Unraveling the Architecture of Classic Hodgkin Lymphoma One Cell at a Time

Classic Hodgkin lymphoma (cHL) is unique among hematopoietic cancers in that the tumor mass is primarily composed of nonmalignant immune cells, which are secondary to the much rarer malignant cells (∼1% of total cells in affected lymph nodes), termed Hodgkin–Reed–Sternberg (HRS) cells. This situation can be compared biologically to a reactive lymph node (RLN), where the presence of a pathogenor other inflammatory stimulus triggers local immune-cell migration to and proliferation in the affected lymph node. In cHL, the inflammatory stimulus is the presence of the HRS cells themselves. The HRS cells, however, not only engage nonmalignant immune cells to form a distinctive tumor microenvironment, but also communicate with the cells in this immunologic microenvironment (appropriately described as a multicellular ecosystem) in a manner that is immunosuppressive and favorable to the growth of the tumor. Immune checkpoint inhibitors, particularly the programmed death 1 (PD-1)–blocking antibodies nivolumab and pembrolizumab (1–4) have shown encouraging efficacy in refractory or relapsed cHL, implying the ability to convert the tumor-supporting microenvironment into a tumor-inhospitable one. Notably, HRS expression of PD-L1 and MHC class II has been shown to predict response to PD-1 inhibition with nivolumab (5). Therefore, study of the heterogeneity in cellular composition and immune–antigen expression in cHL could inform future directions to improve cHL treatment.

The innovative study by Aoki and colleagues (6) utilized single-cell RNA sequencing (scRNA-seq) to characterize the nonmalignant immune-cell populations present in cHL lymph nodes, and to distinguish these from cells present in RLNs from healthy donors. From 22 patients with cHL and 5 control RLNs, the authors were able to analyze transcriptomic data from more than 127,000 cells, and thereby group them into 22 phenotypic cell clusters. Treg, Th1, Th2, and Th17 clusters were all amplified in cHL versus RLNs, whereas RLNs were relatively enriched in B-cell and CD8⁺ T-cell groups. The authors identified a distinct LAG3⁺CD4⁺FOXP3⁻ T-cell population in cHL, expressing the cytokines IL10 and TGFβ, which the authors consider to be Tr1-type Tregs, a cell population previously associated with EBV⁺ cHL (7), which has been shown to suppress effector CD8⁺ T-cell function in cHL (8). The authors used an HRS-derived cell line supernatant to induce LAG3⁺ expression in CD4⁺ T cells in vitro and found that these induced LAG3⁺ T cells suppressed proliferation of noninduced CD4⁺ T cells in subsequent coculture, suggesting an immunosuppressive function induced by HRS-derived soluble factors. The antiproliferative effect was confirmed in LAG3⁻ T cells from 4 patients with cHL, cultured ex vivo with or without the patient's LAG3⁺ T cells: LAG3⁺ cells reduced both proliferation and TNF production in LAG3⁻ T cells.

Imaging mass cytometry (9), a novel technique combining the spatial resolution of IHC with the multidimensional single-cell analysis potential of suspension mass cytometry (also known as CyTOF), was utilized to identify spatial relationships of the cells identified in scRNA-seq analysis and the HRS cells. HRS cells in MHC-II–negative cHL (where HRS cells are MHC-II negative) were found to be in direct spatial apposition to LAG3⁺ T cells. Because MHC-II–negative cHLs have been observed to be relatively resistant to anti–PD-1 therapy (5), this histopathologic finding may be relevant to immune checkpoint escape. LAG3 itself was more highly expressed in MHC-II–negative versus MHC-II–positive cHLs within the LAG3⁺ cell population, and LAG3⁺ cells were also more abundant in the MHC-II–negative cHLs. In contrast, neither MHC-I nor EBV status, nor histologic subtype of cHL, appeared to affect LAG3⁺ T-cell abundance or LAG3 expression. In contrast, FOXP3⁺ T cells were much more abundant in MHC-II–positive cHLs, and could be found in contact with MHC-II–positive HRS cells. In a validation cohort of 166 patients treated with first-line chemotherapy and analyzed immunohistologically, abundance of LAG3⁺ cells, and also of CD68⁺ macrophages, consistent with previous studies (10), was found to be a poor prognostic indicator; however, MHC-II negativity itself was not.

This study has several implications: that HRS cells induce LAG3 expression in a population of Tr1 Tregs, by secreted and/or contact-mediated factors; that this primarily occurs in MHC-II–negative cHLs; and that the LAG3⁺ T cells have an antiproliferative effect on LAG3⁻ T cells, which in turn may contribute to local immunosuppression and potential for immune checkpoint escape. The authors advance the hypothesis that MHC-II on MHC-II–positive HRS cells binds to LAG3 on Tr1 cells and downregulates it by negative feedback (Fig. 1). It is not clear whether this hypothesized LAG3-dependent mechanism of LAG3 suppression would be stable, and indeed it is not yet clear whether MHC-II expression on HRS downregulates LAG3 expression on Tr1 cells, or vice versa. Furthermore, Aoki and colleagues observed PD-1 (PDCD1) expression in subsets of multiple lymphocyte populations, including LAG3⁺ T cells, and most prominently in LAG3⁻CD4⁺ T cells, which are present in both MHC-II positive and MHC-II negative cHL (6). Therefore, a mechanism by which LAG3⁺ T cells would promote resistance to PD-1 blockade in MHC-II–negative cHL is not yet evident. Clearly, there remain substantial gaps to be filled in understanding the mechanism by which immune checkpoint escape is more frequent in MHC-II–negative cHLs (5). Furthermore, whether there are in fact independent prognostic effects of MHC-II negativity and abundant LAG3⁺ Tr1 cells, or whether these are interdependent, remains unclear, as does the relationship between LAG3⁺ cell abundance in MHC-II–negative cHLs and FOXP3⁺ cell abundance in MHC-II–positive cHL. By advancing a spatial resolution–based single-cell analysis of cHL, Aoki and colleagues have opened a new window into a previously shadowy room. Somewhere within may reside keys to improved outcomes for those cHLs currently refractory to immunotherapy. Substantial further studies of the same sort will be needed to identify what and where those keys are.

No potential conflicts of interest were disclosed.