Additional file 1: of A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains

Table S1. Age, gender and chronic illness of 20 healthy individuals. Table S2. Numbers of unique reads, reads and nucleotides in TRA reads obtained from PBMCs of 20 healthy individuals. Table S3. Numbers of unique reads, reads and nucleotides in TRB reads obtained from PBMCs of 20 healthy individuals. Table S4. Percentage of mismatched nucleotides in in-frame and out-of-frame TCR sequences. Table S5. Occurrence frequency of out-of-frame unique sequence reads in TRA and TRB. Table S6. Percentage frequency of shared TRA reads between all pairs of individuals. Table S7. Percentage frequency of shared TRB reads between all pairs of individuals. Figure S1. Correlation of gene usage of TRAV, TRAJ, TRBV and TRBJ between healthy individuals. Figure S2. Concordance correlation coefficient in TRAV, TRAJ, TRBV and TRBJ. Figure S3. Comparison of TCR usages between in-frame and out-of-frame reads sequences. Figure S4. Diversity indices of in-frame and out-of-frame TRA and TRB. Figure S5. Correlation of TCR diversity with age. Figure S6. Correlation of TCR usage from a published FACS data with AL-PCR and Multiplex PCR. (DOCX 548 kb

Accumulation of Metal-Specific T Cells in Inflamed Skin in a Novel Murine Model of Chromium-Induced Allergic Contact Dermatitis

<div><p>Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis. However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy. A novel model of Cr allergy was induced by two sensitizations of Cr plus lipopolysaccharide solution into mouse groin followed by single Cr challenge into the footpad. TCR repertoires and nucleotide sequences of complementary determining region 3 were assessed in accumulated T cells from inflamed skin. Cytokine expression profiles and T-cell phenotypes were determined by qPCR. CD3+CD4+ T cells accumulated in allergic footpads and produced increased T helper 1 (Th1) type cytokines, Fas, and Fas ligand in the footpads after challenge, suggesting CD4+ Th1 cells locally expanded in response to Cr. Accumulated T cells included natural killer (NK) T cells and Cr-specific T cells with VA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.</p></div

Characteristics of CDR3 regions of AV11-1, AV14-1, BV8-2 and BV14-1 in Cr-induced ACD mice.

<p>Amino acid sequences of CDR3 regions of VA11-1 (A), VA14-1 (B), VB8-2 (C), and VB14-1 (D) in Cr-induced ACD mouse footpads at 7 days after challenge. Identical CDR3 sequences (III, ¶, Ю, Й, ф, Ё, Ж, €) were obtained from the footpads of different mice (A–C).</p

Histopathology and IHC analyses of CD3 and F4/80 in Cr-induced ICD and ACD mouse footpads.

<p>Histopathology and IHC analyses of F4/80+ and CD3+ cells in footpad tissues. Frozen sections of footpad tissues were prepared from ICD, ACD, and control mice at 1, 3, and 7 days after challenge. Sections were stained with H&E (A-N), CD3 (O-S), and F4/80 (T-X). Scale bar = 40 µm.</p

TCR repertoire analyses in Cr-induced ACD mice.

<p>(A, B) TCRAV and TCRBV repertoires were analyzed from footpads of ACD and control mice by microplate hybridization assay. Spleens from corresponding Cr-injected mice and saline-injected mice were used as controls. Bars and error bars indicate mean ±SD. At 7 days after challenge, percentage frequencies of the expression levels of VA11-1, VA14-1, VB8-2, and VB14-1 were significantly higher in footpads of the ACD mice compared with the control mice (*<i>p</i><0.05, unpaired Student <i>t</i>-test). (C) Increase in the frequencies of VA11-1, VA14-1, VB8-2, and VB14-1 after Cr challenge. TCRAV and TCRBV repertoires were analyzed from RNA samples from footpads of Cr-induced ACD mice obtained at day 1, 3 and 7. The frequencies of VA11-1, VA14-1, VB8-2, and VB 14-1 in the Cr-induced ACD mice were significantly increased at days 1, 3, and 7 compared with control mice (*<i>p</i><0.05, unpaired Student <i>t</i>-test). All experiments were performed in triplicate.</p

Footpad swelling and mRNA expression of inflammatory cell markers in Cr-induced ICD and ACD mice.

<p>(A) Footpad swelling at various time points. ICD, ACD, and control mice were analyzed at 1, 2, 3, 7, 10, and 14 days after challenge. (B) Footpad mRNA expression levels of IL-1β, HDC, CXCL10, CXCR3, CD14, and CD3 were assessed at the indicated days. Bars and error bars indicate mean ± SD. Statistical significance was tested by unpaired Student’s t-test (*<i>p</i><0.05, **<i>p</i><0.005).</p

Expression levels of T cell cytokines, cytotoxic granules, and apoptosis-related genes in Cr-induced ACD mice.

<p>(A) The expression ratio of Th1-related/Th2-related genes. (B) The mRNA expression of cytotoxic granules (Perforin, granzyme A, and granzyme B), apoptosis related genes (Fas and Fas L), and TNF related genes (TNF-α, and TNF-R1) (*<i>p</i><0.05, unpaired Student <i>t</i>-test). Bars and error bars indicate mean ±SD.</p

Footpad swelling and expression of T-cell markers in Pd-induced allergic mice.

<p>(A) Time course of footpad swelling under different conditions of sensitization and challenge. BALB/c mice were sensitized twice with 125µl of 10 mM Pd and 10 µg/ml LPS and then challenged three times with 25 µl of 10 mM PdCl₂ (closed triangle) or PBS (open triangle) (n=7). In addition, mice were challenged three times with Pd (closed circle) or PBS (open triangle) without sensitization (n=10). Changes of footpad thickness were measured before and 1, 3, 7, 10 and 14 days after the last challenge of Pd. Data were indicated mean ± standard deviation (SD). Statistical significance was tested by unpaired Student’s <i>t</i>-test. (B) Expression levels of CD3ε mRNA were dependent upon sensitization condition. Mice were treated with different sensitization and challenge conditions (n=7). CD3ε expression was measured by qPCR 7 days after the last challenge. Bars and error bars indicate mean ± standard deviation. Statistical significance was only detected in mice administered two sensitizations and three challenges. (C) Time course of gene expression for histidine decarboxylase (HDC) and CD3ε in Pd-induced allergy mice. Expression of HDC and CD3ε were measured by qPCR at different time points after the last challenge. Data indicates mean ± SD (n=10). Statistical significance was tested by unpaired Student <i>t</i>-test (*<i>p</i><0.05, **<i>p</i><0.005).</p

T cell clonality and characteristics of CDR3 sequences in TCR clonotypes bearing AV18-1 and BV8-2.

<p>T cell clonality was analyzed by CDR3 size spectratyping and nucleotide acid sequences were determined in TCR clonotypes bearing AV18-1 and BV8-2. RNA was extracted from footpads of Pd-induced allergy mice at 7 days (n=5) and nucleotide acid sequences of CDR3 were determined in 49-52 cDNA clones per mouse. Representative profiles of CDR3 size spectratyping and deduced amino acid sequences of the CDR3 bearing AV18-1 (A) and BV8-2 (B) are shown. (A) For AV18-1, a polyclonal peak pattern (multiple peaks with Gaussian distribution) was obtained from lymph nodes of control mice. In contrast, Pd-induce allergy mice exhibited oligoclonal peak patterns with a dominant peak at the same position among five mice. Identical CDR3 sequences (†, ¶, *, Ω, Φ, ‡, Ψ) were obtained from the footpads of different mice. (B) For BV8-2, Pd-induced allergy mice showed oligoclonal peak patterns but they were different among the mice. CDR3 sequence analysis also showed multiple predominant clones presented in each mouse but they did not share CDR3 sequences among different mice. All experiments were performed in triplicate.</p

Immunohistochemical analyses of footpads from Pd-induced allergic mice.

<p>Histopathology and immunohistochemical (IHC) analyses were undertaken to identify CD3+, CD4+ and CD8+ cells in footpad tissues. Frozen sections of the footpad tissues were prepared from saline-injected mice or Pd-induced allergy mice (two sensitizations–three challenges) at 7 days after the last challenge. Sections were stained with H&E (C–J), anti-mouse CD3ε (K and L), anti-CD4 (M) and anti-CD8 mAbs (N) and visualized by DAB. Representative photomicrographs are shown. Liquefaction degeneration of basal epithelial layer and infiltration of inflammatory cells into the epithelial basal layer and dermis were observed in Pd-induced allergy mice (G-J), but not control mice (A and B). For IHC analyses, epidermotropism of CD3+ cells was present in the epithelial basal layer and dermis of the Pd-induce allergy mice (L), but not control mice (K). CD4⁺ T cells were abundant (M) while a small number of CD8+ T cells existed only around blood vessels (N). Representative IHC images were analyzed by HistoQuest software and the data was shown as scatter grams on the right.</p