CD4+ T cells were decreased. The cell culture supernatants revealed downregulated IL-10 (p<0.000, p=0.004, p=0.001 and p=0.0001, respectively) and upregulated IL-4 levels (p=0.003, p=0.013 and p=0.0001, respectively) in patients, regardless of CPFX stimulation. Intradermal test was positive in only one patient while all patch tests remained negative. Conclusion: Our findings suggest that the increase of IL-2 and IL-4-secreting CD4+ T cells together with the decrease of IL-10 and IFN-γ-secreting CD4+ T cells is related to DTHR seen in patients with delayed-type CPFX allergy. Intracellular cytokine measurement, together with LTT could ease the management of CPFX hypersensitivity when in vivo tests are non-available, remain inconclusive or negative. Keywords: Allergy, CD4+ T cells, ciprofloxacin, hypersensitivity, intracellular cytokine, LTT