Multiple Effector Functions Mediated by Human Immunodeficiency Virus-Specific CD4+ T-Cell Clones

  10.1128/JVI.75.20.9771-9779.2001.

2001, 75(20):9771. DOI:J. Virol. Sykulev, Bruce D. Walker and Eric S. RosenbergHowell F. Moffett, Steven L. Boswell, Tam Nguyen, Yuri Philip J. Norris, Marina Sumaroka, Christian Brander, 

T-Cell Clones+CD4Human Immunodeficiency Virus-Specific Multiple Effector Functions Mediated by

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JOURNAL OF VIROLOGY,0022-538X/01/$04.00�0 DOI: 10.1128/JVI.75.20.9771–9779.2001

Oct. 2001, p. 9771–9779 Vol. 75, No. 20

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Multiple Effector Functions Mediated by Human ImmunodeficiencyVirus-Specific CD4� T-Cell Clones

PHILIP J. NORRIS,1 MARINA SUMAROKA,2 CHRISTIAN BRANDER,1 HOWELL F. MOFFETT,1

STEVEN L. BOSWELL,3 TAM NGUYEN,1 YURI SYKULEV,2 BRUCE D. WALKER,1

AND ERIC S. ROSENBERG1*

Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School,Boston, Massachusetts 021141; Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson

University, Philadelphia, Pennsylvania 191072; and Fenway Community Health Center,Boston, Massachusetts 021153

Received 13 April 2001/Accepted 6 July 2001

Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cellscontribute to effective antiviral control, but their functional characteristics and the precise epitopes targetedby this response remain to be defined. In this study, we generated CD4� T-cell clones specific for Gag fromHIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells.Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG(amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examinedinduced cell proliferation, gamma interferon (IFN-�) secretion, and cytolytic activity. Cytolysis was abrogatedby concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediatedmechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker forcytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate thatT helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be acomponent of the antiviral effect of these cells.

Increasing evidence indicates that virus-specific T helpercells may play an important role in host immune responsesagainst human immunodeficiency virus type 1 (HIV-1) infec-tion (4, 17, 30, 43, 44). An inverse association between HIV-1plasma RNA virus load and Gag-specific T helper cell re-sponses is observed in untreated, chronic infection, suggestinga role in the control of viral replication (22, 44). In treatedacute HIV-1 infection, preserved HIV-specific T helper cellresponses are associated with an enhanced ability to containviremia when antiretroviral therapy is discontinued (43). Stud-ies involving early treatment of simian immunodeficiency virus(SIV) or DNA vaccination with or without interleukin-2 (IL-2)therapy prior to SIV infection demonstrated improved controlof viremia, along with vigorous CD4� T-cell responses (2, 4,17, 30). The central role of T helper cells in maintaining con-trol of viremia is consistent with findings from murine systems.CD4� T-cell-depleted mice are unable to clear lymphocyticchoriomeningitis virus, gammaherpesvirus 68, and Rauschermurine leukemia virus infections (5, 9, 18, 56).

While HIV-1-specific T helper cell responses appear to beassociated with virologic control, the functional characteristicsof these cells and the precise epitopes targeted remain to bedefined. It is hypothesized that lack of appropriate HIV-1-specific T helper cell responses seen in the majority of HIV-1-infected individuals contributes to the waning of virus-spe-cific cytotoxic T cells (CTL) and eventually results in diseaseprogression (5, 13, 23, 35, 40). Another possibility is that CD4�

T cells play a direct role in the suppression of viral replication.

CD4� cytotoxic T cells have been described in a number ofviral infections, including herpes simplex virus (53), hepatitis Bvirus (3), measles virus (20), human herpesvirus 6 (52), andEpstein-Barr virus (6). CD4� T cells with gp120-specific cyto-lytic activity were first described in the cerebrospinal fluid ofpatients with AIDS (46). However, they have been most ex-tensively observed in HIV-1-seronegative individuals vacci-nated with recombinant gp160 (15, 37, 39, 48, 49).

Few data exist at a clonal level on the functional character-istics of HIV-1-specific T helper cells (31, 34). To furthercharacterize HIV-1-specific T helper cells, we cloned thesecells at limiting dilution. Our results reveal multiple discreteepitopes in the HIV-1 Gag protein, including an epitope in thecyclophilin binding domain known to be important for the virallife cycle prior to reverse transcription (RT), following mem-brane binding and fusion (8). Moreover, clones to this andother epitopes were shown to mediate cytotoxic activity as wellas gamma interferon (IFN-�) production.

MATERIALS AND METHODS

Study subjects. Four persons with vigorous p24-specific T helper cell prolif-erative responses were selected for study. Subject CTS-01 is a 50-year-old Afri-can-American male infected with HIV-1 for at least 20 years. Without antiret-roviral therapy his viral load has been always less than 1,000 RNA copies/ml andhis CD4� T-cell count above 500 cells/ml. Subjects AC-01, AC-25, and AC-36were treated with antiretroviral therapy during acute HIV-1 infection (43), andclones were isolated 11 to 18 months after initiation of therapy. Clones fromAC-01 and AC-36 were isolated before a supervised therapy interruption andfrom AC-25 after treatment interruption and reinstitution. Thirty-five HIV-1-seronegative individuals’ peripheral blood mononuclear cells (PBMC) were usedas controls for proliferative responses to p24 protein (43).

Peptides and antibodies. Recombinant p24 protein (amino acids 133 to 373)derived from the NY-5 strain of HIV-1 was produced in a baculovirus expressionsystem with 90 to 95% purity (Protein Science, Meriden, onn.T). Shorter p24

* Corresponding author. Mailing address: Massachusetts GeneralHospital, GRJ 504, 55 Fruit St., Boston, MA 02114. Phone: (617)724-7519. Fax: (617) 726-7416. E-mail: [email protected].

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peptides were generated as free acids with an Advanced ChemTech 396� pep-tide synthesizer (44). Flow cytometry antibodies were obtained from BectonDickinson (San Jose, Calif.).

T-cell clones. Culture media (R�) consisted of RPMI 1640 (Sigma, St. Louis,Mo.) with penicillin-streptomycin (Mediatech, Herndon, Va.), HEPES (Medi-atech), and L-glutamine (Mediatech). T-cell clones were maintained in R� and10% heat-inactivated human AB serum (R10H). Clones were generated bylimiting dilution. Freshly isolated PBMC (107) were suspended in 10 ml of R10Hin a T25 flask and stimulated with p24 (1 �g/ml) and IL-2 (100 U/ml; Hoff-mann-La Roche). Indinavir (Merck, 0.4 �M), zidovudine (AZT; Glaxo Well-come; 0.5 �M), and lamivudine (Glaxo Wellcome; 3 �M) were added to themedia for the first 4 weeks of culture. After 2 weeks the PBMC were restimulatedwith p24 protein (1 �g/ml), IL-2 (100 U/ml), and 107 irradiated (30 Gy), autol-ogous PBMC. Three days after restimulation the PBMC were plated at limitingdilution with 10, 3, or 1 cell per well. Clones were screened in a 2-day prolifer-ation assay using autologous B-lymphoblastoid cell lines (B-LCL) as antigen-presenting cells and p24 protein as the stimulus. Clones that proliferated inresponse to p24 protein were restimulated every 2 to 3 weeks with an anti-CD3-specific antibody 12F6 (obtained from Johnson Wong, Massachusetts GeneralHospital), IL-2 (100 U/ml), and 107 irradiated allogeneic PBMC.

TCR V� chain analysis. In order to determine if the T-cell lines were mono-clonal, T-cell receptor (TCR) V� expression was determined by an establishedmethod (12). Twenty-four sets of primers specific for TCR V� RNA were used.PBMC (105) were used as positive controls, and distilled water was used fornegative controls.

Proliferation assays. To assay T helper cell responses in fresh PBMC, 105 cellswere incubated with p24 protein at 1 �g/ml for 6 days and then pulsed with[3H]thymidine for 6 h before harvesting as previously described (44). To testT-cell clones, antigen was presented by autologous or partially HLA matchedB-LCL. The B-LCL were irradiated (120 Gy) and incubated overnight in R10Hwith the appropriate antigen at 1 �g/ml. The following day B-LCL and T-cellclones were plated in triplicate wells at 50,000 cells/well each in 96-well plates inR10H. After 48 h, 1 �Ci of [3H]thymidine (Dupont NEN, Boston, Mass.) in 50�l of R10H was added per well. Plates were harvested onto glass fiber filters after18 h. Results were expressed as stimulation index, the ratio of counts from wellswith antigen divided by the counts obtained from wells without antigen. Alter-nately, results were shown as net counts per minute, the difference between thecounts. Based on previous studies of HIV-1-specific proliferative responses,stimulation indexes greater than 5 were considered significant (44).

IFN-� Elispot assays. Elispot plates were coated with anti-IFN-� antibody(Endogen, Woburn, Mass.) and incubated overnight at 4°C. The following dayplates were washed six times with phosphate-buffered saline (PBS; Mediatech).B-LCL (5 � 104) and antigen (1 �g/ml) were added in 100 �l of R10H. T-cellclones were added at 100 cells/well in 100 �l of R10H. After overnight incubationthe cells were discarded and plates were washed six times with PBS, and biotin-ylated anti- IFN-� (Endogen) was added for 1.5 h at 25°C. The plates were thenwashed six times with PBS. Streptavidin (Bio-Rad, Hercules, Calif.) (100 �l/well)was added, and plates were incubated 45 min at 25°C. After six more PBS washes100 �l/well of coloring reagent (nitro blue tetrazolium–5-bromo-4-chloro-3-in-dolylphosphate [NBT/BCIP]; Bio-Rad) was added. Once dots appeared, thereaction was stopped with three water washes. Background responses to wellswith no antigen or irrelevant antigen ranged from 0 to 1.5 spots per well, or 0 to15,000 spot-forming cells (SFC) per 106 cells. Responses greater than 50,000 SFCper 106 cells and 5 times maximum background were considered significant.

Cytotoxicity assays. Target cells (B-LCL) were incubated overnight with wholep24 or the cognate 22-amino-acid peptide at 1 �g/ml and Na2

51CrO4 (60 �Ci/ml;Dupont NEN). The following day the targets were washed twice with cold R10Hbefore effectors were added. Effectors and targets were incubated together intriplicate wells for 4 h at 37°C, then the plates were centrifuged at 1,000 rpm for5 min at 4°C. A 25-�l aliquot of reaction supernatant was assayed for 51Crrelease. Maximal release was obtained by mixing the targets with 100 �l of 1%Triton X-100. The percent specific 51Cr release was calculated as 100 � [(cpmexperimental release � cpm spontaneous release)/(cpm maximal release � cpmspontaneous release)]. Some lysis inhibition experiments were performed in thepresence of 2 mM EGTA and MgCl2. These reagents chelate extracellularcalcium and were added at the time clones and targets were combined. We alsoperformed cytolytic assays in the presence of inhibitors with concentrationsvarying from 0 to 10 �g/ml of concanamycin A (Sigma), brefeldin A (Sigma), oranti-Fas antibody (Coulter Immunotech, Miami, Fla.). The effector cells wereincubated with inhibitors for 2 h prior to plating with B-LCL, and the inhibitorsremained present throughout the assay. Spontaneous lysis was less than 30%unless otherwise noted. For the purpose of interpretation, specific lysis of greaterthan 10% was considered significant.

Lytic granule release assay. To quantify lytic granule release, autologousB-LCL were pulsed with various concentrations of cognate peptide for 1 h at37oC and combined with CD4� T cells at an effector-to-target cell (E:T) ratio of1:1 in round-bottomed 96-well plates. The density of the CTL in the mixture was7 � 105 per ml (1.5 � 105 per well). After 4 h at 37oC the culture supernatantwas collected, and activity of serine esterase, an essential component of thesecreted granules, was measured using the BLT (N-�-benzyloxycarboxyl-L-lysin-ethiobensyl ester) substrate in the presence of DTNB [5,5-dithio-bis(2-nitroben-zoic acid)] by reading the optical density (OD) at 405 nm (method communi-cated by Pierre Henkart). In our experiments DTNB was added prior to theaddition of BLT to measure the background OD at 405 nm. The background wasthen subtracted from the final reading for each sample.

Intracellular staining for IFN-� production. Quantitation for intracellularproduction of IFN-� upon antigen stimulation was performed as described else-where (51). Briefly, 5 � 105 clone cells were incubated with B cells pulsed withpeptide at 5 �g/ml and anti-CD28 and anti-CD49 antibodies at 1 �g/ml each for2 h at 37C at a 5° slant in 1 ml of R10H in a fluorescence-activated cell sorting(FACS) tube. At 2 h brefeldin A was added at 10 �g/ml, and incubation wascontinued as above for 4 more h. The samples were then refrigerated overnight.The next day the samples were washed in PBS plus 1% fetal calf serum, thenstained with surface antibodies for 20 min. After washing in PBS the cells werefixed and permeabilized with two 15-min incubations with Caltag reagents ac-cording to the manufacturer’s instructions. The cells were then stained withintracellular IFN-�–fluorescein isothiocyanate (FITC), washed twice in PBS, andanalyzed on the Becton Dickinson FACS machine. For the concanamycin Ainhibition experiments, the cloned cells were first incubated with concanamycinA for 1 h prior to the addition of B-LCL and antigen. At least 50,000 total eventswere counted for each condition.

RESULTS

Proliferative responses of PBMC to p24 Gag protein. Pre-vious studies have shown dominant HIV-1-specific T helpercell responses directed against the p24 Gag protein and thatthese responses are inversely correlated to HIV-1 viral load(22, 44). To determine the breadth of this response, overlap-ping peptides were used to map discrete targeted regions. Anexample is shown for subject CTS-01, an individual with long-term nonprogressive HIV-1 infection and a robust p24-specificT helper cell response. The dominant epitopes targeted weredefined using synthetic 22-amino-acid peptides overlapping by12 amino acids and spanning the p24 protein. Significant pep-tide-specific proliferative responses were detected to 3 of the23 peptides tested (Fig. 1A), indicating a polyclonal response.The dominant response was directed against peptide 9, se-quence DRVHPVHAGPIAPGQMREPRGS (44). Further-more, responses of 10 HIV-1-seronegative controls to the panelof proteins were negligible (Fig. 1B).

Generation of CD4� T-cell clones and mapping. In order todefine functional characteristics of T helper cell responses andfine map the epitopes targeted, clones were established. Lim-iting-dilution cloning of PBMC from subject CTS-01 was per-formed to derive 10 T-cell lines that proliferated to p24 pro-tein. The fine specificity of the proliferative response wasmapped using a set of overlapping p24 peptides. All T-cell linesbut one were found to proliferate exclusively in response topeptide 9, DRVHPVHAGPIAPGQMREPRGS, with stimula-tion indices ranging from 25 to 230, net cpm 1,675 to 9,530(data not shown). Clonality was determined by RT-PCR forV� region T-cell receptor usage. Clone 16 was selected forin-depth analysis because it expressed T-cell receptor V�4exclusively and was presumed to be clonal (Fig. 2A). Theremaining T-cell lines all expressed V�4 but also expressed oneto three additional V� regions and are likely not clonal. T-celllines were all functionally clonal and are referred to as clones

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in the remainder of the article. Each of the T-cell clones was99.2% CD4�, and the majority were less than 0.5% CD8� byFACS analysis (Fig. 2B).

p24-specific cytotoxic responses mediated by CD4� CD8�

T-cell clones. CD4� T cells are classically thought to assist inantiviral control by providing help to both CD8� CTL and Bcells. CD4� T cells have also been shown to secrete a numberof antiviral chemokines, such as IFN-�, RANTES, MIP-1�,and MIP-1�. We queried whether or not these CD4� T-cellclones would also possess direct cytolytic function. The 10CD4� T cell clones derived from subject CTS-01 were assessedfor their ability to lyse B-LCL incubated with peptide 9. All 10clones assayed lysed autologous B-LCL incubated with peptide9 at an E:T ratio of 100:1 (17 to 68% lysis), but not B-LCLincubated without peptide or with an irrelevant control peptide(Fig. 3). Seven of 10 clones also exhibited specific lysis inresponse to whole p24 protein at an E:T ratio of 100:1 (13 to61% lysis; Fig. 3).

Fine epitope mapping of proliferative, cytotoxic, and IFN-�-secreting responses. T helper cell responses are typicallytargeted at exogenously processed proteins 10 to 17 aminoacids in length. Truncations of peptide 9 were synthesized todefine the minimal epitope recognized by the clones fromsubject CTS-01. Three clones were examined, and data for onerepresentative clone are shown. All three clones recognizedthe same minimal 9-amino-acid peptide, VHAGPIAPG (Fig.4A). Deletion of either the N-terminal valine or C-terminalglycine resulted in complete abrogation of the proliferative

response. However, longer peptides containing these residueswere all recognized, and some were associated with strongerproliferative responses.

We next compared proliferation to cytolytic activity andIFN-� secretion using these truncated peptides. Fine mappingof the cytolytic response revealed the same minimal epitope(Fig. 4B). The clone was found to secrete IFN-� in response tostimulation by B-LCL pulsed with cognate peptide (2.8 � 105

SFC/106; Fig. 4C), but not by B-LCL incubated with an irrel-evant peptide (0 to 1.5 � 104 SFC/106; data not shown). Theminimal epitope for stimulation of IFN-� secretion was iden-tical to the minimal epitope required to induce proliferationand cytolysis, VHAGPIAPG.

Clonal responses were DQ7 restricted. The HLA restrictionof clones targeting the minimal peptide VHAGPIAPG wasdetermined using partially matched B-LCL as antigen-present-ing cells in proliferation, cytolytic, and IFN-� Elispot assays.B-LCL that could present antigen effectively in the three assaysall had allele DQ7, while those lacking DQ7 could not presentantigen effectively (Fig. 5, left panel). Multiple other B-LCLwere assayed, and the results consistently identified DQ7 as theonly allele associated with effective antigen presentation (Fig.5 and data not shown). These data thus confirm that the ef-fector functions mediated by these HIV-1-specific T helper cellclones are HLA class II restricted.

CD4� T-cell mediated cytolysis was seen in multiple indi-viduals. We explored whether CD4� T-cell clones from otherHIV-1-infected individuals would also exhibit antigen-specificcytolytic activity. We derived clones from three individuals who

FIG. 1. (A) PBMC from subject CTS-01 were tested in a lympho-cyte proliferation assay for responses to synthetic 22-amino-acid pep-tides spanning HIV-1 p24 protein sequence (peptides 1 to 23). ThesePBMC recognized several epitopes within p24, with the dominantresponse directed against peptide 9, sequence DRVHPVHAGPIAPGQMREPRGS. (B) The average proliferative response (net cpm)of PBMC from 10 HIV-1-seronegative control subjects to each ofpeptides 1 to 23 is shown.

FIG. 2. (A) TCR V� repertoire analysis: cDNA was derived fromclone 16 from subject CTS-01 by RT-PCR as previously described (12).The cDNA was amplified using primer sets specific for different vari-able segments of the TCR V� chain (V�1 to V�24). Only the V�4-specific primer pair yielded a PCR product (lane 4). Each reaction alsocontained T-cell receptor � chain constant region 3 and 5 primersthat produced a 100-bp fragment, included as a positive PCR controlin each lane. Lanes 25 and 27 contained no V�-specific primers (neg-ative control), whereas lane 26 contained a primer pair specific for theV� constant region (positive control). The DNA ladder shown at leftcontained 100-bp incremental fragments. (B) Flow cytometric analysisof clone 16 showed that the clone was 99.57% CD4� and 0.34% CD8�.

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were diagnosed and treated with highly active antiretroviraltherapy during acute HIV-1 infection who generated vigorousT helper cell responses to p24 antigen (43, 44). Three cloneswere derived from one individual, two from a second, and onefrom a third. Each clone recognized a different epitope within

p24 as mapped by overlapping 22-amino-acid peptides (Fig.6A). Clonality was determined by RT-PCR for V� T-cell re-ceptor usage as described previously. Clones 1 to 3 and 6 eachexpressed a unique T-cell receptor V� region, clone 4 ex-pressed two V� T-cell receptor products, and none of thetested V� T-cell receptors was identified for clone 5. The sixclones were tested for p24-specific cytolytic activity at an E:Tratio of 100:1 (Fig. 6B). All six clones exhibited cytolytic activ-ity, confirming that the observation of cytolytic CD4� T-cellclones was not limited to one individual. The six clones werealso tested for cytolytic activity directed against the 22-amino-acid peptide each was found to recognize in a proliferationassay. Four of the six clones maintained levels of specific lysisgreater than 25% at an E:T ratio of 10:1 (Fig. 6C). Theseresults provide evidence at a clonal level that HIV-1-specific Thelper cells possess multiple effector functions, including pro-liferation, IFN-� secretion, and cytolysis.

Cytolytic activity occurred at doses of peptide similar tothose required to induce proliferation and IFN-� production.The in vivo significance of these cytolytic T helper cell clonesis unknown. In order to determine if the cytolytic function ofthese clones was artificially induced by concentrations of anti-gen much higher than those required for proliferation andIFN-� production, we performed a peptide titration. Figure 7Ademonstrates that a peptide concentration of 1 �g/ml was ableto elicit proliferation, IFN-� production, and cytolysis in theclone from subject CTS-01, implying that the cytolysis ob-served was not merely an artifact due to unusually high antigenconcentrations. Identical results were obtained with a clonefrom subject AC-25 (data not shown).

Lysis was mediated by the perforin pathway. The findingthat these Gag-specific T helper cell clones mediated cytolyticactivity prompted us to examine the mechanism of cell lysis.We focused our attention on clone 16 from subject CTS-01 andclone 4 from subject AC-25. The two main mechanisms of

FIG. 3. Ten p24-specific lines from donor CTS-01 were assayed fortheir ability to lyse B-LCL pulsed with p24 protein or peptide 9. Astandard chromium release assay was performed, with results ex-pressed as percent specific lysis. Negative controls included B cellspulsed with no antigen or with a control 22-amino-acid peptide derivedfrom a portion of p24 not recognized by the subject. Lines wereassayed at an E:T ratio of 100:1. Error bars were calculated by deter-mining the standard deviation of assay results performed on at leasttwo separate occasions.

FIG. 4. Fine mapping of the peptide-specific proliferative, cytotoxic, and Elispot responses of clone 16 from subject CTS-01. The peptidesequences are given on the left, followed by the graphs of (A) proliferation, (B) cytotoxicity, and (C) Elispot responses. Peptides that containedthe minimal epitope are in bold. Elispot results were expressed as SFC/106 clone cells added. Antigen concentration was 1 �g/ml for theproliferation and Elispot assays and 10 �g/ml for the cytotoxicity assays. In each assay the minimal epitope recognized was VHAGPIAPG.

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CD4� T-cell cytotoxicity that have been reported include per-forin-mediated and Fas-mediated lysis (21, 32). Additionally,CD4� CTL have been shown to secrete IFN-�, granzyme A,and tumor necrosis factor (TNF)-� and -� (11, 29, 47, 54).Serine esterase granule secretion was measured after specificstimulation in the clone from subject AC-25 (Fig 7B). Peptide4 from p24 was able to elicit serine esterase release as mea-sured by conversion of the substrate BLT, implying that theclones contained and could release lytic granules. Further-more, chelation of extracellular calcium with EGTA has beenused as a method of selectively blocking perforin-mediatedlysis. We examined lysis mediated by the AC-25 clone in thepresence of EGTA and found it to be abrogated, implying thatthe clone utilized the perforin pathway of lysis (Fig. 7C). Per-forin staining of the clone from AC-25 was performed and was6 to 8 times the background staining of B-LCL or staining withcontrol immunoglobulin (data not shown).

Concanamycin A and brefeldin A have been used as inhib-itors of perforin-mediated and Fas-mediated cytotoxicity, re-spectively, to differentiate between these two mechanisms oflysis (24). Concanamycin A, an inhibitor of vacuolar-type H�-ATPase, accelerates degradation of perforin by increasing thepH of lytic granules. Brefeldin A inhibits intracellular glyco-protein transport and was shown to selectively block Fas-me-diated killing. Anti-Fas antibody can also block Fas-mediatedkilling (55). We found that concanamycin A could abrogatelysis in a dose-dependent fashion in the clone from subjectCTS-01 (Fig. 8A). Brefeldin A and anti-Fas antibody over thesame concentration range showed no effect on cytolytic activ-ity. To demonstrate that the clones retained functions otherthan perforin secretion in the presence of concanamycin A, wequantitated IFN-� production in the presence of concanamy-cin A in the clone from subject AC-25 (Fig. 8B). At the highestconcentration of concanamycin A, IFN-� production was ab-rogated. However, IFN-� production was unaffected over a

range of concentrations of concanamycin A that resulted incomplete abrogation of lysis (Fig. 8C), supporting the notionthat perforin secretion was selectively inhibited by concanamy-cin A. These results imply that the Gag-specific T helper cellresponses utilized a perforin-dependent pathway in killing tar-gets.

DISCUSSION

Although HIV-specific T helper cells are a central compo-nent in antiviral control, an in-depth characterization of theseimmune responses has not been performed. We report that aCD4� T-cell clone specific for a discrete 9-amino-acid peptidein the HIV-1 Gag protein responds to antigen by proliferation,IFN-� secretion, and perforin-mediated lysis of p24-pulsedtarget cells. Moreover, we extend these studies to show thatcytolytic activity is a frequently observed in vitro functionalactivity of HIV-1-specific T helper cell clones.

The extent to which cytolytic activity of T helper cells playsa role in vivo in controlling infections is not clear and remainscontroversial. In an experimental system with herpes simplexvirus type 1-specific T-cell lines, cytolytic activity was demon-strated to be mediated solely by natural killer and CD4� CD8�

T cells (45). No herpes simplex virus type 1-specific CD8� Tcells were found, indicating that under certain circumstancesCD4� CTL may exert immune control of viral infections.Given that a number of cells infected by HIV-1 also expressHLA class II molecules, one could speculate that CD4� CTLcould play a role in control of HIV-1 infection. Alternatively,deletion of HIV-1-specific antigen-presenting cells could havea downregulatory effect on the immune response and interferewith the delivery of help to CD8� CTL.

Few data exist showing a direct correlation between CD4�

cytolytic T cells and disease progression. In a murine model ofinfluenza virus infection, protection from a lethal challenge

FIG. 5. HLA restriction of CD4� T-cell clone 16 from subject CTS-01. Antigen was presented to the clone by partially HLA matched B-LCLpulsed overnight with peptide 9. Negative controls included no antigen or peptide 23, a 22-amino-acid peptide from p24 not recognized by thesubject’s PBMC (data not shown). Only B-LCL which expressed the DQ7 allele elicited antigen-specific responses in proliferation, cytotoxicity, andIFN-� Elispot assays. The top scale is for net cpm, and the bottom scale is for percent lysis and SFC/106 cells. An E:T ratio of 10:1 was used forcytotoxicity assays.

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and over 100-fold reduction in lung virus titers was observedwith adoptive transfer of a Th1 influenza virus-specific CD4�

T-cell clone with cytolytic activity (14, 33). Th2-type CD4�

T-cell clones were found to have neither cytolytic ability northe ability to protect from lethal influenza virus challenge inthis system. In a Friend retrovirus infection model, CD4�

T-cell clones were shown to possess cytolytic activity and toinhibit the production of virus in vitro (19). The clones that weisolated secreted IFN-� and possessed cytolytic activity on spe-cific stimulation, consistent with the Th1-type clones identified

in murine studies. HIV-1-specific CD4� CTL have primarilybeen described in the setting of gp160-vaccinated, seronegativevolunteers (15, 37, 39, 48) and were found to be both Th0 andTh1 phenotypes (49). Few reports have demonstrated the ex-istence of HIV-1-specific CD4� CTL in natural infection (31,46), and in some reports HIV-1-specific CD4� T helper cellclones possessed no cytolytic activity (34), while the majority ofthe clones that we isolated possessed HIV-1-specific cytolyticactivity. Efforts to demonstrate HIV-1-specific cytolytic activityfrom PBMC have met with mixed results (16, 28), and there-

FIG. 6. (A) Proliferative responses of each of six CD4� T-cellclones derived from three individuals treated during acute HIV-1infection are shown. The clones are labeled 1 to 6, with the first threederived from subject AC-01, the next two from subject AC-25, and thelast from subject AC-36. Peptides 3 (amino acids 153 to 174), 4 (aminoacids 163 to 184), 10 (amino acids 223 to 244), 13 (amino acids 253 to274), 14 (amino acids 263 to 284), and 19 (amino acids 313 to 344) wererecognized by one or more clones. (B) The six clones were tested forcytolytic ability. Autologous B-LCL were incubated with 51Cr andsoluble p24 and used as targets in a 4-h CTL assay at an E:T ratio of100:1. Spontaneous lysis ranged from 10 to 36%. Negative controlsincluded B-LCL without added antigen. (C) The same six clones wereassayed for their ability to kill B-LCL targets pulsed with the 22-amino-acid peptide within p24 recognized by each clone (see panel A). Theassays were performed at an E:T ratio of 10:1, and negative controlsincluded B-LCL without added antigen.

FIG. 7. (A) Dependence of proliferative, cytolytic, and IFN-� se-cretion activity on peptide concentration. Autologous B cells wereincubated with peptide 9 from p24 or with the control peptide 23 fromp24 (not recognized) from 0 to 10 �g/ml. CD4� T-cell clone responsesfrom subject CTS-01 were measured in each of the three assays andexpressed as stimulation index (SI), percent lysis (CTL), and SFC per100. (B) Elaboration of serine esterase upon antigen-specific stimula-tion of clone 4 from subject AC-25. Autologous B cells were incubatedwith peptide 4 from 0 to 10 �g/ml prior to use as antigen-presentingcells. Serine esterase release was quantified by modification of thesubstrate BLT, and results were expressed as OD405, with higher ODcorresponding to greater granule secretion. The dotted line indicatesthe background release in response to the target cells without thepeptide. (C) Abrogation of lysis mediated by clone 4 from subjectAC-25 in the presence (E) and absence (F) of 2 mM Mg2� and EGTA.Chelation of extracellular calcium caused abrogation of lysis over therange of peptide concentrations.

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fore it is unclear whether the clones that we isolated are aresult of several rounds of in vitro stimulation or whether theyexist in vivo.

The observation that a 9-amino-acid peptide is sufficient for

activation of CD4� T cells is in keeping with recent studiesbased on crystallization of murine major histocompatibilitycomplex (MHC) class II molecule I-Ak with peptide and theT-cell receptor (42). Larger peptides are accommodated in theMHC class II binding pocket, but only 9 amino acids wereshown to directly contact the T-cell receptor and MHC class IIprotein. HLA-DQ7 has been described to bind molecules lack-ing a P1 anchor residue, binding simple alanine octamers andeven hexamers (41). More recent work modeling putative pep-tide-binding motifs predicted four essential pockets that couldinteract with peptides at positions P1, P4, P6, and P9, with P1interacting with the N-terminal portion of the peptide (26). P1was predicted to interact with aromatic or small hydrophobicresidues, P4 with small residues, P6 with small hydrophobic orpolar residues, and P9 with large amide, polar, or small resi-dues. The peptide that we delineated supported the abovemodel, as it matched the motif at all predicted pocket-bindingpositions: P1 (valine), P4 (glycine), P6 (isoleucine), and P9(glycine). HLA-DQ7-restricted clones have been described ina number of other diseases, such as melanoma (38), humanpapillomavirus type 1 (50), and hepatitis C virus (10). Epide-miological studies have linked HLA-DQ7 with clearance ofinfection with hepatitis C virus (1, 36) and persistence of hep-atitis B virus. While DR13 and DQ6 have been associated withdelayed progression to AIDS (25), DQ7 has not been corre-lated with disease progression in HIV-1.

The epitope recognized by the clones derived from subjectCTS-01, VHAGPIAPG, is contained within the binding se-quence of Gag p24 to cyclophilin A, thought to be crucial to anearly step in the HIV-1 viral life cycle (7). The epitope recog-nized by these clones is from a relatively conserved region ofGag (27). The critical interaction of Gag and cyclophilin Acould provide pressure against viral mutation in this region ofthe genome, making immune escape more difficult at thisepitope. Further studies will be needed to address sequencevariation of autologous viral isolates from subject CTS-01 andother persons who target this epitope. Likewise, fine charac-terization of other epitopes may define additional targetedepitopes within functional HIV-1 domains.

The CD4� T-cell clones we studied here exhibited antiviralactivity via proliferation, IFN-� secretion, and p24-specific cy-tolysis that appears to be perforin mediated. Further studieswill be required to determine if CD4� T cells can play a directrole in antiviral control. The demonstration of effector activityin CD4� T-cell clones provides further impetus to includeCD4� T-cell epitopes in future vaccines. Fine epitope mappingand HLA restriction of responses provide an important firststep in identifying areas to target in immunotherapeutic inter-ventions.

ACKNOWLEDGMENTS

This work has been supported by NIH grants AI01698–01, AI01541,and AI40873. P.J.N. is grateful for funding from Cable Positive. P.J.N.,E.S.R., and B.D.W. are supported by the Doris Duke Charitable Foun-dation. B.D.W. is a Doris Duke Distinguished Clinical Science Profes-sor.

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FIG. 8. (A) Effects of concanamycin A, brefeldin A, and anti-Fasantibody on cytolytic activity of the clone from subject CTS-01. T cellswere incubated with various concentrations of concanamycin A, brefel-din A, or anti-Fas antibody for 2 h preceding a standard 4-h chromiumrelease assay. The E:T ratio was 10:1, and B-cell targets were pulsedwith 10 �g/ml of peptide 9 overnight prior to the assay. Similar resultswere obtained with clone 4 from subject AC-25. Spontaneous lysisranged from 25 to 35%. (B) IFN-� secretion after stimulation withpeptide 4 was measured in clone 4 from subject AC-25 in the presenceof various concentrations of concanamycin A (CMA). B-LCL werepulsed with peptide 4 and added at an E:T ratio of 100:1. The per-centage of CD4� T cells secreting IFN-� is listed in the upper right ofeach dot plot. (C) Cytolysis was measured in clone 4 from subjectAC-25 in the presence of various concentrations of concanamycin Aand compared to IFN-� production (from panel B). B-LCL werepulsed with peptide 4 and added at an E:T ratio of 10:1 for the CTLassay. While abrogation of IFN-� secretion was seen at the highestconcentration of concanamycin A, lysis was completely inhibited atconcentrations of concanamycin A that had no effect on IFN-� secre-tion. The left axis gives percent IFN-� production and percent lysis.

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