Behind the Paper

Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction

Lanqi Gong Apr 07, 2023

Background

Nasopharyngeal carcinoma (NPC), as an immunologically ‘hot’ tumor, exhibits an unsatisfied response rate to anti-PD-1 monotherapy, particularly in its advanced stage. As shown in our recent single-cell paper1, a high infiltration of regulatory T cells (Tregs) might lead to robust immunosuppression that impedes the efficacy of immune-checkpoint blockade in NPC patients. Even though our finding echoes other papers conducted by our peers at different medical centers2,3, the mechanism of how Tregs are developed and exert their suppressive activity during NPC progression has not been deciphered. Thus, we are enthusiastic about digging out hidden treasures in the NPC multicentral single-cell datasets. Our curiosity leads to this paper, where we utilize multi-omics approaches integrated with pre-clinical models to overcome immunotherapy resistance in NPC by targeting Tregs. 

Key Findings

We collected and re-analyzed single-cell datasets from our own cohort and two other cohorts from Guangzhou, China, generating a multicentral single-cell cohort with 50 patient samples (Figure 1). Consistent with our previous finding, the NPC microenvironment is Treg-enriched, while the nonmalignant nasopharyngeal microenvironment is CD4+ naive T cell-enriched. Thus, we considered NPC cells might be able to regulate CD4+ naive T cell-to-Treg development and Treg activation, as supported by pseudotime trajectory analysis. Then, the first research question we asked was how NPC cells influenced Treg development and activation: whether via cytokine communication or contact-based interaction? The transwell and direct co-culture assays between NPC cells and CD4+ naive T cells proved that it was the latter case. Subsequently, based on cell-cell interaction analysis on single-cell and newly generated spatial transcriptome data, we identified that NPC enhanced Treg development and suppressive activity via CD70-CD27 interaction. In vitro allogeneic and antigen-specific assays and the humanized mouse model revealed that more CD70-activated Tregs secreted more immunosuppressive factors, including IL-10, TGF-β, and adenosine, which significantly inhibited cytotoxicity and proliferation of CD8+ T cells. 

Cusatuzumab is a high-affinity anti-CD70 monoclonal antibody that has shown as an effective treatment in multiple hematopoietic malignancies in phase I and II clinical trials4. Hence, we wondered if cusatuzumab can be used to treat NPC cells to inhibit CD70-mediated Treg development and activation. The efficacy of cusatuzumab treatment was systematically evaluated in NPC cell line, patient-derived xenograft, and xenograft-derived organoid models, exhibiting a synergistic effect with PD-1 blockade camrelizumab, which was clinically approved for NPC patient treatment in China. Since there exists a negative association between Tregs and the clinical response of anti-PD-1 treatment, we foresee a big opportunity for cusatuzumab in treating CD70-high malignancies, not only including NPC, but also renal clear-cell carcinoma and melanoma with high Treg infiltration. In this section of our paper, we would like to particularly and sincerely thank Prof. George Sai-Wah Tsao for sharing the valuable NPC xenografts with us. Prof. Tsao passed away in December 2022 after his long-time fight with cancer, and we deeply appreciate his contribution to this paper and to the whole scientific community, particularly in NPC research. 

To further investigate the downstream molecular mechanism of CD70-CD27 interaction, we applied to single-cell RNA sequencing to peripheral mononuclear cells (PBMCs) co-cultured with CD70-control and CD70-knockout NPC cells. Mechanistically, CD70 knockout inhibits a collective lipid signaling network in CD4+ naïve and Tregs involving mitochondrial integrity, cholesterol homeostasis, and fatty acid metabolism, as extensively validated by mass spectrometry-based metabolomic sequencing and metabolic assays. Considering CD70-CD27 interaction as a metabolic switch that turns on lipid reprogramming in CD4+ naïve and Tregs, we further demonstrated that lipid depletion could abolish the Treg-promoting effect of CD70+ NPC cells, and enhance anti-tumor immunity. This part of our study added another brick to our existing molecular understanding of CD70-CD27 interaction in Tregs. 

In the end, we were interested in pursuing how CD70 was constantly upregulated in NPC patients. EBV infection was our focus because it was strongly associated with NPC pathogenesis and could induce many epigenetic modifications contributing to NPC growth, therapeutic resistance, and immune evasion. Through our collaboration with Dr. Wei Dai's lab at the University of Hong Kong, we analyzed ATAC-seq results on EBV+ NPC cells, EBV- NPC cells and normal nasopharyngeal epithelial cells. We delineated that CD70 upregulation was caused by EBV-enhanced chromatin accessibility at the CD70 promotor region, which could be effectively bound by NFKB2. NFKB2 knockdown and inhibition downregulated both mRNA and protein expression of CD70 in NPC cells, leading to higher tumor cell death when co-cultured with PBMCs. Overall, the EBV-NFKB2-CD70 axis is a new immune escape mechanism identified in NPC (Figure 2).

 

Discussion

The mechanism for tumor cells to escape from immune surveillance is multifaceted. In NPC, many scientists believe EBV infection is the most important driver for immune evasion. Our paper shows that EBV infection can significantly upregulate CD70 expression in NPC cells that remodels a CD4+ T cell-enriched microenvironment into a Treg-enriched one to facilitate NPC progression and therapeutic resistance. We believe our finding is critical to answering the question about NPC pathogenesis: how can NPC cells initiate and continuously progress in a harsh immune-inflamed microenvironment. While we have shown that anti-CD70 treatment can stop NPC progression and synergize with PD-1 blockade by inhibiting Tregs in pre-clinical models, we should keep in mind that not all NPC patients are responsive to such therapy, as shown in a small-scale clinical trial in Belgium5. It reminds us to keep finding molecular characteristics for patient stratification and optimizing anti-CD70 treatment stepwise before it turns into a safe and reliable strategy for NPC patients.

References

  1. Gong, L., et al. Comprehensive single-cell sequencing reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of nasopharyngeal carcinoma. Nat Commun 12, 1540 (2021).
  2. Chen, Y.P., et al. Single-cell transcriptomics reveals regulators underlying immune cell diversity and immune subtypes associated with prognosis in nasopharyngeal carcinoma. Cell Res 30, 1024-1042 (2020).
  3. Liu, Y., et al. Tumour heterogeneity and intercellular networks of nasopharyngeal carcinoma at single cell resolution. Nat Commun 12, 741 (2021).
  4. Riether, C., et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat Med 26, 1459-1467 (2020).
  5. De Meulenaere, A., et al. An open-label, nonrandomized, phase Ib feasibility study of cusatuzumab in patients with nasopharyngeal carcinoma. Clin Transl Sci 14, 2300-2313 (2021).