Treatment with Soluble CD24 Attenuates COVID-19-Associated Systemic Immunopathology

ABSTRACTBACKGROUNDSARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy.METHODSWe studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19.FINDINGSTwenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity.INTERPRETATIONOur data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.FUNDINGNIH

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Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01222-y ◽

2022 ◽

Vol 15 (1) ◽

Author(s):

No-Joon Song ◽

Carter Allen ◽

Anna E. Vilgelm ◽

Brian P. Riesenberg ◽

Kevin P. Weller ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Systemic Inflammation ◽

Lung Epithelial Cells ◽

Phase Iii ◽

Immune Homeostasis ◽

Spectral Flow ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

The Impact

Abstract Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain–containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. Methods Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. Results Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. Conclusions Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

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Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

10.21203/rs.3.rs-1059931/v1 ◽

2021 ◽

Author(s):

Bo Li ◽

Chunmei Yang ◽

Gui Ja ◽

Yansheng Liu ◽

Na Wang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Effective Utilization ◽

Blood Mononuclear Cells ◽

Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

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Adiponectin/AdipoR1 Axis Promotes IL-10 Release by Human Regulatory T Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.677550 ◽

2021 ◽

Vol 12 ◽

Author(s):

Patricia Ramos-Ramírez ◽

Carina Malmhäll ◽

Omar Tliba ◽

Madeleine Rådinger ◽

Apostolos Bossios

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Foxp3 Expression ◽

P38 Map Kinase ◽

Adiponectin Receptor ◽

Peripheral Blood Mononuclear ◽

Cytokine Milieu

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

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

10.17504/protocols.io.byrppv5n ◽

2021 ◽

Author(s):

Alessia Furgiuele ◽

Emanuela Rasini ◽

Maria Giulia Albizzati ◽

Alessandra Luini ◽

Marco Ferrari ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Brefeldin A ◽

Intracellular Cytokine ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Ifn Γ

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.

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Adoptive Therapy with Cord Blood Regulatory T Cells Can Treat Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-129395 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1940-1940

Author(s):

Hongbing Ma ◽

Ke Zeng ◽

Mitsutaka Nishimoto ◽

Mi-Ae Lyu ◽

Meixian Huang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Research Funding ◽

Adoptive Therapy ◽

Host Disease ◽

Graft Vs Host Disease ◽

The Impact

Background Adoptive therapy with regulatory T cells (Tregs) has already been established as a promising strategy for prevention of graft vs. host disease (GVHD) in clinical trials. Our group at MD Anderson Cancer Center has previously shown that a significantly lower dose of cord blood (CB) Tregs as compared to conventional T cells (Tcon) in the donor graft is able to prevent GVHD while preserving the graft vs. leukemia (GVL) effect. Therefore, we now examined the efficacy of using CB Tregs in the treatment of GVHD. Method: Xenogenic GVHD mouse model was established using NOD/SCID/IL2Rgnull (NSG) mice were sublethally irradiated at 300 cGy followed by injection of 1x107 peripheral blood (PB) mononuclear cells on day 0, as previously described. Ex vivo expanded CB Tregs were injected on day -1 (for prophylaxis) or at different days post PBMC injection for treatment. Mice were serially examined for appearance, weight, posture, GVHD score and survival. Serial peripheral blood sampling for flow cytometry and serum cytokine analysis. CB Tregs were also analyzed by flow cytometry. In order to understand the impact of the routine immunosuppressive agents on the function of CB Tregs, we incubated the CB Tregs in culture with cyclosporine (200ng/ml) or sirolimus (20 ng/ml) from day 8 to day 14. Cells were harvested on day 14 and analyzed by flow cytometry and CellTrace Violet suppression assay. Result: A single dose of 1x107 CB Tregs injected at day +7 did not result in a survival difference compared to the control arm (data not shown). Therefore, we froze multiple aliquots of expanded CB Tregs to be injected at different intervals post-transplant. Thawed CB Tregs showed stable phenotype of CD4+25+127lo: 94.7%; intracellular Helios+: 98.5% and intracellular FOXP3+: 99.4% and were able to suppress 87% of the proliferating conventional T-cells (Tcons). In order to compare the efficacy of the CB Tregs for GVHD treatment, we set up 3 arms: i) Control: PBMC alone; ii) Prophylaxis: 1x107 CB Tregs injected on day -1 and iii) Treatment: 1x107 CB Tregs injected on day +4, +7, +18 and +25. The mice in the prophylaxis and treatment arm retained their weight as compared to the control arm (p<0.003) (Fig 1A) and showed significantly better overall survival (P=0.01) (Fig 1B), which correlated with the decrease in circulating inflammatory cytokines including TNFa (Fig 1C). Since the standard of care for acute GVHD still remains high dose steroids, we evaluated the effect of continued exposure to steroids (prednisone-100ug/ml) for a period of 96 hours on the viability of CB Tregs. When compared to CB Tcons, 90.3% CB Tregs remained alive and viable compared to 64.7% of Tcons (Fig 1D). No differences were observed in the intracellular expression of FOXP3 or Helios in the control vs. cyclosporine or sirolimus exposed cells (Fig 1E). Similarly, no significant impact was observed on their suppressor function (Fig 1F). Conclusions: Multiple injections with CB Tregs can effectively treat GVHD. Combination therapy of CB Tregs with the commonly used GVHD treatments can be explored. Figure 1 Disclosures Iyer: Genentech/Roche: Research Funding; Incyte: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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B Cells of HIV-1–Infected Patients Bind Virions through Cd21–Complement Interactions and Transmit Infectious Virus to Activated T Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.5.637 ◽

2000 ◽

Vol 192 (5) ◽

pp. 637-646 ◽

Cited By ~ 133

Author(s):

Susan Moir ◽

Angela Malaspina ◽

Yuexia Li ◽

Tae-Wook Chun ◽

Tomeka Lowe ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Lymphoid Tissues ◽

Activated T Cells ◽

Peripheral Blood Mononuclear ◽

The Impact ◽

Hiv 1 ◽

Hiv Negative

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4+ cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

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Alteration in mononuclear cell subpopulations in dogs immunized with gentamicin-attenuatedLeishmania infantum

Parasitology ◽

10.1017/s0031182012001187 ◽

2012 ◽

Vol 139 (13) ◽

pp. 1689-1696 ◽

Cited By ~ 5

Author(s):

HAMID DANESHVAR ◽

FARNAZ SEDGHY ◽

SHAHRIAR DABIRI ◽

HOSSEIN KAMIABI ◽

MOHAMMAD M. MOLAEI ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Mononuclear Cells ◽

Lower Percentage ◽

Wild Type ◽

Peripheral Blood Mononuclear ◽

Mhc Ii ◽

Blood Mononuclear Cells ◽

Cell Subpopulations ◽

The Impact

SUMMARYThe impact of immunization with gentamicin-attenuatedLeishmania infantum(H-line) on the immunophenotypic profile of popliteal lymph node (PLN) and peripheral blood mononuclear cells (PBMCs) of dogs was assessed by flow cytometry and immunohistochemistry. Compared with the dogs infected withL. infantumwild-type (Group WT), there was a significantly higher percentage of CD4+, CD44+T cells and CD14+, MHC-II+cells and a lower percentage of CD4+CD25+regulatory T cells in PLN of the immunized dogs withL. infantumH-line (Group H). The percentage of CD4+and CD8+T cells in PBMCs of immunized dogs was higher than that in dogs of Group WT. The CD4:CD8 ratio in PLN of dogs of Group H was significantly higher than that in dogs of Group WT. A significantly higher percentage of CD21+B cells and a lower percentage of CD79b+cells were found in PLN of the immunized dogs compared with dogs of Group WT. Immunohistochemical investigation showed no parasites in the PLN of immunized dogs whereas there were parasites in the PLN of 60% of dogs infected withL. infantumWT. In this study, the immunophenotypic profile of mononuclear cells of the immunized dogs correlates with cellular immunity.

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Persistent Systemic Inflammation in Patients With Severe Burn Injury Is Accompanied by Influx of Immature Neutrophils and Shifts in T Cell Subsets and Cytokine Profiles

Frontiers in Immunology ◽

10.3389/fimmu.2020.621222 ◽

2021 ◽

Vol 11 ◽

Author(s):

Patrick P. G. Mulder ◽

Marcel Vlig ◽

Bouke K. H. L. Boekema ◽

Matthea M. Stoop ◽

Anouk Pijpe ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Inflammatory Response ◽

Systemic Inflammation ◽

Peripheral Blood ◽

Burn Injury ◽

Severe Burn ◽

And Shifts ◽

Severe Burn Injury

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1β, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1–2.8 × 106/ml after burn vs. 5 × 103/ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4+ T cell population, elevated numbers of CCR4+CCR6- and CCR4+CCR6+ cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications.

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