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Journal: mBio
Article Title: A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells
doi: 10.1128/mBio.02121-20
Figure Lengend Snippet: Nanoparticle-based therapeutic vaccination synergizes with a PD-L1 blockade to increase retrovirus-specific CD8 + T cell immunity. (A) Chronically FV-infected mice were treated twice with αPD-L1 or an isotype control (Iso) antibody, starting at 6 weeks postinfection. Groups of mice received therapeutic vaccination with CpG and GagL 85–93 -functionalized CaP nanoparticles alone or in addition to the PD-L1 blockade at the beginning of the treatment. Seven days after the initial treatment, the CD8 + T cell response was analyzed. (B and D) Numbers of total CD8 + T cells (B) or percentages of GagL 85–93 -specific tetramer + CD8 + T cells (D) were determined in the spleen by counting viable cells using trypan blue staining. Cell counts were applied to viable-cell populations in a flow cytometric analysis. (C) Representative dot plots from flow cytometry showing the frequencies of Gag-specific tetramer + CD8 + T cells. (E) Representative histogram from flow cytometry showing GzmB-expressing CD43 + tetramer + CD8 + T cells. (F) Mean fluorescent intensity (MFI) for GzmB gated on CD43 + tetramer + CD8 + T cells. (G) Ratio between GzmB-expressing CD43 + tetramer + CD8 + T cells and Foxp3-expressing CD4 + regulatory T cells in the spleen. (H) Seven days after initial treatment, an in vivo cytotoxicity assay was performed to determine the killing capacity of Gag-specific CD8 + T cells. Representative histograms of CD45.1 gated donor cells from the spleen showing in vivo killing of target cells loaded with FV GagL peptide. (I) Elimination of donor CD45.1 + cells in the spleen and blood of chronically infected mice after treatment. (J) Representative dot plots from flow cytometry showing the frequencies of IFN-γ- and TNF-α-producing tetramer + CD8 + T cells. (K) Frequencies of IFN-γ- and TNF-α-expressing tetramer + CD8 + T cells after treatment. Splenocytes were restimulated in vitro for 4 h with PMA and ionomycin in the presence of BFA. (L) Frequencies of proliferating tetramer + CD8 + T cells indicated by Ki67 expression. Results are pooled from two independent experiments. (M) Viral load was determined in the spleen 7 days after treatment started. Results are pooled from three independent experiments. Data shown are means ± standard errors of the means (SEM). Statistics were done by one-way ANOVA with Tukey’s multiple-comparison posttest.
Article Snippet: The following monoclonal antibodies were used. αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCD80 (clone 16-10A1),
Techniques: Infection, Staining, Flow Cytometry, Expressing, In Vivo, Cytotoxicity Assay, In Vitro
Journal: mBio
Article Title: A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells
doi: 10.1128/mBio.02121-20
Figure Lengend Snippet: Professional APCs experience maturation by combination therapy. Chronically FV-infected mice were treated twice with αPD-L1 or an isotype control (Iso) antibody with or without therapeutic NPV. Splenocytes were analyzed 7 days after the initial treatment. Mean fluorescent intensity (MFI) data are shown for the expression of CD80 by CD11c + F4/80 – DCs (A) and F4/80 + macrophages (B). The results of 2 independent experiments are illustrated. Data represent means ± SEM. Statistics were done by one-way ANOVA with Tukey’s multiple-comparisons posttest.
Article Snippet: The following monoclonal antibodies were used. αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCD80 (clone 16-10A1),
Techniques: Infection, Expressing
Journal: mBio
Article Title: A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells
doi: 10.1128/mBio.02121-20
Figure Lengend Snippet: Differentiated effector cells are induced by therapeutic vaccination during chronic retrovirus infection, but not by a PD-L1 blockade. Chronically FV-infected mice were treated twice with αPD-L1 or an isotype control (Iso) antibody with or without therapeutic NPV. CD8 + T cell immunity was analyzed 7 days after the initial treatment. (A) Representative flow cytometry dot plots for the identification of SLECs by Klrg1 and CD127 expression. (B) Frequencies of Klrg1 + CD127 − tetramer + CD8 + SLECs are depicted. (C) Frequencies of Klrg1 – CD127 + tetramer + CD8 + cells are shown. The results of 2 independent experiments are illustrated. Data represent means ± SEM. Statistics were done by one-way ANOVA with Tukey’s multiple-comparison posttest. T EX , exhausted CD8 + T cells.
Article Snippet: The following monoclonal antibodies were used. αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCD80 (clone 16-10A1),
Techniques: Infection, Flow Cytometry, Expressing
Journal: mBio
Article Title: A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells
doi: 10.1128/mBio.02121-20
Figure Lengend Snippet: Enhanced reactivation of differentiated exhausted CD8 + T cells. Chronically FV-infected mice were treated twice with αPD-L1 or an isotype control (Iso) antibody with or without therapeutic NPV. CD8 + T cell immunity was analyzed 7 days after the initial treatment in the spleen. (A) Percentages of PD-1 expression by CD8 + T cells during chronic FV infection. (B) PD-1 expression by tetramer + CD8 + T cells after treatment. (C) Percentages of Ki67-expressing TOX + or TOX – Tet + CD8 + T cells. (D) Frequencies of TOX-expressing cells among PD-1 + tetramer + CD8 + T cells. (E) Representative flow cytometry dot plots illustrate the expression of TOX by the two PD-1 + subpopulations of Tet + CD8 + T cells. Percentages of TOX expression by PD-1 lo and PD-1 hi tetramer + CD8 + T cells were analyzed. (F) Frequencies of GzmB-expressing PD-1 + tetramer + CD8 + T cells were analyzed. (G) GzmB-expressing PD-1 hi tetramer + CD8 + T cells were analyzed. Representative flow cytometry dot plots demonstrate the gating on PD-1 hi -expressing Tet + CD8 + T cells. (H) PD-1 hi -expressing CD8 + T cells were isolated from the spleens of chronically infected mice 7 days after the initial treatment and cultured ex vivo with Gag peptide-loaded and unloaded CFSE-stained target cells. The specific killing of peptide-loaded target cells is depicted. The results from 2 independent experiments were pooled. Bars represent means ± SEM. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison posttest.
Article Snippet: The following monoclonal antibodies were used. αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCD80 (clone 16-10A1),
Techniques: Infection, Expressing, Flow Cytometry, Isolation, Cell Culture, Ex Vivo, Staining
Journal: mBio
Article Title: A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells
doi: 10.1128/mBio.02121-20
Figure Lengend Snippet: The timing of therapeutic vaccination is critical for effective combination therapy with a PD-L1 blockade. (A) Chronically FV-infected mice were treated twice with αPD-L1 or an isotype control (Iso) antibody starting at 6 weeks postinfection. Groups of mice received either therapeutic vaccination with CpG- and GagL 85–93 -functionalized CaP nanoparticles alone or in addition to a PD-L1 blockade 4 days after the treatment start. Seven days after therapeutic vaccination, the CD8 + T cell response was analyzed. (B) Viral load was determined in the spleen 7 days after therapeutic vaccination. (C) Percentages of Gag-specific tetramer + CD8 + T cells are shown. (D) Frequencies of IFN-γ- and TNF-α-expressing tetramer + CD8 + T cells after treatment. Splenocytes were restimulated in vitro for 4 h. (E) Frequencies of GzmB-expressing CD43 + tetramer + CD8 + T cells. (F) Frequencies of GzmB-expressing PD-1 hi tetramer + CD8 + T cells. Numbers of CXCR5 + (G) or CXCR5 – (H) tetramer + CD8 + T cells are shown. The results of 2 independent experiments were pooled. Bars represent means ± SEM. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple-comparison posttest.
Article Snippet: The following monoclonal antibodies were used. αCD4 (clone RM4-5), αCD8 (clone 53-6.7), αCD80 (clone 16-10A1),
Techniques: Infection, Expressing, In Vitro