Search

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.

PDF file - 36K

Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival.Experimental Design: We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline “R-CHOP” therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS).Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%–79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%–88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%–76.6%) versus 72.6% (95% CI, 58.8%–82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV+ DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules.Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820–8. ©2016 AACR.

Individual results of TCR sequencing for all DLBCL and non-diseased nodal tissues.

Purpose: Candidate circulating disease response biomarkers for classical Hodgkin lymphoma (cHL) might arise from Hodgkin–Reed–Sternberg (HRS) cells or nonmalignant tumor-infiltrating cells. HRS cells are sparse within the diseased node, whereas benign CD163+ M2 tissue-associated macrophages (TAM) are prominent. CD163+ cells within the malignant node may be prognostic, but there is no data on serum CD163 (sCD163). The HRS-specific serum protein sTARC shows promise as a disease response biomarker. Tumor-specific and tumor-infiltrating circulating biomarkers have not been compared previously.Experimental Design: We prospectively measured sCD163 and sTARC in 221 samples from 47 patients with Hodgkin lymphoma and 21 healthy participants. Blood was taken at five fixed time-points prior, during, and after first-line therapy. Results were compared with radiological assessment and plasma Epstein-Barr virus DNA (EBV-DNA). Potential sources of circulating CD163 were investigated, along with immunosuppressive properties of CD163.Results: Pretherapy, both sCD163 and sTARC were markedly elevated compared with healthy and complete remission samples. sCD163 better reflected tumor burden during therapy, whereas sTARC had greater value upon completion of therapy. sCD163 correlated with plasma EBV-DNA, and associated with B symptoms, stage, and lymphopenia. Circulating CD163+ monocytes were elevated in patients, indicating that sCD163 are likely derived from circulating and intratumoral cells. Depletion of cHL CD163+ monocytes markedly enhanced T-cell proliferation, implicating monocytes and/or TAMs as potential novel targets for immunotherapeutic manipulation.Conclusion: The combination of circulating tumor-infiltrate (sCD163) and tumor-specific (sTARC) proteins is more informative than either marker alone as disease response biomarkers in early and advanced disease during first-line therapy for cHL. Clin Cancer Res; 19(3); 731–42. ©2012 AACR.

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.

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.

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.

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.

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.

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.

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.

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.

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 CD56(dim) NK cell subset characterized by the lack of expression of DNAX accessory molecule-1 (DNAM-1). Compared with CD56(bright) and CD56(dim)DNAM-1(pos) NK cell subsets, CD56(dim)DNAM-1(neg) NK cells displayed reduced motility, poor proliferation, lower production of interferon-γ, and limited killing capacities. Soluble factors secreted by CD56(dim)DNAM-1(neg) NK cells impaired CD56(dim)DNAM-1(pos) NK cell–mediated killing, indicating a potential inhibitory role for the CD56(dim)DNAM-1(neg) NK cell subset. Transcriptome analysis revealed that CD56(dim)DNAM-1(neg) NK cells constitute a new mature NK cell subset with a specific gene signature. Upon in vitro cytokine stimulation, CD56(dim)DNAM-1(neg) NK cells were found to differentiate from CD56(dim)DNAM-1(pos) 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 CD56(dim)DNAM-1(pos)/CD56(dim)DNAM-1(neg) NK cell ratios and reduced cytotoxic activity of CD56(dim)DNAM-1(pos) 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.

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor 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 CD226hi 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.

Preclinical studies have suggested that purified populations of CD1c (BDCA-1) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c BDC and administered successfully to 12 patients. Each patient received between 1 and 5 × 10 fresh CD1c BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1-2 fever, pain, or injection-site reactions. Vaccination with CD1c BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

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.

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.

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.

The analysis of regulatory T cells (T-reg(s)) is becoming an increasingly important consideration in the development of novel immunotherapeutic strategies. Accurate quantification of T-regs during treatment protocols is crucial, particularly where the therapeutic strategy is targeting T-regs. The TruCOUNT™ method has utility for enumerating different immune cells but has not been used to detect T-regs. We have utilized this technology to develop an assay to enumerate human T-regs in whole blood, based on CD127 expression. The mean number of CD4(+)CD25(+)CD127(lo) T-regs per μl of whole blood was 48±16.9 with a range of 18 - 79 (n=22) and the average percentage was 6.1±1.9% (range 2.2-10.4%). The percentages of CD4(+)CD25(+)CD127(lo) T-regs were similar when detected in whole blood or density-gradient separated PBMC, and were comparable to those distinguished using the T-reg marker FoxP3. The assay was robust and reliable for enumeration of the lower frequency T-regs, with CV's for intra-assay repeatability and inter-assay precision of9% and35%, respectively. The CV's for the detection of total CD4(+) T lymphocytes using this assay were2% for intra-assay repeatability and18% for inter-assay precision, providing further evidence for reproducibility. This assay has a number of advantages over current methods, including small sample volume, the ability to determine absolute cell counts, and no need for hematology cell analyzers. This assay will simplify clinical trial immune monitoring and can be used to provide crucial data on patient T-reg numbers before, during, and after therapeutic interventions.

Purpose: Candidate circulating disease response biomarkers for classical Hodgkin lymphoma (cHL) might arise from Hodgkin–Reed–Sternberg (HRS) cells or nonmalignant tumor-infiltrating cells. HRS cells are sparse within the diseased node, whereas benign CD163+ M2 tissue-associated macrophages (TAM) are prominent. CD163+ cells within the malignant node may be prognostic, but there is no data on serum CD163 (sCD163). The HRS-specific serum protein sTARC shows promise as a disease response biomarker. Tumor-specific and tumor-infiltrating circulating biomarkers have not been compared previously. Experimental Design: We prospectively measured sCD163 and sTARC in 221 samples from 47 patients with Hodgkin lymphoma and 21 healthy participants. Blood was taken at five fixed time-points prior, during, and after first-line therapy. Results were compared with radiological assessment and plasma Epstein-Barr virus DNA (EBV-DNA). Potential sources of circulating CD163 were investigated, along with immunosuppressive properties of CD163. Results: Pretherapy, both sCD163 and sTARC were markedly elevated compared with healthy and complete remission samples. sCD163 better reflected tumor burden during therapy, whereas sTARC had greater value upon completion of therapy. sCD163 correlated with plasma EBV-DNA, and associated with B symptoms, stage, and lymphopenia. Circulating CD163+ monocytes were elevated in patients, indicating that sCD163 are likely derived from circulating and intratumoral cells. Depletion of cHL CD163+ monocytes markedly enhanced T-cell proliferation, implicating monocytes and/or TAMs as potential novel targets for immunotherapeutic manipulation. Conclusion: The combination of circulating tumor-infiltrate (sCD163) and tumor-specific (sTARC) proteins is more informative than either marker alone as disease response biomarkers in early and advanced disease during first-line therapy for cHL. Clin Cancer Res; 19(3); 731–42. ©2012 AACR.

Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival. Experimental Design: We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline “R-CHOP” therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS). Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%–79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%–88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%–76.6%) versus 72.6% (95% CI, 58.8%–82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV+ DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules. Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820–8. ©2016 AACR.

Epstein-Barr virus (EBV) is implicated in a range of B-cell malignancies and expresses unique microRNAs (EBV-miRNAs). Due to the requirements for high-quality RNA, studies profiling EBV-miRNA in EBV-positive lymphomas have been restricted to cell-lines or frozen samples. However, the most commonly available archived patient material is paraffin-embedded formalin-fixed (FFPE) tissue. This has impeded the widespread profiling of EBV-miRNA expression in clinical samples. The requirements for accurate EBV-miRNA real-time RT-PCR quantitation in FFPE tissues representing a broad-spectrum of EBV-positive lymphomas were determined systematically, including where the neoplastic cells are sparse relative to the non-malignant infiltrate. The level of cellular EBV-load correlated strongly with the sum of EBV-miRNA expression and the number of EBV-miRNAs detectable. As calibrators for cellular EBV-load, the sum EBV-miRNA was optimal to EBV-genome copy number and EBER2 expression level, with the added advantage of not requiring additional assays. EBV-miRNA was profiled reliably within archival FFPE tissue in 14/23 patients, but not in tissues with low abundance EBV. This method enabled specific and simultaneous detection of numerous EBV-miRNAs in FFPE lymphoma samples that contain EBV at high to medium levels, making it as a useful tool for studies of EBV-miRNA in the majority of diagnostic biopsies.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Evaluation of: Lin Y, Gustafson MP, Bulur PA, Gastineau DA, Witzig TE, Dietz AB: Immunosuppressive CD14+HLA-DRlow/- monocytes in B-cell non-Hodgkin’s lymphoma. Blood 117, 872–881 (2011). The mechanisms of systemic immunosuppression in B-cell non-Hodgkin’s lymphoma (NHL) are poorly characterized. Lin and colleagues collected blood from 40 NHL patients prior to therapy. Monocytes from NHL patients suppressed T-cell proliferation, were unresponsive to Toll-like receptor stimulation by CpG and resistant to maturing into CD83+ dendritic cells. This suppression was mediated in part through arginine metabolism, as exogenous arginine supplementation reversed this, and NHL patients had elevated arginase I in their plasma. These cells had decreased HLA-DR and TNF-α receptor II (CD120b) expression compared with controls. Patients with increased ratios of CD14+HLA-DRlow/- monocytes had more advanced disease and suppressed immune functions, indicating that CD14+HLA-DRlow/- monocytes are a pivotal and profoundly effective contributor to systemic immunosuppression in NHL.

Success in discovering bioactive peptide mimetics is often limited by the difficulties in correctly transposing known binding elements of the active peptide onto a small and metabolically more stable scaffold while maintaining bioactivity. Here we describe a scanning approach using a library of pyranose-based peptidomimetics that is structurally diverse in a systematic manner, designed to cover all possible conformations of tripeptide motifs containing two aromatic groups and one positive charge. Structural diversity was achieved by efficient selection of various chemoforms, characterized by a choice of pyranose scaffold of defined chirality and substitution pattern. A systematic scanning library of 490 compounds was thus designed, produced, and screened in vitro for activity at the somatostatin (sst(1-5)) and melanin-concentrating hormone (MCH(1)) receptors. Bioactive compounds were found for each target, with specific chemoform preferences identified in each case, which can be used to guide follow-on drug discovery projects without the need for scaffold hopping.

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.

Follicular Lymphoma (FL) is the most common indolent Non-Hodgkin Lymphoma. Despite generally favorable survival outcomes, 20% of FL patients experience 'Progression of Disease within 24 months' (POD24) and subsequently have poor long-term overall survival (OS) (Casulo, JCO 2015). Unfortunately, POD24 has limited clinical value because it cannot guide up-front clinical decisions. Accurate pre-therapy prognosticators are vital for clinical trial design and are also increasingly being mandated by funding agencies for stratification of patients to emerging front-line treatments. The new 'state-of-the-art' prognosticators 'm7-FLIPI' and POD24-PI' (Pastore, Lancet Oncol 2015; Jurinovic, Blood 2016) supplement clinical parameters with genetic mutational status. However, their applicability to population based cohorts including early-stage and asymptomatic patients remains unknown. Furthermore, there is significant heterogeneity of outcome within these prognostic groupings. The established biological and prognostic importance of the tumor microenvironment (TME) in FL suggests that prognosis would be enhanced by incorporating information on host immunity (Scott, Nat Rev Can 2014). Forty-five pre-treatment FL biopsies were categorized into 'hot' or 'cold' immune nodes by multiplex immunofluorescent imaging and respectively characterized by concordant high or low expression of multiple immune effector and checkpoint-associated proteins. (Fig 1A). Consistent with these findings, gene expression using the Nanostring platform showed that immune effectors (CD4/CD8/TNFa/CD137/CD56) positively correlated with immune checkpoints (PD-1/PD-L1/PD-L2/TIM3/LAG3/CD163/CD68) indicative of an adaptive immune response. Additionally, high-throughput unbiased TCRb sequencing showed the intratumoral TCR repertoire was more clonal in 'hot' compared to 'cold' FL samples (p=0.024), indicative of a skewed T-cell immune response (Fig 1B). We then applied these findings to an independent population-based cohort of 175 cases of FL from the rituximab era with long-term follow-up (median ~7 years), including advanced (n=137) and localized cases (n=38). The aims were to: a) identify new targetable immune parameters of prognostic importance in the rituximab-era; and b) compare and contrast these with published prognostic tools: FLIPI, FLIPI-2, m7-FLIPI, POD24-PI and 'immune survival score' ('ISS', Dave, NEJM 2004). OS was not only inferior in those experiencing POD24 (HR 4.88, p2-fold increase in 5-year patient health costs. Hence, POD24, as well as FFS and TT2T were therefore chosen as the primary outcome measures. M7 mutation frequencies were similar to those previously published (Pastore, Lancet Oncol 2015). However, the prognostic utility of the m7-FLIPI could not be demonstrated, whereas the FLIPI, FLIPI-2, and POD24-PI retained their prognostic value. The POD24-PI was most predictive of FFS (p We have validated the TME in predicting outcome in a population based cohort of FL patients with long-term follow-up treated in the rituximab era. Furthermore, we describe the role of PD-L2 as well as several additional pertinent, clinically-actionable markers of the TME which predict survival to conventional therapies in FL. Low expression of PD-L2 appears to be a surrogate of a broadly co-ordinated downregulation of the intratumoural response. These immune scores are independent of and additive to additive to the FLIPI and POD24-PI. Development of new prognostic models require the incorporation of host immunity along with clinico-genetic features to further improve the specificity, and to accurately risk stratify FL patients. Disclosures No relevant conflicts of interest to declare.

T-cell infiltration of the tumor microenvironment (TME) in DLBCL is a key determinant of response to chemo-immunotherapy (Keane, Lancet Haem 2015). We have previously shown that greater diversity of the T-cell receptor (TCR) repertoire within the TME is correlated with improved survival following R-CHOP in DLBCL (Keane, CCR 2017). There are limited data on the impact of the intratumoral TCR repertoire on interim-PET (iPET), the relationship between intratumoral and circulating TCRs, and on dynamic changes of the TCR during therapy. In this study, we interrogated the TCR repertoire in a subset of DLBCL patients treated on the prospective Australasian Leukaemia Lymphoma Group NHL21 study (Hertzberg, Haematologica 2017), in which all patients had 4x RCHOP prior to iPET risk stratification. The CDR3 region of TCRβ chain underwent high-throughput unbiased TCRβ sequencing (Adaptive Biotechnologies). Metrics included: productive templates (total functional T-cells), productive rearrangements (functional T-cells with distinct specificity), productive clonality (repertoire unevenness due to clonal expansions), and maximal frequency clones (% most dominant single clone). Matched intratumoral diagnostic samples, blood at pre-therapy and post-cycle 4 (at the time of iPET) were tested. 42 patients (enriched for iPET+ cases) had sufficient material for testing. Median age was 55 (range 22-69) years and 72% were males. IPI was low/intermediate/high in 13/63/25% respectively. Cell of origin (COO) by Lymph 2CX method (nanoString) was ABC in 30%, and GCB in 44%. 40% were iPET+. In tissue, there was a median of 4652 productive templates, translating into 2998 productive rearrangements identified. Notably, the clonal repertoire of intratumoral TCRs in iPET+ patients was larger than iPET-ve patients (productive clonality 8.1 vs 5.1 x10-2, p=0.04), whereas the numbers of functional T-cells did not vary between groups. Comparing the tumor with the blood samples showed a high, but variable, degree of overlap between peripheral blood and the TME - TCR repertoire. Median number of top 100 tumor tissue clones shared in peripheral blood was 53.5 (range, 1-97) in pre-therapy and 39.5 (range, 0-93) in post-therapy blood, indicating that the both the circulation and the tumor likely contribute to immune-surveillance. In pre-therapy blood, the median productive templates and productive rearrangements were 44,950 (range, 6,003-273,765) and 29,090 (range, 5,190-152,706), and the median clonality was 8.5 (1.46-45.3) x 10-2. There were no differences between iPET+ and iPET-ve patients in these parameters. However, there was a marked change in T-cell composition between time points. Interestingly, in iPET-ve patients clonality measures were increased, with productive clonality 9.4 vs 14.4 x10-2, p=0.03; and % maximum productive frequency 3.39 vs 5.89, p=0.04. These findings demonstrate that the intratumoral TCR repertoire, and sequential blood sampling provide important information on outcome in DLBCL treated with RCHOP. A highly clonal T-cell repertoire in the TME was associated with iPET positivity after 4 cycles of R-CHOP. In line with findings in solid cancers treated with checkpoint blockade, development of clonal responses in peripheral blood was associated with iPET negativity. These findings indicate that clones expanded during therapy may be important in tumor clearance but that highly clonal T-cell responses in the tumor at diagnosis may hinder expansion of other T-cell responses to neoantigens. The circulating TCR composition is representative of the TME. These findings will assist the rationale design and therapeutic monitoring of novel immuno-therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Understanding the biology of the DC and its pivotal role in immune response initiation and regulation has enabled new effective anti-cancer therapies to be developed for treatment of a wide range of tumor types. Many studies on DC-based cancer vaccine development have focused on the development of methods that can effectively deliver tumor Ags to DCs. Numerous methods have been developed or evaluated for the delivery of defined and undefined tumor Ags to DCs for processing and presentation to T lymphocytes. This review provides a brief summary of these methods, the techniques that are used in each, how they have -- or could -- be applied clinically, as well as the advantages and disadvantages of each method.

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the non-rejector combination of DA donors into PVG recipients. In the present study, a polyclonal anti-LSF-1 antibody was used to detect the lymphoid cell populations expressing LSF-1 epitopes in several transplant models. In this study we examined cell samples acquired from rats that had undergone OLT in syngeneic, rejecting or non-rejecting combinations. Flow cytometry indicated that the polyclonal antibody reacts specifically with an epitope present on a CD4 CD11b positive cell of recipient origin. In the first 3 weeks after transplant there is a large increase in the number of these cells isolated from the spleens and livers of rats in tolerogenic OLT model, which correlates with prolongation of allograft survival. In the rejector and isograft models of OLT there is a minimal change in the expression of the LSF-1 N-terminal pep epitope. These results suggest that these recipient CD4 CD11b cells may have a critical role in the formation of transplant tolerance by interfering with the indirect pathway of allorecognition via as yet undetermined mechanisms. The increased expression of the LSF-1 N-terminal pep epitope on liver leukocytes 14 days after partial hepatectomy indicates a natural role for LSF-1 in the process of liver regeneration after insult or injury.

PD-L1/PD-L2 are immunomodulatory molecules that engage with the PD-1 receptor on immune effector T and NK-cells to inhibit anti-lymphoma immunity. PD-1/PD-L1/PD-L2 axis molecules are prognostic in Hodgkin Lymphoma (HL, Roemer et al J Clin Oncol 2016) and Diffuse Large B-cell Lymphoma (DLBCL, Keane et al Lancet Haem 2015). Importantly, blockade of the axis is associated with particularly potent clinical responses in relapsed/refractory HL (Ansell et al NEJM 2015), as well as response in DLBCL (Armand et al J Clin Oncol 2013). Focus has been on the interaction of PD-L1 on malignant B-cells with PD-1 on HLA-class I restricted CD8+ effector T-cells. This is despite considerable evidence that: A) malignant B-cells in HL and DLBCL frequently lack the ability to present HLA-class I due to mutations in b2M and associated antigen presenting molecules (Challa-Malladi et al Cancer Cell 2013). This makes them insensitive to direct lysis by CD8+ T-cells (Zaretsky et al NEJM 2016) but potentially enhances their sensitivity to NK-cells; B) PD-L1/PD-L2 are expressed by inhibitory CD163+ monocytes/macrophages as well as by malignant B-cells (Chen et al CCR 2013). Here, we seek to establish the contribution of NK-cells and inhibitory CD163+ expressing monocytes/macrophages in the setting of HL and DLBCL. CD163/PD-1/PD-L1/PD-L2 gene expression was quantified by nanoString in 194 patients and was elevated in HL relative to DLBCL tissues (P The NK-cell marker CD56 were higher in HL compared to DLBCL tissues (P An in-vitro functional model of TAM-like monocytes was developed to demonstrate the potential impact of inhibitory CD163+ expressing monocytes/macrophages on NK-cells in HL and DLBCL. Monocytes were cultured with the M6 TAM inducing cytokine cocktail of M-CSF and IL-6. Consistent with an inhibitory phenotype, M6 cultured monocytes were highly enriched for CD163 (P We describe a hitherto unrecognised immune evasion strategy mediated via skewing towards an exhausted PD-1 enriched CD16-CD56hiCD3- NK-cell phenotype. In addition to inhibition of NK-cells by the malignant B-cell, suppression of NK-cells occurs by PD-L1/PD-L2 expressing tumor associated macrophages. This mechanism is more prominent in HL than DLBCL. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

A synthetic peptide conjugate based on the N-terminal sequence of a 10 000 MW immunosuppressive serum protein (reOLT 4) was used to immunize female Lewis rats prior to mating, in order to determine whether blocking this protein had an effect on pregnancy. The N-terminal sequence of (reOLT 4) has close sequence homology to the beta-chain of rat haemoglobin so a peptide conjugate based on the N-terminal sequence of this protein was also used to immunize female Lewis rats. Controls included animals that were not immunized and animals that received the peptide carrier, diphtheria toroid (DT). No statistical differences were found in gestation time or litter sizes in these groups. Differences were, however, evident between these groups and animals that received DT-(reOLT 4) (group 3) or the DT-beta-chain haemoglobin (group 5), There were no statistical differences in litter size or gestation time for group 4 when compared with group 5. Enzyme-linked immunosorbent assay and dot-blot analysis revealed that rats from both group 4 also had strong responses against DT. the peptide conjugate they were immunized with and the corresponding full-length protein. In both cases, animals from group 3 and group 5 had weak responses to the peptide that they did not receive, together with lower erythrocyte counts and haematocrits, and elevated heart to body weight ratios. Additionally, antibody purified on a (reOLT 4) immunoaffinity column was capable of binding to rat erythrocytes, A second investigation comparing anaemia prior to fertilization and maintained anaemia over the gestation period revealed that only the latter was; capable of decreasing litter size to the same degree as obtained for groups 3 and 5. We conclude that for groups 4 and 5 it is the autoimmune effect of continual anaemia over the gestation period, mediated by autoantibodies. which results in the observed lower litter size.

We investigated the effect of a prostacyclin analogue (OP 2507) on PVG (RT1c) recipients subjected to an extended anhepatic phase (AH) and transplanted orthotopically with PVG livers. All of the animals that underwent orthotopic liver transplantation (OLT) with a 20-min AH survived for 1 week with or without OP 2507, (OP) treatment (10/10, 100%). When the AH was lengthened to 45 min, the 1-week survival rate of recipients was poor in the OP-untreated groups (1/10, 10%). Treatment of the recipient with OP 2507, 0.15 micrograms/kg per minute for 30 min, prior to the 45-min AH substantially improved 1-week survival (5/6, 83.3%, P0.05). The serum TNF-alpha level at day 1 in OP-treated animals that underwent OLT with a 45-min AH was significantly lower than that in animals with 45-min AH OLT without OP treatment. We conclude that OP 2507 treatment has potential usefulness as a perioperative treatment when the AH is extended during OLT.

Liver grafts between certain rat strain combinations, such as DA (RT1(a))-into-PVG (RT1(c)), are accepted without the use of immunosuppressive agents. To explore the nature and role of serum proteins in liver-induced immunosuppression, we have developed a retransplantation model of rat liver grafting, In this procedure, orthotopic liver transplantation (OLT) is carried out in the DA-into-PVG combination; two days later the DA liver is removed and a new PVG Liver implanted into the same recipient (re-OLT). Serum hem re-OLT rats was immunosuppressive when tested in mixed lymphocyte reactions (MLR). Three novel proteins were detected in re-OLT serum by SDS-PAGE, with sizes of 180 kD, 87 kD, and 10 hll. The N-terminal sequences of these were distinct and did not match protein sequences in the computer databases, although there was some homology between the 10 kD sequence and the beta-chain of rat hemoglobin Purified 87 kD and 10 kD proteins were immunosuppressive in MLR; in both cases suppression was dose-dependent and non-stimulator-specific. Production of the 180 kD and 87 kD molecules required the presence of the recipient spleen We conclude that re-OLT serum contains novel immunosuppressive proteins, which may be products of immune recognition and associated with the immediate termination of graft rejection.

High sensitivity and low background, which are the significant features of the procedure of Johansson and Skoog (J. Biochem. Biophys. Methods. 1987, 14, (Suppl.) 33) for silver staining of nucleic acids in polyacrylamide gels, have been improved by excluding ethanol from all solutions and increasing the length of washing steps after sensitisation and staining. Including a fixation step before sensitisation increased the contrast of the bands even at high concentration of nucleic acid, producing a shift in the tone of colour from black to grey instead of gold to yellow bands. This technique is superior in terms of sensitivity and background to a number of other silver staining protocols, three which utilise silver diammine and three which utilise silver nitrate ions.

Despite the Revised International Prognostic Index's (R-IPI) undoubted utility in diffuse large B-cell lymphoma (DLBCL), significant clinical heterogeneity within R-IPI categories persists. Emerging evidence indicates that circulating host immunity is a robust and R-IPI independent prognosticator, most likely reflecting the immune status of the intratumoral microenvironment. We hypothesized that direct quantification of immunity within lymphomatous tissue would better permit stratification within R-IPI categories. We analyzed 122 newly diagnosed consecutive DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemo-immunotherapy. Median follow-up was 4 years. As expected, the R-IPI was a significant predictor of outcome with 5-year overall survival (OS) 87% for very good, 87% for good, and 51% for poor-risk R-IPI scores (P

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122+ cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8+ T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8+ T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8+ T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio-process to purify CMRF-56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF-56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.

The efficient antigen-presenting function of dendritic cells (DC) makes them an attractive cellular adjuvant for clinical immunotherapeutic protocols aimed at eradicating minimal residual disease after conventional treatment of multiple myeloma (MM) and other malignancies. We used single-step positive immunoselection with biotinylated CMRF-56 monoclonal antibody to generate a CD11c(+) blood DC (BDC) enriched antigen-presenting cell population, which, after exposure to activation stimuli for as little as 2 hours, displayed a mature costimulatory BDC phenotype and secreted inflammatory cytokines. Of the activation stimuli tested, granulocyte macrophage colony-stimulating factor (GM-CSF) provided optimal activation of the CMRF-56 immunoselected preparations and primed efficient cytotoxic T cell (CTL) responses using MART-I peptide as a model tumor-associated antigen. In addition, GM-CSF activated CMRF-56 immunoselected cells cross-presented MM cell lysate and improved the MM-specific polyclonal CTL response (no activation 18.8% +/- 4.3% vs. GM-CSF activation 40.9% +/- 7.3%, P = 0.051). CMRF-56 immunoselected BDC migrated in vitro both spontaneously and specifically toward the secondary lymphoid chemokine CCL21. Their migration was also significantly improved by GM-CSF and prostaglandin E-2 activation and a greater percentage of activated BDC migrated specifically compared with monocyte-derived DC. These results indicate that the CMRF-56 immunoselected BDC preparations can cross-present antigen for effective anti-MM CTL responses and that limited exposure to maturation stimuli can produce phenotypically and functionally mature migrating DC. CMRF-56 immunoselected cells are suitable for use as part of an immunotherapeutic anti-MM vaccine.

The effect of serum from orthotopic liver retransplanted rats (re-OLT serum) on graft-versus-host disease (GVHD) was studied in rats. In the re-transplantation model of rat liver, orthotopic liver transplantation (OLT) was carried out in the DA (RT1) into PVG (RT1(c)) combination; two days later the DA liver was removed and a new PVG liver implanted into the same recipient (re-OLT). In the in vivo GVHD model, male PVG rats were sublethally irradiated and injected intravenously with 3 x 10 DA or BN (RT1(n)) spleen cells through the penial vein. Within 1 h of the inoculation, rats of the experimental group were injected with 1 ml of re-OLT serum taken at postoperative day (POD) 7. Rats in the control group received 1 ml of normal PVG serum or syngeneic re-OLT serum (PVG-PVG, PVG-PVG). All PVG rats in the control groups died of GVHD within 21 days after the inoculation of DA or BN spleen lymphocytes. However, when the animals were treated with re-OLT serum, 100% (6/6) of the rats survived more than 60 days, following inoculation with DA lymphocytes but not with BN lymphocytes. The POD 7 re-OLT serum showed a strong inhibition against DA anti-PVG mixed lymphocyte reaction (MLR), although re-OLT serum did not contain soluble DA class I antigens, anti-DA class I or II antibody. The potential GVHD inhibitory factors in re-OLT serum may be two unique immunosuppressive proteins, which have been detected by SDS PAGE and reported previously. We conclude that re-OLT serum has immunosuppressive factors, which, at least in part, prevented the induction of GVHD in rats.

Lymphoproliferative disorders, including follicular lymphoma (FL), multiple myeloma (MM) and chronic lymphatic leukaemia (CLL), are slowly progressive malignancies which remain incurable despite advances in therapy. Harnessing the immune system to recognise and destroy tumours is a promising new approach to treating these diseases. Dendritic cells (DC) are unique antigen-presenting cells that play a central role in the initiation and direction of immune responses. DC loaded ex vivo with tumour-associated antigens and administered as a vaccine have already shown promise in early clinical trials for a number of lymphoproliferative disorders, but the need for improvement is widely agreed. Recent advances in the understanding of basic DC biology and lessons from early clinical trials have provided exciting new insights into the generation of anti-tumour immune responses and the design of vaccine strategies. In this review we provide an overview of our current understanding of DC biology and their function in patients with lymphoproliferative disorders. We discuss the current status of clinical trials and new approaches to exploit the antigen presenting capacity of DC to design vaccines of the future.

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5 x 10(8) splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.

Carbohydrates play a critical role in many biological processes and disease states including cancer, inflammation and infection. The development of carbohydrates as therapeutics continues to gain interest, as the biological roles of these biopolymers are further elucidated and understood. However, many carbohydrates display poor affinity, stability and bioavailability characteristics, which has led to a widely held view that this class of molecules make poor drugs. As there are already a significant number of carbohydrate-based drugs presently being employed by physicians, it is clear that some carbohydrates do make effective drugs. Recent advances in (a) the understanding of carbohydrate specific transport mechanisms, and (b) the development of novel carbohydrate based bioactives which may overcome many of the previous limitations of stability and bioavailability, suggest that carbohydrate-based compounds may provide a rich source of new drug candidates for a variety of diseases.

Diff use large B-cell lymphoma (DLBCL) is clinically heterogeneous, encompassing a variety of diagnostic presentations and responses to therapy. Yet despite, or perhaps because of, its crudity, the international prognostic index (IPI) still remains by far the most widely adopted prognosticator. With increasing numbers of new therapies on the horizon, it is critical that new ways to stratify this disease are validated. Such strategies must take into account the lymphoma ’ s biological characteristics. Gene expression profi ling (GEP) of DLBCL has proved most successful, and has been used to defi ne two distinct diseases, based on the putative malignant B-cell ’ s cell of origin (COO). Germinal center B-cell-like (GCB) DLBCL has a superior prognosis following chemo-immunotherapy to that of activated B-cell-like (ABC) DLBCL [1]. As yet, GEP has not had wide applicability outside of the research setting, due to prohibitive costs and the lack of a robust platform that could be applied to paraffi n-embedded sections. Attempts have been made to develop more “ user-friendly ” diagnostic tests, using proteins that are diff erentially expressed in the two types of DLBCL. Several immunohistochemical algorithms have been proposed to approximate to GEP (with mixed results), including that proposed by Hans and colleagues [2].