Kinetics of Ca²⁺ flux in CTL stimulated with pMHC/QD or pMHC/Streptavidin containing cognate and noncognate or inactive pMHC ligands at various ratios.

<p>A. Time-dependent Ca²⁺ accumulation in the cytoplasm of Fluo-3-labeled 68A62 CTL induced by Streptavidin-based oligomers presenting IV9-HLA-A2 (cognate, red) and Tax-HLA-A2 (noncognate, yellow) ligands at various ratios, i.e., 4∶0, 2∶2, 1∶3, 0∶4, respectively. Corresponding Ca²⁺ flux traces are designated as 4 (4∶0, red), 2 (2∶2, blue), 1(1∶3, green), 0(0∶4, grey). B, C, D. Time-dependent Ca²⁺ accumulation in the cytoplasm of Fura Red-labeled 68A62 CTL induced by QD-based oligomers presenting IV9-HLA-A2 (cognate, red), Tax-HLA-A2 (noncognate, yellow) or Tax-HLA-A2_mut (inactive, grey) ligands at various ratios. One series of pHLA-A2/QD conjugates being tested displayed 4, 2, 1 or 0 cognate (red) ligands per dot and 6, 8, 9 or 10 noncognate (yellow) ligands per dot keeping the total number of pHLA-A2 molecules per dot to be 10 (B). In another series of tested pHLA-A2/QD conjugates, the number of the cognate (red) ligands was the same, i.e., 4, 2, 1 or 0 per dot and the number of the noncognate (yellow) ligands per dot was 0, 2, 3, or 4, respectively, while the number of the inactive (grey) ligands and the total number of pHLA-A2 ligands per dot was kept 6 and 10, correspondingly (C). In the latter series (C), the density of noncognate Tax-HLA-A2 ligands on the conjugates was lower as compared to the conjugates of the former series (B). In the third series, QD presenting 1 cognate (red) along with either 9 noncognate (yellow) or with 9 inactive (grey) pMHC-I per dot were tested. QD presenting 10 inactive GL9-HLA-A2_mut proteins per dot were used as a negative control (D). Corresponding Ca²⁺ flux profiles are designated as in A.</p

The dependence of cognate-noncognate pMHC-I cooperation on the nature of noncognate peptide. A, B.

<p>Ca²⁺ mobilization in Fura Red-labeled CER43 CTL stimulated with QD(520) that display different noncognate pMHC-I proteins (10 per dot) (<b>A</b>) or 1 cognate and various noncognate pMHC (9 per dot) (<b>B</b>) is presented. <b>C</b>. Normalized values of MFI of CER43 cells interacting with either cognate or various noncognate pMHC-I are shown. The nature of cognate and noncognate pMHC-I ligands is designated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041466#s4" target="_blank">Material and Methods</a> for details).</p

Noncognate pMHC assembled on QD but not on Streptavidin scaffold stain CD8 CTL.

<p><u>Left panel:</u> Staining of 68A62 with cognate (bold solid line) and noncognate (dotted line) pMHC oligomers is shown; staining with the noncognate oligomers containing HLA-A2 with A245V mutation in nonpolymorphic domain (solid line) was used as a negative control. <u>Right panel:</u> Normalized MFI of 68A62 CTL incubated with cognate IV9-HLA-A2/QD(520) and IV9-HLA-A2/Streptavidin are compared with that of noncognate Tax-HLA-A2/QD(520) and Tax/Streptavidin oligomers. Data represent mean ± s.d.</p

Comparison of the binding of various cognate and noncognate pMHC oligomers to CD8 CTL. A.

<p>Binding of cognate (IV9-HLA-A2, bold solid line) and noncognate (Tax-HLA-A2, dotted line) pMHC ligands assembled on different scaffolds to 68A62 CTL as established by flow cytometry. The CTL were incubated with each oligomer at 20 nM for 30 min prior to the analysis. Mutant HLA-A2(A245V) loaded with noncognate peptide (Tax) was used to produced oligomers which were utilized as negative controls (solid line). <b>B.</b> Binding of different oligomers carrying cognate peptide (IV9) in association with either intact (bold solid line) of mutated HLA-A2 (A245V) (dashed line) to 68A62 CTL was evaluated. Binding of cognate dextramers in the presence (dashed) or absence (bold solid line) of blocking anti-CD8 antibodies was examined. Other conditions are as in <b>A</b>. Data represent mean ± s.d. <b>C.</b> Normalized MFI of 68A62 bound to cognate or noncognate pMHC oligomers containing intact HLA-A2 protein. The nature of oligomers and the number of pMHC ligands per oligomer are indicated. Data represent mean ± s.d. <b>D.</b> Normalized MFI of 68A62 bound to cognate oligomers or cognate oligomers containing pMHC_mut with mutated HLA-A2 (A245V) protein. The tested oligomers are as in <b>C</b>.</p

The dependence of TCR-mediated signaling kinetics upon the density of cognate and noncognate pMHC displayed on QD of different sizes.

<p>Time-dependent accumulation of intracellular Ca²⁺ in Fura Red-labeled 68A62 CTL in response to stimulation with 10 nM QD of different sizes presenting various combinations of cognate, noncognate and inactive pMHC ligands. The comparison of the stimulatory potency of (IV9-HLA-A2)₄₀/QD(620) (A) or (IV9-HLA-A2)₁₀(GL9-HLA-A2_mut)₃₀/QD(620) (B) or (IV9-HLA-A2)₁₀(GL9-HLA-A2)₃₀/QD(620) (C) versus (IV9-HLA-A2)₁₀/QD(520) is shown.</p

Distribution of separating distances between IV9-HLA-A2 proteins assembled on QD and Streptavidin scaffolds.

<p>Fluorescence decay of AF594-IV9 peptide bound to HLA-A2 protein (donor) in the presence or absence of AF647-IV9-HLA-A2 protein (acceptor) displayed on the surface of QD(520) (<b>A</b>) or Streptavidin scaffold (<b>B</b>). Fitting the experimental data to the model (Eqs. 1–7) yielded satisfactory fit (χ²<1.2) allowing to evaluate distribution of separating distances between IV9-HLA-A2 molecules displayed on QD (<b>C</b>) or Streptavidin (<b>D</b>).</p

Correlation between the viral load and A3G mRNA, protein in B cells or AID.

<p>Correlation between PVL or CVL and A3G mRNA in PBMC (A,B), AID (C, D), A3G proteins in CD20⁺ B cells (E,F) and A3G in CD4⁺CD95⁺CCR7⁻ effector memory T cells (G,H). AID and A3G were assayed after the 4^th immunization, whereas CVL was calculated as the “area under the curve”. The two protected macaques are shown by open circles. Pearson's correlation coefficient was used for statistical analysis. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

Membrane-associated (ma) IL-15 of DC and CD40L expression of CD4⁺ T cells in 5 groups of macaques.

<p>(A) Membrane-associated (ma) IL-15 of DC and (B) CD40L expression on CD4⁺ T cells in 5 groups of macaques pre- and post-4^th immunization, presented as % mean (±sem). The significance between pre- and post-immunization was analysed by paired “t” test and differences between the 5 groups after immunization was analysed by ANOVA.</p

AID and A3G expression in CD20⁺ B cells pre- and post-2^nd immunization and their correlation.

<p>Comparative investigation of (A) AID and (B) A3G expression in CD20⁺ B cells before and after the 2^nd immunization in the 4 groups of immunized macaques; group 5 unimmunized controls remained unchanged (data not presented). Correlation between A3G and AID expression in CD20⁺ B cells in the 5 groups after 2^nd immunization is presented in (C). Representative flow cytometry of AID and A3G in pre- and post 2^nd immunization is shown in (D). * p<0.05 and ** p<0.01. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p

A3G in CD20⁺ and CD27⁺ memory B cells pre- and post-immunization in the 4 groups.

<p>Expression of A3G in (A) CD20⁺ B cells and (B) CD20⁺CD27⁺ memory B cells in 5 groups of macaques before and after the 4^th immunization assayed by flow cytometry with MAb to A3G, CD20 and CD27 and (C) representative illustration; (n = 8 per group, except gp4 n = 6). * p<0.05. In all figures the 2 uninfected macaques in group 3 are indicated by a solid circle.</p