Targeting Tissue Resident Memory CD8~+ T Cells In The Kidney Is A Potential Therapeutic Strategy To Ameliorate Podocyte Injury And Glomerulosclerosis

Tissue-resident memory T(TRM)cells,a recently identified non-circulating memory T cell population,play a crucial role in tumor immunity and preventing pathogen invasion.The formation and maintenance of TRM cell are influenced by numerous factors,including inflammation,local antigen triggering and tissue-specific cues.Recent studies have found that TRM cells are also present in the kidney and other non-barrier tissues,participate in bacterial infection-induced glomerulonephritis and aggravate renal autoimmune diseases.However,most studies so far mainly focus on TRM cells and their relevance for immediate protection against pathogens upon local reinfection,the composition,localization,effector function,and specificity of TRM cells in the kidney and their relevance for chronic kidney disease remain unknown.Podocytes,highly differentiated glomerular epithelial cells,are essential for the maintenance of glomerular filtration barrier.Podocyte injury is considered to be an important factor in the development of glomerular diseases,including focal segmental glomerulosclerosis,diabetic nephropathy and lupus nephritis.Although some progress has been made in the understanding of pathophysiological mechanisms of podocyte injury,there is still a lack of specific and effective therapies.Current studies have suggested that podocyte injuries are closely associated with disruption of immune homeostasis.Meanwhile,inflammatory cytokines produced by activated T cells such as IFN-y can also induce the expression of MHC-Ⅰ,MHC-Ⅱ,CD80 and CD86 on podocyte surface,which promotes podocyte present antigen and podocyte apoptosis.Podocyte injury is the key mediator in the pathogenesis of glomerular disease mice models,such as ADR induced FSGS and db/db mice.The pathogenesis of T cell immunology in podocyte injury remains unclear.The systemic lupus erythematosus mouse model,as the positive control,is characterized by excessive activation of the immune system and thereby results in glomerulonephritis and podocyte injury.Therefore,a comprehensive understanding of the relationship between activated T cells and podocyte injury is necessary for the development of tissue-resident cell-based immunotherapies for glomerular diseases.In this study,we firstly found that the proportion of renal CD8+TRM cells was significantly increased in the kidney from patients and mice with glomerular diseases.IL-15 promoted CD8+TRM cell formation and activation,thereby promoting podocyte injury and glomerulosclerosis.Interestingly,Sparsentan,the dual angiotensin Ⅱ receptor and endothelin Type A receptor antagonist,can also reduce TRM cell responses by intervening IL-15 signaling,exploring its new pharmacological functions.This study further enriches and develops the understanding of the biological functions of TRM cells and also provides a new target for the prevention and treatment of glomerular diseases.Objective1.To identify the composite of immune cells in renal microenvironment and the change of renal TRM cells under physiological and pathological conditions.2.To discuss the mechanisms of renal CD8+TRM cell formation and activation under pathological conditions.3.To evaluate the effects of targeting renal CD8+TRM cells on glomerulosclerosis and podocyte injury.4.To elucidate the mechanisms by which Sparsentan alleviates podocyte injury by targeting renal CD8+TRM cell formation and activation.Methods and ResultsPart 1 Identify the characteristics and changes of renal TRM cells under physiological and pathological conditions1.1 Identify the characteristics of renal TRM cells under physiological conditionWe performed a 42-antibody panel for mass cytometry to build a composite of human and mouse renal CD45+leukocytes in the steady state.The immune landscape indicated that T lymphocytes were enriched in the kidney,and almost all of renal CD69+T cells exhibited memory cells phenotype:CD45RA’ CCR7-in human and CD44+in mouse.In vivo labeling assay showed that renal CD69+CD44+T cells were protected from intravenous injection(i.v.)antibody labeling(CD45-)and they were bona fide resident population.Phenotypic analysis showed that renal CD69+CD44+ T cells highly expressed tissue homing chemokine receptor CXCR3 but lowly expressed lymph node homing receptor CD62L.Furthermore,they produced more effector molecular IFN-y and perforin than renal CD69-CD44+T cells,which indicated that renal CD69+CD44+T cells were TRM cells.1.2 Identify the characteristics and changes of renal TRM cells under pathological conditionsTo construct mouse model of FSGS,8 week-old male Balb/c mice were selected randomly,and 10 mg/kg of ADR was injected by the tail vein.To construct mouse model of DN,heterozygote BKS db/m mice and homozygote BKS db/db mice were purchased.To construct mouse model of LN,female systemic lupus erythematosus mice MRL/lpr mice were purchased.We found that the proportion of CD8+TRM cells was significantly increased in these three mice models.Meanwhile,the proportion of CD4+TRM cells in MRL/lpr mice was also increased,but no changes in mice with ADR treatment and db/db mice compared to their normal controls.These results indicated that CD8+TRM cells may play a universal role in different glomerular diseases.By flow cytometry,we found that renal CD8+TRM cells expressed more IFN-y and perforin under these pathological conditions,indicating that TRM cells were activated.Immunofluorescent results showed that the number of CD8+TRM cells was increased in the tubulointerstitium surrounding the glomeruli in renal biopsies from patients with FSGS,DKD and LN.The amount of CD4+TRM cells in renal biopsies from patients with LN was also increased,but no changes from patients with FSGS and DKD compared to healthy controls.Part 2 The formation and activation of CD8+TRM cells in pathological conditions and its role in podocyte injury2.1 Explore the source of increased renal CD8+TRM cells under pathological conditionsFTY720,a SIP inhibitor that prevents lymphocyte circulation was used for the pre-treatment of mice with ADR treatment.By flow cytometry,there was no more proliferating renal TRM cells in mice with ADR treatment after the FTY720 treatment.It was found that more CD69+T cells were present in the kidney from ADR-treated nude mice that were transfused with CD8+CD69-CD44+T cells and highly expressed TRM cells markers such as CXCR3 and lowly expressed CD62L.2.2 The effects of IL-15 on CD8+TRM cell formation and activationWe performed ELISA assay to detect the level of IL-15 in renal cortex in mice with ADR treatment.Urine was collected for the detection of UACR.The correlations between the level of IL-15 and UACR were also analyzed by the software Graphpad Prism.ELISA assay showed that the level of IL-15 in the kidney was significantly increased in ADR-treated mice and positively correlated with the UACR.We isolated splenic mononuclear cells and sequentially exposed to rIL-15 and rIL-2.After 3 days,the expression of CD69 in CD8+CD44+T cells the proportion of IFN-y in CD8+TRM cells were analyzed by flow cytometry.It was found that rIL-15 induced CD69+T cells development.As a negative control,IL-2,had no effects on CD69 expression.Furthermore,IL-15 could directly promote IFN-y production in CD8+TRM cells,indicating that IL-15 can activate renal CD8+TRM cells.CD 122 is an integral part of the receptor complex for IL-15 and it was highly expressed in renal CD8+TRM cells,and anti-CD122 antibody inhibited IL-15-induced CD8+TRM cell formation and activation in vitro.2.3 The effects of different renal activated CD8+memory T cells on podocyte fateWe sorted renal CD8+CD69-CD44+T cells and CD8+TRM cells from ADR-treated mice and then transferred them to normal nude mice.After 3 weeks,TUNEL assay and WT-1 staining were applied to detect the number of apoptotic podocytes in nude mice.TUNEL assay and WT-1 staining showed that only renal CD8+TRM cells in ADR-treated mice triggered podocyte apoptosis in vivo.We co-cultured renal CD8+CD69-CD44+T cells and CD8+TRM cells with normal murine podocytes by transwell assay.In vitro co-culture system,it was found that only renal CD8+TRM cells from ADR-treated mice had cytotoxic effects on podocytes including enhanced apoptosis,reduced nephrin and podocin expression,induced actin cytoskeleton derangement and mitochondria damage.2.4 The effects of activated renal CD8+TRM cells on glomerular mesangial cells and endothelial cellsWe co-cultured the activated renal CD8+TRM cells with glomerular cells such as glomerular mesangial cells and endothelial cells by transwell assay.RT-qPCR showed that activated renal CD8+TRM cells had no effects on the expression of collagen 4A and fibronectin in glomerular mesangial cells.Flow cytometry analysis showed that activated renal CD8+ TRM cells cannot mediate glomerular mesangial cells and endothelial cells apoptosis.2.5 The effects of activated renal CD8+TRM cells with pretreatment with IL-15 signaling blockade on podocyte fateBy flow cytometry and RT-qPCR,we found activated renal CD8+TRM cells with pretreatment with IL-15 or CD 122 antibody had less cytotoxicity and recovered the expressions of podocin in podocytes.2.6 The effects of IL-15 signaling blockade on glomerulosclerosis and podocyte injury in vivoSplenic CD8+CD69-CD44+T cells in normal mice were sorted by flow cytometry.We injected anti-IL-15 or anti-CD 122 antibody to ADR-treated nude mice which were transfused with CD8+CD69-CD44+T cells.Anti-IL-15 or anti-CD 122 antibody treatment significantly inhibited renal CD8+TRM cell formation and activation.The urine was collected to detect the UACR by the automatic biochemical instrument.PAS staining was performed to detect the glomerular morphology;TEM was used to observe the foot process injury;Immunofluorescence staining was applied to detect the expression of nephrin and podocin in glomeruli.Anti-IL-15 or anti-CD122 antibody-treated mice showed lower UACR,less glomerular and podocyte injuries compared to their controls.In addition,IL-15 signaling blockade recovered the expressions of nephrin and podocin.In db/db mice,the level of IL-15 was also increased and positively correlated with the proteinuria.Consistently,in anti-IL-15 or anti-CD 122 antibody treated db/db mice,the formation and activation of renal CD8+TRM cell were markedly inhibited.Lower urinary albumin excretion,less glomerulosclerosis and podocyte injury were also observed in db/db mice after anti-IL-15 or anti-CD 122 antibody treatment.2.7 Explore the effects of renal NK cells in glomerular injury in ADR-treated and db/db miceWe injected ADR-treated and db/db mice with anti-NK1.1 antibody.By flow cytometry,NK cells in the kidney were effectively depleted in ADR-treated and db/db mice.The glomerular injuries in ADR-treated and db/db mice were not alleviated with deficiency of NK cells by UACR and PAS staining.Part 3 The role and regulation of Sparsentan in renal CD8+TRM cells and glomerular injury3.1 The effects of Sparsentan on renal CD8+TRM cells and glomerular injury in ADR-treated mice and db/db miceWe injected Sparsentan to ADR-treated mice and db/db mice and isolated the renal mononuclear cells.By flow cytometry,we found that Sparsentan could reduce the amount of renal CD8+TRM cells in mice with ADR treatment and mice.Moreover,Sparsentan reduced the production of IFN-y and perforin in isolated renal CD8+TRM cells,alleviated proteinuria,mesangial matrix expansion and podocyte injury.3.2 The effects of activated CD8+TRM cells in Sparsentan-treated ADR-treated mice on podocyte fateWe sorted activated renal CD8+TRM cells in Sparsentan-treated ADR-treated mice and co-cultured them with normal murine podocytes.Sparsentan alleviated podocyte injury as evidenced by reduced apoptosis,recovered podocin expression,attenuated cytoskeleton and mitochondria injury when we sorted renal CD8+TRM cells from Sparsentan treated ADR-treated mice and co-cultured with podocytes.3.3 Explore the correlation between the levels of Ang Ⅱ,ET-1 and IL-15 in kidneyWe performed ELISA assay to detect the levels of Ang Ⅱ,ET-1 and IL-15 in renal cortex in ADR treated mice and db/db mice.We found that the levels of Ang Ⅱ and ET-1 were enhanced in the kidney from mice with ADR treatment and db/db mice by ELISA assay,and the levels of Ang Ⅱ and ET-1 were positively correlated with the level of IL-15.We further found that primary renal parenchymal cells secreted more IL-15 after Ang Ⅱ or ET-1 treatment.In addition,Sparsentan inhibited IL-15 production in the kidney from mice with ADR treatment and db/db mice.Conclusion and innovation1.Renal tissue displays high abundance of TRM cells under physiological conditions and the proportion and activation of CD8+TRM cells were significantly increased in the kidney from patients and mice with glomerular diseases.CD8+TRM cells play the universal role of in glomerular injury.2.IL-15 promotes CD8+ TRM cell formation and activation under pathological conditions and IL-15 signaling blockade inhibits renal CD8+TRM cells immune response.Functionally,activated renal CD8+TRM cells promote podocyte mitochondrial damage and apoptosis and thereby cause the podocyte injury and glomerulosclerosis.3.Sparsentan inhibits CD8+TRM cell formation and activation by intervening IL-15 signaling and then alleviates podocyte injury,exploring its new pharmacological functions.