Epstein-Barr virus and immune status imprint the immunogenomics of non-Hodgkin lymphomas occurring in immune-suppressed environments

Abstract

Non-Hodgkin lymphomas (NHL) commonly occur in immunodeficient patients, both those infected by human immunodeficiency virus (HIV) and those who have been transplanted, and are often driven by Epstein-Barr virus (EBV) with cerebral localization, raising the question of tumor immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 immunodeficient (34 post-transplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were large B-cell lymphoma and 25% were primary central nervous system lymphoma, while 40% were EBV+. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden, tumor landscape and tumor microenvironment and prediction of tumor neoepitopes. Both tumor mutational burden (2.2 vs. 3.4/Mb, P=0.001) and numbers of neoepitopes (40 vs. 200, P=0.00019) were lower in EBV+ than in EBV- NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations observed exclusively in EBV+ and immunodeficient NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV- cases but in only half of the EBV+ ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The tumor microenvironment analysis showed higher CD8 T-cell infiltrates in EBV+ versus EBV- NHL, together with a more tolerogenic profile composed of regulatory T cells, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T-cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV- patients, offering potential opportunities for future T-cell-based immune therapies.

Figure 1.

The tumor mutational burden is lower in Epstein-Barr virus-positive non-Hodgkin lymphoma than in Epstein-Barr virus-negative cases. (A) Tumor mutational burden (TMB), defined as the number of mutations per megabase; log₁₀, according to Epstein-Barr virus (EBV) status among 68 patients with non-Hodgkin lymphoma (NHL) on the left, 51 immunodeficient NHL patients in the middle, and the 49 patients with large B-cell lymphoma on the right. Red and green denote EBV-negative and -positive NHL respectively. (B) TMB according to immune status. Salmon, blue and yellow colors denote immunocompetent patients, transplant recipients and patients infected with human immunodeficiency virus, respectively. (C) TMB according to disease localization. Pink and blue denote systemic and central nervous system localization, respectively. Wilcoxon test. (D, E) Overall survival depending on TMB >3/Mb among EBV^– (D) and EBV⁺ (E) cases. Kaplan-Meier analysis. ID: immunodeficient; LBCL: large B-cell lymphoma; CNS: central nervous system.

Figure 2.

The mutational landscape of non-Hodgkin lymphoma differs by Epstein-Barr virus and immune status. (A) Co-oncoplot of the most recurrently mutated genes (≥15%) within the 49 samples of large B-cell lymphoma (LBCL) according to Epstein-Barr virus (EBV) status (EBV^– on the left, EBV⁺ on the right). TP53, MYD88 and HNRNFP were differently mutated between the two groups but these results lost their significant value after false discovery rate (FDR) correction was applied. (B) Co-oncoplot of the most recurrently mutated genes (≥15%) within the 49 LBCL samples according to immune status (immunocompetent on the left, immunodeficient on the right). STAT 3, TYW1 and MYD88 were differently mutated between the two groups but these results lost their significant value after FDR correction was applied. Fisher exact test was used to compare categorical data. CNS: central nervous system; PTLD: post-transplant lymphoproliferative disorder; DLBCL: diffuse large B-cell lymphoma; ID: immunodeficient; IC: immunocompetent; HIV: human immunodeficiency virus.

Figure 3.

The number of neoepitopes is lower in Epstein-Barr virus-positive non-Hodgkin lymphoma than in Epstein-Barr virus-negative cases. (A) Number of predicted neoepitopes (log10) from the non-Ig variants within the 31 RNA samples, according to Epstein-Barr virus (EBV) status on the left and immune status (in EBV^– non-Hodgkin lymphoma) on the right. Wilcoxon test. (B) Number of predicted neoepitopes (log10) from the non-Ig variants within the 66 whole-exome sequencing samples (2 samples were excluded because of absence of germline assessment) according to EBV status. Wilcoxon test. (C) Correlation study between the number of predicted neoepitopes from the RNA and the whole-exome sequencing data (top) and between the number of predicted neoepitopes from the RNA data and the tumor mutational burden (bottom). Spearman correlation. (D) Number of predicted neoepitopes (log10) from Ig variants within the 24 RNA samples (7 samples were excluded because of dominant IgH clone <15%). Wilcoxon test. NHL: non-Hodgkin lymphoma; ID: immunodeficient; IC: immunocompetent; HIV: human immunodeficiency virus; WES: whole-exome sequencing.

Figure 4.

Neoepitope-specific T cells were detected in 71% cases, including responses directed against Ig-derived neoepitopes. Number of interferon (IFN)-γ spots (/10⁶ cells) after peptide stimulation for the 14 tested patients (represented on the x axis). Green squares denote responses directed against the complete pool, red circles denote responses directed against Ig-derived neoepitopes and black circles denote responses directed against individual non-Ig neoepitopes. Thawed peripheral blood mononuclear cells were co-cultured with personalized pooled peptides for 10 days and then tested for reactivity using IFN-γ enzyme-linked immunospot (ELISPOT) assays. Patients were all tested for their personalized pooled peptides (named “complete pool”) and eventually for each individual peptide if the numbers of cells were adequate (named as the mutated gene). The mean numbers of spot-forming cells (SFC) from triplicate assays were normalized to the number of IFN-γ spots detected per 1x10⁶ peripheral blood mononuclear cells after background subtraction. The threshold for ELISPOT-IFN-γ positivity was 50 SFC/10⁶ cells. EBV: Epstein-Barr virus; ID: immunodeficiency; IC: immunocompetent; HIV: human immunodeficiency virus; CNS: central nervous system; PTLD: post-transplant lymphoproliferative disorder; DLBCL: diffuse large B-cell lymphoma; TMB: tumor mutational burden.

Figure 5.

The intra-tumoral T-cell receptor repertoire diversity does not differ between Epstein-Barr virus-positive and -negative non-Hodgkin lymphomas. The number of unique productive T-cell receptor-β clonotypes according to Epstein-Barr virus status (left), immune status (middle) and disease localization (right). Wilcoxon test. TCR: T-cell receptor; EBV: Epstein-Barr virus; NHL: non-Hodgkin lymphoma; immunodeficient; IC: immunocompetent; CNS: central nervous system.

Figure 6.

Epstein-Barr virus drives the tumor microenvironment in non-Hodgkin lymphoma in immunosuppressed and immunocompetent patients. (A) Cell type abundance assessed with CIBERSORTx, according to Epstein-Barr virus (EBV) status. Wilcoxon test. (B) Correlation study between tumor mutational burden and memory resting CD4 T cells and CD8 T cells within EBV⁺ non-Hodgkin lymphoma (green circles, upper) and EBV^– non-Hodgkin lymphoma (red circles, lower). Spearman correlation. NHL: non-Hodgkin lymphoma; NK: natural killer.

Figure 7.

Gene expression profiling tends to differ between Epstein-Barr virus-positive and -negative non-Hodgkin lymphoma. Single sample gene set enrichment analysis (ssGSEA) scores of negative immune stimulation (left panel), positive immune stimulation (middle panel) and T-cell function (right panel). ssGSEA calculates separate enrichment scores for each pairing of a sample and gene set. Each ssGSEA enrichment score represents the degree to which the genes in a particular gene set are coordinately up- or down-regulated within a sample. Wilcoxon test. EBV: Epstein-Barr virus; NHL: non-Hodgkin lymphoma.