Additional file 3: of Human leukocyte antigen class 1 genotype distribution and analysis in persons with active tuberculosis and household contacts from Central Uganda

Table S3. Comparison of allelic frequencies between active TB patients and household contacts in the study. Table comparing the allele frequencies for HLA-A, HLA-B and HLA-C alleles between active TB patients and household contacts in the study. (XLS 49 kb

Additional file 2: of Human leukocyte antigen class 1 genotype distribution and analysis in persons with active tuberculosis and household contacts from Central Uganda

Table S2. Total allelic frequencies of the study subjects from Kawempe, Kampala. Table of the total allele frequencies for HLA-A, HLA-B and HLA-C alleles of both active TB patients and household contacts in the study. (XLS 28 kb

Alignment of the major histocompatibility complex (MHC) class I-binding epitopes with the amino acid sequence of Rv1886c.

<p>Epitopes identified for HLA-A*02:01 are shown in light blue, for A*24:02 in dark blue, for A*30:01 in dark green, peptides for A*30:02 in light green, for A*68:01 in yellow, for B*07:02 in orange, for B*58:01 in brown and for C*07:01 in red. Alignments of MHC class II restricted epitopes (HLA-DR1 – dark grey, DR2 – medium grey and DR4 – light grey) that have previously been reported in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058309#pone.0058309-Gaseitsiwe2" target="_blank">[32]</a> are included as well. The signal sequence of Rv1886c is shown in red and the rest of the protein in black.</p

Heat-map comparing epitope-MHC affinity, epitope-MHC dissociation rate and antigen-specific CD8+ T-cell recognition.

<p>The affinity was divided into the following groups <10 nM (dark red), 10–70 nM (light red), 70–500 nM (orange), 500–1000 nM (dark yellow) and >1 µM (light yellow). The dissociation-rate (off-rate) were divided according to >10 h (dark red), 6–10 h (light red), 3–6 h (orange), 1.5–3 h (dark yellow) and <1.5 h (light yellow). Finally, the T-cell recognition was grouped as follows; >1% antigen-specific CD8+ T-cells recognizing the epitope (dark red), 0.6–1% (light red), 0.4–0.6% (orange), 0.2–0.4% (dark yellow) and <0.2% (light yellow).</p

MHC class I binding, affinity and off-rate data for the variant peptide-epitopes derived from Rv0288 (TB10.4).

<p>?Amino acid substitutions within an epitope are marked in bold italic text. <i>??</i>Positive binding epitopes are marked in bold. Binding is reported as percent relative the binding of a positive control peptide, affinity is reported as an ED50 value (M), and off-rate is reported as a <i>t</i>_1/2 value (h), as described in material and methods. MHC class I-peptide complexes were constructed for the epitopes: A02-Rv0288_AMLGHAGDM, A02-Rv0288_{AML<b><i>D</i></b>HAGDM}, A02-Rv0288_MLGHAGDMA, A02-Rv0288_{ML<b><i>D</i></b>HAGDMA}, A24-Rv0288_MYNYPAMLG and A24-Rv0288_{MYNYP<b><i>T</i></b>ML<b><i>D</i></b>}. *Binding, affinity and off-rate have previously been reported in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058309#pone.0058309-AxelssonRobertson2" target="_blank">[24]</a>. **Binding, affinity and off-rate have previously been reported in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058309#pone.0058309-AxelssonRobertson1" target="_blank">[17]</a>.</p

MHC class I binding, affinity and off-rate data for peptide-epitopes derived from Rv0288 (TB10.4).

*<p>Promiscuous epitopes are marked in italic. <i>**</i>Positive binding epitopes are marked in bold. Binding is reported as percent relative the binding of a positive control peptide, affinity is reported as an ED50 value (M), and off-rate is reported as a <i>t</i>_1/2 value (h), as described in material and methods. MHC class I-peptide complexes were constructed for the epitopes: A68-Rv0288_HAMSSTHEA and A68-Rv0288_ANTMAMMAR.</p

MHC class I binding, affinity and off-rate data for peptide-epitopes derived from Rv3875 (ESAT-6).

*<p>Promiscuous epitopes are marked in bold or italic (italic – binding >1 allele, bold – binding to >2 alleles). <i>**</i>Positive binding epitopes are marked in bold. Binding is reported as percent relative the binding of a positive control peptide, affinity is reported as an ED50 value (M), and off-rate is reported as a <i>t</i>_1/2 value (h), as described in material and methods. Multimer MHC-peptide complexes were constructed for the epitopes: A02-Rv3875_LLDEGKQSL, A02-Rv3875_AMASTEGNV, A24-Rv3875_AYQGVQQKW and A3002-Rv3875_AMASTEGNV.</p

Percentage multimer positive CD8+ T-cells divided into different compartments.

<p>(A) Percentage antigen-specific recognition of the 45 individual multimers used in this study, each dot represents the staining in PBMCs from one patient and the different restricting alleles are shown in different colors (HLA-A*02:01 – black, A*24:02 – red, A*30:01 – orange, A*30:02 – green, A*68:01 – blue, B*07:02 – purple, B*58:01 – pink and C*07:01 – turquoise), each individual multimer within an allele is shown by different shapes of dots. (B) Average detection in all patients of antigen-specific T-cells divided per antigenic protein and per restricting allele (A*02:01 – black squares, A*24:02 – red triangles, A*30:01/A*30:02 – green triangles, A*68:01 – blue diamonds, B*07:02 – purple circles, B*58:01 – pink crosses and C*07:01 – turquoise crosses). (C) Individual detection of antigen-specific T-cells specific for the ‘super-epitope’ (QI)MYNYPAM(LG), each dot represents the staining in an individual patient, different colors represents the different restricting allele (A*02:01 – black, A*24:02 – red, A*30:01 – orange and A*30:02 – green).</p

Frequencies of total CD8+ T-cells as well as different compartments of antigen-specific CD8+ T-cells expressing differentiation and maturation markers.

<p>(A) Total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black) belonging to the different phenotypic compartments; naïve/precursor (CD45RA+CCR7+) (circles), central memory (CD45RA−CCR7+) (triangles), effector memory (CD45RA−CCR7−) (diamonds) and terminally differentiated cells (CD45RA+CCR7−) (squares). (B) Total CD8+ and antigen-specific T-cells belonging to the terminally differentiated compartment (CD45RA+CCR7−) and (C) effector memory compartment (CD45RA−CCR7−) divided per restricting MHC class I allele (Total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). (D) Expression of the degranulation marker CD107a in total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black). (E) Total CD8+ and antigen-specific T-cells expressing CD107a divided per restricting MHC class I allele (total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). (F) Expression of the survival marker CD127 in total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black). (G) Total CD8+ and antigen-specific T-cells expressing CD127 divided per restricting MHC class I allele (total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). Each dot represents an individual multimer in one individual TB patient. Student's two-sided t-test was performed and significant values were calculated based on the following p-values: *p<0.05, **p<0.01, ***p<0.001.</p

Antigen-specific recognition of the immunogenic epitopes from the H37Rv reference strain as well as the same epitopes containing naturally occurring amino acid substitutions.

<p>(A) Percent antigen specific CD8+ T-cell recognizing the A*02:01 restricted epitopes (AML(G/<b>D</b>)HAGDM) (circles) and (ML(G/<b>D</b>)HAGDMA) (squares) and the A*24:02 restricted epitope (MYNYP(A/<b>T</b>)ML(G/<b>D</b>) (diamonds), wild-type epitopes (closed symbols) and variant epitopes (open symbols). (B) Representative figures of multimer staining regarding the A*24:02 restricted epitope (MYNYP(A/<b>T</b>)ML(G/<b>D</b>)). The x-axis shows the staining of the wt epitope and the y-axis staining the variant epitopes. Numbers of events for the wt/mut epitopes regarding patient 6088 were +– 162, –+ 181 and ++ 2. A similar T-cell distribution could be detected regarding patient 5905 (+– 17, –+ 21 and ++ 1).</p