Feline CD8+ cells induced with FIV infected, irradiated T cells produce multiple anti-FIV factors

Developmental and Comparative Immunology 29 (2005) 809–824 www.elsevier.com/locate/devcompimm

Feline CD8C cells induced with FIV infected, irradiated T cells produce multiple anti-FIV factors Zhongxia Li, Anagha Phadke, Eric A. Weaver, Judith M. Ball, Ellen W. Collisson* Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, USA Received 7 October 2004; revised 7 January 2005; accepted 14 January 2005 Available online 17 March 2005

Abstract Feline immunodeficiency virus (FIV) infection in cats is the only non-primate, small animal model for HIV-AIDS. Replication of FIV has been shown to be optimally suppressed by soluble factors produced by inducer cell-stimulated feline CD8C cells from FIV-infected cats. The nature of this dose-dependent suppression of FIV was examined. Antiviral factors, produced in serum-free medium, were shown to be either heat stable or heat labile. Suppressing activity was identified in a heparin-bound fraction and the non-bound fraction and in fractions separated by reverse-phase HPLC. The FIV suppression could not be correlated with IFN type I or II. Neither a nor b chemokines were likely candidates because molecular size exclusion centrifugation indicated that the major factors were larger than 50 kD. Identified qualitative differences in the properties of the soluble suppressive activity generated from feline lymphocytes indicated that multiple factors are responsible for the non-cytolytic CD8C T cell suppression of FIV replication. q 2005 Elsevier Ltd. All rights reserved. Keywords: FIV; CD8C cell antiviral activity; multiple factors

1. Introduction Feline immunodeficiency virus (FIV), a T lymphotropic lentivirus in the family Retroviridiae, was first reported by Pedersen et al. [1] to cause an AIDS-like illness in cats. FIV infection in cats initially causes a transient illness characterized by fever, lymphadenopathy and neutropenia followed by chronic asymptomatic infection prior to its association with clinical AIDS [2–5]. Because of the similarity of the virus to * Corresponding author. Tel.: C1 979 845 1697; fax: C1 979 862 1088. E-mail address: [email protected] (E.W. Collisson).

0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.01.003

HIV and the similarity of its feline pathogenesis to HIV infection in humans, FIV provides a relevant, small animal model for HIV associated AIDS [6,7]. Maintenance of asymptomatic infection with primate and feline lentiviruses has been associated with activities of CD8C T lymphocytes [8–16]. Both lentiviral specific, MHC restricted CD8C CTL responses and CD8C T cell non-cytolytic responses have been described in cats [3,17–20]. Optimal FIVsuppressive, non-cytolytic activity can be demonstrated from CD8C T cells following stimulation with inducer cells infected ex vivo with FIV [17], as was similarly observed with CD8C anti-HIV activity in infected and uninfected human individuals [21,22].

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Although a number of cytokines, including interleukins and chemokines, have been associated with non-cytolytic antiviral activity, the putative CD8C T cell antiviral factor (CAF) remains an unidentified component of innate immune responses to these viruses in cats [8,9,17,23,24]. While exploitation of these suppressing activities may have potential future therapeutic applications, little has been reported on the nature of the soluble suppressing activity generated by feline CD8C T cells. In this study, biochemical and physical characteristics are described indicating that multiple factors present in supernatants are responsible for the induced non-cytolytic anti-FIV activity.

2. Materials and methods 2.1. Experimental animals Specific pathogen-free (SPF) cats purchased from Harlan Sprague-Dawley (Madison, WI) or Liberty (Liberty Corner, NJ) were serologically negative for feline leukemia virus and FIV. They were housed in an SPF environment at the Laboratory Animal Research and Resources Facility, Texas A & M University, College Station as approved by the Texas A & M University Laboratory Animal Use Committee. Cats AUO2, AUO3, AWF1, AZV2, E238, E284 and A306 were chronically infected with doses from 50 to 1250 TCID50 of FIV-PPR strain, while cats OAE5, and OLQ4 were sham inoculated [3,17,25]. Cats M165 and M242 had also been similarly inoculated more than 3 years prior to these studies with 50 TCID50 of the FIV-PPR strain. 2.2. Virus FIV-PPR was propagated in feline PBMC. After 7– 10 days of infection, virus expression was evaluated with an FIV capsid antigen detection ELISA kit (IDEXX, Portland, Maine). Supernatants with maximal optical density were collected, and these stocks were stored at K80 8C before titrating on fresh PBMC. 2.3. Cell culture Feline PBMC were isolated from EDTA (K3)treated whole blood by Histopaque-1077 (Sigma, St

Louis, MO) density gradient centrifugation. PBMC were cultured as described previously with RPMI 1640 (Gibco BRL, Grand Land, NY), supplemented with 10% fetal bovine serum (FBS), 50 mg of gentamicin (Gibco BRL) per millilitre, 5!10K5 M 2-mercaptoethanol (Gibco BRL), and 2 mM L-glutamine (Gibco BRL), and with 100 U of human recombinant IL-2 (hr IL-2) (Gibco BRL) per millilitre unless cultured with inducer cells or otherwise specified [17]. PBMC were also grown in serumfree medium HB 101 (Irvine Scientific, Inc., Santa Ana, CA) supplemented with 50 mg of gentamicin (Gibco BRL) per millilitre, 5!10K5 M 2-mercaptoethanol (Gibco BRL), 2 mM L-glutamine (Gibco BRL), 1 mM sodium pyruvate, 15 mM Hepes, and as specified with 100 U of human recombinant IL-2 (hr IL-2) (Gibco BRL) per millilitre (Gibco BPL). The media were changed every 3–4 days. FIV replication in the culture supernatants was evaluated by the FIV capsid antigen ELISA (IDEXX, Portland, ME). 2.4. Stimulation of effector cells Inducer cells were prepared as follows; feline PBMC were stimulated with 5 mg/ml of concanavalin A (Con A) for 3 days, and the lymphoblastoid cells were infected with 0.5 ml of FIV-PPR stock for 60 min at 37 8C and cultured in complete RPMI containing 100 U of human recombinant IL-2 (hr IL2, Gibco BRL) per millilitre at 37 8C in a humidified CO2 incubator. After six days, virus expression was monitored by the capsid antigen ELISA kit. These FIV-infected cells were stored in liquid nitrogen, prior to using as inducer cells for the stimulation of autologous effector cells. FIV-infected cells were irradiated with 11,000 rads from a 60Co source (Nuclear Science Center, Texas A & M University) and used as autologous inducer cells by coculturing with effector cells [19]. The irradiated FIV-infected inducer cells were monitored for the FIV-inactivitation by culturing them with FIV-negative PBMC for 10 days in the presence of human recombinant IL-2, followed by detection with the FIV capsid ELISA. Exposure to 11,000 rad inactivated all detectable virus in the cells whereas cell viability was O90%. Effector cells (106 cells /ml) were stimulated every 3–4 days with inducer cells alone (without ConA or hr IL-2) either in RPMI 1640 or HB101 medium.

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2.5. FIV suppression assay Cell-free supernatants were collected from inducer cell-stimulated PBMC of FIV-PPR-infected cats. Following stimulation, the supernatants were centrifuged at 110,000!g for 2 h (Beckman Instruments, Palo Alto, CA) to pellet the residual viral particles. The supernatants were then sterilized by passing through a 0.2 mm-pore size filter. Cultured T cells (2!104 cells/well), infected in vitro with FIV-PPR, were cultured for 7–10 days with the supernatant at a ratio of 1:1 with complete RPMI [17]. Equivalent amounts of fresh supernatants and media were added to the infected cells every 3–4 days. FIV replication was determined by the FIV capsid (p24) antigen ELISA. 2.6. Interferon assay A cytopathic effect (CPE) inhibition assay was used to detect interferon [26]. Briefly, Crandell feline kidney (CrFK) cells were cultured overnight in a 96-well plate to form confluent monolayers. The feline interferon a standard (PBL, Inc., New Brunswick, NJ) and supernatants from stimulated PBMC were serially diluted in DMEM containing 10% FBS before adding 100 ml to each well. After 6 h, 500 PFU per well of vesicular stomatitis virus (VSV) in 0.05 ml of media were added. Virus and cell controls were included. The plate was incubated at 37 8C until the virus control wells displayed a CPE of close to 100%. The medium was aspirated from each well, and cell monolayers were fixed with 10% formaldehyde in PBS for 1 h before washing and staining for 5 min with 0.1 ml of 0.5% (wt/vol) crystal violet. After decanting the staining solution, the plate was washed and air-dried. The interferon titer was read as the reciprocal of the dilution represented in the well in which 50% of the cell monolayer was protected. Further confirmation of feline IFN type I was based on the stability of suppressing activity after treating at pH 2 [27]. IFN-gamma ELISA was performed using anti-feline IFN-gamma antibodies (E6D4A5 and E4D1A9) described elsewhere [28] (kind gift from Dr G.A. Dean, North Carolina State University). Briefly, 50 ul of E6D4A5 (20 mg/ml in PBS) was used to coat NUNC Maxi Sorp plates (Nalge Nunc Inc., Naperville, IL) overnight in a humid container. After washing and blocking, samples were added into the wells and

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incubated for 2 h at room temperature. Biotin-labeled E4D1A9 were then added into each well, and incubated for 30 min. After washing, 200 ul of streptavidinperoxidase conjugate was added to each well at a dilution of 1:5000 (Sigma, St Louis, MO), and incubated at room temperature for 30 min. TMB substrate was added after washing (KPL, Gaithersburgm MD), and the plate was read with ELISA reader at 650 nm. The recombinant feline IFN-gamma (developed in our laboratory) was used as a standard control. 2.7. Heparin column chromotography The pooled supernatants collected from lymphocytes with strong suppressing activity at day 3 or 7 were passed through a HiTrap Heparin Sepharosee HP column (Amersham Pharmcia Biotech AB, Uppsal, Sweden) according to the manufacturer’s instructions. After washing with buffer (10 mM sodium phosphate, pH 7.0), the bound factors were eluted with 1.5 ml of increasing concentrations of NaCl; 0.1, 0.25, 0.5, 1.0, 1.5, and 2.0 M. The eluants were dialyzed against three changes of serum-free HB 101 medium. After filtering through 0.2 mm-pore size filters, the dialyzed fractions were tested for their capacity to suppress FIV replication in T cells grown in serum-free medium. 2.8. Estimation of the molecular weight cut-off Supernatants from cells grown in serum-free HB101 medium were centrifuged at 110,000!g for 2 h (Beckman Instruments, Palo Alto, CA) to pellet the residual viral particles, and then placed in 15-ml tubes with a Centriplus 50 (Millipore, Bedford, MA) centrifugal ultrafiltration membrane with a molecular weight cutoff (MWCO) of 50 kD, and centrifuged at 3000!g for 60 min at 4 8C. The filtrate was then centrifuged in 15-ml aliquots through a Centriplus 10 with a MWCO of 10 kD. The final filtrate was centrifuged in 15-ml aliquots through a Centriplus 3 with a MWCO of 3 kD. The retentate and filtrate fractions were then subjected to the FIV suppression assay. 2.9. Enrichment for cell phenotype Negative selection for enrichment or depletion of CD8C T cells from inducer cell-stimulated effectors of FIV-PPR -infected cats was performed by the panning

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method as described elsewhere [17,25]. The enriched CD8C T cells were obtained by the depletion with antifeline CD4 monoclonal antibody, whereas the depleted CD8 T cells were prepared by the depletion with antifeline CD8 antibody (Southern Biotechnology Associates, Birmingham, AL). Following panning, the CD8-depleted or CD8-enriched preparations were cultured with IL-2 (100 U/ml) or with inducer T cells for an additional 4 days as described above. The purity of the enriched and depleted cells by this method was confirmed by the FACscaliber flow cytometer (BectonDickinson, San Jose, CA) at the Veterinary Pathobiology Core Facility (Director, Dr Roger Smith, Texas A & M University, College Station). 2.10. Reverse phase HPLC Supernatants in serum-free media collected from PBMC stimulated by 60Co-irradiated inducer cells (FIV infected) were subjected to ultracentrifugation at 110,000!g for 2 h at 4 8C and filtered through a 0.2 mm-pore size filter, before applying to a C18 column (Beckman, Fullerton, CA) that had been equilibrated with H2O containing 0.1% trifluoroacetic acid (TFA). Proteins bound to the column were eluted with a 60-min linear gradient of aqueous acetonitrile (0–60%) containing 0.1% TFA. The flow rate was maintained at 0.5–1.0 ml/min. The resulting fractions were freeze-dried and reconstituted in serum-free media before testing for suppressing activity. 2.11. Statistical analysis The differences in FIV replication and cell viability were analyzed with a two-tailed Student’s t-test. FIV replication was determined by an FIV capsid (p24) ELISA and virus expression was expressed as the relative percentage of the sample optical density divided by that of the positive control. Positive control was FIV-infected PBMC cultured in complete RPMI 3

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in the absence of effector cell supernatants. Suppression was considered to be positive when relative percentage of FIV capsid in samples was %65% of those in the untreated controls. Data are representative of at least two experiments. 3. Results 3.1. Effector cells continued to make anti-viral activity for at least 7 days Biochemical and physical properties of the suppressor activity were examined. Our previous studies described the antiviral activity of supernatants accumulated following induction of CD8C T cells with irradiated, FIV infected T cells (inducer cells). Neither inducer cells nor targets for antiviral activity need be MHC matched with the CD8C T effector cells [17,25]. Suppression was determined from supernatant collected each day within the first 7 days after addition of inducer cells, in order to determine the kinetics of induction. Irradiated inducer cells were added to PBMC at days 0 and 3. The supernatant was then cultured with infected target cells, followed by detection of FIV capsid protein using an ELISA. The relative percentage of FIV capsid detected in supernatant was calculated by dividing the OD values of treated samples by the OD of the medium controls cultured without supernatant. Our predetermined upper limit of relative percent FIV capsid compared to those in the absence of effector cell supernatant was 65% [17]. Any level of relative percentage of FIV capsid below 65% was considered significant suppression. The absolute ELISA OD650 values and the relative percentage of FIV capsid as observed from the supernatants of PBMC collected from infected cat AWF1 are shown in Fig. 1A and B, respectively. Effector cells of infected cat AWF1 continued to suppress FIV replication significantly until at least day 7 of stimulation with inducer cells (p! 0.05). Greater suppression was observed during

Fig. 1. Kinetics and dose-response of the anti-FIV activity of supernatants from PBMC of FIV infected cats. The absolute FIV capsid OD650 values (A), relative FIV capsid (%) (B) and dose-response (C) were shown from each supernatant. Inducer cells were added on days 0 and 3. Supernatants in complete RPMI 1640 were collected and replenished each day for 1 week. For the dose-response study, the supernatant was two-fold serially diluted, and tested for their ability to suppress FIV replication. Target T cells (2!104 cells/well) infected in vitro with FIVPPR were cultured for 6 days with media consisting of 50% supernatant, or media alone without supernatant (positive control), or cultured with supernatant from inducer cells alone. FIV replication was tested by an FIV capsid ELISA. Relative FIV capsid (%) was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. Each bar represents meansGstandard errors of the mean of triplicate samples. The asterisks (*) indicate statistically significant differences (p!0.05).

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the first 2 days after addition of inducer cells. Similar to the untreated controls with only infected cells, the supernatant from inducer alone controls (i.e. in the absence of PBMC) did not suppress FIV replication. Supernatants accumulated during the first 3–4 days of induction were used for further characterization. 3.2. Supernatant suppression was dose-dependent The dose response of suppressing activity from cat AWF1 effector cells was quantified with serial twofold dilutions of supernatants collected 3 days after stimulation with autologous, infected inducer cells. The capacity of supernatant dilutions of effector cells to suppress FIV replication was examined with FIV-PRR infected lymphoblastoid target cells. The inhibition of FIV-PPR replication by supernatants from effector cells was dose-dependent (Fig. 1C), demonstrating more than 50% suppression even at a dilution of 1:64 (1.6%, vol/vol). The supernatants from these stimulated cells positively suppressed FIV replication up to the 1:128 dilution, using 65% as the maximal level of relative percentage of viral capsid protein detected. However, more than 90% suppression (i.e. 10% relative FIV capsid) was observed only from supernatant in the 1:2 and 1:4 dilutions. 3.3. Induction with inducer cells was required for optimal FIV suppression To determine the extent to which optimal suppression was dependent on stimulation by inducer cells, the supernatants were collected at days 3 and 7 from PBMC cultured with or without autologous, FIV-infected inducer cells, and evaluated for their capacity to suppress replication of FIV. Fig. 2 shows the absolute FIV capsid OD650 (Fig. 2A) and the relative percentage of FIV capsid protein detected (Fig. 2B) in the cultures of target cells with various supernatants collected at days 3 and 7 in the presence or absence of inducer stimulation. Independent of the day collected, supernatants from PBMC stimulated with the inducer cells always had significantly lower OD650 values and a lower relative percentage of detectable FIV capsid than those without inducer cell stimulation (p!0.05). Supernatants from stimulated PBMC of both cats collected at day 3 had significantly greater suppression than at day 7 (p!0.05), whereas

supernatants from the unstimulated group on day 3 did not suppress FIV replication (pO0.05). The suppressive activity of the PBMC supernatants from cat M242 was characteristic of those from other cats examined. In contrast, suppression was also observed under unstimulated conditions for cat AUO3. However, even cat AUO3 PBMC required exposure to inducer cells to generate maximal suppressive activity. 3.4. Both heat stable and heat labile suppressing activity could be detected Because the presence of 10% FBS in complete RPMI interferes with attempts to determine heat stability and to purify suppressing activity, PBMC from infected cats were cultured and stimulated in serum-free HB101 medium. Autologous, irradiated inducer cells were added on days 0 and 3, and the supernatants were collected at days 3 and 7, in order to characterize the heat stability of their anti-FIV activity (Table 1). Suppression was observed with the supernatants collected on days 3 and 7 from induced PBMC of all the cats examined. Although varying amounts of suppression were lost, significant suppression was also maintained after heat treatment of supernatants from cells of all animals (p!0.05), with the exception of the supernatant of cat M242 collected on day 7 after induction of cells. 3.5. Production of IFN does not correlate with suppressing activity Interferon-a, a type 1 interferon, is known to inhibit HIV-1 and FIV replication [29–32]. The presence of IFN was determined with a viral CPE inhibition assay. Functional feline IFN was detected only in the supernatant of cat A306 (Table 2), and the IFN type 1 nature of the IFN was confirmed by its antiviral activity following treatment at pH 2. Because IFN could not be detected in stimulated cells from the remaining cats examined, it was not considered a candidate cytokine responsible for overall suppression. However, the observed production of IFN-a in the single cat indicates that differences exist among animals and multiple antiviral factors can be generated in CD8C T

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Fig. 2. The effect of induction with inducer cells on the production of suppressing activity. Absolute OD650 values (A) and relative FIV capsid (%) (B) were shown. PBMC stimulated with inducer T cells were compared with PBMC cultured in the absence of inducer cells, and supernatants collected at days 3 and 7 were examined for their ability to suppress FIV replication. FIV replication was tested by an FIV capsid ELISA. Relative FIV capsid (%) was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. Each bar represents meansGstandard errors of the mean of triplicate samples. The asterisks (*) indicate statistically significant differences (p!0.05).

cells. IFN-g was not detected in any supernatant as determined by ELISA (data not shown). 3.6. Heparin column chromotography Heparin sepharose is a versatile tool for the purification of many proteins, such as growth factors, coagulation proteins, steroid receptors, chemokines, and interferon molecules [33–37]. Both a- and b-chemokines have been reported to inhibit HIV replication [38,39]. To determine whether chemokines were involved in the generated anti-viral activity,

the supernatants collected at days 3 or 7 were diluted and passed through a heparin column. After washing the column, the fractions were eluted with varying concentrations of NaCl. These eluted fractions were dialyzed and examined for their ability to suppress FIV replication. Fractions eluted with 0.25 and 0.5 M NaCl demonstrated significant viral suppression (p!0.05) (Fig. 3A and B). However, suppression was also observed in the flow-through, in spite of a calculated dilution factor of 3. Identifiable activity in both heparin bound and unbound fractions again suggests the influence of multiple factors with antiviral activity.

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Table 1 Comparison of FIV-suppression from supernatants collected from inducer cell-stimulated PBMC of FIV positive catsa Days 0–3 supernatanta

Cell source of supernatant AWF1 inducer cell AWF1 effectorsd AZV2 effectors M165 effectors M242 effectors A306 effectors Medium only

c

Days 4–7 supernatant

Without heat treatment

Heat treatmentb

Without heat treatment

Heat treatment

91G1.8 1.7G0.9* 1.1G0.1* 0.3G0.4* 4G1.9* 0.8G0.1* 100G3.9

105.7G9.6 3.4G0.4* 3.5G0.6* 1.8G0.7* 14.3G3.7* 0.8G0.4* 100G2.6

93.7G1.8 4.8G1.5* 12.4G4.4* 11G4.6* 45.4G6.9* 5.7G2.8* 100G3.9

87.6G3.7 11.7G0.4* 24.5G3.8* 34.6G0.6* 67.9G2.8 19.5G3.9* 100G2.6

*Statistically significant at a p value of !0.05. a % FIV capsid in target cells exposed to supernatants of PBMC for 3 and 7 days. FIV replication in infected target lymphoblastoid cells was determined by the FIV capsid ELISA and relative FIV capsid release (%) was calculated by dividing the OD values of samples by those of the medium controls (100%) cultured without supernatant. The data are shown as meansGstandard errors of the mean, each performed in triplicate. b Supernatants were heated at 100 8C for 10 min prior to adding to target cells. c Supernatant from inducer cells in HB101 media without PBMC. d Supernatant from PBMC cultured with infected inducer cells.

3.7. The molecular weight of anti-viral activity is greater than 10 kD To determine the approximate molecular weight of molecules responsible for the suppressing activity, the supernatants were initially size-fractionated by centrifuging through ultrafiltration membranes of varying molecular weight limits (MWCO). The filtrate of the 50-kD MWCO membrane lost FIV suppressing activity (70.1% capsid protein detected compared to supernatants of untreated infected cells), whereas the suppression in the retentate of the 50-kD MWCO membrane was significantly enhanced (p!0.05) (Fig. 4). Following the centrifugal ultrafiltration through a 10-kD MWCO membrane, the retentate of the 10-kD MWCO membrane had greater detectable suppression (54.4% capsid protein detected), whereas the filtrate of the 10-kD MWCO membrane did not suppress FIV replication (90.7% capsid protein detected). This suggests that the molecule(s) with optimal activity may be greater than 50 kD under the applied conditions. However, lower levels of suppressing activity were detected for the molecules between the 10 and 50 kD limits. 3.8. Bulk CD8C T cells generated anti-FIV activity To determine the effect of CD8C T cells on the anti-FIV activity, CD8-enriched and CD8-depleted

Table 2 Heat stable IFN type I can be detected only in the supernatant collected from inducer cell-stimulated PBMC of one FIV positive cat Cell source of supernatant FIV (C) E238 inducerc E238 effectord AWF1 effector AUO3 effector A306 effector AZV2 effector AUO2 effector FIV(K) OAE5 effector OLQ4 effector Media control

FIV capsid (%) (meanGSEM)a

IFN-I titer in supernatant (units/ml)b

101.6G2.7 15.6G0.3* 10.4G0.9* 38.3G3.1* 3.6G1.9* 55.9G5.5* 34.5G7.2*

0 0 0 0 360* 0 0

35.9G1.5* 69.1G3.6 100G4.1

0 0 0

*Statistically significant at a p value of !0.05. a PBMC from FIV-positive or FIV-negative cats were stimulated by autologous irradiated inducer cells at day 0, and supernatants in complete RPMI were collected at day 3. Cultured lymphoblastoid cells (2!104 cells/well) infected in vitro with FIV-PPR were cultured for 9 days with addition of supernatant on days 0 and 4 at a ratio of 1:1 with media. FIV replication was determined by an FIV capsid ELISA. Relative FIV capsid release (%) was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. The data are shown as meansG standard errors of the mean, each performed in triplicate. b The feline IFN-I titers in supernatants were determined with VSV CPE inhibition assay in CrFK cells. c Supernatant from inducer cells without effector cells. d Supernatant from PBMC cultured with infected inducer cells.

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Fig. 3. The relative percent of FIV capsid produced following exposure of infected cells to the eluted fractions from day 3 (A) or day 7 (B) pooled supernatant following heparin column chromatography. The pooled supernatants were passed through a heparin column after dilution. After washing, the column was eluted with 1.5 ml of varying concentrations of NaCl and the collected fractions were dialyzed against serum free HB101 medium before examining for antiviral activity. FIV replication was tested by an FIV capsid ELISA. Relative FIV capsid (%) was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. Each bar represents meansGstandard errors of mean of triplicate samples. The asterisks (*) indicate statistically significant differences (p!0.05).

cells were prepared from cultivated T cells or PBMC using the panning method [17,25]. As indicated by FACS analyses, the purity of CD8C population in CD8C cell-enriched preparations (following panning with anti-CD4 antibody) from these three cats was O90% and panning procedures used to remove the CD8C cells resulted in !0.1% CD8C cells in CD8C cell-depleted preparations (data not shown). After initial stimulation of PBMC with inducer cells followed by panning, the CD8C cell-enriched preparations were cultured in the presence of inducer cells (cat A306). Similarly selected from cats E238 and M165 were cultured in the absence of inducer cells

but in the presence of recombinant human (rh) IL-2. The relative percentages of FIV capsid protein detected in supernatants of CD8C cell-enriched preparations from the three FIV-infected cats examined were significantly lower than the controls (p!0.05), indicating the generation of strong antiFIV activity by these cells (Fig. 5). In contrast, the relative percentage of detectable FIV capsid following exposure to supernatants from CD8C-depleted cell preparations was similar to that of the medium controls cultured without supernatant. Because CD8C-depleted preparations of the three FIVinfected cats examined did not generate suppressing

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Fig. 4. Molecular size fractionation of FIV suppressing activity from supernatant of induced effector cells. The indicated fractions were examined for their suppression of FIV replication following exclusion centrifugal ultrafiltration. FIV replication was tested by an FIV capsid ELISA. The relative percent of FIV capsid was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. Each bar represents meansGstandard error of mean of triplicate samples. The asterisks (*) indicate statistically significant differences (p!0.05).

Fig. 5. Anti-FIV suppressing activity was generated by bulk CD8C cells. After 7 days of stimulation of PBMC with autologous inducer cells, CD4C or CD8C cells were depleted by the panning method (17, 25). The selected CD8C cell-enriched or -depleted cells were stimulated again with inducer cells for 4 days (cat A306) or cultured for 4 days only with IL-2 (cats E238 and M165) in serum-free medium. The supernatants were collected and tested for their ability to suppress FIV replication by culturing with target FIV-infected T cells for 8–10 days. FIV replication was determined by an FIV capsid ELISA. The percent of FIV capsid was calculated by dividing the OD values of samples by those of the medium controls cultured without supernatant. Each bar represents meansGstandard errors of the mean of triplicate samples. The data are representative of two separate experiments. The asterisks (*) indicate statistically significant differences (p!0.05).

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activity, CD8C cells were likely responsible for the production of the anti-FIV activity. 3.9. Reverse phase-HPLC identified more than one hydrophobic antiviral fraction To further characterize the activity in these supernatants, reverse-phase HPLC was used to separate the fractions with or without suppressing activity from the supernatant of cat AWF1 lymphocytes, stimulated for 4 days in the presence of irradiated, FIV infected inducer cells. Significantly lower amounts of FIV capsid were detected by ELISA in Fractions 12 and 14–21 which were located in the hydrophobic region eluted with 40–60% acetonitrile (p!0.05) (Fig. 6A). Of the 21 fractions collected, 12, 14, 15 and 16, only had !10% of the detectable capsid compared to that of the positive medium control. Therefore, these fractions resulted in O90% suppression of the FIV replication (Fig. 6A). The suppressing activity in fraction 12 was cytolytic and heat labile, whereas that in fractions 14–16 was noncytolytic and retained most of its suppressing activity following heat treatment (Fig. 6B). Another two fractions with heat labile activity (19 and 20) resulted in non-cytolytic suppression, less than 20% of FIV capsid expression from infected target cells (Fig. 6A and B). However, no fraction from the irradiated T lymphoblast alone controls (that is, the inducer cells) was identified with antiviral activity (Fig. 6C). Similar to fractions collected from induced PBMC supernatants, non-cytolytic suppressing activities were identified in the corresponding fractions collected from the supernatants of the enriched CD8C T lymphocytes stimulated by inducer cells (data not shown). Therefore, again CD8C T cells produced the multiple suppressing factors identified by distinct biochemical and physical characteristics.

4. Discussion The complex nature of the physical and chemical properties of non-cytolytic, anti-FIV activity consistently indicated that multiple factors were responsible for the suppression generated from feline inducer cellstimulated CD8C T cells although the distinguishing

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properties could be a result of differences in protein processing, such as proteolytic cleavage. The depletion of CD8C T cells from either PBMC or the cultured T cells abolishes this anti-FIV non-cytolytic activity, confirming other reports that the production of the anti-FIV factors was limited to CD8C T cells [8,9,17,23,24]. Similar to the CD8C cell antiviral factor (CAF) described in humans [40–42], the antiFIV factors from supernatant was dose-dependent, partially heat labile, and produced exclusively by CD8C T cells. However, the activity differed from the influenza A virus-stimulated factor generated from PBMC, which was also generated by the non-CD8C T cells [30]. Here we confirmed that CD8C cells from our animals were clearly the major, if not sole, source of the observed anti-FIV activity. Noncytolytic mechanisms play an innate-like role in FIV cellular immunity. CD8C T cells were reported to produce some cytokines and beta-chemokines, such as interleukin-16, macrophage-inflammatory proteins -1 alpha and beta, RANTES, interferons, macrophage -derived chemokine, I-309, granzymes A and B, and stromal-derived factor (SDF-1), or possible alphadefensins [38–40,43–45], but these proteins were found to lack identity with the human CAF [46–58]. Human SDF-1 was reported to suppress CrFK-tropic FIV infection of CrFK cells, but not of MYA-1 cells [59,60], and IFN-a to partially inhibit HIV replication by the supernatant from influenza virus A-stimulated PBMC [30]. Of the cats that produced suppressing activity in our study, only cat A306 produced detectable feline IFN-a, again suggesting that multiple patterns of antiviral factors or possible synergetic cytokine effects can account for the overall anti-FIV suppression. Each evaluation described indicated that multiple factors are responsible for the non-cytolytic suppression of FIV replication in the supernatant of effector cells stimulated by inducer cells. First, the antiviral supernatant in serum-free media seems to be mostly heat stable although some activity also remained after heat treatment. Second, the fractions eluted from a heparin column with 0.25 and 0.5 M NaCl had suppressing activity. However, the flow-through portion, which did not bind to the heparin column, also demonstrated impressive suppressing activity. Third, while reverse phase HPLC demonstrated suppressing activity in the hydrophobic regions,

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Fig. 6. The OD650 values of reverse-phase HPLC fractions from supernatant collected after 4 days stimulation of PBMC with autologous inducer cells from cat AWF1. The reverse phase-HPLC fractions were collected following elution with increased concentrations of acetonitrile,

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the nature of viral suppression following the denaturing conditions of RF-HPLC and lyophilisation indicated that heat stable and heat labile fractions could be detected from separate peaks. Silver staining following SDS-PAGE demonstrated that multiple bands were visible after heparin column chromatography and reverse-phase HPLC (data not shown), again suggesting the existence of multiple factors. Inconsistencies were reported on the molecular weight of human CAF [21,57,61]. The activity of human CAF identified in CD8C T cell supernatant was associated with two heat-labile proteins, a O50 kD heparin-bound fraction and a ! 50 kD heparin-unbound fraction [21]. The heparin-binding protein appeared to be a modified form of antithrombin III with a usual size of w53 kD. The molecular weight of fractions with strong anti-FIV activity in our study is close to 50 kD, and definitely greater than 10 kD. Therefore, the lower MW chemokines and alpha defensins (both !10 kD) are not likely anti-FIV candidates. The anti-FIV factors described in our studies may, at least partially, overlap in molecule size with the 10–50 kD, heat stable, human CD8C T cell CAF (61), but differ with the small heat stable protein (w6 kD) from human CD8C T cell supernatant described by Mosoian et al. [57]. Since the human CD8C cell non-cytolytic antiHIV response can be blocked by protease inhibitor [51], Levy [61] proposed that one major protein along with the presence of several other antiviral factors may be involved in CAF. He also hypothesized that the CD8C T cells produce a protease and a precursor to CAF which is cleaved by the protease, possibly at the cell surface, to become active as an antiviral protein. Geiben-Lynn [62] and Levy [61] suggested that the suppressing activity was likely present in low concentrations. Our results also suggest that the multiple factors may act together at low concentrations to control retroviral replication. In doseresponse experiments, dose-dependence of

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supernatants from the one cat source indicated 50% suppression of the FIV replication at a 1:64 dilution, although, similar to the culture fluid with CAF in HIV suppression [53], 90% suppression of the FIV replication was observed at only the 1:2 or 1:4 dilution. The low concentration needed for suppression may explain difficulties in isolating the responsible molecules. Although the composition of the anti-FIV factors may differ depending on the means of stimulation, we have consistently demonstrated greater, statistically significant suppression when exposing effector cells to irradiated FIV-infected inducer cells rather than through mitogen stimulation [17]. We have also shown that skin fibroblasts, expressing a Semliki Forest virus vectors, can function as inducer cells for CD8C T lymphocyte secretion of antiviral activity [25]. Culturing of fresh PBMC in the absence of external induction could induce lower levels of suppressing activity, but only when lymphocytes were from the highest responder cats (such as AUO3). Possibly the presence of a small amount of internal inducer cells in PBMC could account for the detectable suppression in the supernatants from PBMC cultured without any added stimulation. The CD8C T cells from the highest responder cats may be in a continuous state of activation. The CD8C non-cytolytic antiviral response is consistently detected in asymptomatic HIV- or FIVinfected and poorly if at all present in uninfected individuals [17,21]. This non-cytolytic activity is significantly correlated with the clinical status in infected patients [54,63]. Our study suggests that the anti-FIV non-cytolytic activity may not necessarily parallel with cytolytic activity. Cat E238 showed impressive non-cytolytic anti-FIV activity at 3 years post-initial infection, but not when the activity was first determined at less than 6 months p.i. [17]. However, the cytotoxic T lymphocyte (CTL) response of E238 at that time was more vigorous than the other animals infected with the PPR strain

3 and their suppression of FIV replication examined after freeze-drying and resuspending in HB101 serum-free medium. (A) These supernatant fractions were reconstituted from the lyophilized fractions, without further heat treatment, before determining the anti-FIV activity. (B) Aliquots of the reconstituted fractions were heat treated at 100 8C for 5 min, prior to examining for suppressive activity. (C) Fractions were collected and reconstituted from irradiated AWF1 inducer cell alone controls. The error bars represent the standard errors of the mean of triplicate samples and may not be discernible where they are small. The asterisks (*) indicate statistically significant differences (p!0.05).

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[17]. The fact that this cat was initially infected with the highest dose (1250 TCID50) of FIV-PPR, may account for the level of CTL response early in infection as similarly described in HIV infection [64,65]. With long-term persistent infection, the noncytolytic activity seemed to be pre-dominant even in cat E238. Thus far, we have not been able to demonstrate optimal suppressor activity within the first 2 months post-infection with FIV (unpublished observations). Our results agree with the reports of Jeng et al. [9] that cats with asymptomic chronic infection of the NCSU1 isolate of FIV had vigorous, easily detectable, non-cytolytic suppressing activity. Viral load of infected cats in our colony can be inversely correlated with in vitro anti-viral activity (manuscript in preparation). In summary, the characterizations of suppressing activities in supernatants collected from stimulated feline effector cells clearly indicated the presence of multiple, distinct factors. Because of the inherent, redundant nature of the immune system and the multitude of cytokines that have been shown to affect lentiviral replication, it is not surprising that more than one antiviral factor can be described. The importance of this mechanism for controlling viral load in the persistently infected animal is worth identifying as it could be exploited for maintaining clinical health in the continued presence of the viable provirus.

Acknowledgements This study was supported in part by Morris Animal Foundation (#96FE-09), the Baylor Center for AIDS Research Core through NIH grant number AI502342 and the National Institute of Health grant number AI36360-01. We thank Nuclear Science Center, Texas A & M University for providing the 60Cobalt irradiation service. We also thank Ms Anna Mata, Lynda Case and Angel Tapia for helping with sample collections.

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