106/ml CD8+ T cells were labelled using a DiD lipophilic tracer (Molecular Probes) according to manufacturers instructions and the cells washed three times in colourless X-VIVO 15 (BioWhittacker). influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8C1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients. Introduction The outcomes for patients with leukaemia have improved considerably over the last 30 years due to enhancements in supportive care, expertise and the development of haematopietic stem cell transplants. Most patients now achieve a first remission following combinational chemotherapy protocols, but the best treatment BW 245C option for patients for whom it is applicable remains allo-transplantion, an option which depends on meeting health criteria BW 245C and donor availability. First remission is expected to be the optimal time-point for immunotherapy to be administered, when residual disease loads are low. Tumour antigens have been shown to induce specific T-cell responses in patients and thus act as targets for immunotherapy treatments [1C3]. A number of end-point assays have been developed to assess both cellular (Enzyme-Linked ImmunoSpo (ELISpot), cytotoxic T-lymphocyte (CTL) assays) and humoral (enzyme-linked immunosorbent assay (ELISA)) immune responses induced in patients during immunotherapy clinical trials, for use as indicators of clinical efficacy. The development of peptideCMHC tetramers (pMHC)  and variants thereof [5,6] have allowed the visualization of antigen-specific T cells through flow cytometry and the use of  approaches. Such analyses have enabled specific T-cell enumeration, assessment of cytokine production/secretion (flow based assays, ELIspot) and the expansion of T-cell populations for functional analysis (CTL analysis). However flow cytometry is technically difficult, time consuming, expensive and, until recently, limited to only one or a very few antigen specificities per sample . This changed with the description of pMHC microarrays by Soen in 2003 . Soen demonstrated the potential of the pMHC arrays to detect OVA-antigen specific populations from both T-cell receptor (TCR) transgenic and wild type mice. Subsequently Chen et al  used the pMHC arrays to detect multiple T-cell populations in 11 human leukocyte antigen (HLA)-A*0201 positive BW 245C patients with resected stage IIC/III and IV melanoma who were undergoing a melanoma-associated peptide vaccine trial. In addition to showing the presence of specific T-cell populations that could recognise epitopes within independent antigens, the authors demonstrated functionality through the co-spotting of pMHCs with a panel of cytokines. Similar methods such as the Fluorescence-activated cell sorting (FACS)-based combinatorial encoding approach [8,11], despite being elegant and robust, are limited in the number of T-cell populations they can detect (25 and 15 different T-cell populations in a single sample, respectively). Hadrup et al  suggested that the analysis of more than 100 T-cell populations per sample may be possible in the future through the use of a larger number of fluorochromes and multi-dimensional combinatorial encoding while Newell et al  stated that 31 and 63 populations could be analysed with five- and six-colour FACs. Recently Newell et al  demonstrated that through combining Mouse monoclonal to Mcherry Tag. mCherry is an engineered derivative of one of a family of proteins originally isolated from Cnidarians,jelly fish,sea anemones and corals). The mCherry protein was derived ruom DsRed,ared fluorescent protein from socalled disc corals of the genus Discosoma. combinatorial [8,11] and mass cytometryCbased pMHC staining approaches  that they could detect more than 100 specific T-cell populations as well as 20C30 phenotypic markers, in a single blood or intestinal lymphocyte sample. However combinatorial approaches require expensive reagents (quantum dots ) and/or complex multifactorial analysis, with four or more multi-laser FACS analysis being essential. We have developed a pMHC array to analyse T cells from leukaemia patients to determine epitope-specific recognition from a range of cancer-testis (CT) and leukaemia-associated antigens (LAAs). In contrast with previous studies, these samples were taken either at disease presentation prior to treatment or at relapse following conventional therapies and none of the samples were stimulated to expand specific T-cell populations prior to pMHC array analysis. Materials and Methods.