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γERK Activation and Costimulates IFN- T Cells Promotes Akt and+ CD4+CD45RO

Factor-Mediated Signaling in Human Cutting Edge: Vascular Endothelial Growth

and David M. BriscoeEdelbauer, Maria P. Stack, Katiana Calzadilla, Soumitro Pal Aninda Basu, Andre Hoerning, Dipak Datta, Monika

http://www.jimmunol.org/content/184/2/545doi: 10.4049/jimmunol.0900397December 2009;

2010; 184:545-549; Prepublished online 11J Immunol 

Referenceshttp://www.jimmunol.org/content/184/2/545.full#ref-list-1

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Cutting Edge: Vascular Endothelial GrowthFactor-Mediated Signaling in Human CD45RO+ CD4+ TCells Promotes Akt and ERK Activation and CostimulatesIFN-g ProductionAninda Basu,1 Andre Hoerning,1 Dipak Datta, Monika Edelbauer, Maria P. Stack,Katiana Calzadilla, Soumitro Pal, and David M. Briscoe

In this study, we find that CD45RO+ memory popula-tions of CD4+ T lymphocytes express the vascular endo-thelial growth factor (VEGF) receptorsKDRandFlt-1 atboth the mRNA and protein levels. Furthermore, byWestern blot analysis, we find that VEGF increases thephosphorylation and activation of ERK and Akt withinCD4+CD45RO+ T cells. These VEGF-mediated signal-ing responses were inhibited by a KDR-specific smallinterfering RNA in a VEGF receptor-expressing JurkatT cell line and by SU5416, a pharmacological KDR in-hibitor, in CD4+CD45RO + T cells. We also find thatVEGF augments mitogen-induced production of IFN-gin a dose-dependent manner (p , 0.001) and signifi-cantly (p , 0.05) increases directed chemotaxis of thisT cell subset. Collectively, our results for the first timedefine a novel function for VEGF and KDR inCD45RO+ memory T cell responses that are likely ofgreat pathophysiological importance in immunity.The Journal of Immunology, 2010, 184: 545–549.

Vascular endothelial growth factor (VEGF), a well-established angiogenesis factor, has been found tohave potent proinflammatory properties, including

an ability to mediate leukocyte trafficking into sites of cell-mediated immunity (1–5). Some of its proinflammatoryproperties are dependent on direct interactions with its re-ceptors expressed on monocytes (6), and some are thought tobe related to the ability of VEGF to induce endothelial acti-vation and chemokine production (3). Furthermore, a rela-tively underappreciated aspect of VEGF biology is that thereis also evidence that VEGF has direct biological effects onT cells. For instance, VEGF has been observed to augmentAg-induced cytokine production, including both Th1 (7),Th2 (8), and Th17 (9) responses. However, the mechanism(s)and basis for these interactions are poorly understood.

The biological activities of VEGF are mediated by itsreceptors Flt-1 (VEGF receptor [VEGFR]1), KDR (VEGFR2,also called Flk1 in the mouse), and neuropilin-1 (NRP-1) (10,11). All VEGFRs are expressed by endothelial cells, and selectreceptors are reported to be expressed by cells of the immunesystem (6, 7, 12–14). Flt-1 and NRP-1 are expressed on hu-man monocytes and APCs (6, 15), and Flt-1 and KDR/Flk-1have been identified on murine populations of T cells (13) andhuman leukemic T cell lines (14, 16). Furthermore, a recentreport indicated that murine CD4+CD25+FoxP3+ T regula-tory cells (but not effector cells) express NRP-1, which wasfound to function in Ag presentation (12). Moreover, otherstudies have suggested that NRP-1 is expressed on populationsof human naive T cells where it functions in the initiation of Tcell activation and in primary immune responses (17). Thus,there are several reports indicating that VEGFRs are expressedon different T cell subsets, suggesting the potential importanceof VEGF–VEGFR interactions in immunity.In this study, we observed that the VEGFRs, KDR and Flt-1,

are expressed on the CD45RO+ subset of human CD4+ Tcells. Furthermore, we demonstrate that VEGF stimulatesKDR-induced signals within this subset of T cells, cos-timulates IFN-g production, and mediates chemotaxis. Ourresults define a novel function for VEGF and KDR in theimmune response and provide for the intriguing possibilitythat overexpressed VEGF at sites of chronic inflammationmay facilitate the peripheral homing and local reactivation ofmemory populations of T cells.

Materials and MethodsReagents

Human rVEGF and IFN-g–inducible protein of 10 kDa (IP)-10 were obtainedfrom R&D Systems (Minneapolis, MN), and anti-CD3 and anti-CD28 wereobtained from BD Pharmingen (San Diego, CA). For Western blotting, anti–phospho-ERK1/2 (polyclonal anti-Thr202/Tyr204) and anti–phospho-Akt (poly-clonal anti-Ser473) were purchased from Cell Signaling Technology (Danvers,

Transplantation Research Center and the Division of Nephrology, Department of Med-icine, Children’s Hospital Boston; and Department of Pediatrics, Harvard MedicalSchool, Boston, MA 02115

1A.B. and A.H. contributed equally to these studies.

Received for publication February 10, 2009. Accepted for publication November 19,2009.

This work was supported by National Institutes of Health Grant RO1 HL074436 (toD.M.B.) and by fellowship grants from the Deutsche Forschungsgemeinschaft (to A.H.)and the Fonds zur Forderung der Wissenschaftlichen Forschung Austrian Science Fund(to M.E.).

Address correspondence and reprint requests to Dr. David M. Briscoe, Division ofNephrology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mailaddress: david.briscoe@childrens.harvard.edu

Abbreviations used in this paper: IP-10, IFN-g–inducible protein of 10 kDa; NRP-1,neuropilin-1; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor;VEGFR, VEGF receptor.

Copyright� 2010 by TheAmerican Association of Immunologists, Inc. 0022-1767/10/$16.00

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MA), Abs to total ERK, total Akt (clone C-20), NRP-1 (clone C-19), and Flt-1(clone C-17) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA),and anti-KDR(clone 55B11)was purchased fromCell SignalingTechnology. ForFACS analysis, PE-conjugated anti-KDR (clone 89106) and anti–Flt-1 (clone49560) were purchased fromR&DSystems, anti–NRP-1 (clone AD5-17F6) wasobtained from Miltenyi Biotec (Auburn, CA), and isotype controls were pur-chased from BD Pharmingen. The pharmacological KDR inhibitor SU5416 andpertussis toxin were obtained from Sigma-Aldrich (St. Louis, MO), and the Aktsignaling inhibitor LY294002 and the ERK signaling inhibitor PD 98059 werepurchased from Calbiochem (San Diego, CA).

Cell culture

CD4+CD45+RO+ memory T cells were isolated from the blood of healthyvolunteers using a negative isolation kit (Miltenyi Biotec, Auburn, CA) ac-cording to the manufacturer’s protocol. The purity of T cells was determinedby FACS after each isolation. For occasional experiments, pooled populationsof CD4+ T cells were purified using the Dynal positive isolation kit (In-vitrogen, Carlsbad, CA), and RO+ and RA+ subsets were identified by FACS.Human T cells were cultured in RPMI 1640 (Lonza, Walkersville, MD) andsupplemented with 10% FBS. Jurkat T cells were grown in modified RPMI1640 medium from the American Type Culture Collection (Manassas, VA)supplemented with 10% FBS (HyClone, Logan, UT).

Transfection

A validated KDR small interfering RNA (siRNA) (sense-r [CGC UGA CAUGUACGGUCUA] dTdT antisense-r [UAGACCGUACAUGUCAGCG]dTdT) and control (AllStars negative control siRNA, number 1027280) werepurchased from Qiagen (Valencia, CA) and were transfected (100 nM) intoJurkat T cells using Lipofectamine 2000 (Invitrogen). After 96 h, transfectedcells were treated with VEGF (5–20 ng/ml) for 10 and 15 min and harvested.The efficiency of siRNA for knockdown was assessed by PCR and/or Westernblot analysis in control cells.

Western blot analysis

Protein samples were lysedwith ice-cold radioimmunoprecipitation assay buffer(Boston Bioproducts, Ashland, MA) were separated on a SDS-polyacrylamidegel and transferred to a polyvinylidene difluoride membrane (Millipore, Bed-ford, MA). Using standard methodology, proteins were detected in eachmembrane by chemiluminescence (Pierce, Rockford, IL).

PCR

Total RNA was prepared using the RNeasy isolation kit (Qiagen). cDNAsynthesis and PCRwere performed using the SuperScript one-step RT-PCR kit(Invitrogen) andgene-specific primers according to themanufacturer’s protocol.The oligonucleotide primers used were as follows: human KDR, forward, 59-AAA GAC TAC GTT GGA GCA ATC CCT-39, and reverse, 59-CTG GATTGT GTA CAC TCT GTC AAA-39; human Flt-1, forward, 59-ATG GCTCCC GAA TCT ATC TTT GAC-39, and reverse, 59-GCC CCG ACT CCTTAC TTT TAC TGG-39; and human GAPDH, forward, 59-ACC ACAGTCCAT GCC ATC AC-39, and reverse, 59-TCC ACC ACC CTG TTG CTGTA-39. Quantitative real-time PCR was performed using the 7300 real-timePCR system and the Assays-on-Demand Gene Expression Product (TaqMan,MGC probes; Applied Biosystems, Foster City, CA). Gene-specific primers forthe analysis of human KDR and GAPDH by real-time PCR were obtainedfrom Applied Biosystems. Ct values for the evaluation of KDR expression werecalculated.

Flow cytometry

Cell suspensions were stained with FITC- or PE-conjugated Abs, or isotypecontrols using standard techniques. Stained cells were subsequently analyzedusing a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA),CellQuest, and FlowJo software.

ELISPOT

Briefly, multiscreen-IP microtiter plates (Millipore, Bedford, MA) were coatedwith anti-human IFN-g (BD Pharmingen) and anti-CD3 (0.1–1 mg/ml).CD45RO+ memory CD4+ T cells (5 3 104 cells/well) were subsequentlycultured in the plates alone, or with VEGF (10–20 ng/ml), or with VEGF andSU5416 (1 and 5 mM) for 24 h The cells were removed by washing and ELI-SPOT was performed using standard methodology (3). Negative controls wereunstimulated cells, and positive controls were PHA (1 mg/ml)-treated cells.

Chemotaxis assays

Live-time chemotaxis assays were performed across 3-mm pore membraneFalcon FluoroBlok Transwell inserts (BD Biosciences, Franklin Lakes, NJ) in24-well culture plates, similar to that described by others (18). Cell culturemedium (180 ml) was added into the lower chamber in the absence or presenceof VEGF (10–50 ng/ml). Addition of the T cell chemoattractant IP-10 (50 ng/ml) into the lower chamber was used as a positive control. Negatively isolatedCD4+CD45RO T cells were labeled with 5 mM CFSE (Molecular Probes,Carlsbad, CA) and were added (13 105 labeled cells/well in duplicate) into theupper chamber of each Transwell in a total volume of 180 ml of cell culturemedium. Lymphocyte migration was monitored in the lower chamber of theTranswell by the assessment of increasing fluorescence every 15 min using anautomated plate reader (Victor; Wallac, Turku, Finland).

Chemotaxis assays were also performed across type 1 collagen coated 3-mmpore polycarbonate filters using the standard Boyden Chamber assay, ac-cording to the manufacturer’s instructions (Neuro Probe, Gaithersburg, MD).Negatively isolated CD4+CD45RO T cells (1 3 105 cells/well) were addedinto the upper wells, in duplicate, in a total volume of 50 ml of cell culturemedium. VEGF and IP-10 were added into the lower wells (also in 50 ml ofmedium), and the chamber was incubated for 3 h at 37˚C in 5% CO2. Cellswere removed from the upper surface of the filters, and they were fixed inmethanol and stained with Wright–Giemsa stain (Richard-Allan Scientific,Waltham, MA). The number of transmigrated stained T cells on the un-dersurface of the filter was counted by light microscopy in a mean of six fields(at 3400 magnification)/condition.

ResultsVEGFRs are expressed on recently activated and memory subsets ofhuman CD4+ T cells

We initially evaluated the expression of VEGFRs on humanCD4+ T cell subsets and a Jurkat T cell line. By FACS analysis,we found that NRP-1, KDR, and Flt-1 were expressed at lowlevels on our Jurkat T cells (Fig. 1A), but in multiple experi-ments, we failed to find notable expression of any VEGFR onunactivated pooled populations of human CD4+ T cells or onnaive CD45RA+CD4+ human T cell subsets (data not shown).In contrast, although NRP-1 was at low negligible levels, wefound that the expression of KDR and Flt-1 was notable at theprotein level by FACS and byWestern blot analysis on purifiedCD45RO+ populations of CD4+ T cells (Fig. 1B–D). BothKDR and Flt-1 were also expressed at the mRNA level in theseT cells (Fig. 1E). However, in multiple experiments, we foundthat there were variations in baseline levels of expression ofKDR on CD4+CD45RO+ T cells, and in occasional donors,KDR was expressed at high levels comparable to that observedin endothelial cells (data not shown).We used quantitative real-time PCR to evaluate whether induced T cell expression ofKDRwas related to the activation status of the CD4+ T cells. Asillustrated in Fig. 1F, we found a 10-to 20-fold increase in KDRmRNA expression following stimulation of pooled populationsof CD4+ T cells with anti-CD3. Thus, VEGFRs are expressedon recently activated and memory subsets of human T cells.

VEGF–VEGR interactions result in the activation of the MAPK andPI3K-Akt signaling pathways in human CD4+ T cells

VEGFiswell established toactivate protective, proliferative, andmigratory signaling in endothelial cells via the MAPK and thePI3K-Akt pathways (11, 19).We initially cultured our Jurkat Tcell line with increasing concentrations of VEGF (0–20 ng/ml),and we subsequently evaluated the activation of these classicalVEGF-inducible signaling pathways by Western blot analysis.We found that the treatment of Jurkats with VEGF (for timeperiods from 2–15 min) resulted in the phosphorylation ofERK and Akt (Fig. 2 and data not shown).BecauseKDR iswell established to function inVEGF-induced

activation of ERK and Akt in endothelial cells (19), we next

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transfected our Jurkat T cells with siRNA to KDR (or controlsiRNA) prior to treatment with VEGF. We found that VEGFincreased the expression of pERK (Fig. 2A) and pAkt (Fig. 2B) incontrol siRNA-transfected cells, and the response was signifi-cantly reduced following transfection with KDR siRNA (p ,0.05, n = 3 experiments; Fig. 2A, 2B). We also cultured purifiedpopulations of CD4+CD45RO+ T cells with VEGF in the ab-sence or presence of SU5416, a pharmacological KDR signalinginhibitor. As illustrated in Fig. 2C–E, we found that the treat-

ment of these cells with VEGF also resulted in activation/phos-phorylation of ERK andAkt, and furthermore, we found that theresponse was reduced in the presence of SU5416 (Fig. 2D, 2E).TCR-mediated signaling also activates the MAPK and PI3K-

Akt pathways. To determine whether there is cross talk betweenVEGF-andTCR-inducedresponses,wenextculturedourJurkatT cells with anti-CD3 in the absence or presence of VEGF, andwe subsequently evaluated the expression of pERK by Westernblot analysis. As expected, we found that TCR-mediated signals

FIGURE 1. Expression of VEGFRs on Jurkat T cells and CD4+CD45RO+ human memory T cells. The expression of NRP-1, Flt-1, and KDR was examined by

FACS on Jurkat T cells (A) and on CD4+CD45RO+ T cells (B and C ). B illustrates the purity of CD4+CD45RO+ T cells following negative isolation from

PBMCs. A and C, The difference (D) in mean fluorescence staining (experimental minus isotype control) is shown within each FACS plot. D, Western blot

analysis for VEGFR expression. M, memory CD4+ CD45RO+ T cells; J, Jurkat T cells. E, RT-PCR for KDR and Flt-1 in Jurkat T cells (lane 2) and

CD4+CD45RO+ T cells (lane 3). Lane 1 represents RT-PCR of non-reverse-transcribed RNA from the CD4+CD45RO+ T cells as a negative control. F, ThemRNA expression of KDR was evaluated by quantitative real-time PCR in pooled populations of CD4+ cells, either unactivated or following mitogenic activation

(anti-CD3, 1 mg/ml). A–C are representative of at least 10 experiments, and each PCR is representative of two with similar findings.

FIGURE 2. VEGF-inducible signaling in human CD4+CD45RO+ T cells. Jurkat T cells or purified populations of CD4+CD45RO+ T cells (negatively isolated

from PBMCs) were stimulated with VEGF (10 ng/ml) for 2, 10, and 15 min and were subsequently subjected to Western blot analysis. A and B illustrate the

expression of pERK and pAkt or total ERK and total Akt in Jurkat T cells transfected either with KDR siRNA or control siRNA, prior to stimulation with VEGF.

Densitometry illustrating the relative expression of pERK/ERK (A) and pAkt/Akt (B) is illustrated above each Blot. In C, CD4+CD45RO+ T cells were treated

with VEGF alone, and in D and E, CD4+CD45RO+ T cells were treated with VEGF in combination with SU5416, a pharmacological KDR signal inhibitor (1

mM). Following treatment, the expression of pERK/ERK and/or pAkt/Akt was evaluated by Western blot analysis. F, Western blot analysis of pERK/ERK

expression in Jurkat T cells stimulated with VEGF alone, anti-CD3 (0.3 mg/ml) alone or both in combination as indicated. Densitometry is illustrated above the

blot. All illustrated blots are representative of at least three with similar results.

The Journal of Immunology 547

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markedly induced the expression of pERK (Fig. 2F). Moreover,we observed that the treatment of Jurkats with both VEGF andanti-CD3 in combination resulted in an additive effect onpERKexpression. Collectively, these findings define a potential role forVEGF in the costimulation and/or the reactivation of humanmemory subsets of CD4+ T cells.

VEGF–VEGFR interactions result in the costimulation of IFN-gproduction by CD4+CD45RO+ T cells

To next determine whether VEGF elicits functional reac-tivation responses, purified populations of CD4+CD45RO+

T cells were stimulated with increasing concentrations ofVEGF alone or in combination with anti-CD3. As illustratedin Fig. 3A–C, although we found differences in baseline IFN-g responses to anti-CD3 in different donors, VEGF consis-tently enhanced anti-CD3–stimulated production of IFN-g(p , 0.001, n . 10 experiments). VEGF also increased IL-2production, but its effect was not as marked as that observedfor IFN-g, and the addition of VEGF to anti-CD3–treatedcultures failed to costimulate IL-4 production in memory Tcell subsets (data not shown).Because we find that VEGF-KDR interactions mediate cell

signaling responses in our T cells (Fig. 2), we also wished to testwhether they costimulate IFN-g production. CD4+CD45RO+

T cells were cultured with anti-CD3 and VEGF in combina-tion, in the absence or presence of SU5416 (Fig. 3C ). We ob-served that SU5416 inhibited VEGF-inducible IFN-gproduction to baseline levels, indicating that VEGF–KDR in-teractions play a major role in the costimulation of cytokineproduction.

VEGF mediates migratory responses in human CD4+CD45RO+

memory T cells

To evaluate whether VEGF is functional for T cell chemotaxis,CFSE-labeled CD4+CD45RO+ T cells were placed in theupper chamber of FluoroBlok Transwells, and the response toVEGF was assessed live-time every 15 min, for up to 90 minusing an automated assay. As illustrated in Fig. 4A, we foundthat the migratory response to VEGF was significantly higherthan controls at all time points examined. Using the Boydenchamber chemotaxis assay, we also found a marked migratoryresponse to VEGF (Fig. 4B, p , 0.05) suggesting that VEGFmediates motility response(s) in this T cell subset. To testwhether VEGF-inducible activation of the Akt and/or the ERKpathways function in the chemotaxis response, we next culturedT cells with LY294002 (to inhibit Akt signaling) or PD98059(to inhibit ERK signaling) prior to, and during, the chemotaxisassay. We used pertussis toxin, a well-established G protein-coupled receptor signaling inhibitor as a control. As illustratedin Fig. 4B, we found that VEGF elicited a migratory responsein the cells treated with pertussis toxin. However, the cellstreated with either LY294002 or PD98059 failed to migrate inresponse to VEGF. As expected, there was a marked chemotacticresponse to IP-10, which was significantly reduced in the cellstreated with pertussis toxin (Fig. 4C). Collectively, these ob-servations indicate that VEGF functions to elicit migratory re-sponses in human CD4+CD45RO+ subsets of T cells, and

FIGURE 3. VEGF-induced costimulation of IFN-g production in human

CD4+CD45RO+ T cells. A, CD45RO+ populations of CD4+ T cells were

negatively isolated from human PMBCs and were treated with anti-CD3 (0.1

and 0.3 mg/ml) and VEGF (10 and 20 ng/ml) as indicated. After 24 h, ELI-

SPOT was performed to evaluate IFNg production. B shows representative

wells in triplicate from the experiment shown in A. The spot count in each wellis illustrated. C, T cells were cultured with anti-CD3 (0.3 mg/ml), VEGF (20

ng/ml), and the pharmacological KDR signal inhibitor SU5416 (SU1, 1 mM)

and SU5 (5 mM) as illustrated. ELISPOT was again performed for IFN-g

production after 24 h. Each illustrated experiment is representative of at least

four with similar results. p Values were calculated using the Student t test.

FIGURE 4. VEGFmediates chemotaxis of humanCD4+CD45RO+memory

T cells.A, Cell culturemediumwas added into the lower chamber of 3-mmpore

FluoroBlok Transwells in the absence or presence of VEGF (50 ng/ml) or IP-10

(50 ng/ml, as a positive control). Subsequently, CFSE-labeledCD4+CD45RO+

T cells (13 105 cells) were placed in the upper chamber of each Transwell (in

duplicate wells), and migration into the lower chamber was monitored via the

assessment of fluorescence.T0 represents the time in between plate setup and the

first fluorescence reading (∼5–7min). T15–90 represent automated fluorescence

readings in the lower chamber every 15 min for a total of 90 min. B and C,CD4+CD45RO+ T cells (1 3 105 cells) were placed in the upper chamber of

a microchemotaxis Boyden chamber and migration into the lower chamber was

assessed after 3 h, as described in Materials and Methods. T cells were used

untreated or following 2-h pretreatment with the Akt inhibitor LY294002, the

Erk inhibitor PD98059, or pertussis toxin. VEGF or IP-10 was added into the

lower chambers, as indicated.The illustrated experiments are representative of at

least three performed in duplicate, all with similar results. p Values were cal-

culated using the Student t test (*p , 0.05).

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furthermore, that this effect involves VEGF-inducible activationof the Akt and ERK signaling pathways.

DiscussionVEGFRs are expressed on select subpopulations of T cells,indicating potential role(s) for VEGF in the regulation ofimmune responses. In this study, we demonstrate that KDRand Flt-1 are expressed at both the mRNA and protein levelson CD45RO+ memory subsets of human CD4+ T cells.However, similar to others (12), we failed to find detectablelevels of the VEGFR NRP-1 on effector/memory CD45RO+

T cells. NRP-1 is reported to be expressed on murineCD4+CD25+FoxP3+ T regulatory cells and on human sub-populations of naive T cells (17). These observations suggestthat NRP-1 may be functional in naive and T regulatoryresponses. Our observations in this report indicate that KDRand Flt-1 but not NRP-1 are functional in VEGF-mediatedactivation of CD45RO+ T cells.We observed that VEGF mediates the activation of both the

ERKandAkt signalingpathways inhumanTcells. Interestingly,it has been demonstrated that there is an intricate cross talkbetween these pathways to regulate the outcome of the signalingresponse (20). Akt activation may negatively regulate ERKsignaling, which may serve, for instance, to maintain a physio-logical balance in terms of proliferation and/or survival. Thus, itis possible that the effect of VEGF tomediate ERK activity maybe limited due to simultaneous Akt activation.Our findings, as well as those by others (13) indicate that

VEGF has direct chemoattractant effects on T cells. Further-more, we find that VEGF-inducible activation of ERK and Aktsignaling within T cells mediate this migratory response. Be-cause VEGF is expressed at high levels in many chronic in-flammatory disease processes (1, 2), the implications of ourobservations are that targeting of T cell VEGF or KDR, or itssignaling partners, may be therapeutic to inhibit the traffickingand/or the peripheral localization of human memory T cells.On the other hand, augmenting KDR-induced signaling inT cells could have implications, for instance, in the directedmigration of T cell subsets into tumors.Collectively, our studies defined in this report indicate that

VEGF has direct effects on human memory T cells and mayqualitatively and quantitatively regulate migratory and reac-tivation responses. Thus, a novel implication of our studies isthat VEGF, a classical endothelial cell mitogen and growthfactor, has potent effects on the inflammatory response.

AcknowledgmentsWe thank Debabrata Mukhopadhyay, Ph.D., for ongoing constructive com-

ments and Olivier Dormond, M.D., Ph.D., for helpful discussions about the

techniques used in this report.

DisclosuresThe authors have no financial conflicts of interest.

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