CD28-deficient Mice Research Articles

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis.

Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown. We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis. Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

Viral-vectored boosting of OmcB- or CPAF-specific T-cell responses fail to enhance protection from Chlamydia muridarum in infection-immune mice and elicits a non-protective CD8-dominant response in naïve mice

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T cell memory. We tested the ability of intramuscular immunization with modified vaccinia virus Ankara (MVA) or Chimpanzee Adenovirus (ChAd) expressing chlamydial outer membrane protein, OmcB, or the secreted protein, CPAF, to enhance T cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972, and to elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold respectively but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a CD8-dominant T cell response that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, while CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden compared to wild-type controls. These data reinforce the essential nature of the CD4 T cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses.

Hybrid Membrane-Camouflaged Biomimetic Immunomodulatory Nanoturrets with Sequential Multidrug Release for Potentiating T Cell-Mediated Anticancer Immunity.

Ascorbic acid (AA) has been attracting great attention with its emerging potential in T cell-dependent antitumor immunity. However, premature blood clearance and immunologically "cold" tumors severely compromise its immunotherapeutic outcomes. As such, the reversal of the immunosuppressive tumor microenvironment (TME) has been the premise for improving the effectiveness of AA-based immunotherapy, which hinges upon advanced AA delivery and amplified immune-activating strategies. Herein, a novel Escherichia coli (E. coli) outer membrane vesicle (OMV)-red blood cell (RBC) hybrid membrane (ERm)-camouflaged immunomodulatory nanoturret is meticulously designed based on gating of an AA-immobilized metal-organic framework (MOF) onto bortezomib (BTZ)-loaded magnesium-doped mesoporous silica (MMS) nanovehicles, which can realize immune landscape remodeling by chemotherapy-assisted ascorbate-mediated immunotherapy (CAMIT). Once reaching the acidic TME, the acidity-sensitive MOF gatekeeper and MMS core within the nanoturret undergo stepwise degradation, allowing for tumor-selective sequential release of AA and BTZ. The released BTZ can evoke robust immunogenic cell death (ICD), synergistically promote dendritic cell (DC) maturation in combination with OMV, and ultimately increase T cell tumor infiltration together with Mg2+. The army of T cells is further activated by AA, exhibiting remarkable antitumor and antimetastasis performance. Moreover, the CD8-deficient mice model discloses the T cell-dependent immune mechanism of the AA-based CAMIT strategy. In addition to providing a multifunctional biomimetic hybrid nanovehicle, this study is also anticipated to establish a new immunomodulatory fortification strategy based on the multicomponent-driven nanoturret for highly efficient T cell-activation-enhanced synergistic AA immunotherapy.

Cell adhesion molecule CD44 is dispensable for reactive astrocyte activation during prion disease

Prion diseases are fatal, infectious, neurodegenerative disorders resulting from accumulation of misfolded cellular prion protein in the brain. Early pathological changes during CNS prion disease also include reactive astrocyte activation with increased CD44 expression, microgliosis, as well as loss of dendritic spines and synapses. CD44 is a multifunctional cell surface adhesion and signalling molecule which is considered to play roles in astrocyte morphology and the maintenance of dendritic spine integrity and synaptic plasticity. However, the role of CD44 in prion disease was unknown. Here we used mice deficient in CD44 to determine the role of CD44 during prion disease. We show that CD44-deficient mice displayed no difference in their response to CNS prion infection when compared to wild type mice. Furthermore, the reactive astrocyte activation and microgliosis that accompanies CNS prion infection was unimpaired in the absence of CD44. Together, our data show that although CD44 expression is upregulated in reactive astrocytes during CNS prion disease, it is dispensable for astrocyte and microglial activation and the development of prion neuropathogenesis.

CD38 deficient mice are not protected from atherosclerosis

CD38 is a multifunctional enzyme implicated in chemotaxis of myeloid cells and lymphocyte activation, but also expressed by resident cells such as endothelial and smooth muscle cells. CD38 is important for host defense against microbes. However, CD38's role in the pathogenesis of atherosclerosis is controversial with seemingly conflicting results reported so far. To clarify the discrepancy of current literature on the effect of CD38 ablation on atherosclerosis development, we implanted a shear stress modifier around the right carotid artery in CD38−/− and WT mice. Hypercholesterolemia was induced by human gain-of-function PCSK9 (D374Y), introduced using AAV vector (serotype 9), combined with an atherogenic diet for a total of 9 weeks. Atherosclerosis was assessed at the aortic root, aortic arch and the right carotid artery. The findings can be summarized as follows: i) CD38−/− and WT mice had a similar atherosclerotic burden in all three locations, ii) No significant differences in monocyte infiltration or macrophage content could be seen in the plaques, and iii) The amount of collagen deposition in the plaques were also similar between CD38−/− and WT mice. In conclusion, our data suggest that CD38−/− mice are neither protected against nor prone to atherosclerosis compared to WT mice.

CD73-dependent generation of extracellular adenosine by vascular endothelial cells modulates de novo lipogenesis in adipose tissue.

Next to white and brown adipocytes present in white and brown adipose tissue (WAT, BAT), vascular endothelial cells, tissue-resident macrophages and other immune cells have important roles in maintaining adipose tissue homeostasis but also contribute to the etiology of obesity-associated chronic inflammatory metabolic diseases. In addition to hormonal signals such as insulin and norepinephrine, extracellular adenine nucleotides modulate lipid storage, fatty acid release and thermogenic responses in adipose tissues. The complex regulation of extracellular adenine nucleotides involves a network of ectoenzymes that convert ATP via ADP and AMP to adenosine. However, in WAT and BAT the processing of extracellular adenine nucleotides and its relevance for intercellular communications are still largely unknown. Based on our observations that in adipose tissues the adenosine-generating enzyme CD73 is mainly expressed by vascular endothelial cells, we studied glucose and lipid handling, energy expenditure and adaptive thermogenesis in mice lacking endothelial CD73 housed at different ambient temperatures. Under conditions of thermogenic activation, CD73 expressed by endothelial cells is dispensable for the expression of thermogenic genes as well as energy expenditure. Notably, thermoneutral housing leading to a state of low energy expenditure and lipid accumulation in adipose tissues resulted in enhanced glucose uptake into WAT of endothelial CD73-deficient mice. This effect was associated with elevated expression levels of de novo lipogenesis genes. Mechanistic studies provide evidence that extracellular adenosine is imported into adipocytes and converted to AMP by adenosine kinase. Subsequently, activation of the AMP kinase lowers the expression of de novo lipogenesis genes, most likely via inactivation of the transcription factor carbohydrate response element binding protein (ChREBP). In conclusion, this study demonstrates that endothelial-derived extracellular adenosine generated via the ectoenzyme CD73 is a paracrine factor shaping lipid metabolism in WAT.

The protective role of CD73 in periodontitis: preventing hyper-inflammatory fibroblasts and driving osteoclast energy metabolism.

Periodontitis is an immune-mediated inflammatory disease affecting almost half of the adult population and is the leading cause of tooth loss in the United States. The role of extracellular nucleotide signaling including nucleotide metabolizing enzyme CD73 adds an important layer of interaction of purine mediators capable of orchestrating inflammatory outcomes. CD73 is able to catabolize 5'-adenosine monophosphate into adenosine at the extracellular level, playing a critical role in regulating many processes under physiological and pathological conditions. Here, we explored the role of CD73 in ligature-induced periodontitis in vivo comparing wild-type C57Bl/6J and CD73-deficient mice. We assessed gingival levels of inflammatory cytokines in vivo and in murine gingival fibroblasts in vitro, as well as bone loss, and RANKL-induced osteoclastogenesis. We have also analyzed CD73 mRNA in samples derived from patients diagnosed with severe periodontitis. Our results in mice show that lack of CD73 resulted in increased inflammatory cytokines and chemokines such as IL-1β, IL-17, Cxcl1 and Cxcl2 in diseased gingiva relative to the healthy-controls and in comparison with the wild type. CD73-deficient gingival fibroblasts also manifested a defective healing response with higher MMP-13 levels. CD73-deficient animals also showed increased osteoclastogenesis in vitro with increased mitochondrial metabolism typified by excessive activation of oxidative phosphorylation, increased mitochondrial membrane potential and accumulation of hydrogen peroxide. Micro-CT analysis revealed that lack of CD73 resulted in decreased bone mineral density, decreased trabecular bone volume and thickness as well as decreased bone volume in long bones. CD73 deficiency also resulted in increased alveolar bone loss in experimental periodontitis. Correlative studies of gingival samples from severe (Grade C) periodontitis showed decreased levels of CD73 compared to healthy controls, further supporting the relevance of our murine results. In conclusion, CD73 appears to play a protective role in the gingival periodontal tissue and bone homeostasis, regulating hyper-inflammatory state of stromal fibroblasts and osteoclast energy metabolism and being an important candidate for future target therapies to prevent or control immune-mediated inflammatory and osteolytic diseases.

Gab3 Plays an Essential Role in CD8 + T-Cell Expansion through Regulating IL-2-Mediated Activation of PI3K/AKT/mTOR and Erk/FoxO Pathways

Grb2-associated binding protein 3 (Gab3) is a member of the Gab family of scaffolding proteins. Its role in T cells is largely unknown. In the current study, we investigated its in vivo function in CD8 + T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong infection model. We created BM chimera mice with a specific deficiency of Gab3 in CD8 + T cells. This was achieved by transplanting a mixture of BM cells obtained from CD8-deficient mice and Gab3-deficient (Gab3 -/-) mice (1) into lethally irradiated CD45.1 mice. The control chimera mice were lethally irradiated CD45.1 mice transplanted with a mixture of BM cells from CD8-deficient mice and WT mice. Twelve weeks after the BM transplantation, the Gab3 -/- and control BM chimera mice were infected with Armstrong. On day eight following infection, Gab3 -/- CD8 + T cells exhibited reduced expansion compared to WT CD8 + T cells. Importantly, Gab3 -/- LCMV antigen specific CD8 + T cells, GP33 + and NP396 + CD8 + T cells, were significantly reduced compared to their WT counterparts. Furthermore, CD8 + T cells in Gab3 -/- BM chimera mice showed significant increase of apoptosis compared to that in WT BM chimera mice following LCMV infection. To explore the mechanism underlying the observed phenotype, we used a primary CD8 + T-cell culture system, in which we stimulated CD8 + T cells with anti-CD3 and anti-CD28 followed by expanding the cells in IL-2. We observed that Gab3 deficiency severely impaired IL-2-induced CD8 + T cell expansion. Gab3 -/- CD8 + T cells in this culture system displayed increased apoptosis and reduced proliferation compared to wild-type (WT) controls, which may account for the reduced CD8 + T cell expansion. We then performed Seahorse analysis of the cultured CD8 + T cells after IL-2 stimulation. We found that IL-2-mediated extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were largely impaired in Gab3 -/- CD8 + T cells compared to WT CD8 + T cells. This data indicates a crucial role of Gab3 in both glycolysis and mitochondrial respiration. We further investigated how Gab3 regulates the IL-2 signaling pathway by conducting transcriptomic and phosphoproteomic analysis. RNA-seq analysis revealed that genes associated with mTOR signaling, glucose metabolism, cell cycle, and Erk/MAPK targets were highly enriched in WT CD8 + T cells, whereas genes related to apoptosis, autophagy, and FoxO3 targets were highly enriched in Gab3 -/- CD8 + T cells. In contrast, the gene set enrichment analysis (GSEA) showed that IL-2-induced Stat5 target gene expression was comparable between WT and Gab3 -/- CD8 + T cells, indicating an important role of Gab3 specifically in the activation of the mTOR and Erk/MAPK pathways downstream of IL-2 signaling. Consistent with that observed in the transcriptomic analysis, phosphoproteomics analysis revealed a significant reduction in IL-2-induced phosphorylation of key components in ERK/FOXO signaling pathway, including RafA, MAPK1, FoxO3, AMPK, and mTOR downstream molecule Eif4B in Gab3 -/- CD8 + T cells compared to WT CD8 + cells. Furthermore, phosphorylation of Stat5 at different sites was comparable between Gab3 -/- and WT CD8 + T cells, further supporting an important role of Gab3 specifically in IL-2 mediated mTOR and Erk/MAPK activation. Finally, we validated the results obtained from the transcriptomic and phosphoproteomic analyses using western blot analysis. Our findings revealed that in Gab3 -/- CD8 + T cells, the IL-2-induced phosphorylation of signaling molecules in the mTOR pathway (S6K, S6, 4EBP1, AKT) and the Erk pathway (Erk) was impaired. Notably, the phosphorylation of Stat5 remained intact despite the absence of Gab3. In summary, our studies have demonstrated that Gab3 plays an essential role in CD8 + T-cell expansion by promoting cell proliferation and prohibiting cell apoptosis through regulating IL-2-mediated activation of PI3K/AKT/mTOR and Erk-FoxO3 pathways. (1) We gratefully acknowledge that Gab3-deficient mice were originally generated by Dr. Larry Rohrschneider at Fred Hutchinson Cancer Research Center ( Mol Cell Biol 2003 Apr;23(7):2415-24. doi: 10.1128/MCB.23.7.2415-2424.2003.). The mice were subsequently backcrossed to C57BL/6.

GBS hyaluronidase mediates immune suppression in a TLR2/4- and IL-10-dependent manner during pregnancy-associated infection.

Bacteria such as GBS can cause infections during pregnancy leading to preterm births, stillbirths, and neonatal infections. The interaction between host and bacterial factors during infections in the placenta is not fully understood. GBS secretes a hyaluronidase enzyme that is thought to digest host hyaluronan into immunosuppressive disaccharides that dampen TLR2/4 signaling, leading to increased bacterial dissemination and adverse outcomes. In this study, we show that GBS HylB mediates immune suppression and promotes bacterial infection during pregnancy that requires TLR2, TLR4, and IL-10. Understanding the interaction between host and bacterial factors can inform future therapeutic strategies to mitigate GBS infections.

CD45 limits early Natural Killer cell development.

The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumor cells. To fully harness the tumor killing ability of NK cells, we need to improve NK cell persistence and to overcome suppression of NK cell activation in the tumor microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with the genetic loss of CD45 in mice resulting in increased numbers of mature NK cells. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, we demonstrate here that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development, compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy.

CD45 alleviates airway inflammation and lung fibrosis by limiting expansion and activation of ILC2s

Group 2 innate lymphoid cells (ILC2s) are critical for the immune response against parasite infection and tissue homeostasis and involved in the pathogenesis of allergy and inflammatory diseases. Although multiple molecules positively regulating ILC2 development and activation have been extensively investigated, the factors limiting their population size and response remain poorly studied. Here, we found that CD45, a membrane-bound tyrosine phosphatase essential for T cell development, negatively regulated ILC2s in a cell-intrinsic manner. ILC2s in CD45-deficient mice exhibited enhanced proliferation and maturation in the bone marrow and hyperactivated phenotypes in the lung with high glycolytic capacity. Furthermore, CD45 signaling suppressed the type 2 inflammatory response by lung ILC2s and alleviated airway inflammation and pulmonary fibrosis. Finally, the interaction with galectin-9 influenced CD45 signaling in ILC2s. These results demonstrate that CD45 is a cell-intrinsic negative regulator of ILC2s and prevents lung inflammation and fibrosis via ILC2s.

CD38 deficiency promotes skeletal muscle and brown adipose tissue energy expenditure through activating NAD+-Sirt1-PGC1α signaling pathway.

Obesity is a metabolic syndrome characterized by abnormal lipid deposition and energy imbalance. CD38 is a single-chain transmembrane glycoprotein widely expressed in a variety of cell types. The roles of skeletal muscle and brown fat in CD38 deficiency under HFD-induced obesity remain unknown. In this study, we established obesity model with HFD and examined the changes in metabolites with metabonomics. Our results showed that CD38 expression was increased in muscle and brown fat after HFD treatment. Moreover, the results of metabonomics showed that CD38 deficiency significantly altered the metabolites in energy metabolism, cofactor generation, and redox homeostasis. Furthermore, CD38 deficiency reduced the expressions of NADPH oxidase 2 and FASN in mRNA level. We found that the expressions of Sirt1, Sirt3, and PGC1α were upregulated in CD38-deficient muscle tissue. In brown fat, the Sirt1-3, cell death inducing DFFA-like effector A, ELOVL3, and Dio2 expressions were increased in CD38-deficient mice. Our results showed the uncoupling protein 1 expression was upregulated. And NAD+ supplementation increased the expression of Sirt1 and PGC1α after palmitic acid treatment. Taken together, our results demonstrated that the protection of CD38 deficiency on HFD-induced obesity was related to the inhibition of oxidative stress and increasing energy expenditure via activating NAD+/Sirtuins signaling pathways in muscle and brown fat.

Microbiome modulation after severe acute kidney injury accelerates functional recovery and decreases kidney fibrosis

Targeting gut microbiota has shown promise to prevent experimental acute kidney injury (AKI). However, this has not been studied in relation to accelerating recovery and preventing fibrosis. Here, we found that modifying gut microbiota with an antibiotic administered after severe ischemic kidney injury in mice, particularly with amoxicillin, accelerated recovery. These indices of recovery included increased glomerular filtration rate, diminution of kidney fibrosis, and reduction of kidney profibrotic gene expression. Amoxicillin was found to increase stool Alistipes, Odoribacter and Stomatobaculum species while significantly depleting Holdemanella and Anaeroplasma. Specifically, amoxicillin treatment reduced kidney CD4+T cells, interleukin (IL)-17 +CD4+T cells, and tumor necrosis factor-α double negative T cells while it increased CD8+T cells and PD1+CD8+T cells. Amoxicillin also increased gut lamina propria CD4+T cells while decreasing CD8+T and IL-17+CD4+T cells. Amoxicillin did not accelerate repair in germ-free or CD8-deficient mice, demonstrating microbiome and CD8+T lymphocytes dependence for amoxicillin protective effects. However, amoxicillin remained effective in CD4-deficient mice. Fecal microbiota transplantation from amoxicillin-treated to germ-free mice reduced kidney fibrosis and increased Foxp3+CD8+T cells. Amoxicillin pre-treatment protected mice against kidney bilateral ischemia reperfusion injury but not cisplatin-induced AKI. Thus, modification of gut bacteria with amoxicillin after severe ischemic AKI is a promising novel therapeutic approach to accelerate recovery of kidney function and mitigate the progression of AKI to chronic kidney disease.

Differential response of HBV Envelope-specific CD4+ T cells is related to HBsAg loss after stopping nucleos(t)ide analogue therapy.

Long-term maintenance of viral control, even HBsAg loss, remains a challenge for chronic hepatitis B (CHB) patients undergoing nucleos(t)ide analogue (NA) discontinuation. This study aimed to investigate the relationship between HBV-specific T cell responses targeting peptides spanning the whole proteome and clinical outcomes in CHB patients after NA discontinuation. Eighty-eight CHB patients undergoing NA discontinuation were classified as responders (remained relapse-free up to 96wk) or relapsers (relapsed patients who underwent NA re-treatment for up to 48 weeks and re-achieved stable viral control). HBV-specific T cell responses were detected at baseline and longitudinally throughout follow-up. We found responders had a greater magnitude of HBV Polymerase (Pol)-specific T cell responses than relapsers at baseline. After long-term NA discontinuation, simultaneously enhanced HBV Core- and Pol-induced responses were observed in responders. Particularly, responders with HBsAg loss possessed enhanced HBV Envelope (Env)-induced responses after short and long-term follow-up. Notably, CD4+T cells accounted for the predominance of HBV-specific T cell responses. Correspondingly, CD4-deficient mice showed attenuated HBV-specific CD8+T cell responses, reduced HBsAb-producing B cells, and delayed HBsAg loss; in contrast, in vitro addition of CD4+T cells promoted HBsAb production by B cells . Additionally, interleukin (IL)-9, rather than PD-1 blockade, enhanced HBV Pol-specific CD4+T cell responses. HBV-specific CD4+T cell responses induced by the targeted peptide possess specificities for long-term viral control and HBsAg loss in CHB patients undergoing NA discontinuation, indicating that CD4+T cells specific for distinct HBV antigens may endow with divergent antiviral potential.

Mechanism of CD38 via NAD+ in the Development of Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease globally, and it can proceed to cirrhosis and hepatocellular carcinoma, as well as cardiovascular disease, chronic renal disease, and other complications, resulting in a massive economic burden. At the moment, nicotinamide adenine dinucleotide (NAD+) is thought to be a possible treatment target for NAFLD, besides Cluster of differentiation 38(CD38) is the primary NAD+ degrading enzyme in mammals and may play a role in the pathophysiology of NAFLD. For example, CD38 regulates Sirtuin 1 activity and hence affects inflammatory responses. CD38 inhibitors enhance glucose intolerance and insulin resistance in mice and lipid accumulation in the liver is greatly decreased in CD38-deficient mice. This review describes the role of CD38 in the development of NAFLD in terms of Macrophage-1, insulin resistance, and abnormal lipid accumulation in order to offer recommendations for future NAFLD pharmacological trials.

Tolerance to 2,4-Dinitrofluorobenzene‒Induced Contact Hypersensitivity Is Mediated by CD73-Expressing Tissue-Homing Regulatory T Cells

Regulatory T cells (Tregs) express CD73, an ectonucleotidase that converts adenosine (Ado) monophosphate to Ado, which has been shown to suppress immune reactions. To investigate the role(s) of CD73+ Tregs during the induction of tolerance, we used a 2,4-dinitrofluorobenzene‒driven contact hypersensitivity model, in which tolerance can be induced by pretreating wild type mice with 2,4-dinitrothiocyanobenzene. CD73-deficient mice were unable to acquire tolerance. Likewise, transfer of CD73‒/‒ Tregs failed to suppress 2,4-dinitrofluorobenzene‒induced ear swelling in wild type mice, whereas transfer of wild type‒derived Tregs into CD73‒/‒ mice re-established tolerance. This indicates a crucial role of CD73+ Tregs for skin-induced tolerance. Furthermore, we found that 2,4-dinitrothiocyanobenzene induces more activated CD73+ tissue-homing Tregs (marked by Ki-67, CTLA4, CCR4, CD103, CCR6, and CD49b expression) in draining lymph nodes and blood, eventually accumulating in the skin. The application of anti-CD73 antibodies that block CD73-derived Ado production as well as the injection of Ado deaminase, which degrades Ado in tissues, abrogated tolerance induction. Thus, our data indicate that CD73+ Ado-producing Tregs are crucial for the regulation of contact hypersensitivity reactions and tolerance induction in the skin and that manipulating the function(s) of CD73 in tissues may offer a tool to influence autoimmunity and inflammation invivo.

Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte CD40 in the aging hematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in bone marrow during aging and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40 deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases.

Abstract C007: Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

Abstract Current immune checkpoint therapies including anti-CTLA-4 and anti-PD1 have limited efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC), largely due to a paucity of effector T cells within the tumor microenvironment (TME). In our study, we show that vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, is over-expressed in human PDAC tumors. Thus, we hypothesized that paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP-receptor (VIP-R) expressing T cells. We first evaluated whether inhibiting VIP-R signaling by peptide-based VIP-R antagonist affects the growth of PDAC cells in vitro. Treatment of PDAC cells with increasing concentrations of VIP-R antagonist did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 albeit not significant. Furthermore, depletion of VIP receptor 2 (VPAC2) in a PDAC cell line, Panc02, by CRISPR-Cas9 KO had similar growth rates in vitro with a slight growth delay in vivo compared to the parental cell line. These data suggest that the expression of VPAC2 on PDAC cells may not confer a significant autocrine effect on tumor growth impacted by VIP signaling. On the contrary, human T cells cultured with VIP-R antagonists had enhanced activation, decreased proportions of Tregs, and exhausted T cells co-expressing PD-1, Tim-3, and Lag-3. Next, we performed pharmacological inhibition with VIP-R antagonists in tumor-bearing mice to model T cell homing, activation, and antitumor responses in preclinical murine models of PDAC. Daily subcutaneous injections of VIP-R antagonists had synergistic antitumor activity when combined with anti-PD-1 therapy with decreased tumor burden and improved survival. In contrast to the limited single-agent activity of anti-PD1 or VIP-R antagonists, combination therapy eliminated tumors in up to 40% of animals. Anti-tumor responses were T cell-dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. In addition, tumor-free mice were resistant to tumor re-challenge, indicating the generation of anti-cancer immunological memory. In correspondent to these data, combination therapy led to enhanced T cell activation in vivo with increased antigen-specific T cells, decreased frequencies of Tregs as well as enhanced T cell infiltration to the tumor site as observed by infiltration of adoptively transferred GFP+ T cells into PDAC tumors. Taken together, our findings show that VIP-VIPR signaling is a targetable mechanism of immune escape in PDAC. Inhibiting VIP-R signaling improves T cell effector properties and enhances the responsiveness of PDAC to immune checkpoint therapy. Further pharmacokinetic and toxicology studies are underway. Citation Format: Tenzin Passang, Sruthi Ravindranathan, Jian-Ming Li, Shuhua Wang, Michael Brandon Ware, Hanwen Zhang, Gregory B. Lesinski, Edmund K. Waller. Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C007.

CD14 signaling mediates lung immunopathology and mice mortality induced by Achromobacter xylosoxidans.

Objective and designOur research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model.MethodsC57Bl/6 or CD14-deficient mice were infected intratracheally with non-lethal inoculum of A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. In another set of experiments, C57Bl/6 or CD14-deficient mice were inoculated with lethal inoculum and the survival rate determined.ResultsCD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load. The lungs of CD14-deficient mice presented a smaller area of tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and the levels of sCD14 in BALF and serum of C57Bl/6-infected mice.ConclusionsIn summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-022-01641-8.

Extracellular vesicles from pristane-treated CD38-deficient mice express an anti-inflammatory neutrophil protein signature, which reflects the mild lupus severity elicited in these mice.

In CD38-deficient ( Cd38-/- ) mice intraperitoneal injection of pristane induces a lupus-like disease, which is milder than that induced in WT mice, showing significant differences in the inflammatory and autoimmune processes triggered by pristane. Extracellular vesicles (EV) are present in all body fluids. Shed by cells, their molecular make-up reflects that of their cell of origin and/or tissue pathological situation. The aim of this study was to analyze the protein composition, protein abundance, and functional clustering of EV released by peritoneal exudate cells (PECs) in the pristane experimental lupus model, to identify predictive or diagnostic biomarkers that might discriminate the autoimmune process in lupus from inflammatory reactions and/or normal physiological processes. In this study, thanks to an extensive proteomic analysis and powerful bioinformatics software, distinct EV subtypes were identified in the peritoneal exudates of pristane-treated mice: 1) small EV enriched in the tetraspanin CD63 and CD9, which are likely of exosomal origin; 2) small EV enriched in CD47 and CD9, which are also enriched in plasma-membrane, membrane-associated proteins, with an ectosomal origin; 3) small EV enriched in keratins, ECM proteins, complement/coagulation proteins, fibrin clot formation proteins, and endopetidase inhibitor proteins. This enrichment may have an inflammation-mediated mesothelial-to-mesenchymal transition origin, representing a protein corona on the surface of peritoneal exudate EV; 4) HDL-enriched lipoprotein particles. Quantitative proteomic analysis allowed us to identify an anti-inflammatory, Annexin A1-enriched pro-resolving, neutrophil protein signature, which was more prominent in EV from pristane-treated Cd38-/- mice, and quantitative differences in the protein cargo of the ECM-enriched EV from Cd38-/- vs WT mice. These differences are likely to be related with the distinct inflammatory outcome shown by Cd38-/- vs WT mice in response to pristane treatment. Our results demonstrate the power of a hypothesis-free and data-driven approach to transform the heterogeneity of the peritoneal exudate EV from pristane-treated mice in valuable information about the relative proportion of different EV in a given sample and to identify potential protein markers specific for the different small EV subtypes, in particular those proteins defining EV involved in the resolution phase of chronic inflammation.