Terahertz Spectroscopy and Imaging of Frozen Biological Samples

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Synthetic Peptides Used for Vaccination May Fail to Faithfully Mimic the Naturally Processed Antigens

<p>The TCR repertoire specific for the natural tumor antigen (N) contains a group of CD8⁺ T cells that recognize N with high functional avidity and display high tumor reactivity (TCR-A, focused). TCR-A is stimulated by the natural ligand and expands during spontaneous responses to the tumor in some patients with antigen-expressing tumors. A larger group of CD8⁺ T cells able to recognize N also exists in the naïve T cell repertoire (TCR-B). TCR-B recognizes N with decreased avidity and shows low to undetectable tumor reactivity (unfocused). Heteroclitic peptides (H) are analogs of N that contain modifications that increase their immunogenicity. They are selected on the basis of their increased recognition by T cells from the TCR-A group. When used as immunogens, they elicit a group of CD8⁺ T cells specific for H (TCR-C). The reactivity of TCR-C will be variable and will depend on the extent of overlapping between the TCR-A, -B, and -C groups. In Lee and colleagues' study [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0010026#pmed-0010026-b9" target="_blank">9</a>], most of the elicited CD8⁺ T cells belong to the TCR-B group (unfocused), suggesting a large overlap between TCR-C and TCR-B and a more limited overlap of TCR-C with TCR-A. This phenomenon can be explained by the structural difference between N and H. Other factors that may differ between natural and peptide-induced immune responses, including the density of the peptide on antigen-presenting cells and the mode of presentation (i.e., the nature of antigen-presenting cells), could also contribute to the outcome.</p

Isolation of XAGE-1b specific CD4⁺ T cells and generation of specific clonal populations.

<p>(A) XAGE-1b specific CD4⁺ T cells were isolated by IFN-γ capture assay. Numbers indicated in the upper right region of dot plots show the percentages of IFN-γ producing cells among the CD4⁺ T cells, after stimulation in the absence or in the presence of the peptide pool. IFN-γ producing cells were sorted and cloned under limiting dilution conditions. (B) CD4⁺ T-cell clones were obtained and screened to assess reactivity to XAGE-1b. Clones were stimulated in the presence or the absence of the peptide pool and specificity to XAGE-1b was determined by ELISA. Responses were considered significant when the amount of IFN-γ detected in the presence of peptide was at least 3-fold higher than that detected in the absence of peptide. (C) Aliquots of XAGE-1b specific CD4⁺ T-cell clones were stimulated in the absence or presence of the peptide pool or of individual single peptides and the amount of IFN-γ was measured in 24-hour culture supernatants by ELISA. (D) Reactivity to XAGE-1b single peptides was assessed in 14 clones and 4 fine specificities were identified.</p

XAGE-1b peptides.

XAGE-1b peptides.</p

Assessment of serological responses to XAGE-1b and NY-ESO-1 NSCLC patients.

<p>(A) Summary of the ELISA for the 141 NSCLC patients and 60 healthy donors (HD). The Optical density (OD) cut-off between responders and non-responders was determined as the mean OD obtained with the sera from the 60 HD + 5 standard deviations (SD) (dotted line). (B) Sera titration from the 12 XAGE-1b seropositive patients and from 1 HD was performed in a range of dilutions, from 1/100 to 1/100,000. Antibody titers were determined as the serum dilution corresponding to 50% of the maximal OD response.</p

Serological responses to XAGE-1b and NY-ESO-1 in relation to NSCLC histological types.

<p>Serological responses to XAGE-1b and NY-ESO-1 in relation to NSCLC histological types.</p

Assessment of the HLA restriction of XAGE-1b-specific CD4⁺ T-cell clones.

<p>(A) Identification of MHC II restricting molecules. The left panel shows an example of an HLA-II restriction assay clone, B2F8. Peptide recognition was assessed by ELISA, incubating XAGE-1b specific CD4⁺ T-cell clones exhibiting each of the 4 fine specificities either in the absence or in the presence of αDP, αDQ, αDR and αDR52b specific antibodies. Peptide recognition was restricted by HLA-DR in all cases, as summarized in B. The HLA-DR restricting allele was identified by incubating XAGE-1b specific CD4⁺ T-cell clones with different APC including untransfected (3T3) or DR1-transfected mouse fibroblast (LDR1) and DR13-expressing EBV-cell lines (EBV149, EBV 156). Each APC was pre-incubated with the reactive peptides and the restricting allele was determined by assessing their capacity to present the peptides to the corresponding CD4⁺ T-cell clones. Peptide recognition was assessed by measurement of IFN-γ secretion in culture supernatants, in the presence of each APC. As summarized, we found that peptide recognition was restricted by HLA-DR13 in all cases.</p

Assessment of circulating CD4⁺ and CD8⁺ T cell responses to XAGE-1b in NSCLC patients.

<p>(A, C) The presence of XAGE-1b specific CD4⁺ (A) and CD8⁺ (C) T cell responses was assessed in peptide pool stimulated cultures by intracellular cytokine staining, with cytokine specific antibodies αIFN-γ and αIL-17, after stimulation in the absence or the presence of the XAGE-1b peptide pool. The percentages of IFN-γ producing cells are shown in the upper left quadrants of each dot plot, showing increased IFN-γ production in peptide stimulated samples. The data shown are an example of a CD4⁺ and CD8⁺ T cell responder patient. (B, D) Summary of the CD4⁺ and CD8⁺ T cell responses are shown for all the 12 seropositive patients. As shown in (B), CD4⁺ responses are detected for all seropositive patients, whereas a significant CD8⁺ response was identified only in 1 patient, NA703 (D). Values at least 3-fold higher than baseline (no peptide) were considered significant.</p

Differential expression of Janus kinase 3 (JAK3), matrix metalloproteinase 13 (MMP13), heat shock protein 60 (HSP60), and mouse double minute 2 (MDM2) in human colorectal cancer progression using human cancer cDNA microarrays

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Circulating CD4 T cells secreting IL-10 <i>ex vivo</i>, alone or with IL-17, include FOXP3^- cells and FOXP3⁺ Helios^- pTreg.

<p><i>Ex vivo</i>–isolated CD4⁺ T cells were stimulated with PMA/ionomycin, stained with anti-CD45RA, -IL-10, -IL-17, -FOXP3, and -Helios mAbs and analyzed by flow cytometry. (A) Dot plots depicting FOXP3 expressing cells within cytokine secreting populations are shown for one donor. The proportion of FOXP3 expressing cells within each cytokine secreting populations and their mean fluorescence intensity (MFI) are summarized for all donors (<i>n</i> = 8). (B) The proportions of cells co-secreting IL-2 or IFN-γ within the indicated populations are summarized for all donors (<i>n</i> = 4). (C) The proportions of Helios expressing cells within FOXP3⁺ Treg in the indicated populations are summarized for all donors (<i>n</i> = 8). Statistical analyses were performed using the Mann—Whitney <i>U</i> test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, *** <i>p</i> < 0.001, ns, Non-significant.</p