Neuropilin-1 Expression Is Induced on

Neuropilin-1Expression Is Induced on Tolerant Self-Reactive CD8+T Cells but Is Dispensable for the Tolerant Phenotype

Stephanie R.Jackson1,Melissa Berrien-Elliott1,Jinyun Yuan1,Eddy C.Hsueh2,3,Ryan M.Teague1,3*

1Saint Louis University School of Medicine,Department of Molecular Microbiology and Immunology,St.Louis,Missouri,United States of America,2Saint Louis University School of Medicine,Department of Surgery,St.Louis,Missouri,United States of America,3Saint Louis University Cancer Center,St.Louis,Missouri,United States of America

Abstract

Establishing peripheral CD8+T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues.However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction.Thus,understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health.Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant.For example,induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8+T cells are integral in the induction of tolerance.We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process.We discovered that,in addition to other known regulatory receptors,tolerant self-reactive CD8+T cells distinctly expressed the surface receptor neuropilin-1(Nrp1).

Nrp1was highly induced in response to self-antigen,but only modestly when the same antigen was encountered under immune conditions,suggesting a possible mechanistic link to T cell tolerance.We also observed a similar Nrp1expression profile on human tumor infiltrating CD4+and CD8+T cells.Despite high expression on tolerant CD8+T cells,our studies revealed that Nrp1had no detectable role in the tolerant phenotype.Specifically,Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells,lacking in vivo cytolytic potential,IFN c production,and antitumor responses.While reporting mostly negative data,our findings have therapeutic implications,as Nrp1is now being targeted for human cancer therapy in clinical trials,but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.

Citation:Jackson SR,Berrien-Elliott M,Yuan J,Hsueh EC,Teague RM(2014)Neuropilin-1Expression Is Induced on Tolerant Self-Reactive CD8+T Cells but Is Dispensable for the Tolerant Phenotype.PLoS ONE9(10):e110707.doi:10.1371/journal.pone.0110707

Editor:Taishin Akiyama,University of Tokyo,Japan

Received August7,2014;Accepted September15,2014;Published October24,2014

Copyright:?2014Jackson et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Data Availability:The authors confirm that all data underlying the findings are fully available without restriction.All relevant data are found within the paper.

Additional gene array data have been deposited in the Gene Expression Omnibus(GEO)with accession code GSE58722.

Funding:Research reported in this publication was supported by the National Institute of Allergy and Infectious Disease/National Institutes of Health (R01AI087764)to RMT,by a Cancer Research Institute Investigator Award to RMT,and by a National Cancer Institute/National Institutes of Health fellowship (F30CA180375)to SRJ.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The corresponding author,Ryan Teague,is a PLOS ONE Editorial Board member.This does not alter the authors’adherence to PLOS ONE Editorial policies and criteria.

*Email:rteague@https://www.sodocs.net/doc/a317340442.html,

Introduction

Activated cytotoxic T cells represent a powerful branch of the adaptive immune system,capable of detecting cellular abnormality and protecting the human host from microbial threats and malignancy.These cells are armed with a plethora of effector mechanisms,including cytolytic molecules and proinflammatory cytokines.While critical for host defense,CD8+T cell responses can be detrimental or even fatal when deregulated[1,2,3,4,5]. Thus,T cell activation following antigen engagement must be tightly controlled.One mechanism of control is peripheral T cell tolerance,which is critical in preventing immunopathology mediated by excessive CD8+T cell activity,and is especially important to limit the activation of self-reactive T cells harbored in the periphery of healthy individuals[6].However,tolerance also presents a formidable barrier to eliciting anti-tumor immune responses since many cancer antigens are also expressed in healthy self-tissue[7].In an effort to improve treatment options for patients with cancer,extensive work has gone into characterizing the factors that lead to T cell tolerance and the development of strategies that break tolerance toward tumor/self-antigens to augment immunotherapy[8].

We and others have reported that CD8+T cell tolerance is regulated in part by the coinhibitory surface receptors PD-1,LAG-3and CTLA-4[9,10,11].In our studies,these proteins were upregulated after antigen priming,particularly under tolerant conditions where these molecules proved fundamental for the dysfunctional phenotype[9].Although not characterized as a coinhibitory receptor,a similar pattern of expression was also observed for the surface molecule,neuropilin-1(Nrp1),implying a possible link between Nrp1and the induction or maintenance of CD8+T cell tolerance.

Nrp1is a type-I transmembrane glycoprotein originally discovered for its role in neuron axon guidance and embryonic

vessel formation[12,13,14,15].Its expression has subsequently been reported on malignant cells and several immune cell subsets including dendritic cells(DC),conventional T cells,and regulatory T cells(Treg)[16,17,18].Nrp1has three extracellular domains important for ligand binding and receptor dimerization,and a short cytoplasmic tail that lacks a kinase domain.To support downstream signaling,Nrp1dimerizes with other surface proteins such as plexin molecules,VEGFR2,TGFB-R,EGFR,HGFR, and PDGFR-a,allowing strong interactions with the multiple ligands.These varied binding partners permit Nrp1ligation to modulate a variety of signaling pathways,contributing to the remarkable diversity in the physiological activities attributed to Nrp1[16].

The first description of a possible role for Nrp1in the immune system showed homotypic interactions between Nrp1on mature DC and human T cells in the initiation of the primary T cell immune response[18].Subsequent studies focused primarily on Nrp1in murine Treg,as antigen engagement selectively supports Nrp1expression on Treg versus conventional CD4+T cells[17]. In Treg,Nrp1promotes synapse formation with DC and longer, more stable interactions leading to enhanced suppression[19]. The first in vivo reports found that Nrp1helped suppress autoreactive CD4+T cells in a murine experimental autoimmune encephalomyelitis model[20].More recently,Nrp1was identified as a marker of natural versus induced Treg and shown to play a role in contact-independent suppression mediated by these cells [21,22].Mechanistic studies by Delgoffe and colleagues demon-strated a role for Nrp1in the stability and suppressive activity of Treg,involving phosphatase PTEN recruitment to the immuno-logical synapse via association with the PDZ-protein interaction domain encoded in the cytoplasmic tail of Nrp1expression[23]. Thus,there is a building consensus that Nrp1is important for activation,synapse formation,and suppressive activity of CD4+ Treg.

Few reports have explored the biology of Nrp1on CD8+T cells. In a thorough analysis of the genes involved in CD8+T cells memory formation,Kaech et al.reported modest Nrp1upregula-tion(2-fold higher)on effector and memory CD8+T cells relative to naive cells[24].In2012,Hansen et al comment briefly that a minor population of CD8+T cells in the spleen expressed Nrp1 (1%),which contrasted with80%of Foxp3+Treg and23%of CD4+T cells[25].Nrp1has been suggested as a potential marker for liver-sinusoidal endothelium-primed CD8+T cells that escape deletional tolerance[26].These Nrp1+T cells formed a distinct memory cell population that lacked cytokine responsiveness, representing the first correlation between Nrp1expression and CD8+T cells with a dysfunctional phenotype.However,these reports were correlative,and the functional relevance of Nrp1in regard to CD8+T cell immunity and tolerance has not yet been determined.

In this study,we investigated the expression profile and functional role of Nrp1on CD8+T cells under naive steady state conditions,and within distinct in vivo environments where antigen was encountered within immune,inflammatory,or tolerant contexts.We report that Nrp1was selectively induced and highly expressed on CD8+T cells engaging self-antigen,both in mice and in human melanoma infiltrating T cells.Despite this unique expression pattern,Nrp1appeared to play no part in the dysfunctional phenotype of murine self-reactive T cells.This was confirmed in Nrp1-deficient T cells,which performed similarly to wild-type T cells within both immune and tolerant environments, and when used in adoptive immunotherapy for cancer.These results support Nrp1as a potential biomarker for dysfunctional self-reactive CD8+T cells,which is dispensable for the induction and maintenance of T cell tolerance.

Results

Nrp1is highly expressed on tolerant CD8+T cells primed by self-antigen in healthy hepatocytes

To define the intrinsic pathways regulating CD8+T cell tolerance versus immunity,the gene expression profiles of Gag-specific CD8+T cells(TCR Gag)were defined after transfer into normal B6mice(naive),B6mice with an immunogenic Gag-positive FBL tumor(immune),or Alb:Gag mice that express the same Gag antigen under control of the Albumin promoter in healthy hepatocytes(tolerant)[27].We previously reported that recognition of Gag in the immune context leads to CD8+T cell expansion,acquisition of effector function,and memory forma-tion.However,in the tolerant context of an Alb:Gag host,these same T cells proliferate briefly but fail to acquire effector function and are largely deleted8days after transfer[9,28].These tolerant T cells were characterized by high expression of multiple inhibitory receptors(e.g.CTLA-4,PD-1,LAG-3)vital for their dysfunctional phenotype[9].Subsequent gene array analysis revealed that expression of the gene that encodes Nrp1mirrored that of these co-inhibitory receptors on tolerant T cells. Specifically,like PD-1(encoded by pdcd1),nrp1gene expression was elevated upon engagement of antigen,but significantly upregulated in T cells engaging antigen under tolerant conditions (Fig.1A).This expression profile was reflected on the surface of tolerant T cells relative to immunized T cells(Fig.1B),which was evident as early as2days after T cell transfer into the tolerant environment(Fig.1C).Collectively,these data demonstrate that Nrp1marks tolerant T cells primed by a self-antigen in vivo,and compel further analysis of its possible role in the biology of tolerant CD8+T cells.

Nrp1expression promotes optimal CD8+T cell proliferation in response to self-antigen

One of the earliest reported roles for Nrp1in the immune system is the regulation of T cell proliferation.In these first reports,Nrp1expression on human dendritic cells and CD4+T cells promoted prolonged T cell:DC interactions and more extensive proliferation of resting T cells[18].However,these and other studies were limited only to in vitro analysis[19].In the murine EAE model of multiple sclerosis,the exact opposite trend was observed,with Nrp1-deficient CD4+T cells proliferating more than Nrp1+cells[20].Thus,there is still uncertainty surrounding the role of Nrp1in T cell proliferation.More importantly,the impact of Nrp1expression on CD8+T cell proliferation has not been analyzed.In our model,tolerance induction following engagement of antigen in the liver commences with extensive proliferation of responding CD8+T cells[28].To examine the contribution of Nrp1here,we evaluated the proliferation of transgenic Gag-specific T cells deficient for the gene that encodes Nrp1via Lck-cre mediated gene deletion(Nrp1f/f Lck-cre TCR Gag),referred to hereafter as nrp1f/f.

WT Gag-specific CD8+T cells(CD90.1+)were co-transferred with nrp1f/f Gag-specific CD8+T cells(CD90.1+/90.2+)into normal B6recipients or Alb:Gag recipients.After4days in Alb:Gag hosts,surface expression of Nrp1was evident on WT cells,but expression on nrp1f/f T cells was similar to naive T cells (Fig.2A).This same nrp1expression pattern was reflected at the gene expression level in identically treated cells(Fig.S1).While it is unlikely that Nrp1expression was completely absent in all nrp1f/f T cells,we consistently observed a5-10fold reduction in surface

protein expression relative to WT T cells (Fig.S1).Both sets of transferred T cells underwent several rounds of cell division regardless of genotype but fewer nrp1f/f T cells went into cell cycle and those that did underwent fewer rounds of division relative to WT T cells (Fig.2B and 2C).These data demonstrate that Nrp1is not essential for CD8+T cell proliferation,but may be required for optimal responses,contributing modestly to the extent of CD8+T cell activation during early induction of peripheral self-tolerance.

Nrp1does not contribute to deficiencies in effector function by tolerant T cells

Negative regulatory receptor signaling has been shown to trigger dysfunction of tolerant CD8+T cells [9].If Nrp1expression also contributes to poor effector activity,Nrp1-deficient T cells should have improved cytokine responses following self-antigen encounter.In support of this,studies by Bottcher et al .identified a distinct population of Nrp1+CD8+T cells lacking cytokine

responsiveness that correlated with high levels of Nrp1expression [26].To evaluate whether Nrp1expression directly regulated effector function of tolerant CD8+T cells,WT Gag-specific T cells (CD90.1+)were co-transferred with nrp1f/f CD8+T cells (CD90.1+/90.2+)into Alb:Gag hosts with or without acute Listeria monocytogenes infection.In agreement with our previous results [28],IFN c production was not elicited in WT T cells within Alb:Gag recipients,but could be induced in these same hosts when accompanied by inflammation during an acute Listeria infection (Fig.3).However,the same was true for Nrp1-deficient T cells,suggesting Nrp1was not involved in the regulation of effector cytokine production by tolerant CD8+T cells,nor was Nrp1required for self-reactive CD8+T cells to acquire effector functions during Listeria infection.

To further characterize the contribution of Nrp1to the functional defects associated with tolerant self-reactive T cells,in vivo cytolytic activity was assessed.WT or nrp1f/f T cells

were

Figure 1.Nrp1is highly expressed on tolerant CD8+T cells primed by a self-antigen in healthy liver.Naive Gag-specific CD8+T cells (CD90.1+)were transferred into B6mice (naive),B6mice bearing an immunogenic FBL tumor (immune),Alb:Gag mice (tolerant).(A)Two days after transfer,T cells were purified by cell sorting and RNA isolated for gene expression by microarray.Graphs displays nrp1and pdcd1relative gene expression pooled from biological triplicate samples,and error bars represent SD.(B)Nrp1protein expression on T cells 3days after adoptive transfer into the naive,immune or tolerant environment.Data are representative of 3experiments,each with 3recipient mice per group.(C)Nrp1protein upregulation on T cells 2and 4days after transfer into Alb:Gag recipients.Data are representative of 2time course experiments.doi:10.1371/journal.pone.0110707.g001

transferred separately into Alb:Gag recipient mice in the presence or absence of Listeria ,followed 3days later by an infusion of fluorescently labeled peptide-pulsed target cells.Consistent with our previous data [28],WT Gag-specific T cells failed to kill Gag-pulsed target cells within the tolerant environment,but specific cytolytic activity was induced by self-antigen when encountered in conjunction with Listeria (Fig.4).Cytolytic activity by Nrp1-deficient T cells was essentially identical to WT T cells under these same conditions.Likewise,blockade of Nrp1by in vivo administration of anti-Nrp1antibodies had no impact on WT

T

Figure 2.Nrp1expression corresponds with a modest increase in CD8+T cell proliferation in response to self-antigen.Naive WT or Nrp1-deficient CD8+T cells were labeled with efluor670cytoplasmic dye and co-transferred into B6(naive)or Alb:Gag (tolerant)mice.(A)Nrp1expression on WT and nrp1f/f T cells 3days after transfer is compared in the overlaid histograms.(B)Dilution of efluor670dye in transferred T cells was assessed 3days after transfer and is displayed in overlayed histograms.(C)Geometric mean fluorescent intensity of efluor670in T cells from either WT or Nrp1-deficient T cells (lower dye expression corresponds to more proliferation)is pooled from 4separate experiments,each with 3mice per group.Error bars are standard error of the mean (SEM)with P value indicated.doi:10.1371/journal.pone.0110707.g002

cell effector function (data not shown).These data further support that Nrp1neither hinders nor facilitates the cytolytic potential of self-reactive CD8+T cells.

Nrp1does not influence peripheral deletion of self-reactive CD8+T cells

In tumor cells,ligation of Nrp1can either promote cell survival or induce apoptosis,depending on the specific co-receptor and ligand involved [15].Nrp1ligation is reported to protect Treg against apoptotic cell death and induce a transcriptional profile enriched in pathways promoting survival and stability [23].In our model,Nrp1expressing self-reactive T cells are deleted by Bim-mediated apoptosis approximately 8days after infusion into Alb:Gag recipients [9].To identify any potential role of elevated

Nrp1expression in the elimination of these T cells,WT (CD90.1+)and nrp1f/f (CD90.1+/90.2+)Gag-specific CD8+T cells were co-transferred into normal B6or Alb:Gag host mice.Eight days later,the persistence of these transferred T cells was assessed in spleen,lymph node,bone marrow and liver.Under naive steady-state conditions,nrp1f/f CD8+T cells persisted slightly better than WT T cells in many of these tissues,but both cell types were deleted within the tolerant environment regardless of genotype (Fig.5).Similarly,in vivo blockade by administration of anti-Nrp1antibodies was also ineffective at preventing WT T cell deletion (data not shown).It was also evident that Nrp1expression did not affect T cell trafficking,as the relative proportion of WT to nrp1f/f CD8+T cells remained equivalent in all tissues analyzed.

These

Figure 3.Nrp1expression does not contribute to the lack of effector function in tolerant CD8+T cells.Naive WT (CD90.1+)and Nrp1-deficient (CD90.1+/90.2+)Gag-specific CD8+T cells were transferred into Alb:Gag recipients with (lower)or without (upper)a Listeria infection.Three days later,production of IFN c and TNF a by transferred T cells was measured after overnight restimulation with Gag peptide.(A)Plots display T cell frequency (left)and IFN c and TNF a production (right).Inset numbers are the percent of total splenocytes within the inscribed square region (left).Numbers in each quadrant represent the percent of gated CD8+CD90.1+T cells (right).(B)The percent of gated CD8+CD90.1+or CD8+CD90.1+/90.2+T cells that express IFN c in differentially treated Alb:Gag recipients was graphed,with each circle representing individual mice pooled from 4separate experiments.Horizontal bars represent the average for each group (ns =not significant).doi:10.1371/journal.pone.0110707.g003

results suggest that Nrp1had no role in peripheral deletion of self-reactive CD8+T cells following encounter with self-antigen.

Ablation of Nrp1does not improve adoptive T cell immunotherapy for leukemia

Together,our data suggest that Nrp1does not have an important role in the regulation of CD8+T cell function or survival,and has no real influence on the dysfunctional phenotype of tumor/self-reactive CD8+T cells.To definitively assess whether Nrp1expression impacts CD8+T cell function over time,we

directly compared WT and nrp1f/f T cells for efficacy in our model of adoptive T cell immunotherapy for cancer.A disseminated and progressive murine FBL leukemia was established in normal B6or Alb:Gag host mice by i.v.injection,as previously described [28].One week later,WT or nrp f/f Gag-specific T cells were infused i.v.into the same recipients,which were monitored for health out to 40days.Because WT T cells become tolerant in Alb:Gag recipients [9,28],adoptive immunotherapy is less effective and these recipients died of progressive tumor in less than 20days (Fig.6).Transfer of nrp1f/f T cells provided a similarly low level

of

Figure 4.The cytotoxic potential of tolerant CD8+T cells is not regulated by Nrp1.WT or Nrp1-deficient CD90.1+Gag-specific CD8+T cells were transferred into Alb:Gag recipients with or without Listeria infection.Three days after transfer,recipients were infused with a 1:1ratio of Gag (eFluor 670low )and control (eFluor 670high )peptide-pulsed target cells.Twenty hours later (day 4),recipient spleens were harvested and the frequency of transferred Gag-specific T cells was determined by flow cytometry (upper panels).Inset numbers are the percent of total splenocytes within the inscribed regions.Target cell frequency is displayed as histograms with the percentage of total eFluor 670-positive cells inset above the indicated regions (lower panels).(B)Graph displays relative target cell killing (%eFluor 670high /%eFluor 670low )pooled from 3independent experiments.Error bars represent standard deviation (ns =not significant).doi:10.1371/journal.pone.0110707.g004

therapeutic benefit to tumor-bearing Alb:Gag recipients.Con-versely,in tumor-bearing B6recipients,transferred Gag-specific T cells are activated by the immunogenic FBL tumor [9].Here,such T cells were capable of overcoming established leukemia in a majority (.75%)of recipient mice regardless of Nrp1expression (Fig.6).These results reinforce the notion that Nrp1has no bearing on the in vivo persistence,trafficking,or function of CD8+T cells despite high expression on tolerant versus immunized cells (Fig.1).

Neuropilin-1is expressed on human tumor infiltrating T cells

Expression of Nrp1has been described on a variety of human tumor cells [16],tumor infiltrating Tregs [29,30,31],and peripheral blood lymphocytes [16,18].However Nrp1expression on human tumor-infiltrating CD8+T cells has not been reported.Results from our mouse studies suggest that Nrp1is expressed on tumor/self-reactive T cells,particularly under conditions that favor T cell dysfunction or tolerance.Despite a lack of involvement in the tolerant phenotype,Nrp1may represent a valuable biomarker for such T cells in cancer patients.We evaluated tumor-infiltrating lymphocytes (TIL)derived from patients undergoing resection of metastatic melanoma.Nrp1was not detected on the surface of either CD4+or CD8+T cells from the peripheral blood of patients or healthy donors (Fig.7A).In contrast,Nrp1was expressed on CD4+and even more so on CD8+TIL in a majority of patient tumor samples (Fig.7A and 7B).Nrp1expression on TIL corresponded with an antigen-experienced CD45RO +phenotype (Fig.7A),implying a possible connection with tumor/self-antigen reactive T cells.Although Nrp1expression on tolerant mouse T cells did not contribute to the dysfunction,it did provide a unique marker of tumor/self-reactive T cells displaying a tolerant phenotype.To extend this observation to human T cells,ex vivo proliferation of CD8+TIL and peripheral blood lymphocytes (PBL)derived from the

same

Figure 5.Deletion of self-reactive CD8+T cells is not mediated by expression of Nrp1.Gag-specific CD8+T cells from WT (CD90.1+)and Nrp1-deficient (CD90.1+/90.2+)donor mice were co-transferred into B6or Alb:Gag recipients.The frequency of transferred T cells in recipient tissues was assessed after 8days.The total number of WT or Nrp1-deficinet T cells in the indicated tissue is displayed graphically and shows pooled data from 4independent experiments,with each circle representing data from one mouse.Horizontal bars represent the average for each group and P values are indicated (ns =not significant).

doi:10.1371/journal.pone.0110707.g005

Figure 6.Nrp1expression on adoptively transferred CD8+T cells does not influence the efficacy of immunotherapy for leukemia.B6or Alb:Gag recipient mice were inoculated with FBL leukemia.Seven days later,tumor-bearing recipients were infused with WT or Nrp1-deficient Gag-specific CD8+T cells.Recipient survival was tracked for 40days,and results pooled from 2separate experiments are depicted in the graph showing percent survival (y -axis)over time in days (x -axis),with a total 10mice in each treatment group.Data from B6and Alb:Gag groups were compared and P values are indicated (ns =not significant).

doi:10.1371/journal.pone.0110707.g006

patients was compared following stimulation with anti-CD3/ CD28beads.Data from2representative patients clearly demonstrates less proliferation from Nrp-1+tumor-infiltrating CD8+T cells relative to Nrp1-negative T cells isolated from PBL (Fig.7C).Thus,while a case can be made that Nrp1expression correlates with dysfunctional or tolerant T cells,the precise molecular mechanisms that lead to elevated Nrp1under these conditions,and how Nrp1contributes,if at all,in the process remains unknown.

Discussion

The receptor Nrp1has been widely studied for its role in regulatory CD4+T cells[17,19,23,32].However,the precise mechanism by which Nrp1is involved in immunosuppression remains unclear.Nrp1is also expressed on other cells of the immune system,but how Nrp1contributes to the biology in these diverse populations is unknown.In our study of CD8+T cell tolerance,the negative regulatory receptors PD-1,CTLA-4,and LAG-3were uniquely upregulated on CD8+T cells engaging self-antigen[9].Similarly,Nrp1was highly upregulated on the same T cells.Thus,we hypothesized that Nrp1,like these other molecules, might have an important role in the induction and/or mainte-nance of peripheral CD8+T cell tolerance.

Tolerant Nrp1+T cells have characteristic defects in the ability to produce effector cytokines,perform cytolytic functions,and are typically deleted from the periphery within8days of primary self-antigen encounter[9,28].Therefore,we expected that a deficiency in Nrp1might alter the induction of tolerance similar to what has been described when cells engage antigen in the absence of other negative regulatory receptors[33].Instead,lack of Nrp1 expression had no impact on self-reactive T cells,which still failed to acquire effector capabilities and were deleted by apoptosis identically to WT self-reactive T cells.Additionally,Nrp1 deficiency did not affect the ability to boost self-reactive CD8+T cell activation under inflammatory conditions.Thus,in our model, Nrp1held no perceivable influence over the phenotype and function of tolerant self-reactive CD8+T cells.

Coinhibitory molecules play an immunoregulatory role in controlling the balance between T cell activation and tolerance. Manipulation of these coinhibitory pathways provides a means to enhance immune responses to promote anti-tumor immunity[34]. Although antitumor immunity is often limited by self-tolerance, the outcome of antigen engagement within tumor versus healthy self-tissue can be quite different due to differences in costimulatory and coinhibitory signals received.The additional inflammatory and immunosuppressive mediators present in a tumor environ-ment might alter the threshold for tumor/self-reactive T cell activation.Conversely,a role for Nrp1expression on CD8+T cells that is distinct from tolerance might emerge under conditions in which tumor is present.To assess a potential role for Nrp1in CD8+T cell function under these conditions,we evaluated the ability of Nrp1-deficient T cells to provide effective adoptive T cell immunotherapy for cancer.This is of special interest since anti-Nrp1antibodies have recently completed phase I(NCT00747734) and phase Ib(NCT00954642)trials for patients with advanced solid tumors[35,36].Thus,characterizing the immune subsets that anti-Nrp1might target is important for evaluating potential mechanism of action and off-target toxicities.In our murine immunotherapy experiments,Nrp1expression on adoptively transferred T cells had no influence on recipient survival outcomes,with treated mice uniformly succumbing to tumor under tolerant conditions but able to overcome the same tumors under immune conditions regardless of Nrp1expression.Collec-tively,these results support that expression of Nrp1on CD8+T cells does not play a role in the tolerant phenotype that normally limits responses during adoptive T cell immunotherapy for cancer in mice.

In evaluations of Nrp1on human CD8+T cells,we observed that some melanoma tumor-infiltrating T cells displayed signifi-cantly upregulated Nrp1relative to T cells from normal adjacent skin tissue or peripheral blood.Thus,a correlation between Nrp1 expression and tumor/self-reactive could be made.However,the role Nrp1plays in the biology of human CD4+and CD8+TIL remains unknown.Additionally,the tumor-derived factors that lead to elevated Nrp1in some but not all patients with similar cancers have not been defined and warrant further investigation. While we did not identify a role for Nrp1in the regulation of CD8+T cell function or survival in a tolerant environment,our results should not be interpreted to imply a lack of utility for Nrp1 blockade in cancer therapy.Nrp1function is complex,in part due to the multiplicity of co-receptors and ligands that Nrp1can interact with,and its broad expression on a variety of cell types [17,18,37].The impetus for using Nrp1antibody blockade in cancer derives from its direct role in tumor cell survival and proliferation[38,39],its role in VEGF ligand-mediated angiogen-esis[15,40,41,42],and also in the augmentation of Treg suppression[16,19,32].Thus,Nrp1may represent a direct tumor target and also a target for immunotherapy by modulation of Treg activity.

Our study evaluated the role of Nrp1on tumor/self-reactive CD8+T cells,which has previously been underexplored.We conclude that the expression of Nrp1,although highly elevated on T cells engaging self-antigen in a tolerant context,does not regulate CD8+T cell tolerance.This has important clinical repercussions,as Nrp1is being targeted therapeutically in cancer patients but the mechanism by which this strategy might enhance anti-tumor immunity is not completely understood.Additionally, while our results suggest Nrp1may not represent a viable target for manipulation of self-reactive CD8+T cells during cancer immunotherapy,Nrp1could represent a useful target in other scenarios,such as autoimmunity[20,43],or as a potential biomarker to help identify autoreactive T cells in patients. Materials and Methods

Mice

Rag12/2TCR Gag transgenic mice have been previously described[9,28].Alb:Gag mice express the H-2b-restricted Friend murine leukemia virus-derived Gag glycoprotein in healthy hepatocytes under control of the Albumin promoter,as previously demonstrated[27].C57BL/6(B6)and Lck-cre mice were purchased from The Jackson Laboratory.Nrp1f/f mice were graciously provided by Dr.David Ginty and have previously been described[44].Nrp1f/f and Lck-cre mice were crossed with rag12/2TCR Gag transgenic mice.To generate Gag-specific CD8+T cells deficient for neuropilin,Nrp1f/f x TCR Gag mice were crossed with Lck-cre x TCR Gag mice to generate Nrp1f/f x Lck-cre x TCR Gag mice,which are rag1sufficient.All animals were maintained under specific pathogen-free conditions and used in accordance with our animal protocol approved by the Animal Care Committee of the Department of Comparative Medicine, Saint Louis University School of Medicine(Saint Louis,MO). Cell lines,peptides,and antibodies

FBL is a murine erythroleukemia cell line of B6origin that expresses the H-2b-restricted Friend murine leukemia virus-derived Gag glycoprotein as a tumor associated antigen[9,28].

Figure7.Nrp1expression is increased on human tumor infiltrating T cells and corresponds with antigen experience.CD45+ lymphocytes were analyzed from the blood(PBL)and tumor(TIL)of patients undergoing surgical resection of metastatic melanoma,and compared to lymphocytes from the blood of healthy donors.(A)The frequency of CD4+and CD8+T cells from the indicated tissues was compared(left)and co-expression of CD45R0and Nrp1assessed on these gated T cell populations(right).(B)The frequency of Nrp1+T cells among TIL,patient PBL,normal

skin tissue adjacent to tumor,or healthy donor PBL is displayed graphically.Results are pooled from8–20independent samples,with each circle

Gag peptide(CCLCLTVFL)and ovalbumin(SIINFEKL)control peptides were purchased from Pi Proteomics.Cell culture was conducted in complete RPMI-1640with10%FBS(Sigma). Fluorochrome-conjugated antibody to mouse CD8(53–6.7)was purchased from eBiosciences.Fluorochrome-conjugated mouse antibodies to CD90.1(OX-7),IFN c(XMG1.2),TNF a(MP6-XT22),and human antibodies to CD4(RPA-T4),CD8(RPA-T8), and CD45RO(UCHL1)were purchased from BD Biosciences. Fluorochrome conjugated antibodies to mouse neuropilin-1 (761705)and human neuropilin-1(446921)were purchased from R&D Systems.efluor-670cell dye was purchased from Invitrogen. Anti-mouse Nrp-1A blocking antibody was provided by Genen-tech and administered intraperitoneally at10mg/kg every other day,similar to studies previously described[45].

T cell transfer,tumor inoculation,infection

Gag-specific T cells were isolated from spleens and lymph nodes of rag12/2TCR Gag or Nrp1f/f Lck-cre TCR Gag donors.Whole-cell suspensions containing26106V a3-TCR+CD8+cells were intravenously injected into sex and age(6–8week)matched recipients.In some experiments,transferred cells were labeled with efluor670proliferation dye(eBioscience)before infusion according to the manufacturer’s protocol.To provide an immunogenic environment,56106FBL cells were established intraperitoneally in B6recipients3days before T cell transfer.Infected recipients received26106CFU attenuated(D ActA)Listeria monocytogenes intravenously immediately prior to T cell transfer.

Gene array

Naive Gag-specific T cells were sorted or transferred into B6 mice with established FBL tumor(immune)and Alb:Gag mice (tolerant).Two days after T cell transfer,recipient spleen and lymph node were harvested and pooled.Transferred cells were sorted on a FACSAria(BD Biosciences).RNA was isolated using RNeasy Plus Mini Kit(Qiagen).Total RNA was used to generate aRNA(Affymetrix)and hybridized to the GeneChip Mouse Genome430 2.0Array(Affymetrix)and analyzed with a GeneChip scanner30007G(Affymetrix).Data were obtained from3biological replicates per condition,and has been deposited in the Gene Expression Omnibus(GEO)with accession code GSE58722.

Flow cytometry

Recipient spleen and peripheral lymph nodes were harvested for analysis at indicated time points.Tissues were homogenized into single-cell suspensions before cell culture or staining for flow cytometry.Cell suspensions were stained for extracellular markers at4u C for30minutes.Ex vivo cytokine production was assessed following overnight stimulation with4m g/mL Gag or Ova peptide in the presence of GolgiPlug(BD Biosciences).For intracellular staining of IFN c and TNF a,cells were fixed and permeabilized in Cytofix/Cytoperm buffer(BD Biosciences),and proteins stained in Perm/Wash buffer(BD Biosciences)for30minutes at4u C according to the manufacturer’s protocol.All flow cytometry was conducted using either an LSR II or FACSCanto II(BD Biosciences),and resulting data analyzed using FlowJo software (Tree Star).In vivo killing assay

Recipient mice received adoptive T-cell transfers and infections as described above.Three days after T cell transfer,B6splenocytes (targets)were pulsed with10m g/ml Gag or control Ova peptide, and differentially labeled with1m M or5m M eFluor670, respectively.Targets were then washed twice in phosphate buffered saline,combined at a1:1ratio,and injected into recipient mice intravenously.Twenty hours later,the frequency of eFluor high versus eFluor low targets from recipient spleens was assessed by flow cytometry.

Immunotherapy assay

One week prior to treatment,FBL leukemia was established in B6or Alb:Gag mice by intravenous injection of0.96105viable FBL tumor cells.Seven days after tumor inoculation,tumor-bearing mice received adoptive transfer of26106Gag-reactive CD8+T cells by intravenous injection.Recipient survival was tracked for a minimum of40days with daily health monitoring, and recipients were euthanized upon moribund appearance. Quantitative RT-PCR

Transferred T cells were sorted to better than95%purity using a FACSAria III(BD Biosciences).Total RNA was isolated from sorted cells using RNeasy Mini Kit(Qiagen)and cDNA was synthesized using Transcriptor First Strand cDNA synthesis kit (Roche).Real-time PCR was performed with SYBR Select Master Mix(Life Technology)on a7500Real-Time PCR System (Applied Biosystems).Relative amplification values were calculated by normalizing to amplification of beta actin.The following primers were used:Nrp1sense primer:59-CACCAACCCCACA-GATGTTGT-39,Nrp1antisense primer:59-CCAACATTCCA-GAGCAAGGAT-39.

Statistical analysis

The Kruskal–Wallis test was used for statistical comparison (GraphPad Prism4)of total cell numbers between different treatment groups.A one-way ANOVA was used for statistical comparison of cell frequencies between multiple treatment groups. Survival data were analyzed with a Mantel-Cox log-rank test.P values less than0.05were considered significant.

Patients and normal donors

Following a research protocol approved by the Saint Louis University School of Medicine Institutional Review Board(IRB), peripheral blood,tumor,and normal tumor-adjacent tissue were obtained from patients undergoing resections for metastatic melanoma.Blood samples were also obtained from normal healthy volunteers as separate controls.All subjects provided informed written consent by completion of an IRB approved consent form prior to participation in the study.

Isolation of human TIL and PBMC

Heparinized blood was diluted1:1(v/v)with PBS before Ficoll density centrifugation.The buffy coat containing PBMC was harvested,and washed twice in cold PBS and subjected to cell surface staining and flow cytometry analysis.Fresh pieces of tumor and normal adjacent tissue were minced into1-mm-size pieces and

representing data from one patient/donor.Horizontal bars represent the average for each group and P values are indicated(ns=not significant).(C) Proliferation of CD8+T cells from patient PBL or TIL was directly compared following3day in vitro stimulation with anti-CD3/CD28beads.Histograms show relative dilution of efluor670dye in stimulated(black line)versus non-stimulated(grey filled peaks)for2representative patients.Inset numbers are the percent of cells within the indicated region.

doi:10.1371/journal.pone.0110707.g007

tissue subjected to mechanical dissociation over40m m cell strainer.The resulting cellular suspension was diluted1:1(v/v) with PBS before Ficoll density centrifugation.The buffy coat containing TIL was harvested,washed twice in cold PBS,stained for expression of cell surface markers,and subjected to flow cytometry analysis.

Supporting Information

Figure S1Nrp1expression is significantly compro-mised in nrp1f/f T cells.Naive WT or Nrp1-deficient CD8+ T cells were transferred into B6(naive)or Alb:Gag(tolerant)mice.

(A)After3days,WT and nrp1f/f T cells were sorted to better than 95%purity and mRNA isolated for quantitative RT-PCR analysis. Relative nrp1gene expression in WT and nrp1f/f T cells from the indicated environments is shown.Samples were performed in triplicate and error bars represent standard deviation(B)Naive WT or nrp1f/f CD8+T cells were transferred into B6(naive)or Alb:Gag(tolerant)mice.The geometric mean fluorescent intensity of anti-Nrp1-FITC(closed circles)and isotype-Ig(open circles) surface staining is shown for each of3separate mice per group. Horizontal bars represent the average for each group and P values are indicated(ns=not significant).

(TIF)

Acknowledgments

The authors would like to thank Sherri Koehm and Joy Eslick for technical assistance with flow cytometry and cell sorting.Collin Chen and Maureen Donlin provided assistance with the gene array studies identifying differences in Nrp1gene expression.The authors thank Dr.David Ginty for providing the conditional nrp1f/f mouse.

Author Contributions

Conceived and designed the experiments:SRJ RMT.Performed the experiments:SR JY MBE RMT.Analyzed the data:SRJ RMT. Contributed reagents/materials/analysis tools:ECH.Wrote the paper: SRJ RMT.

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零件用平键和轴套螺母紧固使之与轴成为一体。整个转子由两端轴承支承在泵壳体上，转子部分的叶轮数目是根据泵的级数而定。 3、轴承部分 本D、DG型泵轴承有滑动轴承和滚动轴承两种，按型号不同而定，均不承受轴向力。泵在运行中应当允许转子部分在泵壳体中轴向游动，不能采用向心轴承。 4、泵的密封 泵的前段、中段、后段之间密封面均采用二硫化钼润滑脂密封，转子部分与固定部分之间靠密封环、导叶套、填料密封，当密封环和导叶套的磨损程度已影响泵的工作和性能时应予及时更换。 5、平衡机构 平衡机构由平衡环、平衡盘、平衡套等组成，平衡机构用于平衡泵的轴向力。

在下述操作程序中，请注意“警告”、“小心”、“注意”标记词，这些词旨在强调人身安全和恰当的操作方法及维修方面的重点，其词义解释如下： 警告：操作程序、习惯等若违背，可能引起人身伤亡事故 小心：操作程序、习惯等若违背，可能引起对设备的损坏 注意：操作程序、条件等应引起高度重视 四、到货检查 到货后，应立即对设备进行验收，因装运引起的任何缺陷应立即报告给承运人。说明书文本和其它部件（如电机）的说明书及装箱单应存放在一个安全方便的地方以供随时参考。 五、泵的装配和拆卸 1、泵的装配本D、DG型泵装配质量的好坏直接影响泵能否正常运行，并影响泵的使用寿命和性能参数；影响机组的振动和噪音，装配中应注意以下几点： a、固定部分各零件组合后的同心度靠零件制造精度和装配质量来保证，应保证零件的加工精度和表面粗糙度，不允许碰、划伤。作密封剂用的二硫化钼应干净。紧固用的螺钉、螺栓应当受力均匀。 b、叶轮出口流道与导叶进口流道的对中性是依靠各零件的轴向尺寸来保证。流道对中性的好坏直接影响泵的性能，故泵的尺寸不能随意调整。 c、泵装配完毕后，未装填料前，用手转动泵转子，检查泵转子在壳体内旋转是否灵活，轴向窜动是否达到规定要求。 d、上述检查符合要求后，在泵两端轴封处加入填料，注意填料环在填料室中的相对位置。 2、泵的拆卸 a、拆卸要与装配相反的顺序进行，拆卸时应严格保护零件的制造精度不

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50-泵吸入口径（mm） 32-泵排出口径（mm） 160-叶轮名义直径（mm） S1-轴封形式（S1为单端内装型机械密封） 303-过流零件的材料代号（ZG1Cr18Ni9） IH系列泵是单级、单吸、悬臂式离心泵，是机械部确定取代F型离心泵的节能产品。适合于输送化工流程中有腐蚀性、粘度类似于水的液体。可广泛用于化工、石油、冶金、电力、造纸、食品、制药、合成纤维等行业。介质温度一般为-20℃～15℃，若采用适当的冷却措施，可输送更高温度的介质。 本系列泵共33个基本型，每种型号都可以通过切割叶轮获得较低的流量和扬程。 DYK DGK YD YDS型多级离心输液泵 DYK100-30*5

DYK--------- 100----------设计流量（M3/h） 30------------单级设计扬程（m） 5--------------级数 DYK、DGK、YD、YDS型泵适用于油田集输、厂矿企业排水和锅炉给水，输送介质为不带固体颗粒的原油、清水及物理、化学性质类似的液体，输送介质的温度低于105℃ 25CDLF2-260 25--------吸入口直径 CDL------轻型立式多级离心泵 F----------普通型略，过流部件为不锈钢304或316

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文档编号:TSS_PUMP.DOC 离心泵单元仿真培训系统 操作说明书 北京东方仿真软件技术有限公司 二〇〇六年十月

目录 一、工艺流程说明2 1、离心泵工作原理基础2 2、工艺流程简介4 3、控制方案5 4、设备一览6 二、离心泵单元操作规程6 1、开车操作规程6 2、正常操作规程8 3.停车操作规程9 4、仪表及报警一览表11 三、事故设置一览12 四、仿真界面14 附：思考题17

一、工艺流程说明 1、离心泵工作原理基础 在工业生产和国民经济的许多领域，常需对液体进行输送或加压，能完成此类任务的机械称为泵。而其中靠离心作用的叫离心泵。由于离心泵具有结构简单，性能稳定，检修方便，操作容易和适应性强等特点，在化工生产中应用十分广泛，据统计超过液体输送设备的80%。所以，离心泵的操作是化工生产中的最基本的操作。 离心泵由吸入管，排出管和离心泵主体组成。离心泵主体分为转

动部分和固定部分。转动部分由电机带动旋转，将能量传递给被输送的部分，主要包括叶轮和泵轴。固定部分包括泵壳，导轮，密封装置等。叶轮是离心泵中使液体接受外加能量的部件。泵轴的作用是把电动机的能量传递给叶轮。泵壳是通道截面积逐渐扩大的蜗形壳体，它将液体限定在一定的空间里，并将液体大部分动能转化为静压能。导轮是一组及叶轮旋转方向相适应，且固定于泵壳上的叶片。密封装置的作用是防止液体的泄漏或空气的倒吸入泵内。 启动灌满了被输送液体的离心泵后，在电机的作用下，泵轴带动叶轮一起旋转，叶轮的叶片推动其间的液体转动，在离心力的作用下，液体被甩向叶轮边缘并获得动能；在导轮的引领下沿流通截面积逐渐扩大的泵壳流向排出管，液体流速逐渐降低，而静压能增大。排出管的增压液体经管路即可送往目的地。及此同时，叶轮中心因为液体被甩出而形成一定的真空，因贮槽液面上方压强大于叶轮中心处，在压力差的作用下，液体不断从吸入管进入泵内，以填补被排出的液体位置。因此，只要叶轮不断旋转，液体便不断的被吸入和排出。由此，离心泵之所以能输送液体，主要是依靠高速旋转的叶轮。 离心泵的操作中有两种现象应当避免：气缚和气蚀。 气缚是指在启动泵前泵内没有灌满被输送的液体，或在运转过程中泵内渗入了空气，因为气体的密度小于液体，产生的离心力小，

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