Early alterations in stem-like/resident T cells, innate and myeloid cells in the bone marrow in preneoplastic gammopathy

Abstract

Preneoplastic lesions carry many of the antigenic targets found in cancer cells but often exhibit prolonged dormancy. Understanding how the host response to premalignancy is maintained and altered during malignant transformation is needed to prevent cancer. In order to understand the immune microenvironment in precursor monoclonal gammopathy of undetermined significance (MGUS) and myeloma, we analyzed bone marrow immune cells from 12 healthy donors and 26 MGUS/myeloma patients by mass cytometry and concurrently profiled transcriptomes of 42,606 single immune cells from these bone marrows. Compared to age-matched healthy donors, memory T cells from both MGUS and myeloma patients exhibit greater terminal-effector differentiation. However, memory T cells in MGUS show greater enrichment of stem-like TCF1/7hi cells. Clusters of T cells with stem-like and tissue-residence genes were also found to be enriched in MGUS by single-cell transcriptome analysis. Early changes in both NK and myeloid cells were also observed in MGUS. Enrichment of stem-like T cells correlated with a distinct genomic profile of myeloid cells and levels of Dickkopf-1 in bone-marrow plasma. These data describe the landscape of changes in both innate and adaptive immunity in premalignancy and suggest that attrition of the bone-marrow-resident T cell compartment due to loss of stem-like cells may underlie loss of immune surveillance in myeloma.

Keywords: Adaptive immunity; Cancer; Immunology; Oncology.

Figure 1. Bone marrow mononuclear cells from healthy donors (n = 7), MGUS (n = 8), and myeloma (n = 10) were characterized using single-cell mass cytometry.

(A) Central memory (CD45RO⁺CCR7⁺) CD8⁺ and CD4⁺ T cells as percentage of total memory CD8⁺ and CD4⁺ T cells. (B) CD8⁺ and CD4⁺ effector T cells (Tefs) (effector memory cells, Tems: CD45RO⁺CCR7^–; and terminal effectors, Ttes: CD45RO^–CCR7^–) as percentage of total memory CD8⁺ and CD4⁺ T cells. (C) CD8⁺ Tems and Ttes as percentage of CD8⁺ Tefs. (D) CD4⁺ Tems and Ttes as percentage of CD4⁺ Tefs. (E) Median expression of TCF1 and GATA-3 transcription factors in CD8⁺ memory T cells. (F) Median expression of Eomes and T-bet in memory CD8⁺ T cells. (G) Gating strategy for defining cells that express high levels of TCF1 (TCF^hi) and intermediate levels of TCF1 (TCF^int) and those that do not express TCF1 transcription factor (TCF1^neg). A representative dot plot from a patient with MGUS. (H) Percentage of memory CD8⁺ T cells that express TCF1^hi or TCF^int or lack TCF1 expression (TCF1^neg). (I) Percentage of TCF1^hi in CD8⁺ Tems, Tcms, and Ttes. (J) Characteristics of the TCF1^hi, TCF1^int, and TCF1^neg CD8⁺ memory T cells. All bar graphs show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney test.

Figure 2. Bone marrow myeloid cells were identified using single-cell mass cytometry analysis of bone marrow mononuclear cells from healthy donors (n = 4), MGUS (n = 8), and myeloma (n = 8) (gray, blue, and black, respectively).

Gating strategy of myeloid cells is described within Supplemental Figure 3. (A) Biaxial plots of CD11b versus CD95, CD86, CD155, PD-L1, c-KIT, and HLA-DR of healthy, MGUS, and myeloma donor myeloid cells. (B) Bulk myeloid cells’ median intensity for CD95, CD86, CD155, PD-L1, c-KIT, and HLA-DR. All bar graphs show mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3. scRNA–Seq to characterize 42,606 bone marrow mononuclear immune cells from 33 samples (8 healthy donor, 14 MGUS, and 11 myeloma).

(A) t-Distributed stochastic neighbor embedding (t-SNE) plot with 15 distinct cell populations determined by k-nearest neighbor unsupervised clustering. (B) Heatmap of zero-centered average gene expression of highly differentially expressed genes (selected by most extreme P value, Wilcoxon’ rank-sum test) for each cluster identified in A compared with all other clusters. (C) t-SNE plot distinguishing cells by disease state. (D) Cells in each cluster as a percentage of total cells by disease state (*P < 0.005, χ² test).

Figure 4. Characterization of gene expression differences in T cell and myeloid populations from scRNA-Seq.

(A) T cells in each cluster as a percentage of total T cells. T2 cluster is enriched in MGUS (χ² P < 0.0001), and T3 cluster is enriched in MM (χ² P < 0.0001). (B) Volcano plot of differential gene expression between clusters T2 and T3. (C) Volcano plot of differential gene expression in myeloid cells between myeloma and MGUS. (D) Volcano plot of myeloid differential gene expression between samples with higher-than-mean population of cluster T2 stem-like/resident T cells and samples with lower-than-mean population of cluster T2 T cells. (E) DKK1 was measured in the plasma of MGUS and MM patients. Percentage of CD8⁺ memory cells that are TCF1⁺ in patients with DKK1 levels below median (DKK1^lo: n = 10) and patients with DKK1 levels above median in the group (DKK1^hi: n = 10). Bar graph shows mean ± SEM. *P < 0.05.