Table_2_Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease.xls

BackgroundWe previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression.MethodsGene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology.ResultsBM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival.ConclusionsOur results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression.</p

Image_1_Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease.tif

BackgroundWe previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression.MethodsGene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology.ResultsBM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival.ConclusionsOur results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression.</p

Table_1_Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease.xls

BackgroundWe previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression.MethodsGene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology.ResultsBM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival.ConclusionsOur results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression.</p

Image_2_Gene Expression Analysis of the Bone Marrow Microenvironment Reveals Distinct Immunotypes in Smoldering Multiple Myeloma Associated to Progression to Symptomatic Disease.tif

BackgroundWe previously reported algorithms based on clinical parameters and plasma cell characteristics to identify patients with smoldering multiple myeloma (SMM) with higher risk of progressing who could benefit from early treatment. In this work, we analyzed differences in the immune bone marrow (BM) microenvironment in SMM to better understand the role of immune surveillance in disease progression and to identify immune biomarkers associated to higher risk of progression.MethodsGene expression analysis of BM cells from 28 patients with SMM, 22 patients with monoclonal gammopathy of undetermined significance (MGUS) and 22 patients with symptomatic MM was performed by using Nanostring Technology.ResultsBM cells in SMM compared to both MGUS and symptomatic MM showed upregulation of genes encoding for key molecules in cytotoxicity. However, some of these cytotoxic molecules positively correlated with inhibitory immune checkpoints, which may impair the effector function of BM cytotoxic cells. Analysis of 28 patients with SMM revealed 4 distinct clusters based on immune composition and activation markers. Patients in cluster 2 showed a significant increase in expression of cytotoxic molecules but also inhibitory immune checkpoints compared to cluster 3, suggesting the presence of cytotoxic cells with an exhausted phenotype. Accordingly, patients in cluster 3 had a significantly longer progression free survival. Finally, individual gene expression analysis showed that higher expression of TNF superfamily members (TNF, TNFAIP3, TNFRSF14) was associated with shorter progression free survival.ConclusionsOur results suggest that exhausted cytotoxic cells are associated to high-risk patients with SMM. Biomarkers overexpressed in patients with this immune gene profile in combination with clinical parameters and PC characterization may be useful to identify SMM patients with higher risk of progression.</p

Co-culture of CB MNCs with IL-2 and aAPCs yields significantly greater expansion of NK cells than culture with IL-2 alone.

<p>A. Mean fold growth of CD56⁺/CD3⁻ NK cells from 8 fresh and 6 frozen cord blood expansions with aAPCs and IL-2 versus 3 expansions with IL-2 alone (14 day culture). B. Time course of NK cell growth over 14 day culture between all 3 conditions. By day 7, the fresh CB aAPC-containing culture demonstrated greater NK cell growth than culture with IL-2 alone (p<0.05). The frozen CB showed a similar trend at day 7, which did not reach statistical significance (p = 0.06). C. All three culture conditions yielded comparable, low percentages of CD3⁺ cells:. 0.44%, 0.74% and 0.66% CD3⁺ cells from the culture with IL-2 alone, fresh CB MNCs with aAPC feeders or frozen CB MNCs with aAPC feeders respectively (p>0.5 for all comparisons). Mean +/− SD is shown for each figure. P<0.05 where indicated (*).</p

Phenotype of CB-NK cells cultured with aAPCs.

<p>A. Over the 14-day expansion, CB-NK cells cultured with aAPC feeder cells demonstrated a progressively pure, CD56⁺/CD3⁻ population, (representative dot plots of 17 expansions). B. aAPC-expanded CB-NK cells maintained Eomesodermin^hi and T-bet^hi phenotype after expansion. Representative histograms from 3 different CB-NK expansions; cells are gated on the live CD56⁺ population. C. CB MNCs from the same CB unit were expanded with aAPCs +IL-2 or IL-2 alone (n = 3 separate CB units). Representative dot plots of NK cell surface receptor expression on day 14 are shown. D. By median fluorescence intensity (MFI), aAPC-expanded CB-NK demonstrated a decreased surface expression of the NCRs NKp30, NKp46 and NKp44. However there was a similar expression between the conditions of the KIR antigens, inhibitory receptor NKG2A, co-receptor CD94 and activating receptor NKG2C) (n = 3 paired expansions, mean +/− SD is shown, p≤0.05 where indicated).</p

aAPC-expanded CB-NK cells delay development of myeloma in a NSG murine model.

<p>1×10⁶ GFP firefly luciferase-transduced ARP-1 cells (Clone 24) were given IV on day -1. In the CB-NK treated group, 10×10⁶<i>ex vivo,</i> aAPC-expanded CB NK cells were given retro-orbitally on days 0, 12 and 19 with IL-2, 2000 IU (IP) three times per week. Serial BLI and kappa ELISA measurements were acquired until day 18. Results represent mean values of n = 5 mice in each group until day 18, by which time 1 mouse in the ARP-1 alone group had died. A. Serial BLI images demonstrate impaired myeloma development in mice receiving CB-NK cells. B. Signal intensity (p/s) was significantly greater in mice receiving Clone 24 ARP-1 cells alone versus those receiving both Clone 24 ARP-1 cells and CB-NK cells. Region of interest (ROI) is indicated by rectangles superimposed on each mouse from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076781#pone-0076781-g005" target="_blank">Figure 5A</a>, p≤0.05 at days 8–22. C. Serum kappa levels (ng/mL) were significantly higher in mice treated with Clone 24 ARP-1 cells versus those treated with Clone 24 ARP-1 cells and CB-NK cells, p≤0.01 at each time point. D. By Kalpan-Meier method, there was a significant difference in survival of the mice, (p = 0.003) in favor of the NK-treated group. The mice who received Clone 24 ARP-1 cells alone had a median survival of 31 days versus 38 days for the mice who received Clone 24 ARP-1 cells and CB-NK cells.</p

aAPC-expanded CB-NK cells form immunological synapses with and are cytotoxic against myeloma targets.

<p>A. CMAC-labeled tumor targets (blue) were incubated at a 1∶1 ratio with aAPC-expanded CB-NK cells for 15 minutes. Conjugates were then fixed, permeabilized and stained for NK effector cell F-actin with rhodamine-phalloidin (red). Confocal and brightfield images were acquired; representative images from each slide are shown. aAPC-expanded CB-NK cells form immune synapses with the classic NK target K562 as well as a variety of MM cell lines. B. aAPC-expanded CB-NK cells were co-incubated in triplicate for 4 hours with ⁵¹Cr-labeled target cells at ratios as shown. Supernatants were then harvested and analyzed the next day for ⁵¹Cr content. % Cytotoxicity = (sample value-spontaneous lysis)/(max-lysis-spontaneous lysis) x 100%. CB-NK cells demonstrate dose-dependent cytotoxicity against K562 (classic NK cell target) and MM cells lines RPMI 8266, ARP-1 and U266 (representative of n>3 assays for each cell line). C. aAPC-Expanded CB-NK cells displayed equal or more cytotoxicity against K562 cells versus CB-NK cells expanded with IL-2 alone (representative from n = 4 assays).</p

Culture of CB-NK cells.

<p>Unselected CB MNCs were cultured for 7 days in a GP500 bioreactor with IL-2 (100 IU/mL) and aAPCs at 2∶1 aAPC:MNC ratio. Cells were immunomagnetically CD3-depleted on Day 7 and re-cultured in same conditions for an additional 7 days. On day 7 cells were again CD3-depleted and subject to phenotypic and functional studies.</p