Raltegravir Inclusion Decreases CD4 T-Cells Intra-Cellular Viral Load and Increases CD4 and CD28 Positive T-Cells in Selected HIV Patients

Raltegravir (RLT) prevents the integration of HIV DNA in the nucleus, but published studies remain controversial, suggesting that it does not decrease proviral DNA. However, there are only a few studies focused on virus-targeted cells. We aimed our study on the impact of RLT inclusion on total intra-cellular viral DNA (TID) in cellular subsets and immune effects in patients with newly acquired undetectable plasmatic viral load (UVL). Six patients having UVL using an antiretroviral combination for 6 months and CD4 T-cells > 350/mL and <500/mL were selected to receive RLT for 3 months from M0 to M3. Patients had 7 sequential viro-immunological determinations from M-1 to M5. Immune phenotypes were determined by flow cytometry and TID quantification was performed using PCR assay on purified cells. TID (median values) at the initiation of RLT in CD4 T-cells was 117 copies/millions of cells, decreased to 27.5 on M3, and remained thereafter permanently under the cut-off (<10 copies/millions of cells) in 4 out of 6 patients. This was associated with an increase of CD4 and CD4 + CD28+ T-cells and a decrease of HLA-DR expression and apoptosis of CD4 T-cells. RLT inclusion led to decreases in the viral load along with positive immune reconstitution, mainly for CD4 T-cells in HIV patients.

Current Immunotherapeutic Strategies Targeting the PD-1/PD-L1 Axis in Non-Small Cell Lung Cancer with Oncogenic Driver Mutations

Treatment strategies targeting programed cell death 1 (PD-1) or its ligand, PD-L1, have been developed as immunotherapy against tumor progression for various cancer types including non-small cell lung cancer (NSCLC). The recent pivotal clinical trials of immune-checkpoint inhibiters (ICIs) combined with cytotoxic chemotherapy have reshaped therapeutic strategies and established various first-line standard treatments. The therapeutic effects of ICIs in these clinical trials were analyzed according to PD-L1 tumor proportion scores or tumor mutational burden; however, these indicators are insufficient to predict the clinical outcome. Consequently, molecular biological approaches, including multi-omics analyses, have addressed other mechanisms of cancer immune escape and have revealed an association of NSCLC containing specific driver mutations with distinct immune phenotypes. NSCLC has been characterized by driver mutation-defined molecular subsets and the effect of driver mutations on the regulatory mechanism of PD-L1 expression on the tumor itself. In this review, we summarize the results of recent clinical trials of ICIs in advanced NSCLC and the association between driver alterations and distinct immune phenotypes. We further discuss the current clinical issues with a future perspective for the role of precision medicine in NSCLC.

Dissection of anti-inflammatory and immunomodulatory activity of Mangifera indica L. reveals the modulation of mPGES-1/PPARγ axis and Th17/Treg ratio.

Background and Purpose: In the context of inflammation and immunity, there are fragmented and observational studies relating to the pharmacological activity of Mangifera indica L. and its main active component mangiferin. We, therefore, aimed to evaluate the potential beneficial effects of this plant extract (MIE, 90% in mangiferin) in a mouse model of gouty arthritis, dissecting the cellular immune phenotypes and the biochemical mechanism/s beyond its activity. Experimental Approach: Gouty arthritis was induced by the intra-articular administration of MSU crystals (200 μg 20 μl-1). MIE (0.1-10 mg kg-1) or corresponding vehicle (DMSO/saline 1:3) were orally administrated concomitantly to MSU (time 0), 6 and 12 h after the stimulus. Thereafter, knee joint score and oedema were evaluated in addition to western blot analysis for several components of mPGES-1/PPARγ pathway. Moreover, the analysis of pro/anti-inflammatory cyto-chemokines coupled to the assessment of the cellular infiltrate’s phenotype was investigated. Key Results: Treatment with MIE revealed a dose-dependent reduction in joint inflammatory scores with maximal inhibition observed at 10 mg kg-1. MIE significantly reduced leukocyte infiltration and activation and the expression of different pro-inflammatory cyto-chemokines in inflamed tissues. Furthermore, biochemical analysis revealed that MIE modulated COX-2/mPGES-1 and mPGDS-1/PPARγ pathways. Flow cytometry analysis also highlighted a prominent modulation of infiltrating inflammatory monocytes (CD11b+ve/CD115+ve/LY6Chi), and (both infiltrated and circulating) Treg cells (CD4+ve/CD25+ve/FOXP3+ve) after MIE treatment. Conclusion and Implications: Collectively, the results of this study demonstrate a novel function of MIE to positively affect the local and systemic inflammatory/immunological perturbance in the onset and progression of gouty arthritis.

Study of Bone Marrow Mesenchymal Stem Cells Regulating NK Cells Function through Tigit/ CD226 in Patients with Multiple Myeloma

Introduction：Bone marrow microenvironment plays a significant supporting role in the proliferation, differentiation, migration, survival and drug resistance of myeloma cells. To detect the expression of the co-inhibitory receptor TIGIT and the co-stimulatory receptor CD226 on the surface of natural killer cells (NK cells) in multiple myeloma (MM) patients, and changes in the immune phenotypes and killing function of NK cells. Furthermore, to explore the underlying mechanism of bone marrow mesenchymal stem cells (BMSCs) regulating NK cells function through TIGIT/CD226 in patients with MM. The expressions of TIGIT, CD226, activated molecules NKG2D and CD107a and functional molecules IFN-γ and Perforin on the NK cells in bone marrow or peripheral blood were detedted by FCM. BMSCs and NK cells were co-cultured at a ratio of 1:4 in vitro, which was grouped according to different monoclonal antibodies (mAbs) added. After co-cultured, the changes in the expression of NKG2D, NKp30, NKp44 and CD69 of NK cells and IFN-γ concentration were examined by FCM. NK cells and U266 were co-cultured with different E:T ratio and the apoptosis of U266 was detected to evaluate the killing function of NK cells by FCM. The expressions of CD155 on BMSCs in the bone marrow were detected by FCM. The correlations between CD155 expression on BMSCs and the production of IFN-γ and Perforin in bone marrow NK cells, as well as clinical characteristics were analyzed. Results: 1. In bone marrow, expression of TIGIT was significantly higher in patients with NDMM than those in patients with CR and HC (both P values &lt;0.01). CD226 was significantly lower in NDMM patients than those in patients with CR and HC (both P values &lt;0.01). NK cell surface activated molecules NKG2D and CD107a, and functional molecules IFN-γ and Perforin were significantly decreased in NDMM (Fig. A). In peripheral blood, TIGIT, CD226 and above immune phenotype expression levels were basically consistent with the trend in bone marrow. 2. In co-culture experiments of BMSCs and NK cells, only added anti-TIGIT mAbs, compared to both added anti-TIGIT and CD226 mAbs, the immune phenotypes of NK cells significantly increased (Fig. B). And the concentration of IFN-γ in the co-culture supernatants also increased (Fig. C). 3. After co-cultured with BMSCs, NK cells were co-cultured again with U266 in vitro, the apoptosis level of U266 in TIGIT mAbs group increased than those in TIGIT+ CD226 mAbs group at different E: T ratio (Fig. D). 4. The expression of CD155, the ligand of TIGIT and CD226, increased notably on the surface of BMSCs in MM patients(P&lt;0.01) (Fig. E). 5. The expression of CD155 on BMSCs was negatively correlated with the production of IFN-γ and perforin in bone marrow NK cells with NDMM patients. And it has correlation with clinical characteristics including β2-MG, LDH, ALB, Hb, ISS stage and R-ISS stage in patients with MM. Summary: BMSCs may regulate NK cells through TIGIT/CD226. High expression of TIGIT on NK cells may mediate the inhibitory effect of BMSCs in MM patients. Blocking TIGIT could restore NK cells activation and killing function. CD155, a common ligand of TIGIT and CD226, was overexpressed on BMSCs in NDMM patients, but was low expressed on bone marrow MM cells. And it has correlation with NK cells function and clinical characteristics, indicating that CD155 may be involved in the regulation of NK cells function by BMSCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Application of AI-Based Analytical Approaches for Multiplex Immunophenotyping of SF3B1-Mutant Myelodysplastic Syndrome Subtype

Background: Somatic mutations in splicing factor 3B subunit 1 (SF3B1) occur in 25% of all myelodysplastic syndromes (MDS) cases and identify a condition characterized by ring sideroblasts, ineffective erythropoiesis, and indolent disease course in lower-risk (LR) MDS. The impact of SF3B1 mutations on erythroid dysregulation has become apparent but little is known about cellular immune phenotypes. Identification of patient characteristics downstream of the pathogenic mutations in SF3B1 may reveal immunological determinants of disease phenotype and response to therapeutic interventions (e.g. luspatercept). In the present study, we applied multiplex immunophenotyping technologies in combination with artificial intelligence (AI)-based analytical approaches to identify genotype-immunophenotype correlations that may affect disease course and clinical outcomes in the SF3B1-MDS subtype. Methods: We performed transcriptomic immune profiling on bone marrow (BM) mononuclear cells (MNCs) from 12 SF3B1-MUT and 10 SF3B1-WT LR-MDS patients using the NanoString nCounter PanCancer Immune Profiling Panel. Besides, viably frozen PBMC samples (8 SF3B1-MUT; 4 SF3B1-WT) were thawed and stained for CyTOF with a 35-marker MaxPar Immune Profiling Panel (Fluidigm) for deep immune profiling. CyTOF data were analyzed with an in-house developed pipeline (ImmunoCluster, Opzoomer et al. 2021) incorporating the FlowSOM algorithm for unsupervised clustering (K=70). We also retrieved flow cytometric data on fresh BM and peripheral blood (PB) samples acquired independently as part of the diagnostic work-up and re-analyzed them using the T-REX pipeline (Barone and Paul et al. 2020). Results: On the transcriptomic level, the SF3B1-MUT subtype showed a distinct inflammatory signature compared to SF3B1-WT (59 DE genes at FDR &lt; 0.05, Fig. 1A & B). GO/KEGG pathway analysis indicated that downregulated genes in SF3B1-MUT patients are involved in antigen processing/presentation (HLA class II transcript, CD8a), regulation of proinflammatory interleukin-1 beta secretion (IL1B, NLRP3), and cellular response to interferon-gamma (HLA class II transcripts, IRF4/8, CCL5, CCL20). Genes that were upregulated in SF3B1-MUT BM MNCs are involved in cell cycle (CDK1, CCND3), proliferation/survival (AXL), iron homeostasis (TFRC), and erythroid differentiation (TAL1). Unbiased interrogation of cellular phenotypes in PB through FlowSOM analysis of CyTOF data revealed an increased abundance of a CD4 + T cell metacluster resembling a central memory (CM) phenotype in SF3B1-MUT compared to SF3B1-WT PBMCs (7.9 vs 4.5% of CD45 +). Independent automated T-REX-based analysis of clinical flow data supported this finding and further indicated expansion of a cluster resembling CM CD8 + T cells in SF3B1-MUT PB. Lower IL1B expression in SF3B1-MUT and the implication of the IL-1/IL-1RAP axis in the inflammatory leukemic niche (De Boer et al. 2020) prompted us to re-analyze clinical flow cytometric data on IL-1RAP expression using the T-REX pipeline. T-REX revealed differences in IL-1RAP-expressing clusters between SF3B1-MUT and SF3B1-WT BM samples (9 SF3B1-MUT; 9 SF3B1-WT). Specifically, we noticed a cluster of IL-1RAP + CD34 - cells (HLA-DR + CD123 +) present in SF3B1-WT but greatly contracted in SF3B1-MUT MDS (Fig. 1C & D). Our data suggest that proinflammatory signaling via the IL-1/IL-1RAP axis is reduced in SF3B1-MUT BM. Aberrant marker expression on SF3B1-MUT monocytes has been reported (Duetz et al. 2020). T-REX analysis of BM CD14 + cells revealed distinct monocyte clusters with differential expression of CD123, CD11b, and HLA-DR. Our initial analysis showed that SF3B1-MUT-specific clusters displayed lower expression of CD123 and CD11b, suggesting that mutations in SF3B1 directly or indirectly affect monocyte phenotypes and most likely function. Conclusions: We provide evidence that the SF3B1-MDS subtype is associated with a distinct inflammatory signature characterized by lower IL1B expression, changed IL-1RAP + clusters, distinct monocyte phenotypes, and preservation of T cell homeostasis. Further investigation of the immune signature in SF3B1-mutated MDS subtype may lead to using personalized immunotherapy strategies in the future. This work also exemplifies the potential of unsupervised strategies for interrogating cellular immune phenotypes in MDS subtypes within clinical flow cytometry datasets. Figure 1 Figure 1. Disclosures Harrison: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Platzbecker: Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria; AbbVie: Honoraria; Geron: Honoraria; Takeda: Honoraria. Kordasti: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Alexion: Honoraria; Beckman Coulter: Honoraria.

Gene Signatures and Cancer-Immune Phenotypes Based on m6A Regulators in Breast Cancer

The N6-methyladenosine (m6A) has been considered as a new layer of epitranscriptomic regulation on mRNA processing, stability, and translation. However, potential roles of m6A RNA methylation modification in tumor immune microenvironment (TIME) of breast cancer are yet fully understood. In this study, we comprehensively evaluated the genetic variations and transcript expressions of 15 m6A regulators in 1,079 breast cancer samples from the Cancer Genome Atlas (TCGA) database. We validated major regulators had significantly differential mRNA and protein expression in tumor tissue compared to normal tissues from 39 pairs of clinical breast cancer samples with different molecular subtypes, and especially high expression of m6A readers YTHDF1 and YTHDF3 predicted poor survival. Two clusters of breast cancer patients identified by the 15 m6A regulators’ pattern showed distinct overall survival, immune activation status, and immune cell infiltration, and clinical samples confirmed the diversity of lymphocytic infiltration. The profiles of these two clusters accorded with that of two classical cancer-immune phenotypes, immune-excluded and immune-inflamed phenotypes, it suggested that m6A regulators-based patterns might serve as crucial mediators of TIME in breast cancer. Moreover, the m6A phenotype-related gene signatures could also be survival predictor in breast cancer. Therefore, comprehensive evaluation of tumor m6A modification pattern will contribute to enhance our understanding of the characterization of immune cell infiltration in the tumor microenvironment and promote the responsiveness of breast cancer to immunotherapy.

921 Comparison of PI3K/AKT/mTOR pathway profiles amongst three immune phenotypes classified by artificial intelligence-powered H&E analyzer in non-small cell lung cancer

BackgroundThe phosphatidylinositol 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway plays a significant role in both tumorigenesis and progression of disease in non-small cell lung cancer (NSCLC).1 Increased activation of the pathway, whether in tumor or immune cells, results in an immunosuppressive tumor microenvironment.2 Therefore, we looked into how this pathway differs in three distinct NSCLC immune phenotypes.MethodsLunit SCOPE IO (Lunit, Seoul, Republic of Korea), a deep learning-based hematoxylin and eosin (H&E) image analytics tool, identifies lymphocytes and quantifies lymphocyte density within the cancer epithelium (CE-Lym), stroma (CS-Lym), and combined area (C-Lym). We applied Lunit-SCOPE IO to H&E-stained tissue images of 965 NSCLC samples from The Cancer Genome Atlas (TCGA). Tumors in the lowest tertile of C-Lym were labeled as immune-desert, and the remaining tumors were classified as inflamed and immune-excluded according to the median of the ratio of CE-Lym to CS-Lym.Utilizing RNA-sequencing data from TCGA, gene set enrichment analysis (GSEA) was conducted to analyze the differences in mTORC1 and PI3K/Akt/mTOR signaling between the subtypes.3 We obtained mutational data related to the PI3K/Akt/mTOR pathway from cBioPortal to compare the ratio of functional mutations between the immune phenotypes.4ResultsThe mTORC1 signaling gene set was consistently enriched in immune-excluded, whether compared to inflamed (padj < 0.01, normalized enrichment score [NES]: 2.3) or immune-desert (padj < 0.01, NES: 1.6). However, PI3K/Akt/mTOR signaling gene set enrichment did not show statistically significant differences between the immune phenotypes.Within the three immune phenotypes, we analyzed three functional mutations: PIK3CA, PTEN, and Akt1 (figure 1). Of the total 112 samples showing the functional mutations of the PI3K/Akt/mTOR pathway, 53 were immune-excluded, 31 inflamed, and 28 immune-desert. The relation between mutation frequency and the immune subtypes was significant (X2 (2) = 11.1979, p < .01). The immune-excluded was more likely than the other subtypes to have functional PI3K/Akt/mTOR mutations.Abstract 921 Figure 1The landscape of functional mutation and immune phenotypes regarding PI3K/Akt/mTOR pathwayConclusionsThe three tissue phenomic subtypes showed different PI3K/Akt/mTOR pathway profiles, with immune-excluded having the most mutation samples and the greatest enhancement of mTORC1 signaling gene set. Likewise, tissue H&E based tumor microenvironment classification by Lunit SCOPE IO can be applied to other hallmark pathways and tumor types, and such further investigation of the tumor microenvironment can provide insights into novel therapeutic avenues.ReferencesTan AC. Targeting the PI3K/Akt/mTOR pathway in non-small cell lung cancer (NSCLC). Thorac Cancer 2020;11(3):511–8.O’Donnell JS, Massi D, Teng MWL, Mandala M. PI3K-AKT-mTOR inhibition in cancer immunotherapy, redux. Semin Cancer Biol 2018;48:91–103.Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The molecular signatures database hallmark gene set collection. Cell Systems 2015;1(6):417–25.Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2(5):401–4.

91 Impact of ultra-fast ‘FLASH’ radiotherapy on single cell immunogenomics in diffuse intrinsic pontine glioma (DIPG)

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of 90 Gy/second and 2 Gy/minute, for the FLASH and CONV groups, respectively. Four days post-RT, mice brainstems were harvested, homogenized, stained for CD45 and tagged with a hashtag antibody specific to each group. CD45+ immune cells were isolated and sequenced using the 10X Genomics chromium single-cell 3’ platform. After processing and alignment of the reads using CellRanger with default parameters, the data was quality checked and filtered before hashtag demultiplexing, unsupervised clustering and downstream analysis was implemented following the Seurat R package. Differential expression evaluated based on the non-parametric Wilcoxon rank sum test. Key genes determine by an adjusted p value of < 0.05 based on bonferroni correction and |avg log2FC| > 0.8.ResultsPreliminary analysis identifies 15 clusters with distinct CD45 immune phenotypes (figure 1). Differential gene expression analysis by hashtag antibody (treatment group) reveals 14 clusters differentially expressing key genes, including 3 clusters upregulated in DIPG compared to NML, and 2 clusters upregulated in irradiated tumors compared to SHAM and NML (figure 2). Notably, analysis demonstrates an individual cluster upregulated in FLASH versus all other groups (p = 3.07E-171). Further deconvolution of specific immune phenotypes represented by each cluster is ongoing.Abstract 91 Figure 1tSNE plot based on clustering of RNA signatures, grouped by RNAAbstract 91 Figure 2tSNE plot based on clustering of RNA signatures, grouped by hashtag antibodyConclusionsOur preliminary analysis shows differential immune responses among DIPG tumors compared to NML. We also find several immune cell subsets that are unique to DIPG treated with CONV or FLASH compared to unirradiated samples. Most notably, we identify a single immune cell subset that is exclusive to FLASH alone, indicating that FLASH elicits a unique immune response in murine DIPG.

Comprehensive of N1-Methyladenosine Modifications Patterns and Immunological Characteristics in Ovarian Cancer

Backgroundrecently, many researches have concentrated on the relevance between N1-methyladenosine (m1A) methylation modifications and tumor progression and prognosis. However, it remains unknown whether m1A modification has an effect in the prognosis of ovarian cancer (OC) and its immune infiltration.MethodsBased on 10 m1A modulators, we comprehensively assessed m1A modification patterns in 474 OC patients and linked them to TME immune infiltration characteristics. m1Ascore computed with principal component analysis algorithm was applied to quantify m1A modification pattern in OC patients. m1A regulators protein and mRNA expression were respectively obtained by HPA website and RT-PCR in clinical OC and normal samples.ResultsWe finally identified three different m1A modification patterns. The immune infiltration features of these m1A modification patterns correspond to three tumor immune phenotypes, including immune-desert, immune-inflamed and immune-excluded phenotypes. The results demonstrate individual tumor m1A modification patterns can predict patient survival, stage and grade. The m1Ascore was calculated to quantify individual OC patient’s m1A modification pattern. A high m1Ascore is usually accompanied by a better survival advantage and a lower mutational load. Research on m1Ascore in the treatment of OC patients showed that patients with high m1Ascore showed marked therapeutic benefits and clinical outcomes in terms of chemotherapy and immunotherapy. Lastly, we obtained four small molecule drugs that may potentially ameliorate prognosis.ConclusionThis research demonstrates that m1A methylation modification makes an essential function in the prognosis of OC and in shaping the immune microenvironment. Comprehensive evaluation of m1A modifications improves our knowledge of immune infiltration profile and provides a more efficient individualized immunotherapy strategy for OC patients.