31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Selective Role of RGS9-2 in Regulating Retrograde Synaptic Signaling of Indirect Pathway Medium Spiny Neurons in Dorsal Striatum

In the striatum, medium spiny neurons (MSNs) are heavily involved in controlling movement and reward. MSNs form two distinct populations expressing either dopamine receptor 1 (D1-MSN) or dopamine receptor 2 (D2-MSN), which differ in their projection targets and neurochemical composition. The activity of both types of MSNs is shaped by multiple neuromodulatory inputs processed by GPCRs that fundamentally impact their synaptic properties biasing behavioral outcomes. How these GPCR signaling cascades are regulated and what downstream targets they recruit in D1-MSN and D2-MSN populations are incompletely understood. In this study, we examined the cellular and molecular mechanisms underlying the action of RGS9-2, a key GPCR regulator in MSNs implicated in both movement control and actions of addictive drugs. Imaging cultured striatal neurons, we found that ablation of RGS9-2 significantly reduced calcium influx through NMDARs. Electrophysiological recordings in slices confirmed inhibition of NMDAR function in MSNs, resulting in enhanced AMPAR/NMDAR ratio. Accordingly, male mice lacking RGS9-2 displayed behavioral hypersensitivity to NMDAR blockade by MK-801 or ketamine. Recordings from genetically identified populations of striatal neurons revealed that these changes were selective to D2-MSNs. Surprisingly, we found that these postsynaptic effects resulted in remodeling of presynaptic inputs to D2-MSNs increasing the frequency of mEPSC and inhibiting paired-pulse ratio. Pharmacological dissection revealed that these adaptations were mediated by the NMDAR-dependent inhibition of retrograde endocannabinoid signaling from D2-MSNs to CB1 receptor on presynaptic terminals. Together, these data demonstrate a novel mechanism for pathway selective regulation of synaptic plasticity in MSNs controlled by GPCR signaling.SIGNIFICANCE STATEMENT This study identifies a role for a major G-protein regulator in controlling synaptic properties of striatal neurons in a pathway selective fashion

GABAA receptor ɛ-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat

Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABAA receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex.In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 μm) did not Significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 μm) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50–100 μm) was effective in enhancing GABAA-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM; 1 or 10 μm), failed to depress GABA-induced currents in the cNTS.Microinjections of midazolam (10 and 100 μm solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 μm) significantly and reversibly depressed it (gain decrease to 53 ± 11 % of control, P < 0.01).Reverse transcriptase polymerase chain reaction revealed the presence of α1, α2, β2, β3 and γ2 GABAA receptor subunit mRNA in the cNTS. No alternatively spliced variants of the α1- and γ2-subunits were revealed. Moreover, GABAAɛ-subunit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts.Immunocytochemical analysis revealed numerous GABAAɛ-subunit-positive neurones within the cNTS with significantly fewer ɛ subunit-positive cells in the rNTS.As incorporation of the ɛ-subunit in recombinant GABAA receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of ɛ-subunit expression. This is the first demonstration of a possible physiological impact of the ɛ -subunit on native GABAA receptors