Figure 1.
Identification of CD1a+/CD3+ cells from LCH lesions by flow cytometry.
Representative plots showing anti-CD1a-FITC [NA 1/34] and anti-CD3-APC-H7 [SK7] staining of lesional cells and peripheral blood from LCH patient #1, and cells extracted from control Tonsil #2. The unstained (left) plot shows approximately 500 live cells. Remaining plots display 30,000 live cells or greater. Live cells are defined as those cells remaining after doublet and propidium iodide positive cells were excluded. The quadrant numbers indicate the percentage of each population within the live cell gate.
Table 1.
Clinical details of LCH patients.
Table 2.
Experimental details for LCH patients.
Table 3.
The mean percentages of CD1a+ cells, CD1a+/CD3+ cells and CD3+ cells in the live cell population of lesional cells from six LCH patients.
Table 4.
Experimental details for control patients.
Figure 2.
Representative plots from LCH patient #1 showing the gates used to exclude cell doublets.
Figure 3.
CD1a+/CD3+ cells from LCH lesions are morphologically and phenotypically polyclonal T-cells.
(A) Relative size of CD1a+/CD3− LCs (mean forward scatter intensity = 1.7×105), CD1a+/CD3+ T-cells (1.1×105) and CD1a−/CD3+ T-cells (1.1×105) on flow cytometric profiles from patient #1 (B-cells were excluded). Plots show staining for anti-CD1a-FITC [NA 1/34] and anti-CD3-APC-H7 [SK7]. (B) (Left) Immunocytochemical staining of a cytospin from a single cell suspension prepared from the lesion of patient #1 using anti-CD1a (DAB+/brown) and anti-CD3 (Fast Red), counterstained with Mayer’s hematoxylin and mounted in Dako Ultramount (Scale bar = 5 µm). Image shows a typical lymphocyte (TA), a T-cell with CD1a staining (TB), and a Langerhans cell (LC). (Right) H&E stain showing typical lymphocyte morphology of FACS-sorted CD1a+/CD3+ cells (Scale bar = 5 µm). (C) Phase contrast image (top left) and fluorescent images (Scale bar = 5 µm). Double immunofluorescence labeling of a CD1a+/CD3+ T-cell from a single cell suspension prepared from the lesion of patient #1, using anti-CD1a-AlexaFluor 488 (green), anti-CD3-AlexaFluor 594 (red). The nucleus is stained with DAPI (blue). The filter used for the bottom left image allows simultaneous viewing of all colors. Microscopy was performed on a Leica DMLB microscope (Leica Microsystems). Images were captured with a Leica DC300F digital camera (Leica Microsystems).
Figure 4.
CD1a expression on T-cells is not restricted to either CD4+ or CD8+ subsets.
The flow cytometry plots are from one experiment using lesional cells from LCH patient #1. Live cells were gated into quadrants based upon CD1a and CD3 antibody intensity and the percentages of cells were calculated. Live cells are defined as those cells remaining after doublet and propidium iodide positive cells were excluded. CD1a+/CD3+ cells were then gated to identify CD1a+/CD3+/CD4+ cells and CD1a+/CD3+/CD8+ cells. Similarly, CD1a−/CD3+ cells were gated to identify CD1a−/CD3+/CD4+ cells and CD1a−/CD3+/CD8+ cells. Plots show staining for anti-CD1a-APC [HI149], anti-CD3-APC-H7 [SK7], anti-CD4-V450 [RPA-T4] and anti-CD8-PE [HIT8a].
Table 5.
CD1a expression is not restricted to CD4+ or CD8+ T-cell subsets.
Table 6.
Percentage of CD1a+ T-cells that express additional CD markers.
Figure 5.
CD1a+ T-cells are not restricted to either naïve or memory T-cells.
Expression of the T-cell activation markers CD45RA and CD45RO in CD1a+ and CD1a− T-cells in flow cytometry plots of lesional cells from LCH patients #1 and #4. Plots show staining for anti-CD1a-FITC [NA 1/34], anti-CD3-APC-H7 [SK7], anti-CD45RA-PE-Cy7 [HI100] and anti-CD45RO-PE [UHCL-1].
Figure 6.
Expression of CD1a and CD3 mRNA in CD1a+/CD3+ cells sorted by FACS from LCH lesions.
(A) Plot showing anti-CD1a-FITC [NA 1/34] and anti-CD3-APC-H7 [SK7] staining of LCH lesional cells from patient #1 by flow cytometry. The boxed area shows the gate used to sort CD1a+/CD3+ cells. This plot is representative for all patients examined. RNA was extracted from the CD1a+/CD3+ sorted cells and reverse transcribed for PCR amplification. (B) Agarose gel electrophoresis of RT-PCR products generated using primers specific for CD1a, CD3 and β-actin. PCR was initially performed with sorted cells from patient #1 and was later performed with sorted cells from patients #3, #4 and #6. PCR was not possible on sorted cells from patients #2 and #5. The positive control (+ve) was a cDNA sample previously shown to contain the amplicon and the negative control (-ve) was a reaction with no cDNA added.
Figure 7.
Comparisons of antibody binding specificities.
(A) We used anti-CD3-APC-H7 [SK7] in all FACS analyses except for Vβ repertoire analyses, where anti-CD3-PC5 [UCHT1] was used as per IOTest Beta Mark TCR Vβ Repertoire Kit (Beckman Coulter) recommendation. A comparison between anti-CD3-APC-H7 [SK7] (left) and anti-CD3-PC5 [UCHT1] (right) using peripheral blood demonstrates that antibodies have a similar specificity. Plots show 100,000 events and frequency is expressed as a percentage of live lymphocytes. (B) Anti-CD3-APC-H7 [SK7] was used in combination with two different anti-CD1a antibodies for all FACS analyses excluding Vβ repertoire analyses. A comparison between anti-CD1a-FITC [NA 1/34] (left) and anti-CD1a-APC [HI149] (right) using a mix of peripheral blood and Jurkat cells shows that these antibodies have a similar specificity. Plots show 10,000 events and frequency is expressed as a percentage of live cells.
Table 7.
Primer sequences for reverse transcription-PCR.