Characterization of Cytotoxic Macrophages in a Pre-Clinical Model of Epstein-Barr Virus (EBV)-Driven Lymphoproliferative Disease

Epstein-Barr Virus (EBV) is an oncogenic herpes virus associated with the development of malignancies associated with poor outcomes. Current treatments lead to further immune suppression, increasing the risk of EBV reactivation and other opportunistic infections. Strategies aimed at reversing the immune-suppressive microenvironment are likely to result in more durable responses while avoiding deleterious effects of chemotherapy. It is well-established that macrophages play an important role in inhibiting the anti-tumor response as evidenced by poor prognosis associated with increasing tumor-associated macrophage (TAM) density or lower ratio of cytotoxic T lymphocytes. Here we describe a pre-clinical model showing the expansion of cytotoxic TAMs and potential mechanisms to prevent this immunosuppressive environment. To assess soluble factors produced by the interaction of tumor on immune effector cells, we used a co-culture (CoCx) system of EBV-transformed human B cell lymphoblastoid cell lines (LCL) cultured with autologous peripheral blood mononuclear cells (PBMC). Serum-free medium collected after 48 hrs showed increased myeloid-associated factors which were absent from either the LCL or PBMC cultures, suggesting the interaction of tumor cells with immune effectors was leading to the production of chemokines known to attract myeloid cells. When purified autologous CD14+ monocytes were incubated in the presence of CoCx conditioned medium (CM) they generated proliferative foci that were absent in the LCL or PBMC CM alone. To better understand which cellular subsets were necessary for the release of these soluble factors, immune subsets were removed from PBMC prior to incubation with LCL or purified and incubated with LCL. We found that T cells, (CD8+ and CD4+), when incubated in the presence of LCL were necessary and sufficient to stimulate the proliferation of myeloid cells. CD56+, CD14+, or CD19+ subsets within the CM did not appear to contribute to the proliferation of purified myeloid cells. We next used flow cytometry to characterize the monocyte-derived population that expanded when introduced to CoCx CM. We again isolated autologous CD14+ monocytes from PMBC and incubated them for 6 days with CoCx CM followed by immunophenotyping. The monocyte-derived population retained CD14 and CD11b in addition to showing pronounced increases in both canonical M1 and M2 macrophage markers (HLA-DR, and CD163 respectively), and PD-L1, indicating activation consistent with a TAM phenotype. To characterize the functional consequence of this cell population, CoCx CM-derived macrophages were incubated with autologous T cells that were either activated non-specifically with anti-CD3/C28 or left inactivated. Surprisingly, we noted a marked decrease in viable activated T cells (both CD4+ and CD8+) when exposed to conditioned macrophages. However, the inactivated T cell populations were only mildly effected. Together, with previously described experiments, this suggests a mechanism by which exposure of T cells to EBV+ lymphoblastic tumor lines results in cytokine release, polarizing monocytes to TAMs that, in turn, exhibit cytotoxic activity to tumor-specific T cells. The precise mechanism of cytotoxicity is under investigation. We previously reported the eIF4A translational inhibitor silvestrol potentiated a potent immune-mediated anti-tumor response against EBV-driven lymphoma mainly by promoting expansion and activity of EBV-specific cytotoxic T cells. We next questioned if macrophages were important to this mechanism. We found that with silvestrol, when added to CoCx, the resultant CM did not drive the proliferation of TAMs, and Immunophenotype of CoCx showed silvestrol treatment to result in the ablation of CD14+ cells from culture. Interrogation by transcriptome analysis indicated a potential role of aryl hydrocarbon receptor (AhR). Protein analysis by western blotting indicated an increase of AhR in isolated monocytes when exposed to CoCx CM, which was abrogated by addition of silvestrol. Together these results suggest AhR may play a role in the polarization of macrophages within the tumor microenvironment leading to elimination of tumor-specific T cells. While full mechanistic characterization is ongoing, these preliminary results provide a potentially novel approach to modulate tumor specific immunity in this challenging group of EBV-related malignancies. Disclosures Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Large-Scale Production of Autologous CD14+-Monocyte Derived Dendritic Cells Co-Electroporated with Amplified Total Tumour mRNA and Human CD40L mRNA in Patients with B-Cell Chronic Lymphocytic Leukemia.

BACKGROUND: B-cell chronic lymphocytic leukemia (CLL) is currently incurable with conventional chemotherapeutic approaches. The induction of specific anti-CLL immune responses in vitro using autologous tumour-antigen loaded dendritic cell (DC)-based approaches has been previously demonstrated. The aim of this study was to optimize and validate large-scale production of an autologous DC vaccine prior to initiation of a planned Phase I/II clinical trial for patients with previously treated CLL. STUDY DESIGN: Following informed consent, 8 patients with confirmed CLL had 11 leukapheresis collections for mononuclear cells performed between April 2005 and May 2006. Methods for vaccine production were optimized during an initial cohort of CLL patients (N=7); production methods ‘at scale’ were subsequently confirmed with a validation cohort (N=3). METHODS: Leukemic B-cells were positively selected (CD19+/CD5+) and preserved in RNAlater™ prior to overnight shipment to Argos Therapeutics Inc (Durham, NC); amplified patient specific total tumour mRNA and huCD40L mRNA were subsequently supplied by Argos for final vaccine formulation. CD14+ monocytes were positively selected using a CliniMACS device (Miltenyi Biotech, Germany). Purified monocytes were subsequently cultured for 5 days in growth medium containing IL-4 and GM-CSF; DCs were matured with addition of TNF-β, IFN-γ, and PGE2 on day 5. DCs were harvested on day 6, co-electroporated with autologous total tumour RNA and huCD40L RNA, and cryopreserved for subsequent analysis. RESULTS: Mean patient age was 62 years, 6/8 were male, and all had Rai clinical stage I, previously untreated disease; mean peripheral white blood cell count was 17.2 × 109/L (range 7.4 – 26.7 × 109/L). Mean CD14+ monocyte yield post-CliniMACS selection was 2.42 × 109 cells (range 1.51 – 3.53 × 109) and monocyte purity was high (mean 96.5%, range 80 – 99.7%). In the validation cohort, day 6 immunophenotype (mean, range), measured 4 hours post-electroporation, was consistent with mature, activated DCs: CD14+ 2.5% (1.21–5.00), CD80+ 98.2% (98.0–99.7), CD83+ 87.5% (78.0 – 94.7), and CD86+ 99.5% (99.0–99.9). Mean CD40L expression, a surrogate marker for electroporation efficiency, was 80.3% (range 70.2 – 88.0); mean CD209 expression was 97.7% (95–99.7). Absolute numbers of DCs generated post-electroporation ranged from 1.26 × 108/L to 3.24 × 108/L and post-thaw DC viability ranged from 71–94%. T-cell co-culture experiments confirmed the generation of specific, autologous cytotoxic T-lymphocyte (CTL) responses to CLL targets, demonstrated by: (1) MHC class I restricted autologous CLL induced INF-γ response (intracellular staining), and (2) statistically significant functional MHC class I restricted CTL response (chromium-release assay). CONCLUSIONS: This study confirms the feasibility of generating large numbers of autologous CD14+-derived dendritic cells co-electroporated with patient specific total tumour RNA and huCD40L RNA from CLL patients. These data provide justification for a currently accruing Phase I/II clinical trial designed to evaluate this treatment in CLL patients that have a stable, low disease burden following at least one course of systemic chemotherapy.

Dendritic Cell Immaturity during Infancy Restricts the Capacity To Express Vaccine-Specific T-Cell Memory

ABSTRACT The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with “matured” dendritic cells (DC) cultured from autologous CD14+ precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P < 0.0001) and plasmacytoid DC (P < 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants.