Classic Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells within a T-cell-rich tumor microenvironment (TME). Epstein-Barr virus (EBV) is present in ∼25% of cases, but its contribution to pathogenesis and immunomodulation remains unclear due to technical barriers. Using complementary spatial proteomics and transcriptomics across multi-institutional cohorts, we systematically map key EBV-linked TME reorganization. EBV-positive cHL exhibits distinct immunological features, including memory CD8 T cell enrichment, heightened T cell dysfunction spatially correlated with HRS proximity, and terminally exhausted T cell signatures contrasting with progenitor-exhausted patterns in EBV-negative disease. We identify EBV-encoded LMP1 as a factor in T cell dysfunction through enhanced HRS:CD8 interactions, and its expression level correlates with T cell terminal exhaustion in a distance-dependent manner. This spatial framework dissects viral-mediated immune evasion in the cHL TME, highlighting potential therapeutic opportunities to target virus-associated T cell dysfunction for precision immunotherapy in virus-associated malignancies.

Introduction: Newly diagnosed (ND) diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that includes at least 5 molecular clusters (C1-C5) defined by distinct genetic signatures and responses to standard induction therapy. The clusters with inferior responses to frontline R-CHOP include: ABC-enriched C5 DLBCLs, characterized by frequent MYD88L265Pand CD79B mutations, 18q copy number gain, and extranodal tropism; GCB-predominant C3 tumors with BCL2 translocations, including a subset with concurrent MYC translocations, and mutations in chromatin modifiers, B-cell transcription factors, and PI3K pathway components; and cell-of-origin independent C2 DLBCLs with biallelic TP53 alterations and associated genomic instability (AGI). In contrast, C1 and C4 DLBCLs have more favorable responses to R-CHOP. We recently developed a neural network-based probabilistic molecular classifier, DLBclass, to prospectively assign tumors to their respective clusters (Chapuy et al., Blood 2025). Despite the interest in matching targeted therapies with molecularly defined subsets of ND DLBCL, novel agents are initially evaluated in patients (pts) with relapsed (R) tumors that have incompletely characterized genetic signatures. Herein, we investigate the molecular substructure of R DLBCL using the DLBclass framework. Methods: We performed whole exome sequencing and obtained the comprehensive genetic signatures (mutations [SNVs and indels], somatic copy number alterations [SCNAs], structural variants [SV] including translocations) and DLBclass assignments for 122 first-relapsed DLBCLs including 30 central nervous system (CNS) R, and a subset (42) of matched diagnostic (Dx) specimens from pts who were treated with anthracycline-based induction chemo-immunotherapy and had full clinical annotation.Our previously characterized cohort of ND DLBCLs and the NIH series with defined genetic signatures, DLBclass calls,and known clinical outcomes were used for comparison. Results: We used MutSig2CV to identify recurrent mutations in R DLBCL and found that ~80% of the alterations were DLBclass-defined features of ND DLBCL. This suggested that the propensity to relapse may be largely predetermined at diagnosis and provided the rationale for applying DLBclass in the R setting. In comparison to ND DLBCLs, R DLBCLs were enriched for C2 tumors (28.5% [ND] vs 41.8% [R], p=0.007), with a paucity of C1 lymphomas (15.5% [ND] vs 5.7% [R], p=0.004). Extranodal (EN) Rs, including CNS Rs, were more likely than nodal (N) Rs to be C5 DLBCLs (32.2% [EN] vs 3.65% [N], p=0.002). In contrast, N Rs were more likely to be C3 tumors (11.8% [EN] vs 30.8% [N], p=0.01). These findings link specific patterns of tissue tropism with distinct genetic programs. Of interest, late R (>24 mos) were enriched for C5 DLBCLs in comparison to early R (<12 mos) (35.3% vs 18.9%, p=0.06). Among pts with paired Dx and R samples, 31/42 (74%) retained their original molecular cluster IDs at R, whereas 8 of the remaining 11 acquired features of increased TP53-AGI and C2 cluster IDs. The higher frequency of TP53-AGI in R DLBCLs prompted us to further characterize the genes perturbed by focal CNAs in this setting. The more frequent focal CNAs at R included known immune response modulators and targets of inactivating mutations – CD70 (19p13.3del), B2M (15q15.3del), MHCI (6p21.33del), CD58 (1p13.1del), and FAS (10q23.31del). These findings potentially link TP53-AGI with additional CNA-dependent mechanisms of immune evasion at R. We also identified Dx features associated with an increased risk of R using the previously characterized ND DLBCLs from our earlier analyses, the NIH cohort and this series, annotated for outcome (R [250 tumors] vs no R [249 DLBCLs]). Chromosome 17pdel, TP53mut, and 6p23.33del (MHCI) were significantly more frequent in Dx tumors of pts who subsequently relapsed, further emphasizing the importance of TP53-AGI and MHCI loss. In a multivariate Cox model adjusted for DLBclass IDs, concurrent MYC and BCL2 SVs (HR=3.39) and MYC SVs (HR=2.33) retained adverse prognostic significance, highlighting the additional impact of these alterations on outcome. Conclusions: Relapse in DLBCL is largely driven by genetic programs present at diagnosis and defined by DLBclass. These signatures influence the clinical course of relapse, including tissue tropism, timing and immune escape, providing a framework for improved risk assessment and therapeutic targeting.

Hodgkin Reed-Sternberg (HRS) cells of classic Hodgkin lymphoma (cHL), like many solid tumors, elicit ineffective immune responses. However, patients with cHL are highly responsive to PD-1 blockade, which largely depends on HRS cell-specific retention of MHC class II and implicates CD4+ T cells and additional MHC class I-independent immune effectors. Here, we utilize single-cell RNA sequencing and spatial analysis to define shared circulating and microenvironmental features of the immune response to PD-1 blockade in cHL. Compared with non-responders, responding patients have more circulating CD4+ na & iuml;ve and central memory T cells and B cells, as well as more diverse CD4+ T cell and B cell receptor repertoires. Importantly, a population of circulating and tumor-infiltrating IL1 beta+ monocytes/macrophages is detectable in patients with cHL but not healthy donors, and a proinflammatory, tumor-promoting signature of these circulating IL1 beta+ monocytes is associated with resistance to PD-1 blockade in cHL. Altogether, our findings reveal extensive immune rewiring and complementary roles of CD4+ T cells, B cells and IL1 beta+ monocytes in the response to PD-1 blockade and suggest that these features can be captured with a peripheral blood test.

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in 25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial pro-teomics on 6 EBV-positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tu-mor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV-positive and EBV-negative cases that differently impact HRS cell proliferation, tumor-immune interactions, and mecha-nisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular fac-tors of virus-associated tumors, with potential for targeted therapeutic strategies.

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4 + T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA and scT-cell receptor (TCR) sequencing to characterize the peripheral immune response to PD-1 blockade and define non-CD8 + T-cell dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL who were treated with PD-1 blockade on the CheckMate 205 clinical trial (at cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed (ND), previously untreated cHL and 13 healthy donors. We first analyzed circulating CD4 + T cells given their likely importance in the response to PD-1 blockade in cHL. We found that patients with R/R cHL had markedly lower baseline TCR diversity than healthy donors across the entire CD4 + naïve/central memory (CM) T-cell space. Additionally, patients with the most favorable responses to PD-1 blockade had significantly increased CD4 + naïve/CM TCR diversity at baseline (C1D1) and more abundant naïve/CM subsets on treatment (C4D1). These response-related differences likely reflect a continued capacity to mount CD4 + T-cell responses to neoantigens. We also identified several circulating CD4 + T-cell populations that were significantly more abundant in patients with cHL than in healthy donors, including follicular helper-like T cells, IFN-stimulated cytotoxic T cells and actively proliferating CTLA4 + LAG3 + T cells. Compared to healthy donors, all evaluated patients with cHL had significantly reduced numbers of classically defined circulating NK cells and an expanded population of IFN-stimulated NK cells with likely reduced cytotoxic potential. In addition to marked deregulation of the NK cell compartment, all patients with cHL had decreased numbers of circulating B cells at all stages of differentiation. We used the scRNA sequencing data to reconstruct individual B-cell receptor (BCR) sequences with the TRUST4 algorithm and found that the numbers of total and unique BCRs, as well as chao1 diversity, were significantly decreased in patients with ND cHL in comparison to healthy donors. Additionally, patients with the most favorable responses to PD-1 blockade had significantly higher baseline BCR diversity and circulating B-cell numbers. Altogether, these data underscore the likely importance of B cell-mediated immune responses in cHL, either directly or via Fc-binding of innate effectors. Finally, the most abundant circulating CD3 - population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumor-promoting cytokines/chemokines, as well as PD-L1 and SIRPa. These inflamed IL1β + monocytes were virtually absent from the blood of healthy donors. Using RNAscope, we also detected macrophages with similar features in the intact cHL tumor microenvironment, in the immediate proximity of malignant HRS cells. To assess the broader significance of the inflamed IL1β + monocytes, we interrogated a single-cell compendium of monocytes and macrophages from multiple solid tumors and identified a population of monocytes/ macrophages with a similar transcriptional signature. These results suggest that similarly programmed inflammatory monocytes/macrophages are detectable in multiple cancers. We next assessed the clinical significance of circulating inflamed IL1β + monocytes in patients with R/R cHL who were treated with PD-1 blockade. Patients who did not respond to PD-1 blockade expressed significantly higher levels of the inflamed IL1β + monocyte transcriptional signature which led to the development of a predictive assay. We then identified a comparable circulating monocyte population with the same transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor, renal cell carcinoma, which underscored the broad significance of these findings. Taken together, our findings highlight the likely complementary role of CD4 + T cells, B cells and cancer-associated monocytes in the response to PD-1 blockade in cHL.

Diffuse large B-cell lymphoma (DLBCL) not otherwise specified is the most common aggressive non-Hodgkin lymphoma and a biologically heterogeneous disease. Despite the development of effective immunotherapies, the organization of the DLBCL tumor-immune microenvironment (TIME) remains poorly understood.We interrogated the intact TIME of 51 de novo DLBCLs with triplicate sampling to characterize 337 995 tumor and immune cells using a 27-plex antibody panel that captured cell lineage, architectural, and functional markers. We spatially assigned individual cells, identified local cell neighborhoods, and established their topographical organization in situ. We found that the organization of local tumor and immune cells can be modeled by 6 composite cell neighborhood types (CNTs). Differential CNT representation divided cases into 3 aggregate TIME categories: immune-deficient, dendritic cell-enriched (DC-enriched), and macrophage-enriched (Mac-enriched). Cases with immune-deficient TIMEs have tumor cell-rich CNTs, in which the few infiltrating immune cells are enriched near CD31+ vessels, in keeping with limited immune activity. Cases with DC-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD11c+ DCs and antigen-experienced T cells also enriched near CD31+ vessels, in keeping with increased immune activity. Cases with Mac-enriched TIMEs selectively include tumor cell-poor/immune cell-rich CNTs with high numbers of CD163+ macrophages and CD8 T cells throughout the microenvironment, accompanied by increased IDO-1 and LAG-3 and decreased HLA-DR expression and genetic signatures in keeping with immune evasion. Our findings reveal that the heterogenous cellular components of DLBCL are not randomly distributed but organized into CNTs that define aggregate TIMEs with distinct cellular, spatial, and functional features.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin and Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4+ T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA sequencing to characterize the peripheral immune response to PD-1 blockade and more broadly define non-CD8+ dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL treated with PD-1 blockade (nivolumab) on the CheckMate 205 clinical trial (cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed, previously untreated cHL and 13 healthy donors. Unlike healthy donors, all evaluated patients with cHL had circulating IFN stimulated adaptive and innate populations, including several distinct CD4+ T-cell effectors and an NK cell subset with reduced cytotoxic potential, and decreased numbers of B cells at all stages of differentiation. Patients with the most favorable responses to PD-1 blockade had: 1) significantly increased CD4+ T-cell receptor diversity and more abundant naïve/ central memory subsets; and 2) significantly higher B-cell receptor diversity and increased numbers of circulating B cells. The most abundant circulating CD3- population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumorigenic cytokines and chemokines, PD-L1 and SIRPa. This newly identified monocytic population was virtually absent from the blood of healthy donors. RNAscope analysis of the intact tumor microenvironment localized these tumor-infiltrating monocytes/macrophages to the immediate proximity of HRS cells. Monocytes from patients whose disease progressed following PD-1 blockade expressed significantly higher levels of immunosuppressive cytokine/chemokine signature which led to the development of a predictive transcriptional assay. We identified a comparable circulating monocyte population and transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor underscoring the broad-based significance of these findings.

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1(+) T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types.

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation.

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.

Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin-Sca1+C-kit+CD150-CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

PURPOSE:Axicabtagene ciloleucel (axi-cel) was approved by the Food and Drug Administration for relapsed aggressive B-cell non-Hodgkin lymphoma in part on the basis of durable remission rates of approximately 40% in a clinical trial population. Whether this efficacy, and the rates of toxicity, would be consistent in a postcommercial setting, with relaxed eligibility criteria and bridging therapy, is unknown. This study describes the efficacy and safety correlates and outcomes in this setting. PATIENTS AND METHODS:One hundred twenty-two patients from 7 medical centers in the United States were treated with axi-cel and were included in a modified intent-to-treat (mITT) analysis. Seventy-six patients (62%) were ineligible for the ZUMA-1 trial. Response and toxicity rates, duration of response (DOR), survival, and covariates are described on the basis of the mITT population. Correlative studies on blood and tumor samples were performed to investigate potential biomarkers of response and resistance. RESULTS:Median follow-up was 10.4 months. In the mITT population, the best overall and complete response (CR) rates were 70% and 50%, respectively. Median DOR and progression-free survival (PFS) were 11.0 and 4.5 months in all patients and were not reached (NR) in CR patients. Median overall survival (OS) was NR; 1-year OS was 67% (95% CI, 59% to 77%). Although response rates were similar in the ZUMA-1-eligible and ZUMA-1-ineligible groups (70% v 68%), there was a statistically significant improvement in CR rate (63% v 42%, P = .016), DOR (median, NR v 5.0 months; P = .014), PFS (median, NR v 3.3 months; P = .020), and OS (1-year OS, 89% v 54%; P < .001) in patients who were ZUMA-1 eligible. Rates of grade ≥ 3 cytokine release syndrome and neurotoxicty were 16% and 35%, respectively. CONCLUSION:Axi-cel yields similar rates of overall response and toxicity in commercial and trial settings, although CR rates and DOR were more favorable in patients eligible for ZUMA-1.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.