PMC7841290_01
Case presentation. (A) Timeline of disease diagnosis and treatment. (B) Magnetic resonance imaging (MRI) obtained at baseline (top panel) and after 2 cycles of nivolumab treatment (bottom panel). T1 post-contrast and FLAIR sequences are shown in the left and right column, respectively. Red and blue circles indicate the location of tissue acquisition from the enhancing (red) and non-enhancing (blue) regions. (C) Immunohistochemistry analysis showing H&E staining and reactivity of immune cell markers CD68, CD4, and CD8 (10x magnification) from the resected tissue following nivolumab treatment.
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AO, WHO grade III nineteen years prior to this study. As illustrated in Figure 1A, he received radiation therapy (RT) after the initial surgery to remove the tumor. An MRI scan after two cycles of treatment (nivolumab 240 mg intravenously, every two weeks on a 28 day-cycle) showed that the enhancing lesions in the right insular region increased in size significantly, while the fluid-attenuated inversion recovery (FLAIR) signal in the right frontal and temporal lobe remained largely unchanged from the baseline, suggesting worsening high grade glioma. (Figure 1B). The pathologic exam showed a mixture of tumor cells and immune cell infiltration which suggested treatment effects (Figure 1C). CD4+ and CD8+ T cell infiltration. CD68+ cells, representing macrophage and microglia cell populations, were also identified on the tumor slides (Figure 1C). Although we discovered that T cells in non-enhancing lesion expressed higher levels of PRF1, GZMM, GZMH, and GZMB when compared to that in the enhancing lesion, the significance of this is unknown and more studies are needed to define this in the context of immune therapy (Figure S1). Like CD8+ T cells, NK cells in the non-enhancing region expressed a higher cytolytic score compared to that of enhancing region (Figure 3C, fold change 1.52; Wilcoxon test, p = 4.136 x 10-14), though the percentages of NK cells within each sample were similar.
Single-cell analysis of resected tissue from enhancing and non-enhancing lesions (A) Uniform Manifold Approximation and Projection (UMAP) representation of ten identified cell types in enhancing and non-enhancing samples from the resected tissue. (B) Percentages of each cell type of all cell populations. (C) Percentages of each cell type of the enhancing and non-enhancing samples.
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We identified ten major cell types based on their gene expression profiles (Figure 2A). Though the tissues were expected to be composed of mostly tumor, only 3.5% of the cells were classified as oligodendroglioma cells (Figure 2B). Most of the cell clusters were identified as immune cell types, including microglia (34.2%), macrophages/monocytes (14.4%), NK cells (9.8%), T cells (24.6%), and a small amount of B cells (0.9%) (Figure 2B). Over half (51.5%) of the cells in non-enhancing lesion were composed of microglia compared to only 10.4% of the cells in enhancing tissue (Figure 2C). Interestingly there were many more T cells in the enhancing region (Figure 2A). By analyzing differential gene expression (DGE), we found that SH2D2A, encoding T cell Specific Adapter protein (TSAd), which mediates the activation of T cells, increased in the T cells of non-enhancing lesion (Figure S2). GNLY, which encodes granulysin, a protein presents in the cytotoxic granules that are released by the activated CTLs, was found to be upregulated in T cells from non-enhancing tumor area (Figure S2). The DGE analysis revealed that RNASET2, which mediates the M1-like polarization and suppresses M2-like polarization, was upregulated in microglia and macrophages/monocytes of the non-enhancing lesion (Figure S2).
Subclustering analysis of T and NK cells. (A) UMAP plot showing three distinct T cell subpopulations, CD4+, CD8+, and Tregs (CD4+/FOXP3+). (B) UMAP representation of cytolytic score of T cells. (C) Violin plots summarizing the cytolytic score of NK cells and T cells from enhancing and non-enhancing samples. The average cytolytic scores of T cells were 1.87 in enhancing and 2.50 in non-enhancing lesions (Wilcoxon test p = 3.084 x 10-6), and the average cytolytic scores of NK cells were 3.06 in enhancing and 4.66 in non-enhancing (Wilcoxon test p = 4.136 x 10-14).
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T cells identified CD8+, CD4+, and regulatory T cell (Treg, CD4+/FOXP3+) subpopulations (Figure 3A). The CD8+ T cells, as expected, expressed the highest cytolytic score of T cells while the Tregs expressing at the lowest cytolytic score (Figure 3B). When the cytolytic score per cell was calculated, a fold-change of 1.33 in non-enhancing lesion versus enhancing lesion was found (Wilcoxon test p = 3.084 x 10-6) (Figure 3C). Like CD8+ T cells, NK cells in the non-enhancing region expressed a higher cytolytic score compared to that of enhancing region (Figure 3C, fold change 1.52; Wilcoxon test, p = 4.136 x 10-14), though the percentages of NK cells within each sample were similar. NFKB1, which regulates the maturation and effector function of NK cells, was the only gene upregulated in NK cells of the enhancing lesion (Figure S3).
Analysis of macrophages/monocytes and microglia. (A) UMAP showing the distinct populations of macrophage/monocyte and microglia in enhancing and nonenhancing lesion. (B) CD68+ cells in the macrophage/monocyte and microglia populations in enhancing and non-enhancing lesion. (C) M1-like markers expression distribution in macrophage/monocyte and microglia populations in enhancing and non-enhancing lesion. CD80 (left) and CD86 (right). (D) M2-like markers expression distribution in macrophage/monocyte and microglia populations in enhancing and non-enhancing lesion. CD163 (left) and MRC1 (right).
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The majority of the microglia were found in the non-enhancing lesion, while macrophages/monocytes have a similar distribution in both enhancing and non-enhancing lesion (Figure 4A). As expected, both cell clusters expressed CD68, consistent with the identity of the cell types (Figures 4B). The expression of the M1-like (pro-inflammatory) cell marker CD80 was minimal throughout, while the CD86+ cells were much more enriched in both non-enhancing and enhancing lesions (Figure 4C). Both M1-like and M2-like markers were found to be expressed in macrophages/monocytes to a similar extent in the enhancing and non-enhancing regions (Figure 4D). Since the most recent surgery, he has remained radiographically and clinically stable for at least 12 months (Figure S4).