Generation of endogenous soluble PD-1 as a potential therapeutic strategy for cancer treatment

The Programmed cell death (PD-1) pathway is an immunoinhibitory T cell pathway that is involved in maintaining peripheral T cell tolerance and regulation of inflammation. PD-1 binds to its ligand, PD-L1 which is expressed normally on a variety of cells and aberrantly on cancer cells. Binding of PD-1 to PD-L1 is a dominant immune checkpoint pathway operative in the tumour microenvironment; its normal function is to control immune homeostasis but is exploited by the cancer cells to evade immune attack. Targeting this pathway by blocking antibodies (nivolumab and pembrolizumab) has given impressive anti-cancer responses in patients with a range of cancer types. Blocking of these pathways have successfully shown to restore the function of exhausted T cells. PD-1 is alternatively spliced to form two types of protein, a transmembrane signalling receptor (fl-PD-1) that mediate T cell death by binding to the ligand, PD-L1 and an alternatively spliced variant, soluble PD-1 that lacks the transmembrane domain and is secreted by the T cells and could inhibit PD-L1 from acting on the receptor. The soluble form of PD-1 is generated by skipping exon 3 from the PD-1 mRNA by alternative splicing. These splicing decisions in the cells are made by RNA binding proteins called SR Splicing Factors (SRSFs). SRSF1, among the other SRSFs, is a key player of constitutive and alternative splicing. Nuclear localisation and activation of SRSF1 for performing its role in alternative splicing requires its phosphorylation by SR protein kinases (SRPKs). The aim of this study is to understand the factors and mechanisms that are involved in the regulation of PD-1 splicing. RT-PCR analysis of Jurkat T cells showed the expression of both fl-PD-1 and sPD1. Our bioinformatics analysis indicated multiple potential binding site motifs of SRSF1 adjacent to PD-1 splicing sites. However, RNA immunoprecipitation of SRSF1 followed by RT-PCR showed pull down of only fl-PD-1 indicating the role of SRSF1 in promoting splicing of fl-PD-1. Jurkat cells transfected with the nuclear localised SRSF1 plasmid resulted in the increased expression of fl-PD-1. Site directed mutagenesis of the important SRSF1 splicing regulatory regions in the PD-1 gene resulted in reduced PD-1 expression. Furthermore, inhibition of SRPK1 by lentiviral knockdown and by small molecule inhibitor resulted in reduced mRNA expression of fl-PD-1 and increased production of IL-2 by Jurkat cells. In conclusion, switching of the splicing decision from fl-PD-1 to sPD1 by targeting SRPK1 could represents a potential novel mechanism of immune checkpoint inhibition for cancer therapy

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC).METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry.RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.</p

Generation of endogenous soluble PD-1 as a potential therapeutic strategy for cancer treatment

The Programmed cell death (PD-1) pathway is an immunoinhibitory T cell pathway that is involved in maintaining peripheral T cell tolerance and regulation of inflammation. PD-1 binds to its ligand, PD-L1 which is expressed normally on a variety of cells and aberrantly on cancer cells. Binding of PD-1 to PD-L1 is a dominant immune checkpoint pathway operative in the tumour microenvironment; its normal function is to control immune homeostasis but is exploited by the cancer cells to evade immune attack. Targeting this pathway by blocking antibodies (nivolumab and pembrolizumab) has given impressive anti-cancer responses in patients with a range of cancer types. Blocking of these pathways have successfully shown to restore the function of exhausted T cells. PD-1 is alternatively spliced to form two types of protein, a transmembrane signalling receptor (fl-PD-1) that mediate T cell death by binding to the ligand, PD-L1 and an alternatively spliced variant, soluble PD-1 that lacks the transmembrane domain and is secreted by the T cells and could inhibit PD-L1 from acting on the receptor. The soluble form of PD-1 is generated by skipping exon 3 from the PD-1 mRNA by alternative splicing. These splicing decisions in the cells are made by RNA binding proteins called SR Splicing Factors (SRSFs). SRSF1, among the other SRSFs, is a key player of constitutive and alternative splicing. Nuclear localisation and activation of SRSF1 for performing its role in alternative splicing requires its phosphorylation by SR protein kinases (SRPKs). The aim of this study is to understand the factors and mechanisms that are involved in the regulation of PD-1 splicing. RT-PCR analysis of Jurkat T cells showed the expression of both fl-PD-1 and sPD1. Our bioinformatics analysis indicated multiple potential binding site motifs of SRSF1 adjacent to PD-1 splicing sites. However, RNA immunoprecipitation of SRSF1 followed by RT-PCR showed pull down of only fl-PD-1 indicating the role of SRSF1 in promoting splicing of fl-PD-1. Jurkat cells transfected with the nuclear localised SRSF1 plasmid resulted in the increased expression of fl-PD-1. Site directed mutagenesis of the important SRSF1 splicing regulatory regions in the PD-1 gene resulted in reduced PD-1 expression. Furthermore, inhibition of SRPK1 by lentiviral knockdown and by small molecule inhibitor resulted in reduced mRNA expression of fl-PD-1 and increased production of IL-2 by Jurkat cells. In conclusion, switching of the splicing decision from fl-PD-1 to sPD1 by targeting SRPK1 could represents a potential novel mechanism of immune checkpoint inhibition for cancer therapy

PEPITEM modulates leukocyte trafficking to reduce obesity-induced inflammation

Dysregulation of leukocyte trafficking, lipid metabolism and other metabolic processes are the hallmarks that underpin and drive pathology in obesity. Current clinical management targets alternations in life-style choices (e.g. exercise, weight loss) to limit the impact of the disease. Crucially, re-gaining control over the pathogenic cellular and molecular processes may offer an alternative, complementary strategy for obese patients. Here we investigate the impact of the immunopeptide, PEPITEM, on pancreas homeostasis and leukocyte trafficking in mice on high-fed obesogenic diet (HFD). Both prophylactic and therapeutic treatment with PEPITEM alleviated the effects of HFD on the pancreas, reducing pancreatic beta cell size. Moreover, PEPITEM treatment also limited T-cell trafficking (CD4+ T-cells and KLRG1+ CD3+ T-cells) to obese visceral, but not subcutaneous, adipose tissue. Similarly, PEPITEM treatment reduced macrophage numbers within the peritoneal cavity of mice on HFD diet at both 6 and 12 weeks. By contrast, PEPITEM therapy elevated numbers of T and B-cells were observed in the secondary lymphoid tissues (e.g., spleen, inguinal lymph node) when compared to the untreated HFD controls. Collectively our data highlights the potential for PEPITEM as a novel therapy to combat the systemic low-grade inflammation experienced in obesity and minimise the impact of obesity on pancreatic homeostasis. Thus, offering an alternative strategy to reduce the risk of developing obesity-related co-morbidities, such as type 2 diabetes mellitus, in individuals at high risk and struggling to control their weight through life-style modifications

Partial substitution of exogenously applied phosphatic fertilizers by phosphate solubilizing bacteria in maize under calcareous soil

Phosphorus (P) availability is the major constrain in obtaining optimum crop yield in calcareous soils due to its precipitation as dicalcium and octacalcium phosphate by reacting with Ca+2 and Mg+2. Therefore, we explored the role of phosphate solubilizing bacteria (without and with PSB @ 2 kg ha−1) in optimizing maize yield and P availability from soluble and insoluble P sources applied @ of 100 kg P2O5 into calcareous soil. PSB inoculation significantly improved maize plant height (5.6%), 1,000 grain weight (11%), dry matter (7.5%), stover (10.8%) and grain yield (6.8%), plant P concentration (10.1%) and uptake (18.6%), extractable P (3.1%), agronomic (48%) and uptake (53%) P use efficiency over uninoculated plots. Phosphorus application significantly improved maize yield, soil health and agronomic P use efficiency (4.84 times over control); however, its impact was more pronounced when applied as 50% P each from farmyard manure (FYM) and single super phosphate (SSP). On the basis of overall performance, the sources were ranked as 50% FYM + 50% SSP >50% rock phosphate (RP) + 50% SSP > 100% SSP > 75% FYM + 25% SSP > 75% RP +25% SSP > 100% FYM > 100 RP > control. Interactively, a significant and maximum increased over absolute control in most of the soil and plant tested characteristics were observed when 100 kg P2O5 ha−1 was supplemented 50% each as FYM and SSP along with PSB inoculation which was followed by 50% P each as FYM and SSP demonstrating that PSB were effective in enhancing RP solubilization under calcareous soil. Maximum value cost ratio of 3.1 was observed for 50% P each as FYM and SSP + PSB which was similar to 100% P as FYM + PSB and 75% FYM+ 25% SSP + PSB. Therefore, in calcareous soil P shall be amended 50% each as organic and mineral sources with PSB for its efficient utilization and obtaining optimum yield of maize

Comparing the phosphorus use efficiency of pre-treated (organically) rock phosphate with soluble P fertilizers in maize under calcareous soils

In calcareous soils, phosphorus (P) availability to plant is impaired due to the formation of insoluble complexes with calcium and magnesium. Therefore, this study was executed to compare the P use efficiency (PUE) of four different P sources [rock phosphate (RP), acidulated rock phosphate (ARP), single super phosphate (SSP) and di ammonium phosphate (DAP)] alone or pre-treated with organic amendments (farm yard manure (FYM) enriched compost, simple compost and humic acid (HA)) along with control in maize crop under calcareous soils. All treatments irrespective of P sources received 90 kg P2O5 ha−1. Phosphorus application regardless of its sources and combination with organic amendments significantly improved maize growth, yield as well as P uptake and PUE. Rock phosphate when applied alone was recorded inferior but its performance significantly improved with compost or its pre-addition with FYM and HA, that further enhanced upon acidulation. Maize grain yield increased by 21, 22.2, 67.9 and 94% with RP, ARP, ARP enriched compost and ARP+ compost respectively, over control. Similarly, PUE of DAP improved from 31.7 to 43.1 and 39 with sample and enriched compost correspondingly. Post-harvest soil and grain P were at par for SSP, ARP and DAP alone or in conjugation with organic amendments when averaged across the amendments. These results suggested that pretreatment of P sources with organic amendments is an economical and more feasible approach to improve maize yield and PUE. Moreover, on-farm acidulation of RP may give at par results with SSP and DAP with cheaper rate and hence recommended for P management in maize in alkaline calcareous soils