APECED patients with skin features have increased anti-melanoma antibody levels.

<p>Cell lines were stained with serum obtained from APECED patients (2.D.G, 3.D.G., 4.U.G., 5.U.C., 6.M.R., 8.S.S., 12.F.M., 14.U.T.) diluted 1/500. Mm9, mm25, sk.mel23, sk.mel28, A2058 are human melanoma lines, TE671 a human rhabdomyosarcoma, and B16F10 murine melanoma cell line. Background using normal human serum was substracted and mean fluorescence indicated as follows −<0.5, +0.5–0.75, ++0.75–1, +++>1. The secondary antibodies used were <b>A</b> anti-human IgM FITC and <b>B</b> anti-human IgG FITC.</p

AIRE^−/− mice have increased overall CD4 and CD8 responses after tumour challenge.

<p>Mice of indicated genotype were primed and challenged with B16F10, then splenocytes were isolated, and stained for CD62L or CD44. <b>A</b> Percentage of CD62L^low cells among CD4⁺ or CD8⁺ T lymphocytes ex vivo <b>B</b> Percentage of CD44⁺ cells among CD4⁺ or CD8⁺ T lymphocytes ex vivo <b>C</b> Percentage of CD62L^low cells among CD4⁺ or CD8⁺ T lymphocytes after in vitro culture with IFNγ stimulated B16F10. * p value Mann-Whitney test comparing % of cells with low CD62L among CD4⁺ CD3⁺ T lymphocytes. This result is representative of two repeats.</p

AIRE^−/− mice do not hyperreact to foreign antigen but respond better to certain melanocyte antigen than AIRE^+/+ littermate controls.

<p>Mice of the indicated genotype were injected s.c at the base of the tail with 4×10⁶ pfu of lentivirus expressing the indicated peptides. Serial samples of blood cells (left panels) were stained for CD8 and MHC class I tetramers specific for T cells responding to the injected peptides (example at peak of response in right panels). <b>A</b> LCMV-derived foreign gp33 presented by H-2D^b<b>B</b> mouse TRP-2 presented by H-2K^b<b>C</b> gp100 presented by H-2D^b.</p

Expression of melanocyte antigens in medullary thymic epithelial cells.

<p>Medullary or cortical thymic epithelial cells were sorted by flow cytometry. <b>A</b> RT-PCR was performed on both types of cells using primers for GAPDH, gp100 and TRP-2. <b>B</b> Real time qPCR was performed using sorted populations. Expression relative to gp100 expression in AIRE^+/+ mTECs is shown for gp100 and TRP-2. Error bars are from duplicates.</p

AIRE^−/− mice reject tumour more efficiently than AIRE^+/+ littermate controls mice after priming.

<p><b>A</b> Mice of indicated genotype were challenged with B16F10, and tumour growth monitored over 75 days. <b>B</b> As treated in <b>A</b>, but mice were primed with irradiated B16F10 4 weeks prior to challenge. Curves were compared with Logrank test with p values indicated. Similar trends were found in at least 5 different experiments. In each of these experiments between 5 and 10 mice/group were included.</p

Anti-B16F10 antibodies serum levels are elevated.

<p>Mice of the indicated genotype were primed with 5×10⁶ irradiated B16F10 and challenged with 2.5×10⁵ live B16F10. <b>A</b> At time of sacrifice (when tumours reached 10 mm² or in the case of tumour-free mice after 50 days), serum was obtained. 1/500 diluted serum was used to stain B16F10. Secondary antibody was an anti-mouse pan Ig APC. <b>B</b> Serum was obtained before priming, 4 weeks after priming, or 16 days after live tumour challenge and used to stain B16F10. Naïve littermate controls or unprimed but challenged controls were included were indicated. Secondary antibodies used were anti-mouse IgM FITC (upper panel) or anti-mouse IgG FITC (lower panel). * p value by Mann-Whitney test comparing mean fluorescence of serum staining B16F10. These findings were reproduced three times with similar results with n>10 in each group.</p

MAA-specific CD8⁺ T cells present in TILs and draining LNs.

<p><b>A</b> Wildtype mice were injected with 10⁶ live B16F10 and 10 days later, TILs were isolated and stained for CD8 and TRP-2-specific TCR using tetramers. <b>B</b> and <b>C</b> Mice of indicated genotypes were primed and challenged with B16F10 cells, then frequencies of gp100, TRP-1 or TRP-2 tetramer positive cells in the CD8⁺ T cell population were plotted. Representative of two similarly conducted experiments.</p

Depletion of regulatory T cells leads to improved tumour rejection in both AIRE^−/− and AIRE^+/+ mice.

<p>Mice of the indicated genotype were primed and challenged with B16F10. Three days and one day prior to challenge, mice were treated twice with 0.5 mg of control antibody GL113 (<b>A</b>), or with regulatory T cell-depleting antibody PC61 (<b>B</b>). Seven days after depletion with PC61 but not GL113 antibodies, CD4⁺CD25⁺ cells were reduced by 60% in the blood. Appearance of tumours was measured for 121 days.</p

Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.</p