el404  (R&D Systems)

Journal: Journal of Clinical Investigation

Article Title: Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance

doi: 10.1172/jci179709

Figure Lengend Snippet: Figure 2. Intra-graft gene expression within the CD8+ T cell clusters in accepted kidney allografts show changes from a cytotoxic to an exhausted/regulatory-like phenotype. (A and B) UMAP plot of CD8+ T cell cluster subsets at each time point in accepted kidney allografts (A) and rejecting kidney allografts at 1 week (B). (C and E) Violin plots of cytotoxic (red) and exhaustion/regulatory (green) genes in CD8+ T cell clusters in accepted kidney allografts (C) and accepted recipient’s spleen (E). The vertical axis indicates log-ranked gene expression levels. Mean expression levels are indicated in black points. (D) Density plot of CD8a+Fgl2+Il2rb+ cells in accepted kidney allografts at 3 weeks post-transplant. (F) Bar graph shows ELISPOT analysis of IFN-g production in T cells and CD8 T cells obtained from accepted kidney allograft and recipient’s spleen at 24 weeks post-transplant. Data are represented as mean ± SD, compared by 2-way ANOVA test. (G) Violin plot and dot plot of cytotoxic (red) and exhaustion/regulatory (green) genes in CD8+ T cell clusters, comparing rejecting grafts at 1 week and accepted kidney allografts at 1 and 3 weeks. The black bar indicates the mean value of expression levels in violin plot. (H) Heatmap of top differentially expressed genes in CD8+ T cell clusters at each time point in accepted kidney allografts.

Article Snippet: ELISPOT IFN-g-producing cells were quantified using the mouse IFN-g ELISpot kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Gene Expression, Expressing, Enzyme-linked Immunospot

Journal: Journal of Clinical Investigation

Article Title: Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance

doi: 10.1172/jci179709

Figure Lengend Snippet: Figure 4. Increased IFN-g expression and production within the T cell population in accepted kidney allografts, but FGL2 and CD8+ cells are not needed for the induction and maintenance of accepted kidney allografts. (A) Density plot of Ifng+ cells in total cells in accepted kidney allografts at each time point. (B) Violin plot and dot plot show levels and percentages of Ifng expression in T cell subset in accepted kidney allografts at each time point. (C) Bar graph shows ELISPOT analysis of IFN-g production in T cells obtained from accepted kidney allograft and recipient's spleen at 4 weeks post-transplant. Data are represented as mean ± SD, compared by 2-way ANOVA test. (D) Graft survival curve of kidney transplantation to IFN-g KO (n=3) or WT (n=3) (p=0.007). (E) Graft survival curve of kidney transplantation to Fgl2 KO (n=6) or WT (n=6) (p=0.138). (F) Line graph of serum levels of creatinine (Cr) and blood urea nitrogen (BUN) in long-term survived Fgl2 KO recipients (n=3). Data represent the mean ± SEM. (G) Graft survival curve of kidney transplantation to CD8 KO (n=5); WT (n=5). (H) Graft survival curve of heart transplantation to CD8 KO (n=3); WT (n=3) (p=0.432). (I and J) Pathological findings of kidney allografts obtained from CD8 KO. (I) H&E staining shows rTLOs formation perivascularly. (J) Immunohistochemistry of CD8 staining shows the absence of CD8 cell infiltration in kidney allografts taken from CD8 KO recipients. (K) Graft survival curve of kidney transplantation to PD-1 KO (n=5); WT (n=5) (p=0.002). (L) H&E staining of kidney allografts obtained from PD-1 KO shows signs of rejection. Statistical significance was determined by Log-rank test (D, E, H, and K). The scale bar indicates 100 µm (I, J, and L).

Article Snippet: ELISPOT IFN-g-producing cells were quantified using the mouse IFN-g ELISpot kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunospot, Transplantation Assay, Staining, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance

doi: 10.1172/jci179709

Figure Lengend Snippet: Figure 5. Reprogramming of donor reactive T cells following adoptive transfer in the presence of an accepted kidney allograft. (A) Schematic of experimental design. (B) ELISPOT assay of donor-sensitized T cells prior to transfer (n=3). Data represent the mean ± SD. (C) Frequency of CD8+ T cells in donor-sensitized T cells and levels of PD-1, CD122, Eomes, and Foxp3 expression in CD8+CD45.1+ donor-sensitized T cells prior to transfer. (D and E) Pathological findings of kidney allografts collected from adoptive transfer (AT) after kidney transplant (KTx) recipients at 14 days post-transfer. H&E stain (D) and Immunohistochemistry of CD45.1 staining (E). The scale bar indicates 100 µm. (F) Frequency of CD8+CD45.1+ T cells and CD8-CD45.1+ T cells in kidney, spleen, and lymph node (LN) obtained from adoptive transfer after kidney transplant recipients. (G) Frequency of the percentage of PD-1 positive cells per CD8+CD45.1+ adoptively transferred cells and CD8+CD45.2+ recipient endogenous cells. (F and G) Data are represented as mean ± SEM, compared by 1-way ANOVA test.

Article Snippet: ELISPOT IFN-g-producing cells were quantified using the mouse IFN-g ELISpot kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Enzyme-linked Immunospot, Expressing, Staining, Immunohistochemistry

Journal: Journal of Clinical Investigation

Article Title: Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance

doi: 10.1172/jci179709

Figure Lengend Snippet: Figure 7. Reprogramming of donor reactive T cells does not occur in alloreactive T cells that lack IFN-gR (IFNGR). (A) Schematic of experimental design. (B) ELISPOT assay of donor-sensitized T cells prior to transfer (n=3). Data represent the mean ± SD. (C-G) Pathological findings of kidney allografts collected from adoptive transfer (AT) after kidney transplant (KTx) recipients at 2 to 5 weeks post-transplant (C-E) and from WT.B6 without AT at 3 weeks (F) and 6 weeks post-KTx (G). H&E staining (C-G). The black arrow indicates rTLOs (F and G). The scale bar indicates 100 µm. (H) Frequency of CD8+CD45.1- T cells and CD8-CD45.1- T cells in kidney, spleen, and lymph node (LN) obtained from AT of IFNGR KO alloreactive cells after KTx recipients. Data are represented as mean ± SEM, compared by 1-way ANOVA test. (I) Frequency of PD- 1+CD122+, PD-1+Eomes+, and PD-1+Foxp3+ cells per CD8+CD45.1- T cells and CD8- CD45.1- T cells in rejecting kidney allografts from AT of IFNGR KO alloreactive cells after KTx recipients. Data are represented as mean ± SEM.

Article Snippet: ELISPOT IFN-g-producing cells were quantified using the mouse IFN-g ELISpot kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, Adoptive Transfer Assay, Staining

Journal: Frontiers in Immunology

Article Title: RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine

doi: 10.3389/fimmu.2022.974016

Figure Lengend Snippet: Source of reagents and kits used in the study.

Article Snippet: Mouse IL-4 ELISPOT kit , RnD systems , EL404.

Techniques: In Vivo, Luciferase, Enzyme-linked Immunospot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Oncotarget

Article Title: The novel complex combination of alum, CpG ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo

doi: 10.18632/oncotarget.17504

Figure Lengend Snippet: ( A ) The 4-h NK assay against YAC-1 targets was determined at various effector:target ratios. Error bars represent mean + SEM. ** p < 0.01, *** p < 0.001. ( B ) The cytolytic activity of splenocytes against NY-ESO-1 + B16 cells was measured using a 4 h 51 Cr-release assay at various E:T ratios. Error bars represent mean + SEM. *** p < 0.001. ( C ) The representative graphs of spot-forming cells for IFN-γ and IL-4 at one week post-immunization in the ELISpot assay. ( D ) The average number of IL-4/IFN-γ secreting splenocytes was calculated (n = 3, three independent experiments). Error bars represent mean + SEM. * p < 0.05, ** p < 0. 01. ( E ) T cell analysis in splenocytes after vaccination. Intracellular staining of IFN-γ in CD4 + and CD8 + T cells was analyzed by FACS.

Article Snippet: The mouse IFN-γ/IL-4 Dual-Color ELISpot kit (R&D Systems, Minneapolis, USA) was used for ELISpot assay.

Techniques: Activity Assay, Release Assay, Enzyme-linked Immunospot, Cell Analysis, Staining

Journal: Menopause

Article Title: Repurposing ospemifene for potentiating an antigen-specific immune response

doi: 10.1097/gme.0000000000000776

Figure Lengend Snippet: FIG. 6. Effects of ospemifene (OSP) on IFN-g immune response and cytotoxic T lymphocyte activity after chronic and intermittent dosing schedules. Antigen-specific IFN-g immune response was assessed by ELISpot 48 hours after the final dose of tecemotide peptide vaccine (PV). (A) Average spot forming cells (SFC/3.0 105 cells þ SEM) and (B) average target cell apoptosis percentages (þSEM) after 9 weeks of chronic daily OSP (50 mg/kg; n ¼ 16) compared with control (n ¼ 22), two cycles of PV alone (n ¼ 6), and OSP þ PV (n ¼ 11) (P < 0.01, P < 0.0001 compared with control unless indicated otherwise; two-way ANOVA). (C) Average SFC/5.0 105 cells þ SEM after treatment with one cycle of PV (n ¼ 10) or PV combined with intermittent (16 d) OSP 10, 50, or 100 mg/kg (n ¼ 11 each combination) (P < 0.05, two-way ANOVA). (D) Average target cell apoptosis percentages (þSEM) were assessed in control (n ¼ 10), one cycle of PV (n ¼ 11), short-course (16 d) OSP at 10 and 50 mg/kg (n ¼ 11), and OSP þ PV combination (n ¼ 11) treatment groups (P < 0.05, P < 0.01; one-way ANOVA). ANOVA, analysis of variance; IFN, interferon.

Article Snippet: A Mouse IFN-g/IL-4 dual color ELISpot Kit (R&D Systems, Minneapolis, MN) or eBioscience (San Diego, CA) Mouse IFN-g ELISpot Ready-SET-Go kit were used to perform the analysis according to the manufacturer’s instructions.

Techniques: Activity Assay, Enzyme-linked Immunospot, Control

Journal: Menopause

Article Title: Repurposing ospemifene for potentiating an antigen-specific immune response

doi: 10.1097/gme.0000000000000776

Figure Lengend Snippet: FIG. 7. Antitumor effects and serum IFN-g immune response to tecemotide peptide vaccine (PV) immunotherapy after treatment with control, PV alone, ospemifene (OSP) 100 mg/kg alone or OSP þ PV. Each of four weekly 100-mg s.c. injections of PV was preceded by three daily oral doses of OSP (100 mg/kg). All mice were implanted with breast cancer allografts on day 0, and serum and spleens were collected on day 28 after 4 weeks of treatment. Tumor volumes were measured twice each week. (A) Average tumor volumes (þSEM) are shown for days 22 and 25 along with (B) serum IFN-g levels as assessed on day 28 (n ¼ 8, all groups) (P < 0.05 compared with both control and OSP 100 mg/kg; two-tailed Student’s t test). (C) Representative ELISpot results are displayed for the scrambled and BP25 peptides for each treatment group along with the range of spot forming cells (SFC)/5.0 105 total cells and the number of subjects (n ¼ 5-6) with more than 300 and 1000 SFCs, respectively, in response to the BP25 peptide. IFN, interferon.

Article Snippet: A Mouse IFN-g/IL-4 dual color ELISpot Kit (R&D Systems, Minneapolis, MN) or eBioscience (San Diego, CA) Mouse IFN-g ELISpot Ready-SET-Go kit were used to perform the analysis according to the manufacturer’s instructions.

Techniques: Control, Two Tailed Test, Enzyme-linked Immunospot

Journal: Menopause

Article Title: Repurposing ospemifene for potentiating an antigen-specific immune response

doi: 10.1097/gme.0000000000000776

Figure Lengend Snippet: FIG. 8. Dose and schedule-dependent effects of ospemifene (OSP) on serum IFN-g, lymphocyte IL-6 expression, antigen-specific immune response, and T-cell activation. Mice were treated with OSP at 25 or 50 mg/kg for 1, 2, or 3 days before tecemotide peptide vaccine (PV) administration each week for 3 weeks (n ¼ 6, all groups). (A) Average serum IFN-g (SEM) was analyzed by multiplex (P < 0.05 compared with control; two-tailed Student’s t test). (B) Average spot-forming cells (SFC/1.0 106 cells þ SEM) after ELISpot analysis (P < 0.05, P < 0.001; two-tailed Student’s t test). (C) Mean intracellular IL-6 (þSEM) in spleen-derived lymphocytes was analyzed by qRT-PCR and normalized against the expression of mouse b2-microglobulin (B2M) (P < 0.001, P < 0.0001 compared with control; one-way ANOVA). Average expression (þSEM) of CD69 (D) and CD27 (E) in spleen-derived lymphocytes as assessed by flow cytometry (P < 0.05, P < 0.01 compared with control; one-way ANOVA). ANOVA, analysis of variance; IFN, interferon; IL, interleukin; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: A Mouse IFN-g/IL-4 dual color ELISpot Kit (R&D Systems, Minneapolis, MN) or eBioscience (San Diego, CA) Mouse IFN-g ELISpot Ready-SET-Go kit were used to perform the analysis according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Multiplex Assay, Control, Two Tailed Test, Enzyme-linked Immunospot, Derivative Assay, Quantitative RT-PCR, Flow Cytometry, Real-time Polymerase Chain Reaction