Genetic variation in the chemokine system is likely to affect responses to infection, and influence the course of autoimmune and inflammatory disease. We and others have shown that the human beta-chemokine CCL3-L1, unlike its related non-allelic isoform CCL3, has high affinity for the chemokine receptors D6, CCR3 and CCR5. Moreover, CCL3-L1, but not CCL3, is susceptible to cleavage by CD26, creating a truncated -2 form with enhanced affinity for CCR1 and CCR5. Strong interaction with CCR5 means that CCL3-L1, and particularly its -2 variant, are by far the most potent natural HIV entry inhibitors described to date. Here, using real-time PCR we have shown that CCL3-L1 and a novel CCL4 isoform (termed CCL4-L1) can vary from 1-6 copies per diploid genome (pdg) in Caucasians and are occasionally completely absent. The other isoforms (CCL3 and CCL4) remain at two copies per dpg. Importantly, in a model system of pro-inflammatory chemokine production (LPS-activated monocytes)higher gene copy number correlates with an increased ratio of CCL3-L1 versus CCL3 mRNA, and enhanced chemokine production. Supernatants from samples with high copy number are able to more potently chemoattract CCR5-expressing cells, an effect blocked with anti-CCL3/CCL3-L1 antibodies. As a result of these studies, we hypothesize that genetic variation in CCL3-L1 gene copy number may affect the susceptibility to, or the progression or severity of, diseases in which this chemokine plays a role.

The role of CC chemokines in protection against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission is not well understood. It was observed that mitogen-induced production of CCL3 and CCL4 by cord-blood mononuclear cells was increased among infants born to HIV-positive compared with HIV-negative mothers, and that a deficiency in production of CCL3 was associated with increased susceptibility to intrapartum HIV-1 infection. CCL3-L1 gene copy number was associated with CCL3 production and with vertical transmission. However, at equivalent CCL3-L1 gene copy numbers, infants who acquired HIV-1 infection relative to their exposed but uninfected counterparts had lower production of CCL3, suggesting that they may harbour some non-functional copies of this gene. Nucleotide changes that may influence CCL3 production were evident in the CCL3 and CCL3-L1 genes upstream of exon 2. Our findings suggest that infants who display a deficient-production phenotype of CCL3 are at increased risk of acquiring HIV-1, indicating that this chemokine in particular plays an essential role in protective immunity.

Immunotherapy is a promising strategy for the treatment of various types of cancer. An antibody that targets programmed death ligand-1 (PD-L1) pathway has been shown to be active towards various types of cancer, including melanoma and lung cancer. MPDL3280A, an anti-PD-L1 antibody, has shown clear clinical activity in PD-L1-overexpressing bladder cancer with an objective response rate of 40-50%, resulting in a breakthrough therapy designation granted by FDA. These events pronounce the importance of targeting the PD-L1 pathway in the treatment of bladder cancer. In the present study, we investigated the prognostic significance of the expression of three genes in the PD-L1 pathway, including PD-L1, B7.1 and PD-1, in three independent bladder cancer datasets in the Gene Expression Omnibus database. PD-L1, B7.1 and PD-1 were significantly associated with clinicopathological parameters indicative of a more aggressive phenotype of bladder cancer, such as a more advanced stage and a higher tumor grade. In addition, a high level expression of PD-L1 was associated with reduced patient survival. Of note, the combination of PD-L1 and B7.1 expression, but not other combinations of the three genes, were also able to predict patient survival. Our findings support the development of anti-PD-L1, which blocks PD-L1-PD-1 and B7.1-PD-L1 interactions, in treatment of bladder cancer. The observations were consistent in the three independent bladder cancer datasets consisting of a total of 695 human bladder specimens. The datasets were then assessed and it was found that the expression levels of the chemokine CC-motif ligand (CCL), CCL3, CCL8 and CCL1L1 expression level, while ADAMTS13 was differentially expressed in patients with a different survival status (alive or deceased). Additional investigations are required to elucidate the role of these genes in the PD-L1-mediated immune system suppression and bladder cancer progression. In conclusion, findings of this study suggested that PD-L1 is an important prognostic marker and a therapeutic target for bladder cancer.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV(+) individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).

Pretreatment tumor PD-L1 expression has been shown to correlate with response to anti-PD-1/PD-L1 therapies. Yet, most patients with PD-L1(+) tumors do not respond to treatment. The current study was undertaken to investigate mechanisms underlying the failure of PD-1-targeted therapies in patients with advanced renal cell carcinoma (RCC) whose tumors express PD-L1. Formalin-fixed, paraffin-embedded pretreatment tumor biopsies expressing PD-L1 were derived from 13 RCC patients. RNA was isolated from PD-L1(+) regions and subjected to whole genome microarray and multiplex quantitative (q)RT-PCR gene expression analysis. A balance between gene expression profiles reflecting metabolic pathways and immune functions was associated with clinical outcomes following anti-PD-1 therapy. In particular, the expression of genes involved in metabolic and solute transport functions such as UGT1A family members, also found in kidney cancer cell lines, was associated with treatment failure in patients with PD-L1(+) RCC. Conversely, tumors from responding patients overexpressed immune markers such as BACH2, a regulator of CD4(+) T-cell differentiation, and CCL3 involved in leukocyte migration. These findings suggest that tumor cell-intrinsic metabolic factors may contribute to treatment resistance in RCC, thus serving as predictive markers for treatment outcomes and potential new targets for combination therapy regimens with anti-PD-1. Cancer Immunol Res; 4(9); 726-33. ©2016 AACRSee related Spotlight by Ohashi, p. 719.

BACKGROUND

Anti-CTLA-4 immune checkpoint blockade is associated with immune-related adverse events (irAEs). Grade 3-4 diarrhea/colitis is the most frequent irAE requiring treatment discontinuation. Predicting high-risk diarrhea/colitis patients may facilitate early intervention, limit irAE severity, and extend treatment duration. No biomarkers currently predict for anti-CTLA-4 immunotherapy related severe diarrhea.

METHODS

Whole-blood was collected pre-treatment and 30 days post-treatment initiation from patients with stage III or IV unresectable melanoma who received 15 mg/kg tremelimumab at 90 day intervals in two clinical trials. The discovery dataset was a phase II study that enrolled 150 patients between December 2005 and November 2006. The validation dataset was a phase III study that enrolled 210 patients between March 2006 and July 2007. RT-PCR was performed for 169 genes associated with inflammation, immunity, CTLA-4 pathway and melanoma. Gene expression was correlated with grade 0-1 versus grade 2-4 diarrhea/colitis development.

RESULTS

Pre-treatment blood obtained from the discovery dataset (N = 150) revealed no gene predictive of diarrhea/colitis development (p < 0.05). A 16-gene signature (CARD12, CCL3, CCR3, CXCL1, F5, FAM210B, GADD45A, IL18bp, IL2RA, IL5, IL8, MMP9, PTGS2, SOCS3, TLR9 and UBE2C) was identified from 30 days post-tremelimumab initiation blood that discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis. The 16-gene signature demonstrated an AUC of 0.814 (95% CI 0.743 to 0.873, p < 0.0001), sensitivity 42.9%, specificity 99.2%, positive predictive value (PPV) 90.0%, and negative predictive value (NPV) 91.4%. In the validation dataset (N = 210), the 16-gene signature discriminated patients developing grade 0-1 from grade 2-4 diarrhea/colitis with an AUC 0.785 (95% CI 0.723 to 0.838, p < 0.0001), sensitivity 57.1%, specificity 84.4%, PPV 57.1% and NPV 84.4%.

CONCLUSIONS

This study identifies a whole-blood mRNA signature predictive of a clinically relevant irAE in patients treated with immune checkpoint blockade. We hypothesize that immune system modulation induced by immune checkpoint blockade results in peripheral blood gene expression changes that are detectable prior to clinical onset of severe diarrhea. Assessment of peripheral blood gene expression changes in patients receiving anti-PD-1/PD-L1 immunotherapy, or combination anti-CTLA4 and anti-PD-1/PD-L1 immunotherapy, is warranted to provide early on-treatment mechanistic insights and identify clinically relevant predictive biomarkers.

BACKGROUND

Clinicaltrials.gov , NCT00257205 , registered 22 November 2005.

IL-33 is an IL-1-related cytokine that can act as an alarmin when released from necrotic cells. Once released, it can target various immune cells including mast cells, innate lymphoid cells and T cells to elicit a Th2-like immune response. We show here that bone marrow-derived mast cells produce IL-13, IL-6, TNF, GM-CSF, CCL3 and CCL4 in response to IL-33 stimulation. Inhibition of the p38 MAPK, or inhibition or knockout of its downstream kinases MK2 and MK3, blocked the production of these cytokines in response to IL-33. The mechanism downstream of MK2/3 was cytokine specific; however, MK2 and MK3 were able to regulate TNF and GM-CSF mRNA stability. Previous studies in macrophages have shown that MK2 regulates mRNA stability via phosphorylation of the RNA-binding protein TTP (Zfp36). The regulation of cytokine production in mast cells was, however, independent of TTP. MK2/3 were able to phosphorylate the TTP-related protein Brf1 (Zfp36 l1) in IL-33-stimulated mast cells, suggesting a mechanism by which MK2/3 might control mRNA stability in these cells. In line with its ability to regulate in vitro IL-33-stimulated cytokine production, double knockout of MK2 and 3 in mice prevented neutrophil recruitment following intraperitoneal injection of IL-33.

OBJECTIVE

In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times.

METHODS

3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 μg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed.

RESULTS

Cells activated by increasing doses of LPS upregulated TNF-α expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXC<em>L1</em>2/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α and CXC<em>L1</em>2 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 °C. The chemokines MIP-1α/CCL3 and MIP-1β/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments.

CONCLUSIONS

These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

CCL-L1 binds more potently to CCR5 than any other chemokine; it inhibits HIV-1 infection in vitro, and its gene occurs in variable copy numbers, some individuals failing to produce it. We analysed the frequency of the absence of CCL3-L1 in 268 Caucasian Australian HIV-1 patients and 260 uninfected individuals. A CCL-L1-negative genotype frequency of 2.3% was found in HIV-1 negative individuals. No association was found between the absence of CCL3-L1 and susceptibility to, or rate of progression of, HIV-1 infection.

OBJECTIVE

Iron participates in several mechanisms involving inflammation and innate immunity, yet the dysregulation of its homeostasis is a major cause of metabolic syndrome. Adipocytes should play a major role in iron metabolism, as an impairment in iron turnover is closely related to insulin resistance, obesity, and type 2 diabetes. The aim of this study was to investigate the role of iron in an in vitro-inflamed adipocyte model.

METHODS

Gene expression of tumor necrosis factor-α, interleukin-6, inflammatory chemokines (CCL3, CCL4, and CXCL1L1 cell model. Cells underwent treatment with FeSO4 heptahydrate and lipopolysaccharide (LPS) stimulation. Toll-like receptor 4 (TLR4) membrane expression, lipid droplet immunohystochemistry, and lipolysis were also evaluated.

RESULTS

Iron sulphate heptahydrate elicited gene expression of hepcidin, hemojuvelin, and ferroportin at different time courses. Additionally, it activated lipolysis but did not trigger any adipokine gene expression. When cells treated with physiological doses of iron were also stimulated with LPS, an enhancement in the LPS-induced gene expression of cytokines and chemokines was observed. The enhancement occurred with different patterns depending on different time courses and investigated genes, showing its maximal effect for IL-6 gene expression.

CONCLUSIONS

FeSO4 heptahydrate at a relatively physiological dose, induced gene expression of iron modulatory proteins and also enhanced RNA transcripts of several inflammatory cytokines and chemokines through a priming/synergistic mechanism involving membrane TLR4.

Aims: The advent of immune checkpoint inhibitor therapy has proven beneficial in a subset of high-grade urothelial carcinomas (HGUC) of the bladder. Although treatment selection is currently largely determined by programmed death-ligand 1 (PD-L1) status, multiple factors in the immune system may modulate the host immune response to HGUC and immunotherapy. In this pilot study, we used a transcriptomic approach to identify the immune milieu associated with PD-L1 expression to enhance our understanding of the HGUC immune evasion network.

Methods: The immune transcriptome of 40 HGUC cystectomy cases was profiled using the NanoString nCounter Human V.1.1 PanCancer Panel. All cases were assessed for associated PD-L1 status (SP263) using whole tissue sections. PD-L1 status was determined as high or low using 25% tumour and/or immune cell staining.

Results: The most significantly differentially expressed gene was PD-L1 messenger RNA (CD274), which strongly correlated with protein expression (r=0.720, p<0.001). The sensitivity, specificity, positive and negative predictive values of CD274 for PD-L1 expression were 85%, 96%, 92% and 93%, respectively. The PD-L1 associated gene signature also included complement components C1QA and CD46 and NOD2 (innate immune system), proinflammatory cytokines CXCL1L1L1 and OSM along with the immune response mediator SMAD3, among others. Pathway analysis determined enrichment of these genes in interleukin-10 production, lymphocyte chemotaxis and aberrant IFNγ, NF-κB and ERK signalling networks.

Conclusions: We report key genes and pathways in the immune transcriptome and their association with PD-L1 status, which may be involved in immune evasion of HGUC and warrants further investigation.

Keywords: immunohistochemistry; pathology, molecular; urinary bladder; urologic neoplasms.

Nuclear factor-ĸB (NF-ĸB) transcription factor is a family of essential regulators of the immune response and cell proliferation and transformation. A typical factor is a heterodimer made of either p50 or p52, which are limited processing products of either p105 or p100, respectively, and a member of the Rel family of proteins, typically p65. The transcriptional program of NF-ĸB is tightly regulated by the composition of the dimers. In our previous work, we demonstrated that the ubiquitin ligase KPC1 is involved in ubiquitination and proteasomal processing of p105 to generate p50. Its overexpression and the resulting high level of p50 stimulates transcription of a broad array of tumor suppressors. Here we demonstrate that additional mechanisms are involved in the p50-mediated tumor-suppressive effect. p50 down-regulates expression of a major immune checkpoint inhibitor, the programmed cell death-ligand 1 (PD-L1), both in cells and in tumors. Importantly, the suppression is abrogated by overexpression of p65. This highlights the importance of the cellular quantities of the two different subunits of NF-ĸB which determine the composition of the dimer. While the putative p50 homodimer is tumor-suppressive, the "canonical" p50p65 heterodimer is oncogenic. We found that an additional mechanism is involved in the tumor-suppressive phenomenon: p50 up-regulates expression of the proinflammatory chemokines CCL3, CCL4, and CCL5, which in turn recruit into the tumors active natural killer (NK) cells and macrophages. Overall, p50 acts as a strong tumor suppressor via multiple mechanisms, including overexpression of tumor suppressors and modulation of the tumor microenvironment by recruiting active immune cells.

Keywords: NF-ĸB p50; PD-L1; chemokines; tumor suppression; ubiquitin ligase KPC1.

Objective: PD-L1 is an immune checkpoint molecule that regulates immune and inflammatory responses. While cells of periodontal tissues express PD-L1, its presence in GCF is not known. The purpose of this study was to measure the PD-L1 values in GCF and correlate values with the presence of chemokine and cytokine values from periodontally diseased subjects and periodontally healthy subjects.

Results: PD-L1 values (pg/30 s), determined in triplicate using a fluorescent microparticle-based immunoassay ranged from 0.04-31.65 pg/30 s. PD-L1 correlated with 15 out of 22 chemokine and cytokine responses. In 85 healthy sites in 31 subjects, PD-L1 values were negatively correlated with IL6, CXCL8, IL1L1 values were positively correlated with CCL1L1A, IL1B, IL2, IL7, IL1L1L1 in Th1 and Th2 activation and T-cell exhaustion signaling canonical pathways. PD-L1 values were correlated with the expression of chemokines and cytokines, which likely regulates immune cell trafficking and protects the periodontium from uncontrolled immune responses to pathogens and inflammation-induced tissue damage.

Keywords: Cytokine; PD-1; PD-L1; Periodontal disease; Periodontitis.

Obese visceral adipose tissue (AT) inflammation is driven by adipokine-mediated cross talk between CD8⁺ T cells and adipocytes, a process mitigated by long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) but underlying mechanisms and ensuing effects on macrophage polarization status are unknown. Using an in vitro co-culture model that recapitulates the degree of CD8⁺ T cell infiltration reported in obese AT, 3T3-L1 adipocytes were co-cultured for 24 h with purified splenic CD8⁺ T cells from C57Bl/6 mice consuming either a 10% w/w safflower oil (control, CON) or 7% w/w safflower oil + 3% w/w fish oil (FO) diet for 4 weeks (n=8-10/diet). Co-cultured cells were in direct contact or in a contact-independent condition separated by a Transwell permeable membrane and stimulated with lipopolysaccharide (10 ng/ml) to mimic in vivo obese endotoxin levels. In contact-dependent co-cultures, FO reduced inflammatory (IL-6, TNFα, IFN-γ) and macrophage chemotactic (CCL2, CCL7, CCL3) mRNA expression and/or secreted protein, NF-κB p65 activation, ROS accumulation, NLRP3 inflammasome priming (Nlrp3, Il1β mRNA) and activation (caspase-1 activity) compared to CON (P<.05). The anti-inflammatory action of FO was reproduced by the addition of a TNF-α neutralizing antibody (1 μg/ml) to CON co-cultures (CON/anti-TNF-α), albeit to a lesser degree. Conditioned media from FO and CON/anti-TNF-α co-cultures, in turn, reduced RAW 264.7 macrophage mRNA expression of M1 polarization markers (iNos, Cd11c, Ccr2) and associated inflammatory cytokines (Il6, Tnfα, Il1β) compared to CON. These data suggest that inflammatory CD8⁺ T cell/adipocyte cross talk is partially attributable to TNF-α signaling, which can be mitigated by LC n-3 PUFA.

The Wnt/β-catenin pathway, which is associated with disease progression, is activated in many cancers. Tankyrase (TNKS) has received attention as a target molecule for Wnt/β-catenin pathway inhibition. We identified K-476, a novel TNKS inhibitor, a dual pocket binder that binds to both the nicotinamide and ADP-ribose pockets. In a human colon cancer cell line, K-476 specifically and potently inhibited TNKS and led to stabilization of the Axin protein, resulting in Wnt/β-catenin pathway suppression. Aberrant Wnt/β-catenin pathway activation was recently reported as a possible mechanism of ineffectiveness in immune checkpoint inhibitor (ICI) treatment. Because the Wnt/β-catenin pathway activation causes dendritic cell inactivation and suppresses chemokine production, resulting in a paucity of CD8⁺ T cells in tumor tissue, which is an important effector of ICIs. Thus, TNKS inhibitors may enhance the efficacy of ICIs. To examine whether K-476 enhances the antitumor effect of anti-PD-L1 antibodies, K-476 was administered orally with an anti-PD-L1 antibody to melanoma-bearing C57BL/6J mice. Although K-476 was ineffective as a monotherapy, it significantly enhanced the antitumor effect in combination with anti-PD-L1 antibody. In mice, intra-tumor infiltration of CD8⁺ T cells was increased by combination treatment. K-476 upregulated the chemokine expression (e.g., Ccl3 and Ccl4), which attracted CD8⁺ T cells. This was considered to contribute to the increased CD8⁺ T cells in the tumor microenvironment. Furthermore, while the potential gastrointestinal toxicity of TNKS inhibitors has been reported, it was not observed at effective doses. Thus, K-476 could be an attractive therapeutic option to enhance the efficacy of ICIs.

Keywords: PD-L1; Wnt/β-catenin; immune checkpoint inhibitor; immunotherapy; tankyrase inhibitor.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non-small cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune related proteins in fine-needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD-<em>L1</em> expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody-based profiling by proximity extension assay (PEA). PEA profiling employed proteins panels relevant to immune- and tumor signaling, and was followed by Qlucore® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA-profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, chemokines <em>CCL3</em> and CCL23) were identified and expression of several of these proteins (e.g. <em>CCL3</em> and CCL23) correlated to PD-<em>L1</em> expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD-<em>L1</em> expression. Our identified protein signatures related to stage included, among others, CXC<em>L1</em>0 and I<em>L1</em>2RB1. We conclude that transthoracic FNA allows for extensive immune- and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

Keywords: Fine Needle aspiration; Immune signaling; Non-small cell lung cancer; PD-L1; biomarkers; proximity extension assay.

Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy; however, only ~22% of patients achieve a complete response, and resistance mechanisms are poorly understood. The role of inflammation and immune cell biology in this setting is under-investigated. In this study, we profiled the inflammatory protein secretome of normal (non-cancer) (n = 8) and malignant rectal tissue (n = 12) pre- and post-radiation in human ex vivo explant models and examined the influence of these untreated and treated secretomes on dendritic cell biology (n = 8 for cancer and normal). These resultant profiles were correlated with patient clinical characteristics. Nineteen factors were secreted at significantly higher levels from the rectal cancer secretome when compared to the normal rectal secretome; Flt-1, P1GF, IFN-γ, IL-6, IL-10, CCL20, CCL26, CCL22, CCL3, CCL4, CCL1L1p < 0.05). Radiation was found to have differential effects on normal rectal tissue and rectal cancer tissue with increased IL-15 and CCL22 secretion following radiation from normal rectal tissue explants (p < 0.05), while no significant alterations were observed in the irradiated rectal cancer tissue. Interestingly, however, the irradiated rectal cancer secretome induced the most potent effect on dendritic cell maturation via upregulation of CD80 and PD-L1. Patient's visceral fat area correlated with secreted factors including CCL20, suggesting that obesity status may alter the tumour microenvironment (TME). These results suggest that radiation does not have a negative effect on the ability of the rectal cancer TME to induce an immune response. Understanding these responses may unveil potential therapeutic targets to enhance radiation response and mitigate normal tissue injury. Tumour irradiation in this cohort enhances innate immune responses, which may be harnessed to improve patient treatment outcome.

Keywords: dendritic cells; inflammation; radiotherapy; rectal cancer; tumour immunology.

Streptococcus mitis colonizes all niches of the human oral cavity from early infancy and throughout life. Monocytes patrol blood vessels, lymphoid and non-lymphoid tissues and migrate into infected tissue where they participate in the inflammatory cascade and immune regulation. Here, we studied the effect of S. mitis on monocytes. Transcriptome analysis of monocytes exposed to S. mitis (SmMo) revealed increased transcription of chemotactic factors (CCL2, CCL3, CCL20, CXCL1, CXCL2) and cytokines (IL1A, IL1B, IL6, IL23, IL36G, TNF), indicating that S. mitis may trigger recruitment of leucocytes and initiate inflammation. Increased transcription in SmMo of IL1B, IL6 and IL23 indicated that S. mitis may participate in the induction of Th17 responses and agreed with our earlier findings of S. mitis-mediated memory Th17 reactivity. Furthermore, S. mitis inhibited tetanus toxoid-specific CD4 T cell proliferation. This can be due to the increased secretion of IL-10 and expression of PD-L1 that was observed in SmMo. PGE2 can modulate IL-10 and PD-L1 expression, concomitant with that of CCR7, IL-12 and IL-23 that also were changed. This, along with increased SmMo transcription of PTGS2 (COX2) and PTGER4 (EP4), pointed to a role of PGE2. Measurement of PGE2 secretion by SmMo showed indeed a marked increase, and chemical inhibition of PGE2 production lowered the PD-L1 expression on SmMo. In conclusion, our findings show that S. mitis may trigger immune modulation by recruiting immune cells to the site of infection, while at the same time dampening the severity of the response through expression of IL-10, PGE2 and PD-L1.

Galectin-1 contains a carbohydrate-recognition domain (CRD) as a member of the lectin family. Here, we investigated whether galectin-1 regulates adipogenesis and lipid accumulation. Galectin-1 mRNA is highly expressed in metabolic tissues such as the muscle and adipose tissues. Higher mRNA expression of galectin-1 was detected in white adipose tissues (WATs) of mice that were fed a high-fat diet (HFD) than in those of mice fed a normal-fat diet (NFD). Protein expression of galectin-1 also increased during adipocyte differentiation. Galectin-1 silencing inhibited the differentiation of 3T3-L1 cells and the expression of lipogenic factors, such as PPARγ, C/EBPα, FABP4, and FASN at both mRNA and protein levels. Lactose, an inhibitor by the binding with CRD of galectin-1 in extracellular matrix, did not affect adipocyte differentiation. Galectin-1 is localized in multiple cellular compartments in 3T3-L1 cells. However, we found that DMI (dexamethasone, methylisobutylxanthine, insulin) treatment increased its nuclear localization. Interestingly, galectin-1 interacted with PPARγ. Galectin-1 overexpression resulted in increased PPARγ expression and transcriptional activity. Furthermore, we prepared galectin-1-knockout (Lgals1^-/-) mice and fed a 60% HFD. After 10 weeks, Lgals1^-/- mice exhibited lower body weight and gonadal WAT (gWAT) mass than wild-type mice. Fasting glucose level was also lower in Lgals1^-/-mice than that in wild-type mice. Moreover, lipogenic genes were significantly downregulated in the gWATs and liver tissues from Lgals1^-/- mice. Pro-inflammatory cytokines, such as CCL2, CCL3, TNFα, and F4/80, as well as macrophage markers, were also drastically downregulated in the gWATs and liver tissues of Lgals1^-/- mice. In addition, Lgals1^-/-mice showed elevated expression of genes involved in thermogenesis in the brown adipose tissue. Collectively, galectin-1 exacerbates obesity of mice fed HFD by increment of PPARγ expression and activation. Our findings suggest that galectin-1 could be a potential therapeutic target for obesity and needed further study for clinical application.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of TCR-initiated signal transduction. Both the HPK1^-/- mice and the genetically engineered mice with a point mutation that disrupts the catalytic activity of HPK1 possess enhanced antitumor immunity, especially when these mice are treated with anti-PD-L1 immune checkpoint Ab. Because CD4⁺FOXP3⁺ regulatory T cells (Tregs) play an important role in suppressing tumor immunity, we investigated whether the loss of HPK1 expression could result in the reduction of Treg functions. We found that the number of HPK1^-/- Tregs is elevated relative to the number found in wild-type C57/BL6 mice. However, HPK1^-/- Tregs lack the ability to carry out effective inhibition of TCR-induced proliferative responses by effector T cells. Furthermore, HPK1^-/- Tregs respond to TCR engagement with an elevated and sustained Erk MAPK and p65/RelA NF-κB phosphorylation in comparison with wild-type Tregs. Also, a multiplex cytokine analysis of HPK1^-/- Tregs revealed that they demonstrate an aberrant cytokine expression profile when stimulated by anti-CD3ε and anti-CD28 crosslinking, including the uncharacteristic expression of IL-2 and antitumor proinflammatory cytokines and chemokines such as IFN-γ, CCL3, and CCL4. The aberrant HPK1^-/- phenotype observed in these studies suggests that HPK1 may play an important role in maintaining Treg functions with wider implications for HPK1 as a novel immunotherapeutic target.

Background: Recurrence or de novo infection of hepatitis C virus (HCV) after liver-transplantation has been associated with progressive graft hepatitis that can be improved by treatment with novel direct-acting antivirals. Cases of rejection episodes have been described during and after HCV treatment. The evolution of innate and adaptive immune response during and after cure of hepatitis C after liver-transplantation is unknown.

Methods: We here studied 74 protein biomarkers in plasma of liver-transplanted patients receiving antiviral therapy. In addition, deep immune-phenotyping of both the myeloid and lymphoid immune cell subsets in peripheral blood mononuclear cells was performed.

<strong class="sub-title"> Results: </strong> We found that liver-transplanted patients with active HCV infection displayed distinct alterations of inflammatory protein biomarkers such as CXC<em>L1</em>0, CASP-8, CCL20, CC<em>L1</em>9, IFN-gamma, CDCP1, IL-18R1, CXC<em>L1</em>1, <em>CCL3</em>, IL8, I<em>L1</em>2B, TNFB, CXCL6, OPG, I<em>L1</em>0, Flt3L, HGF, uPA, NT-3, CCL4, IL6, TNFRSF9, PD-<em>L1</em>, I<em>L1</em>8 and MCP-1 and enrichment of peripheral immune cell subsets unlike transplanted patients without HCV infection. Interestingly, patients that cleared HCV post liver-transplantation did not normalize the altered inflammatory milieu nor did the peripheral immune cell subsets normalize to what would be seen in the absence of HCV recurrence.

Conclusion: Overall, these data indicate that HCV-specific imprints on inflammatory analytes and immune cell subsets post liver-transplantation are not completely normalized by therapy-induced HCV elimination. This is in line with the clinical observation that cure of HCV post-transplantation did not trigger rejection episodes in many patients.

Keywords: Graft hepatitis C; HCV recurrence; Liver transplantation; Protein biomarkers; Sustained virologic response; immune correlates.

In classical Hodgkin Lymphoma (cHL), immunoediting via protein signaling is key to evading tumor surveillance. We aimed to identify immune-related proteins that distinguish diagnostic cHL tissues (=diagnostic tumor lysates, n = 27) from control tissues (reactive lymph node lysates, n = 30). Further, we correlated our findings with the proteome plasma profile between cHL patients (n = 26) and healthy controls (n = 27). We used the proximity extension assay (PEA) with the OlinkTM multiplex Immuno-Oncology panel, consisting of 92 proteins. Univariate, multivariate-adjusted analysis and Benjamini-Hochberg's false discovery testing (=Padj) were performed to detect significant discrepancies. Proteins distinguishing cHL cases from controls were more numerous in plasma (30 proteins) than tissue (17 proteins), all Padj < 0.05. Eight of the identified proteins in cHL tissue (PD-L1, IL-6, CCL1CCL3, IL-13, MMP12, TNFRS4, and LAG3) were elevated in both cHL tissues and cHL plasma compared with control samples. Six proteins distinguishing cHL tissues from controls tissues were significantly correlated to PD-L1 expression in cHL tissue (IL-6, MCP-2, CCL3, CCL4, GZMB, and IFN-gamma, all p ≤0.05). In conclusion, this study introduces a distinguishing proteomic profile in cHL tissue and potential immune-related markers of pathophysiological relevance.

Keywords: Hodgkin lymphoma; Immunology; LAG3 CCL1L1; biomarkers; proteomics; proximity assays; tumor microenvironment.