Effect of dimethyl sulfoxide (DMSO) on the binding of anti-HLA (clone W6/32) and anti-Ig antibodies binding to microarray spots printed with pHLA A2-Ig dimers. From left to right: PE-labeled W6/32 (20 μg/ml) contacted with spots printed with 0.1mg/ml unloaded pHLA A2-Ig dimer in PBS solution, in 0.33% (v/v) DMSO/PBS solution, and with 66.7μg/ml CMVpp65-loaded pHLA A2-Ig dimer in 0.33% (v/v) DMSO/PBS solution, and FITC-labeled anti-Igλ (20 μg/ml) contacted with spots printed with 0.43mg/ml unloaded pHLA A2-Ig dimer in PBS solution and with 276 μg/ml CMVpp65-loaded pHLA A2-Ig dimer in 1.4% (v/v) DMSO/PBS solution. Mean spot fluorescence intensities are normalized to the mean spot fluorescence intensities for the unloaded dimer in PBS solution for each antibody staining, with the percentages shown above the bars corresponding to these relative mean spots intensities. Insets: scanned fluorescence images of subarrays for the two printing solutions designated by the arrows.

Quantified mean spot fluorescence intensities of cells captured from suspensions containing different concentrations of CMVpp65-specific CTLs incubated with either the CMVpp65-loaded dimer or the unloaded dimer. A CMVpp65-enriched cell population containing ~10% CMVpp65-specific CTL, as determined by FACS, was first labeled with CFSE, and incubated with either CMVpp65-loaded pHLA A2-Ig dimer (loading at 24°C for 2 days) or the unloaded dimer, and then serially diluted in unlabeled autologous CD8 T-cell-depleted PBMCs before contacting with the microarray. Concentrations of CMVpp65-specific CTL in the different suspensions were: 0% (100% CD8 T-cell-depleted PBMCs), 0.01%, 0.1%, 1% and 10% (undiluted) of the total cell number. Each suspension is contacted with a 10 × 10 subarray of 100 anti-Igλ antibody spots. Individual spots are printed with ~ 333pl of anti-Igλ antibody/BSA solution, and are 150μm in diameter. Cell seeding conditions: 50μl of 2 × 10⁶/ml total cells on an area of 6.8 × 6.8 mm². Error bars in the graph represent the standard deviation across the 100 spot replicates. The standard deviation for 0% frequency of CMVpp65-specific cells incubated with the CMVpp65-dimer is 10.0 (mean spot intensity = 60.7) compared to the standard deviation for 0% frequency of CMVpp65-specific cells incubated with unloaded dimer of 6.5 (mean spot intensity = 62.1). The horizontal dashed and dotted line designate the mean spot intensities corresponding to cells captured from the 0% CMVpp65-specific cell suspension incubated with the CMVpp65-loaded pHLA A2-Ig dimer and the unloaded dimer, respectively. Statistical differences between mean spot intensities for CMVpp65-specific and non-specific cell capture at each CTL frequency computed based on the student-t distribution produced the following p-values: p<0.0001 at 10%; p=0.0129 at 1%; p=0.1209 at 0.1%; and p=0.2175 at 0.01%.

Detection of M1.58-, NA.75-, and PA.46-specific CTL in CD8+ CTL isolated from peripheral blood of A2-positive human donor A8 by pHLA A2-Ig cellular microarrays and FACS. For each assay, the level of non-specific binding to the unloaded dimer was subtracted before plotting. Antigen-specific CTL frequencies in the microarray assay are quantified as the sum of spot intensities for only those spots displaying bound cells, normalized by the sum of anti-CD3 spot intensities. Individual spots of the 5 × 5 subarray were printed using 333pl of anti-Igλ antibody/BSA solution, and are 500 μm in diameter with a spot center-to-center distance of 1 mm. Cell seeding conditions: 50μl of ~2 × 10⁶/ml total cells.

Schematic depiction of the cellular microarray assay in which soluble peptide-loaded HLA A2-Ig dimers are incubated with T cells in solution. The antigen-specific sub-population of cells are identified by the surface-bound pHLA A2-Ig dimers and subsequently captured on the microarray by binding to printed anti-Igλ antibodies that recognize the immunoglobulin (Ig) portion of the dimer.

Comparison of pHLA A2-Ig dimer microarray and FACS assays of the frequency of antigen-specific CTLs in enriched T-cell populations. (a) Representative images of cell capture on 5 × 5 subarrays of 25 anti-Igλ spots and anti-CD3 spots. From left to right: CMVpp65-enriched CTLs incubated with the unloaded dimer, CMVpp65-enriched CTLs incubated with CMVpp65-loaded dimer, M1.58-enriched CTLs incubated with M1.58-loaded dimer, PA.46-enriched CTLs incubated with PA.46-loaded dimer, and CMVpp65-enriched CTLs on anti-CD3 spots. (b) Mean spot fluorescence intensities normalized by the mean fluorescence intensities for binding to the anti-CD3 spots on the microarray compared to the frequency of antigen-specific CTLs determined by FACS. For the microarray assay, the mean spot intensity for the unloaded dimer was subtracted before normalizing. For the FACS assay, the frequency measured for the unloaded dimer was subtracted beforehand. Error bars for the microarray assay represent standard errors for the 25 spot replicates. Dimer loading conditions: 15 days at 4°C for CMVpp65, 11 days at 24°C for M1.58, and 16 days at 24°C for PA.46. Individual spot are printed from 10 nl of solution and are 500μm in diameter. Spot center-to-center distance is ~ 1mm. Cell seeding conditions for each subarray: 50μl of ~0.8 × 10⁶/ml total cells.

Individual spot intensities corresponding to CFSE-labeled M1.58-specific T-cell capture on a 10 × 10 subarray of 100 anti-Igλ antibody spots as a function of M1.58-specific CTL frequency. (a) Spot intensities corresponding to cells captured on subarrays contacted with cell suspensions containing CFSE-labeled M1.58-specific CTL at frequencies of 0% (100% CD8 cell-depleted autologous PBMCs), 0.01%, 0.1%, 1%, and 10%. Individual spot intensities are shown as open circles, vertically aligned for each frequency. Fluorescence intensities of spots contacted with the 0% M1.58-specific CTL suspension define the cutoff intensity for spots with captured cells. Spots with intensities below the cutoff are considered to have no captured cells and are shown as black circles, whereas spots with intensities above the cutoff are considered to have captured cells and are shown as red circles. For each frequency, the number to the left of the vertically aligned circles designates the number of spots with captured cells. (b) Sum of spot fluorescence intensities corresponding to the red circles in (a) as a function of the frequency of M1.58-specific CTL in the suspension. The linear dependence is log₁₀[intensity] = 5.304+0.9886°log₁₀[% freq.] with R²=0.998. Experimental conditions are the same as in Figure 4.