Protective Cellular Retroviral Immunity Requires ... - Journal of Virology

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RICHARD C. HOM,"2 ROBERT W. FINBERG,12 STEVEN MULLANEY,3 AND ... and Division of Cancer Pharmacology,3 Dana-Farber Cancer Institute, and.
Vol. 65, No. 1

JOURNAL OF VIROLOGY, Jan. 1991, p. 220-224

0022-538X/91/010220-05$02.00/0 Copyright © 1991, American Society for Microbiology

Protective Cellular Retroviral Immunity Requires both CD4+ and CD8+ Immune T Cells RICHARD C. HOM,"2 ROBERT W. FINBERG,12 STEVEN MULLANEY,3 AND RUTH M. RUPRECHT23*

Laboratory of Infectious Diseases' and Division of Cancer Pharmacology,3 Dana-Farber Cancer Institute, Department of Medicine,2 Harvard Medical School, Boston, Massachusetts 02115

and

Received 18 June 1990/Accepted 9 October 1990

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 107 T cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 107 T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge. as

While immunization for human immunodeficiency virus type 1 is still under investigation and has not yet been proven efficacious in humans, various experimental approaches have led to successful vaccinations against different murine retroviruses. With one exception, which involved purified Friend murine leukemia virus (FV) envelope glycoprotein (11), all successful vaccination protocols involved whole viruses, either killed, live attenuated, or live recombinant attenuated. In 1959, Friend used Formalin-inactivated cell extracts from FV-infected mice for immunization; upon challenge, vaccinated mice survived longer than unvaccinated mice (9). Fink and Rauscher demonstrated that Formalin-inactivated spleen homogenates from mice infected with Rauscher murine leukemia virus (RLV), combined with Freund's adjuvant, completely protected immunized animals against disease following RLV challenge; passive immunization with immune serum, however, yielded only a 50% protection (7). Immunization with Formalin-inactivated FV led to 80% protection against FV-induced leukemia (9). Vaccination with formaldehyde-inactivated Moloney murine leukemia virus led to detectable levels of neutralizing antibodies in resistant mice but not in susceptible strains (20). Subunit vaccination with purified gp85 of FV was found to be successful (11); detection of serum antibodies against gp85 correlated with protection against FV viremia and disease. Mice inoculated with live recombinant vaccinia virus expressing FV envelope proteins had an envelopespecific T-cell-proliferative response. When challenged with FV complex, these animals were protected against leukemia. Their immune response was characterized by neutralizing antibodies as well as cytotoxic T cells (6). The exact roles of T cells and their subsets in providing immunity against viremia and murine leukemia virus-induced disease, however, have not been well defined. In this work, we have examined the role played by T cells in preventing RLV infection, with particular emphasis on CD4+ and CD8+ T-cell subsets. RLV is a murine leukemia

retrovirus complex which consists of several components (30): (i) a replication-competent helper virus which by itself causes B-cell lymphoma late in life; (ii) a replication-defective spleen focus-forming virus which causes massive splenomegaly and erythroleukemia accompanied by anemia, thrombocytopenia, and liver infiltration by blast cells which lead to death 4 to 6 weeks postinoculation; and (iii) mink cell focus-forming viruses, whose role in the pathogenesis of the disease is unclear (30). Murine infections with the RLV complex have been used in the study of pharmacological treatment strategies (23-26). In our previous experiments we have demonstrated that high-dose single-agent 3'-azido-3'-deoxythimidine (AZT) (26) or combination therapy with AZT plus recombinant human alpha A/D interferon (rHuIFN-aA/D) can prevent viremia and disease after acute virus exposure (23, 24). The combination regimen of AZT and rHuIFN-axA/D was highly synergistic in vivo and nontoxic (23). After a 3-week course of therapy started 4 h postinoculation, 100% of RLV-exposed mice given combination therapy were free of viremia and disease. Most importantly, over 90% of these animals were capable of resisting subsequent rechallenge with live RLV in the absence of therapy, thus demonstrating a highly effective acquired immunity to RLV (25). In these experiments, live, pathogenic RLV was blocked pharmacologically in its replication but was still able to elicit a fully protective immune response. This immunity was seen only in animals that had been exposed to RLV at time zero but not in mock-inoculated controls given the same therapy with AZT and rHuIFN-aA/D. For this work, we have exploited our unexpectedly successful vaccination protocol with live RLV in inbred mice to analyze the nature of the protective T-cell response. MATERIALS AND METHODS Mice. Female BALB/c mice were purchased from Taconic Laboratory, Germantown, N.Y., and were used at 4 to 6 weeks of age. Virus. The RLV strain used, RVB3, was derived from the

* Corresponding author. 220

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original stock (26). RLV was prepared by tail vein injection 104 PFU of RLV into BALB/c mice. After 20 days, the of mice were sacrificed, and single-cell suspensions of the spleens were obtained in a volume of 0.2 ml/g of spleen in media supplemented with 20% fetal calf serum. Supernatants of the cells were obtained, filtered, and frozen. The number of PFU was determined via XC plaque assay described elsewhere (26). RLV immunization. To obtain immune mice, naive female BALB/c mice at 4 to 6 weeks of age were inoculated at time zero via tail vein injection with104 PFU of RLV in a volume of 0.2 ml. Antiviral therapy with AZT (0.1 mg/ml in the drinking water; Burroughs Wellcome Co., Research Triangle Park, N.C.), combined with 10,000 U of rHuIFN-aA/D (Hoffmann-LaRoche, Nutley, N.J.) given via intraperitoneal injection once daily, was started 4 h later and continued for 20 days. On day 25, immunoblots of peripheral blood were obtained, and the mice were rechallenged with the same dose of RLV and left untreated for 21 days. On day 45 or later, the mice were sacrificed and tested for anti-RLV immunity as defined by the lack of viremia on day 25 and after RLV rechallenge. Viremia was measured by immunoblot analysis of blood or spleen samples. T-cell subset separation. Spleen cells were isolated from mice and were used for adoptive T-cell transfer studies. The cells were washed in L15 media supplemented with 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 3% bovine calf serum (Hazleton) and incubated over a nylon wool column (Cellular Products, Buffalo, N.Y.) at 37°C. Nonadherent cells were eluted with warm supplemented L15 media. To remove CD4+ cells, T cells were resuspended in GK1.5 supernatant (a rat monoclonal antibody directed against the murine CD4; American Type Culture Collection) at a density of 2 x107 cells per ml, incubated for 30 min over ice, and then washed with cold media. Similarly, to remove CD8+ cells, supernatant from the 53-6.72 hybridoma (a rat monoclonal antibody directed against the murine CD8; American Type Culture Collection) was used. Cells were added to washed immunomagnetic beads coated with goat anti-rat immunoglobulin G antibody (Advanced Magnetics, Cambridge, Mass.) at a density of 0.5 x 109 cells per 10 ml of beads in a 75-cm2 tissue culture flask. The flasks were gently agitated for 30 min at 37°C. Bound cells were removed with a magnet (BioMag Separator; Advanced Magnetics) placed on the surface of the flasks. The cells were then washed, counted, and resuspended in phosphate-buffered saline (PBS) at a density of 4 x 107 cells per 0.2 ml prior to tail vein injection. For the experiment in which T cells were fractionated into both CD4+ and CD8+ subsets and subsequently recombined, the CD4+/CD8+ ratio of the unfractionated cells was determined by fluorescence-activated cell sorting (FACS) to be 1.9. The separated CD4+ and CD8+ T cells were recombined at the same ratio and injected into eight naive recipients, which were then subjected to RLV challenge with 104 PFU intravenously 1 h later. The viability of the T cells after T-cell subset separations was determined by exclusion of trypan blue (GIBCO) and by proliferative response of 105 cells per well to phytohemagglutinin A (Sigma) and concanavalin A (Sigma) at 10 jg/ml in a 96-well round-bottom plate (Costar). The cells were pulsed with 1 ,uCi of tritiated thymidine (New England Nuclear) 3 days later for 8 h and then harvested on a PHD cell harvester (Cambridge Technology, Cambridge, Mass.). FACS. All cells used in staining for the T-cell subsets were first separated from the erythrocytes using lymphocyte separation medium (Organon-Teknika, Durham, N.C.). The

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CD8+

relative CD4+ and populations were determined by FACS on an EPICS V cell sorter (Coulter) before and after fractionation of the cells. Fluorescein-conjugated antibody against murine CD8 (Lyt2) and phycoerythrin-conjugated antibody against murine CD4 (L3T4) (Becton-Dickinson, Mountain View, Calif.) were used for FACS. These antibodies were derived from the 53-6.72 and GK1.5 hybridomas, respectively. The appropriate T-cell subsets were depleted to .95%. The histograms remained unchanged when fluorescein isothiocyanate-labeled anti-rat immunoglobulin G were used to stain for any cells bound with the CD4 or CD8 antibodies which were not removed with the magnetic beads (data not shown). Immunoblot analysis for RLV. Spleens were homogenized in RIPA buffer (50 mM Tris hydrochloride [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% sodium desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride), and total protein was measured by Bradford's method. Twenty micrograms of total protein was loaded onto an SDS-10% polyacrylamide gel and transblotted onto an Immobilon-P membrane (Millipore, Bedford, Mass.). The primary antibody used was a goat anti-RLV antiserum (National Institutes of Health Repository no. or 75S000297); the secondary antibody was a 75S000294 horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin G (Bio-Rad, Richmond, Calif.). Specific binding was demonstrated after the blot was developed with the substrate 4-chloro-1-naphthol (Bethesda Research Laboratories, Gaithersburg, Md.). Spleens were scored as positive by the presence of the p30, p1SE, and the env bands. RESULTS

Immune T cells protect mice against RLV viremia and disease. T cells were isolated from mice immunized more than 40 days earlier with live RLV whose replication was blocked by combination therapy with AZT and rHuIFNaA/D. Antiviral coverage was started 4 h after virus exposure and administered continuously for 20 days. Five to ten days after cessation of therapy, all animals were tested for the presence of viremia by immunoblot analysis and then rechallenged with live virus in the absence of therapy. The and CD8+ T-cell subsets were isolated by negative CD4+ selection by using antibodies directed against the respective phenotypic cell surface markers. The results of adoptive T-cell transfer experiments are shown in Fig. 1. When 4 x 107 unfractionated T cells were adoptively transferred to naive recipient mice, the animals were protected not only against RLV-induced splenomegaly but also against viremia as determined by immunoblot analysis of both spleen and serum, where no RLV-related antiger: were detected. T cells do not protect and Separated immune against RLV viremia and disease. Next, we examined the role of isolated T-cell subsets in conferring protective immunity CD8- and CD4to RLV. T cells were separated into FACS histograms demselection. subsets by negative CD8+ onstrated that our separations resulted in a depletion efficiency of > 95% of the relevant subsets, leaving a population of negatively selected cells consisting primarily of either T cells and natural killer (NK) cells (data not CD4+ orInCD8+ shown). contrast to the transfer of unfractionated immune T cells, when 4 x 107 (CD4-depleted) or CD4+ were (CD8-depleted) immune T cells per naive recipient transferred separately, only partial protection against RLV was found as determined by spleen sizes (Fig. 1.) and

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FIG. 2. Immunoblot analysis of spleen cell homogenates from selected recipients of T cells. All recipients except A had been challenged with RLV 1 h after adoptive cell transfer. M, Molecular weight markers (Bethesda Research Laboratories); + and -, RLVinfected and negative-control spleens, respectively; R, sucrosebanded RLV. Lanes: A, recipient given total immune T cells but not challenged with RLV; B, C, and D, recipients given unfractionated immune T cells; E, recipient given 4 x 107 total T cells which had been fractionated into CD4+ and CD8+ subpopulations and remixed at the ratio found prior to fractionation; F and G, recipients given 4 107 CD4+ cells; H and I, recipients given 4 x 107 CD8+ cells. In general, samples judged negative by the immunoblot assay were negative by XC plaque assay (25). In 49 murine samples (spleen or serum) analyzed, we found only one discordance between spleen immunoblot analysis and XC plaque assay of serum. The virus titer in that particular serum sample was approximately 10 to 60 PFU/ml. x

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FIG. 1. Adoptive cell transfer of CD4+ or CD8+ T cells from RLV-irmmune mice does not protect mice against RLV infection. Mice were injected intravenously 1 h after receiving unfractionated or fractionated T cells with 104 PFU of RLV. Prior experiments established that 4 x 107 T cells were sufficient to provide immunity (data not shown). The number of cells used in the transfer of CD4+ cells to determine protection by those cells was 4 x 107, while those used in the CD8+ protection experiment varied from 1 x 107 to 4 x 107 cells. Twenty days after the cell transfers and RLV inoculations, the mice were bled and sacrificed to determine the presence of virus in the sera or spleens by immunoblot analysis and spleen weights. The numbers above the bars are the numbers of mice with positive immunoblots divided by the numbers of mice per group. El, Recipient mice were not challenged with RLV. (A) Spleen sizes of recipient mice given 4 x 107 unfractionated CD4+ or T cells from naive or immune donor mice. (B) Spleen sizes of recipient mice receiving 4 x 107 immune T cells or 4 x 107, 2 x 107, or 1 x 107 CD8+ T cells from RLV-immune mice.

protection (Fig. 3). These experiments demonstrate clearly that the in vitro depletion procedure did not diminish the T-cell function appreciably. No viremia was detected in these recipient animals, as judged by immunoblot analysis (Fig. 2). Only the control group of mice receiving no T cells had detectable viral antigens. None of the six animals receiving unfractionated T cells and none of the eight animals receiving recombined CD4+ and CD8+ T cells were positive. We also note that in all of these experiments, recipient mice not challenged with RLV and receiving only cells from donors previously exposed to RLV did not develop viremia or splenomegaly, indicating that no infectious virus was transferred with the cells. Proliferation of the E

immunoblot analysis. Only 1 of 10 mice receiving CD4+ immune T cells was virus free on immunoblot analysis (Fig. 1 and 2), while none of 10 mice challenged with RLV after receiving the same number of unfractionated immune T cells reacted positively on immunoblot analysis (data not shown). Control mice receiving either naive unfractionatd or naive CD8-depleted cells prior to RLV challenge developed massive splenomegaly with positive immunoblots. When CD8+ immune T cells were transferred at 1 x 107, 2 x 107, and 4 x 107 cells (12 mice each), only 2 of 36 recipients had negative immunoblots. Both of the immunoblot-negative mice had received 4 x 107 cells. Recombining separated CD4+ and CD8' immune T cells protects against RLV viremia and disease. Recombining CD4+ and CD8+ immune T cells at the same ratio as in unfractionated immune T cells, as determined by FACS analysis, and transferring 4 x 107 T cells again conferred full

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FIG. 3. Recombining immune CD4+ and CD8+ T cells protects against viremia and disease. Spleen sizes of recipient mice receiving no T cells, unfractionated T cells, or remixed T cells from fractionated CD4+ and CD8+ subsets are shown. Symbols: mice injected with RLV; recipient mice not challenged with RLV. The numbers at the top of the bars are the number of mice with positive immunoblots divided by the number of mice per group. -,

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fractionated and recombined T-cell subsets did not differ from that of unfractionated T cells, as determined by tritiated thymidine incorporation. DISCUSSION We have demonstrated that immune T cells derived from mice vaccinated with live RLV, whose replication was blocked by postexposure prophylaxis with AZT plus rHuIFN-aoA/D, can mediate immunity against RLV challenge. Neither the immune CD4+ nor the immune CD8+ T-cell subset alone was sufficient for protection of naive mice against acute RLV exposure. In contrast, the combination of immune CD4+ cells with CD8+ cells reconstituted at the same ratio as in unfractionated splenic T cells was capable of protecting naive mice against viremia and disease. The immune T cells generated in our vaccination protocol are likely to act on cells expressing RLV antigens. Thus, adoptive T-cell transfer is expected to result in the killing of virus-producing cells early in the course of infection and in the killing of virus-induced tumor cells late in the course of disease. We are presently examining whether the immune T cells generated by our vaccination protocol completely eliminated all cells harboring RLV provirus or suppressed virus production, leading to latency. Several previous studies have analyzed the role of T cells against murine leukemia virus-induced neoplasia as opposed to viremia only (2, 3, 5, 8, 12, 15, 18, 19). While adoptive transfer of immune T cells may induce recovery from FV leukemia when given early after infection with FV (3), the role of T-cell subsets was not defined. Klarnet et al., however, found that CD4+ and CD8+ T cells from mice immunized with FV-induced leukemia were capable of recognizing distinct antigens encoded by FV (12). BALB/c mice immunized with live RLV attenuated by tissue culture passage developed active immunity against challenge with Moloney murine leukemia virus-induced leukemic cells (20). In a different retrovirus system, Leclerc and Cantor found both T-cell subsets to be necessary for preventing tumor formation with murine sarcoma virus-transformed cells as well as with Moloney murine leukemia virus-induced lymphoma (15). It is possible that interactions between the two T-cell subsets, such as the elaboration of lymphokines by one subset, are needed to enhance the activity of the other one (5, 8, 18, 31). In our experiments, however, an absolute requirement for both CD4+ and CD8+ T cells was shown for prevention of viremia and subsequent virus-induced disease, in contrast to therapy for established malignancies. The necessity for various T-cell subsets for protection against viral diseases was studied in other systems involving nonretroviral agents such as herpes simplex, vesicular stomatitis, lymphocytic choriomeningitis, and influenza viruses (1, 4, 13, 14, 17, 21, 29). In experiments involving transfer of CD8+ T-cell clones, the administration of exogenous lymphokines resulted in more efficient antiviral responses (22, 27, 29). In a different approach, Leist et al. used in vivo depletion of CD4+ T cells to demonstrate that CD8+ cytotoxic T lymphocytes depend upon the simultaneous presence of CD4+ T cells for optimal antiviral response (16). In contrast to these studies, which involved either T-cell clones or single T-cell subsets, our data, from adoptive cell transfer experiments, give direct evidence for the need for both T-cell subsets for a secondary cellular immune response against a retrovirus. One might argue that our separation protocols do not result in depletion of NK cells, which thus might mediate

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antiviral protection. This, however, is unlikely, since NK cells, which do not require prior antigenic priming, did not protect recipients given unfractionated nonimmune T cells. Likewise, no protection was conferred by either immune T-cell subset, both of which were likely to have been NK-cell enriched. Since our negative-selection procedures were specific for either CD4+ or CD8+ subpopulations, NK cells were not removed (resulting in an increased percentage of NK cells in the transferred population). Although our prior experiments have shown that a large inoculum of immune murine antiserum can protect about 60% of naive recipients against RLV challenge (25), the experiments demonstrating the lack of protection by adoptive transfer of up to 4 x 107 immune CD4+ T-helper cells suggest that B cells may not play a crucial role in protecting animals from RLV infections. Data have shown that antibodies may have a detrimental role in human immunodeficiency virus type 1 infections (10, 28). In this work, we have demonstrated that a cellular response alone is sufficient for protection against challenge with a retrovirus. We conclude that development of vaccines against retroviral infections may require an emphasis on the generation of cellular immunity and that it may be necessary to stimulate more than one type of T-cell response to confer full protection against a retroviral challenge. ACKNOWLEDGMENTS

Insightful suggestions and comments by Baruj Benacerraf, Joyce Fingeroth, and Abebe Haregewoin are gratefully acknowledged. We

thank Sandra Nusinoff-Lehrman (Burroughs Wellcome) for the gift of AZT and I. Sim (Hoffmann-LaRoche) for the gift of rHuIFNotA/D. This research was supported by the NIAID and a Faculty Research Award from the American Cancer Society to R.M.R. REFERENCES 1. Bonneau, R. H., and S. R. Jennings. 1989. Modulation of acute and latent herpes simplex virus infection in C57BL/6 mice by adoptive transfer of immune lymphocytes with cytolytic activity. J. Virol. 63:1480-1484. 2. Bookman, M. A., R. Swerdlow, and L. A. Matis. 1987. Adoptive chemoimmunotherapy of murine leukemia with helper T lymphocyte clones. J. Immunol. 139:3166-3170. 3. Britt, W. J., and B. Chesebro. 1983. H-2D control of recovery from Friend virus leukemia: H-2D region influences the kinetics of the T lymphocyte response to Friend virus. J. Exp. Med.

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