Subject demographics and clinical data.

<p>* information unknown due to limited clinical history or lack of viraemic plasma sample</p><p>^{^} includes a single individual co-infected with GT1 and GT3 strains</p><p>^# predominately IDU and Tattoo</p><p>Subject demographics and clinical data.</p

Examples of likely GT-specific responses using ELISpot analysis.

<p>The two panels (A) and (B) represent GT1-epitopes that elicited responses from GT3-infected subjects. Peptides representing GT1 peptides tested in the original ELISpot screen and in a subsequent ELISpot with the alternative GT3 peptide(s). Original screen result is shown on the front row (grey) and subsequent ELISpot assay on the second row (black; mean SFU/10⁶ PBMCs from duplicates). Each panel represents data from a single subject.</p

Chronic non-GT1-infected individuals are able to mount T-cell responses to GT1 epitopes.

<p>The SFU/million PBMCs are shown for subjects with known GT1 infection and non-GT1 infection. Although subjects may have responded to more than one peptide covering an epitope, in this figure only the highest response was used.</p

Breakdown of HLA-specific T-cell targets tested and number eliciting a T-cell response based on an IFN-γ ELISpot assay.

<p>Predicted T-cell targets include published HCV T-cell targets that contain a site associated with a specific HLA allele with the same restriction. Number in bracket indicates number of subjects tested that carry the particular HLA type.</p

table_7_Urinary Peptides As a Novel Source of T Cell Allergen Epitopes.XLSX

<p>Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.</p

AIM assay detects tetanus-specific CD4⁺ T cells.

<p>(A) Representative flow cytometry plots of CD25⁺OX40⁺ upregulation by CD4⁺ T cells in naïve (CD45RA⁺CCR7⁺; lower panel) and antigen-experienced memory (excluding naïve cells; upper panel) cells after stimulation with TT-megapool [TT (MP)], Tetanus Toxoid [TT (ag)], Dengue virus-megapool [DV (MP)], or PHA as a positive control. (B) Median CD25⁺OX40⁺ expression by CD4⁺ memory T cells after 24 hours. Each dot represents one donor originally primed with DTwP vaccine and not recently boosted. Kruskal-Wallis multiple comparison test, *, <i>p</i><0.05. (C and D) Percentage IFNγ- (C) or IL-4-producing (D) CD4⁺ memory T cells in response to TT megapool, or PMA/Ion.</p

table_4_Urinary Peptides As a Novel Source of T Cell Allergen Epitopes.XLSX

<p>Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.</p

AIM Assay detects tetanus-specific CD4⁺ effector memory T cell expansion.

<p>(A) Representative flow cytometry plots of CD4⁺CD25⁺OX40⁺ T cells gated on CD45RA and CCR7 expression for central memory (CD45RA^- CCR7⁺), effector memory (CD45RA^- CCR7^-), naïve (CD45RA⁺ CCR7⁺), and TEMRA (CD45RA⁺ CCR7^-) cells after 24 hours of TT-megapool stimulation for a donor originally primed with DTwP vaccine and not boosted (no boost) or after Tdap booster vaccination (boost). The CD4⁺ cell response to TT-megapool (B) or TT whole protein (C) shows a significant increase after boosting of the effector memory population (<i>p</i> = 0.009 and <i>p</i> = 0.017 respectively) and concomitant decrease of the central memory population (<i>p</i> = 0.028 and <i>p</i> = 0.006 respectively). Two-tailed Mann-Whitney test. Median ± interquartile range is shown. Each dot represents one donor.</p

table_1_Urinary Peptides As a Novel Source of T Cell Allergen Epitopes.XLSX

<p>Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.</p

table_3_Urinary Peptides As a Novel Source of T Cell Allergen Epitopes.XLSX

<p>Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.</p