Serotonin inhibits axonal regeneration of identifiable descending neurons after a complete spinal cord injury in lampreys

Classical neurotransmitters are mainly known for their roles as neuromodulators, but they also play important roles in the control of developmental and regenerative processes. Here, we used the lamprey model of spinal cord injury to study the effect of serotonin in axon regeneration at the level of individually identifiable descending neurons. Pharmacological and genetic manipulations after a complete spinal cord injury showed that endogenous serotonin inhibits axonal regeneration in identifiable descending neurons through the activation of serotonin 1A receptors and a subsequent decrease in cyclic adenosine monophosphate (cAMP) levels. RNA sequencing revealed that changes in the expression of genes that control axonal guidance could be a key factor determining the serotonin effects during regeneration. This study provides new targets of interest for research in non-regenerating mammalian models of traumatic central nervous system injuries and extends the known roles of serotonin signalling during neuronal regeneration. This article has an associated First Person interview with the first author of the paper

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

Beta-catenin negatively regulates peripheral T cell activation (35.36)

Abstract β-catenin signaling has been shown to play a role in T cell development. However, the function of β-catenin in peripheral T cell regulation is not understood. Activation of T cells via TCR/CD28 ligation resulted in low-level stabilization of β-catenin which was augmented with proteasome inhibition. To determine the consequences of increased β-catenin expression, CAR Tg T cells were transduced with an adenoviral vector encoding a non-degradable β-catenin, which inhibited proliferation and cytokine secretion. Experiments were performed to determine the mechanism of this inhibition. Decreased IL-2 production was confirmed at the mRNA level, and surface expression of TCR and CD28 were not altered. IL-2 production was rescued with PMA+Ionomycin, arguing for a defect in proximal TCR signaling. TCR-induced phosphorylation of Lck, LAT, Akt, and ERK were intact in β-catenin-expressing T cells. However, phosphorylation of PLC-γ1 was significantly reduced, as was Ca++ flux. Conversely, deletion of the β-catenin gene from primary T cells using CAR Tg conditional knockout mice and a Cre-adenovirus caused augmented cytokine production and proliferation. Our results suggest that β-catenin negatively regulates TCR-mediated activation or peripheral T cells via a novel mechanism.</jats:p

β-catenin inhibits T cell activation by selective interference with LAT-PLC-γ1 phosphorylation. (50.12)

Abstract The functional role of β-catenin in post-thymic T cells is poorly understood. We observed that β-catenin protein was constitutively degraded in peripheral T cells. Introduction of a stabilized mutant of β-catenin into primary T cells resulted in reduced proliferation and cytokine secretion following TCR/CD28 stimulation, and interfered with effector cell differentation. The biochemical mechanism of blunted T cell activation was investigated. Stable β-catenin was predominantly localized to the cytosol, and cytokine production was rescued with PMA+Ionomycin, arguing for an inhibitory effect on early TCR signaling. Analysis of specific signaling events revealed a selective inhibition of LAT phosphorylation on tyrosine 136, which was associated with defective PLC-γ1 phosphorylation and calcium signaling but normal ERK activation. Stable β-catenin was detected constitutively in regulatory T cells, and conditional deletion of β-catenin in these cells caused rapid cell death. Contrary to a recent report using GSK-3 inhibitors, we saw no effect on T cell memory differentiation with direct genetic manipulation of β-catenin. Our findings indicate that β-catenin negatively regulates effector T cell activation via a unique biochemical mechanism. Functionally, our observations suggest that conditions under which β-catenin might be inhibited in vivo would potentiate T cell-based immunity at the level of both effector and regulatory T cells.</jats:p

Beyond PD-1: Investigating the Therapeutic Potential of TIGIT Blockade in DLBCL

Background: The PD-1/PD-L1 axis is a dominant cancer immune escape pathway, and PD-1 blockade therapy has greatly benefited patients with select solid tumors and lymphomas. Unfortunately, anti-PD-1 monotherapy has limited efficacy against relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) - a disease where new therapies are needed. Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial checkpoint blockade therapy (CBT) would enhance the activity of PD-1-based therapy in r/r DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a recently identified co-inhibitory receptor expressed on dysfunctional tumor-infiltrating T cells. PD-1 and TIGIT co-blockade therapy has demonstrated impressive activity in pre-clinical solid tumor and myeloma models. However, the degree to which TIGIT is involved in mediating T cell suppression in DLBCL is not fully known. Methods: TIGIT expression on lymphoma-infiltrating T cells (LITs) from 18 fresh lymphoma samples was analyzed by flow cytometry. Multiplex IHC on tissue microarrays (TMAs) was also performed to investigate TIGIT expression in DLBCL samples. The syngeneic murine A20 B cell lymphoma model was employed to study: 1) the kinetics of TIGIT and other co-receptor expression on LITs, 2) the association of TIGIT expression with effector function among LITs, and 3) the effectiveness of anti-TIGIT mono- and combination CBT in mice with established A20 lymphomas. A20 lymphoma tumors were established in groups of Balb/c mice by subcutaneous (SQ) injections of 5 x 106 cells. Expression of TIGIT and other co-receptors in A20 LITs was examined by flow cytometry at various time points. Function of TIGIT+ LITs was assessed by examining cytokine production following ex vivo stimulation with PMA and ionomycin. To test the efficacy of TIGIT blockade, mice received intraperitoneal injections of anti-TIGIT, anti-PD-1, anti-4-1BB, or combinations of these antibodies. Treatments began once tumors reached a diameter of 10 mm and were continued every 3 days for 5 doses. Tumor growth was monitored and compared to that in A20-bearing mice treated with isotype control antibodies. In some experiments, mice that achieved complete tumor rejection following single or dual CBT were re-challenged with A20 cells to investigate immunological memory responses. Results: Across a variety of human lymphomas, flow cytometric analysis revealed that TIGIT was broadly upregulated on LITs, including regulatory T cells and conventional CD4+and CD8+ T cells (Figure A and B). TIGIT expression on LITs in DLBCL was particularly high. Nearly all TIGIT+ LITs were also PD-1+, suggesting that these receptors co-orchestrate a T cell dysfunctional state in the lymphoma environment. Multiplex immunofluorescence staining of DLBCL samples demonstrated that TIGIT was most highly expressed on CD8+ T cells and that TIGIT+ T cells tended to be localized near, and in some cases, surrounding CD20+ lymphoma cells. Consistent with observations in human lymphomas, LITs isolated from murine A20 lymphoma commonly co-expressed TIGIT and PD-1, and the degree of expression correlated directly with tumor volume. This correlation was also present for other co-receptors, including 4-1BB, TIM3, and CTLA-4. Ex vivo restimulation of A20 LITs revealed that TIGIT+ T cells produced lower levels of effector cytokines, such as TNF-α, compared with TIGIT- T cells. In mice with established A20 lymphomas, both TIGIT and PD-1 mono-blockade led to modest delays in tumor outgrowth compared with mice treated with isotype control antibodies. Strikingly, however, combined PD-1 and TIGIT blockade resulted in complete rejection of A20 lymphomas in most mice and led to significantly prolonged survival compared to mice treated with single agent CBT (Figure C). Combination TIGIT and 4-1BB CBT was also remarkably effective in driving rejection of A20 lymphomas, and led to remarkable memory responses. Conclusions: TIGIT promotes immune tolerance in the DLBCL environment. While TIGIT monotherapy has anti-lymphoma activity, combinatorial CBT incorporating anti-TIGIT antibodies drives extremely potent rejection of established lymphomas in mice. These results provide rationale for further study of TIGIT blockade as a therapeutic strategy in r/r lymphomas, including DLBCL. Disclosures Sunseri: iTeos Therapeutics: Research Funding. Wald:iTeos Therapeutics: Employment. Preillon:iTeos Therapeutics: Employment. Smith:Portola Pharmaceuticals: Research Funding. Driessens:iTeos Therapeutics: Employment. Kline:Merck: Honoraria; Merck: Research Funding. </jats:sec

TIG-007: Study of EOS884448/GSK4428859A Alone, and in Combination with Iberdomide with or without Dexamethasone, in Participants with Relapsed or Refractory Multiple Myeloma

Abstract Background: T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor expressed on subsets of T cells and NK cells. In multiple myeloma (MM), TIGIT expression increases as the disease progresses and correlates with defective T cell effector functions. Higher TIGIT expression was observed in MM bone marrow CD8+ T cells in mice and patients compared to other immune checkpoint inhibitors, including PD-1, TIM-3, LAG-3, or CTLA-4 (Guillerey, C. et al, Blood 2018; Minnie, S. A. et al . Blood 2018). EOS884448/GSK4428859A (EOS-448) is a potent and highly selective fully human antagonist IgG1 antibody targeting TIGIT. Preclinically, anti-TIGIT Ab elicits superior anti-tumor immune responses compared to anti-PD1 mAbs (Guillerey, C. et al, Blood 2018). In murine Vk*Myc MM models, Fc-enabled a-TIGIT Ab elicits effective control of MM disease progression after autologous stem-cell transplant (ASCT), while Fc-disabled version is inactive. Anti-tumor activity is seen with monotherapy after ASCT at high T cell doses and provides significant synergistic activity when combined with an Immunomodulatory imide drug (IMiD) if T cell doses are suboptimal (Minnie, S. A. et al, Abstract submitted ASH 2021). Iberdomide (also known as CC-220) is a novel potent cereblon (CRBN) E3 ligase modulatory compound (CELMoD) that regulates multiple transcription factors within immune cells (Gandhi, A. K. et al . Brit J Haematol 2014). Iberdomide has shown notable clinical activity and acceptable tolerability in heavily pre-treated patients with relapsed or refractory multiple myeloma (RRMM), including those refractory to prior IMiDs (Lonial, S. et al. J Clin Oncol 2019). Given the dominant role of TIGIT in the immune suppression associated with MM, we hypothesize that TIGIT represents an ideal checkpoint to target clinically. EOS-448 alone or the synergistic combination of EOS-448 with iberdomide may provide a therapeutic opportunity to amplify myeloma-specific T-cell anti-tumor responses in difficult to treat RRMM patients previously exposed to IMiD, proteasome inhibitors (PIs) and anti-CD38. Methods: This phase I/II, open-label, multicenter, dose-escalation/expansion study will assess the safety, tolerability and preliminary activity of EOS-448 as monotherapy and in combination with iberdomide with or without dexamethasone in up to 158 adults with RRMM, who have progressed after prior treatments with IMiDs, PI and anti-CD38. EOS-448 will be infused intravenously on Day 1 of 28-day cycles. In Part 1, the safety and tolerability of EOS-448 as monotherapy and in combination with iberdomide with or without dexamethasone will be assessed in cohorts of up to 18 participants to identify the maximum tolerated dose and recommended phase II dose (RP2D) in each of the 3 treatment arms. In Part 2, the safety and anti-cancer activity of the RP2D will be assessed in up to 102 RRMM participants. Primary endpoints are treatment emergent adverse events, laboratory abnormalities, dose-limiting toxicities and clinical activity according to the International Myeloma Working Group (IMWG) Uniform Response Criteria. Secondary endpoints include overall response rates, duration of response, PK, and antidrug antibodies. Exploratory biomarkers including study treatment-mediated pharmacodynamic (PD) effects, PK-PD correlations, and correlative analyses of predictive and PD measurements with response, toxicity, and resistance will be investigated. Minimal residual disease (MRD) status with therapy will also be assessed as clinically indicated. The study is planned to open in November 2021 in North America and Europe. Disclosures Moreau: Abbvie: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Celgene BMS: Honoraria; Oncopeptides: Honoraria. Holmberg: Janssen: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Sanofi: Research Funding; Millennium-Takeda: Research Funding; Bristol Myers Squibb: Research Funding; Up-To-Date: Patents &amp; Royalties. Meuleman: iTeos Therapeutics: Consultancy. Graham: iTeos Therapeutics: Current Employment. De Henau: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company; Bristol-Meyer-Squibb: Current equity holder in publicly-traded company. Driessens: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. McGrath: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company; Norgine: Other: Spouse Current Employment. Lager: iTeos Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hill: NeoLeukin Therapeutics: Consultancy; Applied Molecular Transport: Research Funding; Syndax Pharmaceuticals: Research Funding; NapaJen Pharma: Consultancy; iTeos Therapeutics: Consultancy, Research Funding; Compass Therapeutics: Research Funding; Generon Corporation: Consultancy; Roche: Research Funding. </jats:sec

Inhibition of Tryptophan-Dioxygenase Activity Increases the Antitumor Efficacy of Immune Checkpoint Inhibitors

Abstract Tryptophan 2,3-dioxygenase (TDO) is an enzyme that degrades tryptophan into kynurenine and thereby induces immunosuppression. Like indoleamine 2,3-dioxygenase (IDO1), TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here, we described a new small-molecule inhibitor of TDO that can modulate kynurenine and tryptophan in plasma, liver, and tumor tissue upon oral administration. We showed that this compound improved the ability of anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we used TDO-KO mice and found that anti-CTLA4 or anti-PD1 induced rejection of MC38 tumors in TDO-KO, but not in wild-type mice. As MC38 tumors did not express TDO, we related this result to the high systemic tryptophan levels in TDO-KO mice, which lack the hepatic TDO needed to contain blood tryptophan. The antitumor effectiveness of anti-PD1 was abolished in TDO-KO mice fed on a tryptophan-low diet that normalized their blood tryptophan level. MC38 tumors expressed IDO1, which could have limited the efficacy of anti-PD1 in wild-type mice and could have been overcome in TDO-KO mice due to the high levels of tryptophan. Accordingly, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type, but not in TDO-KO, mice. These results support the clinical development of TDO inhibitors to increase the efficacy of immunotherapy of TDO-expressing tumors and suggest their effectiveness even in the absence of tumoral TDO expression. See article by Hoffmann et al., p. 19</jats:p

Inhibition of Tryptophan-Dioxygenase Activity Increases the Antitumor Efficacy of Immune Checkpoint Inhibitors

Tryptophan 2,3-dioxygenase (TDO) is an enzyme that degrades tryptophan into kynurenine and thereby induces immunosuppression. Like indoleamine 2,3-dioxygenase (IDO1), TDO is considered as a relevant drug target to improve the efficacy of cancer immunotherapy. However, its role in various immunotherapy settings has not been fully characterized. Here, we described a new small-molecule inhibitor of TDO that can modulate kynurenine and tryptophan in plasma, liver, and tumor tissue upon oral administration. We showed that this compound improved the ability of anti-CTLA4 to induce rejection of CT26 tumors expressing TDO. To better characterize TDO as a therapeutic target, we used TDO-KO mice and found that anti-CTLA4 or anti-PD1 induced rejection of MC38 tumors in TDO-KO, but not in wild-type mice. As MC38 tumors did not express TDO, we related this result to the high systemic tryptophan levels in TDO-KO mice, which lack the hepatic TDO needed to contain blood tryptophan. The antitumor effectiveness of anti-PD1 was abolished in TDO-KO mice fed on a tryptophan-low diet that normalized their blood tryptophan level. MC38 tumors expressed IDO1, which could have limited the efficacy of anti-PD1 in wild-type mice and could have been overcome in TDO-KO mice due to the high levels of tryptophan. Accordingly, treatment of mice with an IDO1 inhibitor improved the efficacy of anti-PD1 in wild-type, but not in TDO-KO, mice. These results support the clinical development of TDO inhibitors to increase the efficacy of immunotherapy of TDO-expressing tumors and suggest their effectiveness even in the absence of tumoral TDO expression.See article by Hoffmann et al., p. 19.status: publishe