6) 268ndash284wwwelseviercomlocateyviro

Virology 352 (200

CXCR4 tropic human immunodeficiency virus type 1 induces anapoptotic cascade in immature infected thymocytes that resembles

thymocyte negative selection

Shailesh K Choudhary a1 Russell M Walker b Douglas M Powell b Vicente Planelles cCraig Walsh d David Camerini a

a Department of Molecular Biology and Biochemistry Center for Immunology and Center for Virus Research University of California Irvine CA 92697-3900 USAb Millennium Pharmaceuticals Cambridge MA 02139 USA

c Department of Cellular Biology and Immunology Department of Pathology School of Medicine University of Utah Salt Lake City UT 84112 USAd Department of Molecular Biology and Biochemistry and Center for Immunology University of California Irvine CA 92697-3900 USA

Received 1 December 2005 returned to author for revision 5 January 2006 accepted 27 April 2006Available online 14 June 2006

Abstract

HIV-1 often replicates in the thymus of infected individuals causing thymocyte depletion and thymic dysfunction Nevertheless themechanisms by which thymocyte depletion occurs are not clear Here we report that HIV-1 infection induced apoptosis primarily in productivelyinfected thymocytes aldrithiol-2 or Efavirenz treatment largely abrogated HIV-1-induced apoptosis Moreover X4-HIV-1 induced apoptosisprimarily in immature CD4+ CD8+ (DP) thymocytes whereas most mature CD4 or CD8 single-positive (SP) thymocytes were resistant to X4 HIV-1-induced apoptosis despite infection Consistent with this we observed significant induction of several genes involved in negative selection ofDP thymocytes Furthermore treatment of thymocytes with cycloheximide abrogated HIV-1-induced apoptosis implying a requirement for denovo protein synthesis Our results suggest that HIV-1-induced apoptosis of thymocytes requires the activation of caspases and the participation ofmitochondrial apoptosis effectors which serve to amplify the apoptotic signal a process similar to that elaborated during thymocyte negativeselectioncopy 2006 Elsevier Inc All rights reserved

Keywords HIV-1 Thymocyte Apoptosis Negative selection

Introduction

HIV-1 infection is characterized by progressive depletion ofCD4+ T cells immune dysfunction and eventually suscepti-bility to opportunistic infections and malignancies HIV-1evades immune defenses in part by inducing the destruction ofboth infected and uninfected cells of the immune system(Gandhi et al 1998 Grivel et al 2000 Grivel and Margolis1999 Holm and Gabuzda 2005 Jekle et al 2003 Miura et al2001 Muro-Cacho et al 1995 Su et al 1995 Yue et al

Corresponding author Fax +1 949 824 8551E-mail address dcamerinuciedu (D Camerini)

1 Current address University of North Carolina at Chapel Hill NorthCarolina NC 27599-7435 USA

0042-6822$ - see front matter copy 2006 Elsevier Inc All rights reserveddoi101016jvirol200604037

2005) HIV-1-mediated apoptosis is largely responsible forCD4+ T cell death although necrotic cell death has also beenreported (Finkel et al 1995 Jamieson et al 1997 Jekle et al2003 Lenardo et al 2002 Muro-Cacho et al 1995 Su et al1995 Yue et al 2005 Zhang et al 1998) The hosts inabilityto adequately replace CD4+ T cells further contributes to thedecline in this essential lymphocyte population (Guadalupeet al 2003 Schacker et al 2002 Stanley et al 1993 Su et al1995)

The thymus is the primary lymphoid organ where T cells aregenerated during fetal and neonatal development In adultshowever homeostatic proliferation of peripheral T cells may besufficient to maintain normal T cell numbers Nevertheless thethymus functions throughout life although at reduced levels inaged individuals and responds to T cell depletion induced byHIV-1 (Douek et al 1998 Jamieson et al 1999 Kalayjian

269SK Choudhary et al Virology 352 (2006) 268ndash284

et al 2005 Poulin et al 1999) HIV-1 infection of thethymus particularly of infants has been documented and isfrequently associated with rapid progression to AIDS (Kourtiset al 1996 Nahmias et al 1998) HIV-1 infection of thethymus causes thymic dysfunction reduced thymic volume andthymocyte depletion in children and adults (Gaulton et al1997 Rosenzweig et al 1993 Vigano et al 2000) BothCXCR4 tropic (X4) and CCR5 tropic (R5) HIV-1 strainsreplicate and deplete thymocytes in severe combine immuno-deficient mice bearing human fetal thymusliver grafts (SCID-hu ThyLiv mice) and in fetal thymic organ culture (FTOC)however X4 HIV-1 is more cyotopathic in these models thanR5 HIV-1 (Berkowitz et al 1998a Bonyhadi et al 1995Camerini et al 2000 Choudhary et al 2005 Duus et al 2001Scoggins et al 2000) Berkowitz et al have shown that variousdevelopmental subsets within the thymus including immature(CD3minus CD4+ CD8minus) intrathymic T progenitor (ITTP) cellsexpress CXCR4 and are therefore targets for X4 HIV-1infection and depletion (Berkowitz et al 1998b Su et al1995) In contrast fewer than 5 of thymocytes express CCR5(Taylor et al 2001) The integrity of the thymic microenviron-ment is essential for T cell development perturbation of thisenvironment by HIV-1 infection of the thymus may causeprofound effects on T cell development and function (Stanley etal 1993 Su et al 1995) HIV-1 infection of thymus-liver(ThyLiv) grafts in SCID mice causes depletion of hematopoi-etic progenitor cells and destruction of thymic epithelial cells(Jenkins et al 1998 Koka et al 1998 Stanley et al 1993)Moreover progenitor cells derived from infants born to HIV-positive mothers have decreased cloning efficiency and generatefewer T cells in FTOC This impaired progenitor cell functionmay be responsible at least in part for lower naive CD4+ T cellcounts and reduced thymic output in HIV-1-negative infants ofHIV-1-positive mothers (Nielsen et al 2001)

The mechanisms by which HIV-1 causes thymocytedepletion are not well known and warrant further systematicstudy Both direct and indirect killing of thymocytes have beenreported in infected SCID-hu ThyLiv organs (Bonyhadi et al1993 Choudhary and Camerini 2006 Jamieson et al 1997Stanley et al 1993 Su et al 1995) Thymocytes withcondensed nuclei fragmented DNA and partial chromosomalloss were observed in HIV-1-infected ThyLiv organs but not inuninfected organs (Bonyhadi et al 1993 1995 Kaneshima etal 1994 Su et al 1995) Jamieson et al (1997) observed arapid phase of CD4+ thymocyte depletion during the peak ofviral replication suggesting direct killing of HIV-1-infectedthymocytes but they observed few apoptotic cells Su et al(1995) in contrast observed apoptotic cell death mostly inuninfected thymocytes arguing for indirect bystander killingMoreover the nature of the signals that initiate HIV-1-mediatedapoptosis in the thymus are not known We infected thymocyteswith the X4 HIV-1 clone NL4-3 at several multiplicities ofinfection (MOI) and observed that NL4-3 induced apoptosisprimarily in productively infected thymocytes aldrithiol (AT-2)-mediated inactivation of HIV-1 or treatment of thymocytecultures with the reverse transcriptase inhibitor Efavirenzlargely abrogated HIV-1-induced apoptosis Moreover imma-

ture CD4+ CD8+ (DP) thymocytes were primary targets of X4-HIV-1-induced apoptosis whereas most mature CD4+ singlepositive (SP) and CD8 SP thymocytes were resistant to X4 HIV-1-induced apoptosis suggesting that infection of maturethymocytes may contribute to the generation of latently infectednaive T cells (Brooks et al 2003 2001) HIV-1 infection ofthymocytes induced a number of genes that have previouslybeen implicated in the apoptotic demise of DP thymocytesduring negative selection including Nurr1 and T cell death-associated gene 8 (TDAG8) (Cheng et al 1997 Tosa et al2003 Woronicz et al 1994 Zhou et al 1996) Our resultssuggest that HIV-1-mediated apoptosis of DP thymocytesinvolves more than one apoptotic signaling pathway andresembles the apoptotic processes elaborated during negativeselection of thymocytes Our results also demonstrate thatmature CD4 SP CD27+ thymocytes are resistant to HIV-1-mediated apoptosis defining a potential source of latentlyinfected naive T cells

Results

HIV-1 induced apoptosis in thymocytes

Thymocytes were isolated from fetal thymus and wereinfected with the X4 HIV-1 molecular clone NL4-3 at amultiplicity of infection (MOI) of 03 based on the titer of theviral stocks measured in activated PBMC Infection ofthymocytes with NL4-3 at this MOI routinely resulted ininfection of more than 80 of thymocytes when measured byinternal staining of gag protein p24 using the MAb KC57-FITC We routinely observed an approximately three-foldhigher infection rate in thymocytes compared to PBMC whichvaried less than 10 dependent upon donor tissue Apoptosiswas measured 24 h post-infection by staining of cells with anantibody to active caspase-3 by quantification of DNAfragmentation using terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) analysis and by determi-nation of phosphatidylserine (PS) externalization by annexin Vbinding in conjunction with 7-AAD staining This time periodwas sufficient for only a single round of infection Weobserved that NL4-3 infection induced pronounced andsignificant activation of caspase-3 in thymocytes (P lt 0001)an average of 72 of thymocytes were anti-active caspase-3reactive following NL4-3 infection whereas in mock-infectedthymocytes only 27 on average were active caspase-3+ (Figs1A and D) Active caspase-3 can cleave many substrates thatare vital for cell survival including anti-apoptotic regulatorsprotein kinases and other signal transduction regulatorscytosolic and nuclear structural proteins repair factors andcell cycle regulators The cleavage of the inhibitor of caspase-activated DNase (ICAD) leads to the release of active CADand DNA fragmentation We measured DNA fragmentation byTUNEL assay which labels DNA strand breaks with FITC-dUTP followed by flow cytometric analysis NL4-3 infectionof thymocytes induced DNA fragmentation approximately80 of thymocytes were TUNEL+ 24 h after NL4-3 infectionwhereas only 32 were TUNEL+ after mock infection (Fig

Fig 1 HIV-1 induced apoptosis in thymocytes Thymocytes were infected centrifugally (spin-infected) with NL4-3 at multiplicity of infection (MOI) of 03 to get 50ndash80 cells infected or were mock infected with media alone Thymocytes were treated with etoposide (50 μM) for 20 h in parallel to induce apoptosis for a comparison(A) Representative histograms of one of four experiments showing that HIV-1 infection induced activation of the executioner caspase caspase-3 at 24 h post-infection(B) Representative dot-plots of one of two experiments showing HIV-1-induced chromatin fragmentation quantified by TUNEL at 24 h post-infection (C)Representative dot-plots of one of four experiments showing that HIV-1 caused translocation of phosphatidylserine (PS) to outer leaflets of the plasma membrane at 24and 48 h post-infection (D) Average percentage of active caspase-3+ thymocytes at 24 h post-infection with error bars indicating the standard deviation of four separateexperiments done in duplicate (E) Average percentage of annexin V+ 7-AADminus thymocytes 24 h post-infection with error bars indicating the standard deviation of fourseparate experiments done in duplicate

270 SK Choudhary et al Virology 352 (2006) 268ndash284

Fig 2 The CXCR4 antagonist AMD3100 abrogated HIV-1-induced apoptosisof thymocytes Thymocytes were pre-treated with AMD3100 (1 μM) for 2 h andthen spin-infected with either NL4-3 (MOI of 03) or mock infected with mediaalone AMD3100 was maintained in the cultures throughout the course of theexperiment Thymocyte apoptosis was measured 24 h post-infection by annexinV binding to exposed PS (A) and activation of caspase-3 (B) Results shown arerepresentative of two experiments each done in triplicate with error barsindicating standard deviations

271SK Choudhary et al Virology 352 (2006) 268ndash284

1B) We also measured PS exposure a marker for phagocytosisof apoptotic cells using annexin V-PE binding in conjunctionwith 7-AAD to distinguish early apoptotic cells from non-viable cells Thymocytes that stained positive for annexin V-PEand negative for 7-AAD were actively undergoing apoptosiswhereas those that stained positive for both annexin V-PE and7-AAD were either at the end stage of apoptosis or undergoingnecrotic cell death We observed a significant increase inannexin V-PE+ 7-AADminus thymocytes at 24 h post-infection(P lt 0001) in mock-infected cultures 8 of thymocytes wereannexin V-PE+ 7-AADminus (average of four experiments) thisincreased to an average of 28 in HIV-1-infected thymocytes(Figs 1C and E) Forty-eight hours post-infection howeverthe annexin V-PE+ 7-AADminus population appeared to transit tothe annexin V-PE+ 7-AAD+ quadrant suggesting that the deathprocess had completed in these cells Because we generatedhigh titer NL4-3 stocks by amplifying the virus in CEM-GFPcells it was formally possible that factors released from theseproducer cells into the culture media might contribute to theapoptosis observed To rule out this possibility we infectedthymocytes in the presence or absence of AMD3100 anantagonist of the CXCR4 chemokine receptor which blocksbinding of HIV-1 to CXCR4 and thus blocks entry of X4 HIV-1 (De Clercq et al 1994) AMD3100 completely abrogatedNL4-3-induced apoptosis in thymocytes (Fig 2) Similarly thefusion inhibitor T-20 also completely abrogated HIV-1-inducedapoptosis (Figs 3E and F) indicating that apoptosis was HIV-1dependent and not due to cytokines or other factors releasedduring production of HIV-1 in CEM-GFP cells

We observed similar results when thymic grafts from SCID-hu ThyLiv mice were used Because we can implant 30ndash40mice with tissue of single donor use of these grafts allowed usto compensate for donor variation and this approach was used insubsequent experiments Only one graft or single fetal thymuswas used for each experiment Because anti-active caspase-3staining and annexin V-PE staining provided reliable quantifi-cation of apoptosis and also permitted four color flowcytometry these assays were used in all subsequentexperiments

HIV-1-induced apoptosis in uninfected and infected thymocytes

Thymocytes were infected with NL4-3 at different MOI andstained for internal p24 and active caspase-3 simultaneously toassay HIV-1-induced apoptosis in infected and uninfectedthymocytes At high MOI we observed that the majority ofthymocytes undergoing apoptosis were infected (Fig 3A) Atlow MOI however apoptosis was observed in cells that werenot expressing detectable levels of p24 This can also beinferred from the non-linear relationship of the percent ofinfected thymocytes to the percent apoptotic thymocytes (Fig3B) This may result from the limit of internal p24 detection byflow cytometry Alternatively thymocytes may be undergoingbystander apoptosis induced by HIV-1 gp120 gp41 or othermolecules on the virion surface Infection at the lowest MOIused (001) resulted in a viral particle to cell ratio of at least 12considering that each HIV-1 virion contains 2000ndash3600 p24

molecules and the viral stocks used had a p24 concentration of1 μgml (Singh et al 2000) This result is therefore consistentwith induction of apoptosis by contact with virions followinglow MOI infection Moreover defective virions which arelikely more abundant than replication competent virions mightsuffice to induce apoptosis

To investigate the contributions of direct and indirect killingby HIV-1 we pre-incubated thymocytes overnight with orwithout Efavirenz and then infected with the X4 HIV-1 cloneNL4-3 or mock infected We observed a significant decrease inHIV-1-mediated thymocyte apoptosis in the presence ofEfavirenz by both active caspase-3 staining (P lt 001) andannexin V binding (P lt 001) approximately 65 fewerthymocytes underwent HIV-1-specific apoptosis 24 h afterNL4-3 infection in the presence of Efavirenz when compared tountreated cultures as measured by both caspase-3 activation andannexin V binding (Figs 3C and D) Efavirenz treatment ofmock-infected cultures however induced cell death inapproximately 7 of thymocytes measured by annexin Vbinding which was significant (P lt 001) Efavirenz treatmentof thymocytes in this experiment yielded a fifty-fold inhibition

Fig 1 HIV-1 induced apoptosis in thymocytes Thymocytes were infected centrifugally (spin-infected) with NL4-3 at multiplicity of infection (MOI) of 03 to get 50ndash80 cells infected or were mock infected with media alone Thymocytes were treated with etoposide (50 μM) for 20 h in parallel to induce apoptosis for a comparison(A) Representative histograms of one of four experiments showing that HIV-1 infection induced activation of the executioner caspase caspase-3 at 24 h post-infection(B) Representative dot-plots of one of two experiments showing HIV-1-induced chromatin fragmentation quantified by TUNEL at 24 h post-infection (C)Representative dot-plots of one of four experiments showing that HIV-1 caused translocation of phosphatidylserine (PS) to outer leaflets of the plasma membrane at 24and 48 h post-infection (D) Average percentage of active caspase-3+ thymocytes at 24 h post-infection with error bars indicating the standard deviation of four separateexperiments done in duplicate (E) Average percentage of annexin V+ 7-AADminus thymocytes 24 h post-infection with error bars indicating the standard deviation of fourseparate experiments done in duplicate

270 SK Choudhary et al Virology 352 (2006) 268ndash284

Fig 2 The CXCR4 antagonist AMD3100 abrogated HIV-1-induced apoptosisof thymocytes Thymocytes were pre-treated with AMD3100 (1 μM) for 2 h andthen spin-infected with either NL4-3 (MOI of 03) or mock infected with mediaalone AMD3100 was maintained in the cultures throughout the course of theexperiment Thymocyte apoptosis was measured 24 h post-infection by annexinV binding to exposed PS (A) and activation of caspase-3 (B) Results shown arerepresentative of two experiments each done in triplicate with error barsindicating standard deviations

271SK Choudhary et al Virology 352 (2006) 268ndash284

1B) We also measured PS exposure a marker for phagocytosisof apoptotic cells using annexin V-PE binding in conjunctionwith 7-AAD to distinguish early apoptotic cells from non-viable cells Thymocytes that stained positive for annexin V-PEand negative for 7-AAD were actively undergoing apoptosiswhereas those that stained positive for both annexin V-PE and7-AAD were either at the end stage of apoptosis or undergoingnecrotic cell death We observed a significant increase inannexin V-PE+ 7-AADminus thymocytes at 24 h post-infection(P lt 0001) in mock-infected cultures 8 of thymocytes wereannexin V-PE+ 7-AADminus (average of four experiments) thisincreased to an average of 28 in HIV-1-infected thymocytes(Figs 1C and E) Forty-eight hours post-infection howeverthe annexin V-PE+ 7-AADminus population appeared to transit tothe annexin V-PE+ 7-AAD+ quadrant suggesting that the deathprocess had completed in these cells Because we generatedhigh titer NL4-3 stocks by amplifying the virus in CEM-GFPcells it was formally possible that factors released from theseproducer cells into the culture media might contribute to theapoptosis observed To rule out this possibility we infectedthymocytes in the presence or absence of AMD3100 anantagonist of the CXCR4 chemokine receptor which blocksbinding of HIV-1 to CXCR4 and thus blocks entry of X4 HIV-1 (De Clercq et al 1994) AMD3100 completely abrogatedNL4-3-induced apoptosis in thymocytes (Fig 2) Similarly thefusion inhibitor T-20 also completely abrogated HIV-1-inducedapoptosis (Figs 3E and F) indicating that apoptosis was HIV-1dependent and not due to cytokines or other factors releasedduring production of HIV-1 in CEM-GFP cells

We observed similar results when thymic grafts from SCID-hu ThyLiv mice were used Because we can implant 30ndash40mice with tissue of single donor use of these grafts allowed usto compensate for donor variation and this approach was used insubsequent experiments Only one graft or single fetal thymuswas used for each experiment Because anti-active caspase-3staining and annexin V-PE staining provided reliable quantifi-cation of apoptosis and also permitted four color flowcytometry these assays were used in all subsequentexperiments

HIV-1-induced apoptosis in uninfected and infected thymocytes

Thymocytes were infected with NL4-3 at different MOI andstained for internal p24 and active caspase-3 simultaneously toassay HIV-1-induced apoptosis in infected and uninfectedthymocytes At high MOI we observed that the majority ofthymocytes undergoing apoptosis were infected (Fig 3A) Atlow MOI however apoptosis was observed in cells that werenot expressing detectable levels of p24 This can also beinferred from the non-linear relationship of the percent ofinfected thymocytes to the percent apoptotic thymocytes (Fig3B) This may result from the limit of internal p24 detection byflow cytometry Alternatively thymocytes may be undergoingbystander apoptosis induced by HIV-1 gp120 gp41 or othermolecules on the virion surface Infection at the lowest MOIused (001) resulted in a viral particle to cell ratio of at least 12considering that each HIV-1 virion contains 2000ndash3600 p24

molecules and the viral stocks used had a p24 concentration of1 μgml (Singh et al 2000) This result is therefore consistentwith induction of apoptosis by contact with virions followinglow MOI infection Moreover defective virions which arelikely more abundant than replication competent virions mightsuffice to induce apoptosis

To investigate the contributions of direct and indirect killingby HIV-1 we pre-incubated thymocytes overnight with orwithout Efavirenz and then infected with the X4 HIV-1 cloneNL4-3 or mock infected We observed a significant decrease inHIV-1-mediated thymocyte apoptosis in the presence ofEfavirenz by both active caspase-3 staining (P lt 001) andannexin V binding (P lt 001) approximately 65 fewerthymocytes underwent HIV-1-specific apoptosis 24 h afterNL4-3 infection in the presence of Efavirenz when compared tountreated cultures as measured by both caspase-3 activation andannexin V binding (Figs 3C and D) Efavirenz treatment ofmock-infected cultures however induced cell death inapproximately 7 of thymocytes measured by annexin Vbinding which was significant (P lt 001) Efavirenz treatmentof thymocytes in this experiment yielded a fifty-fold inhibition

Fig 2 The CXCR4 antagonist AMD3100 abrogated HIV-1-induced apoptosisof thymocytes Thymocytes were pre-treated with AMD3100 (1 μM) for 2 h andthen spin-infected with either NL4-3 (MOI of 03) or mock infected with mediaalone AMD3100 was maintained in the cultures throughout the course of theexperiment Thymocyte apoptosis was measured 24 h post-infection by annexinV binding to exposed PS (A) and activation of caspase-3 (B) Results shown arerepresentative of two experiments each done in triplicate with error barsindicating standard deviations

271SK Choudhary et al Virology 352 (2006) 268ndash284

1B) We also measured PS exposure a marker for phagocytosisof apoptotic cells using annexin V-PE binding in conjunctionwith 7-AAD to distinguish early apoptotic cells from non-viable cells Thymocytes that stained positive for annexin V-PEand negative for 7-AAD were actively undergoing apoptosiswhereas those that stained positive for both annexin V-PE and7-AAD were either at the end stage of apoptosis or undergoingnecrotic cell death We observed a significant increase inannexin V-PE+ 7-AADminus thymocytes at 24 h post-infection(P lt 0001) in mock-infected cultures 8 of thymocytes wereannexin V-PE+ 7-AADminus (average of four experiments) thisincreased to an average of 28 in HIV-1-infected thymocytes(Figs 1C and E) Forty-eight hours post-infection howeverthe annexin V-PE+ 7-AADminus population appeared to transit tothe annexin V-PE+ 7-AAD+ quadrant suggesting that the deathprocess had completed in these cells Because we generatedhigh titer NL4-3 stocks by amplifying the virus in CEM-GFPcells it was formally possible that factors released from theseproducer cells into the culture media might contribute to theapoptosis observed To rule out this possibility we infectedthymocytes in the presence or absence of AMD3100 anantagonist of the CXCR4 chemokine receptor which blocksbinding of HIV-1 to CXCR4 and thus blocks entry of X4 HIV-1 (De Clercq et al 1994) AMD3100 completely abrogatedNL4-3-induced apoptosis in thymocytes (Fig 2) Similarly thefusion inhibitor T-20 also completely abrogated HIV-1-inducedapoptosis (Figs 3E and F) indicating that apoptosis was HIV-1dependent and not due to cytokines or other factors releasedduring production of HIV-1 in CEM-GFP cells

We observed similar results when thymic grafts from SCID-hu ThyLiv mice were used Because we can implant 30ndash40mice with tissue of single donor use of these grafts allowed usto compensate for donor variation and this approach was used insubsequent experiments Only one graft or single fetal thymuswas used for each experiment Because anti-active caspase-3staining and annexin V-PE staining provided reliable quantifi-cation of apoptosis and also permitted four color flowcytometry these assays were used in all subsequentexperiments

HIV-1-induced apoptosis in uninfected and infected thymocytes

Thymocytes were infected with NL4-3 at different MOI andstained for internal p24 and active caspase-3 simultaneously toassay HIV-1-induced apoptosis in infected and uninfectedthymocytes At high MOI we observed that the majority ofthymocytes undergoing apoptosis were infected (Fig 3A) Atlow MOI however apoptosis was observed in cells that werenot expressing detectable levels of p24 This can also beinferred from the non-linear relationship of the percent ofinfected thymocytes to the percent apoptotic thymocytes (Fig3B) This may result from the limit of internal p24 detection byflow cytometry Alternatively thymocytes may be undergoingbystander apoptosis induced by HIV-1 gp120 gp41 or othermolecules on the virion surface Infection at the lowest MOIused (001) resulted in a viral particle to cell ratio of at least 12considering that each HIV-1 virion contains 2000ndash3600 p24

molecules and the viral stocks used had a p24 concentration of1 μgml (Singh et al 2000) This result is therefore consistentwith induction of apoptosis by contact with virions followinglow MOI infection Moreover defective virions which arelikely more abundant than replication competent virions mightsuffice to induce apoptosis

To investigate the contributions of direct and indirect killingby HIV-1 we pre-incubated thymocytes overnight with orwithout Efavirenz and then infected with the X4 HIV-1 cloneNL4-3 or mock infected We observed a significant decrease inHIV-1-mediated thymocyte apoptosis in the presence ofEfavirenz by both active caspase-3 staining (P lt 001) andannexin V binding (P lt 001) approximately 65 fewerthymocytes underwent HIV-1-specific apoptosis 24 h afterNL4-3 infection in the presence of Efavirenz when compared tountreated cultures as measured by both caspase-3 activation andannexin V binding (Figs 3C and D) Efavirenz treatment ofmock-infected cultures however induced cell death inapproximately 7 of thymocytes measured by annexin Vbinding which was significant (P lt 001) Efavirenz treatmentof thymocytes in this experiment yielded a fifty-fold inhibition

Fig 3 HIV-1 induced apoptosis in uninfected and infected thymocytes (A) Thymocytes were infected with NL4-3 at different multiplicities of infection (MOI) asindicated Twenty-four hours post-infection thymocytes were fixed permeabilized and incubated with anti-HIV-1 p24-FITC and anti-active caspase-3-PE to quantifyinfected thymocytes as well as cells undergoing apoptosis respectively Dot plots are representative of two experiments done in duplicate (B) Duplicates from eachexperiment were averaged background was subtracted to plot specific active caspase-3+ thymocytes against the percentage of infected (internal p24+) thymocytes Anon-linear correlation was observed (C D) Thymocytes were pre-incubated with Efavirenz (10 μM) overnight and subsequently infected with NL4-3 (MOI of 015) ormock infected Efavirenz was maintained in the cultures throughout the experiment Thymocytes were also infected with aldrithiol (AT-2)-treated NL4-3 or AT-2-treated media as described in Materials and methods Apoptosis in thymocytes was measured 24 h post-infection by annexin V binding to exposed PS (C) andactivation of caspase-3 (D) The bars represent the average of two experiments done in triplicate (E F) Thymocytes were pre-incubated with T-20 (10 μgml) for 2 hand subsequently infected with NL4-3 (MOI of 015) or mock infected Thymocyte apoptosis was measured 24 h post-infection by annexin V binding to exposed PS(E) and activation of caspase-3 (F) Results shown are the average of three experiments each done in triplicate with error bars indicating standard deviations

272 SK Choudhary et al Virology 352 (2006) 268ndash284

of viral reverse transcription as measured by real-time PCR(data not shown) Therefore it is likely that the low level ofresidual apoptosis in the presence of Efavirenz was due to a lowlevel of productive infection as well as the toxicity of the drugitself Nevertheless thymocyte apoptosis induced by HIV-1

infection in the presence of Efavirenz was not statisticallysignificant when compared to Efavirenz-treated mock-infectedcultures We also inactivated HIV-1 using AT-2 which has beenpreviously shown to completely inactivate HIV-1 withoutcompromising the conformational and functional integrity of

Table 1Changes in gene expression of apoptosis and DNA damage repair genes inNL4-3-infected thymocytes compared to mock-infected thymocytes

Locuslinkaccession

Gene description Fold change a

4 h 12 h 24 h

8795 TRAIL receptor-2 death receptor 5 b 09 58 32

10161 Purinergic receptor P2Y (P2RY5) 14 39 32

1647 Growth arrest and DNAdamage-inducible alpha (GADD45α)

12 31 24

355 Fas 12 27 19

8870 Immediate early response 3 (IER3)IEX-1 b

13 27 21

330 Baculoviral IAP repeat-containing3 (BIRC3) b

15 25 22

4929 Nuclear receptor subfamily 4group A member 2 (NR4A2) NURR1b

17 22 20

8477 G protein-coupled receptor 65(GPR65) T cell death-associatedgene 8 (TDAG8)

14 22 17

9448 Mitogen-activated protein kinasekinase kinase kinase 4 (MAP4K4)HPKGCK-like kinase

09 2 18

5604 Mitogen-activated protein kinasekinase 1 MEK1

1 15 18

5359 Phospholipid scramblase 1 (PLSCR1) 14 2 23

54739 XIAP-associated factor-1 (XAF1) 13 14 25

598 BCL2-like 1 (BCL2L1) Bcl-XL 09 07 05

a Data shown are average of three experiments The numbers of samples usedto obtain the data at 4 12 and 24 h post-infection respectively are as followsn = 2 2 and 3b Denotes genes confirmed by real-time quantitative RT-PCR Statistical

significance as measured by the Cyber-T software (wwwgenomicsbiochemucieduCyberT Baldi and Long 2001) P lt 005

P lt 0005

273SK Choudhary et al Virology 352 (2006) 268ndash284

virion surface proteins (Rossio et al 1998) AT-2 treatment ofvirus resulted in protection of thymocytes from virus-inducedapoptosis (P lt 001 both for annexin V binding and caspase-3activation) a 70 reduction in HIV-1-specific annexin Vbinding and 47 reduction in specific active caspase-3-reactivethymocytes was observed (Figs 3C and D) AT-2 treatmentalone however also increased apoptosis mildly in mock-infected thymocytes but this was not significant whencompared to untreated mock-infected cells or AT-2-treatedNL4-3-infected thymocytes The low level of apoptosis causedby AT-2-treated NL4-3 or AT-2-treated mock infection waslikely due to residual AT-2 left in medium following removal byultrafiltration using a centrifugal filter device (see Materials andmethods) In contrast incubation of thymocytes with the fusioninhibitor T-20 completely abrogated HIV-1-induced apoptosis(Figs 3E and F P lt 001) We did not observe a significantdifference in apoptosis in T-20-treated NL4-3-infected thymo-cytes when compared to either untreated mock-infected or T-20-treated mock-infected thymocytes These experiments collec-tively suggest that HIV-1-induced apoptosis occurred primarilyin productively infected thymocytes This was further con-firmed by the fact that incubation of thymocytes with a similartiter of the R5 HIV-1 molecular clone JR-CSF did not causeapoptosis presumably because less than 5 of thymocytesexpress CCR5 and therefore fewer than 5 of the cells could beinfected with the R5 HIV-1 clone JR-CSF (Taylor et al 2001)

HIV-1 infection of thymocytes induced apoptotic genesinvolved in negative selection of thymocytes

To gain better insight into how HIV-1 promotes apoptosis inthymocytes we used a genomics-based approach We infectedthymocytes with HIV-1 clone NL4-3 at an MOI sufficient toinfect 60ndash90 of the thymocytes RNA was isolated at 4 12and 24 h post-infection and was used to probe AffymetrixU133A human genome chips We observed significantinduction of numerous genes involved in cell-cycle regulationapoptosis transcription cell signaling subcellular traffickingas well as cytokine and MHC genes (data not shown) We foundthat several genes that have been previously implicated inthymocyte apoptosis were significantly induced (Table 1)Nurr1 an orphan member of the steroid receptor family thatincludes Nur77 and NOR1 was significantly induced Thisresult was also confirmed by real-time quantitative RT-PCR atall the three time points Interestingly Nurr1 and Nur77 havebeen implicated in redundantly inducing apoptosis in DPthymocytes during negative selection (Cheng et al 1997Woronicz et al 1994 Zhou et al 1996) TDAG8 whichpreviously has been shown to induce apoptosis in DPthymocytes in transgenic mice was also induced by HIV-1infection (Tosa et al 2003) TDAG8 induces DP thymocyteapoptosis in a dexamethasone-dependent manner and promotesthe activation of caspases-3 -8 and -9 (Malone et al 2004 Tosaet al 2003) In contrast baculoviral inhibitor of apoptosis (IAP)repeat containing protein 3 (BIRC3) of the X-linked IAP family(XIAP) which blocks the active site of caspases and thereforeblocks apoptosis was also induced by X4 HIV-1 infection

(Huang et al 2003 Liston et al 1996 Vucic et al 2005)Expression of XAF1 which antagonizes the anti-caspaseactivity of XIAP was induced whereas BCL-2-like gene 1(Bcl-XL) which blocks cell death by inhibiting caspase-9activation and antagonizing pro-apoptotic members of the Bcl-2family was downregulated It is possible that these inducedgenes may control the fate of HIV-1-infected thymocytes

The c-Jun N-terminal kinase (JNK) is another key proteinimplicated in the apoptosis of DP thymocytes (Behrens et al2001 Rincon et al 1998 Sabapathy et al 1999) MAP4K4and GADD45α which can initiate a JNK activation cascadewere both induced in HIV-1-infected thymocytes (Mita et al2002 Takekawa and Saito 1998) GADD45α has recently beenshown to be induced by HIV-1 infection of PBMC andmacrophages and mediates Vpr-induced apoptosis (Andersen etal 2005 Coberley et al 2004) Among the death receptorfamily both Fas and TRAIL-R2 (DR5) were induced Amoderate increase in TRAIL was also observed (data notshown) a finding which may be important in light of thefindings of Lamhamedi-Cherradi et al (2003) that TRAIL-deficient mouse thymocytes are refractory to negative selectionAlthough the role of TRAIL in promoting DP-negativeselection is controversial these findings indicate that bothextrinsic and intrinsic apoptotic pathways may be active in

Table 1Changes in gene expression of apoptosis and DNA damage repair genes inNL4-3-infected thymocytes compared to mock-infected thymocytes

Locuslinkaccession

Gene description Fold change a

4 h 12 h 24 h

8795 TRAIL receptor-2 death receptor 5 b 09 58 32

10161 Purinergic receptor P2Y (P2RY5) 14 39 32

1647 Growth arrest and DNAdamage-inducible alpha (GADD45α)

12 31 24

355 Fas 12 27 19

8870 Immediate early response 3 (IER3)IEX-1 b

13 27 21

330 Baculoviral IAP repeat-containing3 (BIRC3) b

15 25 22

4929 Nuclear receptor subfamily 4group A member 2 (NR4A2) NURR1b

17 22 20

8477 G protein-coupled receptor 65(GPR65) T cell death-associatedgene 8 (TDAG8)

14 22 17

9448 Mitogen-activated protein kinasekinase kinase kinase 4 (MAP4K4)HPKGCK-like kinase

09 2 18

5604 Mitogen-activated protein kinasekinase 1 MEK1

1 15 18

5359 Phospholipid scramblase 1 (PLSCR1) 14 2 23

54739 XIAP-associated factor-1 (XAF1) 13 14 25

598 BCL2-like 1 (BCL2L1) Bcl-XL 09 07 05

a Data shown are average of three experiments The numbers of samples usedto obtain the data at 4 12 and 24 h post-infection respectively are as followsn = 2 2 and 3b Denotes genes confirmed by real-time quantitative RT-PCR Statistical

significance as measured by the Cyber-T software (wwwgenomicsbiochemucieduCyberT Baldi and Long 2001) P lt 005

P lt 0005

273SK Choudhary et al Virology 352 (2006) 268ndash284

virion surface proteins (Rossio et al 1998) AT-2 treatment ofvirus resulted in protection of thymocytes from virus-inducedapoptosis (P lt 001 both for annexin V binding and caspase-3activation) a 70 reduction in HIV-1-specific annexin Vbinding and 47 reduction in specific active caspase-3-reactivethymocytes was observed (Figs 3C and D) AT-2 treatmentalone however also increased apoptosis mildly in mock-infected thymocytes but this was not significant whencompared to untreated mock-infected cells or AT-2-treatedNL4-3-infected thymocytes The low level of apoptosis causedby AT-2-treated NL4-3 or AT-2-treated mock infection waslikely due to residual AT-2 left in medium following removal byultrafiltration using a centrifugal filter device (see Materials andmethods) In contrast incubation of thymocytes with the fusioninhibitor T-20 completely abrogated HIV-1-induced apoptosis(Figs 3E and F P lt 001) We did not observe a significantdifference in apoptosis in T-20-treated NL4-3-infected thymo-cytes when compared to either untreated mock-infected or T-20-treated mock-infected thymocytes These experiments collec-tively suggest that HIV-1-induced apoptosis occurred primarilyin productively infected thymocytes This was further con-firmed by the fact that incubation of thymocytes with a similartiter of the R5 HIV-1 molecular clone JR-CSF did not causeapoptosis presumably because less than 5 of thymocytesexpress CCR5 and therefore fewer than 5 of the cells could beinfected with the R5 HIV-1 clone JR-CSF (Taylor et al 2001)

HIV-1 infection of thymocytes induced apoptotic genesinvolved in negative selection of thymocytes

To gain better insight into how HIV-1 promotes apoptosis inthymocytes we used a genomics-based approach We infectedthymocytes with HIV-1 clone NL4-3 at an MOI sufficient toinfect 60ndash90 of the thymocytes RNA was isolated at 4 12and 24 h post-infection and was used to probe AffymetrixU133A human genome chips We observed significantinduction of numerous genes involved in cell-cycle regulationapoptosis transcription cell signaling subcellular traffickingas well as cytokine and MHC genes (data not shown) We foundthat several genes that have been previously implicated inthymocyte apoptosis were significantly induced (Table 1)Nurr1 an orphan member of the steroid receptor family thatincludes Nur77 and NOR1 was significantly induced Thisresult was also confirmed by real-time quantitative RT-PCR atall the three time points Interestingly Nurr1 and Nur77 havebeen implicated in redundantly inducing apoptosis in DPthymocytes during negative selection (Cheng et al 1997Woronicz et al 1994 Zhou et al 1996) TDAG8 whichpreviously has been shown to induce apoptosis in DPthymocytes in transgenic mice was also induced by HIV-1infection (Tosa et al 2003) TDAG8 induces DP thymocyteapoptosis in a dexamethasone-dependent manner and promotesthe activation of caspases-3 -8 and -9 (Malone et al 2004 Tosaet al 2003) In contrast baculoviral inhibitor of apoptosis (IAP)repeat containing protein 3 (BIRC3) of the X-linked IAP family(XIAP) which blocks the active site of caspases and thereforeblocks apoptosis was also induced by X4 HIV-1 infection

(Huang et al 2003 Liston et al 1996 Vucic et al 2005)Expression of XAF1 which antagonizes the anti-caspaseactivity of XIAP was induced whereas BCL-2-like gene 1(Bcl-XL) which blocks cell death by inhibiting caspase-9activation and antagonizing pro-apoptotic members of the Bcl-2family was downregulated It is possible that these inducedgenes may control the fate of HIV-1-infected thymocytes

The c-Jun N-terminal kinase (JNK) is another key proteinimplicated in the apoptosis of DP thymocytes (Behrens et al2001 Rincon et al 1998 Sabapathy et al 1999) MAP4K4and GADD45α which can initiate a JNK activation cascadewere both induced in HIV-1-infected thymocytes (Mita et al2002 Takekawa and Saito 1998) GADD45α has recently beenshown to be induced by HIV-1 infection of PBMC andmacrophages and mediates Vpr-induced apoptosis (Andersen etal 2005 Coberley et al 2004) Among the death receptorfamily both Fas and TRAIL-R2 (DR5) were induced Amoderate increase in TRAIL was also observed (data notshown) a finding which may be important in light of thefindings of Lamhamedi-Cherradi et al (2003) that TRAIL-deficient mouse thymocytes are refractory to negative selectionAlthough the role of TRAIL in promoting DP-negativeselection is controversial these findings indicate that bothextrinsic and intrinsic apoptotic pathways may be active in

274 SK Choudhary et al Virology 352 (2006) 268ndash284

thymocytes following HIV-1 infection P2Y a purinergicreceptor which can also induce caspase-dependent apoptosisof BALBc thymocytes was induced as well (Le Stunff et al2004) We confirmed some of these HIV-1-induced genes byreal-time quantitative RT-PCR (Table 1) Considering theseresults it is apparent that numerous genes involved in apoptoticsignaling many of which are also known to promote negativeselection in response to self-peptides in the thymus wereinduced in HIV-1-infected thymocytes We conclude that HIV-1-induced death of these DP cells resembles normal thymocytedeath during negative selection

Immature double positive (DP) thymocytes were the primarytarget of HIV-1-induced apoptosis

Thymocytes were isolated infected with HIV-1 or mockinfected and apoptosis was assayed in the three major subsets ofthymocytes DP CD4SP and CD8SP Thymocytes wereincubated with fluorochrome-conjugated CD4 and CD8 MAbfollowed by internal staining for the HIV-1 capsid protein p24and active caspase-3 CD4SP DP and CD8SP thymocyte

Fig 4 NL4-3 induced apoptosis in immature thymocytes Thymocytes were spin-inTwenty-four hours post-infection thymocytes were incubated with CD8ndashPerCP andFITC and anti-active caspase-3-PE Thymocytes were gated on CD4+ CD8minus CD4+

activation Dot plots shown are representative of four experiments (C D) Thymostaining for active caspase-3 24 h post-infection

subpopulations were then separately analyzed for apoptosis andHIV-1 infection Mock infection and 24 h of culture caused lowlevels of apoptosis in CD4SP (227) and DP (117)thymocytes (Fig 4A) In contrast HIV-1 infection ofthymocytes induced apoptosis largely in infected DP thymo-cytes (Fig 4B) and in a smaller fraction of infected matureCD4SP and CD8SP thymocytes Approximately 68 ofinfected DP thymocytes underwent apoptosis as measured byactive caspase-3 staining The remaining 24 of infected DPthymocytes however were resistant to HIV-1-induced apopto-sis In contrast 33 of infected CD4SP thymocytes underwentapoptosis whereas the remaining 60 of the infected CD4SPthymocytes were resistant to HIV-1-induced apoptosis Most ofthe CD8SP cells had no active caspase-3 and only a smallfraction was weakly HIV-1 positive Mature CD4SP andCD8SP subsets arise through various stages of thymocytedifferentiation and development The CD1+ CD69+ CD27minus DPstage is a transitional stage of a common differentiation pathwaythat generates a population that upregulates CD27 down-regulates CD1 and ultimately upregulates CD45RA to give riseto functional CD4SP or CD8SP thymocytes (Plum et al 2000

fected with NL4-3 (MOI 015) or were mock infected with media alone (A B)CD4ndashAPC and then fixed permeabilized and incubated with anti-HIV-1 p24-CD8+ and CD4minus CD8+ subsets and quantified for viral infection and caspase-3cytes were incubated with CD69ndashFITC and CD27ndashAPC followed by internal

Fig 5 HIV-1-induced apoptosis in thymocytes can be abrogated bycycloheximide (CH) Thymocytes were pre-treated with CH (35 μM) for 2 hand then spin-infected with either NL4-3 (MOI of 015) or mock infected withmedia alone CH was maintained in the cultures throughout the course of theexperiment In a parallel experiment apoptosis was induced in thymocytes byetoposide (50 μM) Thymocyte apoptosis was measured 24 h post-infection byannexin V binding to exposed PS (A) and activation of caspase-3 (B) Resultsshown are the average of four experiments (mock mock + CH NL4-3 NL4-3 + CH) and two experiments (etoposide etoposide + CH) each done intriplicate with error bars indicating standard deviations

275SK Choudhary et al Virology 352 (2006) 268ndash284

Spits 2002) The few HIV-1-positive CD8SP thymocytesdetected may be the consequence of infection at an earlierstage of this differentiation process or they may result frombackground detection of the p24 antigen We observed thatHIV-1 infection induced apoptosis in approximately 40 ofCD69+ thymocytes whereas only 13 of CD27+ thymocyteswere apoptotic (Figs 4C and D) These results suggest thatimmature thymocytes were more prone to HIV-1-inducedapoptosis than mature thymocytes

HIV-1-induced apoptosis required de novo protein synthesis

De novo expression of many genes is essential for apoptosisinduction in DP thymocytes (reviewed in Ashwell et al 2000)Some of these genes are also induced by HIV-1 infection Forexample synthesis of Nurr1Nur77 and TDAG8 mRNA isessential for negative selection of DP thymocytes (Li et al2000 Tosa et al 2003) To test the necessity for de novo proteinsynthesis during HIV-1-induced thymocyte apoptosis wetreated thymocytes with the protein synthesis inhibitor cyclo-heximide Cycloheximide treatment largely abrogated HIV-1-induced apoptosis in thymocytes as measured by activecaspase-3 staining (P lt 001) and PS exposure (P lt 001) at24 h post-infection (Figs 5A and B) However treatment ofthymocytes with cycloheximide might be expected to alsoinhibit the translation of viral proteins thus interfering with thedirect killing by the virus Cycloheximide also inhibitedetoposide-induced thymocyte apoptosis showing that de novoprotein synthesis is also required for etoposide-inducedapoptosis of DP thymocytes These results collectively suggestthat HIV-1-induced apoptosis requires de novo proteinsynthesis which could include apoptotic proteins viral proteinsor both

HIV-1-induced apoptosis of thymocytes involved caspases

Caspases are known to play important roles in propagatingand executing death signals Nevertheless caspase-independentdeath pathways exist Thus we were curious to determine ifcaspase activity was necessary for HIV-1-induced death ofthymocytes Indeed many of the genes induced by HIV-1infection of thymocytes including TDAG8 and P2Y have beenimplicated in caspase-mediated cell apoptosis To assay thecontribution of caspases in HIV-1-induced apoptosis we treatedthymocytes with the broad-spectrum caspase inhibitor Z-VAD-FMK As expected treatment with Z-VAD-FMK blocked thedetection of cleaved caspase-3 (Fig 6A) More importantlyhowever it also significantly inhibited the exposure of PS asassayed by annexin V binding (Fig 6B P lt 001) Z-VAD-FMK did not affect the infectivity of NL4-3 because an averageof 54 and 53 of the thymocytes were positive for internalp24 staining in the absence or presence of Z-VAD-FMK inexperiment 1 respectively and 59 and 61 in experiment 2respectively Moreover Z-VAD-FMK treatment caused a 15ndash35 increase in p24+ active caspase-3minus CD4SP thymocytes inall four separate experiments indicating that it prevented deathof DP or CD4SP cells (data not shown) We also observed an

increase in the CD8SP thymocyte population (20ndash50) whichstained positively for p24 and negatively for active caspase-3 in2 of 4 experiments (data not shown) These data suggest thatcaspase inhibition allowed infected DP thymocytes to surviveand differentiate into mature CD4SP and CD8SP thymocyteswhich otherwise may have died at the DP stage of development

To further elucidate the involvement of caspases we treatedthymocytes with Z-VDVAD-FMK Z-IETD-FMK and Z-LEHD-FMK inhibitors of initiator caspases-2 -8 and -9respectively either alone or in combination and measured HIV-1-initiated apoptosis 24 h post-infection (Cheng et al 2004)We observed that Z-VDVAD and Z-IETD-FMK afforded

Fig 6 Thymocyte apoptosis induced by HIV-1 involves caspases Thymocytes were infected with NL4-3 (MOI = 015) in the presence or absence of the generalcaspase inhibitor Z-VAD-FMK or specific inhibitors of caspases-2 -8 -9 Z-VDVAD-FMK Z-IETD-FMK Z-LEHD-FMK respectively each at 50 μMconcentration Inhibitors were maintained in cultures throughout the course of the experiment Apoptosis was measured 24 h post-infection by activation of caspase-3(A C) or annexin V binding to exposed PS (B D) Bars represent the average of triplicate samples with error bars indicating the standard deviations Panels A and Bare representative of six experiments Panels C and D are representative of two experiments

276 SK Choudhary et al Virology 352 (2006) 268ndash284

greater protection from HIV-1-induced thymocyte apoptosisthan Z-LEHD-FMK in two experiments (data not shown)Initiator caspase inhibitors however worked better in combi-nation for blocking HIV-1-mediated thymocyte apoptosis 24 hpost-infection when compared to any of these inhibitors usedalone (Figs 6C and D) PS exposure was reduced to nearbackground levels with all combinations of these caspaseinhibitors However caspase-2 and caspase-8 inhibitors weremost efficient at inhibiting the cleavage of caspase-3 Althoughthese experiments are not sufficient to determine a pathway ofcaspase activation they provide further evidence that activationof caspases plays an important role in the apoptotic demise ofHIV-1-infected thymocytes

Many caspase cascades are initiated by ligation of deathreceptors by their respective ligands To explore the potentialinvolvement of such death receptors we incubated thymocyteswith a Fas ligand neutralizing MAb (clone NOK-1) or a TRAILneutralizing MAb (clone RIK-2) or with TNF-α neutralizingMAb (clone 28401111 MAb 11) at 20 μgml for 1 h followedby infection with NL4-3 or mock infection (Afford et al 1999Caricchio et al 1998 Miura et al 2003 2001 Scheller et al2002) Thymocytes were cultured for 24 h in the presence orabsence of these antibodies and then analyzed for apoptosis Wedid not observe inhibition of HIV-1-mediated thymocyteapoptosis by incubation with any of these MAb (data notshown) Taken together these data suggest that caspase

activation is essential for efficient induction of thymocyteapoptosis by HIV-1 but that this likely occurs via an intrinsicpathway If an extrinsic pathway is also involved it may use adeath receptor other than Fas TRAIL or the TNF receptors

Cyclosporin A and PD98059 inhibited PS exposure induced byHIV-1 infection

Some of the genes induced by HIV-1 infection ofthymocytes including Nurr1 and TDAG8 exhibit theirapoptotic effects by activating intrinsic pathways of apoptosisTo assay possible mitochondrial involvement in HIV-1-inducedapoptosis we treated thymocytes with either cyclosporin A orbongkrekic acid which respectively target cyclophilin D (cyc-D) and the adenine nucleotide translocator (ANT) presentwithin the mitochondrial inner membrane Both cyc-D andANT are components of the permeability transition porecomplex blocking their action inhibits mitochondrial mem-brane permeabilization and certain forms of apoptosis(reviewed in Green and Kroemer 2004) Bongkrekic acid hadno inhibitory effect on PS exposure or caspase-3 activationinduced by HIV-1 infection of thymocytes (data not shown)Cyclosporin A however inhibited PS exposure in 65ndash70 ofthymocytes following HIV-1 infection which was significant(P lt 001) but it had no effect on caspase-3 activation (Figs 7Aand B) Cyclosporin A is also an inhibitor of calcineurin

Fig 7 Cyclosporin A (CyA) and PD98059 block HIV-1-induced PS exposure in thymocytes Thymocytes were pre-incubated with CyA (250 nM) or PD98059(50 μM) for 2 h and then infected with HIV-1 (MOI = 015) or mock infected Apoptosis was measured at 24 h post-infection by annexin V binding to exposed PS (AC) or activation of caspase-3 (B D) Bars represent the average of triplicate samples with error bars indicating standard deviations

277SK Choudhary et al Virology 352 (2006) 268ndash284

Calcineurin and ERK have been previously implicated ininducing the expression of Nur77 and treatment with cyclos-porin A or PD98059 blocked expression of Nur77 and inhibitedcell death (Castro-Obregon et al 2004 Shin et al 2004)Because we observed higher expression of Nurr1 followingHIV-1 infection of thymocytes and Nurr1 is a functionallyredundant member of the Nur77 family we tested whether ornot the MEK1 inhibitor PD98059 could also inhibit HIV-1-mediated apoptosis Like cyclosporin A treatment of thymo-cytes with PD98059 caused a 65ndash70 inhibition of PSexposure (P lt 001) but had little effect on caspase-3 activation(Figs 7C and D) Our data therefore suggest that both theextrinsic and the intrinsic apoptotic pathways are involved inHIV-1-mediated apoptosis the former activates proximalcaspases whereas the latter induces the release of apoptoticfactors including SmacDiablo Both pathways result inactivation of the executioner caspase caspase-3

To further assay the involvement of the intrinsic apoptoticpathway following HIV-1 infection of thymocytes we stainedthymocytes for an internal 38-kDa mitochondrial membraneprotein (7A6 antigen) which is exposed early in the molecularcascade of apoptosis and is recognized by MAb APO27(Koester et al 1997 Zhang et al 1996) We observed thatNL4-3 infection of thymocytes caused at least a 30 increase inspecific APO27+ cells in three experiments (Fig 8A) IL-7exhibits a strong anti-apoptotic effect on early lymphoid

progenitor cells in part by increasing intracellular Bcl-2 leadingto inhibition of the intrinsic apoptotic pathway (Napolitano etal 2003 Okamoto et al 2002 Von Freeden-Jeffry et al1997) We cultured thymocytes in the presence or absence ofIL-7 and observed that IL-7 increased Bcl-2 expression in bothmock-infected and NL4-3-infected thymocytes (Fig 8B)Furthermore IL-7 treatment of thymocytes reduced X4 HIV-1-induced apoptosis by approximately 50 at 24 h post-infection (Figs 8C and D) The inhibitory effect of IL-7 on HIV-1-induced thymocyte apoptosis was significant when measuredby either caspase-3 activation or PS exposure on both SP andDP thymocytes (P lt 001 in all cases) although the effect wasparticularly pronounced for DP thymocytes At 48 h post-infection however IL-7 failed to provide further protection andcells underwent apoptotic death These results implicate theinvolvement of an intrinsic apoptotic pathway that can beprevented by anti-apoptotic Bcl-2 family members in HIV-1-induced death of infected thymocytes

Discussion

In this report we investigated the role of HIV-1-mediatedapoptosis in thymocyte depletion Thymocytes were infectedwith the X4 HIV-1 molecular clone NL4-3 at an MOI highenough to obtain 60ndash90 infection Similar HIV-1 levels ofinfection were used by Holm and Gabuzda (2005) to study HIV-

Fig 8 HIV-1-induced thymocyte apoptosis includes the intrinsic pathway (A) Thymocytes were spin infected with NL4-3 (MOI = 015) or mock infected and stainedfor exposure of the mitochondrial membrane protein 7A6 using APO27-PE monoclonal antibody 24 h post-infection A representative histogram of one of threeexperiments is shown (BndashD) IL-7 induced Bcl-2 expression and delayed HIV-1-induced apoptosis in thymocytes Thymocytes were infected with HIV-1(MOI = 015) or mock infected and then cultured in the presence or absence of IL-7 (10 ngml) Expression of Bcl-2 was measured 24 h post-treatment using theCytofixCytopermtrade kit (BD Biosciences Pharmingen San Diego CA) (B) Apoptosis was measured at 24 and 48 h post-infection by annexin V binding to exposedPS (C) or activation of caspase-3 (D) Bars represent the average of triplicate samples with error bars indicating standard deviations Results from one of tworepresentative experiments are shown

278 SK Choudhary et al Virology 352 (2006) 268ndash284

1-mediated apoptosis of CD4+ T cells In FTOC and SCID-hu(ThyLiv) mice X4 HIV-1 reaches a similar high viral load andcan completely deplete CD4+ thymocytes indicating that theMOI used here is relevant to infection of the thymus Moreoverin HIV-1-infected individuals severe thymocyte loss occursindicating that HIV-1 likely achieves very high viral load in thethymus of patients (Gaulton et al 1997 Rosenzweig et al

1993) We observed apoptosis as quantified by a number ofmarkers including active caspase-3 staining DNA fragmenta-tion by TUNEL assays and PS exposure using annexin Vstaining NL4-3 infection caused a three-fold increase in activecaspase-3+ thymocytes when compared to mock-infected cellsA higher percentage of TUNEL-positive thymocytes than activecaspase-3+ thymocytes however was observed This difference

279SK Choudhary et al Virology 352 (2006) 268ndash284

of about 5 may represent the thymocyte populations that wereproliferating or rearranging their T cell receptor genes and weretherefore artifactually TUNEL positive We did not observe asrobust an increase in the proportion of annexin V-PE+ 7-AADminus

thymocytes as we did with active caspase-3+ or TUNEL+

thymocytes at 24 h post-infection PS exposure marks cells forphagocytosis and is insensitive to Z-VAD-FMK when apoptosisis induced by glucocorticoids suggesting that PS exposure doesnot necessarily require caspase activation It is thereforepossible that proteins involved in PS exposure may have beendownregulated or activated late in the process of apoptosisresulting in a lower percentage of V-PE+ 7-AADminus thymocytes at24 h post-infection when compared to those that were activecaspase-3+ or TUNEL+

The results presented here suggest that apoptosis plays a keyrole in HIV-1-mediated thymocyte depletion consistent withthe findings of McCune and colleagues (Bonyhadi et al 1993Kaneshima et al 1994 Su et al 1995) Our results howeverare in contrast with those of Jamieson et al (1997) whoobserved few apoptotic cells at the peak of viral replication inSCID-hu ThyLiv infections suggesting a role for necrosis inHIV-1-induced thymocyte depletion It may be important tonote however that the mitochondrial electron transportcomplex I is itself a substrate of caspase-3 and its destructionresults in the shut down of ATP production (Ricci et al 20032004) This may lead to intracellular ATP exhaustion andmitochondrial rupture causing necrotic cell death even thoughcell death was initiated by apoptotic signaling It has beenreported recently that mitochondrial electron transport complexI activity is impaired during HIV-1 infection and contributes toT cell apoptosis (Ladha et al 2005) We observed an increasedproportion of active caspase-3+ thymocytes a characteristic ofapoptosis in HIV-1-infected thymocytes Treatment of thymo-cytes with z-VAD-FMK a broad-spectrum caspase inhibitorinhibited PS exposure at 24 h post-infection suggesting theinvolvement of caspase proteolytic activity in HIV-1-mediatedapoptosis

X4 HIV-1 induced apoptosis primarily in productivelyinfected immature thymocytes Treatment of thymocytes withthe reverse transcriptase inhibitor Efavirenz resulted in a 15-logdecrease in viral reverse transcription and a correspondingdecrease in apoptosis was measured in high multiplicityinfections both by activation of caspase-3 and annexin Vstaining AT-2 treatment of HIV-1 which inactivated the viruswithout compromising the conformational and functionalintegrity of virion surface proteins also resulted in inhibitionof apoptosis Presence of the fusion inhibitor T-20 duringinfection which allowed interaction of gp120 with receptor andco-receptor but blocked viral entry also resulted in abrogationof HIV-1-induced thymocyte apoptosis Moreover HIV-1 didnot induce apoptosis in most CD4SP or CD8SP thymocytesThese results indicate that direct killing of immature DPthymocytes via an apoptotic pathway was the primary cause ofthymocyte depletion following high multiplicity infection byX4 HIV-1 Moreover these results are in accordance with thoseof Jamieson et al (1997) who observed a rapid phase of CD4+

thymocyte depletion during the peak of viral replication which

was ascribed to the direct killing of HIV-1-infected thymocytesSimilarly HIV-1 causes a rapid depletion of susceptible CD4+ Tcells in gut lymphoid tissue during the acute phase of HIV-1infection suggesting that the direct killing of infected cells maybe an important pathogenic mechanism in this setting as well(Brenchley et al 2004 Guadalupe et al 2003) Our resultshowever are in contrast to those of Su and colleagues whichsuggested that apoptosis occurred primarily in uninfectedthymocytes (Su et al 1995) The integrity of the thymicmicroenvironment is essential for T cell development and it istherefore possible that HIV-1 may perturb this microenviron-ment sufficiently during the chronic phase of infection to induceapoptosis in uninfected thymocytes (Stanley et al 1993)

X4 HIV-1 infection induced apoptosis primarily in the DPthymocyte subpopulation whereas most mature CD4SP andCD8SP thymocytes were resistant to apoptosis This isconsistent with the profile of genes induced by HIV-1 infectionof thymocytes Expression of several genes including TDAG8Nurr1 and upstream regulators of JNK which are involved innegative selection and apoptosis of DP thymocytes wasinduced by X4 HIV-1 infection of thymocytes (Cheng et al1997 Rincon et al 1998 Tosa et al 2003 Woronicz et al1994 Zhou et al 1996) TDAG8 which is glucocorticoidresponsive was induced at 4 12 and 24 h post-infection byHIV-1 Expression of TDAG8 has been previously shown to besufficient for induction of caspase-dependent apoptosis in DPthymocytes because such apoptosis could be blocked byrepression of endogenous TDAG8 using RNA interference(Malone et al 2004 Tosa et al 2003) We observed a similarcaspase-dependent form of apoptosis in HIV-1-infected DPthymocytes because death was blocked by the broad-spectrumcaspase inhibitor Z-VAD-FMK or combinations of specificcaspase inhibitors Z-VDVAD-FMK Z-IETD-FMK and Z-LEHD-FMK

Expression of Nur77 has also been implicated in theinduction of thymocyte apoptosis (Calnan et al 1995 Sebzdaet al 1999 Zhou et al 1996) Nur77 gene-deficient micehowever did not display an apparent defect in thymocyteselection suggesting that either Nur77 was not required for thisprocess in vivo or that a closely related molecule may substitutefor its action in knockout mice (Lee et al 1995) We observedincreased transcription of Nurr1 following HIV-1 infectionNurr1 is a member of the Nur77 family which also inducesapoptosis in DP thymocytes (Cheng et al 1997 Zhou et al1996) It is likely that similar to Nur77 mitochondrial targetingof Nurr1 may play an essential role in apoptotic demise ofthymocytes (Li et al 2000) Intriguingly caspase-dependentapoptosis of thymocytes infected with HIV-1 was not rescuedby incubating these cells with neutralizing MAbs against FasLTRAIL and TNF These data suggest that HIV-1 induces anintrinsic form of apoptosis that depends on caspase activity butdoes not require death receptor signaling via FasL TRAIL orTNF

JNKSAPK activity also contributes to apoptosis in humanlymphoid cell lines and mouse thymocytes (Behrens et al2001 Herr et al 1999 Rincon et al 1998 Sabapathy et al1999) JNK can inactivate Bcl-2 by phosphorylation releasing

280 SK Choudhary et al Virology 352 (2006) 268ndash284

pro-apoptotic members of the Bcl-2 family as well ascytochrome c and SmacDiablo thereby inducing caspaseactivation and apoptosis in a manner that is not blockedeffectively by FADD-DN CrmA or Bcl-2 (Yamamoto et al1999) We observed increased transcription of GADD45α aswell as MAP4K4 which functions near the beginning of theMAP kinase signal cascade GADD45α interacts with MEKK4MTK and activates the JNKp38 signaling pathway therebyinducing apoptosis (Mita et al 2002 Takekawa and Saito1998) Moreover Andersen et al (2005) observed increasedexpression of GADD45α induced by HIV-1 Vpr leading to G2M arrest and apoptosis These HIV-1-induced apoptotic genesinvolved in negative selection and DP thymocyte apoptosis allrequire the involvement of the mitochondrial pathway ofapoptosis Consistent with this profile of induced genes weobserved increased exposure of the mitochondrial protein7A6 suggesting involvement of the intrinsic pathway ofapoptosis Furthermore IL-7 which induced Bcl-2 expressionin thymocytes delayed HIV-1-mediated apoptosis furtherimplicating mitochondrial involvement in the apoptotic demiseof HIV-1-infected thymocytes We propose that HIV-1-inducedTDAG8 Nurr1Nur77 and MAPK all promote the permeabi-lization of the mitochondrial outer membrane leading torelease of cytochrome c and SmacDiablo as well as caspaseactivation and culminating in the apoptotic demise of DPthymocytes Pharmacological inhibitors of caspases arecurrently under clinical investigation Our results suggest thatthe use of such inhibitors in HIV-1-infected individuals maylimit the detrimental effect of HIV-1 infection on thymopoiesisand thereby increase the level of circulating CD4+ T cells inpatients

Materials and methods

Antibodies and reagents

CD8ndashfluorescein isothiocyanate (FITC) CD4ndashallophyco-cyanin (APC) annexin Vndashphycoerythrin (PE) CD27ndashFITCCD69ndashAPC and appropriate isotype control monoclonalantibodies (MAbs) were obtained from Caltag Laboratories(Burlingame CA) CD8ndashperidinin chlorophyll protein (PerCP)was obtained from BD Biosciences (San Jose CA) Rabbit anti-active caspase-3-PE MAb TNF-α neutralizing MAb (cloneMAb11) and anti-Bcl-2-FITC (clone 100) were obtained fromBD Biosciences Pharmingen (San Diego CA) Fas ligandneutralizing MAb (clone NOK-1) and TRAIL neutralizingMAb (clone RIK-2) were obtained from e-Bioscience (SanDiego CA) To identify HIV-1-infected cells we used anti-p24MAb KC57-FITC from Coulter (Miami FL) APO27 PE wasobtained from ImmunotechCoulter (Marseille France) Thefollowing inhibitors were purchased from Calbiochem (SanDiego CA) general caspase inhibitor Z-VAD-FMK caspase-2inhibitor Z-VDVAD-FMK caspase-8 inhibitor Z-IETD-FMKcaspase-9 inhibitor Z-LEHD-FMK topoisomerase inhibitoretoposide phosphate MAP kinase kinase (MEK) inhibitorPD98059 and mitochondrial permeability transition poreinhibitors bongkrekic acid triammonium salt and cyclosporin

A 7-aminoactinomycin D (7-AAD) was obtained from Molec-ular Probes (Eugene OR) Dimethyl sulfoxide (DMSO) andTNF-α neutralizing MAb (clone 28401111) were obtainedfrom Sigma (St Louis MO) Efavirenz T-20 and AMD-3100were obtained from the NIH AIDS Research and ReferenceReagent Program (Rockville MD)

Preparation and titration of HIV-1 stock

Viral stocks were generated by transient transfection of 293Twith the plasmid molecular clone NL4-3 by CaPO4 co-precipitation High titer viral stocks were generated byamplifying NL4-3 on CEM-GFP cells Virus containingsupernatants were aliquoted and frozen at minus80 degC until neededThe titer of virus in each supernatant was assayed by limitingdilution infection of 2-day PHA and IL-2 stimulated healthydonor PBMC followed by assay of supernatant reversetranscriptase activity 1 week later

Inactivation of virus

Frozen viral stocks were quickly thawed at 37 degC in a waterbath and treated with aldrithiol-2 (AT-2 Aldrich MilwaukeeWI) at a concentration of 1 mM for 1 h at 37 degC to inactivate thevirus (Rossio et al 1998) At the end of treatment AT-2 wasremoved by ultrafiltration using a centrifugal filter device with a100-kDa cutoff (Centriplus YM-100 Millipore Bedford MA)by three changes of media 12 ml each Filtration was done at4 degC and more than 100-fold dilution of AT-2 was achieved Inparallel AT-2-treated media was also filtered similarly in orderto check effects of residual AT-2 on thymocyte apoptosis Viruswas also heat inactivated at 56 degC in a water bath for 2 h withfrequent mixing

Preparation and maintenance of SCID-hu thymusliver mice

Human fetal thymus and liver of 20ndash24 weeks of gestationwas obtained from Advanced Bioscience Resources (AlamedaCA) SCID-hu thymus liverliver mice were created byimplantation of human fetal thymus and liver fragments asdescribed by McCune et al (1988) Briefly 6- to 8-week-oldmice were implanted with a 1-mm3 piece of human fetal thymussandwiched between two 1-mm3 pieces of fetal liver under theleft kidney capsule using a 16-gauge cancer implant needle set(Popper and Sons New Hyde Park NY) The grafts were used4ndash6 months after implantation SCID and SCID-hu mice weremaintained in microisolator cages on racks with HEPA-filteredair blown into each cage

Thymocyte culture and HIV-1 infection

Fresh human fetal thymus or a ThyLiv graft from a SCID-humouse was washed and sheared using two scalpels in Hanksbalanced salt solution without phenol red (HBSS MediatechInc Herndon VA) The thymic tissue was then incubated with02 mgml of collagenase B (Roche Indianapolis IN) and100 Uml of DNase (Sigma) for 45 min at room temperature in

281SK Choudhary et al Virology 352 (2006) 268ndash284

order to isolate both thymocytes and thymic epithelial cells(TEC) The cell suspension was vigorously pipetted and filteredthrough 70-μm nylon mesh to remove large clumps Cells werewashed and seeded at 25 times 106 cellswell in a 48-well flatbottom plate at a density of 107 cellsml in Iscoves mediumsupplemented with 10 fetal bovine serum 1times minimalessential medium vitamin solution 50 μgml gentamicin (allfrom Life Technologies Rockville MD) and insulintransferrinsodium-selenite media supplement (Sigma St Louis MO)Cells were either mock spin-infected or spin-infected with NL4-3 in the presence of polybrene (4 μgml) at 1210timesg for 90 min at22 degC Following spin infection cells were washed two timesand cultured at 107 cellsml in 48-well plates at 37 degC with 5CO2

Apoptosis assays

Annexin V stainingCells were washed once in HBSS containing 002 sodium

azide (HBSSA) and incubated with annexin V-PE 7-AADCD8ndashFITC and CD4ndashAPC MAbs in 100 μl of HBSSAcontaining 2 FBS for 30 min at 4 degC in the dark Cells werewashed twice and fixed in 200 μl of HBSS with 2formaldehyde overnight at 4 degC in the dark prior to flowcytometry Cell populations analyzed were defined based ontheir low angle and 90deg light scattering properties Unstainedcells were used to set markers defining positive reactivity

Active caspase-3 staining and internal p24 stainingCells were first stained for surface marker if desired by

incubating with CD8ndashPerCP and CD4ndashAPC MAb in 100 μl ofPBS containing 002 sodium azide (PBSA) and 2 FBS for30 min at 4 degC in the dark Following surface staining cellswere washed two times in PBSA then fixed and permeabilizedusing the CytofixCytopermtrade kit (BD Biosciences Pharmin-gen San Diego CA) for 20 min at room temperature Cellswere washed and stained in PermWash buffer with PE-conjugated rabbit anti-active caspase-3 MAb and KC57-FITCanti-p24 MAb for 30 min at RT in the dark Followingincubation cells were washed in PermWash buffer two timesre-suspended in the same buffer and analyzed by flowcytometry

Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)

HIV-1-induced DNA fragmentation was measured using theAPO-Directtrade kit (BD Biosciences Pharmingen San DiegoCA) as described by the manufacturer Briefly 2 times 106

thymocytes were fixed in 1 (WV) paraformaldehyde inPBS on ice for 60 min washed two times with PBS re-suspended in 70 (VV) ice-cold ethanol and stored overnightat minus20 degC Cells were washed in PBS then labeled with FITCndashdUTP in reaction buffer containing terminal deoxynucleotidyltransferase (TdT) enzyme Cells were incubated for 60 min atroom temperature washed in rinse buffer stained withpropidium iodideRNase staining buffer and analyzed by flowcytometry

APO27 StainingExposure of the 38 kDa mitochondrial membrane protein

7A6 antigen was detected with the APO27 antibody(ImmunotechCoulter Marseille France) as described by themanufacturer Briefly cells were permeabilized with digitonin(40 μgml) in PBS containing 2 FBS on ice Cells werewashed and stained in PermWash buffer (BD BiosciencesPharmingen San Diego CA) with PE-conjugated APO27MAb for 30 min at RT in the dark Following incubation cellswere washed in PermWash buffer two times re-suspended inthe same buffer and analyzed by flow cytometry

cDNA labeling and DNA array hybridization

Labeling and hybridization protocols closely match thosefound in the Affymetrix GeneChip expression analysis manualTotal RNA was reverse transcribed using oligo-dT primer tocreate first strand cDNA followed by a subsequent secondstrand cDNA synthesis step After phenolchloroform extractionand ethanol precipitation the cDNAwas amplified using biotin-labeled dNTPs and after a clean-up step the biotin-labeledcDNA was fragmented Fragmented cDNAs were thenhybridized to Affymetrix human genome DNA array U133Achips for 16 h after which the chips were washed and stainedusing FS400 fluidic stations These chips were then scanned onAffymetrix Gene Array 2500 Scanners and the signal intensitieswere calculated using Affymetrix Microarray Suite 50 (MAS)software

Real-time quantitative PCR and real-time quantitative RT-PCR

Genomic DNA was isolated by lysing cells in urea lysisbuffer [7 M urea 2 (wv) SDS 035 M NaCl 1 mM EDTA10 mM TrisndashHCl (pH 80)] followed by phenolchloroformextraction and ethanol precipitation of nucleic acids PCRamplification was performed using SYBR Green PCR Mastermix (PE Applied Biosystems Foster City CA) and primer setM667M661 specific for the completion of reverse transcription(Zack et al 1990) The signals were detected using an ABIPrism 7700 apparatus and sequence detector version 171software (Applied Biosystems) The following reaction condi-tions were used initial incubation for 2 min at 50 degC and 10 minat 95 degC followed by 40 cycles of 95 degC for 15 s and 60 degC for1 min Primers specific for the human β-globin gene were usedto determine the number of cells in each sample RNA waspurified using the QIAGEN mini-prep kit following themanufacturers instructions and treated with DNase I on aQIAGEN mini-column cDNA was generated using Taqmanreverse transcription (RT) reagents (Applied Biosystems)following the manufacturers instructions The reaction mixcontained 1times RT buffer 55 mM MgCl2 2 mM dNTPs 50 μMrandom hexamers 25 μM oligo-dT 40 U RNase inhibitor125 U Multiscribe RT and RNA in a 100-μl volume Thereaction was run at 25 degC for 10 min 42 degC for 60 min and thenstopped at 95 degC for 5 min cDNAwas then diluted 110 in H2Oand stored at 4 degC PCR primers and Taqman probes weredesigned using Primer Express software (Applied Biosystems

282 SK Choudhary et al Virology 352 (2006) 268ndash284

Supplementary Table 1) Real-time quantitative PCRs wereperformed using Universal PCR Master Mix (PE AppliedBiosystems Foster City CA) TaqManreg TAMRAtrade Probes(ABI) and DNA oligonucleotide primers (IDT) on an ABIPrism 7700 apparatus The following reaction conditions wereused initial hold at 50 degC for 2 min followed by denaturation at95 degC for 10 min then 40 cycle at 95 degC for 15 s and 60 degC for1 min Target gene probes were labeled with 6FAM whereas the18S and B2-microglobulin control genes were labeled withVIC The cycle number (Rn) value corresponded to the variationin reporter fluorescence intensity during each PCR cyclenormalized to the fluorescence of an internal passive referenceA specific Ct was determined for each PCR The Ct was definedas the cycle number at which a significant increase in thefluorescence signal was first detected ABI software was used todetermine the number of cycles needed for the 6FAM or VICreporters to cross a threshold where a significant change influorescence is detected (Ct value) Relative RNA expressionwas determined using the following formula ΔCt = Average Ct

(target gene) minus Average Ct (18S rRNA gene) ΔΔCt = ΔCt

(sample) minus ΔCt (no template control) relative expression =(2minusΔΔCt) times 1000

Statistical methods

We used analysis of variance to determine the statisticalsignificance of the results and Tukeys test for all pairwisecomparisons When results involved only two groups Studentst test was performed assuming independent variance

Acknowledgments

We thank Neelima R Choudhary Lesley White and JessicaDeLeon for help with SCID-hu mice We also thank J DenisHeck UCI DNA and Protein MicroArray Facility Manager forhelp with statistical analysis of Affymetrix gene array data Thefollowing reagents were obtained through the NIH AIDSResearch and Reference Reagent Program Division of AIDSNIAID NIH Efavirenz T-20 and JM-2987 (hydrobromide saltof AMD-3100)

This work was supported by NIH grants AI 47729 and AI55385 awarded to DC

Appendix A Supplementary data

Supplementary data associated with this article can be foundin the online version at doi101016jvirol200604037

References

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Andersen JL Zimmerman ES Dehart JL Murala S Ardon O BlackettJ Chen J Planelles V 2005 ATR and GADD45alpha mediate HIV-1Vpr-induced apoptosis Cell Death Differ 12 (4) 326ndash334

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Baldi P Long AD 2001 A Bayesian framework for the analysis ofmicroarray expression data regularized t-test and statistical interference ofgene changes Bioinformatics 17 (6) 509ndash519

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Bonyhadi ML Su L Auten J McCune JM Kaneshima H 1995Development of a human thymic organ culture model for the study of HIVpathogenesis AIDS Res Hum Retroviruses 11 (9) 1073ndash1080

Brenchley JM Schacker TW Ruff LE Price DA Taylor JHBeilman GJ Nguyen PL Khoruts A Larson M Haase ATDouek DC 2004 CD4+ T cell depletion during all stages of HIVdisease occurs predominantly in the gastrointestinal tract J Exp Med200 (6) 749ndash759

Brooks DG Kitchen SG Kitchen CM Scripture-Adams DD Zack JA2001 Generation of HIV latency during thymopoiesis Nat Med 7 (4)459ndash464

Brooks DG Hamer DH Arlen PA Gao L Bristol G Kitchen CMBerger EA Zack JA 2003 Molecular characterization reactivation anddepletion of latent HIV Immunity 19 (3) 413ndash423

Calnan BJ Szychowski S Chan FK Cado D Winoto A 1995 A role forthe orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection Immunity 3 (3) 273ndash282

Camerini D Su HP Gamez-Torre G Johnson ML Zack JA Chen IS2000 Human immunodeficiency virus type 1 pathogenesis in SCID-hu micecorrelates with syncytium-inducing phenotype and viral replication J Virol74 (7) 3196ndash3204

Caricchio R Reap EA Cohen PL 1998 FasFas ligand interactions areinvolved in ultraviolet-B-induced human lymphocytes apoptosis JImmunol 161 (1) 241ndash251

Castro-Obregon S Rao RV del Rio G Chen SF Poksay KS RabizadehS Vesce S Zhang XK Swanson RA Bredesen DE 2004Alternative nonapoptotic programmed cell death mediation by arrestin 2ERK2 and Nur77 J Biol Chem 279 (17) 17543ndash17553

Cheng LE Chan FK Cado D Winoto A 1997 Functional redundancy ofthe Nur77 and Nor-1 orphan steroid receptors in T cell apoptosis EMBO J16 (8) 1865ndash1875

Cheng H Chung S Sukumar S 2004 HOXA5-induced apoptosis in breastcancer cells is mediated by caspase 2 and 8 Mol Cell Biol 24 (2)924ndash935

Choudhary SK Camerini D 2006 Animal and organ culture models ofHIV-1 mediated apoptosis In Badley AD (Eds) Cell death during HIVinfection Taylor and Francis Boca Raton pp 293ndash315

Choudhary SK Choudhary NR Kimbrell KC Colasanti J Ziogas AKwa D Schuitemaker H Camerini D 2005 R5 human immunodefi-ciency virus type 1 infection of fetal thymic organ culture induces cytokineand CCR5 expression J Virol 79 (1) 458ndash471

Coberley CR Kohler JJ Brown JN Oshier JT Baker HV Popp MPSleasman JW Goodenow MM 2004 Impact on genetic networks inhuman macrophages by a CCR5 strain of human immunodeficiency virustype 1 J Virol 78 (21) 11477ndash11486

De Clercq E Yamamoto N Pauwels R Balzarini J Witvrouw M DeVreese K Debyser Z Rosenwirth B Peichl P Datema R ThorntonD Skerlj R Gaul F Padmanabhan S Bridger G Henson G Abrams

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Douek DC McFarland RD Keiser PH Gage EA Massey JMHaynes BF Polis MA Haase AT Feinberg MB Sullivan JLJamieson BD Zack JA Picker LJ Koup RA 1998 Changes inthymic function with age and during the treatment of HIV infection Nature396 (6712) 690ndash695

Duus KM Miller ED Smith JA Kovalev GI Su L 2001 Separation ofhuman immunodeficiency virus type 1 replication from nef-mediatedpathogenesis in the human thymus J Virol 75 (8) 3916ndash3924

Finkel TH Tudor-Williams G Banda NK Cotton MF Curiel T MonksC Baba TW Ruprecht RM Kupfer A 1995 Apoptosis occurspredominantly in bystander cells and not in productively infected cells ofHIV- and SIV-infected lymph nodes Nat Med 1 (2) 129ndash134

Gandhi RT Chen BK Straus SE Dale JK Lenardo MJ Baltimore D1998 HIV-1 directly kills CD4+ T cells by a Fas-independent mechanismJ Exp Med 187 (7) 1113ndash1122

Gaulton GN Scobie JV Rosenzweig M 1997 HIV-1 and the thymus Aids11 (4) 403ndash414

Green DR Kroemer G 2004 The pathophysiology of mitochondrial celldeath Science 305 (5684) 626ndash629

Grivel JC Margolis LB 1999 CCR5- and CXCR4-tropic HIV-1 are equallycytopathic for their T cell targets in human lymphoid tissue Nat Med 5 (3)344ndash346

Grivel JC Malkevitch N Margolis L 2000 Human immunodeficiencyvirus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in exvivo-infected human lymphoid tissue J Virol 74 (17) 8077ndash8084

Guadalupe M Reay E Sankaran S Prindiville T Flamm J McNeil ADandekar S 2003 Severe CD4+ T cell depletion in gut lymphoid tissueduring primary human immunodeficiency virus type 1 infection andsubstantial delay in restoration following highly active antiretroviral therapyJ Virol 77 (21) 11708ndash11717

Herr I Wilhelm D Meyer E Jeremias I Angel P Debatin KM 1999JNKSAPK activity contributes to TRAIL-induced apoptosis Cell DeathDiffer 6 (2) 130ndash135

Holm GH Gabuzda D 2005 Distinct mechanisms of CD4+ and CD8+ Tcellactivation and bystander apoptosis induced by human immunodeficiencyvirus type 1 virions J Virol 79 (10) 6299ndash6311

Huang Y Rich RL Myszka DG Wu H 2003 Requirement of both thesecond and third BIR domains for the relief of X-linked inhibitor ofapoptosis protein (XIAP)-mediated caspase inhibition by Smac J BiolChem 278 (49) 49517ndash49522

Jamieson BD Uittenbogaart CH Schmid I Zack JA 1997 High viralburden and rapid CD4+ cell depletion in human immunodeficiency virustype 1-infected SCID-hu mice suggest direct viral killing of thymocytes invivo J Virol 71 (11) 8245ndash8253

Jamieson BD Douek DC Killian S Hultin LE Scripture-Adams DDGiorgi JV Marelli D Koup RA Zack JA 1999 Generation offunctional thymocytes in the human adult Immunity 10 (5) 569ndash575

Jekle A Keppler OT De Clercq E Schols D Weinstein M GoldsmithMA 2003 In vivo evolution of human immunodeficiency virus type 1toward increased pathogenicity through CXCR4-mediated killing ofuninfected CD4 T Cells J Virol 77 (10) 5846ndash5854

Jenkins M Hanley MB Moreno MB Wieder E McCune JM 1998Human immunodeficiency virus-1 infection interrupts thymopoiesis andmultilineage hematopoiesis in vivo Blood 91 (8) 2672ndash2678

Kalayjian RC Spritzler J Pu M Landay A Pollard RB Stocker VHarthi LA Gross BH Francis IR Fiscus SA Tebas P Bosch RJValcour V Lederman MM 2005 Distinct mechanisms of T cellreconstitution can be identified by estimating thymic volume in adultHIV-1 disease J Infect Dis 192 (9) 1577ndash1587

Kaneshima H Su L Bonyhadi ML Connor RI Ho DD McCune JM1994 Rapid-high syncytium-inducing isolates of human immunodeficiencyvirus type 1 induce cytopathicity in the human thymus of the SCID-humouse J Virol 68 (12) 8188ndash8192

Koester SK Roth P Mikulka WR Schlossman SF Zhang C BoltonWE 1997 Monitoring early cellular responses in apoptosis is aided by the

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Koka PS Fraser JK Bryson Y Bristol GC Aldrovandi GM Daar ESZack JA 1998 Human immunodeficiency virus inhibits multilineagehematopoiesis in vivo J Virol 72 (6) 5121ndash5127

Kourtis AP Ibegbu C Nahmias AJ Lee FK Clark WS Sawyer MKNesheim S 1996 Early progression of disease in HIV-infected infants withthymus dysfunction N Engl J Med 335 (19) 1431ndash1436

Ladha JS Tripathy MK Mitra D 2005 Mitochondrial complex I activity isimpaired during HIV-1-induced T cell apoptosis Cell Death Differ 12 (11)1417ndash1428

Lamhamedi-Cherradi SE Zheng SJ Maguschak KA Peschon J ChenYH 2003 Defective thymocyte apoptosis and accelerated autoimmunediseases in TRAILminusminus mice Nat Immunol 4 (3) 255ndash260

Lee SL Wesselschmidt RL Linette GP Kanagawa O Russell JHMilbrandt J 1995 Unimpaired thymic and peripheral T cell death in micelacking the nuclear receptor NGFI-B (Nur77) Science 269 (5223)532ndash535

Lenardo MJ Angleman SB Bounkeua V Dimas J Duvall MGGraubard MB Hornung F Selkirk MC Speirs CK Trageser COrenstein JO Bolton DL 2002 Cytopathic killing of peripheral bloodCD4(+) T lymphocytes by human immunodeficiency virus type 1 appearsnecrotic rather than apoptotic and does not require env J Virol 76 (10)5082ndash5093

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Li H Kolluri SK Gu J Dawson MI Cao X Hobbs PD Lin B ChenG Lu J Lin F Xie Z Fontana JA Reed JC Zhang X 2000Cytochrome c release and apoptosis induced by mitochondrial targeting ofnuclear orphan receptor TR3 Science 289 (5482) 1159ndash1164

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Nahmias AJ Clark WS Kourtis AP Lee FK Cotsonis G Ibegbu CThea D Palumbo P Vink P Simonds RJ Nesheim SR 1998Thymic dysfunction and time of infection predict mortality in humanimmunodeficiency virus-infected infants J Infect Dis 178 (3) 680ndash685

Napolitano LA Stoddart CA Hanley MB Wieder E McCune JM2003 Effects of IL-7 on early human thymocyte progenitor cells in vitro andin SCID-hu ThyLiv mice J Immunol 171 (2) 645ndash654

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Okamoto Y Douek DC McFarland RD Koup RA 2002 Effects ofexogenous interleukin-7 on human thymus function Blood 99 (8)2851ndash2858

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Poulin JF Viswanathan MN Harris JM Komanduri KV Wieder ERinguette N Jenkins M McCune JM Sekaly RP 1999 Directevidence for thymic function in adult humans J Exp Med 190 (4)479ndash486

Ricci JE Gottlieb RA Green DR 2003 Caspase-mediated loss ofmitochondrial function and generation of reactive oxygen species duringapoptosis J Cell Biol 160 (1) 65ndash75

Ricci JE Munoz-Pinedo C Fitzgerald P Bailly-Maitre B Perkins GAYadava N Scheffler IE Ellisman MH Green DR 2004 Disruptionof mitochondrial function during apoptosis is mediated by caspase cleavageof the p75 subunit of complex I of the electron transport chain Cell 117 (6)773ndash786

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Rosenzweig M Clark DP Gaulton GN 1993 Selective thymocytedepletion in neonatal HIV-1 thymic infection Aids 7 (12) 1601ndash1605

Rossio JL Esser MT Suryanarayana K Schneider DK Bess Jr JWVasquez GM Wiltrout TA Chertova E Grimes MK Sattentau QArthur LO Henderson LE Lifson JD 1998 Inactivation of humanimmunodeficiency virus type 1 infectivity with preservation of conforma-tional and functional integrity of virion surface proteins J Virol 72 (10)7992ndash8001

Sabapathy K Hu Y Kallunki T Schreiber M David JP Jochum WWagner EF Karin M 1999 JNK2 is required for efficient T cellactivation and apoptosis but not for normal lymphocyte development CurrBiol 9 (3) 116ndash125

Schacker TW Nguyen PL Beilman GJ Wolinsky S Larson M ReillyC Haase AT 2002 Collagen deposition in HIV-1 infected lymphatictissues and T cell homeostasis J Clin Invest 110 (8) 1133ndash1139

Scheller C Sopper S Chen P Flory E Koutsilieri E Racek T LudwigS ter Meulen V Jassoy C 2002 Caspase inhibition activates HIV inlatently infected cells Role of tumor necrosis factor receptor 1 and CD95J Biol Chem 277 (18) 15459ndash15464

Scoggins RM Taylor Jr JR Patrie J vant Wout AB Schuitemaker HCamerini D 2000 Pathogenesis of primary R5 human immunodeficiencyvirus type 1 clones in SCID-hu mice J Virol 74 (7) 3205ndash3216

Sebzda E Mariathasan S Ohteki T Jones R Bachmann MF OhashiPS 1999 Selection of the T cell repertoire Annu Rev Immunol 17829ndash874

Shin HJ Lee BH Yeo MG Oh SH Park JD Park KK Chung JHMoon CK Lee MO 2004 Induction of orphan nuclear receptor Nur77gene expression and its role in cadmium-induced apoptosis in lungCarcinogenesis 25 (8) 1467ndash1475

Singh SP Lai D Cartas M Serio D Murali R Kalyanaraman VSSrinivasan A 2000 Epitope-tagging approach to determine the stoichi-ometry of the structural and nonstructural proteins in the virus particlesamount of Vpr in relation to Gag in HIV-1 Virology 268 (2) 364ndash371

Spits H 2002 Development of alphabeta T cells in the human thymus NatRev Immunol 2 (10) 760ndash772

Stanley SK McCune JM Kaneshima H Justement JS Sullivan MBoone E Baseler M Adelsberger J Bonyhadi M Orenstein J FoxCH Fauci AS 1993 Human immunodeficiency virus infection of thehuman thymus and disruption of the thymic microenvironment in the SCID-hu mouse J Exp Med 178 (4) 1151ndash1163

Su L Kaneshima H Bonyhadi M Salimi S Kraft D Rabin L McCuneJM 1995 HIV-1-induced thymocyte depletion is associated with indirectcytopathogenicity and infection of progenitor cells in vivo Immunity 2 (1)25ndash36

Takekawa M Saito H 1998 A family of stress-inducible GADD45-likeproteins mediate activation of the stress-responsive MTK1MEKK4MAPKKK Cell 95 (4) 521ndash530

Taylor Jr JR Kimbrell KC Scoggins R Delaney M Wu L CameriniD 2001 Expression and function of chemokine receptors on humanthymocytes implications for infection by human immunodeficiency virustype 1 J Virol 75 (18) 8752ndash8760

Tosa N Murakami M Jia WY Yokoyama M Masunaga T Iwabuchi CInobe M Iwabuchi K Miyazaki T Onoe K Iwata M Uede T 2003Critical function of T cell death-associated gene 8 in glucocorticoid-inducedthymocyte apoptosis Int Immunol 15 (6) 741ndash749

Vigano A Vella S Saresella M Vanzulli A Bricalli D Di Fabio SFerrante P Andreotti M Pirillo M Dally LG Clerici M Principi N2000 Early immune reconstitution after potent antiretroviral therapy in HIV-infected children correlates with the increase in thymus volume Aids 14 (3)251ndash261

von Freeden-Jeffry U Solvason N Howard M Murray R 1997 Theearliest T lineage-committed cells depend on IL-7 for Bcl-2 expression andnormal cell cycle progression Immunity 7 (1) 147ndash154

Vucic D Franklin MC Wallweber HJ Das K Eckelman BP Shin HElliott LO Kadkhodayan S Deshayes K Salvesen GS FairbrotherWJ 2005 Engineering ML-IAP to produce an extraordinarily potentcaspase 9 inhibitor implications for Smac-dependent anti-apoptotic activityof ML-IAP Biochem J 385 (Pt1) 11ndash20

Woronicz JD Calnan B Ngo V Winoto A 1994 Requirement for theorphan steroid receptor Nur77 in apoptosis of T cell hybridomas Nature 367(6460) 277ndash281

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Yue FY Kovacs CM Dimayuga RC Gu XX Parks P Kaul ROstrowski MA 2005 Preferential apoptosis of HIV-1-specific CD4+ Tcells J Immunol 174 (4) 2196ndash2204

Zack JA Arrigo SJ Weitsman SR Go AS Haislip A Chen IS 1990HIV-1 entry into quiescent primary lymphocytes molecular analysis revealsa labile latent viral structure Cell 61 (2) 213ndash222

Zhang C Ao Z Seth A Schlossman SF 1996 A mitochondrial membraneprotein defined by a novel monoclonal antibody is preferentially detected inapoptotic cells J Immunol 157 3980ndash3987

Zhang ZQ Notermans DW Sedgewick G Cavert W Wietgrefe SZupancic M Gebhard K Henry K Boies L Chen Z Jenkins MMills R McDade H Goodwin C Schuwirth CM Danner SA HaaseAT 1998 Kinetics of CD4+ T cell repopulation of lymphoid tissues aftertreatment of HIV-1 infection Proc Natl Acad Sci USA 95 (3)1154ndash1159

Zhou T Cheng J Yang P Wang Z Liu C Su X Bluethmann HMountz JD 1996 Inhibition of Nur77Nurr1 leads to inefficientclonal deletion of self-reactive T cells J Exp Med 183 (4)1879ndash1892