PR1-CTL of all differentiation phenotypes proliferate.

<p>Lin- CD8+ cells from patient 4 were sorted into effector memory (EM), central memory (CM), terminally differentiated (TD) and naïve (N) populations, then labeled with CFSE and stimulated with OKT3 + anti-CD28 antibodies. After 80 hours, cells were labeled with PR1/HLA-A2 tetramer and analyzed for evidence of proliferation by CFSE dilution. (A) The upper panel shows the frequency of proliferating PR1-CTL compared to total CD8+ T cells, and (B) the lower panel shows proliferation indices of PR1-CTL compared to total CD8+ T cells.</p

Loss of functional PR1-CTL precedes cytogenetic relapse in patient 2.

<p>Staining with the PR1/HLA-A2 tetramer was done to determine the percentage of high- and low-avidity PR1-CTL as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011770#s2" target="_blank">methods</a>. In the top panel, PR1-CTL are defined as CD8+ Lin- PR1/HLA-A2-tetramer+. The center panel shows the percentage of CD8+ lymphocytes that produce IFNγ in response to stimulation with 0.2 µM PR1 peptide after subtracting the percentage of CD8+ cells that produced IFNγ in the absence of stimulation. Non-stimulated cells were used to adjust background. Stimulation with SEB superantigen was used to confirm that the lymphocytes were not anergic. The lower panel shows the frequency of CD8+ cells that produce IFNγ in response to SEB stimulation.</p

PR1-CTL persist in patients that maintain remission after IFN withdrawal.

<p>For each patient, the panel shows the total percentage of CD8+ cells that were identified with the PR1/HLA-A2 tetramer at time points after IFN withdrawal.</p

Patient disease responses and PR1-CTL immune responses.

<p>*Patient receiving imatinib 400 mg daily.</p><p>−Denotes that the result that could not be determined due to an insufficient number of cells.</p><p>NA Not applicable since IFN was not withdrawn in these patients.</p><p>BCR-ABL transcripts were determined with quantitative real-time PCR (RQ-PCR) of peripheral blood, as expressed on the International Scale (IS). PR1/HLA-A2 tetramers were used to enumerate PR1-CTL in PBMC, and PBMC were left unstimulated or stimulated with either the PR1 peptide or with SEB and analyzed for IFNγ production by flow cytometry. Shown are the frequencies of CD8+ T cells that produce IFNγ, after subtracting percentage of CD8+ cells that stain positive for IFNγ without any stimulation. The highest frequency of CD8+ cells that produce IFNγ when stimulated with PR1 are shown.</p

Longitudinal disease burden of CML patients in complete cytogenetic remission (CCR) after withdrawal of IFN treatment.

<p>Disease characteristics of seven HLA-A2+ patients in whom IFN treatment was withdrawn. Patients received IFN starting from the time of diagnosis (left-most time point) until it was stopped (denoted as time = 0). For each patient, closed bars denote time points when any Ph+ chromosome was detected in bone marrow, open bars denote time points when no Ph+ chromosomes were detectable (complete cytogenetic remission, CCR) but BCR-ABL transcripts were detectable by QR-PCR, and the solid line denotes time points when BCR-ABL transcripts were not detectable (complete molecular remission). Arrows denote when certain patients received imatinib monotherapy.</p

PR1-CTL express all T cell differentiation phenotypes.

<p>A cocktail of antibodies with 8 fluorochromes was used to label PBMC with anti-CD8, lineage markers (Lin; anti-CD4, anti-CD14, anti-CD16, anti-CD19), CCR7, CD27, CD45RA, CD28, CD57 and PR1/HLA-A2 tetramer. The data was analyzed using the FlowJo analysis software. (A) Gating was done on CD8+ Lin- lymphocytes in the upper panel and on PR1-CTL in the center and lower panels. The center and lower panels show the representative phenotype of naïve and memory PR1-CTL from patient 3. The percentage of PR1/HLA-A2 tetramer-negative and PR1/HLA-A2 tetramer-positive CD8+ cells that express (B) central memory or (C) naïve phenotype averaged from patients 1 to 7 is shown. P-values were calculated using a two-tailed t-test and results are shown as mean ± SEM.</p

Baseline patient characteristics.

1<p>The Sokal score is based on age, spleen size, peripheral blood platelet count, and blast count. Patients are classified as being low-risk (Sokal score <0.8), intermediate-risk (0.8 to 1.2), or high-risk (>1.2).</p>2<p>The Eastern Cooperative Oncology Group (ECOG) performance status is graded on a scale of 0 to 5, with 0 indicating that the patient is fully active and able to carry on all pre-disease activities without restriction and 5 indicating that the patient has died.</p><p>Characteristics of patients at the time of newly diagnosed Philadelphia chromosome-positive (Ph+) chronic-phase CML.</p