B7-DC XAb-induced Tumor Protection is Dependent on CD40.

<p>Wild type or CD40−/− mice were implanted with B16 or WEHI tumor (5×10⁵ cells) and received control antibody or B7-DC XAb by intravenous administration. The mice were monitored for tumor growth and were euthanized if tumor size reached 17×17 mm. Tumor-free B16-implanted mice were monitored regularly for more than 90 days; tumor-free WEHI-implanted mice were monitored regularly for more than 60 days. The scores indicate the number of mice in the indicated treatment group remaining free of tumor. n.s. = not significant.</p

CD40 is required for the DC^XAb-induced generation of anti-tumor CTL responses in vivo.

<p>Wild type or CD40^−/− mice were engrafted with B16 melanoma (A) or WEHI-3 leukemia (B) and treated intravenously with 30 µg B7-DC XAb or control antibody. On day 7, cells from draining lymph nodes were used as effectors in CTL assays. (A) CTL against ⁵¹Cr-labeled B16 tumor targets (Left) or unrelated EL-4 controls (Right). Filled squares show CTL for wild-type mice receiving B7-DC XAb. (B) CTL against ⁵¹Cr-labeled WEHI-3 tumor targets (Left) or unrelated, MHC-matched P815 controls (Right). Filled triangles show the CTL response from wild type mice receiving B7-DC XAb. Symbols and lines for all other treatment groups are nearly superimposable. The results shown are representative of 2 experiments.</p

CD40 is rapidly recruited into complexes containing MHC MHC class II on dendritic cells treated with B7-DC XAb.

<p>(A) DCs were pre-stained with APC-labeled antibody against MHC class II and PE-labeled antibody against CD40 for 15 min. An aliquot of cells was analyzed by flow cytometry (0 min time point). The remaining cells were then treated with 10 µg/ml IgM control antibody (filled histograms) or B7-DC XAb (open histograms). Cells were sampled at the indicated time points and analyzed for a FRET (FL3 channel). (B) Lysates were prepared from untreated DC (0′) or DC treated with control antibody or B7-DC XAb for the indicated times and subjected to immunoprecipitation using an antibody against MHC class II. The resultant complexes were resolved by SDS-PAGE, transferred to PVDF membrane, and probed for CD40. IgH serves as a loading control. The results shown are representative of 2 experiments.</p

Identification of potential mechanistic associations of immune phenotypes using the combination of relational data and cell counts.

<p>Immune phenotype values were measured using the TBNK and T cell-2 protocols from 64 healthy volunteers. The percentages of PD-1, CTLA4, and CD28 positive CD8 or CD4 cells were plotted against either total (A) CD8 cells/ml and (B) CD4 cells/ml. Populations were tested for statistical significance using the Spearman correlation test.</p

Phenotypic characterization of the most common myeloid subsets.

<p>¹: This is a subset of CM1, CM2 and CM3 where DR has been lost. Often this designation is currently grouped independent of the designation of CD16. However, CM1, CM2 and CM3 have been shown to have unique positive effects on immunity. Thus, an alternative designation for the suppressive version of CM1, CM2 and CM3 is CM1-DRneg, etc.</p><p>²: Termed suppressive monocytes (Refs. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121546#pone.0121546.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121546#pone.0121546.ref018" target="_blank">18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121546#pone.0121546.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121546#pone.0121546.ref029" target="_blank">29</a>]).</p><p>³: MDSC were only classified as MO-MDSC and GR-MDSC without clear designation of what these cells and/or subsets are⁸.</p><p>⁴: GR-MDSCs generally have the same surface markers as granulocytes but are identified by altered density properties in density gradient purifications of mononuclear cells.</p><p>Phenotypic characterization of the most common myeloid subsets.</p

Additional analyses of monocytes reveals diversity of surface expression on classical, intermediate, and non-classical monocytes.

<p>A. Examples of monocyte gating from a healthy volunteer control and DLBCL patient. Mononuclear cells were gated from CD45+ WBCs ([WBCs]). CD14⁺ monocytes were gated from [MNCs]. CD14⁺ cells were either plotted with HLA-DR to identify HLA-DR^lo/neg populations or CD16 to delineate CD14⁺CD16^- classical monocytes, CD14⁺CD16⁺ intermediate monocytes, and CD14^loCD16⁺ non-classical monocytes. B. Histogram overlays of HLA-DR, CD86, TNFR2, and CD40 on a healthy volunteer control and DLBCL patient. Classical monocytes were colored in red, intermediate monocytes colored in black, and non-classical monocytes were colored blue.</p

Radar plots permit simultaneous visualization of leukocyte compartments.

<p>Fresh blood was stained with the TBNK/M/G protocol and analyzed per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121546#pone.0121546.g001" target="_blank">Fig. 1</a>. A. Gated populations from histograms for selected markers were color coded for radar plot analyses. Lymphocytes (orange) and CD14pos (purple) were gated from mononuclear cells ([MNCs]). CD3 (red), CD19 (blue), and CD56 (black) were gated from the Lymphocyte gate. B. Bivariate dot plot and radar plot comparisons. Populations were either gated from the MNC or Lymphocyte gate. Radar plots show populations on 3, 4, and 6 axes. The markers for the 6 axes include CD3, CD4, CD8, CD14, CD19, and CD56. C. Radar plot analyses of longitudinal data from a patient and a healthy control. The arrangement of the 6 axes in each plot is identical for each sample. D. Graphical representations of the longitudinal cell count data collected from 5 DLBCL patients and 10 healthy volunteer controls (HV). * = <i>P</i> value < 0.05.</p

The development of a 10-color protocol to enumerate lymphocytes, monocytes, and granulocytes.

<p>Antibodies corresponding to the TBNK/M/G protocol were added directly to fresh peripheral blood. The red blood cells lysed, and run on the Gallios cytometer with the addition of fluorescent counting beads. A. Fluorescent beads are gated on FL2 by time, and total beads collected by FL4 and forward scatter (FS) B. Gating strategy for the enumeration of leukocyte subsets. The gates for the selected populations are listed above each bivariate dot plot or histogram. [WBCs] = total leukocytes gated by forward and side scatter; [MNCs] = mononuclear cells.</p

Characterization of myeloid marker staining patterns on whole blood.

<p>Peripheral blood from a healthy control sample was processed via the lyse/wash Myeloid protocol. Histograms were generated from each of 9 antibodies used to delineate myeloid populations. In most cases regions (R1, R2, and R3) were created for each peak of expression including peaks with no expression (N). Other regions represent populations of cells falling between the negative/positive peaks or populations that do not have a clear peak. Density gradient plots were created from cells gated from each region plotted by forward and side scatter. A representative example from a healthy volunteer is shown.</p

10-color analysis of myeloid cells reveals complexity of myeloid phenotypes.

<p>Peripheral blood from a healthy control sample was processed via the lyse/wash Myeloid protocol. A. Bivariate plots were used to delineate and color-code myeloid populations. CD33 was used to distinguish myeloid cells from lymphoid cells. LIN2 was also used as a surrogate for CD14. A plot of LIN2 and HLA-DR from the Myeloid gate was used to further separate cells into mature monocytes (LIN2+, [Monocytes]), LIN2+HLA-DR^lo/neg monocytes ([A] in dark green), LIN2-CD33+HLA-DR- myeloid derived suppressor cells (MDSC in black), and circulating dendritic cells (DC in red). Monocytes were further gated into classical monocytes LIN2+CD16- (blue), intermediate monocytes (orange), and non-classical monocytes (purple). Granulocytes were gated from CD45+ high side scatter populations and colored brown. CD33 by CD11b dot plots from mononuclear cells ([MNCs]) and total leukocytes ([CD45]) using the same color coding scheme listed above. B. Radar plots of myeloid cells were generated from MDSCs, DC, Monocytes and total myeloid cells ([Myeloid]). Selected markers and axes were arranged uniquely for each myeloid subset. C. Histograms showing representative examples of CD123 expression on LIN^-HLA-DR^-CD33⁺ ImMC MDSC (black), LIN⁺CD33⁺ monocytes (white), and CD15⁺CD66b⁺ granulocytes (brown) from a healthy volunteer and DLBCL patient. The graph shows the geometric mean fluorescence intensity of CD123 on LIN^-HLA-DR^-CD33⁺ MDSC (ImMC), LIN⁺CD33⁺ monocytes (white), and CD15⁺CD66b⁺ granulocytes and CD123⁺CD11c^- plasmacytoid dendritic cells (pDC) from healthy volunteers (n = 11). * = p<0.05 as determined by two-tailed Wilcoxon matched-pairs signed rank test.</p