MCMV load in CD73^−/− mice.

<p>Time course showing MCMV viral loads in CD73^−/− and C57BL/6 mice 7, 21, 50 and 100 dpi. Viral load analysis performed by qRT-PCR (n = 3, mean±SEM).</p

CD73^−/− does not affect memory inflation of CD8⁺ T-cells induced by Adeno-LacZ.

<p>CD73^−/− and C57BL/6 mice were immunised intravenously (i.v) with 1×10⁹ pfu Ad-LacZ. (A) Histograms show representative CD73 staining on βgal₉₆- (red) and βgal₄₉₇- (blue) CD8⁺ T-cells at 14 and 85 days post immunisation in comparison to total CD8⁺ T-cells from naive mice. (B) CD73 staining on βgal₉₆ and βgal₄₉₇ CD8⁺ T-cells at 14 and 85 days post immunisation in comparison to total CD8⁺ T-cells from naive mice (n = 5, mean±SEM). (C) Time course showing specific CD8⁺ T-cells for βgal₉₆- red/orange and βgal₄₉₇- dark blue/light blue for C57BL/6 and CD73-/- mice respectively. Blood from 0, 14, 21, and 50 days post immunisation were stained with tetramers and analyzed by flow cytometry. Mean percentages of live tetramer-positive CD8⁺ lymphocytes are indicated (n = 5, mean±SEM).</p

Phenotype of specific CD8⁺ T-cells in CD73^−/− mice after MCMV infection.

<p>Time course showing the percentage of cells expressing or MFI of the indicated markers on M38- (red/orange) and M45-specific CD8⁺ T-cells (blue/light blue), in the spleen, liver, lung and blood in comparison with naive CD8⁺ T-cells (n = 6, mean±SEM).</p

Following the proliferative and functional state of specific CD8⁺ T-cells in CD73^−/− mice after MCMV infection.

<p>Time courses showing the percentage of M38- (red/orange) and M45-specific CD8⁺ T-cells (blue/light blue), in the spleen on 0, 7, 21, 50 and 100 days post infection (n = 6, mean±SEM) and the shown stain (A) ICS staining for the proliferation marker Ki67. (B) Percentage CD8⁺ T-cells from the spleen producing effector molecules after stimulation with either the M38 or M45 peptide.</p

Contribution of KK10 to immune control of HIV determined by tet-SAP.

<p>Panels A-C and D-F show results with cells from two different HIV-infected HLA-B*27:05-positive donors. <b>(A)</b> Tetramer staining 48h post-treatment with KK10-SAP to confirm depletion of KK10-specific cells. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+). <b>(B)</b> Viral replication in HLA-B*27:05-expressing H9 cells without or with added untreated bulk, KK10-SAP-treated, KK10-PE-treated or mismatch-tet-SAP-treated CD8+ T-cells. Infected cells were measured by NL4-3-GFP expression. <b>(C)</b> Suppressive capacity of bulk, KK10-SAP-treated or mismatch SAP-treated CD8+ T-cells. <b>(D)</b> Tetramer staining to confirm KK10-SAP-mediated depletion of KK10-specific CD8+ T-cells (48 hours post-treatment) or depletion of KK10-PE-stained cells with anti-PE magnetic beads. <b>(E)</b> Viral replication (as in B) in H9-HLA-B*27:05-positive infected target cells alone or with bulk, KK10-SAP-treated or KK10-bead-depleted CD8+ T-cells. <b>(F)</b> Suppressive capacity of bulk, KK10-SAP-treated or KK10-bead-depleted CD8+ T-cells. B,C,E,F, error bars represent s.e.m. Represents 2 separate experiments with two HLA-B*27:05-positive donors.</p

Definition of antiviral efficacy of different CD8+ T-cell responses using tet-SAP.

<p>All panels show data from one HIV-infected HLA-B*27:05-positive subject. <b>(A)</b> Viral loads, CD4+ T-cell counts with timepoints of assays indicated. <b>(B)</b> IFN-γ ELISPOT CD8+ T-cell responses to HLA-B*27-restricted optimal epitopes. Only responses >50 SFC/10⁶ PBMC are shown. <b>(C)</b> Frequency of antigen-specific cells of three dominant specificities determined by tetramer staining. <b>(D)</b> Proviral sequences of the three dominant responses. Known HLA-B*27:05-associated footprints and the significance of the associations (q value) are shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184496#pone.0184496.ref033" target="_blank">33</a>]. Nd, not done. <b>(E-G)</b> Results from timepoint 48 months post-infection; (H-J) results from timepoint 96 months post-infecition. <b>(E,H)</b> Dot plots showing tetramer staining of existing in this donor HLA-B*27:05-restricted responses at the timepoint assayed. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+). <b>(F,I)</b> Viral replication in H9-HLA-B*27:05-positive infected target cells alone or with bulk CD8+ T-cells or CD8+ T-cells depleted of a particular specificity with tet-SAP. Infected cells were measured by NL4-3-GFP expression. <b>(G,H)</b> Suppressive capacity of bulk or tet-SAP-depleted CD8+ T-cells. F,G,I,J, error bars represent s.e.m. G,J, ANOVA with Dunnett’s Multiple comparisons test to compare suppressive capacity of bulk versus tet-SAP-treated CD8+ T-cells. *p<0.05, **p<0.01, ***p<0.001.</p

Recognition and internalisation of conventional and cytotoxic tetramers.

<p><b>(A-F)</b> Representative dot plots of PBMC staining in an HIV-infected HLA-B*27:05-positive subject with conventional fluorescently labeled tetramer HLA-B*27:05-Gag-KK10-PE <b>(A)</b> or SAP-coupled HLA-B*27:05-Gag-KK10-SAP tetramer, detected with a secondary Alexa Fluor 488 anti-SAP antibody in permeabilised <b>(B)</b> or not permeabilised <b>(C)</b> cells. Absence of non-specific binding of HLA-mismatched tet-PE <b>(D)</b> and tet-SAP <b>(E)</b> and free unconjugated SAP <b>(F)</b> is shown. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+). <b>(G)</b> Kinetics of tetramer binding and internalisation, determined by measurements of total and internal tetramer fluorescence in cells at internalisation-promoting (37°C) or internalisation-inhibiting (4°C) conditions. No further increase in internal fluorescence is observed after 90–120 minutes of internalisation time, suggesting that nearly all cognate metabolically active (37°C) CD8+ T-cells have internalised the tetramer. PBMC from an HIV-negative healthy donor with an EBV-HLA-A*02:01-GL9 response were used here. ‘Total tet-PE’ = surface-bound and internal tetramer fluorescence measured in cells not stripped of any surface-bound tetramer; ‘internal tet-PE’ = internal tetramer fluorescence measured in acid-stripped cells. A-F, representative of 8 independent experiments with PBMC from different donors. G, representative of 2 independent experiments with cells from two individuals.</p

Tet-SAP effectively and specifically depletes cognate CD8+ T-cells.

<p><b>(A)</b> Representative dot plots of a time course, showing depletion of HIV HLA-B*27:05-Gag-KK10-specific CD8+ T-cells mediated by KK10-SAP. At time -2 hours, cells were left untreated or treated with KK10-SAP, mismatch-SAP or free SAP for 2 hours at 37°C, washed and left in media (time = 0 hours). Every 24 hours aliquots of cells from each condition were stained with the relevant fluorochrome-conjugated tetramer and analysed by flow cytometry. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+) assessed at indicated times after treatment. <b>(B)</b> Graphical representation of 4 depletion time courses (performed as in A) with tet-SAP of different HIV, CMV and EBV specificities and restricted by different HLA class I molecules using cells from different individuals with the corresponding specificities. At time -2 hours cells were treated with tetramer-SAP of interest (red lines and symbols): HIV HLA-B*27:05 KK10-SAP (top left), CMV HLA-B*07:02 TM10-SAP (top right), HIV HLA-A*02:01 GL9-SAP (bottom left), or EBV HLA-A*02:01 GL9-SAP (bottom right). Controls included: untreated cells (black lines and symbols), treated with mismatch-SAP tetramer (blue lines and symbols; when sufficient cell numbers were present), or treated with free SAP (green lines and symbols, when sufficient cell numbers were present). % tet+ cells is normalised to baseline pre-treatment levels (time = -2h). Tx = treatment. <b>(C,D)</b> KK10-SAP-mediated depletion (assessed 48 hours post-treatment) of HLA-B*27:05-restricted KK10-specific CD8+ T-cells does not have an off-target effect on HLA-B*27:05-restricted KY9-specific CD8+ T-cells, while KY9-SAP-mediated elimination of KY9-specific cells does not affect KK10-specific cells. A,B, representative of at least 10 separate experiments with cells from different individuals with different HLA types and with tetramers of different specificities. C,D, representative of 3 independent experiments with cells from different HIV-positive HLA-B*27:05-positive donors.</p