Article Figures & Data
Figures
- FIGURE 1.
The peptide-conjugates with NLV- and GIL- CD8+ epitopes boost IFN-γ and TNF-α recall responses of epitope-specific CD8+ T cells in human whole blood. The peptide-conjugate [MTTE]3-NLV (A), containing the CD8 epitope pp65(NLV) from CMV, was incubated at a final concentration of 120 nM in human whole blood from HLA-A*0201+ and CMV+ healthy donors in a circulating blood loop assay. After 2 h, Brefeldin A was added, and after a total of 6 h, aliquots were harvested and stained for T cell markers, tetramer HLA-A*0201 CMV pp65 (NLVPMVATV), and intracellular IFN-γ and TNF-α and analyzed with flow cytometry (B). The IFN-γ release in response to [MTTE]3-NLV was compared with up to ∼100-fold higher SLP (NLV) concentration [(C), n = 3]. The peptide-conjugate [MTTE]3-GIL (480 nM) containing the CD8 epitope GIL from influenza (GILGFVFTL) was run as described above [(D), n = 1].
- FIGURE 2.
The conjugation of at least three MTTE sequences per SLP is required to boost IFN-γ recall responses of Ag-specific CD8+ T cells in human whole blood. The peptide-conjugate [MTTE]3-NLV (Fig. 1A) was incubated at a final concentration of 120 nM in whole blood from HLA-A*0201+ and CMV+ healthy donors in a circulating blood loop assay. After 2 h, Brefeldin A was added, and after a total of 6 h, cells were stained for tetramer HLA-A*0201 CMV pp65 (NLVPMVATV) and intracellular IFN-γ and analyzed with flow cytometry. [MTTE]3-NLV in a conjugated form was compared with the two separate entities ([MTTE]3 + SLP [NLV]) [(A), n = 5 donors run at separate occasions, and the lines represent the mean]. IFN-γ responses induced by conjugates with one, two, or three MTTE sequences were compared, and the percentage of IFN-γ+ NLV–specific cells induced by conjugates with one, two, or three MTTE is shown [(B), n = 4 donors run at separate occasions]. IFN-γ responses induced by [MTTE]3-NLV were compared with that of a conjugate containing a scrambled MTTE sequence [ETTM]3-NLV, showing the mean and SD [(C), n = 2 donors run at separate occasions]. The cold conjugate without an SLP ([MTTE]3 or the scrambled control conjugate [ETTM]3) was preincubated in the blood (from a donor that mounts a strong T cell response against [MTTE]3-NLV) for 10 min before [MTTE]3-NLV was added and analyzed as described above [(D), n = 1]. The IFN-γ response of a donor that mounts a poor T cell response against [MTTE]3-NLV, was analyzed with the addition of a mouse anti-MTTE IgG2a Ab at the final concentration of 40 μg/ml [(E), one representative donor out of three]. The data were analyzed with Friedman test with Dunn correction. **p < 0.01.
- FIGURE 3.
The uptake of [MTTE]3-AF488 by human blood monocytes and B cells is Ab dependent. Alexa Fluor 488–labeled [MTTE]3-SLP, ([MTTE]3-AF488), raMTTE, or in vitro preincubated [MTTE]3-AF488 and raMTTE (preformed IC) were incubated in blood from healthy donors in a circulating blood loop assay. After 1 h (and 4 h where indicated), aliquots were harvested and stained for monocytes (CD14+CD19−) and B cells (CD19+CD14−). The uptake was analyzed with flow cytometry, and the fold change in mean fluorescence intensity (AF488) postloop/preloop (0 time point) is presented in [(A), n = 6]. The surface-bound AF488 (1 and 4 h) was quenched with an anti–Alexa Fluor 488 Ab [(B), 1 h, n = 8 and 4 h, n = 3]. The uptake of [MTTE]3-AF488 and [ETTM]3-AF488 by monocytes [(C), n = 9] and by blood-derived DCs [(E), n = 2]. The uptake pattern by monocytes of [MTTE]3-AF488 and [ETTM]3-AF488 in a titration [(D), n = 1]. The data were analyzed with the Friedman test with Dunn correction (A) or the Wilcoxon signed-rank test (B and C). The lines represent the mean. **p < 0.01.
- FIGURE 4.
Blocking C1q reduces the uptake of [MTTE]3-AF488 by monocytes and affects the IFN-γ recall responses boosted by [MTTE]3-NLV. The uptake of Alexa Fluor 488–labeled [MTTE]3-SLP ([MTTE]3-AF488) by monocytes was blocked with EDTA (n = 5), EGTA (n = 4), Compstatin (n = 3) (A), or a C1q-blocking peptide (n = 4) (C). The blocking agents were preincubated for 10 min before the addition of [MTTE]3-AF488 to blood from healthy donors in a circulating blood loop assay. After 1 h of incubation, aliquots were harvested and stained for blood monocytes (CD14+ and CD19−). The percentage of total uptake was calculated by subtracting the background (medium) mean fluorescence intensity (MFI) (AF488) followed by (MFI of block/MFI of [MTTE]3-AF488) × 100 (A and C). The blocking agents were preincubated for 10 min before [MTTE]3-NLV was added to whole blood from HLA-A*0201+ and CMV+ healthy donors. After 2 h, Brefeldin A was added, and after a total of 6 h, aliquots were harvested and stained for T cell markers, tetramer HLA-A*0201 CMV pp65 (NLVPMVATV), and intracellular IFN-γ and analyzed with flow cytometry. The percentage of total recall response was calculated by dividing (percentage of IFN-γ of block with percentage of IFN-γ of [MTTE]3-NLV) × 100 [(B) and (D), n = 1]. The data were analyzed with the Wilcoxon signed-rank test. The lines represent the mean. *p < 0.05, **p < 0.01.
- FIGURE 5.
Healthy individuals have circulating IgG, but not IgM, Abs against the MTTE peptide. Antitetanus IgG titers of TetaQuin were determined by an in-house ELISA using plates coated with TTd and the detection Abs: polyclonal anti-human IgG and isotype-specific HRP-conjugated Abs [(A), repeated two times with similar results]. The presence of anti-MTTE IgG and IgM in plasma from blood loop donors was determined by coating streptavidin plates with either MTTE-biotin or ETTM-biotin, and the IgG Abs were detected by a polyclonal anti-human IgG HRP-conjugated Ab [(B), n = 10] or anti-human IgM HRP-conjugated Ab [(B), n = 9]. The data were analyzed with the Wilcoxon signed-rank test. The lines represent the mean. **p < 0.01.
- FIGURE 6.
A standard tetanus booster vaccination elevates anti-MTTE IgG1 levels in plasma and enhances IFN-γ recall responses of NLV-specific CD8+ T cells in response to [MTTE]3-NLV. The anti-MTTE IgG and IgM levels in plasma pre- and posttetanus booster vaccination were determined in an in-house ELISA where streptavidin plates were coated with MTTE-biotin (or ETTM-biotin) and the Abs were detected with a polyclonal anti-IgG, polyclonal anti-IgM, or IgG isotype-specific HRP-conjugated Abs [(A), mean, n = 6 and (B), n = 6]. The peptide-conjugate [MTTE]3-NLV was incubated in whole blood from HLA-A*0201+ and CMV+ healthy donors (pre- and posttetanus booster vaccination) in a circulating blood loop assay. After 2 h, Brefeldin A was added, and after a total of 6 h, aliquots were harvested and stained for T cell markers, tetramer HLA-A*0201 CMV pp65 (NLVPMVATV), and intracellular IFN-γ and TNF-α and analyzed with flow cytometry [(C), n = 2]. The data were analyzed with a two-way ANOVA. *p < 0.05, **p < 0.01.
Tables
Abbreviations Sequence [MTTE]3-NLV [FIGITELKKLESKINKVF-AAKYARVRAKC]3 - CORE LINKER- SLP (NLV)a [ETTM]3-NLV [EKLINKLSKIFKGTIEVF-AAKYARVRAKC]3 - CORE LINKER - SLP (NLV) [MTTE]3-AF488 [MTTE]3 - CORE LINKER - AFKSKACB-Alexa Fluor 488 MTTE-Biotin FIGITELKKLESKINKVF-SSSAFADVEAAZO-Biotinb SLP (Irrelevant) DGLQGLLLGLRQRIETLEGK SLP (NLV) AGILARNLVPMVATVQGQNLKYc SLP (GIL) DLEALMEWLKTRPILSPLTKGILGFVFTLTVPc C1q binding peptide CEGPFGPRHDLTFCW Control peptide FPLRAPTFFVRRTIG Donor Total IgG IgG1 IgG4 Before After Before After Before After Donor 1a 1:3200 1:25600 ND 1:200 ND 1:10 Donor 2a 1:6400 1:25600 1:10 1:200 ND 1:10 Donor 3 1:1600 1:6400 1:10 1:10 1:10 1:10 Donor 4 1:100 1:12800 1:10 1:100 ND ND Donor 5 1:800 1:3200 1:10 1:100 1:50 1:200 Donor 6 1:25600 1:12800 1:50 1:100 1:50 1:50 The titers are expressed by the dilution whereby a fold change >1.1 is achieved of the ratio between the MTTE and the control-coated ETTM plate.
↵a The donors are represented in Fig. 6C.
ND, not detected.
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