Sequence alignment of mass peaks matching to ATP5B.

<p>Clustal alignment of the ATP5B sequence with trypsin digestion peptides identified by MS with a matching sequence of confidence 85% or greater.</p

ß-F1-ATPase ELISA.

<p>Levels of antibodies to ß-F1-ATPase were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. The MM patients indicated by grey symbols were previously identified as being immunopositive to ß-F1-ATPase by SEREX. There was no significant difference (ns) between the groups.</p

Immunohistochemicallocalisation of vimentin.

<p>Immunohistochemical staining of representative MM samples using anti-vimentin antibody diluted 1∶500 (A) Positive stained MM tumour sample, arrows indicate representative groups of tumour cells; over 90% of the tumour cells have been stained (B) Non stained MM tumour sample with positively stained stroma. Arrows indicate representative groups of non-staining tumour cells; less than 10% of tumour cells have been stained. (C) Positively stained MM cells present in a pleural effusion sample. Arrows indicate representative positive stained MM cells; over 90% of the tumour cells have been stained. (D) Non stained MM tumour cells in a MM patient pleural effusion. Arrows indicate representative non-staining tumour cells; less than 10% of tumour cells have been stained. Photomicrographs were taken at ×200 magnification.</p

Immunohistochemical tissue localisation of β-F1-ATPpase.

<p>Immunohistochemical staining of representative MM samples using anti-β-F1-ATPpase antibody diluted 1∶100 (A) Positive stained MM tumour sample, arrows indicate representative groups of tumour cells; over 90% of the tumour cells have been stained. (B) Non stained MM tumour sample with positively stained macrophage (indicated by arrow); less than 10% of tumour cells have been stained. (C) Positively stained MM cells present in a pleural effusion sample. Arrows indicate representative positive stained MM cells; over 90% of the tumour cells have been stained. (D) Non stained MM tumour cells in a MM patient pleural effusion; less than 10% of tumour cells have been stained. Photomicrographs were taken at a ×200 magnification.</p

Clinical characteristics and antibody levels for study subjects.

1<p>– Short term survival group; survival <12 months.</p>2<p>– Long term survival group; survival >12 months.</p>3<p>- median ± 95% confidence intervals.</p

Two Dimensional Polyacrylamide Gel Electrophoresis.

<p>The Tris-soluble fraction of the JU77 cell lysate was separated by 2D electrophoresis and proteins stained with Coomassie Blue. Proteins from a parallel gel were transferred to PDVF and incubated with serum from MM Patient 145 diluted 1∶100. Two densely stained IgG reactive spots were defined by the approximate pI range 5 to 7 and molecular weight range 50 to 60 kDa, indicated by the circle. Molecular weights (MW) depicted to the left of figures; isoelectric point (pI) to the top of figures.</p

Vimentin ELISA.

<p>Levels of antibodies to vimentin were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group (excluding the outlier) is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. There was no significant difference (ns) between the groups.</p

Survival of MM patients dichotomized on antibody reactivity.

<p>Kaplan–Meier survival curves of overall survival (months) from diagnosis for patients with (A) anti-vimentin and (B) anti-β-F1-ATPase antibody levels dichotomized below (thick line) and above the median (thin line).</p

Western Immunoblots of MM patient serum with MM tumour cell line lysates.

<p>(A) Representative serum samples from individual MM patients. Patients segregated into short, medium and long term survivors. Patient identification number indicated above each lane. (B) Western immunoblot of cell lysates from five different MM cell lines and the A549 lung adenocarcinoma, U2OS osteosarcoma and HT29 colon carcinoma cell lines incubated with serum from a MM Patient 145. Serum was diluted 1∶100. Molecular weights (MW) depicted to the left of figures.</p

Combination of CTLA-4 blockade and gemcitabine chemotherapy results in the induction of protective T cell memory.

<p>(A) Kaplan-Meier survival plot of mice that had been cured by either anti-CTLA-4 alone or combination therapy and that were subsequently rechallenged with AB1 mesothelioma cells, showing protective immunity in 80% and 92% respectively. T cell subset analysis in tumor-draining lymph nodes in these mice (*p<0.05; **p<0.01***p<0.001): CD44⁺/CD62L⁺/CD4⁺ T central memory cells (B); CD44⁺/CD62L^−/CD4⁺ T effector memory cells (C); CD44⁺/CD62L⁺/CD8⁺ T central memory cells (D); CD44⁺/CD62L^−/CD8⁺ T effector memory cells (E).</p