Status of CD8⁺/IFN-γ⁺ T cell response of recovered HLA-A2⁺ individuals compared to HLA-A2⁻ individuals.

<p>6 out of 19 (31.6%) and 2 out of 15 (13.3%) HLA-A2⁺ recovered individuals responded above cutoff value (horizontal bar in each plot defined as mean + 2SD CD8⁺/IFN-γ⁺ response in HLA-A2⁻ controls) against peptide pools II and IV, respectively. Fischer's exact probability test showed that the response in peptide pool II is statistically significant.</p

The response of individual volunteers to <i>L. major</i> lysate.

<p>Freezed/thawed antigens of <i>L. major</i> were used to stimulate the PBMCs in culture as previous disease indicator. The responses of <i>L. major</i> recovered individuals were potentially detected at CD4 level. Each point represents response of each individual. Horizontal bars represent the median value of CD4⁺/IFN γ⁺ T cells in the related group. Statistical analysis shows a significant difference between R.A2⁺ (HLA-A2⁺ recovered individuals) and R.A2⁻ (HLA-A2⁻ recovered individuals) groups with H.A2⁺ individuals (HLA-A2⁺ healthy donors) with <i>p</i> value<0.05.</p

HLA-A2 super-type binding possibility of selected peptides predicted by NetMHCPan1.1.

<p>*strong binder (Strong binders have an IC₅₀ less than 50 nM).</p><p>**weak binder (Weak binders have an IC₅₀ more than 50 nM and less than 500 nM.</p

<i>L. major</i> specific proteins used as candidate antigens for 9-mer epitope screening.

a<p>The full sequences of the proteins were extracted from GeneDB data of <i>L. major</i> strain MHOM/IL/80/Fredlin.</p

HLA-A2 screening by one step PCR.

<p>One step PCR-SSP method was used to screen for HLA-A2 positives among all samples included (recovered individuals and healthy donors). Lane 1 shows 100 bp DNA ladder marker, lane 4 shows the PCR reaction of T2 cells as positive control, lane 3, 5 and 6 is related to negative samples and lane 2 is related to a positive sample.</p

Enumeration of peptide-specific IFN-γ producing T cells stimulated against peptide pool II by ELISpot assay.

<p>ELISpot analysis (upper row) of a negative responder (HLA-A2⁻ recovered individual) (A) and a positive responder (HLA-A2⁺ recovered individual) (B) against peptide pool II stimulation is depicted compared to flow cytometric results (lower row) of the same samples. PMA/Ion stimulation (C) and culture medium only (D) are used as positive and negative controls, respectively. (a and c are related to un-stimulated controls of each sample, b and d are related to stimulated samples with peptide pool II). Dot plots show CD8 vs. IFN-γ staining. Upper right squares in each plot define the CD8⁺/IFN-γ⁺ region. Numbers represent the percentage of IFN-γ producing CD3⁺/CD8⁺ T cells in the lymphocyte gate.</p

Sequence Specific Primers used in PCR reactions for typing HLA-A2 and sub typing HLA-A*0201 allele.

<p>Sequence Specific Primers used in PCR reactions for typing HLA-A2 and sub typing HLA-A*0201 allele.</p

Characteristics of <i>in silico</i> predicted <i>L. major</i> specific CD8⁺ T cell 9-mer peptides restricted to HLA-A*0201 allele.

a<p>Amino acid position as in the protein sequence.</p>b<p>Threshold set on 5% (percent of whole protein peptides that should be tested).</p>c<p>Specific binding threshold set on 2% (percent of whole protein peptides that should be tested).</p>d<p>Proteasomal cleavage.</p>e<p>Cut off score set on 0.5 (Threshold of binding).</p>f<p>Threshold for epitope selection set on >0.75 (Threshold of binding).</p>g<p>Promiscuous epitope predicted as moderate or high binder.</p

Detection of secreted IFN-γ from PBMCs stimulated against peptide pool II and IV by ELISA in culture supernatants.

<p>IFN-γ production was measured in culture supernatants of HLA-A2⁺ recovered individuals (R.A2⁺), HLA-A2⁻ recovered individuals (R.A2⁻), and HLA-A2⁺ healthy donors (H.A2⁺) stimulated against peptide pools II and IV. Each point represents the net result of individual experiments. Horizontal bars represent the median value of the IFN-γ concentration in the related group. The response of R.A2⁺ individuals was detected higher than that of R.A2⁻ ones in peptide pools II and IV.</p