Longitudinal Analysis of Feline Leukemia Virus-Specific Cytotoxic T Lymphocytes: Correlation with Recovery from Infection

JOURNAL OF VIROLOGY, Mar. 2002, p. 2306 2315 Vol. 76, No. 5 0022-538X/02/$04.00 0 DOI: 10.1128/JVI.76.5.2306 2315.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved. Longitudinal Analysis of Feline Leukemia Virus-Specific Cytotoxic T Lymphocytes: Correlation with Recovery from Infection J. Norman Flynn,* Stephen P. Dunham, Vivien Watson, and Oswald Jarrett Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow G61 1QH, Scotland Received 9 August 2001/Accepted 6 November 2001 Feline leukemia virus (FeLV) is a common naturally occurring gammaretrovirus of domestic cats that is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. Although the majority of cats exposed to FeLV develop a transient infection and recover, a proportion of cats become persistently viremic and many subsequently develop fatal diseases. To define the dominant host immune effector mechanisms responsible for the outcome of infection, we studied the longitudinal changes in FeLVspecific cytotoxic T lymphocytes (CTLs) in a group of naïve cats following oronasal exposure to FeLV. Using 51 Cr release assays to measure ex vivo virus-specific cytotoxicity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 capsid antigen, and proviral DNA in the blood. High levels of circulating FeLV-specific effector CTLs appeared before virus neutralizing antibodies in cats that recovered from exposure to FeLV. In contrast, persistent viremia was associated with a silencing of virus-specific humoral and cell-mediated host immune effector mechanisms. A single transfer of between 2 10 7 and 1 10 8 autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. The results suggest an important role for FeLV-specific CTLs in retroviral immunity and demonstrate the potential to modulate disease outcome by the adoptive transfer of antigen-specific T cells in vivo. * Corresponding author. Mailing address: Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden Rd., Bearsden, Glasgow G61 1QH, Scotland. Phone: 44 141 330 6947. Fax: 44 141 330 5602. E-mail: n.flynn@vet.gla.ac.uk. Cytotoxic T lymphocytes (CTLs) are recognized to be one of the early host defense mechanisms generated in response to viral infection and have been shown to have a role in clearing virus in acute infection and controlling viral replication in persistent infections. In humans the rapid clearance of influenza A virus has been correlated with specific CTL activity (32), and control of primary and latent Epstein-Barr virus and cytomegalovirus infections has also been associated with circulating CTL activity (1, 6, 36). The critical importance of virus-specific CTLs in controlling retroviral replication and in rejection of retrovirus-associated tumors has become apparent in both animal models (9, 15, 39) and humans (8). In particular, the role played by human immunodeficiency virus (HIV)-specific CTLs in controlling virus replication and in determining the outcome from infection has become increasingly apparent. There is a close temporal association between the control of early viremia and the appearance of HIV-specific CTLs (5, 27, 37). More recently, application of peptide-major histocompatibility complex (MHC) tetramer technology (1) has revealed a striking negative correlation between CTL numbers and virus load in chronic HIV infection (34). In vivo CD8 T-cell depletion studies in acute and chronic simian immunodeficiency virus infection in macaques have shown strong dependence on virus-specific CTLs for virus control and slowing disease progression (24, 40). Domestic cats are the natural host for two retroviruses, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Similar to HIV, primary infection with FIV is associated with the rapid emergence of a vigorous virus-specific CTL response (2, 11, 41). Further, several studies have shown that vaccinal protection is critically dependent on high levels of circulating effector FIV-specific CTLs (4, 12, 17, 28). In spite of the quantitatively strong FIV-specific CTL responses typically observed following FIV infection, recovery has never been reported. Both FIV and FeLV infections are associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia (33). In contrast to FIV infection, the majority of cats exposed to FeLV frequently clear circulating virus and recover, yielding a unique opportunity to investigate the host immune effector mechanisms important in retroviral clearance and in determining disease outcome in a natural host species. The host immune effector mechanisms responsible for the outcome following FeLV infection are presently unresolved. Following experimental exposure to FeLV, the majority of cats over 16 weeks of age either recover completely from infection or develop a latent infection (16). A proportion of cats develop a persistent infection, and these animals ultimately develop FeLV-associated diseases. Virus neutralizing antibodies appear to have a role in the recovery of cats from FeLV infection. However, the majority of cats that develop a transient infection recover before virus neutralizing antibodies are detected in the circulation, suggesting that cellular immunity may be important in clearance of the virus. Recently we have shown that inoculation of cats with an FeLV DNA vaccine elicits protective immunity in the absence of a virus-specific humoral response. High levels of FeLVspecific CTLs were detected in the blood and lymphoid tissues of FeLV DNA-vaccinated, protected cats, implicating this mechanism in the observed protection (13). However, there 2306

VOL. 76, 2002 FeLV-SPECIFIC CTL CONTROL OF VIREMIA 2307 are no reports detailing the temporal coevolution of virusspecific cell-mediated immunity and the control of viremia in cats that recover following exposure to FeLV. To define more closely the biology of FeLV infection and determine whether an association exists between the frequency of FeLV-specific CTLs and plasma virus load, we studied the longitudinal changes in FeLV-specific CTLs in a group of naïve cats following oronasal exposure to FeLV. Using uncultured, antigen-specific cytolysis to measure ex vivo CTL activity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 antigenemia, and proviral DNA in the blood. The longitudinal analysis revealed a clear correlation between high levels of circulating FeLV-specific effector CTLs and recovery following exposure to FeLV. Further, a single transfer of autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. MATERIALS AND METHODS Animals, animal care, and experimental exposure. Before exposure to FeLV the 10 12-week-old, outbred, specific-pathogen-free domestic kittens used in this study were free from infectious FeLV, FeLV neutralizing antibodies, and FeLV p27 in the blood. When 16 weeks old, each kitten was infected with 5 10 5 focus-forming units of FeLV-A/Glasgow-1 (18) by introducing 0.1 ml of virus suspension into each nostril and 0.8 ml into the mouth. The kittens were housed together and fed a proprietary brand of cat food twice daily and had ad libitum access to fresh water, in accordance with United Kingdom Home Office guidelines. Detection of infectious virus in blood. The presence of infectious FeLV was determined by inoculation of heparinized plasma samples onto QN10S cells in vitro (19). The presence of FeLV p27 capsid antigen was determined in the plasma using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) (30). Quantification of FeLV provirus loads. Feline buffy coat cells were prepared from blood by addition of red cell lysis buffer (140 mm NH 4 Cl, 1 mm NaHCO 3 ) at 37 C for 3 min, followed by centrifugation. DNA was extracted from the cells using a QIAamp blood mini kit following the manufacturer s instructions (Qiagen GmbH, Hilden, Germany). FeLV provirus was measured by quantitative real-time PCR using an ABI Prism 7700 sequence detection system (Applied Biosystems, Warrington, United Kingdom). Primers specific for FeLV-A env (42) and probe were designed using Primer Express software (Applied Biosystems, Warrington, United Kingdom). The primers used were 5 -GCC CCA AAC GAA TGA AAG C-3 and 5 -AAT CCG TTT GGG ACC CAT G-3. The probe was 5 -FAM-CCC AAG GTC TGT TGC CCC CAC C-TAMRA-3. The 50- l PCRs contained 10 mm Tris (ph 8.3), 50 mm KCl, 200 nm each datp, dctp, and dgtp, 400 nm dutp, 300 nm each primer, 135 nm fluorogenic probe, and 2.5 U of Taq DNA polymerase, to which was added 5 l of sample or standard DNA. After an initial denaturation (2 min at 95 C), amplification was performed with 40 cycles of 15 s at 95 C and 60 s at 60 C. Plasmid DNA containing the env gene of FeLV-A (42) was used as a standard for the PCR. The copy number of this standard was calculated by optical density (OD) measurement at 260 nm and confirmed by agarose gel electrophoresis followed by ethidium bromide staining. A 10-fold dilution series was made in PCR-grade water with 30 g/ml calf thymus DNA (Life Technologies, Paisley, United Kingdom) as a carrier. Preliminary experiments confirmed that this PCR did not detect endogenous retroviral DNA and was sufficiently sensitive to detect one copy of FeLV. Provirus loads were corrected to counts per 10 6 peripheral blood mononuclear cells (PBMC) by performing a further Taqman PCR designed to measure total DNA and thus more accurately estimate cell number. This was performed using primers and probe specific for the rrna gene (20). Standards for this PCR were derived from MYA-1 cell line genomic DNA. The DNA was quantified by OD measurement at 260 nm and a dilution series prepared in PCR-grade water with 30 g/ml yeast RNA (Roche Diagnostics Ltd., Lewes, United Kingdom) as a carrier. To calculate cell number, a DNA content of 6 pg/cell was assumed. Virus neutralizing antibodies. Virus neutralizing antibodies were detected in the plasma by focus reduction of FeLV-A/Glasgow-1 in QN10S cells (19). Detection of FeLV-specific CTL. At regular intervals prior to and following infection, peripheral venous blood was collected into an equal volume of Alsevers solution, and mononuclear cells were prepared by density centrifugation over Ficoll-Paque (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.). FeLVspecific CTL activity was measured directly ex vivo on autologous and allogeneic skin fibroblast target cells infected with recombinant vaccinia viruses expressing either FeLV gag/pro or FeLV env gene products (kind gifts from E. Paoletti, Virogenetics Corporation, Troy, N.J.) or wild-type vaccinia virus as a control. Additionally, the skin fibroblast target cell lines were infected in vitro with FeLV-A/Glasgow-1. These persistently infected cell lines express all FeLV structural antigens, thereby enabling detection of CTL specificities that might be overlooked using recombinant vaccinia viruses to deliver FeLV antigens. Microcytotoxicity assays were performed as described previously (13). In vitro restimulation of lymph node cells. Autologous skin fibroblasts persistently infected with FeLV-A/Glasgow-1 were used as antigen-specific stimulator cells in vitro. To inactivate the viral particles, the FeLV-infected fibroblasts were irradiated for 300 s in a UV-cross-linker (Stratagene) (19) prior to cocultivation with popliteal lymph node lymphocytes at a ratio of approximately 10:1 in complete RPMI medium for 7 days. Antigen-specific lymphoblasts were expanded for a further 7 to 10 days by culture in complete RPMI medium supplemented with 100 IU of human recombinant interleukin-2 (hril-2) per ml (a kind gift from M. Hattori, University of Tokyo). Adoptive transfer of virus-specific lymphoblasts. Single-cell suspensions were prepared from popliteal lymph node biopsy material collected under general anesthesia induced by intravenous injection of 9 mg of alfaxalone and alphadolone acetate (Saffan; Schering-Plough Animal Health, Middlesex, United Kingdom) per kg, from five persistently viremic cats 15 weeks following exposure to FeLV. The cells were restimulated in vitro by coculture with autologous, FeLVinfected, UV-inactivated skin fibroblasts for 7 days, followed by expansion of antigen-specific blasts in hril-2 for a further 7 days. At this time, cats were reinfused intravenously with between 2 10 7 and 1 10 8 autologous lymphoblasts (as shown in Table 2). Cell staining for flow cytometry. To determine the composition of lymphocytes in the cells prepared for adoptive transfer, 2 10 6 lymph node lymphoblasts were incubated with 50 l of mouse monoclonal anti-fcd4 (vpg 34), anti-fcd8 (vpg 9) (43), or anti-cd21 for 30 min at 4 C. Labeled cells were then washed, and bound monoclonal antibody was visualized with a 1:20 dilution of an anti-mouse immunoglobulin-fluorescein isothiocyanate conjugate (DAKO). After a further 30 min at 4 C, the cells were washed again and analyzed on an EPICS (Beckman- Coulter, Emeryville, Calif.). Statistics. Statistical analysis was performed using GraphPad InStat version 3.0 for Windows 95 (GraphPad Software, San Diego, Calif.). RESULTS Cats either recover or develop persistent viremia following FeLV exposure. Cats were infected with FeLV under conditions where it was expected that approximately half would become persistently viremic and the remainder would recover (20). Virus isolation was attempted from the peripheral blood prior to exposure to FeLV and at regular intervals following infection. The results are shown in Table 1. Following exposure to FeLV by the oronasal route, which simulates a natural mode of challenge, virus was detected in 9 of 10 cats by week 7 following exposure. On the basis of the isolation of infectious FeLV in vitro, the cats could be divided into two groups, irrespective of plasma antigenemia. Five cats (1, 6, and 8 to 10) became persistently viremic, with isolation of FeLV possible at all time points examined after week 4. One of these cats (cat 1) died at week 9 following infection due to causes unrelated to the study and was excluded from further investigation. Four cats (2, 4, 5, and 7) developed a transient viremia and recovered. Infectious virus was detected in the blood of these cats on at least one occasion up to 13 weeks postexposure, but was not detected after this time. One cat (cat 3) recovered without the development of viremia.

2308 FLYNN ET AL. J. VIROL. TABLE 1. Isolation of infectious FeLV and p27 capsid antigen in the peripheral blood a Virus and antigen detection at wk post-felv infection: Cat no. 0 1 4 7 10 13 17 21 24 VI p27 VI p27 VI p27 VI p27 VI p27 VI p27 VI p27 VI p27 VI p27 1 2 3 4 5 6 n.d. n.d. 7 8 9 10 a FeLV in peripheral plasma was detected by isolation on QN10S cells which were examined on days 5 to 7 for specific foci of transformation. The presence of FeLV p27 capsid antigen was determined by ELISA. A positive result is recorded where the sample value:positive value (s:p) ratio is 0.200 or greater. Cat 1 died before week 10. Adoptive transfer of autologous lymphoblasts was performed at week 17. VI; virus isolation; n.d., not done. Circulating FeLV capsid antigen is detected 1 week postexposure. Plasma samples were also examined for the presence of FeLV p27 capsid antigen by ELISA (30), in parallel with the virus isolation assays. As described above, cats either became persistently viremic or developed a transient viremia and recovered following exposure to FeLV. As shown in Table 1, viral p27 capsid antigen was first detected in the plasma 1 week following exposure to FeLV, and all of these cats developed a transient viremia following exposure to FeLV and subsequently eliminated infectious virus. In contrast, the cats that became persistently viremic did not exhibit this early peak in plasma viral antigen. Instead, the plasma FeLV p27 capsid antigen became detectable at week 4 following exposure, and the cats remained positive until the termination of the study. FeLV proviral burdens are lower in recovered cats. Provirus load was determined in PBMC preexposure, at 1 week following challenge and at 3-week intervals following challenge by real-time PCR. Proviral DNA was first detected 1 week following exposure. Cats that recovered following FeLV exposure had lower copy numbers of FeLV DNA/10 6 PBMC than cats that became persistently viremic (Fig. 1). However, the peak in FeLV p27 capsid antigen observed between 1 and 4 weeks postexposure in the plasma of cats developing a transient viremia was not reflected in the proviral burdens. In persistently viremic cats, a biphasic pattern was observed, with an initial peak in proviral DNA detectable at 4 weeks postexposure, and a second peak between 10 and 17 weeks postexposure. Virus neutralizing antibodies are detectable in recovered cats. The presence of virus neutralizing antibodies was determined in the peripheral plasma, preexposure and at 3-week intervals following challenge. Throughout the study period, virus neutralizing antibodies were not detected in animals that became persistently viremic (Fig. 2). However, virus neutralizing antibodies were detected in all of the cats that recovered, whether or not they had a transient viremia following exposure to FeLV. The clearance of infectious virus (Table 1) either preceded or coincided with the detection of virus neutralizing antibodies in the peripheral circulation. Rapid induction and maintenance of FeLV-specific CTLs in recovered cats. To assess the involvement of virus-specific cellmediated immunity in the clearance of FeLV, mononuclear cells were prepared from the blood preexposure, 1 week after exposure, and at 3-week intervals thereafter, and FeLV-specific effector CTL responses were measured directly ex vivo. FeLV-specific effector CTL responses were first detected in the blood collected from six of eight cats 1 week after exposure to virus (Fig. 3). By 4 weeks postexposure, virus-specific CTLs were detectable in the blood of all cats. There was negligible recognition of target cells infected with wild-type vaccinia virus, confirming the specificity of the observed responses. Furthermore, allogeneic target cells expressing FeLV antigens were not recognized, indicating the MHC-restricted nature of the response. The cats could be divided clearly into two groups on the basis of the onset of detection of CTL activity, the magnitude of the lysis observed, and the persistence of the CTL activity. Thus, in four of the five cats which recovered following exposure to FeLV, CTLs recognizing target cells expressing FeLV Gag/Pro and Env in addition to FeLV-infected cells were detected from week 1 postexposure and were maintained until completion of the study at week 17. One animal (cat 7) recovered later than the other cats, and did so in the absence of significant ( 10% specific lysis) virus-specific cytolysis. The initial CTL responses observed in the remainder of the cats, which all developed persistent viremia, were not significantly different from those detected in recovered cats. Statistical analysis by one-way analysis of variance of Gag/Pro-, Env-, and FeLV-A-specific lysis confirmed this observation (P values 0.6264, 0.1148, and 0.3267 for weeks 1, 4, and 7 postexposure, respectively). However, by 10 weeks postexposure, the FeLV-specific CTL responses in cats which subsequently recovered were very significantly higher (P value 0.0013) than the responses observed in persistently viremic cats. Further analysis of the antigen specificity of the responses in the two disease outcomes using the Student-Newman-Keuls multiple comparisons test revealed significant differences in the recognition of both Gag/Pro- and Env-expressing targets (P 0.05 for both antigen specificities) at this time. Finally, examination of the specificity of the CTL responses in transiently viremic, recovered cats revealed two overlapping peaks in CTL activity, with Gag/Pro-expressing targets recognized 4 to 7 weeks postexposure and Env-expressing targets

VOL. 76, 2002 FeLV-SPECIFIC CTL CONTROL OF VIREMIA 2309 FIG. 1. FeLV provirus loads in PBMC. FeLV provirus load was determined by quantitative Taqman PCR. DNA was extracted from PBMC at regular intervals prior to and following FeLV exposure. The sensitivity of this technique was determined to be 1 copy per reaction, which typically contained 2.5 10 5 cells. Solid lines, recovered cats; dotted lines, persistently viremic cats. The arrowhead indicates the time of adoptive transfer. recognized slightly later, at 10 to 13 weeks postexposure (Fig. 3). Adoptive transfer of virus-specific T lymphocytes modulates viral burden in vivo. In this study, single-cell suspensions were prepared from popliteal lymph node biopsies collected from four persistently viremic cats 15 weeks following experimental FeLV exposure. The cells were restimulated in vitro by coculture with autologous, FeLV-infected, UV-inactivated skin fibroblasts for 7 days, followed by expansion of antigen-specific blasts in human hril-2 for a further 7 days. At this time, cats were reinfused intravenously with between 2 10 7 and 1 10 8 autologous lymphoblasts (Table 2). Phenotypic characterization of the transferred cells revealed that the cells were of T-cell origin and comprised various proportions of both CD4 and CD8 cells. No B cells were evident, as determined by the expression of CD21. Virus-specific CTL activity of the transferred cells was measured directly ex vivo on autologous and allogeneic fibroblasts infected with recombinant vaccinia virus expressing FeLV Gag/Pro or FeLV Env, or infected with FeLV and therefore expressing all viral antigens. Virus-specific CTL activity was detectable in the lymph node cells used for adoptive transfer in all cats. Although recognition of FeLV Gag/Pro expressing targets predominated, FeLV Env-expressing targets and targets infected with FeLV were also recognized and lysed (Table 2). Postinfusion, the presence of infectious virus was monitored by isolation on QN10S cells, and viral burdens were measured quantitatively by real-time PCR. Retrospective analysis revealed that one cat (cat 7) had recovered naturally and was nonviremic at the time of adoptive transfer (see Table 1). In the three remaining cats (cats 6, 8, and 9), infectious FeLV was isolated from the blood at all time points examined until the termination of the study. Despite the continued presence of infectious virus in these cats, quantitative examination of proviral burdens, as assessed by real-time PCR, decreased following transfusion of lymphocytes. Statistical analysis revealed that the decrease in proviral burden in cats 8 and 9 was highly significant (paired t test, two-tailed P value 0.0046), while the difference observed in cat 6 was not significant (two-tailed P value 0.1351). There was no statistically significant drop in proviral burden observed in cat 10, a persistently viremic control cat which did not receive a transfusion of lymphoblasts. These data indicate that a single transfusion of antigen-activated lymphoblasts with FeLV-specific CTL activity was sufficient to lower the circulating viral burden, but could not completely eliminate infectious virus. Adoptively transferred FeLV-specific CTLs migrate to the lymph nodes. Following adoptive transfer, it was not possible to detect an increase in virus-specific CTL activity in the peripheral blood above those levels observed pretransfer. This may have been due to the dilution of the cytotoxic effector cells

2310 FLYNN ET AL. J. VIROL. FIG. 2. Detection of virus neutralizing antibodies. Virus neutralizing antibodies (VNA) were assayed in the peripheral plasma preexposure and at 3-week intervals following exposure to FeLV by focus reduction of FeLV-A/Glasgow-1 on QN10S cells. Solid lines, recovered cats; dotted lines, persistently viremic cats. The arrowhead indicates the time of adoptive transfer. Note that the values for persistently viremic cats are uniformly negative, so that individual cats are not discernible in the figure. in the adoptive transfer in the circulating blood volume. Alternatively, it may be due to the sequestration of the adoptively transferred cells into the secondary lymphoid organs. To resolve this issue, the lymphoid distribution of FeLVspecific CTL activity was compared in cats which received an adoptive transfer of lymphocytes with that in a persistently viremic control cat which did not receive an infusion of lymphocytes. The results are shown in Fig. 4. Virus-specific effector CTLs were readily detectable in the lymph nodes of the persistently viremic cats which received an adoptive transfer of lymphocytes. This activity was directed predominately to FeLV Gag/Pro-expressing targets; additionally, FeLV-A-infected targets were also recognized. In contrast, no virus-specific effector CTL activity was detectable in the lymph nodes of a persistently viremic control cat which did not receive an infusion of lymphocytes. No significant FeLV-specific CTL activity was detected in the blood of any cat. DISCUSSION Since the first description of FeLV in 1964 (21) considerable progress has been made in understanding the pathology and molecular mechanisms of FeLV-related diseases. It is recognized that cats can fully recover following FeLV infection, an observation that paved the way for the development of several vaccines that are commercially available. However, the immune mechanisms responsible for determining the outcome of FeLV infection and mediating vaccinal immunity remain elusive. While virus neutralizing antibodies are associated with protection, they appear after recovery from infection, and the disappearance of infectious virus from the blood and FeLV DNA vaccination appear to mediate protection in the absence of detectable virus neutralizing antibodies. These observations suggest that virus-specific cellular immune responses may also have a role to play in determining the outcome of infection and in vaccinal immunity. This is consistent with the observations that commercial FeLV vaccines, which protect cats from challenge, do not regularly induce virus neutralizing antibodies, and a DNA vaccine that gives excellent protection induces high levels of CTL activity but no anti-felv antibodies. In the present report, we have correlated the disease outcome, in terms of circulating infectious virus, FeLV p27 antigenemia, and proviral burden, following oronasal exposure to FeLV with the emergence of host virus-specific immune responses. Following oronasal exposure to FeLV, cats either recovered, with or without a transient viremia, or became persistently viremic (see Table 1). The results on isolation of infectious virus from the blood agreed with the detection of FeLV p27 antigenemia and with FeLV proviral burdens, although infectious virus was never isolated from one cat. In persistently infected cats, detection of FeLV proviral DNA in the blood

VOL. 76, 2002 FeLV-SPECIFIC CTL CONTROL OF VIREMIA 2311 FIG. 3. Elicitation of virus-specific CTLs following FeLV exposure. Mononuclear cells were prepared from the peripheral blood of recovered cats (a; n 5) and persistently viremic cats (b; n 4) preexposure and at regular intervals following intranasal and oral exposure to FeLV. FeLV-specific effector CTL activity was measured directly ex vivo on autologous and allogeneic skin fibroblast targets labeled with 51 Cr and infected with recombinant vaccinia viruses expressing either FeLV Gag/Pro (solid bars), Env (hatched bars), or wild-type vaccinia virus as a negative control (consistently below 5% and not shown). Alternatively, targets were infected with FeLV-A/Glasgow-1 in vitro (shaded bars). The release of 51 Cr into the culture supernatant was detected after 4hofincubation at 37 C. Results represent the mean values SEM from triplicate cultures at an effector-to-target cell ratio of 25:1, from which the values for recognition of allogeneic targets have been subtracted. revealed a biphasic pattern, with an initial peak occurring at week 4 and a second peak observed between weeks 10 and 17. Viral p27 capsid antigen was detected earlier following exposure in cats which subsequently recovered following exposure. The earliest time at which viral antigen was detected in the plasma of cats which developed a persistent viremia was 4 weeks postexposure. Was it possible that the earlier exposure of the immune system in the cats that recovered contributed to their ability to eliminate the virus? Was virus-specific immunity primed in these cats? To address this issue, FeLV-specific humoral and cell-mediated immune responses were monitored in the blood of cats at regular intervals following exposure to the virus. Virus neutralizing antibodies were detected in the plasma of all recovered cats. Previous studies have demonstrated that passive transfer of immune serum to kittens can confer protection against challenge (22). However, the delay in the detection of virus neutralizing antibodies until after the clearance of infectious FeLV from the blood of transiently viremic cats observed in this and other studies suggested that the presence of replicating virus may have downregulated this important immune effector mechanism. This silencing of the humoral immune system is also observed in persistently viremic cats, in which virus neutralizing antibodies are either undetectable or at very low levels, as are antibodies to other FeLV proteins (data not shown) (13, 31). Vigorous virus-specific effector CTL responses were observed in all FeLV-exposed cats, irrespective of the outcome of FeLV infection. The CTL activity was virus specific, behaved in an MHC class I-restricted manner, and was detectable directly ex vivo without any prior restimulation of the effector cells. Virus-specific CTLs could be detected as early as 1 week post- TABLE 2. Phenotypic and functional characterization of adoptively transferred lymphocytes and effect of transfer on virus burden a Cat no. No. of cells transferred % Positive cells % CTL activity No. of copies of FeLV/10 6 PBMC fcd4 fcd8 fcd21 FeLV Gag/Pro FeLV Env FeLV-A Pretransfer Posttransfer 6 1 10 8 86.72 18.3 0.34 35.4 3.9 20.8 2,521,627 2,369,672 7 6 10 7 63.33 26.82 0.79 27.3 4.6 21.3 61,227 44,726 8 2 10 7 34.5 19.0 0.1 24.1 14.6 13.9 1,029,395 357,666 * 9 4 10 7 60.4 15.51 0.41 35.8 14.2 16.3 978,047 296,536 * a Following in vitro restimulation and expansion of popliteal lymph node cells, the cells were labeled with monoclonal antibodies recognizing fcd4, fcd8, or fcd21 and analyzed by flow cytometry. The number of cells intravenously reinfused into the autologous donor is also indicated. FeLV-specific CTL activity in the transferred lymphocytes was measured as described earlier. The values shown represent percent lysis from triplicate cultures at an E:T ratio of 25:1, from which the values for recognition of allogeneic targets ( 5%) have been substracted. Virus burdens were measured by real-time PCR. *, statistically very significant by tailed t test.

2312 FLYNN ET AL. J. VIROL. FIG. 4. Adoptively transferred T cells migrate to the lymph nodes. FeLV-specific effector CTL activity was compared in the peripheral lymph nodes and blood of three cats 6 weeks after the infusion of autologous antigen-activated lymphocytes (cats 6, 8, and 9), and in one persistently viremic control cat that did not receive an infusion (cat 10). FeLV-specific CTLs were measured directly ex vivo on autologous (solid lines and symbols) and allogeneic (dashed lines and open symbols) skin fibroblast targets labeled with 51 Cr and infected with recombinant vaccinia viruses expressing either FeLV Gag/Pro ( ), Env (F), or wild-type vaccinia virus as a negative control ( ). Alternatively, targets were infected with FeLV-A/Glasgow-1 in vitro (Œ). The release of 51 Cr into the culture supernatant was detected after 4hofincubation at 37 C. Results represent the mean values from triplicate cultures.

VOL. 76, 2002 FeLV-SPECIFIC CTL CONTROL OF VIREMIA 2313 exposure in the majority of cats which recovered following exposure to FeLV, whereas CTL activity was not evident in most persistently viremic cats until 4 to 7 weeks postexposure. This temporal delay in host virus-specific immune effector mechanisms may have allowed the virus sufficient opportunity to infect a larger reservoir of target cells before immune intervention. Although the level of FeLV-specific CTL activity measured in the recovered cats was higher than that observed in persistently viremic cats during the acute stage of infection (Fig. 3), this difference did not become statistically significant until week 10 postexposure (P value 0.0013). Further, the virusspecific CTLs were maintained in the recovered cats until after infectious virus was cleared from the blood, whereas the CTL responses waned in persistently viremic cats. Examination of the antigen specificity of the responses revealed two overlapping peaks in CTL activity, with recognition of Gag/Pro-expressing targets occurring 4 to 7 weeks postexposure and recognition of Env-expressing targets occurring slightly later, at 10 to 13 weeks postexposure. This pattern of recognition was observed irrespective of the outcome of infection. Thus, it appeared that significantly higher levels of virusspecific cytolysis were observed earlier in cats which recovered following challenge compared to cats which became persistently viremic. However, one cat did not conform to this rule. Cat 7 cleared infectious virus from the blood in the absence of detectable virus-specific humoral or cell-mediated immune responses. Recovery from FeLV has also been observed in naturally infected cats in the absence of an anti-felv humoral immune response (O. Jarrett, unpublished observations). Clearance of FeLV in cat 7 was not noted until 17 weeks following exposure, a minimum of 4 weeks after clearance of circulating virus in the other recovered cats. However, this cat still had detectable viral p27 antigen in the plasma despite the absence of infectious virus. Such discordance of virus isolation and plasma antigen ELISA results is also recognized in naturally infected cats (23). Presently we are unable to comment on the mechanisms of virus clearance in this animal, although one possibility is that the FeLV-specific CTLs elicited in this cat may have been sequestered to sites of virus replication in the host and were not free in the circulation. Following HIV-1 infection in humans, it is recognized that there is compartmentalization of the virus-specific CTLs to the lymph nodes (5), known to be a site for virus sequestration an replication (10, 35). Future studies will investigate this phenomenon by examining the lymphoid distribution of virus-specific CTL responses following FeLV exposure. We have previously reported that inoculation of cats with DNA encoding the FeLV gag and env genes confers protection against viremia and the development of latency without inducing antiviral antibodies (14). Vaccinal immunity was associated with the detection of higher levels of FeLV-specific effector CTL in the blood and lymphoid organs to FeLV Gag/Pro and Env antigens than those observed in unvaccinated control, persistently viremic cats (13). That study also described the skewing of immune recognition towards target cells infected with FeLV in persistently infected cats. This observation suggested that the CTL response in persistently viremic cats may recognize a different viral antigen(s) or epitope(s) from those expressed by FeLV recombinant vaccinia viruses, for example, reverse transcriptase or integrase. In the present longitudinal study, this effect was not as marked, perhaps reflecting the earlier sampling dates in the present study compared to the retrospective analysis reported previously. However, the levels of lysis observed on FeLV-infected targets were greater than the levels observed on Env-expressing targets throughout the study period. Studies of murine leukemia virus infection have shown that it is possible to control viremia and eliminate virus from the host by the adoptive transfer of virus-specific T cells (44, 45). CD8 T cells are important primary effector cells in host defense against HIV, limiting HIV replication following primary infection and delaying disease progression, but ultimately unable to contain the virus (5, 34, 37). Based on this evidence, human studies are evaluating the potential of the adoptive transfer of autologous virus-specific CTLs isolated from the blood and expanded in vitro as a means to quantitatively boost HIV immunity. The infusion of more than 10 9 CTLs/m 2 achieved frequencies of 1 to 4% in the blood and also increased cytolytic activity (26, 29). The transferred T cells localized to the lymph nodes and accumulated at sites adjacent to CD4 T cells actively replicating HIV (7). However, the HIV-specific CTLs persisted very briefly, resulting in only a transient antiviral effect. It seems likely that this low survival rate was associated with an inadequate endogenous HIV-specific CD4 T-cell response required to sustain the CD8 response (25, 38). The results from the above experiments suggest that the virus-specific CTL response represents a critically important mechanism in the clearance of infectious virus from the blood of transiently viremic cats which then recover from their infection. If these correlative observations are true, then it should be possible to directly modify the infectious viral burden by the adoptive transfer of FeLV-specific T cells to persistently viremic cats in vivo. In cats, the adoptive transfer of peripheral blood lymphocytes from uninfected MHC-matched donor cats vaccinated with formalin-inactivated FeLV did not influence the course of FeLV infection unless concomitant systemic therapy with alpha interferon, with or without zidovudine, was given (46). In a related study, reinfusion of autologous lymph node lymphocytes from FeLV antigen-positive naturally infected cats did result in decreased levels of FeLV antigen in the blood of a proportion of the cats (3). However, the phenotype or antigen specificity of the adoptively transferred lymphoblasts used in these studies was unknown (3). In the present study, adoptive transfer of a single infusion containing between 2 10 7 and 1 10 8 lymphocytes was associated with a decline in virus load in all cats. Phenotypic analysis revealed that animals were infused with a mixed population of CD8 and CD4 T cells, which may prolong any antiviral effects of the transferred lymphocytes in vivo. The transferred cells contained FeLV-specific cytotoxic T cells that preferentially recognized and lysed FeLV Gag/Pro-expressing target cells in vitro. With the exception of one cat that had an extremely high proviral burden at the time of adoptive transfer, the decline in virus load observed posttransfer was very significant (two-tailed P 0.0046). However, a single infusion of cells was not sufficient to completely eliminate circulating in-

2314 FLYNN ET AL. J. VIROL. fectious virus, despite the virus-specific cytotoxic potential of the cells and the presence of CD4 T cells. Thus, a number of repeat infusions may be required to boost and sustain the antiviral activity, since each individual virus-specific CD8 CTL can only kill between 3 and 30 target cells before resting. The absence of any detectable FeLV-specific CTL activity in the blood following adoptive transfer of lymphocytes lends support to this theory. However, virus-specific CTL responses were detectable in the lymph nodes of cats receiving an adoptive transfer of lymphocytes, but not in a control cat which did not receive an infusion of cells. This observation suggests that the transferred cells had migrated to the lymph nodes. Similar observations have been made in the lymph nodes of HIVinfected human patients following adoptive transfer of lymphocytes (7). Whereas HIV is known to be sequestered in the lymph nodes of infected patients, FeLV replicates in dividing cells throughout the host, suggesting that the migration of the transferred T cells to the lymph node may reflect the expression of homing receptors on the transferred T cells rather than a migration driven by viral antigen. In conclusion, oronasal exposure of domestic cats to FeLV resulted in either recovery from infection, with or without a transient viremia, or the development of persistent viremia. Clearance of circulating infectious virus was temporally associated with the evolution of a vigorous FeLV-specific CTL response in the blood in the absence of a virus-specific immune response. In persistently viremic cats, there was a silencing of both cellular and humoral virus-specific immune responses. 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