CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs

Cannabinoid Receptor 2-Mediated Attenuation of CXCR4-Tropic HIV Infection in Primary CD4+ T Cells

Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB 1R and CB 2R) and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gai-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine coreceptor function and regulate HIV-1 infectivity. We found that agonism of CB2R, but not CB1R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB2R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB2R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB2R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB 2R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB2R agonists in the treatment of AIDS symptoms may also exert beneficia

In Vivo HIV-1 Cell-to-Cell Transmission Promotes Multicopy Micro-compartmentalized Infection

HIV-1 infection is enhanced by adhesive structures that form between infected and uninfected T cells called virological synapses (VSs). This mode of transmission results in the frequent co-transmission of multiple copies of HIV-1 across the VS, which can reduce sensitivity to antiretroviral drugs. Studying HIV-1 infection of humanized mice, we measured the frequency of co-transmission and the spatiotemporal organization of infected cells as indicators of cell-to-cell transmission in vivo. When inoculating mice with cells co-infected with two viral genotypes, we observed high levels of co-transmission to target cells. Additionally, micro-anatomical clustering of viral genotypes within lymphoid tissue indicates that viral spread is driven by local processes and not a diffuse viral cloud. Intravital splenic imaging reveals that anchored HIV-infected cells induce arrest of interacting, uninfected CD4+ T cells to form Env-dependent cell-cell conjugates. These findings suggest that HIV-1 spread between immune cells can be anatomically localized into infectious clusters

CB₂ agonism inhibits SDF-1á mediated G-protein coupling to CXCR4.

<p>(A–B) GTPãS binding following addition of the CXCR4 agonist SDF-1á to permeabilized primary CD4+ T cells pretreated with either DMSO or 100 nM of the CB₂ agonist JWH-133 (A) in a representative donor and (B) in multiple donors (mean±SEM; n = 4 donors; **p = 0.0034, *p<0.02). (C–D) GTPãS binding following addition of the CB₂ agonist JWH-133 to permeabilized CD4+ T cells pretreated with either DMSO or 100 nM of the CXCR4 agonist SDF-1á (C) in a representative donor and (D) in multiple donors (mean±SEM; n = 4 donors).</p

Cannabinoid inhibition of CXCR4-tropic HIV-1 infection is dose-dependent and CB₂-selective.

<p>(A) HIV-1 (GFP) expression 4 days post-infection in primary CD4+ T cells pretreated with 100 nM of the CB2-specific agonist JWH-133 and/or the CB₂-specific antagonist AM630 in a representative donor. HIV expression (GFP; FL1) is shown on the x-axis, and an empty channel (FL3) is shown on the y-axis. (B) Dose-dependent inhibition of viral infection by JWH-133 in primary CD4+ T cells is blocked by 100 nM of the CB₂ antagonist AM630 (mean±SEM; n = 7 donors; **p = 0.0032, *p<0.03). (C) Inhibition of CXCR4-tropic (NL4-3) but not CCR5-tropic (JRFL) HIV-1 after 4 days in primary CD4+ T cells pretreated with 100 nM of JWH-133 (mean±SEM; n = 5 donors). (D) Inhibition of viral infection in cells pretreated with the CXCR4 antagonist AMD3100 (which is predicted to block viral binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033961#pone.0033961-Wu3" target="_blank">[57]</a>), or 1 µM of cannabinoid agonists ACEA, Hu210, JWH-133, JWH-015 and Ser016 with or without pretreatment with the CB₁ antagonist AM251 or the CB₂ antagonist AM630. (E–F) Dose-dependent inhibition of NL-G1 infection in primary CD4+ T cells pretreated with (F) 2-arachidonoylglycerol (2-AG) or (G) anandamide (AEA) is blocked by 1ìM of the CB₂ antagonist AM630 (mean±SEM; n = 5 donors; *p<0.03).</p

CB₂ agonism inhibits productive infection of CXCR4-tropic HIV-1, but not cell-associated transfer or viral fusion.

<p>(A–B) Viral fusion, as indicated by â-lactamase cleavage, in primary CD4+ T cells pretreated with either JWH-133 or AMD3100 in (A) a representative donor and (B) in multiple donors (mean±SEM; n = 4 donors). (C) Transfer of HIV (Gag-iCherry) from infected Jurkat donors to memory (CD45RO+) or naive (CD45RO-) primary CD4+ T cells pretreated with 100 nM of the CB2 agonist JWH-133 or the CXCR4 antagonist AMD3100, in a representative donor. (D) Quantification of viral HIV (Gag-iCherry) transfer from Jurkat donors after 3 hours in CD45RO+ and CD45RO- cells pretreated with JWH-133 or AMD3100 (mean±SEM; n = 4 donors). (E–G) Productive infection following HIV transfer from primary CD4+ donors infected with HIV (GFP). Pretreated CD4+ targets were cocultured with infected CD4+ donor for 3 hours, sorted by CD45RO expression, and then activated. HIV (GFP) expression is shown in (E) CD45RO- naive or (F) CD45RO+ memory CD4+ T cells after 4 days of activation with IL-2 and anti-CD3/CD28 or anti-CD3 alone, respectively. (G) Quantification of productive infection post-activation in sorted CD45RO- or CD45RO+ CD4+ T cells pretreated with JWH-133 or AMD3100 (mean±SEM; n = 4 donors; **p = 0.0099; *p<0.05).</p

CB₂ agonism inhibits acute SDF-1á mediated signaling in primary CD4+ T cells.

<p>(A–B) SDF-1á induced phosphorylation of Akt and (C–D) p42/44 MAP kinase is downregulated in CD4+ T cells pretreated with 100 nM of the CB2 agonist JWH-133. (A, C) Representative western blots of phospho-kinase expression following SDF-1á treatment for 0, 0.5, 1, 5, 15 or 30 minutes. (B, D) Quantification of kinase phosphorylation in multiple donors (mean±SEM; n = 3 donors; *p<0.05) taken over basal, defined as signal at time 0.</p

CB₂ agonism at concentrations sufficient to inhibit HIV-1 infection does not significantly alter cell-surface CXCR4 expression or inhibit cellular proliferation in primary CD4+ T cells.

<p>(A–B) Cell surface expression of CXCR4 in CD4+ T cells pretreated with either DMSO or 100 nM of JWH-133 (A) in a representative donor and (B) in multiple donors (mean±SEM; n = 4 donors). (C–E) Proliferation, as indicated by CFSE dilution; and activation, as measured by CD25 expression; in CD4+ T cells, stimulated with cross-linking anti-CD3 and anti-CD28 mAb, 4 days after pretreatment with 100 nM of JWH-133 (C) in a representative donor and (D–E) in multiple donors (mean±SEM; n = 5 donors).</p