The phenotype of bone marrow derived DC is not affected by absence of CTSD.

<p>Bone marrow cells from CTSD −/− and CTSD +/+ donor chimeras were cultured in GM-CSF for seven days, purified on CD11c magnetic beads, and then analysed for expression of surface markers by flow cytometry. Cells were >80% CD11c positive. Shaded histogram: isotype control. Solid line : CTSD −/− DC. Dotted line CTSD +/+ DC. One of three experiments.</p

The aspartic proteinase inhibitor MPC6 inhibits processing of PcMSP1₂₁.

<p>Bone marrow DC (10³) from CTSD +/+ donor chimeras were cocultured with differing concentrations of (A) PcMSP1₂₁ protein or (B) a peptide coding the B7 epitope (1 µg/ml) and MPC6 (5 µM) for three hours and then colcultured with B7 hybridoma cells. p values show the significance value comparing presence and absence of inhibitor (Anova). One of three experiments. (C)_Bone marrow DC from CTSD +/+ donor chimeras were cocultured with iRBC (5 and 30 erythrocytes/DC) for three hours, and then the DC (10³) were cocultured with B7 hybridoma cells. p values show the significance value comparing presence and absence of inhibitor (Anova). One of two experiments.</p

CTSD deficient, malaria infected mice have decreased late parasitaemia and enhanced malaria specific IgG responses.

<p>A) CTSD −/− and CTSD +/+ donor chimeras were infected with <i>P. chabaudi</i>, and percent parasitaemia measured at different days post infection as shown. Geometric mean and standard error of mean is shown (n = 6). Significance was measured using the Mann Whitney test. B) and C. As in A, but plasma was collected after 36 days for ELISA against whole parasite lysate (B) or PcMPS1₂₁ protein (C). Each individual mouse, and the geometric mean and standard error of mean (n = 6) is shown. One experiment of two , each with six mice in each group.</p

Absence of CTSD impairs the ability of DC to process erythrocytes infected with <i>P. chabaudi</i> merozoites in vitro.

<p>A) DC (10³) from CTSD −/− and CTSD +/+ donor chimeras were co-cultured with B7 T hybridoma cells (2×10⁴) and different numbers of iRBC. IL-2 release was measured after 24 hours using the indicator cell line CTLL. B) DC from CTSD −/− and CTSD +/+ donor chimeras were cocultured with iRBC (5 erythrocytes/DC) for three hours, and then different numbers of DC were co-cultured with B7 or B5 hybridoma cells as in A. C) DC (10³) from CTSD −/− and CTSD +/+ donor chimeras were co-cultured with B7 T hybridoma cells (2×10⁴) and different concentrations of MSP1 protein or a peptide coding for the B7 epitope at different concentrations. IL2 was release was measured as above. An additional intermediate concentration of MSP1 (50 µg/ml) was also tested in additional experiments with similar results. P values show the difference between CTSD +/+ and CTSD −/− analysed by Anova. One of four experiments.</p

Normal numbers and class II MHC expression of DC isolated from spleens of <i>P. chabaudi</i> infected CTSD −/− and CTSD +/+ donor chimeras.

<p>DC were isolated from spleens of CTSD −/− and CTSD +/+ donor chimeras using magnetic CD11c beads, and stained for CD11c, CD8 and I-A^d using flow cytometry. Total numbers of cells isolated were similar in both sets of chimeras (3.83×10⁶ in CTSD +/+, and 4.5×10⁶ in CTSD −/− for experiment shown, one representative of two).</p

PDL1 Expression on Plasma and Dendritic Cells in Myeloma Bone Marrow Suggests Benefit of Targeted anti PD1-PDL1 Therapy

<div><p>In this study we set out to investigate whether anti PDL1 or PD–1 treatment targeting the immune system could be used against multiple myeloma. DCs are important in regulating T cell responses against tumors. We therefore determined PDL1 and PDL2 expression on DC populations in bone marrow of patients with plasma cell disorders using multicolour Flow Cytometry. We specifically looked at CD141⁺ and CD141^- myeloid and CD303⁺ plasmacytoid DC. The majority of plasma cells (PC) and DC subpopulations expressed PDL1, but the proportion of positive PDL1+ cells varied among patients. A correlation between the proportion of PDL1⁺ PC and CD141⁺ mDC was found, suggesting both cell types could down-regulate the anti-tumor T cell response.</p></div

Expression of PDL1 on PC and monocytes in myeloma bone marrow.

<p>(A) PDL1 on plasma cells: Bone marrow cells were stained with antibodies against CD45, CD138, CD38, CD19, and CD274 (PDL1). Gates were set on FSC and SSC and doublets and CD19+ cells were excluded. Gating strategy is shown in Fig A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139867#pone.0139867.s002" target="_blank">S2 File</a>. The distribution of % PDL1⁺ PC in the bone marrow of patients (n = 14) is shown. (B) Proportion of PDL1⁺ PC does not increase with tumor load. The % PDL1⁺ gated CD38⁺CD19^- PC versus % bone marrow plasma cells is plotted. Each dot represents one patient. P values were calculated from a Spearman’s test (n = 14). (C) PDL1 on monocytes and DCs: Bone marrow cells were stained with antibodies against lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, and CD11c. The gating strategy is shown in Supplementary S1B Fig. Gates were set on FSC and SSC, doublets excluded, and gates further set on lineage- CD45⁺cells. Figure shows distribution of % PDL1+ monocytes/DC in the bone marrow of patients (n = 14). (D) Correlation of % PDL1+ PC and monocytes/DC; % PDL1⁺CD11c⁺DR⁺ monocytes/DC versus % PDL1⁺CD38⁺CD19^- plasma cells is plotted. Each dot represents one patient. P value was calculated from a Spearman’s test.</p

DC subtypes express PDL1 in myeloma bone marrow.

<p>Bone marrow and blood were stained with antibodies against CD141, lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, CD303, CD1c, and CD11c. The gating strategy is shown in Fig D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139867#pone.0139867.s002" target="_blank">S2 File</a>). Three DC populations were analysed; CD141⁺ (CD141⁺DC) (panels A-C), CD141^- (CD141^-DC) (panels D-F), and CD303⁺DC (pDC) (panels G-I). PDL1 staining on one representative patient (panels A, D, G). Fluorescence minus one (FMO), (dotted line), was used as negative control and the percentage indicates PDL1⁺ cells of the gated DC population. Panels B, E, and H show percentage of PDL1⁺ cells within the (B) CD141⁺ DC, (E) CD141^- DC and (H) CD303⁺ pDC populations in the bone marrow (n = 19), blood (n = 8) from patients, or blood from age matched (median age 61) healthy controls (n = 9). (median age of patients 61). Statistical analysis was performed with Mann Whitney Test. Panels C, F, and I show concomitant expression levels on bone marrow DC subtypes and plasma cells in individual patients. Each dot represents one patient. P values were calculated from Spearman’s tests.</p