Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: Libraries were prepared from CD8+CD45RO+ cells sorted from the peripheral blood as described from 29 patients with T1D and 15 HC. Each well was expanded with irradiated allogeneic PBMC+IL-2, IL-7, and IL-15 as described in Materials and Methods. After 10 days the wells were washed and stimulated with K562 cells that had been pulsed with 6 diabetes peptides, peptides from EBV/flu, or treated with DMSO alone. IFNγ levels in the supernatants were measured by ELISA after 6 days. Data from a representative patient and HC subject are shown in Supplemental Figure 2B. The frequencies of positive wells that were above the threshold, which was mean+3SD of DMSO wells were calculated for each subject. There was a significantly greater proportion of positive wells from CD45RO+ cells from patients with T1D reactive with islet antigens (A)(*p=0.028, t-test with Welch’s correction) compared to HC, but not to peptides from EBV/flu (B). CD45RA+CD8+ cells were sorted from 25 and 13 of the T1D patients and HC respectively. (C,D) There was not a significant difference between the frequency of islet antigen-reactive (C) or EBV/flu reactive cells (D). The relationship between frequency of CD45RO+ CD8+ islet antigen-reactive cells and diabetes duration (E) or age (F) are shown (p=ns, Spearman corr). The orange and green dots represent samples from concordant identical triplets and twins respectively.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) Positive wells from the CD8+ T cell libraries from 4 patients with T1D and 3 HC subjects were further expanded with cytokines and challenged with K562 cells pulsed with each individual peptide used in the original pool. The levels of IFNγ were measured after 6 days. The data are from 11 wells from the 3 patients described in the text (Pt 116, Pt 69, and Pt 63) with T1D and 3 HC subjects (HC1023, 19, PC24). Each graph represents the analysis of positive wells from an individual patient. The bars (black and grey) represent the cytokine responses of different positive wells to the peptides. There were positive responses to ZnT8186–194 in wells from the patients with T1D but responses to IGRP228–236, PPI34–42, PPI15–24, and ZnT8186–194 in the HC subjects. (B) The CD45RO+ cells from one library well without and two library wells with an IFNγ response to the peptide pulsed K562 cells from Pt 63 were expanded in cytokines and stained with tetramers loaded with ZnT8186–194 peptide or control tetramer and analyzed by flow cytometry. The percentages refer to the frequency of tetramer+ cells in the CD8+ gate.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Staining, Control, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) The Vα and Vβ sequences, identified in 5C above from pt 63, were detected, using PCR for the TCR chains, in positive (+) but not negative (−) library wells (selected for IFNγ responses). (DW=control well) There were multiple wells in the CD45RO libraries but a single positive well from the CD45RA+ libraries in which the Vα and Vβ sequences were identified. (B) PBMC isolated in a repeat draw from the same patient were cultured with ZnT8186–194 peptide. IFNγ+CD8+ and IFNγ−CD8+ T cells were identified with a capture assay and sorted. The presence of the TCR Vα and Vβ chains were again identified by PCR (arrow) in the IFNγ+ cells.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Control, Isolation, Cell Culture

Journal: The Journal of general virology

Article Title: Identification of novel HLA-A*0201-restricted CD8+ T-cell epitopes on hepatitis delta virus.

doi: 10.1099/vir.0.80183-0

Figure Lengend Snippet: Fig. 6. IFN-c ELISPOT assay for quantification of cytotoxic responses in patient 1. Stimulated PBMCs were restimulated with peptides HBV core 18–27, HDV 26–34 or HDV 43–51. The number of peptide-specific SFCs in 26104 cells was cal- culated by subtracting the number of spots in the absence of antigen from that in the presence of antigen.

Article Snippet: A human IFN-c ELISPOT assay kit (R&D Systems) was used to determine whether the stimulated PBMCs were functional, by following the manufacturer’s protocol.

Techniques: Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: CD4 T–cell epitopes to Zika envelope protein in HLA class II transgenic mice. Mice transgenic for HLA-DR1 (DRB1*0101) n = 6 ( A ); HLA-DR4 (DRB1*0401) n = 5 ( B ); HLA-DR15:01 (DRB1*1501) n = 4 ( C ) and HLA-DQ8 (DQB1*0302) n = 6 ( D ) were primed with 25 μg of recombinant zika envelope protein (Env). 10 days after immunisation, draining lymph node cells (DLNs) were assayed by IFNγ ELISpot for recall responses to the Env protein and to an overlapping panel of 50 Env peptides. Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). Peptides that were defined as positive epitopes across 3 or more HLA class II transgenic mouse lines are highlighted.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Transgenic Assay, Recombinant, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: T cells responding to ZIKV envelope protein peptide 1 are crossreactive with variant peptides from other flavivirus species. Mice transgenic for ( A ) HLA-DR1 (DRB1*0101) n = 6, ( B ) HLA-DR15:01 (DRB1*1501) n = 6 and ( C ) HLA-DQ8 (DQB1*0301) n = 6 were primed with 25 μg of recombinant ZIKV envelope protein (Env). 10 days after immunisation, DLNs were assayed by IFNγ ELISpot for recall responses to the Env protein, Env peptide 10, Env peptides as shown and the corresponding peptide variants of west nile virus (WNV), yellow fever virus (YFV), dengue virus 1 (D1), dengue virus 2 (D2), dengue virus 3 (D3) and dengue virus 4 (D4). Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). HLA-DR1 ELISpot supernatants were collected prior to assay development and levels of ( D ) IL-17A and ( E ) IL-10 measured by ELISA. Data shown represent mean ± SEM. Statistical significance was determined using an unpaired t-test.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Variant Assay, Transgenic Assay, Recombinant, Enzyme-linked Immunospot, Virus, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses

doi: 10.1038/s41598-017-18781-1

Figure Lengend Snippet: ZIKV virus infection in AG129 mice is associated with a strong T-cell response to ZIKV Env peptide 1 which is crossreactive with variant peptides from other flavivirus species. AG129 mice were (i) mock infected (n = 4) or (ii) infected intraperitoneally with 10 5 FFU of ZIKV (PF13/251013-18) (n = 5). ( A ) Mock (grey circles) and ZIKV infected (black circles) mice were monitored daily for signs of weight loss and culled at 7 days post infection. ( B ) ZIKV RNA load was quantified by real time PCR. Splenocytes from both groups of mice were assayed by IFNγ ELISpot for recall responses to ( C ) ZIKV proteins Env, NS1, NS3 and NS5, ( D ) an overlapping panel of 50 Env peptides and flavivirus variant peptides from west nile virus (WNV), yellow fever virus (YFV), dengue virus 1 (D1), dengue virus 2 (D2), dengue virus 3 (D3) and dengue virus 4 (D4) for peptides 1 ( E ) and 31 ( H ). Data are plotted as number of spot forming cells (SFC) per 10 6 cells for individual mice. Responses were considered positive (+) if the response was greater than 2 SD above the mean of the response in the absence of any antigen (shown as horizontal dotted line). ELISpot supernatants from peptide 1 variants were also collected prior to assay development and levels of IL-17A ( F ) and IL-10 ( G ) measured by ELISA. Data shown represent mean ± SEM.

Article Snippet: The frequency of cells producing IFNγ in response to antigen was quantified by ELISpot using a murine IFN gamma ELISpot set kit (862.031.020, Diaclone; 2B Scientific, Oxon, U.K) – .

Techniques: Virus, Infection, Variant Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: Immunological Analysis of a CCHFV mRNA Vaccine Candidate in Mouse Models

doi: 10.3390/vaccines7030115

Figure Lengend Snippet: ELISPOT assays. ( A ) Splenocyte cells from immunized C57BL/6 were obtained on week four after immunization. IFN-gamma secretion was analyzed by ELISPOT assay. The number of IFN-gamma specific spots in the booster dose group was higher than for the single dose. ( B ) The number of IL-4-specific spots was similar to that for IFN-gamma. This indicates that the booster dose had a stronger effect than the single dose. The data was analyzed using the log-rank test (* P < 0.05; ** P < 0.01; and *** P < 0.001) and presented as mean ± SD. Error bars indicate standard deviations (SD).

Article Snippet: The ELISPOT assays were performed by ELISPOT Mouse IFN-gamma and IL-4 kits (R&D Systems, Minneapolis, MN, USA) based on the established protocol of the manufacturer.

Techniques: Enzyme-linked Immunospot

Journal: Diabetologia

Article Title: Suppressor of cytokine signalling-3 expression inhibits cytokine-mediated destruction of primary mouse and rat pancreatic islets and delays allograft rejection.

doi: 10.1007/s00125-008-1090-0

Figure Lengend Snippet: Fig. 1 Cytokine-mediated mitochondrial impairment, insulin release and apoptosis is inhibited in RIP-Socs3 transgenic mouse islets. Wild- type (white bars) and RIP-Socs3 transgenic (black bars) mouse islets were stimulated with no cytokine (Control) or a mixture of cytokines (IL-1 75 pg/ml+IFNγ 1 ng/ml) for 72 h. The islets were subjected to the MTT assay (a) and the accumulated insulin release in the medium was analysed by ELISA (b). Apoptosis induction was analysed by life–death detection assay (c). Results are presented as mean±SD of three to five individual experiments, *p<0.05; **p<0.01. d Western blot analysis of SOCS3 levels in RIP-Socs3 transgenic (Tg) and wild- type (Wt) islets. Equal amounts of protein (10 µg) were loaded in each lane and analysed using a monoclonal Flag antibody directed against recombinant flag-tagged SOCS3

Article Snippet: After culture, a total of 50 islets/condition were stimulated for 72 h in 200 μl RPMI 1640 glutamax-1 with 0.5% (vol./vol.) human serum and 1% (wt/vol.) penicillin/streptomycin, in the absence or presence of a mixture of 75 pg/ml recombinant mouse IL-1 (1×107 U/mg; BD Pharmingen, San Diego, CA, USA) and 1 ng/ml recombinant rat IFNγ (1×106 U/mg; R&D Systems, Minneapolis, MN, USA).

Techniques: Transgenic Assay, Control, MTT Assay, Enzyme-linked Immunosorbent Assay, Detection Assay, Western Blot, Recombinant