NKG2D⁺ IFN-γ⁺ CD8⁺ T Cells Are Responsible for Palladium Allergy

<div><p>Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8⁺ T cells are responsible for the disease as CD8⁺ T cell-depleted mice and β2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8⁺ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8⁺ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D⁺ CD8⁺ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.</p></div

Pd allergy is induced by hapten-specific responseand transferable through pathogenic T cell transfer.

<p>(A) Ear thickness was measured before challenge and at the indicated time point after challenge. CHS reaction was elicited in Pd plus LPS-sensitized mice (left panel) or DNFB-sensitized mice (right panel), as described. As negative controls (open circles), mice were i.p. injected with PBS (left panel) or vehicle alone (AOO) was applied (right panel) followed by challenge with Pd or DNFB. Data represent means ± SD of 10 ear samples and similar results were observed in two independent experiments. Asterisks (11) indicates statistical significance (<i>P</i><0.01) between Pd-challenged mice and DNFB-challenged mice. Lower panel shows combination of sensitization and challenge. (B) BALB/c nude mice were adoptively transferred with naïve SLN cells (open circle) or 8th Pd-SLN cells (filled square) as described. Seven days after the transfer, recipient mice (n = 5–8) were challenged with 10 nmol of Pd. The ear swelling was measured as described in(A). Data are represented as the mean ± SD. Asterisks (11) indicates statistical significance (<i>P</i><0.01). (C) Ear swelling at 24 hours after Pd challenge was compared between 1st, 4th, and 8th adoptive transfer of Pd-SLN cells. Data are represented as the mean ± SD. Asterisks (11) indicates statistical significance (<i>P</i><0.01).</p

Expression of NKG2D on T cells contribute to the development of metal allergy.

<p>(A) Ninety-six hours after Pd challenge, 8th Pd-SLN cells were prepared and analyzed for expression of CD4, CD8, and NKG2D by flow cytometry. As a control, naïve-SLN cells were analyzed. The dotted line indicates staining of isotype-matched control Ig. Similar results were obtained in eight independent experiments. (B) Fifteen hours after elicitation, 8th Pd-SLN cells were obtained and IFN-γ production by NKG2D⁺ CD8⁺ cells was analyzed. The dotted line indicates staining with isotype-matched control Ig. Data shown is one of representative results. Similar results were obtained in three independent experiments. Percentage of IFN-γ positive cells in NKG2D⁺ CD8⁺ T cells was 83.6±2.1%. (C) Three hours after i.d. administration of PdCl₂ (or PBS as a control) into ear auricles, total RNA was extracted from the ear auricles and cDNA was synthesized. Expression of the NKG2D ligand, pan-H60, transcripts were quantified by real-time PCR and normalized with GAPDH. Data represent means ± SD of 5 ear samples. Asterisks (11) indicate statistical significance (<i>P</i><0.01) between PdCl₂-injected and control samples. Similar results were obtained in three independent experiments. Ear swelling induced by Pd allergy was evaluated in NKG2D ligand transgenic (Tg) mice (D), anti-NKG2D mAb-administered mice (E), and NKG2D positive- or negative-CD8⁺ T cell transferred nude mice (F). Experimental controls in (D) were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g003" target="_blank">Fig. 3B</a>. In (E), rat IgG was administered instead of anti-NKG2D mAb as a control. In (F), naïve-SLN or Pd-SLN cells were transferred into naïve nude mice as negative or positive control, respectively. In (F), 3×10⁵ cells of each sample were adoptively transferred into naïve nude mice. Ear thickness was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g001" target="_blank">Fig. 1A</a>. Data are represented as the mean ± SD of 8–10 ear samples, and similar results were observed in two (D and F) or three (E) independent experiments. Asterisks (11) indicates statistical significance (11<i>P</i><0.01).</p

Increase in disease development by repeat adoptive transfer of lymphocytes from disease-bearing mice.

*<p>SLN cells from naïve BALB/c mice were adoptively transferred into naïve nude mice.</p

IFN-γ from CD8⁺ T cells contributes to the development of metal allergy.

<p>(A) Fifteen hours after Pd challenge, 8th Pd-SLN cells were isolated and analyzed for cytokine production (IL-4, IFN-γ, IL-10, and IL-17). Data presented were obtained from total lymphocytes. (B) IFN-γ⁺ T cell subset analysis. Cells were prepared and stained with anti-CD4, -CD8 and -IFN-γ mAbs, then analyzed by flow cytometry. Percentages of T cell subset in IFN-γ positive lymphocytes were as follows; 7.7±0.1% for CD4⁺ T cells and 72.4±1.4% for CD8⁺ T cells. (C) IFN-γ production from each subset of T cells in the 8th Pd-SLN. The cell population analyzed is indicated in each panel. (D) Comparison of IFN-γ production from CD8⁺ T cells in Pd- or DNFB-challenge of Pd plus LPS-sensitized mice. As a control, unsensitized mice were challenged with PdCl₂. The cell population analyzed is indicated in each panel. Percentages of IFN-γ positive cells in CD8⁺ T cells were as follows; 23.2±0.6% for unsensitized, Pd-challenged mice, 42.3±1.3% for Pd plus LPS-sensitized, Pd-challenged mice, and 23.7±2.0% for Pd plus LPS-sensitized, DNFB-challenged mice. (A, C and D) The dotted line indicates isotype-matched control Ig staining. (A–D) Similar results were obtained in three independent experiments. (E) Pd allergy was induced in IFN-γ-deficient mice (left panel) or perforin-deficient mice (right panel) as described. Experimental controls were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g003" target="_blank">Fig. 3B</a>. Ear swelling was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g001" target="_blank">Fig. 1A</a>. Data represent means ± SD of 10 ear samples. Asterisks (1 or 11) indicate statistical significance (0.01<1<i>P</i><0.05 or 11<i>P</i><0.01, respectively) between sensitized deficient (−/−) mice and sensitized WT mice. Similar results were observed in three (IFN-γ-deficient) or two (perforin-deficient) independent experiments.</p

CD8⁺ T cells are enriched in draining lymph nodes following the development of metal allergy.

<p>(A) Before (Naïve) and after (Induced) induction of metal allergy, the percentage of CD4⁺ or CD8⁺ T cells present in splenocytes or SLN cells from WT mice was analyzed by flow cytometry. Data shown were obtained from TCR β⁺-gated, viable cells. Percentages shown in (A) are one of representative results. Similar results were obtained in three independent experiments. (B) Cell numbers of CD4⁺ or CD8⁺ T cells in (A) were calculated as follows: absolute cell numbers x percentages of each subset (by flow cytmetry analysis). (C) The percentage of CD4⁺ or CD8⁺ T cells present in splenocytes or SLN cells from 1st and 4th adoptive transferred recipient mice was analyzed by flow cytometry. Of note, αβ⁺ T cells were not detected in the SLN or spleen from naïve nude mice. Data shown were obtained from TCR β⁺-gated, viable cells. Percentages shown in (C) are one of representative results. Similar results were obtained in three independent experiments. (D) Cell numbers of CD4⁺ or CD8⁺ T cells in (C) were calculated as follows: absolute cell numbers x percentages of each subset (by flow cytmetry analysis). Asterisks (1 or 11) indicate statistical significance (0.01<1<i>P</i><0.05 or 11<i>P</i><0.01, respectively) between 1st adoptive transferred sample and 4th sequential adoptive transferred samples.</p

CD8⁺ T cells are required for the development of Pd allergy.

<p>(A) Ear swelling induced by Pd allergy was evaluated in anti-CD4 and/or anti-CD8 mAb-administered mice. In these mice, CD4⁺ and/or CD8⁺ T cells were depleted, respectively. As a control, rat IgG was administered instead of anti-CD4 or anti-CD8 mAb. Ear thickness was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g001" target="_blank">Fig. 1A</a>. Data are presented as the mean ± SD of 10 ear samples, and similar results were observed in three independent experiments. Asterisks (11) indicate statistical significance (11<i>P</i><0.01) between anti-CD8 mAb-administered mice and control mice. (B) Pd allergy was elicited in B2m–deficient mice (left panel) or I-A^b-deficient mice (right panel) as described. As experimental controls, C57BL/6 WT mice (n = 5) were sensitized by i.p. injection with 2.5 µmol Pd and 2.5 µg LPS (sensitized) or PBS (unsensitized). Ear thickness was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086810#pone-0086810-g001" target="_blank">Fig. 1A</a>. Data represent the mean ± SD of 8–10 ear samples, and similar results were observed in three (B2m-deficient) or two (I-A^b-deficient) independent experiments. Asterisks (11) indicates statistical significance (<i>P</i><0.01) between sensitized B2m-deficient (−/−) mice and sensitized WT mice or between sensitized I-A^b-deficient (−/−) mice and unsensitized WT mice.</p

Pathogenic T cells are present in a mouse model of metal allergy.

<p>Immunohistochemical analysis of the ear auricles at 24/c mice (A) and 8th Pd-SLN cells-transferred nude mice (C). Ear sections were stained by anti-CD3ε mAb and visualized by DAB. The sections were counterstained with hematoxylin. The scale bars indicate 100 µm. (B) Quantification of CD3ε-positive T cells shown in panel A for WT BALB/c mice and (D) in panel C for 8th Pd-SLN cells-transferred nude mice. Representative images from each group were analyzed and presented as scattergrams using HistoQuest software. X- and Y-axis are intensity of hematoxylin and DAB, respectively. Numbers shown in the scattergrams indicates percentage of CD3ε-positive T cells. Similar results were obtained in three independent experiments. (E) CD3ε-positive T cells infiltrated in ear auricles were statistically analyzed using results from image cytometry. Data represent means ± SD of 6 ear samples. Asterisks (11) indicate statistical significance (<i>P</i><0.01). Similar results were obtained in three independent experiments.</p