scholarly journals Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1232
Author(s):  
Natalia Teresa Jarzebska ◽  
Julia Frei ◽  
Severin Lauchli ◽  
Lars E. French ◽  
Emmanuella Guenova ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Simple Method ◽  
Murine Splenocytes ◽  
T Cell Immune Responses ◽  
Synthetic Mrna ◽  
The Impact

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.

scholarly journals Oxidative Stress Compromises Lymphocyte Function in Neonatal Dairy Calves

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 255
Author(s):  
Wilmer Cuervo ◽  
Lorraine M. Sordillo ◽  
Angel Abuelo
Keyword(s):  
Oxidative Stress ◽  
Immune Responses ◽  
Immune Cell ◽  
Lymphocyte Activation ◽  
Dairy Calves ◽  
Lymphocyte Function ◽  
Newborn Calves ◽  
Ifn Γ ◽  
The Impact

Dairy calves are unable to mount an effective immune response during their first weeks of life, which contributes to increased disease susceptibility during this period. Oxidative stress (OS) diminishes the immune cell capabilities of humans and adult cows, and dairy calves also experience OS during their first month of life. However, the impact that OS may have on neonatal calf immunity remains unexplored. Thus, we aimed to evaluate the impact of OS on newborn calf lymphocyte functions. For this, we conducted two experiments. First, we assessed the association of OS status throughout the first month of age and the circulating concentrations of the cytokines interferon-gamma (IFN-γ) and interleukin (IL) 4, as well as the expression of cytokine-encoding genes IFNG, IL2, IL4, and IL10 in peripheral mononuclear blood cells (PBMCs) of 12 calves. Subsequently, we isolated PBMCs from another 6 neonatal calves to investigate in vitro the effect of OS on immune responses in terms of activation of lymphocytes, cytokine expression, and antibody production following stimulation with phorbol 12-myristate 13-acetate or bovine herpesvirus-1. The results were compared statistically through mixed models. Calves exposed to high OS status in their first month of age showed higher concentrations of IL-4 and expression of IL4 and IL10 and lower concentrations of IFN-γ and expression of IFNG and IL2 than calves exposed to lower OS. In vitro, OS reduced lymphocyte activation, production of antibodies, and protein and gene expression of key cytokines. Collectively, our results demonstrate that OS can compromise some immune responses of newborn calves. Hence, further studies are needed to explore the mechanisms of how OS affects the different lymphocyte subsets and the potential of ameliorating OS in newborn calves as a strategy to augment the functional capacity of calf immune cells, as well as enhance calves’ resistance to infections.

scholarly journals IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Marilin Koch ◽  
Mykola Zdioruk ◽  
M Oskar Nowicki ◽  
Estuardo Aguilar ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Symptomatic Treatment ◽  
Tumor Cell Death ◽  
The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

Sipuleucel-T (sip-T) induced lytic CD8+ T cell responses to target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 162-162
Author(s):  
Emmanuel S. Antonarakis ◽  
David I. Quinn ◽  
Adam S. Kibel ◽  
Daniel Peter Petrylak ◽  
Tuyen Vu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Cell Responses ◽  
Hormone Sensitive ◽  
Ctl Activity

162 Background: Sip-T is an FDA-approved immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic CRPC. Sip-T is manufactured from autologous peripheral blood mononuclear cells cultured with the immunogen PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage colony-stimulating factor. After sip-T, antibody and T cell responses to PA2024 and/or PAP correlate with improved survival. To further elucidate the mechanism of sip-T–induced immune responses, we evaluated the proliferative and lytic ability of PA2024- and PAP-specific CD8+ T cells. Methods: Mononuclear blood cells were labeled with the membrane dye carboxyfluorescein succinimidyl ester (CFSE) and cultured with PA2024 or PAP. In vitro proliferative and lytic CD8+ (cytotoxic T lymphocyte [CTL]) T cell responses to these antigens were evaluated by flow cytometry. For proliferation, progressive dilution of CFSE was measured. For CTL activity, the loss of intracellular granzyme B (GzB), indicating exocytosis of this apoptosis-mediating enzyme, was assessed. Samples were from 2 sip-T clinical trials STAND (NCT01431391) and STRIDE (NCT01981122), hormone-sensitive and CRPC pts, respectively. Results: Six wk after sip-T administration, CD8+ PAP- and PA2024-specific responses were observed (n=14 pts assessed). The magnitude of PA2024-specific CD8+ proliferative responses was greater than that for PAP-specific responses. CD8+ T cells from a subset of pts who exhibited PA2024- and/or PAP-specific proliferative responses were assessed for lytic ability. After in vitro antigen stimulation, CTL activity in all evaluated samples (n=14, PA2024; n=13, PAP) was demonstrated by a significant decrease (p<0.05) in intracellular GzB relative to a no-antigen control. Conclusions: Sip-T induced CD8+ CTL proliferation against the target antigens PAP and PA2024. Moreover, antigen-specific CTL activity provides the first direct evidence that sip-T can induce tumor cell lysis. These antigen-specific CD8+ lytic abilities were observed within 6 wk following sip-T, suggesting rapidly generated immune responses. Clinical trial information: NCT01431391; NCT01981122.

Adipose-derived stem cells regulate fibrosis by altering CD4+ T-cell immune responses in fat grafting in a mouse model

2020 ◽  
Author(s):  
Xinyao Chen ◽  
Yunzi Chen ◽  
Zijue Wang ◽  
Ziqing Dong ◽  
Yao Yao ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Fat Grafting ◽  
Th2 Cells ◽  
Control Group ◽  
T Cell Immune Responses ◽  
Long Term Retention

Abstract Background Autologous fat grafting is becoming increasingly common worldly. However, the long-term retention of fat grafting is still unpredictable due to the inevitable fibrosis that arises during tissue repair. Fibrosis may be regulated by T-cell immune responses that are influenced by adipose-derived stem cells (ASCs). Accordingly, we hypothesized that overly abundant ASCs might promote fibrosis by promoting T-cell immune responses to adipose tissue. Methods We performed 0.3 ml fat grafts with 104/ml, 106/ml and 108/ml ASCs and control group in C57 BL/6 mice in vivo. We observed retention, fibrosis, T-cell immunity, and macrophage infiltration over 12 weeks. In addition, CD4 + T-helper 1 (Th1) cells and T-helper 2 (Th2) cells were co-cultured with ASCs or ASCs conditioned media (CM) in vitro. We detected the ratio of Th2%/Th1% after 24 and 48 hours. Results In vivo, the retention rate was higher in the 104 group, while even lower in the 108 group with significantly increased inflammation and fibrosis than the control group at week 12. There was no significance between control group and the 106 group. Also, the 108 group increased infiltration of M2 macrophages, CD4 + T-cells and Th2/Th1 ratio. In vitro, the ratio of Th2%/Th1% induced by the ASCs-transwell group was higher than the ASCs-CM group and showed concentration-dependent. Conclusions High concentrations of ASCs in adipose tissue can promote Th1–Th2 shifting, and the excess of Th2 cells might promote the persistence of M2 macrophages and increase the level of fibrosis which lead to a decrease in the long-term retention of fat grafts. In addition, we found that ASCs promoted Th1–Th2 shifting in vitro.

Disease activity and the immune set in multiple sclerosis: blood markers for immunotherapy

1998 ◽  
Vol 4 (3) ◽  
pp. 232-238 ◽  
Author(s):  
A J Coles ◽  
M G Wing ◽  
D AS Compston
Keyword(s):  
Multiple Sclerosis ◽  
T Cell ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Myelin Proteins ◽  
Peripheral Blood Mononuclear ◽  
Passive Measurement ◽  
Immunological Surveillance ◽  
Experimental Treatments

There is no established immunological marker of multiple sclerosis activity, which reflects the poor understanding of the immunopathogenesis of multiple sclerosis. Passive measurement of the levels of soluble inflammatory markers, whose half lives are usually measured in minutes and hours, can only indicate the extent of instantaneous inflammation, which is known to fluctuate in multiple sclerosis. We favour measurement of immune responses in vitro. As healthy individuals have T cell reactivities to myelin proteins that are postulated to be pathogenic in multiple sclerosis,1,2we prefer non-antigen specific mitogen and recall antigen assays as immunological markers. We illustrate their use in the treatment of 27 patients with multiple sclerosis using a pulse of humanised anti-lymphocyte (CD52) antibody that caused prolonged T cell depletion. The mitogen-induced proliferation, and secretion of IFN-g, from peripheral blood mononuclear cells in vitro was significantly reduced after treatment, suggesting that immune responses had been modulated. Such observations will only gain credence as an outcome measure if they are shown to correlate with clinical or radiological measures of multiple sclerosis activity. Perhaps more importantly, aspects of the pathogenesis of multiple sclerosis may be revealed by close immunological surveillance of patients undergoing experimental treatments.

5952The involvement and interplay of HMGB1 with soluble MD-2 in dilated cardiomyopathy and its impact in immune cell recruitment

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A Kuemmel ◽  
R Feldtmann ◽  
A Stohbach ◽  
A Riad ◽  
B Chamling ◽  
...  
Keyword(s):  
Hospital Admission ◽  
Dilated Cardiomyopathy ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Early Death ◽  
Monocyte Adhesion ◽  
The Impact ◽  
Death Group

Abstract Objective Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and simultaneous dilatation of the left or both ventricles. Besides other causes, the innate immune system plays a major role in the development and progression of the disease. To uncover links between molecular mechanisms and disease progression our group has focused on the toll like receptor 4 / myeloid differentiation factor-2 (MD-2) system. Purpose We already reported that soluble MD-2 (sMD-2) is a risk factor for survival in patients with DCM. High mobility group box protein 1 (HMGB1) is a potent intrinsic interaction partner of MD-2. In the current study, we quantified HMGB-1 in plasma from patients with DCM at baseline, upon first hospital admission. Furthermore, we studied the impact of different HMGB-1 isoforms on monocyte adhesion in vitro. Methods We included 77 DCM patients divided by median time point of death after first hospital admission into “early death”, “late death” and “alive” group. MD-2 was quantified by means of ELISA. MD-2 and HMGB1 was quantified by means of ELISA. Statistical analysis was performed using a linear regression model. Human umbilical vein endothelial cells (HUVEC; n=6) were treated for 48h with two isoforms of HMGB1 (disulfide (ds) and fully reduced (fr)) alone and in combination with MD-2. Subsequently, those activated HUVEC were incubated with fresh isolated peripheral blood mononuclear cells (PBMCs) for 20 min. Finally, monocyte adhesion was quantified using multicolour FACS. Results At baseline, we found significantly increased sMD-2 level in the “early death” group (591.3±75.5 ng/ml) compared to the “later death” group (369.2±46.5 ng/ml; p=0.015) and the “alive” group (303.2±18.1 ng/ml; p<0.001). Likewise, we could demonstrate significantly increased levels of HMGB1 in the “early death” group (0.93±0.14 ng/ml) compared to the “later death” (0.57±0.17 ng/ml; p=0.04) and the “alive” group (0.49±0.06 ng/ml; p<0.001). In all patients who died during the observation period, sMD-2 and HMGB1 plasma levels showed a positive correlation. In vitro, we could demonstrate a significantly increased monocyte adhesion on HUVECs in the dsHMGB1 and the frHMGB1 group compared to controls (p=0.001; p=0.004). In contrast, the dsHMGB1 MD-2 group showed a significantly decreased monocyte adhesion on HUVECs compared to dsHMGB1 treatment alone (p=0.049). In the frHMGB1 MD-2 group, however, the reduction of the monocyte adhesion was less pronounced and did not reach significance (Fig. 1). Conclusion Our findings give a first hint that the interplay between HMGB1 and MD-2 is particularly involved in the development and progression of DCM. Furthermore, the data suggest that soluble MD-2 is capable of reducing the pro-inflammatory effects of dsHMGB1 but not of frHMGB1

scholarly journals Application of IgG-Derived Natural Treg Epitopes (IgG Tregitopes) to Antigen-Specific Tolerance Induction in a Murine Model of Type 1 Diabetes

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Leslie P. Cousens ◽  
Yan Su ◽  
Elizabeth McClaine ◽  
Xin Li ◽  
Frances Terry ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
T Cell ◽  
Immune Responses ◽  
Murine Model ◽  
Regulatory T Cell ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
The Impact

HLA class II-restricted regulatory T cell (Treg) epitopes in IgG (also called “Tregitopes”) have been reported to suppress immune responses to coadministered antigens by stimulating the expansion of natural Tregs (nTregs). Here we evaluate their impact on human immune responses to islet cell antigensex vivoand on the modulation of type 1 diabetes (T1D) in a murine modelin vivo. Co-administration of Tregitopes and T1D antigens delayed development of hyperglycemia and reduced the incidence of diabetes in NOD mice. Suppression of diabetes could be observed even following onset of disease. To measure the impact of Tregitope treatment on T cell responses, we evaluated the effect of Tregitope treatment in DO11.10 mice. Upregulation of FoxP3 in KJ1-26-stained OVA-specific CD4+T cells was observed following treatment of DO11.10 mice with Tregitopes, along with reductions in anti-OVA Ig and T effector responses. Inex vivostudies of human T cells, peripheral blood mononuclear cells’ (PBMC) responses to GAD65 epitopes in the presence and absence of Tregitope were variable. Suppression of immune responses to GAD65 epitopesex vivoby Tregitope appeared to be more effective in assays using PBMC from a newly diagnosed diabetic subject than for other more established diabetic subjects, and correlation of the degree of suppression with predicted HLA restriction of the Tregitopes was confirmed. Implementation of these defined regulatory T cell epitopes for therapy of T1D and other autoimmune diseases may lead to a paradigm shift in disease management.

scholarly journals CD25+CD4+ Regulatory T Cells from the Peripheral Blood of Asymptomatic HIV-infected Individuals Regulate CD4+ and CD8+ HIV-specific T Cell Immune Responses In Vitro and Are Associated with Favorable Clinical Markers of Disease Status

2004 ◽  
Vol 200 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Audrey L. Kinter ◽  
Margaret Hennessey ◽  
Alicia Bell ◽  
Sarah Kern ◽  
Yin Lin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Cellular Proliferation ◽  
Cell Contact ◽  
Hiv Disease ◽  
T Cell Immune Responses

Human immunodeficiency virus (HIV) disease is associated with loss of CD4+ T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4+ T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25+CD4+ regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25+CD4+ T cells significantly suppressed cellular proliferation and cytokine production by CD4+ and CD8+ T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-β independent. Individuals with strong HIV-specific CD25+ T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4+: CD8+ T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25+CD4+ T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

scholarly journals CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity

2021 ◽  
Author(s):  
Yiru Long ◽  
Jianhua Sun ◽  
Tingting Liu ◽  
Feng Tang ◽  
Xinxin Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Neutralizing Antibodies ◽  
Herd Immunity ◽  
Peptide Vaccine ◽  
Cell Responses ◽  
High Efficient ◽  
T Cell Immune Responses ◽  
Cellular Immune

AbstractSafe, economical and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and halt the pandemic. We have constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains two immunodominant peptides screened from receptor-binding domain (RBD) and is fully chemically synthesized. And the vaccine has optimized nanoemulsion formulation, outstanding stability and safety. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of RBD-specific and protective neutralizing antibodies (NAbs), which were also effective to RBD mutations. CoVac501 was found to elicit the increase of memory T cells, antigen-specific CD8+ T cell responses and Th1-biased CD4+ T cell immune responses in NHPs. More importantly, the sera from the immunized NHPs can prevent infection of live SARS-CoV-2 in vitro.One-Sentence SummaryA novel SARS-CoV-2 vaccine we developed, CoVac501, which is a fully chemically synthesized and self-adjuvanting peptides conjugated with TLR7 agonists, can induce high-efficient humoral and cellular immune responses against SARS-CoV-2.

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