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1.
Figure 5

Figure 5. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

Functional sensitivity for two sets of parallel cultures determined by IFN-γ secretion. (A) TV9-2, (B) TV9p6-2, (C) TV9-8 and (D) TV9p6–8 cultures were stimulated at an E:T ratio of 1:10 with T2 cells pulsed with either TV9 or TV9p6. IFN-γ secretion into the supernatant was quantified by ELISA after 48 hours. (E) EC50 values were calculated for the TV9 and TV9p6 peptides in each case with Graphpad Prism V software. Data are shown as mean ± SEM of triplicate assays and are representative of two independent experiments; in most cases, the error bars are smaller than the plot symbols.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.
2.
Figure 1

Figure 1. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

Scanning a nonapeptide PS-SCL to identify TV9 agonist peptides. (A) Index TV9-1 cytotoxicity elicited by the 180 mixtures of a nonapeptide PS-SCL. Each graph, designated p1–p9, represents a set of 20 mixtures having the defined amino acid listed on the x-axis at a given position. The y-axis denotes the % lysis of T2 cells in the presence of each mixture. The hollow bars represent the native TV9 amino acid sequence. Each mixture was assayed in triplicate and scanning was repeated three times. (B) Design of 48 candidate peptides. The rationale for the selection of a particular active mixture in each position is discussed in the text.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.
3.
Figure 3

Figure 3. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

Specificity and crossreactivity of TV9p6 CTLs monitored over time by tetramer staining. TV9p6-tetramer binding by the CD8+ cultures TV9p6-2, TV9p6-6, TV9p6–7 and TV9p6–8 at three time points is shown in Panels A-a to A–c, B-a to B–c, C-a to C-c and D-a to D-c. Crossreactivity of the TV9p6-cultures was assessed concomitantly by staining with TV9-tetramer (A–d to A–f, B–d to B–f, C–d to C–f and D–d to D–f). For comparison, TV9-tetramer staining of the CD8+ parallel cultures TV9-2, TV9-6, TV9-7 and TV9-8 is shown in Panels A–g, B–g, C–g and D–g, respectively. Numbers in the upper right quadrant represent the percentage of CD8+ tetramer+ stained cells. An irrelevant tetramer was used to gate for specific tetramer binding. Data are representative of at least three independent experiments.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.
4.
Figure 4

Figure 4. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

CD8 dependency of cytotoxic activity mediated by TV9- and TV9p6-CTLs. Parallel cultures to TV9 or TV9p6, established by in vitro immunization using peptide-pulsed DCs, were identified as TV9-Y or TV9p6-Y, where the numeral Y denotes a distinct donor. (A) TV9-2, (B) TV9-6, (C) TV9-7, (D) TV9-8 and parallel (E) TV9p6-2, (F) TV9p6-6, (G) TV9p6–7 and (H) TV9p6–8 CD8+ cultures were assayed for functional sensitivity in a chromium release assay using C1R-A2wt and C1R-A2CD8null targets loaded with a range of concentrations of TV9p6 or TV9 peptide (10−5 to 10−13M) at an E:T ratio of 10:1 (–■–■–,–●–●–: C1R-A2wt pulsed with TV9 and TV9p6, respectively; –□–□–,–○–○–: C1R-A2CD8null pulsed with TV9 and TV9p6, respectively). Lysis of C1R cells alone was subtracted to give percent specific lysis. Data are shown as mean ± SEM of triplicate assays and are representative of two independent experiments; in most cases, the error bars are smaller than the plot symbols.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.
5.
Figure 2

Figure 2. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

Evaluation of the ability of three agonist peptides (TV9p6, TV9p5 and TV9p29) listed in Table I to induce antigen-specific, TV9-crossreactive CTLs by in vitro immunization of naïve CD8+ T cells from two to four healthy seronegative donors. Cultures are labeled as TV9pX-Y, where TV9pX identifies the inducing peptide and the numeral Y denotes a distinct donor. (A) TV9p6-6, TV9p6-2, TV9p6-5 and TV9p6–7; (B) TV9p5-3 and TV9p5-4; and, (C) TV9p29-3 and TV9p29-8. CD8+T cells were assayed for specificity and cross-recognition of TV9 using chromium release assays at effector:target (E:T) ratios ranging from 20:1 to 0.6:1. T2 cells without peptide were included as negative controls in each assay. Data are shown as mean ± SEM of triplicate assays and are representative of two independent experiments; in most cases, the error bars are smaller than the plot symbols.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.
6.
Figure 6

Figure 6. From: Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

TV9p6-CTLs suppress HIV replication at least as efficiently as TV9-CTLs. (A) Percent suppression of NL4-3.1 replication in T1 cells at day 3 post-infection by tetramer+ T cells at different E:T ratios as indicated. Data are shown as mean ± SEM of triplicate assays and are representative of two independent experiments. (B) Suppression of NL4-3.1 virus in T1 cells by TV9- and TV9p6-CTL cultures. The average percent suppression by TV9-2, -6, -7 and -8 T cells at day 3 and 6 are indicated by ▲ and Δ, respectively. Likewise, suppression mediated by TV9p6-2, -6, -7 and -8 T cells at the two time points are denoted by the symbols ● and ○, respectively. The E:T ratio for this assay was 2:1. Data are representative of two independent experiments. The concentrations of p24 in infected T1 cell cultures in the absence of CTLs were 0.3 ± 0.03 ng/mL and 0.8 ± 0.1 ng/mL on day 3 and 93 ± 14 ng/mL and 684 ± 48 ng/mL on day 6. (C) Suppression of HIV JR-CSF replication in two acutely infected, allogeneic HLA-A*0201-matched CD8-depleted PBMC cultures by two parallel CTL cultures (TV9-6 and TV9p6-6; TV9-7 and TV9p6–7) at day 9 post-infection. The concentrations of p24 in infected PBMC-1 and PBMC-2 cultures in the absence of CTLs were 311 ng/mL and 210 ng/mL, respectively. The p values comparing suppression by TV9- and TV9p6-CTL were determined using an unpaired, two-tailed Student T test with Graphpad Prism V software.

Keri L. Schaubert, et al. Eur J Immunol. ;40(7):1950-1962.

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