Changes In CD4 Research Articles

PO-1257 A radiomic signature model to predict chemoradiation-induced change in CD8+ cells in rectal cancer

PO-1257 A radiomic signature model to predict chemoradiation-induced change in CD8+ cells in rectal cancer

A radiomic signature model to predict the chemoradiation-induced alteration in tumor-infiltrating CD8+ cells in locally advanced rectal cancer

Background and purposeRegarding the altered tumor immune status following cytotoxic treatment, this study aims to develop a radiomic signature to predict CD8+ tumor-infiltrating lymphocyte (TIL) density changes in chemoradiotherapy (CRT) of rectal cancer. Materials and methodsWe used the magnetic resonance imaging (MRI) and immunohistochemistry data before and after neoadjuvant CRT. The discovery datasets consisted of pre-CRT dataset A1 (n = 113), post-CRT datasets A2 (n = 32; predominance of tumor) and A3 (n = 20; pure fibrosis). The developed model was validated in dataset B (n = 28). Thirty-eight radiomic features from T2-weighted MRI scans were incorporated into the least absolute shrinkage and selection operator method. ResultsIn pre-CRT dataset A1, the area under the receiver operating characteristic curve (AUC) values of radiomic score for predicting CD8+ TILs were 0.760 and 0.729 for training and validation subsets, respectively. A significant correlation was observed between the signature and CD8+ TIL density in the post-CRT dataset A2 (Pearson’s R = −0.372, P = 0.036), whereas no association was found in dataset A3 (Pearson’s R = −0.069, P = 0.77). The association was also observed in the validation dataset B (Pearson’s R = −0.374, P = 0.049). In dataset A2, the radiomic score difference predicted changes in CD8+ TIL density (AUC = 0.824). ConclusionWe established the MRI-derived radiomic signature for predicting CRT-induced alterations in CD8+ TILs. This study suggests the clinical utility of radiomics-immunophenotype modeling to evaluate tumor immune status following neoadjuvant chemoradiation in rectal cancer.

Abstract 2734: Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer

Abstract Background: The incidence of colorectal cancer (CRC) in those of pre-screening age continues to rise. A deeper understanding of the differences in tumor biology in early age onset (EAO) cancers is needed. Differences in the tumor microenvironment have been understudied in this setting. We previously have identified versican (VCAN), a large matrix proteoglycan, as an important factor in lymphocyte exclusion in CRC. Here we explore the impact of the mutation profile and VCAN status on the infiltration of CD8+ and CD4+ lymphocytes in EAO CRC. Methods: Cancer tissues from 153 patients with CRC were stained via immunohistochemistry for CD4, CD8, and VCAN. CD4 and CD8 stains were quantified by number of positive-staining tumor-infiltrating lymphocytes per high powered field (TILs/HPF). VCAN stains were quantified on an intensity scale from 0-3+. 121 of these patients had tissue that was sequenced using the Qiagen Comprehensive Cancer targeted sequencing panel, and variations were called using Strelka. Patients were split by age into early-onset (EAO; age at diagnosis <50) and later-onset (LAO; age at diagnosis 50 or later). Results: Mutations in BRAF and APC were significantly more common in LAO cancers (p<0.05 for both). TP53 mutations were correlated with significantly lower CD4+ and CD8+ TILs/HPF in the LAO cohort (12.6 CD4+ TILs/HPF for TP53-wild type [WT] vs 3.5 for TP53-mutant, p<0.001; 10.0 CD8+ TILs/HPF for WT vs 3.2 for mutant, p<0.01), but trended opposite, though not statistically significant, in the EAO group (2.4 vs 5.9 CD4+ TILs/HPF; 3.0 vs 5.9 CD8+ TILs/HPF). PIK3CA mutations were correlated with an increase in CD8+ TILs in the LAO cohort (5.3 vs 11.9 CD8+ TILs/HPF; p<0.05) but a decrease in CD4+ TILs in the EAO cohort (4.9 vs 0.8 CD4+ TILs/HPF; p<0.05). TP53-WT tumors are disproportionately low in VCAN in the LAO cohort (p<0.01). Differences in VCAN accumulation could account for the changes in CD8+ and CD4+ T lymphocyte infiltration across the age cohorts and TP53 mutation status. Conclusions: The impacts of TP53 and PIK3CA mutations on immune infiltration in CRC differ in EAO compared to later onset cancers, indicating that the processes controlling immune infiltration vary between age cohorts. Such differences in immune infiltration may be linked to changes in microenvironmental factors such as VCAN. These data warrant further investigation into the relationship between tumor microenvironment and mutation profiles in EAO CRC. Citation Format: Katherine A. Johnson, Philip B. Emmerich, Anna L. Lippert, Cheri A. Pasch, Linda Clipson, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Impact of the mutation profile and versican status on lymphocyte infiltration in early age onset colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2734.

PI3K-δ Inhibition Influences T-Cell Populations and Anti-Tumoral Immune Function in Preclinical Models

Chronic lymphocytic leukemia (CLL) is characterized by continuously activated B-cell receptor (BCR) signaling. Phosphoinositide 3-kinase delta (PI3K-δ) is an important mediator of BCR signaling, which controls cell survival and proliferation through activation of AKT. The PI3K-δ inhibitor idelalisib (IDELA) is a highly effective treatment option for relapsed/refractory CLL patients. In the frontline setting, IDELA in treatment-naïve patients was associated with frequent adverse effects (AEs), such as an increased infection rate, colitis, and pneumonitis. Therefore, understanding the impact of PI3K-δ inhibition on the immune cells in the CLL microenvironment is of importance to identify novel treatment strategies to overcome these AEs.Methods and Results: In this study, we specifically focused on the effect of PI3K-δ inhibition on T-cell function in CLL using the Eµ-TCL1 adoptive transfer (TCL1 AT) model. In brief, Eµ-TCL1 tumor cells were transferred intravenously into syngeneic littermates, followed by continuous treatment after reaching a tumor load of 50% in peripheral blood. GS-649443, a tool compound with more favorable murine pharmacokinetic properties than IDELA, was used for PI3K-δ inhibition in vivo . Tumor load, as well as the immune microenvironment, was analyzed after 2 weeks of continuous treatment of the drug.Since PI3K-δ signaling has been previously demonstrated to be important for CD4+ regulatory T-cell (Treg)-mediated suppression of antitumor immunity, we studied the impact of PI3K-δ inhibition on differentiation and activation of Tregs. GS-649443 treatment resulted in a dramatic reduction in numbers of Foxp3+ CD25+ CD4+ Tregs, as well as a decrease in Treg maturation and activation markers (CD103, GITR, KLRG1, and CD69) compared to vehicle treatment.We further assessed the impact of PI3K-δ inhibition on TCL1 AT-induced changes in CD8+ T-cell differentiation and subset distribution. Notably, treatment with GS-649443 resulted in a dose-dependent decrease in effector T-cell numbers, accompanied by a decrease in activation markers such as CD69 and CD137. In line with these findings, effector cell proliferation, as measured by Ki-67, significantly decreased in GS-649443-treated mice. In accordance with that, degranulation capacity, granzyme B, and IFNγ production by CD8+ T cells was substantially reduced by GS-649443 treatment. These results were further confirmed in a treatment study with PI-3065, another PI3K-δ-specific inhibitor.To investigate a direct impact of PI3K-δ inhibition on CD8+ T cells, we performed in vitro studies with GS-649443. For that purpose, we used splenocytes from Nur77-GFP mice, a reporter mouse model for T-cell receptor (TCR) activity, and stimulated these with aCD3 antibody after pre-treatment with GS-649443 or vehicle. PI3K-δ inhibition led to a profound reduction of TCR signaling, which was accompanied by decreased expression of activation markers like CD69, CD44, CD25, and CD137 on CD8+ T-cells. Furthermore, GS-649443 pre-treatment resulted in significant dose-dependent decrease in CD8+ T-cell proliferation rates, as measured by CFSE dilution. These findings were confirmed using aCD3-stimulated human peripheral blood mononuclear cells that were pretreated with IDELA in vitro, which resulted in an impairment of CD8+ T-cell activation measured by CD69 and CD25 expression, as well as proliferation measured by CFSE dilution.Conclusion: This study clearly shows that PI3K-δ inhibition abrogates the immunosuppressive activity of Tregs in an in vivo model of CLL. Additionally, PI3K-δ inhibition of CD8+ T cells leads to decreased activation and proliferation of these cells, which might contribute to the immune-mediated AEs observed with IDELA when used as frontline treatment. DisclosuresStilgenbauer:Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Changes in T lymphocyte subsets in different tumors before and after radiotherapy: A meta-analysis.

2525 Background: Radiation therapy (RT) induces an immune response, but the relationship of this response with tumor type is not fully understood. Methods: We searched English-language electronic databases including PubMed, EMBASE, and the Cochrane Library to collect studies about the changes in CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes before and after radiotherapy in tumor patients from January 2015 to December 2019. The quality of the included literature was evaluated using the NOS scale provided by the Cochrane Collaboration, and statistical software RevMan 5.4 was used to analyze the included literature. p < 0.05 was considered to indicate statistical significance. Results: A total of 17 studies in 15 articles involving 1735 tumor patients were included. All data were collected within 1 month before or after radiotherapy. Meta-analysis showed that numbers of CD3+ T lymphocytes were significantly reduced after radiotherapy compared with before treatment (standard mean difference [SMD]: -0.76; 95% CI [-1.46, -0.06]; p = 0.03), as were those of CD4+ T lymphocytes (SMD: -0.50; 95% CI: [-0.88, -0.12]; p = 0.01), but there was no statistically significant difference for CD8+ T lymphocytes (SMD: 0.19; 95% CI: [-0.23, 0.62]; p = 0.38). Subgroup analysis showed significant decreases in CD3+ T lymphocytes in liver cancer,esophageal cancer, head and neck cancer, pancreatic cancer and breast cancer after radiotherapy. Numbers of CD4+T lymphocytes increased after radiotherapy in breast cancer,and a decrease was observed in liver cancer, esophageal cancer, colorectal cancer, and head and neck cancer. CD8+ T lymphocyte numbers also increased compared with before radiotherapy in esophageal cancer, lung cancer, and colorectal cancer. But a decrease in liver cancer and head and neck cancer. Conclusions: Numbers of CD3+ and CD4+ T lymphocytes decreased after radiotherapy, whereas CD8+ T lymphocytes showed no significant change. Within 1 month of radiotherapy, the tumor microenvironment showed an immunosuppressive state. The degree of immune response induced by radiotherapy differed between tumor types.

Phase II study of the anti-TGF-β monoclonal antibody (mAb) NIS793 with and without the PD-1 inhibitor spartalizumab in combination with nab-paclitaxel/gemcitabine (NG) versus NG alone in patients (pts) with first-line metastatic pancreatic ductal adenocarcinoma (mPDAC).

TPS4173 Background: Overall survival remains low for pts with mPDAC despite approved therapies, highlighting the need for further innovative treatment options. Intra-tumoral fibrosis that characterizes PDAC has been associated with a state of immune exclusion and may constitute a mechanical obstacle to the penetration of chemotherapy into the tumor as well as contribute to the lack of efficacy of immunotherapy. TGF-β plays a key role in regulating the tumor microenvironment and emerging evidence points to TGF-β as a pivotal activator of cancer-associated fibroblasts, leading to the development of fibrotic networks. Preclinical data in murine models have shown that TGF-β blockade augmented the benefit of both NG and anti-PD-1 therapy, leading to tumor regression. These data provide the rationale for combining TGF-β-targeting agents with immunotherapy and chemotherapy. NIS793 is a human IgG2 mAb that binds to TGF-β. This study investigates NIS793 with and without spartalizumab combined with NG in treatment naïve mPDAC. Methods: This is a phase II open-label, randomized, multicenter study (NCT04390763) beginning with a safety run-in period followed by randomization. Eligible pts are adults with previously untreated mPDAC and an ECOG performance status ≤1. Pts are excluded if they have a tumor histology other than adenocarcinoma or microsatellite instability-high tumor. The safety run-in data will be analyzed after ≥6 pts have received NIS793 (intravenously [IV] 2100 mg Q2W) + spartalizumab (IV 400 mg Q4W) + nab-paclitaxel (IV 125 mg/m2 on Days 1, 8 and 15) + gemcitabine (IV 1000 mg/m2 on Days 1, 8 and 15) for 1 cycle (28 days) to assess the safety and tolerability of the combination and confirm the dose for the randomized part. Pts will be randomized 1:1:1 to NIS793 + spartalizumab + NG (n=50) or NIS793 + NG (n=50) or NG (n=50). Treatment will continue until unacceptable toxicity, disease progression, discontinuation by investigator’s or pt’s choice, or withdrawal of consent. The primary objective is to evaluate the progression-free survival of NIS793 + NG ± spartalizumab versus NG alone. Secondary objectives include safety and tolerability, antitumor activity, overall survival, change in CD8 and PD-L1 status, and characterization of immunogenicity and pharmacokinetics. Efficacy will be assessed locally per RECIST v1.1 and iRECIST at screening, every 8 weeks for 1 year and then every 12 weeks until disease progression. Blood and tumor samples will be taken at baseline and during study treatment for pharmacokinetic, immunogenicity and biomarker assessments. This study is ongoing and will enroll pts from 30 sites across 15 countries. The first pt was treated on October 22, 2020. Clinical trial information: NCT04390763.

Single-cell characterization of a model of poly I:C-stimulated peripheral blood mononuclear cells in severe asthma

BackgroundAsthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods.MethodsTwo complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study.ResultsPBMCs (n = 9414) from five SA (n = 6099) and three HC (n = 3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4 + T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n = 160,000) from the same individuals (SA = 5; HC = 3) demonstrated higher CD8 + and CD8 + effector T cells in SA at baseline, followed by a decrease of CD8 + effector T cells after poly I:C stimulation.ConclusionsSingle-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8 + effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.

Interferon regulatory factor 4 controls effector functions of CD8+ memory T cells

The transcription factor IRF4 is required for CD8+ T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8+ T cell responses. The function of IRF4 in memory CD8+ T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8+ memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8+ memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8+ memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8+ effector memory T cells, CD8+ tissue-resident memory T cells (TRM cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8+ TRM-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8+ memory T cells. Formation and maintenance of CD8+ TRM cells, in contrast, appear to depend on IRF4.

Myeloid cell-derived HOCl is a paracrine effector that trans-inhibits IKK/NF-κB in melanoma cells and limits early tumor progression.

The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity. Upon MDC activation, MPO is secreted into phagosomes where it catalyzes the production of hypochlorous acid (HOCl), a potent chlorinating oxidant. Here, we demonstrated that the myeloid lineage-restricted MPO-HOCl system had antitumor effects in early melanoma growth in aged mice. Orthotopic melanomas grew more slowly in immunocompetent MPO+/+ host mice compared to age-matched syngeneic MPO-/- mice. Real-time intravital tumor imaging in vivo and in cell cocultures revealed a cell-cell proximity-dependent association between MDC-derived MPO enzyme activity and blockade of ligand-induced IκBα degradation in tumor cells. HOCl directly trans-inhibited IκB kinase (IKK) activity in tumor cells, thereby decreasing nuclear factor κB (NF-κB) transcriptional activation and inducing changes in the expression of genes involved in metabolic pathways, cell cycle progression, and DNA replication. By contrast, HOCl induced transcriptional changes in CD8+ T cells related to ion transport and the MAPK and PI3K-AKT signaling pathways that are associated with T cell activation. MPO increased the circulating concentrations of the myeloid cell-attracting cytokines CXCL1 and CXCL5, enhanced local infiltration by CD8+ cytotoxic T cells, and decreased tumor growth. Overall, these data reveal a role for MDC-derived HOCl as a small-molecule paracrine signaling factor that trans-inhibits IKK in melanoma tumor cells, mediating antitumor responses during early tumor progression.

Talimogene laherparepvec upregulates immune-cell populations in non-injected lesions: findings from a phase II, multicenter, open-label study in patients with stage IIIB–IVM1c melanoma

BackgroundTalimogene laherparepvec (T-VEC), an oncolytic virus, was designed to selectively replicate in and lyse tumor cells, releasing tumor-derived antigen to stimulate a tumor-specific immune response.MethodsIn this phase II study in...

Neurokinin-1 receptor signaling induces a pro-inflammatory transcriptomic profile in CD16+ monocytes

Neurokinin-1 receptor (NK1R) signaling can be immunomodulatory and it can lead to preferential transmigration of CD14+CD16+ monocytes across the blood brain barrier, potentially promoting the development of inflammatory neurological diseases, such as neuroHIV. To evaluate how NK1R signaling alters monocyte biology, RNA sequencing was used to define NK1R-mediated transcriptional changes in different monocyte subsets. The data show that NK1R activation induces a greater number of changes in CD14+CD16+ monocytes (152 differentially expressed genes), than in CD14+CD16− monocytes (36 genes), including increases in the expression of NF-κB and components of the NLRP3 inflammasome pathway. These results suggest that NK1R may alter the inflammatory state of CD14+CD16+ monocytes, influencing the development of neuroinflammation.

Modified Hypoxia-Inducible Factor Expression in CD8+ T Cells Increases Antitumor Efficacy.

Adoptive transfer of antitumor cytotoxic T cells is an emerging form of cancer immunotherapy. A key challenge to expanding the utility of adoptive cell therapies is how to enhance the survival and function of the transferred T cells. Immune-cell survival requires adaptation to different microenvironments and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation. In this study, we undertook experiments to define structural determinants of HIF that potentiate antitumor efficacy in cytotoxic T cells. We first created retroviral vectors to deliver ectopic expression of HIF1α and HIF2α in mouse CD8+ T cells, together or individually and with or without sensitivity to the oxygen-dependent HIFα inhibitors Von Hippel-Lindau and factor-inhibiting HIF (FIH). HIF2α, but not HIF1α, drove broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. A specific mutation replacing the hydroxyl group-acceptor site for FIH in HIF2α gave rise to the most effective antitumor T cells after adoptive transfer in vivo In addition, codelivering an FIH-insensitive form of HIF2α with an anti-CD19 chimeric antigen receptor greatly enhanced cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments point to a means to increase the antitumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor.See related Spotlight on p. 364.

No title available] - PubMed

To study the impact of anticoagulant to the quality of umbilical cord blood (UCB). 6060 cord blood units (CBUs) were classified into five groups, such as 28 ml: (10-29) ml, 28 ml: (30-69) ml, 28 ml: (70-109) ml, 28 ml: (110-150) ml and 28 ml: (>150) ml according to volume ratio of anticoagulant and CBVs. The count of pre-cryopreservation total nucleated cell (pre-TNC), the viability of nucleated cell (VNC), the amount of CFU-GM and the ratio changes of CD34+ were evaluated and analyzed statistically. It was found that pre-TNC increased with the growth of volume of CBUs (r=0.9937) under the certain volume of antico-agulant, and the TNC in the minimum UCB volume group was (2.57±0.89)×108; the VNC grew up with the increasing count viability of volume (r=0.9897), and the average viability of the minimum volume group remained over 95%; the CFU-GM climbed up with the increasing of volume (r=0.9024), and the number of CFV-GM in minimum volume group reached to of 89/×105; CD34+% grew up with the increase of volume of CBUs (r=0.9641), and the ratio was (0.30±0.19)% for the minimum volume group. In certain volume of anticoagulant in collection-bag, pre-TNC, VNC, CFU-GM and CD34+% are all dropped with the decrease of CBUs volume , however, all above-mentioned indexes in the minimun random group still meet the requirement for clinical administration.

The Role of lncRNA AF117829.1 in the Immunological Pathogenesis of Severe Aplastic Anaemia.

Severe aplastic anaemia (SAA) is an autoimmune disease with immune tolerance dysfunction mediated by hyperactivated T lymphocytes that target the haematopoietic system. Numerous studies suggest that long noncoding RNAs (lncRNAs) play a significant role in almost every level of gene function/regulation. However, their specific mechanisms in SAA remain undetermined. This study is aimed at determining the role of key lncRNAs in CD8+ T lymphocytes in the mechanisms of SAA. RNA-seq was performed to detect all lncRNAs and mRNAs in peripheral CD8+ T lymphocytes from SAA patients and healthy controls. The lncRNA targets were predicted by bioinformatics, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RT-qPCR was used to verify the expression of key lncRNAs and their predicted targets. We screened lncRNA AF117829.1, which was correlated with autoimmune diseases and downregulated in CD8+ T lymphocytes, and further validated its effects on CD8+ T lymphocytes from SAA patients. We systematically described the lncRNA/mRNA expression changes in CD8+ T lymphocytes in SAA patients and assessed their possible biological functions and signalling pathways. A total of 194 lncRNAs and 2099 mRNAs were changed in SAA patients versus healthy controls. These differentially expressed lncRNAs/mRNAs were associated with organelle components, catalytic activity, the response to stimulation, signal transduction, the immune system and metabolic processes. The downregulated expression of one altered factor, lncRNA AF117829.1, in CD8+ T lymphocytes from SAA patients increased CD8+ T lymphocyte immune function by promoting RIP2 expression. lncRNA AF117829.1 overexpression in CD8+ T lymphocytes reduced perforin and granzyme B expression. The same effect was achieved with GSK583, a RIP2 kinase inhibitor. The proliferation and overactivation of CD8+ T lymphocytes, also known as cytotoxic T cells (CTLs), directly induce bone marrow (BM) failure in SAA patients, but the specific mechanism remains unclear. We found that lncRNA AF117829.1 and its target genes were associated with T cell proliferation, differentiation, and immune dysregulation and that lncRNA AF117829.1 regulated CD8+ T lymphocyte function in SAA patients by promoting RIP2 expression. These findings improve our understanding of the molecular mechanism of immune pathogenesis and provide potential targets for SAA diagnosis and treatment.

Effect of Intensity Modulated Radiotherapy (IMRT) on the immunity, physical status and clinical effect of locally advanced NSCLC patients.

Objectives:To evaluate the clinical value of radiotherapy combined with Camrelizumab in treating locally advanced non-small cell lung cancer (NSCLC) patients.Methods:80 locally advanced NSCLC patients were randomly divided into two groups (n=40). The control group was administered with intensity modulated radiation therapy (IMRT), whereas the experimental group with Camrelizumab in addition to IMRT. All the patients underwent clinical efficacy evaluation in terms of adverse drug reaction (ADR), physical status improvement after the treatment, and changes in T lymphocyte subpopulations (incl. CD3+, CD4+, CD8+, CD4+/CD8+).Results:The efficacy was found to be 70% and 47.5 in experimental group and control group, respectively, with the former being significantly better than the latter (p=0.03). The ADR rates were 50% and 37.5% in the experimental group and control group, respectively; but the difference remained insignificant (p=0.26). As for physical status improvement, experimental group evidently excelled the control group (p=0.04). The post-treatment indicators such as CD3+, CD4+, CD8+, CD4+/CD8+ were significantly more improved in the experimental group than the control group (CD3+, p=0.02; CD4+, p=0.00; and CD4+/CD8+, p=0.01). However, the changes in CD8+ were not significant at all (p=0.46).Conclusions:The combined therapy of IMRT with Camrelizumab appeared effective in dealing with the locally advanced NSCLC patients, as such patients presented significantly better immune state and physical status improvement but not increased ADR. The therapy is both safe and effective.

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells.

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ Tcell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ Tcells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ Tcells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ Tcell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ Tcell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint blockade therapy.

Tumors that develop in the genetic LSL-K-rasG12D murine lung cancer model are resistant to anti-PD-1 antibody treatment. Analysis of tumor-bearing lungs from anti-PD-1-treated mice revealed an up to 2.5-fold increase in IL-17-producing T-cells, with minimal change in CD8+ T-cell activity. Neutralization of IL-17 concurrent with anti-PD-1 treatment on the other hand, resulted in robust CD8+ T-cell activation and a threefold reduction in tumor burden. Loss-of-function studies demonstrated that anti-PD-1 driven activation of CD4+ and γδTCR+ T-cells contributed to IL-17-mediated de-sensitization of CD8+ cytotoxic T-cells (CTL) to therapy; and that CTL activation was critical to tumor eradication. Importantly, post-therapy lung Th17 cell prevalence and activity prognosticated treatment efficacy. Consistent with the murine data, analysis of tumor biopsy samples from non-small cell lung cancer (NSCLC) patients revealed that pre-therapy intratumoral CD8+/RORc+ cell ratio correlated with response to immune checkpoint blockade (ICB). These findings provide the initial evidence for a new mechanism of ICB resistance in lung cancer.

Abstract 16844: Effect of Canagliflozin on Gene Expression and CD34+ Function on Type 2 Diabetes Subjects

Introduction: CD34 +ve Endothelial progenitor cells (EPCs) has been shown to be dysfunctional in subjects with type 2 diabetes mellitus (T2DM) leading to poor endothelial cell function (ECF). Hypothesis: Canagliflozin, an SGLT2 inhibitor when added to other oral antidiabetic medication such Metformin, Insulin, GLP1-agonists, DPP-IV inhibitor, or sulfonylureas, may improve the cardiovascular risk by improving CD34+ EPCs function such as migration, colony formation and maturation towards endothelium. Methods: 29 subjects were enrolled in this 16 weeks, double-blind, two-arm, randomized placebo matched trial, with 100 mg Canagliflozin (Cana, low dose) compared to placebo. T2DM (30-70 years old), HbA1c of 6.5-10%, and all stages of CKD were included. Peripheral blood derived CD34+ cell number, migratory function, mRNA gene expression along with cardiometabolic parameters such as arterial stiffness, biochemistry, resting energy expenditure, and body composition were measured. Data were collected at weeks 0, 8, and 16. A mixed model regression analysis was done with a p-value <0.05 considered significant. Results: We found a statistically significant reduction in systolic (p=0.008) and diastolic (p=0.038) blood pressure. There was a reduction in blood glucose level (p=0.0047) and HbA1c (p=0.03). There was a statistically significant increase in adiponectin (p=0.0062) and trend level reduction in IL6 (p=0.16), indicating an anti-inflammatory effect of canagliflozin. There is also an increase in mRNA expression of genes associated with endothelial function in CD34+ cells such as VEGFA (p=0.049), VEGF receptor KDR (p=0.13), PECAM-1, a mature endothelial marker was also upregulated (p=0.002) and NOS3 (or eNOS) was also upregulated. There were no significant changes in CD34+ numbers by direct counting and parameters tested. We also tested for serum ketones bodies and found no difference at the dose tested. Data on urine podocyte specific exosome is pending Conclusions: Our study indicates that Canagliflozin improves several components of ECF by improving adiponectin, reducing inflammatory cytokines, and by increasing expression of endothelium specific genes.

Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses

Abstract Background Dietary saturated fat (SF) intake is implicated in increasing cardiovascular risk. Studies suggest SF may incite an inflammatory immune response yet the mechanisms for this remain unclear. Circulating blood monocyte subsets (classical “CD16low” and non-classical/intermediate “CD16high”) are thought to differentially contribute to the macrophage foam cell burden in atherosclerotic plaque, yet they are heterogenous and much is still unknown about their response to dietary lipid. Purpose This dietary intervention study hypothesised that a subset-specific monocyte response occurs following acute SF intake in healthy individuals.Top of Form Methods 10 healthy participants were fed a coconut oil, lauric acid-rich meal. Monocyte subsets were purified from fasting and 4h postprandial blood by fluorescence activated cytometry (FACS) into CD16low and CD16high subsets. Surface marker expression was recorded by FACS, gene expression by qPCR, lipid loading by fluorescence microscopy, cytokine responses by ELISA and mass spectrometry was utilised to trace dietary fat into circulating monocytes. Triglyceride rich lipoprotein (TGRL) was obtained through ultracentrifugation of postprandial blood. TLR agonists LPS (TLR4) and R848 (TLR7/8) were used to replicate infectious stimuli to provoke a cytokine response. Results SF meal intake resulted in a 4h postprandial rise in serum triglycerides (TG). Other than a small rise in HLA-DR in postprandial CD16low monocytes, no changes in CD14, CD16 or CD11c levels were noted. Inflammatory gene expression was downregulated although a rise in lipid handling gene ABCA1 was noted in both subsets. Postprandial monocytes accumulated cytoplasmic neutral lipid droplets (larger and more numerate droplets in CD16low). Mass-spectrometry confirmed entry of dietary lipid into circulating monocytes. Postprandial monocyte functional responses were impaired; lipid-loaded postprandial monocytes were less able to migrate towards a chemokine gradient. Following TLR challenge, the inflammatory response was abrogated in postprandial monocytes, further suppressed by in vitro postprandial TGRL addition. Lipidomic analyses confirmed postprandial monocyte lauric acid accumulation in the form of free fatty acid and TG trilaurin, and 9(S)-, 13(S)-HODE and 17(S)-HDoHE, that are metabolites of docosahexanoic acid (DHA) and possess anti-inflammatory properties. The attached graphical abstract summarises the pertinent findings. Conclusions Acute dietary SF intake of a lauric acid-rich meal results in postprandial monocyte suppression of inflammatory TLR responses, a reduction in the ability to migrate towards a chemokine gradient whilst accumulating cytoplasmic neutral lipid droplets. The immunosuppression may be caused by accumulation of anti-inflammatory metabolites of DHA. Graphical Abstract Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation, UK