dmLT Adjuvant Enhances Cytokine Responses to T Cell Stimuli, Whole Cell Vaccine Antigens and Lipopolysaccharide in Both Adults and Infants

Enhancement of mucosal immune responses in children and infants using novel adjuvants such as double mutant heat labile toxin (dmLT) is an important goal in the enteric vaccine field. dmLT has been shown to enhance mucosal IgA responses to the oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. dmLT can enhance IL-17A production from adult T cells, which may increase the production and secretion of mucosal IgA antibodies. However, the adjuvant mechanism remains to be fully elucidated and might differ between infants and adults due to age-related differences in the development of the immune system. The main objective of this study was to determine how dmLT influences antigen presenting cells and T cells from infants compared to adults, and the role of IL-1β for mediating the adjuvant activity. Peripheral blood mononuclear cells (PBMCs) from Bangladeshi infants (6-11 months) and adults (18-40 years) were stimulated with the mitogen phytohaemagglutinin (PHA), the superantigen Staphylococcal enterotoxin B (SEB), ETVAX whole cell component (WCC) or E. coli lipopolysaccharide (LPS) ± dmLT, and cytokine production was measured using ELISA and electrochemiluminescence assays. The adjuvant dmLT significantly enhanced SEB- and PHA-induced IL-17A, but not IFN-γ responses, in PBMCs from both infants and adults. Blocking experiments using an IL-1 receptor antagonist demonstrated the importance of IL-1 signaling for the adjuvant effect. dmLT, ETVAX WCC and LPS induced dose-dependent IL-1β responses of comparable magnitudes in infant and adult cells. Depletion experiments suggested that IL-1β was mainly produced by monocytes. dmLT enhanced IL-1β responses to low doses of WCC and LPS, and the adjuvant effect appeared over a wider dose-range of WCC in infants. dmLT and WCC also induced IL-6, IL-23 and IL-12p70 production in both age groups and dmLT tended to particularly enhance IL-23 responses to WCC. Our results show that dmLT can induce IL-1β as well as other cytokines, which in turn may enhance IL-17A and potentially modulate other immunological responses in both infants and adults. Thus, dmLT may have an important function in promoting immune responses to the ETVAX vaccine, as well as other whole cell- or LPS-based vaccines in infants in low- and middle-income countries.

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TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells in vitro and generate unique cellular immune responses in influenza vaccination

10.1101/2020.07.17.207423 ◽

2020 ◽

Author(s):

Randall Toy ◽

M. Cole Keenum ◽

Pallab Pradhan ◽

Katelynn Phang ◽

Patrick Chen ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Immune Responses ◽

Cellular Immunity ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Mouse Bone Marrow ◽

Peripheral Blood Mononuclear ◽

Flu Vaccination

ABSTRACTAlthough the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection – partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-κB and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination.GRAPHICAL ABSTRACT

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Sorafenib, but not sunitinib, affects function of dendritic cells and induction of primary immune responses

Blood ◽

10.1182/blood-2007-02-075945 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5610-5620 ◽

Cited By ~ 209

Author(s):

Madeleine M. Hipp ◽

Norbert Hilf ◽

Steffen Walter ◽

Daniela Werth ◽

Katharina M. Brauer ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Immune Responses ◽

Map Kinases ◽

Kinase Inhibitors ◽

Mononuclear Cells ◽

Cytotoxic T Cell ◽

Peripheral Blood Mononuclear

AbstractThe tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell responses. These inhibitory effects are mediated by inhibition of PI3 and MAP kinases and NFκB signaling. In contrast, sorafenib had no influence on the phenotype and proliferation of T cells. To analyze the effects of both TKIs on cytotoxic T-cell induction in vivo, C57BL/6 mice were pretreated with sorafenib or sunitinib and immunized with OVA257-264 peptide. Sorafenib, but not sunitinib, application significantly reduced the induction of antigen-specific T cells. Numbers of regulatory T cells were reduced in peripheral blood mononuclear cells from mice treated with sunitinib. These results indicate that sunitinib, but not sorafenib, is suitable for combination with immunotherapeutic approaches for treatment of cancer patients.

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Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts

Nature Communications ◽

10.1038/s41467-021-27674-x ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Rhia Kundu ◽

Janakan Sam Narean ◽

Lulu Wang ◽

Joseph Fenn ◽

Timesh Pillay ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Mononuclear Cells ◽

Memory T Cells ◽

Peripheral Blood Mononuclear ◽

Protective Function ◽

Host Protection ◽

Household Contacts ◽

Significant Difference ◽

Membrane Envelope

AbstractCross-reactive immune responses to SARS-CoV-2 have been observed in pre-pandemic cohorts and proposed to contribute to host protection. Here we assess 52 COVID-19 household contacts to capture immune responses at the earliest timepoints after SARS-CoV-2 exposure. Using a dual cytokine FLISpot assay on peripheral blood mononuclear cells, we enumerate the frequency of T cells specific for spike, nucleocapsid, membrane, envelope and ORF1 SARS-CoV-2 epitopes that cross-react with human endemic coronaviruses. We observe higher frequencies of cross-reactive (p = 0.0139), and nucleocapsid-specific (p = 0.0355) IL-2-secreting memory T cells in contacts who remained PCR-negative despite exposure (n = 26), when compared with those who convert to PCR-positive (n = 26); no significant difference in the frequency of responses to spike is observed, hinting at a limited protective function of spike-cross-reactive T cells. Our results are thus consistent with pre-existing non-spike cross-reactive memory T cells protecting SARS-CoV-2-naïve contacts from infection, thereby supporting the inclusion of non-spike antigens in second-generation vaccines.

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Single-cell RNA sequencing of peripheral blood mononuclear cells from acute Kawasaki disease patients

Nature Communications ◽

10.1038/s41467-021-25771-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhen Wang ◽

Lijian Xie ◽

Guohui Ding ◽

Sirui Song ◽

Liqin Chen ◽

...

Keyword(s):

T Cells ◽

Kawasaki Disease ◽

Immune Responses ◽

Single Cell ◽

Rna Sequencing ◽

Mononuclear Cells ◽

Healthy Controls ◽

Peripheral Blood Mononuclear ◽

Single Cell Rna Sequencing ◽

Blood Mononuclear Cells

AbstractKawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8+ T cells is decreased in acute KD, notably effector memory CD8+ T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD.

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Distribution of ACE2, CD147, cyclophilins, CD26 and other SARS-CoV-2 associated molecules in human tissues and immune cells in health and disease

10.1101/2020.05.14.090332 ◽

2020 ◽

Cited By ~ 2

Author(s):

U. Radzikowska ◽

M. Ding ◽

G. Tan ◽

D. Zhakparov ◽

Y. Peng ◽

...

Keyword(s):

T Cells ◽

Immune Cells ◽

Mononuclear Cells ◽

Human Cells ◽

Bronchial Biopsy ◽

Healthy Children ◽

Peripheral Blood Mononuclear ◽

Altered Expression ◽

Age Related ◽

Study Gene Expression

AbstractBackgroundMorbidity and mortality from COVID-19 caused by novel coronavirus SARS-CoV-2 is accelerating worldwide and novel clinical presentations of COVID-19 are often reported. The range of human cells and tissues targeted by SARS-CoV-2, its potential receptors and associated regulating factors are still largely unknown. The aim of our study was to analyze the expression of known and potential SARS-CoV-2 receptors and related molecules in the extensive collection of primary human cells and tissues from healthy subjects of different age and from patients with risk factors and known comorbidities of COVID-19.MethodsWe performed RNA sequencing and explored available RNA-Seq databases to study gene expression and co-expression of ACE2, CD147 (BSG), CD26 (DPP4) and their direct and indirect molecular partners in primary human bronchial epithelial cells, bronchial and skin biopsies, bronchoalveolar lavage fluid, whole blood, peripheral blood mononuclear cells (PBMCs), monocytes, neutrophils, DCs, NK cells, ILC1, ILC2, ILC3, CD4+ and CD8+ T cells, B cells and plasmablasts. We analyzed the material from healthy children and adults, and from adults in relation to their disease or COVID-19 risk factor status.ResultsACE2 and TMPRSS2 were coexpressed at the epithelial sites of the lung and skin, whereas CD147 (BSG), cyclophilins (PPIA and PPIB), CD26 (DPP4) and related molecules were expressed in both, epithelium and in immune cells. We also observed a distinct age-related expression profile of these genes in the PBMCs and T cells from healthy children and adults. Asthma, COPD, hypertension, smoking, obesity, and male gender status generally led to the higher expression of ACE2- and CD147-related genes in the bronchial biopsy, BAL or blood. Additionally, CD147-related genes correlated positively with age and BMI. Interestingly, we also observed higher expression of ACE2- and CD147-related genes in the lesional skin of patients with atopic dermatitis.ConclusionsOur data suggest different receptor repertoire potentially involved in the SARS-CoV-2 infection at the epithelial barriers and in the immune cells. Altered expression of these receptors related with age, gender, obesity and smoking, as well as with the disease status might contribute to COVID-19 morbidity and severity patterns.

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Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907883116 ◽

2019 ◽

Vol 116 (48) ◽

pp. 24242-24251 ◽

Cited By ~ 30

Author(s):

Kosuke Hashimoto ◽

Tsukasa Kouno ◽

Tomokatsu Ikawa ◽

Norihito Hayatsu ◽

Yurina Miyajima ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Cd4 T Cells ◽

Healthy Aging ◽

Mononuclear Cells ◽

Ex Vivo ◽

Cell Receptor ◽

Peripheral Blood Mononuclear ◽

Age Related

Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

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Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.684670 ◽

2021 ◽

Vol 11 ◽

Author(s):

Marta García-Sánchez ◽

Laura Jiménez-Pelayo ◽

Patricia Vázquez ◽

Pilar Horcajo ◽

Javier Regidor-Cerrillo ◽

...

Keyword(s):

T Cells ◽

Immune Responses ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Neospora Caninum ◽

Response Dynamics ◽

Peripheral Blood Mononuclear ◽

Cellular Immune Responses ◽

Ifn Γ ◽

Cellular Immune

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined by Neospora caninum isolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond to N. caninum infection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated against N. caninum, which may influence the course of infection.

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Potent Immunoregulatory Effects of Salmonella typhi Flagella on Antigenic Stimulation of Human Peripheral Blood Mononuclear Cells

Infection and Immunity ◽

10.1128/iai.67.3.1338-1346.1999 ◽

1999 ◽

Vol 67 (3) ◽

pp. 1338-1346 ◽

Cited By ~ 47

Author(s):

Timothy L. Wyant ◽

Michael K. Tanner ◽

Marcelo B. Sztein

Keyword(s):

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Salmonella Typhi ◽

Soluble Antigen ◽

Peripheral Blood Mononuclear ◽

Whole Cell ◽

Blood Mononuclear Cells

ABSTRACT A key function of monocytes/macrophages (Mφ) is to present antigens to T cells. However, upon interaction with bacteria, Mφ lose their ability to effectively present soluble antigens. This functional loss was associated with alterations in the expression of adhesion molecules and CD14 and a reduction in the uptake of soluble antigen. Recently, we have demonstrated that Salmonella typhiflagella (STF) markedly decrease CD14 expression and are potent inducers of proinflammatory cytokine production by human peripheral blood mononuclear cells (hPBMC). In order to determine whether S. typhi and soluble STF also alter the ability of Mφ to activate T cells to proliferate to antigens and mitogens, hPBMC were cultured in the presence of tetanus toxoid (TT) or phytohemagglutinin (PHA) and either killed whole-cell S. typhi or purified STF protein. Both whole-cell S. typhi and STF suppressed proliferation to PHA and TT. This decreased proliferation was not a result of increased Mφ production of nitric oxide, prostaglandin E2, or oxygen radicals or the release of interleukin-1β, tumor necrosis factor alpha, interleukin-6, or interleukin-10 following exposure to STF. However, the ability to take up soluble antigen, as determined by fluorescein isothiocyanate-labeled dextran uptake, was reduced in cells cultured with STF. Moreover, there was a dramatic reduction in the expression of CD54 on Mφ after exposure to STF. These results indicate that whole-cell S. typhi and STF have the ability to alter in vitro proliferation to soluble antigens and mitogens by affecting Mφ function.

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