Multi-modal profiling and targeting of the tumor microenvironment in central nervous system lymphoma Free

Primary central nervous system lymphoma (PCNSL) is a highly aggressive B-cell lymphoma confined to the CNS and associated with a poor prognosis. Although tumor cells in PCNSL and systemic diffuse large B-cell lymphoma (DLBCL) exhibit similar phenotypes, the effect of tissue of origin on the tumor microenvironment (TME) remains largely unknown. In addition, given the CNS has traditionally been regarded as an immune-privileged site, whether therapeutic strategies targeting the TME in PCNSL are effective has been uncertain.

To address this, we performed multi-modal single-cell profiling, integrating whole-transcriptome, surface proteome, and T/B-cell receptor (TCR/BCR) repertoires on 199,414 cells obtained from 18 DLBCL tumors (8 germinal-center B-cell [GCB]-like and 10 activated B-cell [ABC]-like subtypes) derived from lymph node lesions, 16 CNSL tumors (14 primary and 2 secondary), 5 reactive lymph nodes, and 4 healthy donor-derived peripheral blood samples. CNSL exhibited a distinct TME profile characterized by more pronounced CD8+ T-cell exhaustion and clonal expansion relative to DLBCL. Differentially expressed gene analysis revealed that these clonally expanded and exhausted CD8+ T cells showed enhanced interferon signaling. Bulk TCR sequencing of paired peripheral blood samples from CNSL cases showed no proliferation of the dominant T-cell clones detected in the CNS, suggesting that clonal expansion of T cells occurs within the CNS. In addition, compared with DLBCL, mature NK cells, conventional dendritic cells, and macrophages were increased in CNSL, whereas CD4+ T cells and non-malignant B cells were decreased. Although tumor cells from all CNSL samples showed gene expression profiles characteristic of ABC-like subtype, there were still significant differences in the TME compared with ABC-like DLBCL.

To confirm the impact of tissue of origin on the TME, we established DLBCL and CNSL mouse models by transplanting a murine B-cell lymphoma cell line A20 (BALB/c-derived) into the tail vein or the brain parenchyma of BALB/c mice, resulting in the development of splenic or brain lymphoma, respectively. Multi-modal single-cell profiling of tumor samples from these mice revealed that they successfully recapitulated the differences in the TME between human DLBCL and CNSL, including marked CD8+ T-cell exhaustion and clonal expansion in the CNS. To investigate the functional relevance of CD8+ T cells in CNSL, we depleted CD8+ T cells in the CNSL model and found that anti-CD8 antibody treatment shortened survival. By contrast, adoptive transfer of allogeneic C57BL/6 CD8+ T cells was effective against A20 cells transplanted into the CNS of C57BL/6 Rag2 and Il2rg knockout mice. These results functionally demonstrate the crucial anti-tumor role of CD8+ T cells even in the CNS.

Combined single-cell analyses of both human and mouse tumors revealed that CD8+ T cells in CNSL showed increased mRNA and protein expressions of TNFRSF9, which encodes 4-1BB, a key co-stimulatory molecule associated with T-cell exhaustion. In addition, cell-cell interaction analysis revealed augmented 4-1BB signaling from surrounding myeloid cells into CD8+ T cells in CNSL. Importantly, increased expression of exhaustion markers, including TNFRSF9, was associated with a better prognosis in PCNSL. Moreover, 4-1BB agonist treatment extended the survival of the mice carrying CNSL, highlighting 4-1BB as a potent therapeutic target to enhance the anti-tumor activity of CD8+ T cells in CNSL.

To explore environmental factors that affect the TME in CNSL, we conducted metabolomic profiling of tumors and adjacent normal tissues from these mouse models using capillary electrophoresis-mass spectrometry. CNSL tumors exhibited enhanced glycolytic activity and increased levels of glutarate and lactate. Consistent with this, human and mouse CNSL tumor cells showed increased expression of glycolysis-related genes. In addition, in vitro exposure to glutarate or lactate induced CD8+ T cell exhaustion, suggesting the potential role of these metabolites in modulating T-cell phenotype in the CNS environment.Taken together, our study provides a comprehensive characterization of the TME in CNSL compared to systemic DLBCL, demonstrating the relevance of metabolic environment in shaping the TME in CNSL. In addition, we reveal the therapeutic potential of targeting the TME in CNSL, which may contribute to improving the management strategy for this intractable disease.