The circuitry of the tumor microenvironment in adult and pediatric Hodgkin lymphoma: cellular composition, cytokine profile, EBV, and exosomes

Background: Classical Hodgkin lymphoma (cHL) is a unique lymphoid malignancy with a tumor microenvironment (TME) consisting of a small number of neoplastic—Hodgkin and Reed-Sternberg (H-RS) cells (\u3c1%), surrounded by a large number of nonneoplastic infiltrating immune cells (\u3e90%). The TME of cHL critically depends on immune cells to support tumor growth as H-RS cells cannot survive and proliferate in isolation. Recent Findings: Programmed cell death protein 1 (PD-1) ligand expressed on H-RS cells inhibits the clearance of tumor by causing T-cell exhaustion. Nivolumab and pembrolizumab, PD-1 inhibitors, have been proven to be effective in treating adult and pediatric patients with R/R cHL. Tumor-associated macrophages (TAMs) are a central component of TME and are known to cause poor prognosis in adult HL. However, the prognostic impact of CD68+ TAMs in pediatric HL remains ambiguous. EBV modulates the tumor milieu of HL and plays a strategic role in immune escape by enrichment of the TME with Treg cells and associated immunosuppressive cytokines in adult HL. In contrast, EBV+ pediatric patients have increased infiltration of CD8+ T-cells and show a better therapeutic response suggesting viral-related TME is distinct in childhood HL. The role of CASP3 in apoptosis of H-RS cells and its correlation with response prediction in adult and pediatric HL suggest it may serve as a potential biomarker. In cHL, CD30, EBV, and NF-κB signaling employ exosomes for cell–cell communication that triggers the migration capacity of fibroblasts, stimulate to produce proinflammatory cytokines, and help to create a tumor-supportive microenvironment. Conclusion: The cHL microenvironment is distinct in adult and pediatric HL. Future studies are required to understand the role of interplay between H-RS cells and EBV-associated microenvironment and their clinical outcome. They may present novel therapeutic targets for the development of antilymphoma therapy

Anti-HIV activity of human defensin 5 in primary CD4+ T cells under serum-deprived conditions is a consequence of defensin-mediated cytotoxicity.

We have previously shown that human defensin 5 (HD5) promotes HIV infectivity in both primary CD4+ T cells and HeLa cells expressing CD4 and CCR5. HD5 is induced in response to sexually transmitted infections (STIs) such as Chlamydia trachomatis and Neisseria gonorrhoeae, suggesting it plays a role in STI-mediated enhancement of HIV transmission. In contrast to our findings, a recent study reports that HD5 has an anti-HIV effect in primary CD4+ T cells under serum-deprived conditions. To resolve these apparently contradictory observations, we investigated experimental parameters that might contribute to contrasting effects of HD5.Serum-deprived culture conditions were associated with anti-HIV activity. In contrast to the dependence of the HIV enhancing effect on HD5 structure, the anti-HIV activity in serum-deprived primary CD4+ T cells was independent of HD5 structure as the linear peptide [Abu] HD5 exhibited similar anti-HIV activity. Under serum deprived conditions, HD5 blocked CD4-receptor-independent HIV-1vsv infection before or after viral entry. We found that HD5 and its linear form induced significant cell death in primary CD4+ T cells under serum-deprived culture conditions. HD5-mediated apoptosis was observed as early as 2 h after addition of defensins to serum-deprived primary CD4+ T cells. In contrast to primary CD4+ T cells, HD5 did not induce cytotoxicity and promote HIV infectivity of HeLa-CD4-CCR5 cells under serum-deprived conditions.These results indicate that under serum-deprived culture conditions HD5 is toxic for primary CD4+ T cells, warranting caution in data interpretation

The effect of serum on the HIV enhancing of defensins.

<p>Pseudotyped HIV-1_JR-FL luciferase reporter virus was incubated with or without defensins (5.6 µM) in the presence of FBS or human serum (HS) at various concentrations for 1 h. FBS or HS at a final concentration of 10% was added to the virus-defensin mixture before exposure to HeLa-CD4-CCR5. Luciferase activity was determined 48 h after infection. Results are expressed as fold increase compared to samples with the same concentration of FBS or HS in the absence of defensins. Assays were performed on triplicate cultures; results represent two independent experiments (mean ± SD).</p

HD5-mediated inhibition of HIV in serum-deprived primary CD4+ T cells occurs independently of HIV receptors.

<p>(A) CD4+ T cells from different preparation methods were infected with pseudotyped HIV-1_VSV-G luciferase reporter viruses by spinoculation. Infected cells were cultured under serum-deprived conditions. HIV infection was determined by measuring luciferase activity on day 3 after infection. (B) CD4+ T cells under serum-deprived conditions were treated with HD5 at different concentrations for 1 h at 37°C followed by exposure to serum-free pseudotyped HIV-1_VSV-G luciferase reporter viruses by spinoculation in the presence of HD5. Cells were then cultured in RPMI containing 0.3% human AB serum, ITS supplement, IL-2, and defensins for 3 days followed by measurement of luciferase activity. Data presented are the average ± standard deviation of 3 replicates and represent 2 independent experiments. *P<0.05, defensin-treated samples vs non-treated controls.</p

Anti-HIV activity and cytotoxicity of HD5 are not found in HeLa-CD4-CCR5 cells under serum-deprived condition.

<p>(A) HeLa-CD4-CCR5 cells were exposed to HD5 at indicated concentrations in serum-free RPMI-1640 medium for 2 h at 37°C. The effect of HD5 on HIV-exposed HeLa-CD4-CCR5 cells was also prepared as described in Fig. 6 as a comparison. The cells were then cultured for 24 h in complete medium (10%FBS) or under serum-deprived conditions. Cytotoxicity was measured by CytoTox-Glo cytotoxicity kit. (B) Serum-free HIV-1_JR-FL Env-pseudotyped reporter viruses were incubated with HD5 or [Abu]HD5 for 1 h at 37°C before exposure to HeLa-CD4-CCR5 cells for 2 h at 37°C. Cells were cultured in complete media or under serum-deprived conditions. Data presented are the average ± standard deviation of 3 replicates and represent 2 independent experiments. *P<0.05, defensin-treated samples vs non-treated controls.</p

CD4+ T cells from PHA-activated PBLs are less pure than cells prepared from freshly isolated PBLs.

<p>CD4+ T cells isolated from PHA-activated PBLs (left panel), CD4+ T cells right after isolation from fresh PBLs (middle panel), or CD4+ T cells isolated from fresh PBLs followed by PHA activation for 3 days (right panel) were stained with PE-conjugated mouse anti-CD3 Ab and PerCP-conjugated mouse anti-CD4 Ab. Gated live cells in the scatter plot are shown in the upper panels. The results shown are representative of 3 tested donors, which are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076038#pone-0076038-t001" target="_blank">Table 1</a>.</p

Serum deprivation contributes to anti-HIV activity of HD5 in primary CD4+ T cells.

<p>HIV-1 primary isolate 20635–4 (R5 virus, clade C) was incubated with HD5 at different concentrations for 1 h at 37°C. CD4+ T cells from PHA-activated PBLs (A) and PHA-activated CD4+ T cells (B) were incubated with virus-defensin mixture for 2 h at 37°C (no spinoculation) or 1.5 h at 1250×g spinning (spinoculation). After washing off unbound virus, cells were cultured in RPMI containing 10% FBS and IL-2 or 0.3% human AB serum, ITS supplement, and IL-2. HD5 was added back to cell cultures in the presence of IL-2. The level of p24 protein in culture media was measured by p24 ELISA. Data presented are the average ± standard deviation of 3 replicates. Similar results were observed in 3 independent experiments from different donors; *P<0.05, defensin-treated samples vs non-treated controls.</p

HD5 and linear peptide [Abu]HD5 induced cytotoxicity in primary CD4+ T cells under serum-deprived conditions.

<p>CD4+ T cells from PHA-activated PBLs (A) or PHA-activated CD4+ T cells (B) were exposed to HD5 or [Abu]HD5 at indicated concentrations with or without spinoculation. Cells were then cultured for 24 h under serum-deprived conditions in the presence of IL-2 and defensins. Cytotoxicity was measured by CytoTox-Glo cytotoxicity kit. Data presented are the average ± standard deviation of 3 replicates and represent 3 independent experiments. *P <0.05, defensin-treated samples vs non-treated controls.</p

HD5 induces cytotoxicity in HIV-exposed primary CD4+ T cells under serum-deprived conditions.

<p>HIV-1_JR-FL was incubated with HD5 at different concentrations at 37°C for 1 h. CD4+ T cells were incubated with defensin-virus mixture with or without spinoculation as described in Fig. 2. Cells were washed and then cultured with the original concentration of HD5 in either RPMI-1640 containing10% FBS and IL-2 or RPMI-1640 containing 0.3% human AB serum, 1× ITS supplement, and IL-2. After overnight culture, cytotoxicity was measured by CytoTox-Glo cytotoxicity kit (Promega). Data presented are the average ± standard deviation of 3 replicates; *P<0.05, defensin-treated samples vs non-treated controls. Similar results were observed in 2 independent experiments.</p