Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

Abstract

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-H^bHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4⁺ cells, and this protective effect was observed only in the PBL derived from donors having a CD4⁺ cell count above 200 per mm³. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

HIV has been identified as the etiological agent of AIDS (1, 2), and all possible ways of restricting the replication of HIV should be fully explored for therapeutic intervention (3). Interferon (IFN)-α and -β exert pleiotropic antiretroviral activities and affect many different stages of the HIV infectious cycle in IFN-treated cells (4–6). The clinical effects of IFN administration in HIV-infected individuals, though resulting in some inhibition of virus synthesis, do not reflect the in vitro efficacy that is obtained by pretreating cells with IFN before infection (7–8). We believe that the in vivo efficacy of type I IFNs would be greatly enhanced by establishing the IFN-induced antiviral state before infection takes place. Our approach consists of producing an HIV-resistant state in HIV-target cells through continuous low synthesis of autocrine IFN-β, resulting from transduction with the HMB-H^bHuIFNβ retroviral vector carrying the human IFN-β coding sequence driven by a fragment of the H-2K^b major histocompatibility complex gene promoter (6). In previous work, we showed that the protection of primary lymphocytes derived from uninfected donors can be achieved after transduction by the retroviral vector. In such lymphocytes, virus replication is inhibited and most of the CD4⁺ T cells survive and replicate, in contrast to the untransduced, HIV-infected control cells. In this same study, we showed that IFN-β transduction of peripheral blood lymphocytes (PBL) obtained from four asymptomatic HIV-infected individuals is able to inhibit HIV replication and favor survival of CD4⁺ cells (9).

We have now determined to what extent the spread of endogenous infection can be inhibited in IFN-β-transduced PBL derived from patients at different clinical stages of the HIV infection, and we have examined the effects of low autocrine IFN-β activity, resulting from the expression of the transgene, on the following parameters of lymphocyte function: the expression of cell-surface differentiation markers, the cytokine production, and the stimulation by recall antigens.

MATERIALS AND METHODS

Study Population and Peripheral Blood Mononuclear Cell (PBMC) Preparation.

PBMC derived from three uninfected donors and nine donors infected with HIV-1 were used. On the basis of the absolute counts of CD4⁺ cells, the HIV-infected donors were divided into three groups: group I, early stage disease, more than 500 CD4⁺ T cells per mm³; group II, intermediate stage disease, 200–500 CD4⁺ T cells per mm³; and group III, late stage disease, less than 200 CD4⁺ T cells per mm³ (Table 1). Signed informed consent was obtained from each HIV-infected donor.

PBMC from uninfected donors and from HIV-infected donors were isolated from whole blood by centrifugation over Ficoll/Hypaque density gradients (Pharmacia). Before IFN-β transduction, PBMC were activated, at 10⁶ cells per ml, for 3 days in RPMI medium 1640 (GIBCO/Life Technologies) containing 10% fetal calf serum (HyClone), 5% human serum AB (SAB), 2 μg/ml phytohemagglutinin (PHA; Sigma/Aldrich), 50 units/ml interleukin (IL)-2 (Tebu, Le Perray/Yvelines, France), and 10 μM 3′-azidothymidine (AZT; Sigma). After the 3-day period of culture in the presence of PHA, cultures were split into two, one half to be IFN-β-transduced and one half to serve as untransduced control.

Transduction of PBL.

Activated PBL were transduced with the HMB-H^bHuIFNβ retroviral vector, by coculturing the cells for 3 days on the Ψ-CRIP-HMB-H^bHuIFNβ packaging cells in Iscove’s modified Dulbecco’s medium (GIBCO) supplemented with 10% fetal calf serum, 50 units/ml IL-2, 10 μM AZT, and 3 μg/ml Polybrene (Sigma). The HMB-K^bHuIFNβ vector was constructed as follows. A 660-bp HincII–NdeI fragment containing the human IFN-β coding sequence was excised from the pK^bneoIFNβ plasmid (10), blunt-ended, and inserted into the PUC-H2 (provided by C. Babinet, Institut Curie, France) SmaI site 3′ of the 2-kb fragment of the H-2 K^b gene promoter. The blunted 2.7-kb EcoRI–HindIII fragment containing the IFN-β coding sequence under the control of the H2-K^b gene promoter fragment was cloned in the opposite direction between the EcoRI and BamHI sites of the HMB-neo retroviral vector (11). Control cell populations consisted of PBL transduced by the HMB-neo retroviral vector and untransduced PBL. The PBL transduction efficacy was estimated by PCR amplification, and the absence of murine packaging cells was verified by PCR analysis with a murine α-globin set primer as previously described (12). PBL were cultured, at 10⁶ cells per ml, in RPMI medium 1640/10% fetal calf serum/5% human serum AB containing 50 units/ml IL-2.

HIV Resistance of PBL.

PBL from HIV-infected donors were analyzed every 3 days. Each time, culture supernatants were collected and frozen at −80°C for subsequent assays, and the cells were resuspended in fresh medium, at a concentration of 10⁶ cells per ml. We determined cell mortality by trypan blue staining, the number of HIV DNA copies by PCR amplification, and virus released in the culture supernatants by p24 antigen ELISA (Dupont de Nemours, Les Ulis, France). The production of infectious HIV particles was determined on Hela P4.2 cells as previously described (13).

PCR Analysis of HIV Infection and of Vector Transduction Efficacy.

The number of HIV-1 DNA copies and of the HMB-K^bHuIFNβ or HMB-neo integrations was estimated by resuspending 10⁶ cells per ml of lysing buffer (14) and incubating the cell lysate at 55°C for 1 h. The PCR amplification products (1/1000) were detected in triplicate, by dot-blot hybridization using probes previously described (6, 9), and analyzed by using a PhosphorImager (Molecular Dynamics). The relative intensity of the signals was compared with serial 2-fold dilutions of lysate derived from plasmid-transfected cells that contained one copy of IFN-β transgene per U937 cell (16) or one copy of HIV provirus per J Jhan cell (9).

Fluorescence-Activated Cell Sorter Analysis of Transduced PBL.

The conjugated mAbs used were as follows: anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD56, and anti-CD71 (purchased from Immunotech, Marseille, France); anti-CD25 (from Coulter Coultronics, Margency, France); and anti-CD45 and anti-HLA-DR (from Becton Dickinson). Cells were analyzed by flow cytometry on a Becton Dickinson FACScan. Isotype-matched immunoglobulins served as negative controls for direct staining (Becton Dickinson).

Cytokine Measurements.

Cell supernatants were stored at −80°C until cytokine assays were performed. The results are presented as the mean of duplicate assays. IFN titrations were carried out as previously described (10). The IFNs were characterized as human IFN-γ or human IFN-β by neutralization with a rabbit anti-IFN-γ polyclonal serum and with a rabbit anti-IFN-β polyclonal serum (Lee Biomolecular Laboratories, San Diego). The other cytokines, IL-4, IL-6, IL-10, IL-12, and tumor necrosis factor (TNF)-α, were quantified using ELISA kits purchased from R & D Systems.

In Vitro Antigenic Stimulation Assays.

After coculture with the packaging cells, IFN-β-transduced PBL were washed and maintained for 5 days in RPMI medium 1640 supplemented with 2.5% fetal calf serum, at 5 × 10⁵ cells per ml. Untransduced PBL were also maintained in the same medium for 5 days. After this, the cells were distributed in triplicate wells, in 96-well flat-bottomed plates at 10⁵ cells per well. The PBL were either cultured alone or stimulated with 1 μg/ml concanavalin A (Con A; Sigma); 180 μg/ml cytomegalovirus antigen (Behring, Rueil-Malmaison, France); 50 μg/ml Mycobacterium tuberculosis antigen (purified protein derivative, PPD; Statens Serum Institut, Copenhagen, DK); 50 μg/ml tetanus toxoid (Sanofi-Pasteur, Marnes-la-Coquette, France); or 125 μg/ml Candida albicans (Sanofi-Pasteur). On the sixth day, cultures were pulsed with 1 μCi (1 μCi = 37 kBq) of [³H]thymidine (Amersham), harvested 18 h later, and lysed in buffer containing 0.1 M EDTA and 2.5% Sarkosyl, and radioactivity was measured with a β scintillation counter.

RESULTS

Integration of the Transgene and Expression of Cell Surface Markers in the IFN-β-Transduced PBL.

In the PBL derived from the uninfected and from the three groups of HIV-infected donors, there was no difference in the transduction efficacy, which in each case, ranged from 27% to 62% (Figs. 1A and 2A). Nine days after the onset of PHA activation, IFN-β-transduced PBL secreted IFN-β ranging from 90 to 580 units per 10⁶ cells per 3 days. We observed no significant difference in the level of IFN-β production between PBL derived from uninfected donors and PBL derived from the three groups of HIV-infected donors (Table 2, P = 0.1% as measured by the rank-sum test). This low expression of the IFN-β transgene did not affect cell replication (data not shown). Moreover, in purified CD4⁺ cells derived from group I of HIV-infected donors, we observed no significant difference in the transduction efficacy between whole PBL and purified CD4⁺, 9 days after onset of PHA activation (Fig. 1A).

Figure 1.

Kinetics of transduction and HIV resistance of PBL and of purified CD4⁺ cells derived from two HIV-infected donors of group I (“a” and “b”). ▴, ▪, and • represent untransduced, Neo-transduced, and IFN-β-transduced cells, respectively. ▵ represents untransduced PBL treated by 500 units/ml recombinant IFN-β. □ and ○ represent Neo- and IFN-β-transduced PBL treated by 5,000 units/ml of mAb anti-IFN-β, respectively. (A) Transduction efficacy. (B) Cell mortality expressed as the percentage of the total cells. (C) Number of HIV DNA copies per cell. (D) p24 antigen released into the culture medium. Similar results were obtained with donor c (data not shown).

Figure 2.

Kinetics of IFN-β-transduction and HIV resistance of PBL derived from HIV-infected donors of groups II and III. In untransduced (UT) and IFN-β-transduced (IFN-T) PBL we determined the following every 3 days: (A) Number of IFN-β transgene copies per cell. (B) Cell mortality expressed as the percentage of the total PBL. (C) Number of HIV DNA copies per cell. (D) p24 antigen released into the culture medium.

Analysis of the cell surface markers CD3, CD4, CD8, CD16, CD45, CD56, and HLA-DR, on IFN-β-transduced PBL derived from two uninfected donors, revealed few phenotypic differences (less than 5%) between untransduced and IFN-β-transduced cell populations (data not shown). In activated PBL, a high expression of the activation markers CD25 and CD71 was observed continuously in both IFN-β-transduced and untransduced cells. In contrast, after a 5-day rest period, the expression of the CD25 and CD71 markers declined in the untransduced cells but stayed high in the IFN-β-transduced populations (Table 3).

Inhibition of HIV Replication in IFN-β-Transduced PBL.

Upon cell activation, in the untransduced or Neo-transduced cells of group I, cell mortality increased very rapidly (Fig. 1B) and the percentage of CD4⁺ cells decreased to a value of 5% or lower (Table 4). The number of HIV DNA copies per cell steadily increased until day 15 or 18, to reach an average of 0.35–0.47 copy per cell, depending on the donor (Fig. 1C), and the level of p24 antigen in the supernatant reached peak values on days 15–18 (Fig. 1D). In the IFN-β-transduced cells, cell mortality remained at a steady level of about 10%, and the percentage of CD4⁺ cells at day 18 of the experiment was 22–36% (Table 4). The number of HIV DNA copies and the virus production remained at a low level (Fig. 1D). Moreover, in IFN-β-transduced PBL the production of infectious HIV particles was 10-, 31, and 18-fold less in samples a, b, and c, respectively than in untransduced PBL (Table 4). In PBL treated with recombinant IFN-β we also observed a decrease of cell mortality and p24 production in the supernatant as compared with untreated PBL (Fig. 1), but the recombinant IFN-β treatment was approximately 2-fold less efficient than the transduction by the HMB-K^bHuIFNβ vector (Fig. 1). Furthermore, in IFN-β-transduced PBL treated by anti-IFN-β mAb, cell mortality and p24 production remained at the level obtained with IFN-β-transduced cells (Fig. 1), indicating that the autocrine production of IFN-β was sufficient to induce an anti-HIV state. Interestingly, in IFN-β-transduced purified CD4⁺, we observed a 3-fold increase of IFN-β production (Table 5) and a 2-fold increase in the average number of HMB-K^bHuIFNβ integrations per cell (Fig. 1), indicating selective survival of the IFN producers among HIV-surviving cells.

In the PBL of group II, cell mortality was still lower in the IFN-β-transduced cells (Fig. 2B) as compared with the untransduced cells. At the end of the experiment, the number of HIV DNA copies per cell, in the IFN-β-transduced populations, was lower than at the onset (of the order of about 0.05 copy per cell), indicating that mainly the uninfected cells survived and replicated after IFN-β transduction. Moreover, the percentage of CD4⁺ lymphocytes had not significantly decreased in samples e and f, and the production of HIV infectious particles was 6-, 2.6-, and 2.7-fold less, respectively, in samples d, e, and f transduced by the HMB-K^bHuIFNβ vector, as compared with untransduced cells (Table 4).

The protection conferred by IFN-β transduction was lowest in PBL obtained from donors with less than 200 CD4⁺ cells per mm³. The level of p24 antigen production was still less in the supernatant of IFN-β-transduced cells than that of the untransduced populations (Fig. 2) but no significant difference for the production of infectious HIV particles was detected (Table 4).

IFN-β-Transduction Enhances the Production of Th1-Like Cytokines.

In the PBL derived from uninfected donors, the Th1-like cytokine production was enhanced after IFN-β transduction: the IFN-γ production was 4.5-, 3-, and 1.2-fold more in the X, Y, and Z samples transduced by the HMB-K^bHuIFNβ vector, respectively, as compared with untransduced or Neo-transduced PBL (Table 2). Concurrently, the production of IL-12 was 4- and 2.5-fold more elevated, respectively, in the X and Z samples in the IFN-β-transduced PBL suspension than in the control PBL (Table 2), and similar results were obtained after recombinant IFN-β treatment. However, no significant difference between untransduced and IFN-β-transduced cells was observed in the level of IL-4, IL-6, IL-10, and TNF-α secretion (Table 2 and Fig. 3).

Figure 3.

Effect of IFN-β transduction on production of IL-6, IL-10, and TNF-α by PBL derived from uninfected and from HIV-infected donors. Untransduced (UT) and IFN-β-transduced (IFN-T) PBL were resuspended in fresh medium at a concentration of 10⁶ cells per ml. Every 3 days, culture supernatants were collected and stored at −80°C. The production of cytokine in the culture medium was quantified with ELISA kits 3, 9, 12, and 18 days after the onset of PHA activation.

In the untransduced or Neo-transduced PBL derived from groups I and II, as compared with uninfected PBL, the production of IL-4, IL-6, IL-10, and TNF-α was approximately 2.0-, 3.3-, 2.6-, and 2.2-fold more abundant, respectively, whereas the production of IL-12 was decreased. In contrast, in the IFN-β-transduced PBL of these groups, the level of cytokine production was not significantly different from the production by PBL from uninfected individuals (Table 2 and Fig. 3). In IFN-β-transduced cells as well as in recombinant IFN-β-treated PBL of group I, we observed that the Th1-like cytokine production was more abundant as compared with control cells; it ranged from 6.6- to 8.8-fold for IL-12- and 2.8 to 3.2-fold for IFN-γ. Moreover, after treatment with an anti-IFN-β mAb, the Th1-like production was not significantly modified in IFN-β-transduced PBL. In IFN-β-transduced CD4⁺ cells, the production of Th1-like cytokines was highly enhanced compared with Neo-transduced cells, and the production of IL-4 was not significantly different from that in purified CD4⁺ cells derived from uninfected donors (Table 5).

In group III, a clear-cut difference between untransduced and IFN-β-transduced PBL was observed only for sample g with 134 CD4⁺ T cells per mm³, and the level of cytokine secretion was similar to that of groups I and II in untransduced and IFN-β-transduced PBL. In contrast, for samples h and i, with 68 and 46 CD4⁺ T cells per mm³, respectively, the cytokine production profile was not significantly modified by IFN-β transduction.

IFN-β Transduction Improves the Recall Antigen Responses.

We have examined the effect of IFN-β transduction on stimulation by various antigens (cytomegalovirus antigen, PPD, tetanus toxoid, Candida albicans, and Con A) in PBL derived from uninfected donors and from HIV-infected donors. In the PBL derived from uninfected donors, there was no effect of IFN-β transduction on the degree of antigenic stimulation (Fig. 4). An identical conclusion was reached in the case of PBL derived from HIV-infected donors of groups I and II. In the PBL obtained from donors with less than 200 CD4⁺ T cells per mm³, the untransduced cells in general reacted much less to the PPD, tetanus toxoid, and Candida albicans recall antigens. For example, sample i, had a complete lack of proliferative response to cytomegalovirus antigen, whereas sample h was devoid of response to PPD. In all instances, the proliferative response to the recall antigens was augmented in the IFN-β-transduced PBL, and in some instances, restored to near normal levels (Fig. 4).

Figure 4.

Effect of IFN-β transduction on stimulation by Con A and various recall antigens. Untransduced (white bars) and IFN-β-transduced (black bars) PBL were stimulated with Con A, cytomegalovirus antigen (CMV), Mycobacterium tuberculosis antigen (PPD), tetanus toxoid (teta), or Candida albicans (Cand). Each bar represents the average of three values, with the standard deviation.

DISCUSSION

We have assessed the degree of protection that can be obtained in the IFN-β-transduced PBL derived from HIV-infected individuals at three different clinical stages.

The effect of IFN-β transduction on HIV replication was different in the three groups and dependent on the CD4⁺ cell count of the donors (Fig. 1). The number of infective HIV particles in IFN-β-transduced PBL was correlated with the absolute number of CD4⁺ T cells (Table 4). The apparent lack of protection of PBL derived from donors with a low CD4⁺ T cell count could be due to a decreased efficiency of IFN activity in HIV-infected cells. Lau et al. (15) have shown that the expression of IFN-α/β receptors is down-regulated in donors with AIDS, and that as a consequence, HIV-infected cultured cells show hyporesponsiveness to type I IFN action. As opposed to PBL, in IFN-β-transduced purified CD4⁺ cells, we observed a relative increase of cell mortality and p24 antigen released into the supernatant, indicating a partial protective effect of CD8⁺ cells and implying the involvement of soluble factors produced by CD8⁺ cells (16, 17).

The aim of gene therapy is to protect CD4⁺ cells without interfering with their function, and we studied different parameters to ascertain the possible effects of IFN-β transgene expression on lymphocyte function. Of special interest is the observation that the expression of the HLA-DR marker was not decreased in view of the known inhibitory effects of type I IFNs on IFN-γ-stimulated HLA-DR expression (18). In the cells obtained from the uninfected donors, there was no effect of autocrine IFN-β on the expression of the cell surface differentiation markers tested, with the exception of two activation markers, CD25, the 55-kDa IL-2α receptor chain, and CD71, the transferrin receptor, which were significantly enhanced in the resting IFN-β-transduced PBL. A stimulation of the IL-2α receptor chain in PBMC exposed to IFN-α, IFN-β, or IFN-γ has previously been reported by Onji et al. (19).

In AIDS patients, a shift from Th1 to Th2 type cytokine production has been proposed as instrumental in the disregulation of the immune system, but this concept is subject to debate, and no consensus has been reached as to the general profile of cytokine production in AIDS and its contribution to the progression of the disease (20). The stimulation of IL-12 production by IFN-β in the PBL from uninfected and from HIV-infected donors represents a novel biologic effect of IFN-β, not, to our knowledge, reported before (Table 2). These findings differ from previous reports showing that in the mouse, IFN-γ, but not type I IFNs, influences Th1 cell development (21), and suggest that type I and II IFNs could have different effects on human as compared with mouse T cells. Recently Rogge et al. (22) have demonstrated that IL-12 and type I IFN but not type II IFNs induce expression of the IL-12 β2 chain receptor during in vitro T cell differentiation after antigen receptor triggering. Because IL-12 has previously been shown to induce the production of IFN-γ by T cells (23–25), we believe the stimulation of IL-12 production by IFN-β to be the primary effect, followed by an IL-12-induced stimulation of IFN-γ production. The biologic activities of most type I IFNs display great similarity, and it is therefore highly probable that the shift of T cell development to a Th1 profile as a result of addition of IFN-α, previously described by Parronchi et al. (26), can also be attributed to the induction of IL-12 by this type I IFN. Furthermore, IL-12 has been reported to stimulate the expression of the IL-2α receptor chain (27), which could explain the increased expression of this cell-surface molecule that we observed in the IFN-β-transduced cells. Another aspect of the up-regulation of IL-12 production in the IFN-β-transduced cells of groups I and II is the potential of improving HIV-specific cell-mediated immune responsiveness, since Clerici et al. (28) have shown that addition of IL-12 to PBMC derived from HIV-infected asymptomatic individuals restores T cell proliferation and IL-2 production. IL-4 and IL-10 have been shown capable of inhibiting Th1 cell expansion and, furthermore, a role for these two cytokines in the induction of apoptosis in activated CD4⁺ Th1 cells from HIV-infected persons has been postulated (29). In summary, we observed a double protective mechanism resulting from autocrine IFN-β activity: on the one hand, the direct antiviral effect that decreases HIV production, and on the other hand, a decreased potential for stimulation of HIV replication and for apoptosis as a result of the changed cytokine production profile.

The loss of response to recall antigens is a common occurrence during the development of AIDS (30, 31) and we therefore examined the effect of IFN-β transduction on this immune function. The proliferative response to stimulation with a mitogen, Con A, was nearly identical in the PBL derived from the uninfected and from the HIV-infected donors from the three groups, with no difference between IFN-β-transduced and untransduced cells, which again indicates a lack of effect of autocrine IFN-β activity on cell proliferation (Fig. 4). A similar observation was made with regard to the four recall antigens tested, with the exception of the samples derived from donors of group III. Indeed, in this group, the untransduced PBL of all three donors displayed a marked decrease in their proliferative response to PPD, tetanus toxoid, and Candida albicans. In all instances, the proliferative response to the recall antigens was augmented in the IFN-β-transduced PBL, and in some instances, restored to near normal levels (Fig. 4). This indicates that low autocrine IFN-β as an approach to gene therapy of AIDS may, in addition to directly inhibiting viral replication, have other beneficial effects on immune function.

Table 1.

Characteristics of HIV-infected donors

Donor* (treatment)	Cells/mm3 (%)		Viral load, copies × 10−3/cell
	CD4+	CD8+
Group I
a (no)	875  (31)	1,882  (68)	<1
b (no)	544  (49)	569  (51)	ND
c (no)	517  (37)	862  (62)	<1
Group II
d (no)	426  (38)	683  (62)	11
e (no)	424  (26)	1,226  (75)	2
f (no)	368  (17)	1,376  (62)	ND
Group III
g (AZT)	134  (20)	538  (88)	10
h (AZT + ddI)	68  (10)	623  (87)	8
i (AZT + ddI)	46  (6)	739  (94)	15

^*Patients were classified into three groups according to their absolute CD4⁺ cell count: group I, higher than 500 per mm³, donors a, b, and c; group II, between 200 and 500 per mm³, donors d, e, and f; and group III, lower than 200 per mm³, donors g, h, and i. Clinical treatments: AZT, 3′-azidothymidine; ddI, 2′,3′-dideoxyinosine; ND, not determined.

Table 2.

Production of IFNs, IL-4, and IL-12 by untransduced PBL (UT), Neo-transduced PBL (Neo-T), and IFN-β-transduced PBL (IFN-T), 12 days after onset of PHA activation

Sample	IFN, units/106 cells/72 h		IL, pg/106 cells/72 h
	IFN-β	IFN-γ	IL-4	IL-12
HIV-seronegatives
(X) UT	<5	48	132	122
UT + rIFN	ND	72	113	294
Neo-T	<5	35	127	119
IFN-T + A	<5	40	105	360
(Y) UT	<5	40	ND	ND
IFN-T	580	117	ND	ND
(Z) UT	<5	71	182	125
IFN-T	176	88	171	324
HIV-seropositives
Group I
(a) UT	<5	27	486	28
UT + rIFN	ND	86	252	186
Neo-T	<5	30	503	31
IFN-T	180	210	196	203
IFN-T + A	<5	72	253	185
(b) UT	<5	29	493	18
UT + rIFN	ND	81	140	159
Neo-T	<5	41	512	21
IFN-T	145	168	126	169
IFN-T + A	<5	52	227	157
(c) UT	<5	35	514	20
UT + rIFN	ND	110	236	168
Neo-T	<5	27	438	24
IFN-T	260	210	105	195
IFN-T + A	<5	66	139	187
Group II
(d) UT	<5	36	342	61
IFN-T	185	61	192	145
(e) UT	<5	16	432	118
IFN-T	480	66	200	251
(f) UT	<5	41	364	82
IFN-T	157	78	295	259
Group III
(g) UT	<5	23	425	16
IFN-T	90	35	270	114
(h) UT	<5	<5	190	<10
IFN-T	148	<5	103	14
(i) UT	<5	<5	150	<10
IFN-T	237	50	179	<10

ND, not determined; rIFN, PBL treated by 500 units/ml recombinant IFN-β; A, PBL treated by 5,000 units/ml anti-IFN-β.

Table 3.

Expression of cell surface markers (CD4, CD25, and CD71) by untransduced (UT) and IFN-β-transduced (IFN-T) PBL derived from two uninfected donors (X and Z)

Sample	Phenotypic analysis, %
	Activated PBL			Rested PBL
	CD4	CD25	CD71	CD4	CD25	CD71
(X) UT	59	92	99	53	03	17
IFN-T	65	95	99	58	54	87
(Z) UT	63	87	95	51	07	21
IFN-T	61	91	92	52	61	89

Table 4.

Percentages of CD4⁺ T lymphocytes, CD4⁺/CD8⁺ ratio, and production of infectious HIV particles by untransduced (UT) and IFN-β-transduced (IFN-T) PBL derived from HIV-infected donors

Sample	CD4+, %	CD4+/CD8+ ratio	Infectious HIV particles, ng of p24
			Absolute number	Fold-reduction
Group I
(a) UT	5	<0.1	525	—
UT + rIFN	15	0.2	72	7.3
Neo-T	2	<0.1	497	1.1
IFN-T	22	0.3	52	10.1
IFN-T + A	17	0.2	60	8.7
(b) UT	3	<0.1	837	—
UT + rIFN	15	0.2	99	8.4
Neo-T	4	<0.1	941	0.9
IFN-T	36	0.5	27	31.0
IFN-T + A	23	0.4	51	16.4
(c) UT	1	<0.1	711	—
UT + rIFN	21	0.3	110	6.4
Neo-T	1	<0.1	801	0.9
IFN-T	25	0.4	40	17.8
IFN-T + A	20	0.3	50	14.2
Group II
(d) UT	3	0.1	948	—
IFN-T	23	0.3	156	6.1
(e) UT	<1	<0.1	676	—
IFN-T	13	0.2	258	2.6
(f) UT	6	0.1	684	—
IFN-T	17	0.2	252	1.3
Group III
(g) UT	<1	<0.1	892	—
IFN-T	12	0.1	378	2.3
(h) UT	<1	<0.1	ND	—
IFN-T	<1	<0.1	ND	ND
(i) UT	<1	<0.1	884	—
IFN-T	<1	<0.1	838	1.1

The percentage of CD4⁺ cells and the production of infectious HIV particles were determined at days 18 and 15 respectively. ND, not determined; rIFN, PBL treated by 500 units/ml recombinant IFN-β; A, PBL treated by 5,000 units/ml of mAb anti-IFN-β.

Table 5.

Production of IFNs, IL-4, and IL-12 in Neo-transduced (Neo-T) and IFN-β-transduced (IFN-T) purified CD4⁺ cells, 12 days after onset of PHA activation

Sample	IFN, units/106 cells/72 h		IL, pg/106 cells/72 h
	IFN-β	IFN-γ	IL-4	IL-12
HIV-seronegatives
(X) Neo-T	<5	110	192	320
IFN-T	115	305	202	838
HIV-seropositives
(a) Neo-T	<5	88	972	88
IFN-T	380	460	395	720
(b) Neo-T	<5	67	1,186	97
IFN-T	410	638	291	671
(c) Neo-T	<5	76	1,718	122
IFN-T	325	477	356	572