The authors have declared that no competing interests exist.
Conceived and designed the experiments: JAW SLO GK. Performed the experiments: JAW SLO REP JML JMM. Analyzed the data: JAW SLO JMM CT. Contributed reagents/materials/analysis tools: J-IH SOA. Contributed to the writing of the manuscript: JAW SLO JMM GK.
Langerhans cell histiocytosis (LCH) is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a+/CD3+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs). We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH.
Langerhans cell histiocytosis (LCH) is a complex disease with unpredictable progression and no known cause
LCH was originally defined by the Writing Group of the Histiocyte Society in 1987
The recognition of close similarities between normal epidermal LCs and pathogenic LCs has led to the concept that epidermal LCs are the precursor cells in LCH
Most LCH research has focused on pathogenic LCs. While the accumulation of pathogenic LCs within LCH lesions is considered to be a defining characteristic of LCH
The unresolved role of T-cells in LCH is indicated by the number of conflicting reports relating to the types of T-cells within lesions
CD1a expression on pathogenic Langerhans cells (LCs) in LCH is regarded as a hallmark of this disease, but the role of this highly restricted molecule in LCH has remained uncertain. Normally, CD1a molecules on the surface of LCs present glycolipid antigens to specialized T-cells as part of their role in immunosurveillance
While CD1a is expressed predominantly on LCs, it can be expressed by T-cells under certain conditions. Immature thymocytes express CD1a
Historically, the cellular composition of LCH was determined using archival tissue sections. This approach formed the foundation of what is currently known about this disease, however, these earlier studies were greatly restricted by the number of CD markers that could be detected simultaneously. LCH tissues have been examined by flow cytometry in a limited number of studies
We set out to better define the role of CD1a and T-cells in LCH lesions, using multi-parameter flow cytometry with up to eight cell markers simultaneously. Here we report for the first time, to our knowledge, the expression of CD1a on polyclonal T-cells in LCH lesions. The results provide new insights into the composition of LCH lesions and we anticipate that future studies into the biological function of these cells may lead to an improved understanding of LCH pathogenesis.
Using flow cytometry we characterized the cell marker profiles of LCH lesions from six patients (
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.
Patient | Age | Gender | LCH diagnosis | Tissue examined |
#1 | 9 | M | Eosinophilic granuloma of skull and lymph nodelesion | Lesion Peripheral blood |
#2 | 12 | F | Multifocal eosinophilic granulomas of skull andlymph node lesions | Lesion |
#3 | 58 | F | Multifocal dermal lesions of shin | Lesion Peripheral blood |
#4 | 6 | F | Single eosinophilic granuloma of skull | Lesion Peripheral blood |
#5 | 3 | M | Multifocal eosinophilic granulomas (right tibia,parietal bone, costae) | Lesion Peripheral blood |
#6 | 13 | F | Single eosinophilic granuloma of skull | Lesion |
#7 | 31 | M | Disseminated | Peripheral blood |
#8 | 53 | M | Multifocal (bone, lung) | Peripheral blood |
#9 | 61 | M | Multifocal (bone, lung) | Peripheral blood |
#10 | 1 | M | Disseminated | Peripheral blood |
#11 | 2 | F | Disseminated | Peripheral blood |
Patient | Tissueexamined | No. ofexperimentsperformed | Total no. of livecells examined | CD markers examined by flow cytometry |
#1 | Lesion | 5 | 72,409 | 1a, 3, 4, 8, 14, 16, 19, 25, 31, 34, 45, 45RA, 45RO, 56, 123, 138, 207, TCR Vβ |
PeripheralBlood | 1 | 40,523 | 1a, 3, TCR Vβ | |
#2 | Lesion | 1 | 145,329 | 1a, 3 |
#3 | Lesion | 3 | 9,150 | 1a, 3, 4, 8, 14, 19, 34, 45, 45RA, 45RO, 123, 138, TCR Vβ |
PeripheralBlood | 1 | 9,743 | 1a, 3, TCR Vβ | |
#4 | Lesion | 5 | 105,617 | 1a, 3, 4, 8, 14, 16, 19, 25, 31, 34, 45, 45RA, 45RO, 56, 123, 138, 207, TCR Vβ |
PeripheralBlood | 1 | 9,140 | 1a, 3 | |
#5 | Lesion | 1 | 2,002 | 1a, 3, 4, 8, 19, 45 |
PeripheralBlood | 1 | 10,835 | 1a, 3, TCR Vβ | |
#6 | Lesion | 4 | 25,586 | 1a, 3, 4, 8, 14, 19, 34, 45, 45RA, 45RO, 123, 138 |
#7 | PeripheralBlood | 1 | 42,437 | 1a, 3, TCR Vβ |
#8 | PeripheralBlood | 1 | 22,043 | 1a, 3, 8, 14, 25, 123, 207, TCR Vβ |
#9 | PeripheralBlood | 1 | 60,319 | 1a, 3, 4, 16, 19, 31, 56, TCR Vβ |
#10 | PeripheralBlood | 1 | 202,873 | 1a, 3 |
#11 | PeripheralBlood | 1 | 65,273 | 1a, 3, 4, 16, 19, 31, 56 |
LCH Patient | % CD1a+ | % CD1a+/CD3+ | % CD3+ |
44.7 | 23.8 | 42.0 | |
3.9 | 1.5 | 5.3 | |
6.6 | 5.3 | 29.3 | |
22.5 | 2.3 | 9.4 | |
6.5 | 3.0 | 43.9 | |
31.0 | 13.6 | 45.2 |
CD1a+/CD3+ cells were not detected in normal peripheral blood from six volunteers, in peripheral blood from nine LCH patients or in single cell suspensions (SCS) prepared from the epithelial layer of five tonsils (
Tissue anddescription | No. of experimentsperformed | Total no. ofcells examined | CD markers examined by flowcytometry |
Tonsil #1 | 1 | 7,812 | 1a, 3, 4, 16, 19, 31, 56 |
Tonsil #2 | 1 | 21,581 | 1a, 3, 4, 16, 19, 31, 56 |
Tonsil #3 | 1 | 9,134 | 1a, 3, 4, 45RA, 45RO |
Tonsil #4 | 1 | 11,072 | 1a, 3, 8, 14, 25, 123, 207 |
Tonsil #5 | 1 | 10,417 | 1a, 3, 8, 14, 25, 123, 207 |
Lymph node (LCHpatient with noinvolvement atthis site) | 1 | 6,255 | 1a, 3, 4, 16, 19, 31, 56 |
Peripheral Blood 1 | 1 | 14,989 | 1a, 3, 4, 16, 19, 31, 56 |
Peripheral Blood 2 | 3 | 21,468 | 1a, 3, 4, 8, 14, 16, 19, 25, 31, 45, 56, 123, 207 |
Peripheral Blood 3 | 2 | 7,103 | 1a, 3, 4, 8, 14, 19, 34, 45, 123, 138 |
Peripheral Blood 4 | 1 | 2,776 | 1a, 3, 4, 8, 19, 45 |
Peripheral Blood 5 | 1 | 1,648 | 1a, 3, 4, 8, 19, 45 |
Peripheral Blood 6 | 2 | 15,101 | 1a, 3, 4, 8, 14, 19, 34, 45, 123, 138 |
Peripheral Blood 7(AML) | 1 | 10,966 | 1a, 3, 4, 16, 19, 31, 56 |
Peripheral Blood 8(AML) | 2 | 1,767 | 1a, 3, 4, 8, 14, 16, 19, 25, 31, 56, 123, 207 |
Peripheral Blood 9(CLL) | 1 | 94,100 | 1a, 3, 4, 16, 19, 31, 56 |
Peripheral Blood 10(T-cell lymphoma) | 1 | 248,717 | 1a, 3, 4, 45RA, 45RO |
Tight doublet gates were applied to ensure that CD1a+/CD3+ cells were not two adjoined cells (
Representative plots from LCH patient #1 showing the gates used to exclude cell doublets.
(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).
Since the morphology of CD1a+/CD3+ cells was more typical of lymphocytes than LCs, further experiments were performed to determine their phenotype. The T-cell receptor (TCR) Vβ repertoires of the T-cells within lesions from three LCH patients were examined. Most of the 25 TCR Vβ subsets were detected in lesional CD1a+ T-cells without any obvious bias (data not shown). There was insufficient sample to analyze the remaining three LCH lesions. Because there were multiple TCR subtypes, CD1a+ T-cells could not have arisen from a single parent cell, and were therefore not clonal in origin.
The T-cell subsets within five LCH lesional samples were examined to differentiate between CD4+ helper (TH) T-cells and CD8+ cytotoxic (TC) T-cells. One sample was excluded due to insufficient data. CD1a expression on CD1a+/CD3+ cells was not restricted to TH or TC 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].
LCH Patient | |||||
% of T-cells | #1 | #4 | #5 | #6 | |
56.7 | 24.4 | 6.8 | 30.2 | ||
32.1 | 17.4 | 5.1 | 23.1 | ||
18.4 | 2.2 | 0.5 | 8.5 | ||
43.3 | 80.2 | 93.2 | 70.6 | ||
14.1 | 30.7 | 71.6 | 40.4 | ||
13.6 | 10.2 | 15.5 | 11.0 |
Mean data are expressed as percentages of T-cells from four LCH samples.
Additional CD markers were used where possible, to further phenotype the CD1a+ T-cells (
Additional CD marker | Patient number | |||
#1 | #4 | #5 | #6 | |
CD207 | 26.8 | 11.7 | ND |
ND |
CD45 | 100.0 | 98.4 | 93.3 | 87.4 |
CD16 | 8.5 | 51.4 | ND | ND |
CD25 | 4.1 | 41.5 | ND | ND |
CD56 | 1.4 | 10.8 | ND | ND |
CD14 | 3.1 | 7.4 | ND | 4.8 |
CD34 | 0.0 | 4.1 | ND | 1.1 |
CD123 | 0.1 | 3.3 | ND | 0.3 |
CD138 | 0.0 | 4.9 | ND | 0.9 |
CD19 | 1.0 | 1.9 | 3.3 | 0.6 |
*ND (not determined).
It is known that upon recognition of specific antigen, naïve T-cells (CD45RA+/CD45RO−) reduce their expression of CD45RA and increase their expression of CD45RO, producing memory T-cells (CD45RA−/CD45RO+)
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].
PCR amplification of CD1a and CD3 confirmed our hypothesis that the CD1a+/CD3+ sorted cells from LCH lesions expressed both CD1a and CD3 mRNA in all four LCH lesions examined (
(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.
Amplified CD1a mRNA from CD1a+/CD3+ sorted cells from patient #1 was sequenced. This was performed to confirm that the bands obtained were true CD1a sequence and that the CD1a transcript matched the reference sequence. Normal CD1a sequence was obtained over 1035 nucleotides from position 787-1822 when compared to the 2111 bp NCBI reference sequence (NM_001763.2 GI:110618223). This included 78.5% (728/927) of the mature peptide sequence.
Here we demonstrate for the first time, to our knowledge, the presence of CD1a+ T-cells in all LCH lesions we have studied to date. These cells were not detected in the peripheral blood from LCH patients. Cellular morphology was typical of lymphocytes, and these cells expressed many of the typical T-cell markers. We have shown that sorted CD1a+/CD3+ cells from LCH lesions also expressed both CD1a and CD3 mRNA, indicating that the CD1a was generated within these cells and not transferred as protein by T-cell interaction with LCs.
Previous studies have confirmed that T-cells within LCH lesions are polyclonal
Multi-parameter flow cytometry and cell sorting have aided our capacity to more precisely determine cell phenotypes within LCH lesions. The heterogeneous populations of cells identified in this study clearly show that the cellular composition in LCH lesions is more complex in regards to CD1a expression than was previously recognized
More recent work using cell-specific gene expression profiling compared T-cells from LCH lesions to those of peripheral blood of LCH patients
It is evident from our data that CD1a expression has been induced across a range of T-cell subsets in LCH lesions. The co-expression of T-cell markers and the lack of DC marker expression suggests it is unlikely that CD1a+ T-cells originate from pathogenic LCs. In light of these data, the question must be posed as to how and why this might occur. Peripheral T-cells do not normally express CD1a; however CD1a expression on cortical thymocytes is well documented
T-cells expressing CD1a were recently identified in very low numbers in the lymphoid tissue of tonsils
CD1a expression can be induced by various cytokine combinations
It is known that the LCH micro-environment contains numerous cytokines including GM-CSF and TNFα
Our observation of CD1a+ T-cells in LCH lesions may open up further opportunities for functional studies. Experiments to determine function were beyond the scope of this study. Due to the limited availability of live cells for analysis, any potential mechanisms must remain theoretical. It has been reported that pathogenic LCs in LCH poorly present alloantigens
Debate around the classification of LCH as a neoplastic disease or immune dysfunction has been ongoing
We demonstrate that CD1a is not a unique marker for pathogenic LCs in LCH and a new definition to include CD3− and CD1a+ and/or CD207+ cells or the presence of Birbeck granules is required for the identification of pathogenic LCs in LCH. We hypothesize, that defective cycling of CD1a molecules to the surface of pathogenic LCs may lead to redundancy within the immune system, whereby T-cells can be induced to express CD1a and present antigen within LCH lesions. In summary, our studies have identified for the first time, to our knowledge, the presence of polyclonal CD1a+ T-cells in LCH lesions and the presence of these cells is specific to LCH lesions.
This research was approved by the Ballarat Health Services and St John of God Hospital Ballarat Human Research Ethics Committee, University of Ballarat (now Federation University Australia) Human Research Ethics Committee, and Karolinska Institutet Biobank. Informed consent was written.
We obtained peripheral blood from nine LCH patients and lesional tissue samples from six LCH patients. Four LCH patients provided both peripheral blood and tissue (
Samples were prepared from fresh tissue then stored in liquid nitrogen in 10% DMSO (v/v) or used fresh. LCH lesions, except the dermal sample from patient #3, were prepared as SCS in media (IMDM (Gibco) supplemented with 2 mM L-glutamine, 50 µg/ml kanamycin (Invitrogen) and 10% (v/v) heat-inactivated fetal calf serum (Gibco)). The dermal sample was prepared from a skin biopsy of an LCH lesion as previously described
Cytospins of SCS were fixed with methanol. The DakoCytomation EnVision Doublestain system was used for chromogenic immunocytochemistry. For fluorescent immunocytochemistry, cytospins were blocked with Image-iT FX, incubated with primary antibodies (anti-CD3 [polyclonal], Dako; anti-CD1a [O10], Abcam), washed, labeled with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 596, Invitrogen), washed and mounted with ProLong Gold antifade reagent with DAPI (4,6 diamidino-2-phenylindole) (Invitrogen Molecular Probes). Isotype-matched antibodies were used as negative controls.
LCH biopsy specimens and controls were examined (
(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.
SCS were washed and resuspended in media. The cells were incubated with antibodies in the dark for 30 min at 4°C then washed twice. For phenotypic characterization, the following antibodies were used: anti-CD1a-FITC [NA 1/34], anti-CD1a-APC [HI149], anti-CD3-APC-H7 [SK7], anti-CD3-PC5 [UCHT1], anti-CD4-V450 [RPA-T4], anti-CD8-APC [SK1], anti-CD8-PE [HIT8a], anti-CD14-V450 [MφP9], anti-CD16-PE [3G8], anti-CD19-PE-Cy7 [SJ25C1], anti-CD20-PerCP-Cy5.5 [L27], anti-CD25-PE-Cy7 [M-A251], anti-CD31-AlexaFluor 647 [M89D3], anti-CD34-PE-Cy7 [8G12], anti-CD45-PerCP-Cy5.5 [2D1], anti-CD45RA-PE-Cy7 [HI100], anti-CD45RO-PE [UHCL-1], anti-CD56-PE-Cy5.5 [MEM-188], anti-CD123-PerCP-Cy5.5 [7G3], anti-CD138-PE [DL-101], anti-CD207-PE [DCGM4] and anti-CD303-FITC [AC144]. All antibodies were purchased from BD Biosciences except anti-CD1a-FITC (Dako), anti-CD56-PE-Cy5.5 (Invitrogen), anti-CD3-PC5 and anti-CD207-PE (Beckman Coulter) and anti-CD303-FITC (Miltenyi Biotec).
T-cells were analyzed using the IOTest Beta Mark TCR Vβ Repertoire Kit (Beckman Coulter). Analysis was performed according to recommendations (staining with anti-CD3-PC5 [UCHT1]) but with the addition of anti-CD1a-APC [HI149] (BD Biosciences).
Total RNA was isolated using a miRNeasy Mini kit (QIAGEN) according to manufacturer’s instructions including the optional on-column DNase digestion. Random hexamer primers were used to synthesize cDNA using the Transcriptor First Strand cDNA synthesis Kit (Roche). PCRs were performed using 0.5–5 µl cDNA with gene-specific primers (
GeneName | PrimerBank ID |
Forward PrimerSequence (5′ to 3′) | Reverse Primer Sequence(5′ to 3′) | AmpliconSize (bp) |
CD1a pair 1 | 27764865a1 | 164 | ||
CD1a pair 2 | 27764865a2 | 141 | ||
CD3 | 4502671a1 | 245 | ||
β-actin | Not Applicable |
202 |
Wang and Seed B (2003) Nucleic Acids Res 31(24): e154.
Spandidos et al. (2008) BMC Genomics 9: 633.
Spandidos et al. (2010) Nucleic Acids Res 38(Database issue): D792–D799.
* Abrahamsen et al. (2003) J Molecular Diagnostics 5(1): 34–41.
CD1a was sequenced from 3′-RACE reactions using RNA from CD1a+/CD3+ sorted cells from LCH patient #1 and the CD1a pair 1 forward primer (
Data was analyzed in the GraphPad Prism statistical program (GraphPad Software, San Diego, CA). Non-parametric tests were used due to non-normality of small sample sizes. A Kruskal-Wallis one-way analysis of variance was used to detect differences between the percentage of CD1a+/CD3+ cells in LCH lesions, LCH peripheral blood and control tissues. While two LCH patients provided lesional tissue only, and five provided peripheral blood tissue only, four LCH patients provided both lesional and peripheral blood tissues. Being less powerful than a paired test to detect differences, an independent test was the most suitable option despite some pairing. Two-tailed Mann-Whitney U tests were conducted to detect differences between the percentage of TH cells in the CD1a+/CD3+ and the CD1a−/CD3+ populations, and between the percentage of TC cells in the CD1a+/CD3+ and the CD1a−/CD3+ populations. Data are quoted as means ± standard deviation. Differences were considered statistically significant at an alpha level of 0.05.
We thank Associate Professor Stuart Berzins and Professor Wayne Robinson for their critical review of the manuscript, and Désirée Gavhed and Magda Lourda for their valuable assistance.