mu5ubee rrid ab 11219877 pe cy7 cd279 pd 1 Search Results


mu5ubee rrid ab 11219877 pe cy7 cd279 pd 1  (Thermo Fisher)
94
Thermo Fisher mu5ubee rrid ab 11219877 pe cy7 cd279 pd 1
Mu5ubee Rrid Ab 11219877 Pe Cy7 Cd279 Pd 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pe conjugated cxcr5  (Thermo Fisher)
86
Thermo Fisher pe conjugated cxcr5
Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of <t>CXCR5,</t> BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.
Pe Conjugated Cxcr5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd4  (Becton Dickinson)
90
Becton Dickinson cd4
(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of <t>Ki-67+CD4+</t> and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.
Cd4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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live dead-ir stain  (Thermo Fisher)
90
Thermo Fisher live dead-ir stain
(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of <t>Ki-67+CD4+</t> and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.
Live Dead Ir Stain, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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apc conjugated ccr5 3a9  (Becton Dickinson)
90
Becton Dickinson apc conjugated ccr5 3a9
Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and <t>CCR5</t> expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.
Apc Conjugated Ccr5 3a9, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ifn-γ  (Becton Dickinson)
90
Becton Dickinson ifn-γ
(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific <t>IFN-γ–</t> and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.
Ifn γ, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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perforin  (Becton Dickinson)
90
Becton Dickinson perforin
(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and <t>perforin+</t> (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and <t>Env-specific</t> <t>IFN-γ–</t> and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.
Perforin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ki-67  (Becton Dickinson)
90
Becton Dickinson ki-67
(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of <t>Ki-67+CD4+</t> and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.
Ki 67, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd95  (Becton Dickinson)
90
Becton Dickinson cd95
Plasma SIV RNA viral loads (copies/ml) shown as (A) geometric mean for each group and (B) for individual RMs at initiation of anti–PD-1 Ab or saline infusion. Limit of detection is 60 copies/ml. (C) Kaplan-Meier curve of number of days until viral suppression (<100 copies/ml for at least 2 or more consecutive time points). (D) Plasma SIV RNA viral loads (copies/ml) from chronically SIV-infected RMs administered humanized anti–PD-1 Ab or no Ab during ART initiation in a second study (n = 4 per group). (E) Frequency of Tcm <t>(CD28+CD95+)</t> CD4+ T cells in the rectum. (F) Frequency of IL-17A–producing CD4+ T cells in the rectum after PMA and ionomycin stimulation (saline, n = 9). (G) Frequency of neutrophils in lamina propria (LP) sections after 36 weeks of ART (saline, n = 5; PD-1 Ab treated, n = 6). Bars indicate the mean. Data were not collected for all animals. Shaded gray area depicts ART. Unfilled symbols indicate values from Mamu-A*01 RMs. Specific T cell frequency data are shown as mean ± SEM. *P < 0.05 by Mantel-Cox test (C), 2-way ANOVA (E and F), or 2-tailed Mann-Whitney test (G). n = 10 per group unless otherwise noted.
Cd95, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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tnf-α  (Becton Dickinson)
90
Becton Dickinson tnf-α
(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– <t>and</t> <t>TNF-α–producing</t> CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.
Tnf α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pacblue conjugated cd3 sp-34-2  (Becton Dickinson)
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Becton Dickinson pacblue conjugated cd3 sp-34-2
Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live <t>CD3+</t> CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.
Pacblue Conjugated Cd3 Sp 34 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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nkp44 rea1163  (Miltenyi Biotec)
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Miltenyi Biotec nkp44 rea1163
Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live <t>CD3+</t> CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.
Nkp44 Rea1163, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Isolation, Expressing

CXCR5 expression and localization of PD-1hi CD4 T cells of chronically SIV infected vaccine-controller and non-controller RM. (A) Percentage of CXCR5+ and neg PD-1hi CD4 T cells in the LN and rectum of healthy (n=7 and n=4, respectively), SIV+ vaccine-controller (n=13) and SIV+ non-controller (n=13) RM. (B) Percentage of CXCR5+ and neg PD-1hi CD4 T cells as a frequency of memory CD95+ CD4 T cells in the LN and rectum of healthy, SIV+vaccine-controller, and SIV+ non-controller RM. (C) A representative immunofluorescent staining for PD-1+, CD4+, and CD20+ cells in a rectal lymphoid aggregate of an SIV+ vaccine-controller and SIV+ non-controller RM and quantitative analysis showing the follicle size and intensity and frequency of PD-1+ CD4 T cells in SIV infected vaccine-controller (n=2) and SIV infected non-controller RM (n=3). (D) Expression of Bcl-6 on CXCR5+ and CXCR5- CD95+ CD4 T cells compared to PD-1neg CD95+ CD4 T cells. Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: CXCR5 expression and localization of PD-1hi CD4 T cells of chronically SIV infected vaccine-controller and non-controller RM. (A) Percentage of CXCR5+ and neg PD-1hi CD4 T cells in the LN and rectum of healthy (n=7 and n=4, respectively), SIV+ vaccine-controller (n=13) and SIV+ non-controller (n=13) RM. (B) Percentage of CXCR5+ and neg PD-1hi CD4 T cells as a frequency of memory CD95+ CD4 T cells in the LN and rectum of healthy, SIV+vaccine-controller, and SIV+ non-controller RM. (C) A representative immunofluorescent staining for PD-1+, CD4+, and CD20+ cells in a rectal lymphoid aggregate of an SIV+ vaccine-controller and SIV+ non-controller RM and quantitative analysis showing the follicle size and intensity and frequency of PD-1+ CD4 T cells in SIV infected vaccine-controller (n=2) and SIV infected non-controller RM (n=3). (D) Expression of Bcl-6 on CXCR5+ and CXCR5- CD95+ CD4 T cells compared to PD-1neg CD95+ CD4 T cells. Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Expressing, Infection, Staining

Association between anti-viral CD8 T cells and PD-1hi CD4 T cells in the LN following SIV infection. (A) Representative plots and percentage of Gag CM9+ and Granzyme B+ GagCM9+ CD8 T cells in the Blood, LN, and Rectum of SIV+ vaccine-controller (n=10, 9, 9) and SIV+ non-controller (n=8, 9, 9) RM. (B) Correlations of Gag CM9+ CD8 T cells and Granzyme B+ GagCM9+ CD8 T cells with PD-1hi and CXCR5+ PD-1hi CD4 T cells in the LN of SIV+ vaccine-controller and non-controller RM. (C) In vitro killing assay showing the percentage of PD-1hi CXCR5+ CD4 T cells co-cultured with SIV specific CD8 T cells for 5 days from the LN of SIV+ vaccine-controller RM. For each individual animal, the frequencies of Gag CM9 tetramer+ cells ex vivo prior to in vitro stimulation are shown. Scatter Plots show the median. *, P < 0.05; **, P < 0.01.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Association between anti-viral CD8 T cells and PD-1hi CD4 T cells in the LN following SIV infection. (A) Representative plots and percentage of Gag CM9+ and Granzyme B+ GagCM9+ CD8 T cells in the Blood, LN, and Rectum of SIV+ vaccine-controller (n=10, 9, 9) and SIV+ non-controller (n=8, 9, 9) RM. (B) Correlations of Gag CM9+ CD8 T cells and Granzyme B+ GagCM9+ CD8 T cells with PD-1hi and CXCR5+ PD-1hi CD4 T cells in the LN of SIV+ vaccine-controller and non-controller RM. (C) In vitro killing assay showing the percentage of PD-1hi CXCR5+ CD4 T cells co-cultured with SIV specific CD8 T cells for 5 days from the LN of SIV+ vaccine-controller RM. For each individual animal, the frequencies of Gag CM9 tetramer+ cells ex vivo prior to in vitro stimulation are shown. Scatter Plots show the median. *, P < 0.05; **, P < 0.01.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Infection, In Vitro, Cell Culture, Ex Vivo

(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: RNA Sequencing Assay

Plasma SIV RNA viral loads (copies/ml) shown as (A) geometric mean for each group and (B) for individual RMs at initiation of anti–PD-1 Ab or saline infusion. Limit of detection is 60 copies/ml. (C) Kaplan-Meier curve of number of days until viral suppression (<100 copies/ml for at least 2 or more consecutive time points). (D) Plasma SIV RNA viral loads (copies/ml) from chronically SIV-infected RMs administered humanized anti–PD-1 Ab or no Ab during ART initiation in a second study (n = 4 per group). (E) Frequency of Tcm (CD28+CD95+) CD4+ T cells in the rectum. (F) Frequency of IL-17A–producing CD4+ T cells in the rectum after PMA and ionomycin stimulation (saline, n = 9). (G) Frequency of neutrophils in lamina propria (LP) sections after 36 weeks of ART (saline, n = 5; PD-1 Ab treated, n = 6). Bars indicate the mean. Data were not collected for all animals. Shaded gray area depicts ART. Unfilled symbols indicate values from Mamu-A*01 RMs. Specific T cell frequency data are shown as mean ± SEM. *P < 0.05 by Mantel-Cox test (C), 2-way ANOVA (E and F), or 2-tailed Mann-Whitney test (G). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: Plasma SIV RNA viral loads (copies/ml) shown as (A) geometric mean for each group and (B) for individual RMs at initiation of anti–PD-1 Ab or saline infusion. Limit of detection is 60 copies/ml. (C) Kaplan-Meier curve of number of days until viral suppression (<100 copies/ml for at least 2 or more consecutive time points). (D) Plasma SIV RNA viral loads (copies/ml) from chronically SIV-infected RMs administered humanized anti–PD-1 Ab or no Ab during ART initiation in a second study (n = 4 per group). (E) Frequency of Tcm (CD28+CD95+) CD4+ T cells in the rectum. (F) Frequency of IL-17A–producing CD4+ T cells in the rectum after PMA and ionomycin stimulation (saline, n = 9). (G) Frequency of neutrophils in lamina propria (LP) sections after 36 weeks of ART (saline, n = 5; PD-1 Ab treated, n = 6). Bars indicate the mean. Data were not collected for all animals. Shaded gray area depicts ART. Unfilled symbols indicate values from Mamu-A*01 RMs. Specific T cell frequency data are shown as mean ± SEM. *P < 0.05 by Mantel-Cox test (C), 2-way ANOVA (E and F), or 2-tailed Mann-Whitney test (G). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Infection, MANN-WHITNEY

(A) Ki-67 expression on CD4+ T cells, CD8+ T cells, and GagCM9+CD8+ T cells in blood after first PD-1 Ab infusion (right: saline, n = 2; single anti–PD-1 treated [ST], n = 3; double anti–PD-1 treated [DT], n = 5). Data are shown as the mean ± SEM. (B) Frequency of granzyme B+CD8+ T cells (saline, n = 5; ST, n = 3; DT, n = 10), CXCR5+GagCM9+CD8+ T cells (saline, n = 3; ST, n = 2; DT, n = 5), and CXCR5+CD4+ T cells (saline, n = 5; ST, n = 3; DT, n = 10) in axillary lymph nodes (ALN) at 2 weeks after last PD-1 Ab infusion. Viral outgrowth assay on CD4+ T cells from blood of RMs 2 weeks after the last PD-1 Ab infusion. (C) SIV Gag RNA copies/ml assayed from culture supernatant at days 9, 17, and 25 of culture. (D) Representative p27 gag staining and (E) frequency of p27 gag+ CEM cells from day 25 of viral outgrowth assay. For viral outgrowth assay, saline, n = 4; ST, n = 5; DT, n = 8. Unfilled circles indicate values from Mamu-A*01 RMs. Bars indicate the mean. Exact P values are shown. Two-way ANOVA (A), 2-tailed Mann-Whitney test (B and C), 1-way ANOVA with Dunn’s multiple-comparisons test (B, right), or 2-tailed Student’s t test (E) were used. One saline control animal was interrupted from ART early due to significant weight loss and therefore data are not available for this animal. Unless otherwise noted, saline, n = 4; ST, n = 5; DT, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Ki-67 expression on CD4+ T cells, CD8+ T cells, and GagCM9+CD8+ T cells in blood after first PD-1 Ab infusion (right: saline, n = 2; single anti–PD-1 treated [ST], n = 3; double anti–PD-1 treated [DT], n = 5). Data are shown as the mean ± SEM. (B) Frequency of granzyme B+CD8+ T cells (saline, n = 5; ST, n = 3; DT, n = 10), CXCR5+GagCM9+CD8+ T cells (saline, n = 3; ST, n = 2; DT, n = 5), and CXCR5+CD4+ T cells (saline, n = 5; ST, n = 3; DT, n = 10) in axillary lymph nodes (ALN) at 2 weeks after last PD-1 Ab infusion. Viral outgrowth assay on CD4+ T cells from blood of RMs 2 weeks after the last PD-1 Ab infusion. (C) SIV Gag RNA copies/ml assayed from culture supernatant at days 9, 17, and 25 of culture. (D) Representative p27 gag staining and (E) frequency of p27 gag+ CEM cells from day 25 of viral outgrowth assay. For viral outgrowth assay, saline, n = 4; ST, n = 5; DT, n = 8. Unfilled circles indicate values from Mamu-A*01 RMs. Bars indicate the mean. Exact P values are shown. Two-way ANOVA (A), 2-tailed Mann-Whitney test (B and C), 1-way ANOVA with Dunn’s multiple-comparisons test (B, right), or 2-tailed Student’s t test (E) were used. One saline control animal was interrupted from ART early due to significant weight loss and therefore data are not available for this animal. Unless otherwise noted, saline, n = 4; ST, n = 5; DT, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Expressing, Viral Outgrowth Assay, Staining, MANN-WHITNEY

(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Marker, Expressing, MANN-WHITNEY

Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Isolation, Expressing

Phenotypic characterization of CCR5 expression and infection status of PD-1hi CD4 T cells in the LN and rectum of chronically SIV infected vaccine-controller and non-controller RM. (A) Expression of CCR5 on PD-1neg, int, and hi CD95+ CD4 T cells and the percentage of CCR5+ PD-1hi cells in the LN and rectum of healthy SIV naive (n=6 and n=12, respectively), SIV+vaccine-controller (n=19 and 18, respectively), and SIV+ non-controller (n=17 and 18, respectively) RM and (B) Total frequencies of CCR5+ CD95+ CD4 T cells in the blood, LN, and rectum of healthy SIV naive (n= 9, 6, and 23, respectively), SIV+ vaccine-controller (n=19), and SIV+ non-controller (n = 17, 18, and 16, respectively) RM. (C) Infection status of PD-1 subsets in the LN and rectum of SIV infected RM. Scatter plots show Gag RNA and DNA copies/ng of input RNA and DNA quantified by RT-PCR from FACS sorted naïve (CD95-) and memory (CD95+) PD-1neg, int, and hi CD4 T cells in the mesenteric LN (n=9) and rectum (n=6) and the ratio of RNA copies/DNA copies in PD-1 neg, int and hi subsets (n=4, 7, and 4 respectively). Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Phenotypic characterization of CCR5 expression and infection status of PD-1hi CD4 T cells in the LN and rectum of chronically SIV infected vaccine-controller and non-controller RM. (A) Expression of CCR5 on PD-1neg, int, and hi CD95+ CD4 T cells and the percentage of CCR5+ PD-1hi cells in the LN and rectum of healthy SIV naive (n=6 and n=12, respectively), SIV+vaccine-controller (n=19 and 18, respectively), and SIV+ non-controller (n=17 and 18, respectively) RM and (B) Total frequencies of CCR5+ CD95+ CD4 T cells in the blood, LN, and rectum of healthy SIV naive (n= 9, 6, and 23, respectively), SIV+ vaccine-controller (n=19), and SIV+ non-controller (n = 17, 18, and 16, respectively) RM. (C) Infection status of PD-1 subsets in the LN and rectum of SIV infected RM. Scatter plots show Gag RNA and DNA copies/ng of input RNA and DNA quantified by RT-PCR from FACS sorted naïve (CD95-) and memory (CD95+) PD-1neg, int, and hi CD4 T cells in the mesenteric LN (n=9) and rectum (n=6) and the ratio of RNA copies/DNA copies in PD-1 neg, int and hi subsets (n=4, 7, and 4 respectively). Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction

(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: RNA Sequencing Assay

(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Marker, Expressing, MANN-WHITNEY

(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Marker, Expressing, MANN-WHITNEY

(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: RNA Sequencing Assay

(A) Ki-67 expression on CD4+ T cells, CD8+ T cells, and GagCM9+CD8+ T cells in blood after first PD-1 Ab infusion (right: saline, n = 2; single anti–PD-1 treated [ST], n = 3; double anti–PD-1 treated [DT], n = 5). Data are shown as the mean ± SEM. (B) Frequency of granzyme B+CD8+ T cells (saline, n = 5; ST, n = 3; DT, n = 10), CXCR5+GagCM9+CD8+ T cells (saline, n = 3; ST, n = 2; DT, n = 5), and CXCR5+CD4+ T cells (saline, n = 5; ST, n = 3; DT, n = 10) in axillary lymph nodes (ALN) at 2 weeks after last PD-1 Ab infusion. Viral outgrowth assay on CD4+ T cells from blood of RMs 2 weeks after the last PD-1 Ab infusion. (C) SIV Gag RNA copies/ml assayed from culture supernatant at days 9, 17, and 25 of culture. (D) Representative p27 gag staining and (E) frequency of p27 gag+ CEM cells from day 25 of viral outgrowth assay. For viral outgrowth assay, saline, n = 4; ST, n = 5; DT, n = 8. Unfilled circles indicate values from Mamu-A*01 RMs. Bars indicate the mean. Exact P values are shown. Two-way ANOVA (A), 2-tailed Mann-Whitney test (B and C), 1-way ANOVA with Dunn’s multiple-comparisons test (B, right), or 2-tailed Student’s t test (E) were used. One saline control animal was interrupted from ART early due to significant weight loss and therefore data are not available for this animal. Unless otherwise noted, saline, n = 4; ST, n = 5; DT, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Ki-67 expression on CD4+ T cells, CD8+ T cells, and GagCM9+CD8+ T cells in blood after first PD-1 Ab infusion (right: saline, n = 2; single anti–PD-1 treated [ST], n = 3; double anti–PD-1 treated [DT], n = 5). Data are shown as the mean ± SEM. (B) Frequency of granzyme B+CD8+ T cells (saline, n = 5; ST, n = 3; DT, n = 10), CXCR5+GagCM9+CD8+ T cells (saline, n = 3; ST, n = 2; DT, n = 5), and CXCR5+CD4+ T cells (saline, n = 5; ST, n = 3; DT, n = 10) in axillary lymph nodes (ALN) at 2 weeks after last PD-1 Ab infusion. Viral outgrowth assay on CD4+ T cells from blood of RMs 2 weeks after the last PD-1 Ab infusion. (C) SIV Gag RNA copies/ml assayed from culture supernatant at days 9, 17, and 25 of culture. (D) Representative p27 gag staining and (E) frequency of p27 gag+ CEM cells from day 25 of viral outgrowth assay. For viral outgrowth assay, saline, n = 4; ST, n = 5; DT, n = 8. Unfilled circles indicate values from Mamu-A*01 RMs. Bars indicate the mean. Exact P values are shown. Two-way ANOVA (A), 2-tailed Mann-Whitney test (B and C), 1-way ANOVA with Dunn’s multiple-comparisons test (B, right), or 2-tailed Student’s t test (E) were used. One saline control animal was interrupted from ART early due to significant weight loss and therefore data are not available for this animal. Unless otherwise noted, saline, n = 4; ST, n = 5; DT, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Expressing, Viral Outgrowth Assay, Staining, MANN-WHITNEY

(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Marker, Expressing, MANN-WHITNEY

Plasma SIV RNA viral loads (copies/ml) shown as (A) geometric mean for each group and (B) for individual RMs at initiation of anti–PD-1 Ab or saline infusion. Limit of detection is 60 copies/ml. (C) Kaplan-Meier curve of number of days until viral suppression (<100 copies/ml for at least 2 or more consecutive time points). (D) Plasma SIV RNA viral loads (copies/ml) from chronically SIV-infected RMs administered humanized anti–PD-1 Ab or no Ab during ART initiation in a second study (n = 4 per group). (E) Frequency of Tcm (CD28+CD95+) CD4+ T cells in the rectum. (F) Frequency of IL-17A–producing CD4+ T cells in the rectum after PMA and ionomycin stimulation (saline, n = 9). (G) Frequency of neutrophils in lamina propria (LP) sections after 36 weeks of ART (saline, n = 5; PD-1 Ab treated, n = 6). Bars indicate the mean. Data were not collected for all animals. Shaded gray area depicts ART. Unfilled symbols indicate values from Mamu-A*01 RMs. Specific T cell frequency data are shown as mean ± SEM. *P < 0.05 by Mantel-Cox test (C), 2-way ANOVA (E and F), or 2-tailed Mann-Whitney test (G). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: Plasma SIV RNA viral loads (copies/ml) shown as (A) geometric mean for each group and (B) for individual RMs at initiation of anti–PD-1 Ab or saline infusion. Limit of detection is 60 copies/ml. (C) Kaplan-Meier curve of number of days until viral suppression (<100 copies/ml for at least 2 or more consecutive time points). (D) Plasma SIV RNA viral loads (copies/ml) from chronically SIV-infected RMs administered humanized anti–PD-1 Ab or no Ab during ART initiation in a second study (n = 4 per group). (E) Frequency of Tcm (CD28+CD95+) CD4+ T cells in the rectum. (F) Frequency of IL-17A–producing CD4+ T cells in the rectum after PMA and ionomycin stimulation (saline, n = 9). (G) Frequency of neutrophils in lamina propria (LP) sections after 36 weeks of ART (saline, n = 5; PD-1 Ab treated, n = 6). Bars indicate the mean. Data were not collected for all animals. Shaded gray area depicts ART. Unfilled symbols indicate values from Mamu-A*01 RMs. Specific T cell frequency data are shown as mean ± SEM. *P < 0.05 by Mantel-Cox test (C), 2-way ANOVA (E and F), or 2-tailed Mann-Whitney test (G). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Infection, MANN-WHITNEY

(A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Schematic of PD-1 blockade strategy during phase I and II. (B) Frequency of Ki-67+CD4+ and Ki-67+CD8+ T cells in the blood. (C) Frequency of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD4+ and CD8+ T cells in the blood. (D) Percentage of Ki-67+, granzyme B+, and CXCR5+GagCM9+ CD8+ T cells in the blood (saline, n = 6; PD-1 Ab treated, n = 5). (E) Gene set enrichment analysis (GSEA) of RNA-Seq data from blood at day 10 compared with day 0 following PD-1 blockade during phase I (PD-1 Ab treated, n = 10). Normalized enrichment scores for select upregulated and downregulated gene sets depicted. Dashed line indicates normalized enrichment score cutoff of greater than 1.35 for upregulated gene sets and less than –1.35 for downregulated gene sets with a false discovery rate of less than 0.2. (F) GSEA plots comparing day 10 (D10) to day 0 (D0) of phase I for PD-1 Ab– and saline-treated (n = 5) groups. Leading-edge genes from gene sets are shown as black outlined dots. Shaded gray area depicts ART. Unfilled circles indicate values from Mamu-A*01 RMs. Data in B and C are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by 2-way ANOVA (B and C) or 2-tailed paired Student’s t test (D). n = 10 per group unless otherwise noted.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: RNA Sequencing Assay

(A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Journal: JCI Insight

Article Title: Combination anti–PD-1 and antiretroviral therapy provides therapeutic benefit against SIV

doi: 10.1172/jci.insight.122940

Figure Lengend Snippet: (A) Frequencies of Ki-67+ (3 weeks after ATI; saline, n = 4), Ki-67+CXCR5+ (3 weeks after ATI; saline, n = 4), and perforin+ (4 weeks after ATI) CD8+ T cells in the blood. (B) Frequencies of SIV Gag-specific and Env-specific IFN-γ– and TNF-α–producing CD8+ T cells in the blood at 4 weeks after ATI. Bars indicate the geometric mean. (C) Frequency of Tcm CD4+ T cells as percentage of CD3+ T cells in the blood at 2 weeks after ATI (saline, n = 4). (D) Frequency of Tregs in the blood at 4 weeks after ATI. (E) Ratio of perforin+CD8+ T cells to Tregs at week 4 after ATI. (F) Boolean analysis of marker expression of CXCR5, granzyme B, perforin, and Ki-67 on Tem CD8+ T cells at 3 weeks after ATI. Correlations of frequency of granzyme B+CD8+ T cells from lymph nodes (LNs) of animals at day 0 of ATI with (G) frequency of perforin+CD8+ T cells at 4 weeks after ATI and (H) fold reduction of viral load (VL) from pre-ART set point to week 8 after ATI (single anti–PD-1 treated, n = 3). Unfilled circles indicate values from Mamu-A*01 RMs in A–E. Bars indicate the mean unless otherwise noted. *P < 0.05 by 2-tailed Student’s t test with Welch’s correction (A–D), 2-tailed Mann-Whitney test (E and F), or Spearman’s rank-order correlations test (G and H) were used. r indicates the correlation coefficient. Unless otherwise noted, saline, n = 5; double anti–PD-1 treated, n = 5; single anti–PD-1 treated, n = 10.

Article Snippet: The following Abs were used: CXCR5 (MU5UBEE; eBioscience), CD3 (SP-34-2; BD Biosciences), GagCM9 tetramer (courtesy of the laboratory of Rafi Ahmed), CD28 (CD28.2; eBioscience), CD95 (DX2; BD Biosciences), CD279 (PD-1; EH12.2H7; Biolegend), CD8 (SK1; BD Bioscience), Ki-67 (B56; BD Biosciences), CD4 (L200; BD Biosciences), CD25 (BC96; Biolegend), granzyme B (GB11; BD Biosciences), IFN-γ (B27; BD Biosciences), TNF-α (MAb11; BD Biosciences), perforin (Pf-80/164; BD Biosciences), IL-17A (ebio64-Dec17; eBioscience), and FoxP3 (206D; Biolegend).

Techniques: Marker, Expressing, MANN-WHITNEY

Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Phenotypic characterization of PD-1+ subsets in the blood, lymph node (LN), and rectum of healthy rhesus macaques (RM). (A) Flow cytometric analysis of mononuclear cells isolated from the blood, LN, and rectum of a healthy rhesus macaque. Plots show live CD3+ CD4+ CD95+ PD-1neg, PD-1int, and PD-1hi lymphocytes and scatter plot shows the cumulative data for a group in blood (n=16), LN (n=7), and rectum (n=17). (B) Representative histogram plots of CXCR5, BCl-6, and CCR5 expression overlaying PD-1neg, PD-1int, and PD-1hi CD95+ CD4 T cells in the rectum (n=8) and LN (n= 7, 7, and 6, respectively for CXCR5, BCl-6, and CCR5) of healthy SIV naïve RM. Scatter Plots show median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Isolation, Expressing

Characterization of PD-1hi CD4 T cells in the LN and rectum of chronically SIV infected vaccine-controller and non-controller RM. (A) Flow cytometric analysis of PD-1hi CD95+ CD4 T cells in the blood, LN, and rectum of healthy (n=16, 7, and 23, respectively) SIV+ vaccine-controller (n= 19) and non-controller (n=18) RM. (B) Correlation between PD-1hi CD95+ CD4 T cells and viral load at week 24 post SIV infection in the LN and rectum of SIV+ vaccine-controller (n=19) and non-controller (n=18) RM. (C) Longitudinal frequencies of PD-1hi CD95+ CD4 T cells and CD4 T cells in the rectum of chronically SIV infected RM as a percent of total CD3 T cells. Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

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

Article Title: Diminished Viral Control during SIV Infection is Associated with Aberrant PD-1 hi CD4 T cell Enrichment in the Lymphoid Follicles of the Rectal Mucosa

doi: 10.4049/jimmunol.1401222

Figure Lengend Snippet: Characterization of PD-1hi CD4 T cells in the LN and rectum of chronically SIV infected vaccine-controller and non-controller RM. (A) Flow cytometric analysis of PD-1hi CD95+ CD4 T cells in the blood, LN, and rectum of healthy (n=16, 7, and 23, respectively) SIV+ vaccine-controller (n= 19) and non-controller (n=18) RM. (B) Correlation between PD-1hi CD95+ CD4 T cells and viral load at week 24 post SIV infection in the LN and rectum of SIV+ vaccine-controller (n=19) and non-controller (n=18) RM. (C) Longitudinal frequencies of PD-1hi CD95+ CD4 T cells and CD4 T cells in the rectum of chronically SIV infected RM as a percent of total CD3 T cells. Scatter Plots show the median. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001.

Article Snippet: FITC conjugated Bcl-2 (clone Bcl-2/100; BD Biosciences), PE-conjugated CXCR5 (clone MU5UBEE; eBioscience), PerCP conjugated CD3 (Clone SP-34-2; BD Biosciences), PeCy7 conjugated CD28 (Clone CD28.2; eBiosciences), PE-TR conjugated CD95 (clone; BD Biosciences), Brilliant Violet 421- conjugated CD279 (PD-1; Clone EH12.1; Biolegend), V500 conjugated CD8 (Clone SK1; BD Bioscience), APC conjugated CCR5 (Clone 3A9, BD Biosciences), Live Dead-IR stain (Invitrogen), Alexa700 conjugated Ki-67 (Clone B56; BD Biosciences), FITC conjugated Bcl-6 (Clone K112-91; BD Biosciences), Brilliant Violet 650 conjugated CD4 (Clone OKT4; Biolegend), FITC conjugated IL-17A (Clone eBio64Dec15; ebiosciences), PE conjugated IL-21 (Clone 3A3-N2; BD Biosciences), PerCP-conjugated CD4 (Clone L200; BD Biosciences), PeCy7 conjugated anti-CD279 (PD-1; clone EH12.1; Biolegend), PacBlue conjugated CD3 (Clone SP-34-2; BD Biosciences), APC conjugated IL-2 (Clone MQ1-17H12; BD Biosciences), Alexa700 conjugated IFNγ (Clone B27; BD Biosciences).

Techniques: Infection