Inhibitory checkpoint blockade therapy is an immunomodulatory strategy that results in the restoration of T cell functions, and its efficacy depends on the recognition of tumor cells for destruction. Considering the factors at play, one could propose that anti-tumor responses will not occur if tumor cells are immunologically invisible to T cells. In this study, we tested a strategy based on the modulation of cancer cell's immunovisibility through HDAC inhibition. In a model (heterotopic and orthotopic) of mouse urothelial bladder cancer, we demonstrated that the use of intratumoral or intravesical HDACi in combination with systemic anti-PD-1 was effective at inducing curative responses with durable anti-tumor immunity capable of preventing tumor growth at a distal site. Mechanistically, we determined that protective responses were dependent on CD8 cells, but not NK cells. Of significance, in an in vitro human model, we found that fully activated T cells fail at killing bladder cancer cells unless tumor cells were pretreated with HDACi. Complementary to this observation, we found that HDACi cause gene deregulation, that results in the upregulation of genes responsible for mediating immunorecognition, NKG2D ligands and HSP70. Taken together, these data indicate that HDAC inhibition results in the elimination of the tumor cell's "invisibility cloak" that prevents T cells from recognizing and killing them. Finally, as checkpoint blockade therapy moves into the adjuvant setting, its combined use with locally administrated HDACi represents a new approach to be included in our current therapeutic treatment toolbox.

Histone deacetylase inhibitors (HDACIs) strengthen memory following fear conditioning and cocaine-induced conditioned place preference. Here, we examined the effects of two non-specific HDACIs, valproic acid (VPA) and sodium butyrate (NaB), on appetitive learning measured via conditioned stimulus (CS)-induced reinstatement of operant responding. Rats were trained to lever press for food reinforcement and then injected with VPA (50–200 mg/kg, i.p.), NaB (250–1000 mg/kg, i.p.), or saline vehicle (1.0 ml/kg), 2h before receiving pairings of noncontingent presentation of food pellets preceded by a tone+light cue CS. Rats next underwent extinction of operant responding followed by response-contingent re-exposure to the CS. Rats receiving VPA (100 mg/kg) or NaB (1000 mg/kg) prior to conditioning displayed significantly higher cue-induced reinstatement than did saline controls. Rats that receiving either vehicle or VPA (100 mg/kg) prior to a conditioning session with a randomized relation between presentation of food pellets and the CS failed to show subsequent cue-induced reinstatement with no difference between the two groups. These findings indicate that, under certain contexts, HDACIs strengthen memory formation by specifically increasing the associative strength of the CS, not through an increasing motivation to seek reinforcement.

BackgroundHumans are genetically diverse and possess a rich immunological history. It is logical to consider that these factors may lead to differences in individual immunological responses to therapy when diagnosed with cancer. The successful implementation of immune-based therapies against cancer has brought the need to develop strategies to create meaningful profiles that faithfully depict the patient‘s immunological status. We report an in-depth immunological interrogation methodology, termed ATLAS. This system was designed to generate an accurate representation of the patient‘s immunological landscape that can be used during various time points during immune-checkpoint inhibitor (ICI) therapy.MethodsWe selected data from our prospective registry trial at Loyola University Medical Center to design individual immunological profiles of patients diagnosed with locally advanced or metastatic solid tumors planning to receive ICI. Only metastatic melanoma patients samples pre-ICI therapy are included in this first analysis. Twenty mL of peripheral blood were collected. Giving consideration to scientific rigor and limited sample availability, the assays were designed in miniaturized forms. ATLAS includes classical peripheral blood mononuclear cells (PBMCs) composition and T cell phenotypic and transcriptional analysis. To depict T cell functionality, we examined multiple parameters such as T cell receptor (TCR) signaling threshold, cell proliferation and NF-kB activation, at steady-state and in response to cell activation. To obtain both a broad and T cell-specific view, we quantified circulating chemokines and cytokines in plasma and from activated T cells.ResultsFor this first methodologic demonstration, patient characteristics are depicted in table 1. Data from different ATLAS assays were used to create individual immunological profiles presented as a dashboard for each patient. Distributional plots and measures of center (mean, median) and spread (range, variance) were used to eliminate low-information parameters from the figures. Data visualizations compared individual patients to the sample median for continuous parameters and compared patients‘ percentages to sample average relative abundance. Two patients, P011 shown in figure 1 and P021 shown in figure 2, are depicted using this approach.Abstract 52 Table 1Patient characteristicsAbstract 52 Figure 1Patient 011 dashboardAbstract 52 Figure 2Patient 021 dashboardConclusionsATLAS can be used in real-world conditions to generate comprehensive immunological profiles of cancer patients. Individual profiles indicate that immunological constitution is heterogeneous among patients, even with the same tumor type. We propose that the addition of ATLAS to our clinical and immunological toolbox may help stratify patients to articulate truly personalized oncologic therapies.Ethics ApprovalThe study was approved by Loyola University Medical Center and Loyola University Chicago Ethics Board and Institutional Review Board, approval number 209364.

panel using ddPCR to assess for presence and fractional abundance of the mutation in each sample.RESULTS: To date, 66 MIBC samples have been processed, of which 65 possessed at least one of the mutations within our gene panel, conferring a 98.5% detection rate, with a median of five panel mutations detected per sample (range 0-9). Our use of ddPCR may be accountable for our higher detection rate compared to the 40% predicted from the TCGA, which used Next Generation Sequencing technology. Further analyses are being undertaken to compare samples before and after neo-adjuvant chemotherapy.CONCLUSIONS: This technique utilising a 10 gene panel appears to be sensitive enough to detect mutations in the majority of patients with MIBC. Prospective validation is underway to determine the potential clinical utility of this technique.

Studies have shown the importance of CD62L in leukocyte trafficking. However, the significance of CD62L-expressing cells in concomitant anti-tumor immunity has not been investigated. Using a model of post-surgical tumor immunity, we show that in vivo depletion of CD62L-expressing cells before or immediately after surgical tumor resection enabled the development of anti-tumor concomitant responses against poorly immunogenic B16 (melanoma) or MTE-Ras (head & neck cancer) tumor cells. Anti-CD62L mAb administration depleted the cellular immune components that express CD62L, including NK, NKT, Mo, Ma, B, and T cells. Within the T cell compartment, naïve T cells (CD44lo CD62Lhi), effector (CD44hi CD62Llo), memory (CD44hi CD62Lhi) and regulatory T cells (Treg, CD4 CD25 Foxp3) were also depleted, while effector CD62L-negative T cells were spared in the TDLN. Tumor immunity did not occur in the absence of a primary tumor (sham surgery group) or after CD8 depletion. Whereas anti-CTLA-4 or anti-PD-1 mAbs failed as monotherapies, their administration during CD62L depletion restored their anti-tumor properties. The unified global gene expression analysis of the TDLN shows the significant enrichment of biological processes involved in the CD8 T cell effector phase, IL-2/IL-15, TNF-α, IFN-γ, IFN-α, IL-6, responses, mitotic cell division, glycolysis, and acetylation. Also, genes related to Treg responses were downregulated. These data point to a process in which depletion of CD62L cells in tumor-bearing hosts transforms the TDLN into a highly active immunological site in which tumor-reactive effector CD8 T cells (CD62L-negative) actively divide in the absence of Treg, accompanied by a cytokine response, glucose consumption, and acetylation of transcriptional factors and histones. These results identify an unrecognized role of CD62L-expressing cells in regulating concomitant anti-tumor immunity and restoring anti-tumor responses by checkpoint blockade inhibitors. Our study provides a new framework that could help understand how anti-tumor responses are limited. Citation Format: Brianna Burke, Lourdes Plaza-Rojas, Cynthia Perez, Elena Kostenko, Anna Austin, Justin Boucher, Kushal Prajapati, Michael Delos Reyes, Christine Chung, Jose-Alejandro Guevara-Patino. Concomitant anti-tumor immunity and checkpoint blockade responses are restored by depletion of CD62L expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6414.