A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Abstract

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4(+) T cells from H5N1-immunized HLA-DR1-transgenic mice and LSA-NRC-vaccinated HLA-DR1-positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens.

Figure 1. Sensitivity to cathepsins and identification of rHA1-derived peptides by Mass Spectrometry

(a) Sensitivity to CatB and CatH. HA_306-318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide–DR1αβ complex. (b) Sensitivity of DM and DR1 to Cathepsins. Left: Conventional SDS-PAGE of empty DR1 (lanes 1-2), pre-formed HA_306-318–DR1 (lanes 3-4), and DM (lanes 5-6) incubated in the presence or absence of 200 nM CatB and CatH, rHA1 (lanes 8-9) served as a positive control for digestion by CatB and CatH. Right: Gentle SDS-PAGE (12% Acrylamide, silver-stained) of empty DR1 (lane 3), HA_306-318–DR1 (lane 5), and DM (lane 7) treated with 100 nM CatS. (c-f) Mass spectra of rHA1 derived peptides eluted from DR1 are shown between m/z 1950-2550 Da. b is background for a, and d is background for c. Mass species in bold represent rHA1 fragments eluted from DR1 containing HA_306-318 epitope (Supplementary Fig. 3–4), underlined represent other rHA1-derived peptides, in grey are the background peaks. Experiments were repeated more than three times. (g, h) In vitro proliferation of rHA1-immunized cells as measured by [³H] thymidine incorporation (two individual mice out of five tested).

Figure 1. Sensitivity to cathepsins and identification of rHA1-derived peptides by Mass Spectrometry

(a) Sensitivity to CatB and CatH. HA_306-318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide–DR1αβ complex. (b) Sensitivity of DM and DR1 to Cathepsins. Left: Conventional SDS-PAGE of empty DR1 (lanes 1-2), pre-formed HA_306-318–DR1 (lanes 3-4), and DM (lanes 5-6) incubated in the presence or absence of 200 nM CatB and CatH, rHA1 (lanes 8-9) served as a positive control for digestion by CatB and CatH. Right: Gentle SDS-PAGE (12% Acrylamide, silver-stained) of empty DR1 (lane 3), HA_306-318–DR1 (lane 5), and DM (lane 7) treated with 100 nM CatS. (c-f) Mass spectra of rHA1 derived peptides eluted from DR1 are shown between m/z 1950-2550 Da. b is background for a, and d is background for c. Mass species in bold represent rHA1 fragments eluted from DR1 containing HA_306-318 epitope (Supplementary Fig. 3–4), underlined represent other rHA1-derived peptides, in grey are the background peaks. Experiments were repeated more than three times. (g, h) In vitro proliferation of rHA1-immunized cells as measured by [³H] thymidine incorporation (two individual mice out of five tested).

Figure 1. Sensitivity to cathepsins and identification of rHA1-derived peptides by Mass Spectrometry

(a) Sensitivity to CatB and CatH. HA_306-318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide–DR1αβ complex. (b) Sensitivity of DM and DR1 to Cathepsins. Left: Conventional SDS-PAGE of empty DR1 (lanes 1-2), pre-formed HA_306-318–DR1 (lanes 3-4), and DM (lanes 5-6) incubated in the presence or absence of 200 nM CatB and CatH, rHA1 (lanes 8-9) served as a positive control for digestion by CatB and CatH. Right: Gentle SDS-PAGE (12% Acrylamide, silver-stained) of empty DR1 (lane 3), HA_306-318–DR1 (lane 5), and DM (lane 7) treated with 100 nM CatS. (c-f) Mass spectra of rHA1 derived peptides eluted from DR1 are shown between m/z 1950-2550 Da. b is background for a, and d is background for c. Mass species in bold represent rHA1 fragments eluted from DR1 containing HA_306-318 epitope (Supplementary Fig. 3–4), underlined represent other rHA1-derived peptides, in grey are the background peaks. Experiments were repeated more than three times. (g, h) In vitro proliferation of rHA1-immunized cells as measured by [³H] thymidine incorporation (two individual mice out of five tested).

Figure 1. Sensitivity to cathepsins and identification of rHA1-derived peptides by Mass Spectrometry

(a) Sensitivity to CatB and CatH. HA_306-318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide–DR1αβ complex. (b) Sensitivity of DM and DR1 to Cathepsins. Left: Conventional SDS-PAGE of empty DR1 (lanes 1-2), pre-formed HA_306-318–DR1 (lanes 3-4), and DM (lanes 5-6) incubated in the presence or absence of 200 nM CatB and CatH, rHA1 (lanes 8-9) served as a positive control for digestion by CatB and CatH. Right: Gentle SDS-PAGE (12% Acrylamide, silver-stained) of empty DR1 (lane 3), HA_306-318–DR1 (lane 5), and DM (lane 7) treated with 100 nM CatS. (c-f) Mass spectra of rHA1 derived peptides eluted from DR1 are shown between m/z 1950-2550 Da. b is background for a, and d is background for c. Mass species in bold represent rHA1 fragments eluted from DR1 containing HA_306-318 epitope (Supplementary Fig. 3–4), underlined represent other rHA1-derived peptides, in grey are the background peaks. Experiments were repeated more than three times. (g, h) In vitro proliferation of rHA1-immunized cells as measured by [³H] thymidine incorporation (two individual mice out of five tested).

Figure 1. Sensitivity to cathepsins and identification of rHA1-derived peptides by Mass Spectrometry

(a) Sensitivity to CatB and CatH. HA_306-318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide–DR1αβ complex. (b) Sensitivity of DM and DR1 to Cathepsins. Left: Conventional SDS-PAGE of empty DR1 (lanes 1-2), pre-formed HA_306-318–DR1 (lanes 3-4), and DM (lanes 5-6) incubated in the presence or absence of 200 nM CatB and CatH, rHA1 (lanes 8-9) served as a positive control for digestion by CatB and CatH. Right: Gentle SDS-PAGE (12% Acrylamide, silver-stained) of empty DR1 (lane 3), HA_306-318–DR1 (lane 5), and DM (lane 7) treated with 100 nM CatS. (c-f) Mass spectra of rHA1 derived peptides eluted from DR1 are shown between m/z 1950-2550 Da. b is background for a, and d is background for c. Mass species in bold represent rHA1 fragments eluted from DR1 containing HA_306-318 epitope (Supplementary Fig. 3–4), underlined represent other rHA1-derived peptides, in grey are the background peaks. Experiments were repeated more than three times. (g, h) In vitro proliferation of rHA1-immunized cells as measured by [³H] thymidine incorporation (two individual mice out of five tested).

Figure 2. Identification of type II collagen-derived peptides eluted from DR1

(a, b) Mass spectra of CII derived-peptides eluted from DR1. (a) DR1 was incubated with the following components: Matrix Metalloproteinase 9 (MMP9)-fragmented bovine type II collagen (CII), DM, CatB, and CatH (b) the negative control reactions carried out without including MMP9-fragmented CII. (CII _273-305)_4OH represents the sequence of collagen peptide containing four hydroxylation of Pro or Lys (QTGEP_OHGIAGFKGEQGPK_OHGEP_OHGPAGVQGAP_OHGPAG). The experiment was repeated more than three times. (c) Expanded spectrum of (a) between m/z 2800 and 3500 Da. Peptide modification: Hexose: +162 Da, Hydroxylation: +16 Da, Na⁺ adduct: +22Da (d) Proliferation of cells immunized with native CII protein in CFA incubated with titrating doses of CII_280-294 (AGFKGEQGPKGEPGP), CLIP_89-105, and heat denatured CII protein in vitro as measured by [³H] thymidine incorporation (one out of three representative individual mice tested).

Figure 2. Identification of type II collagen-derived peptides eluted from DR1

(a, b) Mass spectra of CII derived-peptides eluted from DR1. (a) DR1 was incubated with the following components: Matrix Metalloproteinase 9 (MMP9)-fragmented bovine type II collagen (CII), DM, CatB, and CatH (b) the negative control reactions carried out without including MMP9-fragmented CII. (CII _273-305)_4OH represents the sequence of collagen peptide containing four hydroxylation of Pro or Lys (QTGEP_OHGIAGFKGEQGPK_OHGEP_OHGPAGVQGAP_OHGPAG). The experiment was repeated more than three times. (c) Expanded spectrum of (a) between m/z 2800 and 3500 Da. Peptide modification: Hexose: +162 Da, Hydroxylation: +16 Da, Na⁺ adduct: +22Da (d) Proliferation of cells immunized with native CII protein in CFA incubated with titrating doses of CII_280-294 (AGFKGEQGPKGEPGP), CLIP_89-105, and heat denatured CII protein in vitro as measured by [³H] thymidine incorporation (one out of three representative individual mice tested).

Figure 2. Identification of type II collagen-derived peptides eluted from DR1

(a, b) Mass spectra of CII derived-peptides eluted from DR1. (a) DR1 was incubated with the following components: Matrix Metalloproteinase 9 (MMP9)-fragmented bovine type II collagen (CII), DM, CatB, and CatH (b) the negative control reactions carried out without including MMP9-fragmented CII. (CII _273-305)_4OH represents the sequence of collagen peptide containing four hydroxylation of Pro or Lys (QTGEP_OHGIAGFKGEQGPK_OHGEP_OHGPAGVQGAP_OHGPAG). The experiment was repeated more than three times. (c) Expanded spectrum of (a) between m/z 2800 and 3500 Da. Peptide modification: Hexose: +162 Da, Hydroxylation: +16 Da, Na⁺ adduct: +22Da (d) Proliferation of cells immunized with native CII protein in CFA incubated with titrating doses of CII_280-294 (AGFKGEQGPKGEPGP), CLIP_89-105, and heat denatured CII protein in vitro as measured by [³H] thymidine incorporation (one out of three representative individual mice tested).

Figure 3. Identification of DR1 restricted epitope of H5N1 rHA1 by the cell-free antigen processing system

(a–c) Mass spectra of H5N1 rHA1 (A/Vietnam/1203/2004 H5N1 strain, Genebank No. AY651334) derived peptides eluted from DR1. DR1 and DM were incubated with (a) Native H5N1 rHA1, (b) Heat denatured H5N1 rHA1, or (c) no protein antigens, followed by addition of CatB, CatH and CatS, and then immunoprecipitation and peptide elution. Mass species that are underlined represent H5N1-derived HA_259-274, and HA_259-278 peptides eluted from DR1. Background mass species are labeled in grey. (d) MS/MS spectra of HA_259-274 at m/z 1814.82 Da. These spectra represent one of four repeated experiments.

Figure 3. Identification of DR1 restricted epitope of H5N1 rHA1 by the cell-free antigen processing system

(a–c) Mass spectra of H5N1 rHA1 (A/Vietnam/1203/2004 H5N1 strain, Genebank No. AY651334) derived peptides eluted from DR1. DR1 and DM were incubated with (a) Native H5N1 rHA1, (b) Heat denatured H5N1 rHA1, or (c) no protein antigens, followed by addition of CatB, CatH and CatS, and then immunoprecipitation and peptide elution. Mass species that are underlined represent H5N1-derived HA_259-274, and HA_259-278 peptides eluted from DR1. Background mass species are labeled in grey. (d) MS/MS spectra of HA_259-274 at m/z 1814.82 Da. These spectra represent one of four repeated experiments.

Figure 4. Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the minimalist antigen processing system

(a) T cell proliferation of DR1 Tg mice immunized with native H5N1 rHA1 protein in CFA stimulated with HA_259–274 (SNGNFIAPEYAYKIVK), CLIP_89-105, or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (representative of one mouse out of three individual mice tested). (b–c) IL-2 and IFN-γ ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in a. Cell culture supernatants were removed after 24 h, 48 h, and 72 h culture. (d) Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein immunized mice on day 8 and directly stained with either HA_259-274/DR1 or CLIP/DR1 tetramers. Cells from another three mice immunized with PBS/CFA as control were stained with HA_259-274/DR1 tetramers. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers (6 μg ml⁻¹, 3 μg ml⁻¹, or 1.5 μg ml⁻¹) followed by staining with monoclonal antibodies for CD4-FITC, CD8-eFlore605, CD44-Alexa700, F4/80-APC, and B220-APC for additional 20 min at 4 °C. (e) Cells from (d) were expanded with 0.07 μM H5N1 rHA1 for additional 7 d in vitro. Protein stimulated cells were stained with the tetramers (1.5 μg ml⁻¹) and antibodies as described in (d). Numbers represent the percentage of tetramer-positive events among parent CD4⁺ (CD4⁺CD44⁺CD8⁻F4/80⁻ B220⁻7AAD⁻) cells.

Figure 4. Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the minimalist antigen processing system

(a) T cell proliferation of DR1 Tg mice immunized with native H5N1 rHA1 protein in CFA stimulated with HA_259–274 (SNGNFIAPEYAYKIVK), CLIP_89-105, or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (representative of one mouse out of three individual mice tested). (b–c) IL-2 and IFN-γ ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in a. Cell culture supernatants were removed after 24 h, 48 h, and 72 h culture. (d) Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein immunized mice on day 8 and directly stained with either HA_259-274/DR1 or CLIP/DR1 tetramers. Cells from another three mice immunized with PBS/CFA as control were stained with HA_259-274/DR1 tetramers. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers (6 μg ml⁻¹, 3 μg ml⁻¹, or 1.5 μg ml⁻¹) followed by staining with monoclonal antibodies for CD4-FITC, CD8-eFlore605, CD44-Alexa700, F4/80-APC, and B220-APC for additional 20 min at 4 °C. (e) Cells from (d) were expanded with 0.07 μM H5N1 rHA1 for additional 7 d in vitro. Protein stimulated cells were stained with the tetramers (1.5 μg ml⁻¹) and antibodies as described in (d). Numbers represent the percentage of tetramer-positive events among parent CD4⁺ (CD4⁺CD44⁺CD8⁻F4/80⁻ B220⁻7AAD⁻) cells.

Figure 4. Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the minimalist antigen processing system

(a) T cell proliferation of DR1 Tg mice immunized with native H5N1 rHA1 protein in CFA stimulated with HA_259–274 (SNGNFIAPEYAYKIVK), CLIP_89-105, or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (representative of one mouse out of three individual mice tested). (b–c) IL-2 and IFN-γ ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in a. Cell culture supernatants were removed after 24 h, 48 h, and 72 h culture. (d) Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein immunized mice on day 8 and directly stained with either HA_259-274/DR1 or CLIP/DR1 tetramers. Cells from another three mice immunized with PBS/CFA as control were stained with HA_259-274/DR1 tetramers. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers (6 μg ml⁻¹, 3 μg ml⁻¹, or 1.5 μg ml⁻¹) followed by staining with monoclonal antibodies for CD4-FITC, CD8-eFlore605, CD44-Alexa700, F4/80-APC, and B220-APC for additional 20 min at 4 °C. (e) Cells from (d) were expanded with 0.07 μM H5N1 rHA1 for additional 7 d in vitro. Protein stimulated cells were stained with the tetramers (1.5 μg ml⁻¹) and antibodies as described in (d). Numbers represent the percentage of tetramer-positive events among parent CD4⁺ (CD4⁺CD44⁺CD8⁻F4/80⁻ B220⁻7AAD⁻) cells.

Figure 4. Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the minimalist antigen processing system

(a) T cell proliferation of DR1 Tg mice immunized with native H5N1 rHA1 protein in CFA stimulated with HA_259–274 (SNGNFIAPEYAYKIVK), CLIP_89-105, or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (representative of one mouse out of three individual mice tested). (b–c) IL-2 and IFN-γ ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in a. Cell culture supernatants were removed after 24 h, 48 h, and 72 h culture. (d) Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein immunized mice on day 8 and directly stained with either HA_259-274/DR1 or CLIP/DR1 tetramers. Cells from another three mice immunized with PBS/CFA as control were stained with HA_259-274/DR1 tetramers. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers (6 μg ml⁻¹, 3 μg ml⁻¹, or 1.5 μg ml⁻¹) followed by staining with monoclonal antibodies for CD4-FITC, CD8-eFlore605, CD44-Alexa700, F4/80-APC, and B220-APC for additional 20 min at 4 °C. (e) Cells from (d) were expanded with 0.07 μM H5N1 rHA1 for additional 7 d in vitro. Protein stimulated cells were stained with the tetramers (1.5 μg ml⁻¹) and antibodies as described in (d). Numbers represent the percentage of tetramer-positive events among parent CD4⁺ (CD4⁺CD44⁺CD8⁻F4/80⁻ B220⁻7AAD⁻) cells.

Figure 4. Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the minimalist antigen processing system

(a) T cell proliferation of DR1 Tg mice immunized with native H5N1 rHA1 protein in CFA stimulated with HA_259–274 (SNGNFIAPEYAYKIVK), CLIP_89-105, or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (representative of one mouse out of three individual mice tested). (b–c) IL-2 and IFN-γ ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in a. Cell culture supernatants were removed after 24 h, 48 h, and 72 h culture. (d) Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein immunized mice on day 8 and directly stained with either HA_259-274/DR1 or CLIP/DR1 tetramers. Cells from another three mice immunized with PBS/CFA as control were stained with HA_259-274/DR1 tetramers. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers (6 μg ml⁻¹, 3 μg ml⁻¹, or 1.5 μg ml⁻¹) followed by staining with monoclonal antibodies for CD4-FITC, CD8-eFlore605, CD44-Alexa700, F4/80-APC, and B220-APC for additional 20 min at 4 °C. (e) Cells from (d) were expanded with 0.07 μM H5N1 rHA1 for additional 7 d in vitro. Protein stimulated cells were stained with the tetramers (1.5 μg ml⁻¹) and antibodies as described in (d). Numbers represent the percentage of tetramer-positive events among parent CD4⁺ (CD4⁺CD44⁺CD8⁻F4/80⁻ B220⁻7AAD⁻) cells.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.

Figure 5. Identification of DR1 restricted epitope of LSA-NRC and biological validation

(a, b) Mass spectra of LSA-NRC derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC-derived peptides captured by DR1 are underlined. (a) Expanded spectrum between m/z 1700-2430 Da and (b) Spectrum between m/z 3430-3515 Da. The experiments were repeated more than five times. (c) MS/MS spectrum of m/z ∼2278 Da that was identified. (d, e) LSA-NRC immunized cells were incubated with LSA_323-337 (VQYDNFQDEENIGIY), LSA_429-443 (VDELSEDITKYFMKL), LSA_436-449 (ITKYFMKLGGSGSP), CLIP_89–105, and LSA-NRC in vitro. (d) T cell proliferation was measured by [³H] thymidine incorportation (one out of three individual mice tested). (e) Cell culture supernatant from another two mice under similar experimental set up as in (d) were removed 24 h, or 48 h later, and the amount of IL-2 produced was determined by ELISA. (f) PBMC from eight volunteers were obtained pre- or post immunization with LSA-NRC Liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control, different concentrations of LSA_436-449, and CLIP_89-105 for 3 d and were assayed by [³H] thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure.