Figure 3.
Figure 3. Comparative HLA class II ligandome profiling and identification of CML-associated antigens. (A) Saturation analysis of HLA class II peptide source proteins of the CML patient cohort. Number of unique HLA peptide source protein identifications as a function of cumulative HLA ligandome analysis of CML samples (n = 20). Exponential regression allowed for the robust calculation (R2 = 0.9997) of the maximum attainable number of different source protein identifications (dotted line). The dashed red line depicts the source proteome coverage achieved in our CML patient cohort. (B) Overlap analysis of HLA class II peptides of primary CML samples (n = 20), CMLMR samples (n = 15), and hematological benign samples (n = 88), including PBMCs (n = 38), granulocytes (n = 18), CD19+ B cells (n = 9), bone marrow (n = 15), and CD34+ HPCs (n = 8). Comparative profiling of HLA class II peptides (C) and HLA class II source proteins (D) based on the frequency of HLA-restricted presentation in CML and hematological benign ligandomes. The frequencies of positive immunopeptidomes for the respective HLA peptides or source proteins (x-axis) are indicated on the y-axis. To allow for better readability, HLA peptides or source proteins identified on <5% of the samples within the respective cohort are not depicted. The boxes on the left and their magnifications highlight the subset of CML-associated antigens showing CML-exclusive high frequent presentation in CML samples. (E) Hotspot analysis of the protein RB27A by peptide clustering. Identified peptides were mapped to their amino acid positions within the source protein. Representation frequencies of amino acid counts within each cohort for the respective amino acid position (x-axis) were calculated and are indicated on the y-axis. The box on the left and its magnification highlight the identified hotspot with the respective amino acids on the x-axis. (F) Tissue-specific HLA class II peptide length distribution (number of amino acids) of all identified peptides on primary CML samples (n = 20), granulocytes (n = 18), PBMCs (n = 38), CD19+ B cells (n = 9), bone marrow (n = 15), and CD34+ HPCs (n = 8). aa, amino acids; IDs, identifications; npep, number of peptides.

Comparative HLA class II ligandome profiling and identification of CML-associated antigens. (A) Saturation analysis of HLA class II peptide source proteins of the CML patient cohort. Number of unique HLA peptide source protein identifications as a function of cumulative HLA ligandome analysis of CML samples (n = 20). Exponential regression allowed for the robust calculation (R2 = 0.9997) of the maximum attainable number of different source protein identifications (dotted line). The dashed red line depicts the source proteome coverage achieved in our CML patient cohort. (B) Overlap analysis of HLA class II peptides of primary CML samples (n = 20), CMLMR samples (n = 15), and hematological benign samples (n = 88), including PBMCs (n = 38), granulocytes (n = 18), CD19+ B cells (n = 9), bone marrow (n = 15), and CD34+ HPCs (n = 8). Comparative profiling of HLA class II peptides (C) and HLA class II source proteins (D) based on the frequency of HLA-restricted presentation in CML and hematological benign ligandomes. The frequencies of positive immunopeptidomes for the respective HLA peptides or source proteins (x-axis) are indicated on the y-axis. To allow for better readability, HLA peptides or source proteins identified on <5% of the samples within the respective cohort are not depicted. The boxes on the left and their magnifications highlight the subset of CML-associated antigens showing CML-exclusive high frequent presentation in CML samples. (E) Hotspot analysis of the protein RB27A by peptide clustering. Identified peptides were mapped to their amino acid positions within the source protein. Representation frequencies of amino acid counts within each cohort for the respective amino acid position (x-axis) were calculated and are indicated on the y-axis. The box on the left and its magnification highlight the identified hotspot with the respective amino acids on the x-axis. (F) Tissue-specific HLA class II peptide length distribution (number of amino acids) of all identified peptides on primary CML samples (n = 20), granulocytes (n = 18), PBMCs (n = 38), CD19+ B cells (n = 9), bone marrow (n = 15), and CD34+ HPCs (n = 8). aa, amino acids; IDs, identifications; npep, number of peptides.

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