Experimental design.

<p>During acquisition, patients in the conditioned group received desloratadine (US) in combination with the CS. During evocation, the drug was replaced by placebo pills. Sham-conditioned patients received the CS together with placebo pills throughout the study. During 3 subsequent days during acquisition (2–4) and evocation (17–19) patients were instructed to intake the pills together with the drink (CS) at home.</p

Sociodemographic and psychological characteristics.

<p>BDI = Becks Depression Inventory; STAI X2 = Trait anxiety form of the State-trait anxiety inventory.</p>*<p>Results of Chi² Test or univariate ANOVA.</p

Conditioned and sham-conditioned patients show reduced allergic reactions during evocation.

<p><b>A</b> After skin prick test wheal size (mm) was analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in the natural history group. <b>B</b> Symptom scores after nasal provocation were analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in natural history group. Data are presented as mean ±SEM. *p<0.05 **p<0.01 ***p<0.001, comparison against natural history group.</p

Detection of TRP-1_284–298 reactive CD4⁺ T cells in melanoma patients.

<p>Total PBMC (left panel) or CD25⁺-depleted PBMC (right panel) from five HLA-DRB1*03⁺ melanoma patients (<i>A</i>) and four HLA-DRB1*0301⁺ healthy donors (<i>B</i>) were stimulated <i>in vitro</i> with peptide TRP-1_284–298. After 25 days, PBMC were tested for their peptide reactivity by IFN-γ ELISpot assay. All determinations were performed at least in duplicates. The data are presented as mean numbers of IFN-γ spots per 10⁵ cells. Error bars show standard error of the mean.</p

Combinatorial peptide library screening allows detection of individual library peptides containing TRP-1-specific T cell epitopes.

<p><i>A</i>, Composition of the TRP-1-specific library peptide pools X1 to X8 and Y1 to Y8 used for combinatorial screening of specific T cell responses <i>ex vivo</i>. Individual peptides determined by combinatorial screening are highlighted. <i>B</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for recognition of TRP-1-specific library peptide pools. T cell responses of two control mice (Ad5.EGFP) and two Ad5.TRP-1-immunized mice are represented as individual columns in the diagram. Error bars show standard error of the mean of duplicates. Experiments were performed four times, yielding similar results.</p

TRP-1_284–298 represents a HLA-DRB1*0301-restricted CD4⁺ T cell epitope processed by human target cells.

<p>PBMC from melanoma patient VHP were stimulated once with peptide TRP-1_284–298 (10 µg/ml). After 25 days, selected CD4⁺ T cells were tested for their reactivity against different target cells by IFN-γ ELISpot assay. <i>A</i>, Recognition of T2.DR3 target cells pulsed with peptide TRP-1_284–298 and autologous CD3⁺-depleted PBMC (CD3⁻ PBMC) infected with Ad5.TRP-1 or with control virus is depicted. <i>B</i>, Reactivity of T cells against different melanoma cell lines Ma-Mel-103b (HLA-DRB1*0301⁺, TRP-1⁻), Ma-Mel-108 (HLA-DRB1*0301⁺, TRP-1⁺), Ma-Mel-153 (HLA-DRB1*0301⁻, TRP-1⁺) and <i>C</i>, against Ma-Mel-103b cells infected with Ad5.TRP-1 or control virus is presented. <i>A–C</i>, One representative out of two to three independent experiments is given. Error bars show standard error of the mean.</p

Corticosteroids Augment BRAF Inhibitor Vemurafenib Induced Lymphopenia and Risk of Infection

<div><p>We have previously demonstrated an impact of the BRAF inhibitor vemurafenib on patient lymphocyte counts. In the current study, the extent to which concomitant use of corticosteroids in BRAF inhibitor treated patients affects lymphocyte counts and predisposes to infection was investigated. A cohort of 102 patients receiving either the selective BRAF inhibitor vemurafenib or dabrafenib was analyzed. The amount of patients receiving either medication with or without systemic corticosteroids (dexamethasone) was determined and lymphocyte counts before and under therapy assessed. Additionally, the number and severity of infections occurring in these groups was analyzed. Vemurafenib treatment led to a considerable decrease in lymphocyte cell counts, with 62.3% of patients having lymphopenia. Dabrafenib treated patients only rarely demonstrated lymphopenia (12.5%). Dexamethasone co-administration further diminished lymphocyte counts. Lymphopenias were observed in 84.6% of patients receiving vemurafenib and dexamethasone. In our cohort, infections were noted in 9 patients, 4 of these were severe and 2 eventually fatal. All 9 cases with infections demonstrated lymphopenia, 8 of these had received dexamethasone and 7 of these a therapy with vemurafenib. Our findings demonstrate a significant lymphopenia in patients treated with the BRAF inhibitor vemurafenib, which is further augmented by dexamethasone and predisposes to infection. If validated in other studies, risk of infection should be considered when applying corticosteroids in combination with BRAF inhibitors, in particular vemurafenib.</p></div

Phenotypic analysis of TRP-2-specific T cell responses induced in Ad5.TRP-2-immunized HLA-DR3tg mice.

<p><i>A</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-2 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for reactivity against individual TRP-2-specific library peptides determined by combinatorial analysis. T cell responses of two control mice (Ad5.EGFP) and two TRP-2-immunized mice (Ad5.TRP-2) are represented as individual columns in the diagram. Error bars show standard error of the mean. Experiments were performed three times, yielding similar results. <i>B</i>, Selected TRP-2-derived library peptides #8, #19, #22 and #23 are indicated by amino acid (aa) positions and aa sequence. Peptides were analyzed <i>in silico</i> by the SYFPEITHI algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014137#pone.0014137-Rammensee1" target="_blank">[33]</a> for the presence of predicted HLA-DRB*0301 binding sequences (underlined). Prediction scores for HLA-DRB1*0301-restricted epitopes are listed on the right. Known HLA-DRB1*0301-restricted CD4⁺ T cell epitopes are typed in bold and the H2-K^b-restricted CTL epitope TRP-2_180–188 is written in italics. <i>C</i>, HLA-DR3tg mice received i.p. injections of 5×10⁸ pfu Ad5.TRP-2 or Ad5.EGFP (3 mice per group). Two weeks later spleen cells from infected mice were harvested and stimulated <i>in vitro</i> with the indicated peptides. After 6 days, splenocyte cultures were analyzed for the presence of peptide-reactive T cells by intracellular IFN-γ staining. Stained cells were analyzed by flow cytometry for the percentage of IFN-γ⁺ CD4⁺ T cells. Error bars show standard error of the mean of three immunized mice. Experiments were performed three times yielding similar results.</p

Phenotypic analysis of TRP-1-specific T cell responses induced in HLA-DR3tg mice upon immunization with Ad5.TRP-1.

<p><i>A</i>, Spleen cells from HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (2 mice per group) were screened <i>ex vivo</i> by IFN-γ ELISpot assay for reactivity against selected TRP-1-derived library peptides. T cell responses of two control mice (Ad5.EGFP) and two Ad5.TRP-1-immunized mice are represented as individual columns in the diagram. Error bars show standard error of the mean of duplicates. Experiments were performed three times, yielding similar results. <i>B</i>, Selected TRP-1-derived library peptides #8, #9, #36 and #47 are indicated by amino acid (aa) positions and aa sequence. Peptides were analyzed <i>in silico</i> by the SYFPEITHI algorithm for the presence of potential HLA-DRB*0301 binding sequences (underlined) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014137#pone.0014137-Rammensee1" target="_blank">[33]</a>. Prediction scores for HLA-DRB1*0301-restricted epitopes are listed on the right. The potential H2-restricted CTL epitope TRP-1_374–382 is given in italics. <i>C</i>, Spleen cells of HLA-DR3tg mice injected i.p. with 5×10⁸ pfu Ad5.TRP-1 or Ad5.EGFP (3 mice per group) were analyzed for the presence of peptide-reactive T cells by intracellular IFN-γ staining after one round of <i>in vitro</i> re-stimulation with the indicated peptides. Note: The amino acid sequence of these peptides was shifted towards the N-terminus by one residue relative to the predicted epitope sequence. Stained cells were analyzed by flow cytometry for the percentage of IFN-γ⁺ CD4⁺ T cells. Error bars show standard error of the mean of three immunized mice. Experiments were performed twice yielding similar results.</p

Overview of patients’ characteristics with infections under therapy.

<p>LP = lymphopenia; grade 1–4 according to CTCAE criteria version 4.1; BRAFi = BRAF-inhibitor; VEM = vemurafenib; DAB = dabrafenib; DEX = dexamethasone; delta lymphocytes = difference between pretherapeutic lymphocyte count and lymphocyte count under therapy; severe infection = life-threatening infection.</p><p>Overview of patients’ characteristics with infections under therapy.</p