INTRODUCTION

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Many human immunodeficiency virus (HIV)-infected individuals treated with a triple combination therapy, including reverse transcriptase and protease inhibitors, experience drastically reduced plasma viremia and significant immune restoration (2, 14). Three years after the introduction of this potent antiretroviral arsenal we know, however, that the virus is not eradicated and that viremia returns rapidly to basal levels upon discontinuation of therapy (11, 28). These current limitations of HAART might be due to the persistence over years of a reservoir of latently infected memory T cells (12, 45). Additional therapeutic interventions are therefore required that help eradicate the virus. To that purpose we are investigating a gene therapy based on the pleiotropic antiretroviral activities of beta interferon (IFN-), which affects HIV at several stages of its life cycle (10, 13): uptake of viral particles (40), reverse transcription of viral genomic RNA into proviral DNA (3, 18, 34), viral protein synthesis (8), and packaging and release of viral particles (33). In addition, virions released from IFN-treated cells are up to 1,000-fold less infectious than equal numbers of virions released from untreated cells (15). In our approach, HIV target cells are protected by low-level continuous production of IFN-: they are transduced with the HMB-K^bHuIFN- retroviral vector, which carries the human IFN- coding sequence, driven by a murine H-2K^b gene promoter fragment (41). We have previously shown that IFN- transduction of peripheral blood lymphocytes from HIV-free or infected donors strongly inhibits virus replication, favors CD4⁺ T-cell survival, enhances production of Th1-like cytokines, and improves proliferative responses to recall antigens in vitro (39-41). More recently, Matheux et al. (22) have shown that IFN- transducted macaque lymphocytes display an enhanced resistance to SIVmac251 infection in vitro.

Severe combined immunodeficient (SCID) mice xenografted with human lymphoid cells (Hu-SCID mice) are a relevant animal model for HIV infection (24, 26, 27, 38) and have been used to study HIV pathogenesis, therapy, and vaccines (9, 17, 23, 25, 27, 38, 44, 47). Hu-PBL-SCID mice have also proved useful in an in vivo investigation of some HIV-induced immunological dysfunctions (25, 44). The in vivo passage of human T cells into the xenogenic microenvironment profoundly modifies their behavior, however, and after initial activation they become progressively anergic and unable to proliferate or to release interleukin-2 (IL-2) (1, 36, 37). Moreover, HIV infections in this model are usually limited to a 2- or 3-week period because CD4⁺ T cells are rapidly depleted and lack replenishment sources (25).

To evaluate the in vivo protection against HIV that transfers of genetically engineered human CD4⁺ T cells may confer, we developed a new Hu-PBL-SCID mouse model that could support persistent, replicative HIV infection. Through the fourth week after HIV infection, the mice were periodically reinoculated with resting human peripheral blood mononuclear cells (PBMC) mixed with activated CD4⁺ T cells, thereby maintaining both human lymphocyte activation and the in vivo conditions required for HIV replication. We first examined the frequency of engraftment and the level and timing of CD4⁺ T-cell activation and depletion over the 40-day experimental period. We then evaluated the in vivo eradication of HIV-1 conferred by low-level constitutive expression of IFN- obtained with this gene transfer strategy.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Preparation of human CD4⁺ T cells. PBMC, obtained by leukapheresis from four uninfected donors to the blood bank (Hôpital Saint Louis, Paris, France), were purified by density centrifugation in a Ficoll-Hypaque gradient (Eurobio, Les Ulis, France). Human CD4⁺ T-cell subset sorting was performed with immunomagnetic beads coated with mouse anti-human CD4 monoclonal antibodies (MAbs; Dynal, Oslo, Norway) at a bead-to-cell ratio of 3:1 for 30 min at 4°C. Antibody-bead conjugates were removed by incubating the CD4⁺ T-cell subset fraction with Detach-Beads (Dynal) for 1 h at room temperature. The cell fraction purity was determined by fluorescence-activated cell sorter (FACS) analysis.

Vector transduction of human CD4⁺ T cells. PBMC from uninfected donors were activated with 1 µg of phytohemagglutinin (PHA) (Murex Diagnostic, Ltd., Dartford, England) per ml at 10⁶ cells per ml for 3 days in RPMI medium (Gibco Life Technologies, Cergy Pontoise, France) supplemented with 3 µg of glutamine per ml, 1 mM sodium pyruvate, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 10% heat-inactivated human AB serum (SAB), and 20 U of IL-2 (Boehringer GmbH, Mannheim, Germany) per ml. Activated CD4⁺ T cells were then purified and transduced with the HMB-K^bHuIFN- or with the MFG-LacZ retroviral vector by 2 days of coculture on the packaging cell lines (10) in IMDM medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (HyClone), 20 U of IL-2 per ml, and 5 µg of sulfate protamine (Sigma-Aldrich, St. Quentin Fallavier, France) per ml. The vector transduction efficacy was estimated by PCR amplification, as previously described (41).

HIV infection of human CD4⁺ T cells. Human CD4⁺ T cells were seeded at 10⁶ cells/ml and then challenged with 2 × 10⁶ 50% tissue culture infective doses of HIV-1-LAI for 3 h. Every 3 days, the cells were amplified in fresh medium. Twelve days after the onset of HIV infection, the number of HIV DNA copies was estimated by PCR amplification (41), and the virus released in the culture supernatants was quantified by a p24 antigen enzyme-linked immunosorbent assay (ELISA) (Dupont de Nemours, Les Ulis, France).

Establishment of Hu-PBL-SCID mice. A total of 86 6-week-old CB17 female scid/scid mice (Iffa Credo) were used in six separate experiments. Mice were housed in specific-pathogen-free incubators in a biosafety level 3 facility. SCID mice were first engrafted by intraperitoneal (i.p.) injection with 4 × 10⁷ fresh human PBMC, collected by leukapheresis from HIV-seronegative healthy donors. Two weeks later, the mice were injected i.p. with 10⁷ human CD4⁺ PHA-stimulated lymphoblasts (PHA-blasts), with half of the mice receiving HIV-infected blasts and the other half receiving HIV-free blasts. The mice were then reinoculated, either weekly or biweekly, through week 6 after HIV infection with a mixture of autologous resting human PBMC (4 × 10⁷) plus 2 × 10⁷ purified human CD4⁺ PHA-blasts (Fig. 1). Autologous human PHA-blasts (2 × 10⁷) transduced with the LacZ transgene or the IFN- transgene were inoculated weekly with resting human PBMC, according to the same protocol. Groups of mice were euthanized 3 days after the last reinoculation at weekly intervals in three Hu-PBL-SCID mouse experiments (experiments A and B, 12 mice; II, 54 mice) or at week 6 after HIV infection in two other Hu-PBL-SCID mouse series (experiment C, 3 mice; I, 18 mice). At sacrifice, mice were anesthetized, peripheral blood was collected by intracardiac puncture, a peritoneal washing was performed with 1 ml of RPMI medium, and spleens were harvested. Erythrocytes were lysed by repeated exposure to a lysis buffer containing 8.3 g of NH₄Cl, 0.04 g of EDTA, and 1 g of KHCO₃ per liter. Splenic mononuclear cells were gently teased from freshly harvested spleens. The peritoneal and splenic mononuclear cell suspensions were washed twice in RPMI medium.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Experimental design for obtaining Hu-PBL-SCID mice.

FACS analysis of human cells. A three-color FACS analysis was performed on freshly harvested mononuclear cells from three compartments: peritoneal lavages, spleens, and peripheral blood. Isotype-matched immunoglobulin served as the negative control (Becton Dickinson, San Jose, Calif.). Cells were incubated with the appropriate cocktail of antibodies: CD45-PerCP, CD4-FITC, and CD8-PE or CD45-PerCP, CD4-FITC, and HLA-DR-PE (Becton Dickinson, Pont de Claix, France). Erythrocytes were lysed in distilled water. After the washings a minimum of 10,000 cells were analyzed on a flow cytometer FACScan (Becton Dickinson). Results were expressed as percentages of MAb-positive human CD45⁺ cells in the mononuclear cell gate, percentages of CD4⁺ or CD8⁺ human T cells within the CD45⁺ human leukocytes, and percentages of activated HLA-DR⁺ or CD25⁺ CD4⁺ T cells within the human CD4⁺ T cells. The analysis was performed only when a minimum of 200 CD4⁺ human T cells were detectable.

PCR analysis of HIV DNA copies and of vector transduction efficacy. Cell lysate was prepared as previously described (5) and incubated at 55°C for 1 h. DNA extract from 10⁴ cells was amplified by PCR for 30 cycles in the presence of 1 µM ³³P-dCTP (10 mCi/mM; NEN-Life Science Products, Le Blanc Mesnils, France). The same primers as reported previously were used (10). The reaction products were separated on a 4% nondenaturing polyacrylamide gel. Gels were dried and exposed to a PhosphorImager (Molecular Dynamics, Sevenoaks, England) cassette overnight. Serial twofold dilutions of DNA preparations from HIV-1-LAI-infected J.Jhan cells and from plasmid-transfected U937 cells containing one copy of IFN- transgene per cell (20, 41) were used as standards and amplified in each reaction to determine interassay variability and sensitivity.

Viral coculture analysis. For in vitro virus isolation, 2 × 10⁵ cells derived from the peritoneal lavages, from the spleen, and from the peripheral blood of Hu-PBL-SCID mice were cocultured with 2 × 10⁵ uninfected human autologous PHA-blasts in RPMI medium containing 10% human AB serum and 20 U of IL-2 per ml in round-bottom 96-well microtiter plates (Costar). Every 3 days, the cells were resuspended in fresh medium. At day 9 of the culture, we evaluated cell mortality by trypan blue staining, the virus released in the culture supernatant by p24 antigen ELISA (Dupont de Nemours, Les Ulis, France), and the percentage of HIV-infected cells by PCR amplification.

Human cytokine expression in Hu-PBL-SCID mice. Total spleen RNAs of Hu-PBL-SCID mice were isolated with the RNA isolation kit (Stratagene, Montigny-le-Bretonneux, France), and 1 µg of total RNA treated with DNase I (Promega, Charbonniere, France) was used to obtain cDNA products with the First Strand Synthesis kit (Pharmacia Biotech, Orsay, France). One-sixteenth of the cDNA product was amplified by PCR for 30 cycles, in the presence of 1 µM ³³P-dCTP (10 mCi/mM; NEN), to detect the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts as a quantitative control. To estimate human cytokine expression, we used primers specific for the cDNA cytokine sequence, previously described by Zhou and Tedder (48). The reaction products were detected by autoradiography after electrophoresis on 4% nondenaturing polyacrylamide gels, and expression of cytokines was quantified with the PhosphorImager.

Statistics. Analyses were performed by using the Wilcoxon nonparametric and a ² test on Statview Software.

	RESULTS

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A model of persistent and productive HIV-1 infection in Hu-PBL-SCID mice. We first developed a new Hu-PBL-SCID mouse model that supported a persistent, replicative HIV infection for at least 4 weeks: SCID mice were inoculated i.p. with 4 × 10⁷ total human PBMC. Two weeks later mice were reinjected i.p. with a standard dose of autologous PHA-blasts that were either preinfected with the HIV-1-LAI strain or uninfected. To maintain a continuous support of activated human T cells required for HIV replication, the mice were periodically reinoculated with a mixture of autologous resting PBMC and uninfected activated CD4⁺ T cells until the fourth week after HIV infection (Fig. 1). In two preliminary experiments (experiments A and B) human CD45⁺ cells were shown to persist in high proportions: 50% ± 28%, 39% ± 12%, and 20% ± 8% in the peritoneal cavity, spleen, and peripheral blood, respectively, after biweekly reinoculation of human CD4⁺ T cells into grafted mice. Human cells were composed mostly of CD4⁺ T cells (range, 40 to 90%) that were activated in vivo, as indicated by the high level of human CD4⁺ T cells that were positive for HLA-DR and CD25 (rIL-2) (Fig. 2). The proportion of human CD45⁺ cells dropped in the spleens and peripheral blood after HIV infection (4% ± 3% and 6% ± 3%, respectively) but remained unchanged in the peritoneal cavity (48% ± 29%). In two other Hu-PBL-SCID mouse experiments (experiments I and II; Table 1 and Fig. 3), a three- to sixfold decrease in human CD4⁺ T cells and a significant decrease in the CD4/CD8 ratio were observed in each compartment despite the periodic reinoculations of human CD4⁺ T cells. A persistently high proportion of HIV-infected cells was detected by PCR analysis from 6 weeks after inoculation of 66% ± 24% to 100% ± 7% of infected human peritoneal cells (Table 1 [experiment I]). High levels of HIV-infected human cells were also found in spleens and peripheral blood (Fig. 3 [experiment II]). Furthermore, the number of HIV DNA copies per cell increased steadily over time in the human mononuclear cell suspensions in each compartment, reaching an average of 0.4 copy per cell at week 5 and declining thereafter (Fig. 3 [experiment II]). In vivo HIV replication was also assessed by p24 production, which reached plasma levels of 33 to 65 pg/ml (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Kinetics study of human T cells in uninfected and HIV-infected nontransduced Hu-PBL-SCID mice. Experiment A (left panel) was performed on 12 mice, with two animals sacrificed at each time point after grafting. Inoculation of HIV-LAI-infected cells was performed 2 weeks after the grafting in the right-panel samples. FACS analysis of human cells derived from peritoneal lavages was used to determine the percentage of human CD45+ cells within the mononuclear cells. The proportions of CD4+ and CD8+ T cells were determined within the CD45+ human cells, and the proportions of activated HLA-DR+ and CD25+ cells were determined within the fraction of CD45+ CD4+ T cells on at least 200 viable human CD4+ T cells.

View this table: [in this window] [in a new window]   TABLE 1.   Enhancement of HIV resistance in the peritoneal cavity of IFN--transduced Hu-PBL-SCID mice (experiment I)

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Kinetics of IFN- transduction and HIV resistance in Hu-PBL-SCID mice engrafted with human CD4+ T cells (experiment II). Purified human CD4+ cells were transduced by use of the HMB-KbHuIFN- vector as described in Materials and Methods. Nontransduced (UT, circles) or IFN--transduced (IFN, squares) CD4+ T cells were injected i.p. into uninfected (UI, solid symbols) or HIV-infected CD4 SCID (HIV, open symbols) mice. At weekly intervals after HIV infection, three mice (weeks 2 to 5) and six mice (week 6) were sacrificed, and cells were collected. We determined by FACS analysis the percentage of human cells (A) and the percentage of human CD4+ cells (B) and by PCR analysis the percentage of IFN--transduced cells (C) and the number of HIV DNA copies per human cell (D).

In vivo survival of human IFN--transduced CD4⁺ T cells in Hu-PBL-SCID mice. The Hu-PBL-SCID mice were then engrafted with human CD4⁺ T cells transduced by the HMB-K^bHuIFN- vector. Ten days after the ex vivo transduction, 30 to 70% of the T cells expressed the IFN- transgene (data not shown). Mice receiving human IFN--transduced cells had a proportion of human T cells and lymphocyte subset profiles similar to those in control mice that had received nontransduced or lacZ-transduced cells (Table 1). All mice from the IFN- transgene group showed evidence of gene transfer, as detected by PCR amplification of a vector-specific DNA fragment in the three compartments tested. The percentage of human IFN--transduced CD4⁺ T cells ranged from 31 to 75% (Table 1 and Fig. 3). These data show the efficacy of engrafting these mice with IFN--transduced CD4⁺ T cells.

In vivo HIV resistance in Hu-PBL-SCID mice treated with human IFN--transduced CD4⁺ T cells. Four weeks after engraftment, Hu-PBL-SCID mice were infected with HIV-1-LAI and subsequently underwent passive transfers of IFN--transduced CD4⁺ T cells. In experiment I, the percentage of CD45⁺ cells and the CD4/CD8 cell ratio in HIV-infected mice remained similar to that observed in uninfected mice (Table 1). In addition, the proportion of HIV-infected human cells significantly decreased to 10% ± 5% after transfers of IFN--transduced CD4⁺ T cells compared with 60% ± 18% or 24% ± 6% in HIV-infected control mice which had received untransduced or lacZ-transduced cells (Table 1). In experiment II, we studied the kinetics of the HIV-1 resistance in SCID mice engrafted with human IFN--transduced CD4⁺ T cells by sacrificing three mice from each group at weeks 2, 3, 4, and 5 and six mice per group at week 6 (Fig. 3). In SCID mice treated with IFN--transduced cells, the percentage of CD45⁺ cells and the CD4/CD8 cell ratio were similar to values observed in uninfected mice and persisted over time in all compartments tested (i.e., peritoneal lavages, spleens, and blood). In addition, the level of HIV DNA copies per human cell in these compartments remained inferior to 0.05 (Fig. 3). Concomitantly, no p24 antigen was detected in their plasma (<30 pg/ml) (data not shown). These data taken together indicate either the disappearance of or a very low rate of HIV replication after passive transfers of human IFN--transduced CD4⁺ T cells in HIV-infected Hu-PBL-SCID mice.

View this table: [in this window] [in a new window]   TABLE 2.   Survival advantage in in vitro cultures of human lymphocytes derived from IFN--transduced Hu-PBL-SCID mice after HIV infection (experiment II)

Periodic transfers of human IFN--transduced CD4⁺ T cells can eliminate HIV-1 infection. All mice (a total of 14 animals) inoculated with HIV-1-infected cells and receiving periodic transfers of the control-activated nontransduced human CD4⁺ T cells maintained a productive HIV infection in vivo for up to 6 weeks. In contrast, HIV could be recovered in only 9 of the 15 mice that received CD4⁺ T cells genetically engineered with the IFN- vector (P < 0.005). That is, in 40% of the mice engrafted with IFN--transduced CD4⁺ T cells, HIV was undetectable by either PCR analysis or p24 release evaluated ex vivo on freshly harvested mononuclear cells and even after cocultivation of the mouse tissue cells with the autologous human PHA-blasts. This finding suggests that the passive transfers of human IFN--transduced CD4⁺ T cells allow HIV eradication in vivo.

Enhancement of the expression of Th1-like cytokines in IFN--transduced Hu-PBL-SCID mice. Several reports have shown that in vitro type I IFNs favor development of a type I immune response (31, 42). To determine the effect of a low, continuous production of IFN- on cytokine production in vivo, we performed a semiquantitative reverse transcriptase PCR analysis of human cytokine expression in murine spleens at 5 weeks after infection. Similar levels of IL-4, IL-10, and tumor necrosis factor alpha (TNF-) were detected in spleens from uninfected SCID mice receiving IFN--transduced or nontransduced CD4⁺ T cells (Fig. 4). In contrast, the expression of both human IFN- and IL-12 transcripts was 3 ± 1-fold higher in IFN--transduced mice than in their nontransduced counterparts (Fig. 4), indicating that low-level constitutive expression of IFN- enhanced the Th1 cytokine expression in vivo. In HIV-infected mice receiving nontransduced CD4⁺ T cells, the expression levels of human IL-4, IL-10, and TNF- were approximately 12 ± 3-, 11 ± 3-, and 5 ± 1-fold higher, respectively, compared to those in uninfected mice, whereas the levels of human IFN- and IL-12 transcripts were approximately 2 ± 1- and 3 ± 1-fold lower (Fig. 4). In addition, among the mice that received IFN--transduced cells, cytokine expression was similar in uninfected and HIV-infected mice (Fig. 4). These data provide evidence that IFN- blocks the dysregulation in vivo of cytokine observed in HIV infection.

View larger version (82K): [in this window] [in a new window]   FIG. 4.   Detection of cytokine expression in the spleens of IFN--transduced SCID mice by PCR amplification of cDNA. Total RNA was extracted from the spleens of uninfected (UI, lanes 1 to 3 and 7 to 9) or HIV-infected (HIV, lanes 4 to 6 and 10 to 12) SCID mice injected with nontransduced (lanes 1 to 6) or IFN--transduced (lanes 7 to 12) CD4+ T cells. cDNA preparation and PCR amplification were as described in Materials and Methods.

	DISCUSSION

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We evaluated the protection against HIV-1 conferred by periodic reinoculations of IFN--transduced activated CD4⁺ T cells in a new model of Hu-PBL-SCID mice which supports a systemic, persistent, replicative HIV-1 infection. A persistent activation of mature CD4⁺ T cells indeed appears to be important in determining the rate of HIV replication and disease progression (4, 35), but the SCID mouse environment is known to induce in vivo T-cell anergy in engrafted human PBMC (36, 42). In addition, the lack of replenishment of target cells in conventional Hu-PBL-SCID mouse models limits the HIV-1 infection to the short term, with human target cells lost within 2 or 3 weeks after virus inoculation (25). The model used here differs from previously reported models by its periodic reinoculations of activated CD4⁺ T cells that maintain high levels of human cell engraftment in various murine tissues over time and persistence of a human CD4⁺ T-cell activation. Consequently, HIV remained detectable in all of the Hu-PBL-SCID mice grafted with human CD4⁺ T cells for up to 6 weeks after HIV-1 inoculation. This persistent HIV-1 infection profoundly impaired the T-cell status, as indicated by the CD4⁺ T-cell depletion, the upregulation of TNF-, IL-4, and IL-10 and the downregulation of the IFN- and IL-12 expression in a way similar to the immune alterations reported in HIV-infected patients (7).

Current antiretroviral therapies restore most of the Th1 cell functions except against HIV itself, while they profoundly decrease the T-cell activation and the HIV-specific CD8⁺ T-cell responses (2, 29). These new antiretroviral drug regimens control patient virus loads but they do not eliminate the virus. As a consequence, a reservoir of latently infected CD4 T cells might persist for up to 70 years (12, 45). Its eradication may therefore require additional therapies based upon both antiretroviral effects and immune interventions that might help to activate and eliminate the virus-infected cells. Such immune-based strategies should both maintain T-cell activation and enhance the Th1 cell functions required to clear the HIV-infected cells. It has been postulated that activation of resting memory T cells by mixtures of cytokines such as IL-2 plus IL-6 and TNF or by anti-CD3 monoclonal antibodies might help eliminate the reservoir of infected T cells (16). Gene therapy offers potential for developing new therapeutic strategies for acquired disorders (6, 20, 30, 46). We had previously demonstrated in vitro that a low-level continuous production of IFN- in genetically engineered T cells could not only reconstitute antiviral resistance but also improve the CD4⁺ T-cell Th1 functions (41). Indeed, IFN- transduction of peripheral blood lymphocytes and purified CD4⁺ T cells from donors with or without HIV infection inhibited HIV replication, favored CD4⁺ T-cell survival, and improved the proliferative response to recall antigens (39, 41). We now demonstrate that similar results can be obtained in vivo: the HIV levels in all the animals grafted with IFN--transduced CD4⁺ T cells dramatically decreased despite the persistence of grafted human CD4⁺ target cells. In addition, HIV remained undetectable for 4 to 6 weeks after virus inoculation in 40% of these mice, both in vivo and after in vitro cocultures of specimens containing human T cells. In contrast, all mice remained infected for the same period of time in the control groups. Our data therefore suggest that the low-level continuous production of IFN- in the genetically engineered transferred CD4⁺ T cells not only decreased the level of HIV infection but was also capable of eliminating per se the HIV infection in 40% of the infected Hu-PBL-SCID mice.

In addition to these antiviral effects, we found an enhanced survival of human CD4⁺ T cells in HIV-infected mice engrafted with IFN--transduced CD4⁺ T cells, as well as an upregulation of IL-12 and IFN- expression in the spleens of such mice. Our results are in accordance with previous studies showing that type I IFN (IFN- and IFN-) can promote a Th1 cytokine secretion profile, while Th2 development can be prevented by IFN- treatment in HIV-infected individuals suffering from Kaposi's sarcoma (31, 32, 41, 43). Furthermore, Marrack et al. (21) have shown that type I IFNs, as well as IL-2, keep activated T cells alive. In contrast to IL-2, which stimulates the T-cell division, type I IFNs act as survival factors for activated T cells via an unknown mechanism. Such new type I IFN effects might help to reduce the reservoir of HIV-infected cells by keeping alive both the HIV-specific T cells and the CD4 T cells that actively replicate the virus. In this light, the in vivo gene therapy model developed here demonstrates that IFN- transduction of CD4⁺ activated T cells requires consideration as an additional strategy in the antiretroviral drug arsenal for its ability to promote in vivo HIV control and immune restoration.