Introduction: Hodgkin lymphoma (HL) is deficient in major histocompatibility complex class I, rendering it susceptible to antitumoral immunity by natural killer (NK) cells. Despite the functional impairment of PD-1+ NK cells in HL, the underlying mechanisms of NK cell dysfunction remain unclear. Methods: This study involved 14 HL patients and SNK10/KHYG-1 cell lines to assess NK cell activation against cancer cells. Activation was measured through transcript (PCR) and protein expression (flow cytometry). Regulatory mechanisms associated with IRE1α activation were validated through knockdown and luciferase reporter assays. Results: Our findings reveal a novel role for IRE1α-endonuclease in fine-tuning NK cell effector functions by orchestrating the XBP1s/microRNA-34a-5p/PD-1 axis. When NK cells encounter cancer cells, IRE1α endonuclease activates the decay of microRNA-34a-5p, resulting in increased expression of XBP1s and PD-1. IRE1α-endonuclease activation enhances NK cell functions while promoting PD-1 expression. In turn, PD-1 is directly regulated by microRNA-34a-5p, which binds to the 3′UTR of PD-1 transcript to repress PD-1 protein on the NK cell surface. Importantly, IRE1α-pathway activation is impaired in NK cells from HL patients. Conclusion: The IRE1α endonuclease emerges as a key player, simultaneously regulating the XBP1s/microRNA-34a-5p/PD-1 axis in NK cells, a process disrupted in HL. Targeting the IRE1α-pathway holds promise as a therapeutic strategy to optimize NK cell functions in Hodgkin lymphoma treatments.

PDF file - 36K, Sensitivity and specificity of sCD163 and sTARC as biomarkers in cHL. (A-D) Receiver Operating Characteristic (ROC) plots demonstrate high sensitivity and specificity of (A) sCD163 pre-therapy cHL versus healthy participant; (B) sCD163 pre-therapy cHL versus CR six months post-therapy; (C) sTARC pre-therapy cHL versus healthy participants; (D) sTARC pre-therapy cHL versus CR six months post-therapy. AUC, area under the curve.

Activation of the IRE1α-endonuclease is critical for Natural Killer (NK)-cell function. We describe a novel regulatory role for IRE1α-endonuclease in fine-tuning NK-cell effector functions through an inter-connected activation of the transcription factor XBP1s and inhibition of microRNA-34a-5p (miR-34a-5p) to modulate PD-1 immune checkpoint expression. NK-cells, when exposed to cancer cells, activate IRE1α-endonuclease mediated decay of miR-34a-5p. This reduces miR-34a-5p and consequently increases the expression of the target genes XBP1 and PD-1. IRE1α-endonuclease activation not only enhances NK-cell effector function but also promotes PD-1 expression. PD-1 is itself directly regulated by miR-34a-5p, which binds to the 3’UTR of PD-1 messenger RNA to repress PD-1 protein at the NK-cell surface. IRE1α-pathway activation is impaired in the NK-cells of patients with Hodgkin Lymphoma, and miR-34a-5p and PD-1 expression are inversely correlated. The IRE1α-pathway plays a dual role in regulating the XBP1/miRNA-34a axis and PD-1 expression within NK-cells, that is disrupted in cancer patients.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV− HIV− PCNSL and 2 EBV− HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV− PCNSL. As with prior studies, EBV− HIV− PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV− PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV− HIV− PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.

A growing body of evidence suggests that macrophage immune checkpoint molecules are potential targets in the era of cancer immunotherapy. Here we showed that extracellular adenosine, an abundant metabolite in the tumor microenvironment, critically impedes the therapeutic efficacy of anti-CD20 monoclonal antibodies (mAbs) against B-cell lymphoma. Using a syngeneic B-cell lymphoma model, we showed that host deficiency of adenosine 2A receptor (A2AR), but not A2BR, remarkably improved lymphoma control by anti-CD20 mAb therapy. Conditional deletion of A2AR in myeloid cells, and to a lesser extent in NK cells, augmented therapeutic efficacy of anti-CD20 mAb. Indeed, adenosine signaling impaired antibody-mediated cellular phagocytosis (ADCP) by macrophages and limited the generation of anti-lymphoma CD8(+)T cells. Pharmacological inhibition of A2AR overcame the adenosine-mediated negative regulation of ADCP by rituximab in a xeno-transplanted lymphoma model. Moreover, aberrant overexpression of CD39, an apical ecto-enzyme for adenosine generation, showed a negative impact on prognosis in patients with diffuse large B-cell lymphoma, as well as on preclinical efficacy of rituximab. Together, adenosine acts as a "don't eat me signal", and may be a potential target to harness anti-lymphoma immunity.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes antitumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8(+) TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226(hi) TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226(+)CD8(+) T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.

NK cells are a heterogeneous population of innate lymphocytes whose potent anti-cancer properties make them ideal candidates for cellular therapeutic application. However, our lack of understanding of the role of NK cell diversity in anti-tumor responses has hindered advances in this area. In this study, we describe a new CD56dim NK cell subset characterized by the lack of expression of DNAX-accessory molecule 1 (DNAM-1). Compared with CD56bright and CD56dimDNAM-1pos NK cell subsets CD56dimDNAM-1neg NK cells displayed reduced motility, poor proliferation, lower production of IFNγ and limited killing capacities. Soluble factors secreted by CD56dimDNAM-1neg NK cells impaired CD56dimDNAM-1pos NK cell-mediated killing, indicating a potential inhibitory role for the CD56dimDNAM-1neg NK cell subset. Transcriptome analysis revealed that CD56dimDNAM-1neg NK cells constitute a new mature NK cell subset with a specific gene signature. Upon in vitro cytokine-stimulation, CD56dimDNAM-1neg NK cells were found to differentiate from CD56dimDNAM-1pos NK cells. Finally, we report a dysregulation of NK cell subsets in the blood of patients diagnosed with Hodgkin lymphoma and diffuse large B cell lymphoma, characterized by decreased CD56dimDNAM-1pos/ CD56dimDNAM-1neg NK cell ratios and reduced cytotoxic activity of CD56dimDNAM-1pos NK cells. Altogether, our data offer a better understanding of human peripheral blood NK cell populations and have important clinical implications for the design of NK cell-targeting therapies.

PURPOSE Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL. PATIENTS AND METHODS Digital gene expression using a custom code set—five immune effector, six immune checkpoint, one macrophage molecules—was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more. RESULTS Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltrationHI (ie, high PD-L2) FL biopsies from immune infiltrationLO (ie, low PD-L2) tumors. Immune infiltrationHI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltrationLO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile. CONCLUSION Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

Epstein-Barr virus-positive (EBV+) diffuse large B-cell lymphomas (DLBCLs) express high levels of programmed death ligand 1 (PD-L1) and PD-L2. MicroRNA (miR) regulation is an important mechanism for the fine-tuning of gene expression via 3'-untranslated region (3'UTR) targeting, and we have previously demonstrated strong EBV miR expression in EBV+ DLBCL. Whereas the EBV latent membrane protein-1 (LMP1) is known to induce PD-L1/L2, a potential counterregulatory role of EBV miR in the fine-tuning of PD-L1/L2 expression remains to be established. To examine this, a novel in vitro model of EBV+ DLBCL was developed, using the viral strain EBV WIL, which unlike common laboratory strains retains intact noncoding regions where several EBV miRs reside. This enabled interrogation of the relationship among EBV latency genes, cell of origin (COO), PD-L1, PD-L2, and EBV miRs. The model successfully recapitulated the full spectrum of B-cell differentiation, with 4 discrete COO phases: early and late germinal center B cells (GCBs) and early and late activated B cells (ABCs). Interestingly, PD-L1/L2 levels increased markedly during transition from late GCB to early ABC phase, after LMP1 upregulation. EBV miR-BamHI fragment H rightward open reading frame 1 (BHRF1)-2-5p clustered apart from other EBV miRs, rising during late GCB phase. Bioinformatic prediction, together with functional validation, confirmed EBV miR-BHRF1-2-5p bound to PD-L1 and PD-L2 3'UTRs to reduce PD-L1/L2 surface protein expression. Results indicate a novel mechanism by which EBV miR-BHRF1-2-5p plays a context-dependent counterregulatory role to fine-tune the expression of the LMP1-driven amplification of these inhibitory checkpoint ligands. Further identification of immune checkpoint-targeting miRs may enable potential novel RNA-based therapies to emerge.

The upregulation of inhibitory molecules is a major driver of immune escape in cancer. In contrast, less is known about the regulation of T cell activating receptors within the tumor microenvironment. Here, we describe that tumor derived CD155, a transmembrane glycoprotein of the immunoglobulin superfamily, downregulates the activating receptor CD226 (DNAM-1) in mouse and human CD8+ T cells, leading to profound loss of effector function and cancer immune evasion. Mechanistically, CD155 drives internalisation of CD226 through phosphorylation of tyrosine 319 (CD226Y319). Accordingly, in T cells a mutation in Y319 led to increased CD226 surface expression improving the efficacy of cancer immunotherapies in mouse models of cancer. In pre-treatment samples from melanoma patients, CD226+ CD8+ T cells significantly correlated with response to immunotherapy and survival. In summary, we discovered a new paradigm in which tumor cells escape destruction by T cells through targeted downregulation of an activating receptor.

Background The frequent loss of Major Histocompatibility Complex I molecule (MHC-I) on Hodgkin/Reed-Sternberg (HRS)-cells renders them susceptible to Natural Killer (NK) cell-mediated lysis in Hodgkin Lymphoma (HL). Optimal NK cell function involves migration from peripheral blood to sites of disease, formation of an immune synapse (NKIS) between NK cells and HRS cells, and release of effector molecules. We recently showed that PD-1+ NK cells are expanded in the circulation of patients with HL (Vari, F Blood 2018), and that PD-1 blockade enhances their anti-HRS capabilities. However, mechanisms behind the functional impairment of NK cells in HL patients and the impact PD-1 blockade has on NK cell function remain to be established. Although the IRE1-XBP1s pathway, part of the unfolded protein response (UPR) system, has established and fundamental roles in macrophage, B, T and dendritic cells homeostatic function, its involvement in NK cells remains unknown. We hypothesized that IRE1-XBP1s dysfunction contributes to NK cell impairment and tested the impact of PD-1 blockade on individual components of NK cell function, including migration, NKIS formation, and cytokine release. Methods Ex-vivo functional assays were performed on blood from 20 participants. Confocal microscopy, time-lapse imaging, trans-well migration, and functional in-vitro immune assays were utilized on a range of NK and HRS cell lines, with and without IRE1-XBP1s small molecule inhibitors (4µ8c and 6-bromo) and/or PD-1 blockade (pembrolizumab). Results Stimulation of both NK cell lines and primary NK cells, with HRS lines resulted in marked and rapid IRE1-XBP1s pathway activation. This occurred independently of the canonical UPR and was associated with increased NK cell effector function. However, IRE1-XBP1s pathway inhibition resulted in aberrant NK cell morphology, reduced motility and migration, deficient NKIS formation and impaired interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) release. Next, we tested the IRE1-XBP1s pathway in the pre-therapy blood of patients with HL and compared this with healthy age/gender matched controls. Strikingly, following co-culture with an HRS-line the pathway was not activated, but this abnormality was restricted to the CD56brightCD16-ve subset that we have previously shown to be expanded and enriched in PD-1 (as well as downregulation of the lymphoid migratory chemokine CCR7) in patients with HL. In subsequent experiments using in-vitro expanded populations of primary NK cells from HL patients, IRE1-XBP1s pathway inhibition impaired the migration, NKIS formation, CD107a degranulation, and secretion of IFNγ and TNFα. Effects were partially but not completely restored by addition of PD-1 blockade (Fig 1). Conclusion Here, we outline a hitherto unrecognized mechanism involving the IRE1-XBP1s pathway that is pivotal to NK cell function, including the relatively poorly understood processes of migration, NKIS formation, and cytokine secretion. Notably, IRE1-XBP1s pathway activation is dysfunctional within the PD-1 enriched CD56brightCD16- NK cell subset. Although PD-1 blockade appears to have a multi-faceted beneficial role on NK cell migrational/NKIS and cytokine release capabilities, it is still only capable of partial restoration of NK cell effector function. Further understanding of the pathways operative in NK cells may result in improved immunotherapeutic strategies to enhance this arm of the immune response in patients with HL. Disclosures Gandhi: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

Introduction: Primary CNS Lymphomas (PCNSL) with DLBCL histology in the immunosuppressed, e.g. after HIV (HIV+ PCNSL) or organ transplant (EBV+ PCNSL-PTLD: where anti-PD-1 is contraindicated), are characterized by dismal outcome and almost universal EBV positivity. However, incidence is low, biopsy material limited and immunogenetic characterization minimal. We provide detailed data (308 patients) comparing the genetic landscape and tumor microenvironment (TME) of EBV+ PCNSL-PTLD (22); HIV+ PCNSL (24); EBV- PCNSL (41); EBV- systemic (sy) DLBCL (199) and EBV+ syPTLD (22), which led to implementation of a rationally designed combination therapy. Methods: Targeted sequencing, CNV analysis, nanoString (for immune checkpoints and effectors, and macrophage gene expression) and in vitro assays were used. Based on the findings, a novel regimen was administered to patients’ refractory to or unsuitable for frontline therapy. Sequential imaging, pharmacokinetic (PK) and T cell assays were performed. Results: Mutational burden was much lower in EBV+ lymphomas than EBV- lymphomas (p<0.0001). Notably, genetic aberrations in BCR-NFB were rarely observed in EBV+ PCNSL-PTLD and HIV+ PCNSL, and (unlike EBV- PCNSL) in these patients, HLAI/II copy number loss was largely absent. Mutations in CARD11 (which confers ibrutinib resistance) were also rare in EBV+ PCNSL-PTLD and HIV+ PCNSL. By nanoString analysis, EBV+ PCNSL-PTLD expressed higher levels of the immunosuppressive ‘M2’ macrophage marker CD163 and LAG3 and PD-L1/L2 (p<0.001), and high levels of EBV protein LMP1 (known to upregulate PD-L1/L2 and NFB). In vitro co-incubation experiments showed a PCNSL line upregulated CD163+ PD-L1/L2+ M2 polarized monocyte/macrophages relative to a systemic DLBCL line (≥7-fold, p<0.001), and EBV infection of a PCNSL line enhanced NFB activity (Fig 1A). Three immunosuppressed patients (2 frontline, 1 refractory, ages 30-70yrs) with EBV+ lymphoma (2 PCNSL, 1 CNS + systemic) were treated with ibrutinib (starting dose 560mg) and 3rd party partially HLA matched EBV specific CTL. PK confirmed therapeutic CSF levels, including a patient on haemodialysis. CD8 T cells specific for EBV antigens present in PCNSL were detectable post infusion and had an effector memory phenotype (Fig 1B-C). Microchimerism post infusion was confirmed by ultra-sensitive ddPCR. Toxicity was manageable and all 3 are alive (2CR, 1PR). Keywords: Epstein-Barr virus (EBV); post-transplant lymphoproliferative disorders (PTLDs); primary CNS lymphoma (PCNSL). Disclosures: Gandhi, M: Consultant Advisory Role: Celgene, Merck Sharpe & Dohme, Janssen-Cilag, Gilead, Bristol Myers Squibb; Honoraria: Amgen, Janssen-Cilag, Gilead, Roche, Merck Sharpe & Dohme, Takeda; Research Funding: Celgene, Bristol Myers Squibb, Janssen-Cilag, Gilead; Other Remuneration: Roche. Tobin, J: Honoraria: Amgen, Janssen – conference attendance 2017. Trappe, R: Consultant Advisory Role: Abbvie, Atara; Research Funding: Hoffmann-La Roche; Other Remuneration: Travel support from Celgene, Janssen, Gildead, Abbvie. Blyth, E: Consultant Advisory Role: Abbvie, MSD, Novartis. Wight, J: Honoraria: Janssen, Alexion. Keane, C: Consultant Advisory Role: Celgene, Gilead, MSD; Other Remuneration: Roche (Conference Travel).

Natural killer (NK) cells are a heterogeneous population of innate lymphocytes whose potent anticancer properties make them ideal candidates for cellular therapeutic application. However, our lack of understanding of the role of NK cell diversity in antitumor responses has hindered advances in this area. In this study, we describe a new CD56dim NK cell subset characterized by the lack of expression of DNAX accessory molecule-1 (DNAM-1). Compared with CD56bright and CD56dimDNAM-1pos NK cell subsets, CD56dimDNAM-1neg NK cells displayed reduced motility, poor proliferation, lower production of interferon-γ, and limited killing capacities. Soluble factors secreted by CD56dimDNAM-1neg NK cells impaired CD56dimDNAM-1pos NK cell-mediated killing, indicating a potential inhibitory role for the CD56dimDNAM-1neg NK cell subset. Transcriptome analysis revealed that CD56dimDNAM-1neg NK cells constitute a new mature NK cell subset with a specific gene signature. Upon in vitro cytokine stimulation, CD56dimDNAM-1neg NK cells were found to differentiate from CD56dimDNAM-1pos NK cells. Finally, we report a dysregulation of NK cell subsets in the blood of patients diagnosed with Hodgkin lymphoma and diffuse large B-cell lymphoma, characterized by decreased CD56dimDNAM-1pos/CD56dimDNAM-1neg NK cell ratios and reduced cytotoxic activity of CD56dimDNAM-1pos NK cells. Altogether, our data offer a better understanding of human peripheral blood NK cell populations and have important clinical implications for the design of NK cell-targeting therapies.

MicroRNA (miRNA)s are dysregulated in Diffuse large B-cell lymphoma (DLBCL), where they reflect the malignant B-cells and the immune infiltrate within the tumor microenvironment. There remains a paucity of data in DLBCL regarding cell-free (c-f) miRNA as disease response biomarkers. Immunosuppressive monocyte/macrophages, which are enriched in DLBCL, are disease response markers in DLBCL, with miRNA key regulators of their immunosuppressive function. Our aim was to determine whether plasma miRNA that reflect the activity of the malignant B-cell and/or immunosuppressive monocytes/macrophages, have value as minimally-invasive disease response biomarkers in DLBCL. Quantification of 99 DLBCL tissues, to select miRNA implicated in immunosuppressive monocytes/macrophage biology, found miR-494 differentially elevated. In a discovery cohort (22 patients), pre-therapy c-f miR-494 and miR-21 but not miR-155 were raised relative to healthy plasma. Both miR-494 and miR-21 levels 3-6 months reduced post immuno-chemotherapy. The validation cohort (56 patients) was from a prospective clinical trial. Interestingly, in sequential samples both miRNAs decreased in patients becoming Positron Emission Tomography/Computerized Tomography (PET/CT)-ve, but not in those remaining interim-PET/CT+. Patient monocytes were phenotypically and functionally immunosuppressive with ex-vivo monocyte depletion enhancing T-cell proliferation in patient but not healthy samples. Pre-therapy monocytes showed an immunosuppressive transcriptome and raised levels of miR-494. MiR-494 was present in all c-f nanoparticle fractions but was most readily detectable in unfractionated plasma. Circulating c-f miR-494 and miR-21 are disease response biomarkers with differential response stratified by interim-PET/CT in patients with DLBCL. Further studies are required to explore their manipulation as potential therapeutic targets.

Much focus has been on the interaction of programmed cell death ligand 1 (PD-L1) on malignant B cells with programmed cell death 1 (PD-1) on effector T cells in inhibiting antilymphoma immunity. We sought to establish the contribution of natural killer (NK) cells and inhibitory CD163+ monocytes/macrophages in Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL). Levels of PD-1 on NK cells were elevated in cHL relative to DLBCL. Notably, CD3-CD56hiCD16-ve NK cells had substantially higher PD-1 expression relative to CD3-CD56dimCD16+ cells and were expanded in blood and tissue, more marked in patients with cHL than patients with DLBCL. There was also a raised population of PD-L1-expressing CD163+ monocytes that was more marked in patients with cHL compared with patients with DLBCL. The phenotype of NK cells and monocytes reverted back to normal once therapy (ABVD [doxorubicin 25 mg/m2, bleomycin 10 000 IU/m2, vinblastine 6 mg/m2, dacarbazine 375 mg/m2, all given days 1 and 15, repeated every 28 days] or R-CHOP [rituximab 375 mg/m2, cyclophosphamide 750 mg/m2 IV, doxorubicin 50 mg/m2 IV, vincristine 1.4 mg/m2 (2 mg maximum) IV, prednisone 100 mg/day by mouth days 1-5, pegfilgrastim 6 mg subcutaneously day 4, on a 14-day cycle]) had commenced. Tumor-associated macrophages (TAMs) expressed high levels of PD-L1/PD-L2 within diseased lymph nodes. Consistent with this, CD163/PD-L1/PD-L2 gene expression was also elevated in cHL relative to DLBCL tissues. An in vitro functional model of TAM-like monocytes suppressed activation of PD-1hi NK cells, which was reversed by PD-1 blockade. In line with these findings, depletion of circulating monocytes from the blood of pretherapy patients with cHL and patients with DLBCL enhanced CD3-CD56hiCD16-ve NK-cell activation. We describe a hitherto unrecognized immune evasion strategy mediated via skewing toward an exhausted PD-1-enriched CD3-CD56hiCD16-ve NK-cell phenotype. In addition to direct inhibition of NK cells by the malignant B cell, suppression of NK cells can occur indirectly by PD-L1/PD-L2-expressing TAMs. The mechanism is more prominent in cHL than DLBCL, which may contribute to the clinical sensitivity of cHL to PD-1 blockade.

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production in vitro, but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.

Primary CNS Lymphoma (PCNSL) with diffuse large B-cell lymphoma (DLBCL) histology occurring after organ transplantation (EBV+ PCNSL-PTLD) is characterized by extremely poor outcome, almost universal EBV-positivity and late presentation relative to systemic PTLD (Fink, AJT 2012; Evens, AJT 2013). However, as the incidence is low and biopsy material limited, characterization of its immunobiology is minimal. Furthermore, there is no comparative data with EBV+ DLBCL occurring as systemic PTLD (EBV+ syDLBCL-PTLD), or with EBV-ve DLBCL in the non-PTLD setting occurring either as PCNSL (EBV-ve PCSNL) or systemically (EBV-ve syDLBCL).