IL-15 Prevents Renal Fibrosis by Inhibiting Collagen Synthesis: A New Pathway in Chronic Kidney Disease?

Chronic kidney disease (CKD), secondary to renal fibrogenesis, is a public health burden. The activation of interstitial myofibroblasts and excessive production of extracellular matrix (ECM) proteins are major events leading to end-stage kidney disease. Recently, interleukin-15 (IL-15) has been implicated in fibrosis protection in several organs, with little evidence in the kidney. Since endogenous IL-15 expression decreased in nephrectomized human allografts evolving toward fibrosis and kidneys in the unilateral ureteral obstruction (UUO) model, we explored IL-15's renoprotective role by pharmologically delivering IL-15 coupled or not with its soluble receptor IL-15Rα. Despite the lack of effects on myofibroblast accumulation, both IL-15 treatments prevented tubulointerstitial fibrosis (TIF) in UUO as characterized by reduced collagen and fibronectin deposition. Moreover, IL-15 treatments inhibited collagen and fibronectin secretion by transforming growth factor-ß (TGF-ß)-treated primary myofibroblast cultures, demonstrating that the antifibrotic effect of IL-15 in UUO acts, in part, through a direct inhibition of ECM synthesis by myofibroblasts. In addition, IL-15 treatments resulted in decreased expression of monocyte chemoattractant protein 1 (MCP-1) and subsequent macrophage infiltration in UUO. Taken together, our study highlights a major role of IL-15 on myofibroblasts and macrophages, two main effector cells in renal fibrosis, demonstrating that IL-15 may represent a new therapeutic option for CKD.

BMAL1 Knockdown Leans Epithelial-Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.

Cannabinoid receptor 1 is a major mediator of renal fibrosis.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-ß1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

EBV-associated mononucleosis does not induce long-term global deficit in T-cell responsiveness to IL-15.

It has been reported that infectious mononucleosis (IM)-symptomatic primary Epstein-Barr virus infection produces a global down-regulation of interleukin-15 receptor-alpha (IL-15Ralpha) on T cells and natural killer cells associated with a defective IL-15 responsiveness that lasts for many years after the disease episode. In contrast with these results, our data indicate that, in the T-cell compartment derived from remote IM subjects, there is no quantitative or qualitative defect in the expression of the IL-15Ralpha chain and no deficit in T-cell responsiveness to IL-15. We observed efficient signal transduction, survival, and proliferation even in response to low IL-15 concentrations. These data are relevant and shed new light on the immune long-term response in IM subjects because they contradict the hypothesis that defects in Epstein-Barr virus-host immune balance may be correlated with a long-lasting global deficit in T-cell responsiveness to IL-15.

BMAL1 knockdown triggers different colon carcinoma cell fates by altering the delicate equilibrium between AKT/mTOR and P53/P21 pathways.

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

Inhibition of TGF-ß1 Signaling by IL-15: A Novel Role for IL-15 in the Control of Renal Epithelial-Mesenchymal Transition: IL-15 Counteracts TGF-ß1-Induced EMT in Renal Fibrosis.

Renal tubulointerstitial fibrosis is the final common pathway in end-stage renal disease and is characterized by aberrant accumulation of extracellular matrix (ECM) components secreted by myofibroblasts. Tubular type 2 EMT, induced by TGF-ß, plays an important role in renal fibrosis, by participating directly or indirectly in myofibroblasts generation. TGF-ß1-induced apoptosis and fibrosis in experimental chronic murine kidney diseases are concomitantly associated with an intrarenal decreased expression of the IL-15 survival factor. Since IL-15 counteracts TGF-ß1 effects in different cell models, we analyzed whether (1) human chronic inflammatory nephropathies evolving towards fibrosis could be also characterized by a weak intrarenal IL-15 expression and (2) IL-15 could inhibit epithelial-mesenchymal transition (EMT) and excess matrix deposition in human renal proximal tubular epithelial cells (RPTEC). Our data show that different human chronic kidney diseases are characterized by a strong decreased expression of intrarenal IL-15, which is particularly relevant in diabetic nephropathy, in which type 2 tubular EMT plays an important role in fibrosis. Moreover, primary epithelial tubular cultures deprived of growth supplements rapidly produce active TGF-ß1 inducing a "spontaneous" EMT process characterized by the loss of membrane-bound IL-15 (mbIL-15) expression. Both "spontaneous" EMT and recombinant human (rh) TGF-ß1-induced EMT models can be inhibited by treating RPTEC and HK2 cells with rhIL-15. Through a long-lasting phospho-c-jun activation, IL-15 inhibits rhTGF-ß1-induced Snail1 expression, the master inducer of EMT, and blocks TGF-ß1-induced tubular EMT and downstream collagen synthesis. In conclusion, our data suggest that intrarenal IL-15 could be a natural inhibitor of TGF-ß in human kidney able to guarantee epithelial homeostasis and to prevent EMT process. Thus, both in vivo and in vitro an unbalance in intrarenal IL-15 and TGF-ß1 levels could render RPTEC cells more prone to undergo EMT process. Exogenous IL-15 treatment could be beneficial in some human nephropathies such as diabetic nephropathy.

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression.

MDR-1 C3435T polymorphism influences cyclosporine a dose requirement in liver-transplant recipients.

BACKGROUND: Cyclosporine A (CsA) is characterized by high interindividual variations in oral bioavailability and a narrow therapeutic index. CsA is a substrate for P-glycoprotein, a member of the ABC transporter family encoded by the multiple drug-resistant gene MDR1. METHODS: Because MDR1 gene exon 26 C3435T polymorphism influences intestinal P-glycoprotein expression, we investigated whether this polymorphism was correlated with variation in CsA dose requirement and concentration/dose ratio in 44 liver-transplant recipients during 1 month after transplantation. CsA concentration was measured 2 hours after administration (C2), according to international recommendations. RESULTS: The MDR-1 wild-type genotype (3435CC) was observed in 15 patients (34%), whereas 21 (48%) patients were heterozygous (3435CT), and 8 (18%) patients were homozygous for the mutation (3435TT). There was no significant difference between the three groups regarding corticosteroids treatment or renal function during this period. One to 3 days after liver transplantation, when every patient received a similar CsA weight-adjusted dose, the concentration/dose ratio was correlated with exon 26 single nucleotide polymorphism and was significantly higher in subjects homozygous for the mutation (P=0.012). This was confirmed 1 month after transplantation (P=0.049), when the dose was adjusted to maintain the C2 target level of 1,000 microg/L and we observed that TT patients required approximately 50% lower weight-adjusted CsA dose than wild-type patients (P=0,033). CONCLUSIONS: These findings demonstrate that the MDR1 exon 26 C3435T polymorphism is a major determinant of CsA concentration/dose ratio in liver-transplant recipients and is predictive of the dose of CsA to be administered to achieve the target C(2) concentration.

Interleukin-15 plays a central role in human kidney physiology and cancer through the γc signaling pathway.

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαßγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαß heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation).

Interleukin-15 is a major regulator of the cell-microenvironment interactions in human renal cancer.

Primary human epithelial renal cells of normal (HRE), paratumoral (pTEC) and tumoral (RCC) origin display important differences, concerning the expression and biological effects of the IL-15/IL-15R system that deeply influences the evolution of the tumour microenvironment. A major distinguishing feature is represented in RCC and pTEC by the loss of the γc chain leading to the assembly of a IL-15Rαß heterodimer that in response to physiologic concentrations of IL-15 initiates the process of their epithelial-mesenchymal transition (EMT). In contrast, this treatment in HRE cells, which display the IL-15Rαßγc heterotrimer, causes opposite effects inhibiting their drift towards EMT. Thus, IL-15 at physiologic concentrations displays novel functions acting as a major regulator of renal epithelial homeostasis. As second distinguishing feature, RCC and pTEC but not HRE cells express a trans-membrane-bound IL-15 (tmb-IL-15) able to deliver a reverse signal in response to the soluble IL-15Rα chain inducing their EMT. In conclusion, comparison of primary normal (HRE) to primary pathological cells (pTEC and RCC) highlights two major issues: (1) IL-15 is a major regulator of epithelial homeostasis; (2) "apparently normal" pTEC cells, could contribute to organize a generalized "pre-neoplastic" environment committed to favour tumour progression.

Differentiation therapy: targeting human renal cancer stem cells with interleukin 15.

BACKGROUND: Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. METHODS: CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. RESULTS: CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. CONCLUSION: IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

Human renal cancer cells express a novel membrane-bound interleukin-15 that induces, in response to the soluble interleukin-15 receptor alpha chain, epithelial-to-mesenchymal transition.

Although interleukin-15 (IL-15) is a powerful immunomodulatory factor that has been proposed for cancer immunotherapy, its intratumoral expression may be correlated with tumor progression and/or poor clinical outcome. Therefore, neoplasias potentially sensitive to immunotherapy should be checked for their IL-15 expression and function before choosing immunotherapy protocols. Primary human renal cancer cells (RCC) express a novel form of membrane-bound IL-15 (mb-IL-15), which displays three major original properties: (a) It is expressed as a functional membrane homodimer of 27 kDa, (b) it is shed in the extracellular environment by the metalloproteases ADAM17 and ADAM10, and (c) its stimulation by soluble IL-15 receptor alpha (s-IL-15Ralpha) chain triggers a complex reverse signal (mitogen-activated protein kinases, FAK, pMLC) necessary and sufficient to ~induce epithelial-mesenchymal transdifferentiation (EMT), a crucial process in tumor progression whose induction is unprecedented for IL-15. In these cells, complete EMT is characterized by a dynamic reorganization of the cytoskeleton with the subsequent generation of a mesenchymal/contractile phenotype (alpha-SMA and vimentin networks) and the loss of the epithelial markers E-cadherin and ZO-1. The retrosignaling functions are, however, hindered through an unprecedented cytokine/receptor interaction of mb-IL-15 with membrane-associated IL-15Ralpha subunit that tunes its signaling potential competing with low concentrations of the s-IL-15Ralpha chain. Thus, human RCC express an IL-15/IL-15R system, which displays unique biochemical and functional properties that seem to be directly involved in renal tumoral progression.

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

Apoptotic death concurrent with CD3 stimulation in primary human CD8+ T lymphocytes: a role for endogenous granzyme B.

We exposed primary CD8(+) T cells to soluble CD3 mAb plus IL-2 and limited numbers of monocytes (3%). These cells were activated but concurrently subjected to ongoing apoptosis ( approximately 25% were apoptotic from day 2 of culture). However, their costimulated CD4(+) counterparts were much less prone to apoptosis. The apoptotic signaling pathway bypassed Fas and TNFRs, and required the activity of cathepsin C, a protease which performs the proteolytic maturation of granzyme (Gr) A and GrB proenzymes within the cytolytic granules. Silencing the GrB gene by RNA interference in activated CD8(+) T cells prevented the activation of procaspase-3 and Bid, and indicated that GrB was the upstream death mediator. A GrB-specific mAb immunoprecipitated a approximately 70-kDa molecular complex from cytolytic extracts of activated CD8(+) (but not resting) T cells, that was specifically recognized by a nucleocytoplasmic protease inhibitor 9 (PI-9) specific mAb. This complex was also detected after reciprocal immunoprecipitation of PI-9. It coexisted in the cytosol with the 32-kDa form of GrB. As neither were detected in the cytosol of CD4(+) bystander T cells (which poorly synthesized GrB), and as silencing the perforin (Pf) gene had no effect in our system, endogenous GrB was likely implicated. Immunoprecipitation experiments failed to reveal Pf in the cytosol of CD8(+) T cells, and only a tiny efflux of granular GrA was detected by ELISA. We propose that some GrB is released from cytolytic granules to the cytosol of CD8(+) T lymphocytes upon CD3/TCR stimulation and escapes PI-9, thereby mediating apoptotic cell death.