Immune microenvironment dysfunctions enable malignification at the onset of MDS Ganan-Gomez I, Ma F, Chien KS, Yang H, Montalban-Bravo G, Wildeman BE, Kumar B, Kim YJ, Daher M, Takahashi K, Garcia-Manero G and Colla S
1. BM Innate Immune Cells Are Activated in CCUS Patients
Background
• Myelodysplastic syndromes (MDS): clonal stem cell malignancies • Standard therapy not curative, transient responses, poor prognosis • Are prevention or early intervention possible in MDS? • Clonal cytopenias of undetermined significance (CCUS): aging-related premalignant state , low-grade clonal hematologic disorders at high risk of progression to MDS and leukemia • HSC-intrinsic alterations and extrinsic inflammatory factors cooperate to induce abnormal differentiation in CCUS (Ganan- Gomez et al. ASH Meeting 2021). • Characterize early alterations in the bone marrow (BM) immune microenvironment that lead to the expansion of the MDS clone Aim • BM mononuclear cells (MNCs) from patients with CCUS and young and elderly healthy donors (HDs) • Single-cell transcriptomics (scRNA-seq) • In vitro functional assays • Single-cell genomics/antigen expression analysis (Tapestri) Methods
scRNA-seq in BM MNCs
pDCs
cDCs
My/Mono
NK cells
NK cells
E r
HSPCs
CCUS (n=4) Elderly HD (n=2) Young HD (n=2)
B pro
PCs
CD8 + T cells
CD8 + T cells
B cells
CD4 + T cells
CD4 + T cells
0 1020304050 -Log 10 [P val]
Formation of ATP by chemiosmotic coupling Interferon alpha/beta signaling Neutrophil degranulation Mitochondrial translation initiation Cytokine Signaling in Immune system Membrane Trafficking Eukaryotic Translation Elongation Respiratory electron transport, ATP synthesis by chemiosmotic coupling Adaptive Immune System Parasite infection Pathway analysis of significantly upregulated genes in CCUS
Immune microenvironment dysfunctions enable malignification at the onset of MDS Ganan-Gomez I, Ma F, Chien KS, Yang H, Montalban-Bravo G, Wildeman BE, Kumar B, Kim YJ, Daher M, Takahashi K, Garcia-Manero G and Colla S
2. Inhibitory Crosstalk Predicted Between NK and CD8 + T Cells in CCUS
3. CCUS NK/T Cells Are Terminally Differentiated toward Exhaustion
scRNA-seq in sorted CD3 + T and CD56 + NK cells
CellPhoneDBanalysis of DEGs
NK cell activation by CD8 + T cells HLA-E:CD94/NKG2C CD58:CD2 FASLG:TNFRSF1A
CCUS (n=3) Elderly HD (n=2) Young HD (n=2)
Ligand-expressing cells
NK
CD4 T
CD8 T
CytoTRACE differentiation scores Exhaustion marker expression
CCUS Elderly Young
Receptor-expressing cells
Immunosuppresion of CD8 + T cells by NK cells CD48:CD244 TGFB1:TGFBR1
Immune microenvironment dysfunctions enable malignification at the onset of MDS Ganan-Gomez I, Ma F, Chien KS, Yang H, Montalban-Bravo G, Wildeman BE, Kumar B, Kim YJ, Daher M, Takahashi K, Garcia-Manero G and Colla S
4. NK Cells from CCUS Patients Are Irreversibly Dysfunctional
5. NK Cells Are Part of the Mutant CCUS Clone
Joint scDNA-seq and surface protein analysis (Tapestri)
CD8 + T
No expansion In vitro cytolysis assays with leukemic cell lines
Post-7-day NK cell expansion
CD4 + T
Young Healthy Old Healthy CCUS THP1 alone
K562 alone Young HDs Old HDs CCUS
✱✱✱✱
T Prog
✱✱✱
✱✱✱✱
✱✱✱
600
100 150 200 250
NK cells
B cells cDC pDC
400
CD16 + Mono
200
0 50
0 1020304050 0
My/Mono
0
3
6
9
12
Time (h)
Time (h)
• The innate immune repertoire is molecularly and functionally altered in patients with CCUS • Immune evasion of premalignant clones may contribute to clonal evolution to MDS • Rationale for adoptive NK cell therapy in patients with CCUS or low-burden MDS to prevent/arrest MDS progression Conclusions
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