A multidimensional analysis reveals distinct immune phenotypes and the composition of immune aggregates in pediatric acute myeloid leukemia

Abstract

Because of the low mutational burden and consequently, fewer potential neoantigens, children with acute myeloid leukemia (AML) are thought to have a T cell-depleted or 'cold' tumor microenvironment and may have a low likelihood of response to T cell-directed immunotherapies. Understanding the composition, phenotype, and spatial organization of T cells and other microenvironmental populations in the pediatric AML bone marrow (BM) is essential for informing future immunotherapeutic trials about targetable immune-evasion mechanisms specific to pediatric AML. Here, we conducted a multidimensional analysis of the tumor immune microenvironment in pediatric AML and non-leukemic controls. We demonstrated that nearly one-third of pediatric AML cases has an immune-infiltrated BM, which is characterized by a decreased ratio of M2- to M1-like macrophages. Furthermore, we detected the presence of large T cell networks, both with and without colocalizing B cells, in the BM and dissected the cellular composition of T- and B cell-rich aggregates using spatial transcriptomics. These analyses revealed that these aggregates are hotspots of CD8⁺ T cells, memory B cells, plasma cells and/or plasmablasts, and M1-like macrophages. Collectively, our study provides a multidimensional characterization of the BM immune microenvironment in pediatric AML and indicates starting points for further investigations into immunomodulatory mechanisms in this devastating disease.

Fig. 1. Characterizing T cell infiltration in pediatric AML cases and non-leukemic controls.

A Schematic overview of the study population, used techniques, and the digital image analysis pipeline. AML cases are categorized in immune-infiltrated (red) and immune-depleted (blue) groups according to their T cell infiltration levels (above or below median of non-leukemic controls). Representative bone marrow biopsy images from a treatment-naïve pediatric AML case (B) and a non-leukemic control (C) showing H&E staining, CD3⁺ T cells, and CD8⁺ T cells. White lobules indicate adipocytes. Comparison of the normalized abundance of CD3⁺ T cells (D) and CD8⁺ T cells (E) in the bone marrow between pediatric AML cases and non-leukemic controls using the Mann–Whitney test. The dashed lines indicate the median CD3⁺ (D) and CD8⁺ (E) T cell abundance in non-leukemic controls, respectively. Normalized abundance of CD3⁺ (F) and CD8⁺ T cells (G) per cytogenetic subgroup. ‘Normal’ indicates normal karyotype, while ‘Others’ is a merge of cytogenetic abnormalities different from the five defined cytogenetic subgroups. See Table S1. ‘Complex’ indicates cases with complex karyotype AML (≥3 chromosomal abnormalities). The dashed lines shown in Fig. 1D, E are also shown in Fig. 1F-G. H Schematic overview of the TARGET-AML cohort, the additional non-leukemic control group, the performed analysis (CIBERSORTx), and the subsequent categorization of patients into the immune-infiltrated or immune-depleted groups (based on median T- and CD8⁺ T cell abundance in non-leukemic controls). Estimated absolute (ABS) abundance of T- (I) and CD8⁺ T (J) cells in the bone marrow of treatment-naïve pediatric AML cases in the TARGET-AML cohort. The dashed lines shown in Fig. 1I, J indicate the estimated median bone marrow T- and CD8⁺ T cell abundance in four non-leukemic controls.

Fig. 2. Transcriptional differences between immune-infiltrated and immune-depleted pediatric AML.

A Schematic overview of the study population, used techniques, and analyses performed in Fig. 2B–D. B Comparison of the normalized abundance of CD20⁺ B cells in the bone marrow of pediatric AML (CD20 stains available for 69 cases) with CD3⁺ T cell levels above or below the median of non-leukemic controls (later referred to as immune-infiltrated and immune-depleted, respectively; Mann–Whitney test). C Volcano plot of differentially expressed genes between immune-infiltrated (n = 6) and immune-depleted (n = 17) pediatric AML bone marrow biopsies, identified using DEseq2 with an FDR cut-off of 0.05 and minimum fold change (FC) of 2. D Single-sample gene set enrichment analysis of differentially expressed genes between immune-infiltrated and immune-depleted cases using the GO Biological Processes gene set with an FDR cut-off of 0.05. WikiPathways-related results are shown in Table S2. E Schematic overview of the study populations for which gene-expression data was available (primary study cohort and TARGET-AML cohort), and the associated analysis. F Correlation plot of the negative correlation between the M2-predominance score and the normalized number of CD3⁺ T cells, calculated using Spearman correlation. G Comparison of the M2-predominance score between immune-infiltrated (n = 6) and immune-depleted (n = 17) cases using the Mann–Whitney test. H Correlation plot of the negative correlation between the M2-predominance score and the estimated abundance of T cells in the bone marrow of TARGET-AML cases, calculated using Spearman correlation. I Comparison of the M2-predominance score between immune-infiltrated (n = 80) and immune-depleted (n = 79) cases using the Mann–Whitney test.

Fig. 3. T cell networks in the bone marrow of pediatric AML.

A Illustration of the identification of directly interacting T cells (above) and T cell networks (below) using Delaunay Triangulation. B Comparison of the normalized abundance of T cell networks between immune-infiltrated (n = 22), immune-depleted (n = 50), and non-leukemic control biopsies (n = 10; Kruskal-Wallis followed by Dunn’s multiple comparisons test). C Representative image of a T cell network in a treatment-naïve pediatric AML patient. D Schematic of the definition of a large T cell network. E Comparison of the number of T cell networks with at least 100 T cells/network between immune-infiltrated (n = 22), immune-depleted (n = 50), and non-leukemic control biopsies (n = 10; Kruskal-Wallis followed by Dunn’s multiple comparisons test). F Representative image of large T cell networks in a treatment-naïve pediatric AML patient. G Schematic of the definition of a lymphoid aggregate. H, I Representative images of lymphoid aggregates.

Fig. 4. Composition of lymphoid aggregates in immune-infiltrated pediatric AML.

Overview of the different types of regions profiled using spatial transcriptomics in immune-infiltrated biopsies with lymphoid aggregates (A), immune-infiltrated biopsies without lymphoid aggregates (B), and non-leukemic biopsies (C). Green cells indicate T cells, blue cells indicate B cells, and pink/purple cells indicate AML blasts or normal myeloid cells. These examples do not necessarily reflect the actual abundance of these subsets. LA: lymphoid aggregate. D UMAP of transcriptomic profiles of various region types, organized into two separate clusters. Comparison of the expression of the ‘Tfh’ (E), and ‘TLS imprint’ (F) signatures across different region types (Kruskal-Wallis followed by Dunn’s multiple comparisons test). G–L Deconvoluted abundance of various cell subsets and the M2-predominance score compared across different region types (Kruskal-Wallis followed by Dunn’s multiple comparisons test). In case of two p-values, the upper one is associated with the Kruskal-Wallis test, while the lower one reflects the result of Dunn’s multiple comparison test.

Fig. 5. In-depth characterization of T- and B cells in lymphoid aggregates.

A Schematic overview of the analysis approach applied to the spatial transcriptomics dataset and the subsequent multiplex immunofluorescence (IF). scRNA-seq: single-cell RNA-sequencing. B Proportions of memory B cells, plasmablasts, and unassigned B cells in lymphoid aggregates (LA). C Proportions of naïve-, cytotoxic (CTL), mucosal-associated invariant T (MAIT)-, and potentially dysfunctional CD8⁺ T cells in lymphoid aggregate, mixed, and control regions. D Comparison of the deconvoluted proportions of potentially dysfunctional CD8⁺ T cells in lymphoid aggregate, mixed, and control regions (Kruskal-Wallis followed by Dunn’s multiple comparisons test). In case of multiple p-values, the upper one is associated with the Kruskal-Wallis test, while the lower ones reflects the result of Dunn’s multiple comparison test. E Representative image of the multiplex immunofluorescence analysis of a lymphoid aggregate. The names below each image indicate which antibodies are shown. The green boxes on the lower row are zoomed in on the part of the biopsy in the green box in the upper left image. F The proportion of CD3⁺CD8⁺ T cells that expressed two or three inhibitor receptors (IR⁺⁺/⁺⁺⁺) positive (or not) for granzyme B (GZMB).