HLA expression patterns of Hodgkin-Reed-Sternberg cells shape a spatially arranged tumor microenvironment in classical Hodgkin lymphoma
Classical Hodgkin lymphoma (cHL) differs from other malignancies in its histologic architecture: Few malignant Hodgkin-Reed-Sternberg cells (HRSC) recruit an immune cell-rich tumor microenvironment (TME). Importantly, HRSC frequently show loss of human leukocyte antigen (HLA) expression potentially disturbing T-cell interactions. We aimed to understand how the expression of this key structures of HRSC-T-cell interaction affect the spatial cellular composition of the TME.
Material and Methods:
18 lymph node samples of initially diagnosed cHL patients were scored for HLA-I and -II expression. Multiplex immunofluorescence staining for CD30, CD8, CD68, FoxP3 and LAG3 was performed using Akoya Opal Polaris 7-Color Manual IHC Kit. Digital images were analyzed in three equal sized areas near (nTME; including HRSC clusters) and ≥75 μm distant (dTME) to HRSC using QuPath software (Fig. 1). We analyzed cells regarding the expression of one marker, independent of co-expressions.
The content of CD68+ cells, CD8+, and LAG3+ T-cells varied in the proximity of HRSC (nTME) dependent of HLA expression of HRSC. HLA-I+ cases showed higher levels of CD68+, CD8+ and LAG3+ cells compared to HLA-I- cases. However, FoxP3+ cell content in nTME was independent of HLA-I expression. We found only slight differences in cellular composition of nTME comparing HLA-II+ with HLA-II- cases.
To illustrate spatial gradients, we compared cellular composition of nTME with dTME. Considering all cases, we found higher content of FoxP3+, LAG3+ and CD68+ cells in nTME. Of note, the enrichment of these cell types in nTME was slightly more pronounced dependent on HLA-I rather than HLA-II expression of HRSC. Interestingly, we found higher CD8+ cell content in HLA-I+ cases. This finding is consistent with bulk analyses, which showed HLA-I dependent upregulation of cytotoxic genes (see Seifert et al., ISHL12 submission).
In line with previous publications, we demonstrate the HRSC niche to be enriched for FoxP3+, LAG3+ cells, and CD68+ macrophages. The enrichment of certain cell types in HRSC proximity seems to be a general feature of cHL independent of HLA expression, which differs in its composition depending on HLA expression of HRSC. We find that enrichment of CD68+, CD8+ and LAG3+ cells is stronger related to HLA-I than HLA-II expression on HRSC. Together with bulk gene expression data we suggest HLA-I expression of HRSC to be a key determinant of TME composition.