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Results: 5

1.
Fig. 3

Fig. 3. From: Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

Coreceptor-enhanced Q115E pHLA A*0201 tetramers retain cognate binding integrity directly ex vivo. PBMC from two CMV seropositive patients with acute myeloid leukemia were stained with D227K/T228A, wildtype (WT) and Q115E pHLA A*0201 tetramers refolded around the pp65495–503 peptide. Tetramer-positive cells gated as shown in Fig. 2a were sorted by flow cytometry to >98% purity and analyzed for TCRB gene expression as described in the Materials and methods. Shown are the CDR3 amino acid sequences, TCRBV and TCRBJ usage, and the relative frequency of CD8+ T cell clonotypes specific for CMV pp65495–503. Colored boxes denote identical clonotypes.

J. Joseph Melenhorst, et al. J Immunol Methods. ;338(1-2):31-39.
2.
Fig. 5

Fig. 5. From: Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

Coreceptor-enhanced pHLA A*0201 tetramers can identify low avidity antigen-specific CD8+ T cells that are not visible with the corresponding wildtype tetramer directly ex vivo. (a) Bone marrow mononuclear cells from a patient with acute myeloid leukemia were stained with PR1/HLA A*0201 tetramers. The frequencies of D227K/T228A, wildtype and Q115E PR1/HLA A*0201 tetramer-positive and background events, calculated within the CD8+ and CD8 populations respectively after gating as shown in Fig. 2a, are shown as a percentage of the total live CD3+ lymphocyte population. (b) Cells staining brightly (yellow) and dimly (red) with the Q115E PR1/HLA A*0201 tetramer in the experiment shown in (a) exhibit similar phenotypes based on expression of CCR7, CD27, CD45RA and CD57; the tetramer−positive events are almost exclusively CCR7 memory cells, although a few naïve events consistent with background staining are apparent as the gate is extended towards the CD8+ tetramer-negative population. The phenotypic distribution of these PR1-specific CD8+ T cells differs considerably from that of the overall CD8+ T cell population (gray density plot). In all experiments, tetrameric preparations were standardized for concentration and volume, such that conditions differed solely with respect to the pHLA A*0201/CD8 interaction.

J. Joseph Melenhorst, et al. J Immunol Methods. ;338(1-2):31-39.
3.
Fig. 4

Fig. 4. From: Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

Modulation of CD8 coreceptor binding properties differentially influences pMHCI tetramer staining patterns directly ex vivo according to antigen origin. Mean fluorescence intensity (MFI) of distinct populations identified in PBMC (black) and bone marrow mononuclear cells (BMMC; red) with D227K/T228A, wildtype (WT) and Q115E pHLA A*0201 tetramers refolded around CMV (pp65495–503) and LAA (PR1, circles; WT1, triangles) peptides. PBMC from 12 healthy donors were stained with the three CMV pp65495–503/HLA A*0201 tetramers; PBMC from 10 patients and BMMC from 8 patients with acute or chronic myeloid leukemias were stained with the six LAA/HLA A*0201 tetramers (only data from samples with detectable tetramer binding events are shown). The MFIs for three populations are shown in each case: (i) naïve CD8+ T cells (CD27+CD45RO); (ii) memory CD8+ T cells (CD27+CD45RO+; CD27CD45RO+; CD27CD45RO) that did not bind tetramer (tetramer −); and, (iii) memory CD8+ T cells that did bind tetramer (tetramer +). Higher MFI values are apparent in all three populations of CD8+ T cells stained with the various Q115E pHLA A*0201 tetramers compared to the corresponding D227K/T228A and wildtype tetramers. These effects applied to all antigen-specific CD8+ T cell populations, but were generally more pronounced with the CMV pp65495–503 tetramers. In all experiments, tetrameric preparations were standardized for concentration and volume, such that conditions differed solely with respect to the pHLA A*0201/CD8 interaction. Statistical comparisons were conducted using the Wilcoxon signed rank test.

J. Joseph Melenhorst, et al. J Immunol Methods. ;338(1-2):31-39.
4.
Fig. 1

Fig. 1. From: Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

Tetrameric complexes of pHLA A*0201 containing the heavy chain α2 domain Q115E substitution bind low avidity CD8+ T cells. The ILA1 CD8+ T cell clone was stained with wildtype (a) and Q115E-substituted (b) pHLA*0201-PE tetramers bearing different APL at a final concentration of 10 µg/ml with respect to the monomeric pMHCI component. The staining protocol and APL binding/activation properties were described previously (Laugel et al., 2007). As reported, the TCR/pMHCI affinities measured by SPR for the APL interactions shown are as follows: 3G, 3.7 µM; 3G8T, 4.0 µM; 8Y, 22.6 µM; index, 36.6 µM; 5Y, 242.1 µM; 8E,>500 µM. The 3G, 3G8T and 8Y ligands act as superagonists in functional assays relative to index; 5Y and 8E are weak agonists relative to index. (c) Affinity and binding kinetics of the ILA1 TCR/4L HLA A*0201 interaction; SPR experiments were performed as described previously (Laugel et al., 2007). Standard deviation from the mean dissociation constant (KD) of 3 separate experiments is shown. (d) Staining intensities shown in (a) and (b) are plotted against TCR/pMHCI binding affinity (KD) for wildtype (circles) and Q115E (squares) pHLA A*0201 tetramers; APL color codes match those shown in a and b. Curves were fitted as described previously (Laugel et al., 2007). (e–g) Tetramer capture from solution by ILA1 CTL. Concentrations of tetramer were titrated in an attempt to achieve a maximum fluorescence intensity of ~200. This level of staining could not be achieved for the wildtype 4L ligand, and staining only reached an MFI of ~60 in this case. From left to right, panels show % maximal binding over 30 min for ILA1 CTL stained with 0.2 µg/ml 3G8T (e), 1 µg/ml ILAKFLHWL index (f) and 5 µg/ml 4L (g) wildtype (circles) and Q115E (squares) tetramers. Curves in panels (e–g) represent least squares fit to Eq. (20) in van den Berg et al. (2007).

J. Joseph Melenhorst, et al. J Immunol Methods. ;338(1-2):31-39.
5.
Fig. 2

Fig. 2. From: Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

Coreceptor-enhanced pHLA A*0201 tetramers exhibit distinct staining characteristics with preservation of binding specificity directly ex vivo. (a) Successive panels show the gating strategy used to identify antigen-specific CD8+ T cells. First, doublets were distinguished from single cell events on the basis of their forward scatter-area vs. forward scatter-height profile. Live CD3+ T cells were then distinguished from dead cells (ViViD+), monocytes (CD14+) and B cells (CD19+). Subsequently, fluorochrome aggregates were gated out in two consecutive bivariate plots and lymphocytes were further identified according to forward scatter vs. side scatter profile. Histograms were then transformed at the level of the CD4 vs. CD8 plot. (b) Shown is the frequency (top panels) and phenotype (middle and bottom panels) of leukemia-associated antigen PR1-specific CD8+ T cells using D227K/T228A, wildtype and Q115E pHLA A*0201 tetramer analysis in a bone marrow sample from a patient with chronic myeloid leukemia 12 months after hematopoietic stem cell transplantation. Live CD3+ T cells were gated as shown in (a), and tetramer-positive events are displayed in a bivariate plot with CD8. The vertical bar in the D227K/T228A tetramer vs. CD8 plot serves as an orientation point for the positioning of the gate on tetramer-dim cells in subsequent panels. Tetramer-bright (yellow; middle panels) and tetramer-dim (red; bottom panels) CD8+ T cells are overlaid on a CD45RO vs. CD27 density plot for the total CD8+ T cell population; tetramer-bright CD8+ T cells lack naïve cells, whereas tetramer-dim CD8+ T cells are distributed across naïve and antigen-experienced phenotypes. This is most obvious for the Q115E pHLA A*0201 tetramer. (c) As for (b), except that the analysis shows CD8+ T cells specific for CMV pp65495–503 in the peripheral blood of a patient with acute myeloid leukemia. Staining was not observed with any of the three cognate pHLA A*0201 tetrameric forms in CMV seronegative controls (data not shown). In all experiments, tetrameric preparations were standardized for concentration and volume, such that conditions differed solely with respect to the pHLA A*0201/CD8 interaction.

J. Joseph Melenhorst, et al. J Immunol Methods. ;338(1-2):31-39.

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