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The Journal of Immunology, 1998, 161: 513-519.
Copyright © 1998 by The American Association of Immunologists

In Vitro Effects of IL-12 on HIV-1-Specific CTL Lines from HIV-1-Infected Children1

Elizabeth J. McFarland2,*,{dagger},§, Paul A. Harding{dagger}, Samantha MaWhinney{ddagger}, Robert T. Schooley{dagger} and Daniel R. Kuritzkes{dagger}

Department of * Pediatrics, {dagger} Division of Infectious Diseases, and {ddagger} Department of Biometrics and Preventative Medicine, University of Colorado Health Sciences Center and Veterans Affairs Medical Center, Denver, CO 80262; and § Department of Pediatrics, The Children’s Hospital, Denver, CO 80218


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied the in vitro effects of IL-12 on HIV-1-specific CTL lines derived from PBMC of HIV-1-infected children. HIV-1-specific CTL lines were derived by limiting dilution following Ag-specific stimulation of PBMC from HIV-1-infected children and were maintained with repeated anti-CD3 stimulation. Following incubation with IL-12 for 5 to 7 days, HIV-1-specific cytotoxicity was augmented in a dose-dependent fashion (mean increase, 94 ± 83 lytic units; p = 0.0006). Experiments performed with CD3-blocking Abs and MHC-mismatched targets demonstrated that the IL-12-enhanced activity was MHC restricted and dependent on cells bearing CD3. The effect of IL-12 on proliferation of the CTL lines as tested by [3H]TdR uptake was minimal, with stimulation indexes ranging from 1.25 to 4.9. The effects of IL-12 on cytotoxicity were not significantly altered by addition of Ab to the IL-2R (anti-Tac) in quantities sufficient to block exogenous IL-2 (p = 0.15), demonstrating that endogenous IL-2 activity is not required for IL-12-enhanced cytolytic activity. Likewise, addition of neutralizing Ab specific for IFN-{gamma} did not change IL-12-enhanced cytotoxicity (p = 0.61). The in vivo role of IL-12 in the generation and the stimulation of CTL remains to be determined; however, its ability to augment HIV-1-specific CTL in vitro adds additional support for IL-12 as a candidate for immune-based therapy of HIV-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a 70-kDa heterodimeric cytokine predominantly produced by monocytes. It is thought to play an important role in the development of Th1 cells and cell-mediated immune responses (for review, see 1 . However, the role of this cytokine in the generation and stimulation of CTL responses is not fully characterized. IL-12 augments the generation of allogeneic and anti-tumor CTL responses in vitro in both human and murine systems (2, 3, 4, 5). Cytotoxicity of human CD8+ cells against anti-CD3 Ab-coated cells (6) or against anti-CD3-producing hybridoma cells is also enhanced by IL-12 in vitro (7). Less is known about the effects of IL-12 on virus-specific CTL. Stimulation in the presence of IL-12 enhances influenza-specific cytotoxicity in unfractionated PBMC (8). However, administration of high dose IL-12 to mice chronically infected with lymphocyte choriomeningitis virus leads to diminished CD8+ cell expansion and inhibites the lymphocyte choriomeningitis virus-specific CD8+ cell response due to induction of TNF (9).

Alterations in IL-12 physiology may play an important role in the pathogenesis of HIV-1 infection. IL-12 production is diminished in PBMC isolated from HIV-1-infected adults and children even at early disease stages (10, 11, 12). IL-12 augments in vitro several of the immune functions deficient in PBMC from HIV-1-infected individuals, including NK cell lysis (13), IFN-{gamma} production from PBMC (13, 14), lymphocyte proliferative responses to Ag-specific stimulation (13, 14, 15), and the generation of Th1-type CD4+ and Tc1-type CD8+ cells from cloned PBMC (16, 17).

HIV-1-specific CTL are likely to be a critical component of the host immune response to HIV-1 infection. HIV-1-infected adults at advanced stages have lower HIV-1-specific precursor CTL (pCTL)3 frequencies compared with asymptomatic HIV-1-infected adults (18, 19). Cross-sectional and longitudinal studies in infected adults suggest an association of high HIV-1-specific pCTL frequency with a stable disease course (19, 20, 21).

If HIV-1-specific CTL are indeed important to the control of HIV-1 viremia, then cytokines such as IL-12 that enhance CTL may be of therapeutic value. We therefore studied the effect of IL-12 on HIV-1-specific CTL derived from the PBMC of HIV-1-infected children. Children with vertically acquired HIV-1 infection are of particular interest, since circulating activated HIV-1-specific CTL are detected less frequently in children than in infected adults despite the presence in the children of high frequencies of HIV-1-specific pCTL (22, 23). This report shows that IL-12 enhances HIV-1-specific cytotoxicity of CD8+ CTL in a dose-dependent fashion. The enhanced activity is MHC restricted and CD3 dependent and does not require endogenous IFN-{gamma} or IL-2 production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

Subjects were recruited at the Infectious Diseases Clinic of The Children’s Hospital (Denver, CO). HIV-1-specific CTL lines were derived from three children with vertically acquired HIV-1 and one who was transfused with HIV-1-contaminated blood during the perinatal period. The subjects selected had previously been demonstrated to have pCTL specific for at least one HIV-1 Ag. Clinical staging of HIV-1 disease was based on the Centers for Disease Control classification system for children under 13 yr of age (24).

Abs and cytokines

The CD3-specific mAb 12F6 was provided by J. Wong (Massachusetts General Hospital, Boston, MA) (25). mAb specific for CD56 and CD16 (Leu 11b) were purchased from Becton Dickinson (San Jose, CA), polyclonal rat IgG was obtained from Sigma (St. Louis, MO), and human IL-2R {alpha} chain blocking mAb (anti-Tac, anti-CD25) and human IFN-{gamma} neutralizing mAb (anti-IFN-{gamma}) were purchased from Genzyme (Cambridge, MA). Purified human IL-2 was purchased from Schiapparelli Biosystems (Columbia, MD). Recombinant human IL-12 (bioactivity, 1.7 x 107 U/mg) and recombinant human IL-2 were gifts from Genetics Institute (Cambridge, MA) and Hoffmann-La Roche (Nutley, NJ), respectively. IFN-{gamma} was detected by ELISA (R&D Systems, Abingden, U.K.).

Recombinant vaccinia viruses

Vaccinia recombinant vDK1, which expresses HIV-1 Gag, was constructed in our laboratory (26). Recombinant vaccinia vector expressing HIV-1 RT (vCF21) as well as an isogenic control vaccinia recombinant expressing Esherichia coli ß-galactosidase (Lac; vSC8) were provided by B. Moss (National Institute of Allergy and Infectious Diseases, Bethesda, MD).

Target cell lines

B lymphoblastoid cell lines (B-LCL), established by EBV transformation, and the erythroleukemia cell line K562 (CCL 243, American Type Culture Collection, Rockville, MD), which served as a target for NK cells, were maintained in R-20 (RPMI 1640, heat-inactivated 20% FBS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin).

Preparation of stimulator and target cells

Stimulator cells were B-LCL infected with recombinant vaccinia vectors and incubated overnight at 37°C. Cells were washed, suspended in 1 ml of R-10 (R-20 with 10% FBS) containing 10 µg/ml psoralen (4'-aminomethyl-4–5'-8-trimethylpsoralen hydrochloride; Sigma) and exposed to UV light for 10 min. This procedure inactivates viral and cellular DNA and RNA while maintaining antigenicity (27). Target cells were B-LCL infected with recombinant vaccinia viruses and incubated overnight at 37°C with Na2 [51Cr]O4 (50 mCi/ml; DuPont, Boston, MA).

Generation and culture of CTL lines

CTL lines specific for HIV-1 were established by a two-step protocol adapted from Lubaki et al. (27). In brief, PBMC obtained from HIV-1-infected patients were suspended in R-10 containing 5% purified human IL-2 and incubated with stimulator cells at a PBMC:stimulator ratio of 10:1 for 14 days. Cells were fed every 3 to 4 days by partial medium exchange and were tested for HIV-1-specific activity using a standard 51Cr release assay. If HIV-1-specific cytotoxicity was detected, PBMC were seeded at 10, 5, 1, and 0.3/1 cells/well into 96-well flat-bottom plates in R-10–100 (R-10 with 100 U/ml rIL-2) and stimulated with anti-CD3 mAb, 12F6 (0.05 µg/ml), and irradiated (5000 rad) allogeneic PBMC from HIV-1-seronegative donors as feeder cells (2 x 105 cells/well). At 2 wk, cells were restimulated with 12F6 and irradiated feeders cells (1 x 105 cells/well). On day 28, wells were assayed for HIV-1-specific cytotoxicity. Cell lines from positive wells were expanded and maintained during long term propagation in R-10–100 with periodic restimulation by addition of 12F6 and allogeneic, irradiated feeder cells every 10 to 14 days. CTL lines were used for assays at 10 to 14 days after anti-CD3 restimulation, except as otherwise noted.

Preparation of effector cells

In experiments to test the effects of IL-12 on cytotoxic activity, CTL lines were washed and resuspended in R-10 with 12F6 (0.05 µg/ml) in the absence of feeder cells in R-10. IL-12 was added at time zero or after 24 h. 51Cr release assays were performed after 5 days of culture. Preliminary experiments demonstrated that the effects of IL-12 on cytotoxicity were optimal at 5 days. In experiments to assess IL-2 dependency, IL-12 cultures were performed with and without anti-Tac (10–20 µg/ml) or with polyclonal rat IgG as a control for nonspecific inhibition of IL-2 activity. In experiments assessing IFN-{gamma} dependency, cultures were performed with or without anti-IFN-{gamma} (10 µg/ml). This Ab at 0.6 µg/ml will neutralize 50% of the bioactivity due to 5 ng/ml of IFN-{gamma} (28). To determine the blocking effect of anti-CD3 on CTL activity, effector cells were preincubated for 30 min at 37°C with various concentrations of anti-CD3 mAb (12F6) or a control mAb (anti-CD56) just before the 51Cr release assay.

Cytotoxicity assays

Target cells were aliquoted into round-bottom 96-well microtiter plates at 5 x 103 target cells/well. Effector cells were added to wells at three E:T cell ratios in a final volume of 200 µl. Plates were incubated at 37°C for 4 or 6 h, and the release of 51Cr into the supernatant was determined by measuring counts per minute on a beta counter (model 1279, Packard, Meridan, CT). Maximum 51Cr release was determined by detergent lysis of target cells. Spontaneous release was determined by incubating target cells in the absence of effector cells. Spontaneous release was <20% of maximum release in all reported assays. The mean percent cytotoxicity was determined from triplicate wells by the formula: % cytotoxicity = {[(cpmeffector) - (cpmspontaneous)]/[(cpmmaximum) - (cpmspontaneous)]} x 100. HIV-specific cytotoxicity = % HIV cytotoxicity - % control cytotoxicity. Lytic units (LU) were determined as the number of effectors cells required for 20% HIV-1-specific lysis and were expressed per 106 cells.

Proliferation assay

CTL lines suspended in R-10 medium were aliquoted in triplicate into 96-well plates (1 x 105 cells/well). CTL lines stimulated with 12F6 (0.05 µg/ml), were cultured for 5 days with various concentrations of IL-12 or rIL-2. On days 3 and 5, cells were pulsed with 1 µCi of [3H]TdR (DuPont) for 6 h. The cultures were harvested on a Luma unifilter plate (Packard), and incorporated counts per minute were determined using a beta counter.

Phenotypic analysis of cell surface markers

The expression of cell surface markers was analyzed by three-color flow cytometry on an EPICS III flow cytometer (Coulter, Hialeah, FL) using fluorescein-, rhodamine-, or Tri-Color (tandem dye)-conjugated mAb specific for cell surface markers CD3, CD4, CD8, and CD56 (all from Coulter); CD45 (Caltag, San Francisco, CA); and CD28, CD38, and HLA-DR (PharMingen, San Diego, CA).

HLA typing

HLA typing was performed at the University of Colorado Health Science Center Tissue Typing Laboratory using standard serologic techniques.

Statistical analysis

Statistical analyses were performed using Splus statistical software Mathsoft, Seattle, WA). Regression models were used to estimate the mean value for lytic units under various Ab and cytokine treatments. All statistical models controlled for individual clone effects. All tests were two-sided, with a significance level of {alpha} = 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of HIV-1-specific CTL lines derived from PBMC from HIV-1-infected children

We used HIV-1-specific CTL lines to perform a detailed analysis of the effects of IL-12 on HIV-1-specific cytotoxic effector cells. Gag- and RT-specific CTL lines were generated from PBMC of four subjects as described in Materials and Methods. The stimulation Ags, Gag and RT, were selected because pCTL specific for these Ags had been previously demonstrated in PBMC from these subjects. Representative CTL lines from each subject were selected for detailed analysis. Characteristics of the subjects and their CTL lines are given in Table IGo. Subjects included children at disease stages N1, N2, and B3. Specific cytotoxicity against HIV-1 Gag- or RT-expressing autologous B-LCL ranged from 20 to 56% at E:T cell ratios of 5:1. The CTL lines were predominantly (>=89%) CD3+CD8+, except for CTL line P076 OG3, which was a mixture of CD3+CD4+ (23%) and CD3+CD8+ (73%) cells. In this line, all cytolytic activity was mediated by CD3+CD8+ cells, as demonstrated by assays performed after depletion of CD8+ cells (data not shown). Assays using HIV-1-expressing heterologous B-LCL as target cells confirmed that the Gag- and RT-specific cytotoxicity of these CTL lines was MHC restricted (data not shown). HLA A11, B8, and B51 were identified as the restricting alleles for several of the CTL lines.


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  		Table I. Characteristics of subjects and HIV-1 specific cytotoxic T lymphocyte lines

 
IL-12 enhances the cytotoxic activity of HIV-1-specific CTL lines

To determine the effect of IL-12 on the cytolytic activity of HIV-1-specific CTL, cells were stimulated with 12F6 in the absence of feeder cells and were cultured for 5 days with IL-12 at various concentrations or with IL-2 (100 U/ml). Cells cultured in the absence of exogenous cytokine served as a control. IL-12 increased the specific cytotoxic activity of 16 CTL lines from 4 subjects with an average increase of 94 ± 83 LU (p = 0.0006; Fig. 1Go). IL-12 enhanced HIV-1-specific cytotoxicity in a dose-dependent fashion. Maximum increases in HIV-1-specific cytotoxicity were observed at IL-12 concentrations of 1 to 10 ng/ml. Dose-response data from a representative CTL line are shown in Figure 2Go. The maximum increases in cytotoxicity induced by IL-12 were less than the maximum increases induced by IL-2 (data not shown).



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  		FIGURE 1. IL-12 enhances HIV-1-specific cytotoxic activity of CTL lines. Before the 51Cr release assay, CTL lines were restimulated with 12F6 in the absence of feeder cells and cultured in medium with exogenous IL-12 (1 or 10 ng/ml) or without exogenous cytokine for 5 days. Data are shown as lytic units.

 


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  		FIGURE 2. IL-12 enhances the cytotoxicity of HIV-1-specific CTL lines in a dose-dependent manner. Before the 51Cr release assay, HIV-1-specific CTL lines were restimulated with 12F6 in the absence of feeder cells and were cultured for 5 days with various concentrations of IL-12. Data shown for CTL line P079 C10D are representative of three other lines tested.

 
To verify that the cytolytic activity stimulated by IL-12 was MHC restricted and mediated via TCR-dependent interactions, experiments were performed with MHC-mismatched target cells and blocking Abs. Complete inhibition of cytotoxicity was observed in the presence of Ab to CD3 but not with control Ab (Fig. 3Go). To demonstrate that IL-12-enhanced cytotoxicity was MHC restricted, CTL lines were tested against heterologous B-LCL. No HIV-1-specific lysis by IL-12-stimulated CTL lines was observed when B-LCL mismatched at all HLA alleles were used as targets (Fig. 4Go). A limited analysis of cell surface activation markers was performed by FACS. Three lines that responded to IL-12 with a vigorous increase in cytotoxicity (P076 L4E, P076 L9D, P076 J8E) expressed CD28 on 23, 9, and 7% of cells, respectively, after 110 days in culture. These lines were 100% HLA-DR+ and 20 to 30% CD38+.



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  		FIGURE 3. IL-12-enhanced CTL activity is mediated by CD3+ cells. CTL lines were restimulated with 12F6 and cultured with IL-12 for 5 days before use as effectors in a 51Cr release assay. Effectors were preincubated with various concentrations of 12F6 or control Ab (6.25 µg/ml). Data are shown for P005 KE3 and P076 OG3 at E:T cell ratios of 5:1 and 2.5:1, respectively. Error bars designate 1 SE. nd, not done.

 


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  		FIGURE 4. IL-12-enhanced CTL activity is MHC restricted. CTL lines were restimulated with 12F6 and cultured with or without IL-12 for 5 days before use as effector cells in a 51Cr release assay against autologous and MHC-mismatched B-LCL. The CTL line ND11 from subject P076 was tested against autologous B-LCL and B-LCL from subject P005. The CTL line LD11 from subject P005 was tested against autologous and B-LCL from subject HOBR. HLA alleles for the target lines were as follows: P076 (A11, 11; B7, 35), P005 (A1, 2; B8, 44), and HOBR (A3, 26; B7, 35). Data shown are for E:T cell ratios of 2.5:1. Error bars designate 1 SE.

 
Other investigators have noted a blunted response to IL-12 when it is added to the CD8+ lymphocytes >24 h after stimulation of the cells with CD3 mAb (7). To determine whether the effect of IL-12 on the activity of HIV-1-specific CTL lines depended on recent TCR stimulation, CTL lines previously stimulated with 12F6 and cultured with rIL-2 and feeder cells for 13 days were washed, resuspended in fresh medium with or without 12F6, and grown in the presence of IL-12 for an additional 5 days before cytotoxicity assay. An increase in HIV-1-specific cytotoxicity was observed in the presence of IL-12 regardless of the timing of CD3 stimulation (Fig. 5Go).



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  		FIGURE 5. Recent TCR activation is not required for IL-12 enhancement of cytotoxicity. CTL lines, 13 days after anti-CD3 stimulation, were washed and resuspended with (0 day after anti-CD3) or without (13 days after anti-CD3) 12F6. The cells were cultured in the presence or the absence of IL-12 and tested for HIV-1-specific cytotoxicity by 51Cr release assay after 5 days. Data are shown in lytic units for CTL lines L6E ({blacksquare}), AB7 ({diamondsuit}), and CF10 (•).

 
Effect of IL-12 on proliferation and growth of CTL lines

Although IL-12 consistently increased the cytolytic activity of CTL lines, it had a minimal effect on cell proliferation as determined by [3H]TdR uptake. A modest increase in proliferation was observed 3 days after exposure to IL-12 (100 ng/ml) with stimulation indexes ranging from 1.25 to 4.9. No proliferative effect persisted 5 days after IL-12 addition (data not shown). There was no defect in the ability of the CTL lines to proliferate, since marked proliferation was observed when the cells were cultured with rIL-2 (stimulation index, 60–150).

IL-12-enhanced HIV-1-specific cytotoxicity does not require endogenous IL-2 and IFN-{gamma}

To determine whether the effect of IL-12 on CTL activity was dependent on endogenous IL-2, experiments were performed in the presence and the absence of Ab to block the IL-2R {alpha}-chain (anti-Tac). Addition of anti-Tac to CTL lines at the time of IL-12 stimulation resulted in reduced cytotoxicity in some assays, but overall the mean reduction of -78 ± 70 LU was not statistically significant (p = 0.15; Fig. 6Go). By contrast, anti-Tac effectively blocked the action of exogenous IL-2 (1 U/ml), resulting in a mean reduction of HIV-1-specific cytotoxicity of -194 ± 91 LU (p = 0.0012). Anti-Tac also reduced the cytolytic activity of CTL lines grown in the absence of exogenous cytokine, most likely by blocking the action of endogenous IL-2 (data not shown).



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  		FIGURE 6. IL-12 effects on HIV-1-specific cytotoxicity do not require IL-2. CTL lines were restimulated with 12F6 and cultured with exogenous cytokine, IL-12 (10 ng/ml), or IL-2 (1 U/ml) in the presence or the absence of anti-IL-2R (anti-Tac). After 5 days of culture, cells were tested for HIV-1-specific cytotoxicity by 51Cr release assay. Data are shown as lytic units for each assay performed with five lines: J8E ({blacksquare}), L9D ({blacktriangleright}), L4E (•), L6E ({blacktriangleup}), and C10D ({blacktriangledown}).

 
IL-12 is a potent inducer of IFN-{gamma} from T and NK cells (29). We wanted to determine whether the action of IL-12 on cytolytic activity in these cell lines was dependent on endogenous IFN-{gamma} production. Three lines, each with marked increases in cytotoxicity induced by IL-12, were cultured with IL-12 in the presence or the absence of neutralizing Ab for IFN-{gamma} (10 µg/ml). Culture supernatants were collected daily and tested for IFN-{gamma} by ELISA. The lines produced little IFN-{gamma} in response to IL-12, with the peak concentrations ranging from 11 to 31 pg/ml during culture in the absence of anti-IFN-{gamma}. In cultures with neutralizing mAb, no IFN-{gamma} was detected in supernatants. Blocking endogenous IFN-{gamma} had no effect on the IL-12-enhanced HIV-1-specific cytotoxicity (p = 0.61; Fig. 7Go).



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  		FIGURE 7. IFN-{gamma} independence of IL-12 effects on HIV-1-specific cytotoxicity. CTL lines were restimulated with 12F6 and cultured with IL-12 (10 ng/ml) or no exogenous cytokine in the presence or the absence of anti-IFN-{gamma} (10 µg/ml). After 5 days of culture, cells were tested for HIV-1-specific cytotoxicity by 51Cr release assay. Representative data are shown for one of three lines tested. Error bars designate 1 SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that IL-12 increases the cytolytic activity of HIV-1-specific CTL lines derived from PBMC of HIV-1-infected children. The cytolytic activity augmented by IL-12 was mediated by CD3+CD8+ lymphocytes and was MHC restricted. Other studies have demonstrated augmented in vitro cytolytic activity of human CD8+ lymphocytes against allogeneic, anti-CD3-bearing, and influenza virus-infected targets (2, 3, 7, 8). Our results demonstrate that HIV-1-specific CTL responses in human CD8+ lymphocytes are likewise enhanced by stimulation with IL-12 in vitro.

Our subjects were children, aged 3–13 yr. Studies of immune function in children have indicated that most immunologic responses approach adult levels by or before 6 yr of age (30). Thus, the effect of IL-12 on CTL responses observed in our cohort is likely to be similar to that which would be expected in adults. Infected infants show a delayed appearance of HIV-1-specific CTL and have lower levels of circulating, activated HIV-1-specific CTL than adults, but little is known of the mechanisms responsible for that deficiency (22, 23). It would be of interest to determine whether CD8+ lymphocytes isolated from such infants differ from those of older children in their response to IL-12.

We studied the effects of IL-12 on CTL lines presumably derived from circulating HIV-1-specific pCTL. HIV-1-specific pCTL are detected at high frequency in the peripheral blood of infected children and adults until advanced stages of disease (19, 20, 23, 31). Our results do not address the effect of IL-12 on the generation of primary HIV-1-specific CTL responses or the expansion of the pCTL pool. It will be important to perform further investigations into the contribution of IL-12 to the generation of primary HIV-1-specific CTL responses.

HIV-1-specific CTL lines were derived from subjects at various stages of immune deficiency. Although isolation of CTL lines is more difficult from individuals with advanced disease (E. J. McFarland P.A. Harding, and D.R. Kuritzkes, unpublished observations), the response to IL-12 by CTL lines from patient P005 (CD4 lymphocyte count, 39/mm3) was similar to that of CTL lines from patients with earlier stages of disease. This result suggests that there is no intrinsic defect in the IL-12 responsiveness of CTL lines from a patient with advanced disease. By contrast, in vitro proliferative responses to microbial recall Ags were not enhanced by IL-12 when PBMC from patients with CD4 counts <200/mm3 were tested (15).

Although IL-12 consistently augmented HIV-1-specific cytolytic activity of the CTL lines, it had only a modest effect on the proliferation of these cells. Stimulation of cell proliferation by IL-12 was short-lived and was much less than that observed with IL-2. The low level of IL-12-induced proliferation was not an indication of anergy, since the cells proliferated well in the presence of IL-2. Our results are in concordance with those of a previous study in a murine model in which stimulation of allogeneic CTL responses by IL-12 occurred without an increase in cell proliferation (4). However, these results differ from those of several other studies of unfractionated or purified CD8+ lymphocytes in which enhancement of cytolytic activity was associated with increased proliferation (2, 7, 32, 33, 34, 35).

Several factors could account for these differences. Our experiments were performed with cell lines, which may differ from unselected lymphocytes in their ability to proliferate in response to IL-12. Minimal enhanced proliferation was seen in a human Th1 clone cultured with IL-12 (36). Alternatively, it is possible that a stimulatory cofactor is lacking in our system. The proliferative response to IL-12 is markedly enhanced by costimulation of CD28 (37). As observed in other studies of long term cell cultures (38), only a small fraction (7–30%) of the cells in our CTL lines expressed CD28. This may explain the low level of IL-12-induced proliferation observed in these cells lines. The finding that IL-12 enhances cytolytic activity without a significant effect on proliferation suggests that the observed increase in cytotoxicity is most likely not a result of a major expansion of the pCTL pool. However, we cannot rule out the possibility that a small subpopulation of cells within the line is induced to proliferate and is responsible for the enhanced lysis.

Since IL-2 is a potent inducer of cytotoxic activity for the HIV-1-specific CTL lines, it was of interest to examine whether the effects of IL-12 were dependent on endogenous IL-2 production. Ab to the IL-2R had minimal effect on IL-12 enhanced HIV-1-specific cytotoxicity, but efficiently blocked the stimulatory effects of endogenous and exogenous IL-2. Although a previous study found that IL-12 stimulation of mixed lymphocyte reactions in response to allogeneic tumor cells is dependent on IL-2 (2), our results are in agreement with other studies, which showed that stimulation of CTL activity by IL-12 is independent of IL-2 (3, 4, 7, 37).

In experiments with PBMC, IL-12 is a potent inducer of IFN-{gamma} from resting and activated T and NK cells (reviewed in 29 . IL-12 also shifts the in vitro differentiation of CD8+ T cells from HIV-1-infected patients toward clones that produce IFN-{gamma} (i.e., type 1 cytotoxic or Tc1 profile) (17). We observed minimal IFN-{gamma} production induced by IL-12 stimulation of the CTL lines. Moreover, neutralization of endogenous IFN-{gamma} did not reduce the cytolytic activity induced by IL-12, suggesting that the ability of IL-12 to enhance cytotoxicity does not require IFN-{gamma}. Our experiments also suggest that the ability of CD8+ clones from HIV-1-infected patients to shift toward a Tc1 in response to IL-12 may require IL-12 to be present at the time of initial in vitro stimulation.

IL-12 increases the expression of cytolytic components such as perforin, serine esterase, and T cell-restricted intracellular antigen-1 (TIA-1) in MHC-nonrestricted cytotoxic cells (39). This might be the mechanism of action of IL-12 on the HIV-1-specific CTL, as we observed increased granularity by light microscopy after incubation with IL-12 (E. J. McFarland P.A. Harding, and D.R. Kuritzkes, unpublished observations). Other investigators have observed that IL-12 inhibits apoptosis due to IL-2 deprivation in Th1 clones (36). We also noted increased cell survival during culture with IL-12, which may be due to decreased apoptosis, although we did not directly assess apoptosis. However, it is likely that the effects of IL-12 on cytotoxicity are independent of cell survival, since the number of viable cells used in the chromium release assays was equal for effectors generated with or without IL-12. Additional experiments will be needed to determine the specific mechanism of action of IL-12 on HIV-1-specific CTL.

One model for HIV-1 pathogenesis postulates a progressive loss of Th1 responses and an increase in Th2 responses (40), perhaps due to inadequate production of IL-12 (10, 11). Previous studies have shown that IL-12 can enhance Ag-specific proliferative responses (14, 15), NK activity (13), and generation of Th1 clones from cultures (16, 17) of PBMC from HIV-1-infected individuals. Our study demonstrates that IL-12 also augments HIV-1-specific cytotoxicity. These results therefore provide an additional rationale for clinical trials to determine whether the administration of IL-12 leads to significant immune restoration.

In summary, IL-12 enhanced HIV-1-specific cytolytic activity of CTL lines derived from PBMC of HIV-1-infected children. The enhanced lytic activity was mediated by CD3+/CD8+ lymphocytes and was MHC restricted. The activity of IL-12 was IL-2 and IFN-{gamma} independent and did not require significant cell proliferation. Although the in vivo role of IL-12 in the generation and stimulation of CTL remains to be fully determined, its ability to augment HIV-1-specific CTL in vitro adds additional support for IL-12 as a candidate for immune-based therapy of HIV-1.


   Acknowledgments
 
We thank C. Salbenblatt, D. Houts, and A. M. Aycrigg for assistance with specimen collection; K. Helm at the Flow Cytometry Core of the University of Colorado for performing flow cytometry; J. Sypek and S. Wolf of Genetics Institute for the gift of IL-12; M. Gately of Hoffmann-La Roche for the gift of rIL-2; J. Wong, B. Moss, B. Walker, T. Curiel, and E. Connick for the gift of Abs, vaccinia, plasmids, and cell lines; C. Dinarello for helpful discussion; and the patients for donating specimens.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI33299, K08HD01112, AI32915, 5M01RR00069, and 2P30CA46934-09; contributions from Carousel Farms and Celia Thomas and the Colorado Hunter Jumper Association; and a Pediatric AIDS Foundation Scholarship (to E.J.M.). Back

2 Address correspondence and reprint requests to Dr. Elizabeth McFarland, Division of Pediatric Infectious Diseases, Box C227, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Back

3 Abbreviations used in this paper: pCTL, precursor cytotoxic T lymphocyte; B-LCL, B lymphoblastoid cell line; R-20, RPMI 1640, heat-inactivated 20% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin; R-10, RPMI 1640, heat-inactivated 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin; R-10-100, RPMI 1640, heat-inactivated 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin with 100 U/ml recombinant interleukin-2; LU, lytic units; Env, envelop. Back

Received for publication December 23, 1996. Accepted for publication March 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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