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A reproducible method to detect CD8 T cell mediated inhibition of HIV production from naturally infected CD4 cells
Journal oflmmunological Methods, 157 (1993) 181-187 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$06.00
181
JIM06536
A reproducible method to detect CD8 T cell mediated inhibition of HIV production from naturally infected CD4 cells Mary A nn H a u s n e r a, Janis V. Giorgi a and Susan Plaeger-Marshall b Departments of ~ Medicine and b Pediatrics, University of California, Los Angeles, School of Medicine, Los Angeles, CA, USA (Received 22 April 1992, revised received 3 August 1992, accepted 3 August 1992)
CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 ceils to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease. Key words: CD8 T cell; HIV infection; Immune response to virus
Introduction
CD8 T cells are important components of the immune response to many persistent viral pathogens (reviewed in Zinkernagel, 1988). In HIV infection, dramatic alterations in CD8 cell number and surface molecule expression suggest that these cells have a central role (Giorgi and Detels, 1989; reviewed in Lewis and Giorgi, 1990). However, whether the CD8 T cell response in HIV infection reflects antiviral immunity or viral pathogenesis has been difficult to discern because
Correspondence to: S. Plaeger-Marshall, Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90024-1752, USA. Tel.: (310) 206-8296; Fax: (310) 206-1318.
of the tropism of HIV for cells of the immune system. To elucidate the role of CD8 T cells in HIV infection, our laboratory has been studying a unique CD8 cell-mediated in vitro phenomenon, i.e., the inhibition of HIV replication in autologous, naturally infected CD4 cells (PlaegerMarshall et al., 1990). As first described by Walker and colleagues (1986), when cultured peripheral blood mononuclear cells from HIV-infected individuals were depleted of CD8 cells, the amount of virus produced in the cultures increased dramatically. Furthermore, CD8 cells added back to cultures were shown to reduce the production of HIV in a dose-dependent manner, indicating that CD8 T cells were able to control viral replication.
182 Characterization of specific immunological aspects of HIV infection that contribute to control of disease is important for understanding the basic disease process and designing effective therapeutic strategies. Moreover, as an in vitro correlate of protective immunity to HIV, CD8 cellmediated inhibition of HIV may be useful clinically for assessing disease progression or prognosis and therapeutic efficacy. However, study of CD8 cell-mediated inhibition of HIV has been hampered by difficulties in the assay system. In particular, it has been difficult to obtain consistent production of HIV from the cultured CD4 cells of infected subjects with little CD4 cell depletion or disease progression and, presumably, low viral load (Brinchmann et al., 1990). These difficulties have led to the development of different assay systems that are not comparable to each other and may reflect different immunologic mechanisms for viral control. We have developed a reproducible method to demonstrate CD8-mediated suppression of HIV production from autologous naturally infected CD4 cells. We have used a commercial cell separation system in which CD4 cells are captured on a monoclonal antibody-coated culture flask. These flasks yield large numbers of purified CD4 cells, which can be cultured in situ without further manipulation. Anti-CD3 monoclonal antibody (MAb) is used to induce HIV replication in the isolated CD4 cells. CD8 cells are further enriched by negative selection with magnetic immunobeads. The technique has broad applicability in the immunology laboratory for studying HIV infection.
Materials and methods
Patient population A total of 27 HIV-1 seropositive adult males without a diagnosis of AIDS were recruited for these studies from among the UCLA participants in the NIH-sponsored Multicenter AIDS Cohort Study (MACS), a natural history study of HIV infection begun in 1984 (Kaslow et al., 1987). CD4 and CD8 cell numbers were obtained routinely on the participants at 3-6 month intervals,
as described elsewhere (Giorgi et al., 1990). The CD4 cell numbers of the 27 men studied ranged from 1094 to 274 cells/mm 3 at the time of testing in the assay described herein, with 19 of the subjects having CD4 cell counts of > 500/mm 3. The study was approved by the UCLA Human Subjects Protection Committee, and informed consent was obtained from all participants after the nature and possible consequences of the studies had been fully explained.
CD4 and CD8 cell separation Peripheral blood mononuclear cells were separated from fresh heparinized blood by centrifugation over Lymphoprep (Nyegaard, Oslo, Norway). Cells were washed and suspended at a concentration of up to 2 x 107 cells in RPMI 1640 medium (Mediatech, Washington, DC) supplemented with 10% pooled human serum (HS, Gemini Bioproducts, Calabasas, CA). The cell suspension was pipetted onto the culture surface of an activated AIS MicroCELLector CD4 capture flask (Applied Immune Sciences, Menlo Park, CA) consisting of a 25 cm 2 tissue culture-grade flask with anti-CD4 MAb covalently attached to the surface (Morecki et al., 1990). The flask was allowed to sit at room temperature on a stable surface for 1 h. An alternative method for rapid cell adherence was the centrifugation of the flask for 5 min at 95 × g at room temperature, reversal of the flask orientation, and repeated centrifugation. The non-adherent cells were removed by rinsing the flask with seven changes of Hanks' balanced salt solution (HBSS) without divalent cations. To induce production of HIV, the adherent CD4 cells were cultured in the capture flask in RPMI 1640 containing 10% HS, 10% purified human IL-2 (Pharmacia, Silver Springs, MD), 40 U / m l recombinant human IL-2 (rlL-2, Cetus, Emeryville, CA), and either 200 ng/ml OKT-3 (Ortho Pharmaceuticals, Raritan, NJ) or a 1/40 final dilution of PHA-P (Difco Laboratories, Detroit, MI). The purified human IL-2 was found to enhance proliferation of peripheral blood cells in culture (unpublished observation), possibly due to the presence of other undefined growth factors. The recombinant IL-2 at this concentration has been shown to enhance lymphocyte viability in the cultures (Prince and Jensen, 1991). The cells
183
were incubated in a humidified chamber at 37°C in 5% CO 2 for 3-5 days. The non-adherent (i.e., CD8-enriched) cells washed from the CD4 capture flask were further enriched for CD8 cells by negative selection with magnetic immunobeads using the magnetic cell sorter (MACS, Miltenyi Biotec, Cologne, Germany) as described elsewhere (Miltenyi et al., 1990). In brief, cells were washed and resuspended at 6 x 107 cells/ml in phosphate-buffered saline (PBS) with 0.01% sodium azide, without divalent cations. To remove residual CD4 cells, B cells, and natural killer cells from the CD8-enriched population, a cocktail of biotinylated MAbs consisting of anti-CD4 (Gentrak, Plymouth Meeting, PA), anti-CD16 ( L e u l l c , Becton Dickinson Immunocytometry Systems, San Jose, CA), and anti-CD19 (Leu12, Becton Dickinson) was added to the cell suspension. After a 10 min incubation on ice, the cells were washed once and incubated for an additional 10 min on ice with avidin-FITC (Becton Dickinson) at 1 /xg/106 cells. A 1/100 dilution of MACS biotinylated beads was added, and the cells incubated on ice for 5 min. The cell suspension was passed through the magnetized MACS column, and the unbound (i.e., CD8) cells collected. The CD8 cells were resuspended at 106/mi in RPMI 1640 supplemented with 10% HS and 40 U / m l rIL-2, and cultured at 37°C for 3-5 days.
Flow cytometric analysis of cell populations The phenotype of the CD4 and CD8 cells that had been cultured for 3-5 days was determined by flow cytometry to assess the purity of the selected cell populations prior to placing them in the inhibition assay. At this time the CD4 cells had detached from the capture flask surface. Cells were analyzed using standard methodology (Lewis and Rickman, 1992). To describe briefly, the cultured cells were washed, counted, and assessed for viability by trypan blue exclusion. 5 × 105 CD4 or CD8 cells were incubated with a two-color combination of murine monoelonal antibody (MAb) consisting of fluorescein isothiocyanate (FITC)-labeled anti-CD8 and phycoerythrin (PE)-labeled anti-CD4 (Leu2 and Leu3, Becton Dickinson) for 20 min at 4°C. The cells were then washed and analyzed for green (FITC)
and orange (PE) fluorescence using a FACScan instrument (Becton Dickinson) with a single argon laser.
CD8-mediated inhibition of HIV production For functional assessment, the CD4 and CD8 cells that had been cultured separately for 3-5 days were resuspended in fresh medium containing 10% HS, 10% purified human IL-2 and 40 U / m l rIL-2. Cells were pipetted into triplicate wells of 24-well plates (Costar, Cambridge, MA) to achieve ratios of CD4 to CD8 of 0.5:1, 1:1, 1 : 2, or 1 : 4 for a final concentration of 1 x 105 CD4 cells/ml/well. Separate wells of CD4 or CD8 cells were plated as controls. The co-cultures were incubated in a humidified chamber at 37°C and 5% CO 2. 0.5 ml of culture supernatants was collected at days 7, 10, 14 and 21 for assessment of virus production and replaced with 0.5 ml fresh medium. Production of HIV in the supernatants was measured as viral p24 antigen by ELISA assay (Abbott Diagnostics, Abbott Park, IL).
Results
Purity and viability of separated cell fractions The results of flow cytometric analysis from a representative experiment are shown in the scatter plot quadrants in Fig. 1. The whole lymphocyte preparation prior to separation is compared to cultured, separated CD4 and CD8 cells. This experiment showed an initial CD4 fraction of 35%, which was purified to 93%. The initial fraction of CD8 cells was 38% and was purified to 86%. The average percent purity for CD4 cell cultures from 15 subjects on whom extensive assessments were done was 94%, with a range of 89-99%. Contaminating cells tended to be double positive C D 4 / C D 8 cells (quadrant 2) or nonCD8 cells (quadrant 3) (i.e., NK or B cells), with an average of < 2% CD8 cells captured in the CD4 flasks. The viability of the cultured CD4 cells averaged 81%, with a range of 53-95%. Recovery of CD4 cells on the capture flasks could not be accurately determined because the cells were stimulated to proliferate in culture, so that in some instances there were more CD4 cells
184 A
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Fig. 1. Quadrant scatter plots from the flow cytometric analysis of lymphocytes from one subject assessed for expression of CD4 and CD8 antigens before and after cell subset separation. A shows 35% CD4 (quadrant 1) and 38% CD8 (quadrant 4) cells in peripheral blood lymphocytes prior to separation of CD4 and CD8 subsets; B shows 93% CD4 cells obtained from a CD4 capture flask after 3 days of culture; panel C shows cells from a CD8 flask with 86% CD8 cells. r e c o v e r e d t h a n t h e original input. T h e p u r i t y o f C D 8 cells a v e r a g e d 86% with a r a n g e o f 5 1 - 1 0 0 % . T h e a v e r a g e viability o f C D 8 ceils was 76% with a r a n g e o f 2 5 - 1 0 0 % . R e c o v e r y of C D 8 cells negatively s e l e c t e d using i m m u n o b e a d s a v e r a g e d 51% o f the original i n p u t o f C D 8 cells. T h e r e a s o n for t h e low viability o f t h e c u l t u r e d C D 4 a n d C D 8 cells in s o m e subjects is u n k n o w n , b u t m a y b e a r e f l e c t i o n o f t h e a c c e l e r a t e d cell d e a t h d u r i n g in vitro c u l t u r e t h a t has b e e n r e p o r t e d for l y m p h o cytes from H I V - i n f e c t e d individuals ( P r i n c e a n d Czaplicki, 1989).
uals, H I V was u n d e t e c t a b l e in 7-day s u p e r n a t a n t s f r o m P H A - s t i m u l a t e d cultures, w h e r e a s a n t i - C D 3 i n d u c e d m e a s u r a b l e p24 a n t i g e n at t h a t time. B a s e d o n t h e s e results, a n t i - C D 3 M A b was u s e d to activate C D 4 cells in all s u b s e q u e n t exp e r i m e n t s . O f 27 H I V seropositive subjects, two d i d not p r o d u c e d e t e c t a b l e p24 a n t i g e n w h e n t h e i r C D 4 cells w e r e c u l t u r e d with a n t i - C D 3 M A b . T h e s e individuals h a d C D 4 cell counts of 807 a n d 1 0 0 2 / m m 3, which are within n o r m a l
TABLE I
Production of H1V from CD4 cell cultures E x p e r i m e n t s w e r e p e r f o r m e d to c o m p a r e t h e efficiency o f t h e m i t o g e n P H A , which has b e e n u s e d r o u t i n e l y to elicit H I V p r o d u c t i o n f r o m p e ripheral lymphocytes of infected individuals ( G a l l o et al., 1984), versus t h e T cell activation stimulus a n t i - C D 3 M A b (Weiss a n d I m b o d e n , 1987) in i n d u c i n g t h e p r o d u c t i o n o f H I V from t h e C D 4 ceils o f i n f e c t e d subjects. T a b l e I shows the results at days 7 a n d 14 of e x p e r i m e n t s testing nine subjects. F o r t h e first t h r e e subjects, p a i r e d s a m p l e s w e r e t e s t e d s i m u l t a n e o u s l y using t h e two d i f f e r e n t stimuli. Six m o r e subjects w e r e t e s t e d using e i t h e r P H A o r a n t i - C D 3 as an activation stimulus. In all cases, C D 4 cells s t i m u l a t e d with a n t i - C D 3 M A b p r o d u c e d consistently h i g h e r levels of p24 a n t i g e n in c u l t u r e s u p e r n a t e s t h a n d i d those c u l t u r e d with P H A . In t h r e e of the individ-
HIV p24 ANTIGEN IN CULTURES STIMULATED WITH PHA OR ANTI-CD3 Day 7 (pg/ml) PHA
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98 a 0 2,000 98 226 a 936 a 2,000 a 2,000 2,000 571
a The first three results were from paired samples from the same three subjects tested simultaneously with PHA or antiCD3.
185
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Fig. 2. Individual results from six individuals for H I V production from CD4 cells cultured alone (dark bars), or CD4 and CD8 cells co-cultured at ratios of 1 : 1 (hashed bars) or 1 : 2 (vertically striped bars) for 7 days.
reference ranges, indicating little if any CD4 cell depletion, which is suggestive of low viral burden (Schnittman, et al., 1990).
CD8-mediated Inhibition of HIV production Fig. 2 shows results of CD8-mediated inhibition assays from six of the 25 subjects tested who produced p24 antigen from their CD4 cell cultures. Viral p24 antigen production on day 10 of culture is shown for CD4:CD8 ratios of 1:1 or 1:2. It is of note that of the four time points tested, viral p24 antigen production was low but generally detectable by day 7 and peaked by day 14. By day 21, the majority of the cells was dead, and this time point provided little additional information (data not shown). Although the levels of viral p24 antigen produced by CD4 cells varied among the 25 individuals tested, in all individuals, less p24 antigen was detected in the CD4/CD8 co-cultures than from CD4 cells cultured alone. The average percent inhibition for all 25 individuals was 83% (range of 38-100%) at the CD8 : CD4 ratio of 1 : 1 and 90% (range of 59-100%) at 2 : 1. The degree of inhibition of HIV replication did not correlate with the
peripheral blood CD4 cell percent, absolute number, or CD4:CD8 ratio (data not shown).
Discussion
The functional competence of T cells in vitro has been used as an indicator of disease progression and prognosis in HIV infection (Lewis and Giorgi, 1990). The restoration or augmentation of T cell function is a surrogate marker for therapeutic efficacy (Redfield et al., 1991). Hence, assay of CD8 cell function has broad applicability in the laboratory assessment of HIV-infected individuals. Although cytotoxic function of CD8 cells has been studied in HIV infection (Walker et al., 1987) and may provide useful information, the requirements for antigen and class I major histocompatibility complex (MHC) specificity limit the general use of cytotoxic T lymphocyte (CTL) assays. Inhibition of HIV replication by CD8 cells, like CTL function, may be an in vitro correlate of antiviral immunity in HIV infection. We have developed a simple and reproducible assay for
186 CD8 cell-mediated inhibition using commercially available reagents for standardization of cell separation and activation. Several problems inherent in the assay system have been addressed. One critical element is the consistent induction of HIV from cultured CD4 cells, which serves as the read-out for the assay. The complexity of this biologic system suggests that there may be multiple immunologic and virologic factors involved in the production of HIV from naturally infected CD4 cells. In extending their original studies, Walker et al. (1989) found that even small numbers of CD8 cells from some subjects were shown to inhibit the production of HIV in CD4 cultures. In the experiments, CD4 cells were isolated by panning (Wysocki and Sato, 1978). There is no standardization of reagents or methodology for panning, leading to variation in the efficiency of cell separation among investigators using this technique. Our use of a commercially available cell capture system has diminished this variability, while maintaining the technical simplicity of a panning-type method. In a comparison of methods for cell purification in our laboratory (Plaeger-Marshall and Hausner, 1991), we have found the capture flasks to yield cell purity and recovery comparable to or better than other cell separation technologies. It is likely that factors other than the purity of CD4 cells also contributed to the lack of virus production in the cultures of some individuals. We have shown that, even when CD4 cells were highly purified, anti-CD3 MAb induced HIV replication when PHA did not. Brinchmann et al. (1990) have reported that MAb to the T cell antigen receptor (TCR) consistently induced virus production in cell cultures from asymptomatic HIV-infected subjects. The reason for this is not known but may relate to the intracellular activation pathways stimulated by different ligand-receptor interactions. T cell stimuli like anti-CD3 or anti-TCR MAbs more closely mimic the interaction of T cells with specific antigen than do mitogens like P H A that have numerous cell ligands (Valentine et al., 1985; O'Flynn et al., 1985). It is possible that MAbs are more efficient activators of cellular pathways critical to the productive replication of HIV, such as activation of the HIV long terminal repeat (Tong-Starksen et al., 1989).
Although a qualitative deficiency in CD3-mediated cellular activation in HIV infection has been reported (Miedema et al., 1988), the CD3-1inked activation pathway inhibited by HIV may be different from that inducing the metabolic events that trigger viral replication. These issues in the biology of virus-cell interactions remain to be resolved. In summary, we have developed a reproducible and simple assay to measure CD8 T cell mediated suppression of HIV production from the CD4 cells of asymptomatic infected individuals. The standardization of the assay allows its application on a large scale for monitoring HIVinfected individuals. Experiments are in progress to assess the usefulness of the assay for following disease progression and the efficacy of antiviral or immunomodulatory therapies.
Acknowledgements We gratefully acknowledge the participants and staff of the UCLA Multicenter AIDS Cohort Study (MACS), especially Drs. Roger Detels and Barbara Visscher and Ms. Jan Dudley and Mr. Dennis Miles. We thank Ms. Patricia Hultin and Drs. Valentin Isacescu and Negoita Neagos for performance of the laser flow cytometric analyses, and Ms. Jeanne Bertolli for expert editorial assistance. The viral p24 antigen assays were performed by Mr. Amadu Diagne in the U C L A HIV Virology Laboratory directed by Dr. Irvin Chen. This work was supported in part by U.S. Public Health Service grant AI 26098, contract AI 72656, and Center for AIDS Research Grant AI 28697. AIS MicroCELLector flasks were generously supplied by Applied Immune Sciences, Inc.
References Brinchmann, J.E., Gaudernack, G. and Vartdal, F. (1990) CD8 + cells inhibit HIV replication in naturally infected CD4 + T cells: Evidence for a soluble inhibitor. J. Immunol. 144, 2961. Gallo, R.C., Salabuddin, S.Z., Popovic, M., Shearer, G.M., Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J., Safai, B., White, G., Foster, P. and Markham, P.D. (1984) Frequent detection and isolation of cytopathic
187 retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 500. Giorgi, J.V. and Detels, R. (1989) T-cell subset alterations in HIV-infected homosexual men: NIAID multicenter AIDS cohort study. Clin. Immunol. Immunopathol. 52, 10. Giorgi, J.V., Cheng, H.L., Margolick, J.B., Bauer, K.D., Ferbas, J., Waxdal, M., Schmid, I., Hultin, L.E., Jackson, A.L., Park, L., Taylor, J.M.G., and the Multicenter AIDS Cohort Study Group (1990) Quality control in the flow cytometric measurement of T-lymphocyte subsets: the multicenter AIDS cohort study experience. Clin. Immunol. Immunopathol. 55, 173. Kaslow, R.A., Ostrow, D.G., Detels, R., Polk, B.F., and Rinaldo, C.R. (1987) The Multicenter AIDS Cohort Study: Rationale, organization, and selected characteristics of the participants. Am. J. Epidemiol. 126, 310. Lewis, D.E. and Giorgi, J.V. (1990) Immunology of HIV infection. Int. Rev. Immunol. 7, 1. Lewis, D.E. and Rickman, W.J. (1992) Methodology and quality control for flow cytometry In: N.R. Rose, E.C. De Macario, J.L. Fahey, H. Friedman and G.M. Penn (Eds.), Manual of Clinical Laboratory Immunology, 4th edn. American Society for Microbiology, Washington, DC, in press. Miedema, F., Petit, A.J.C., Terpstra, F.G., Schattenkerk, J.K.M.E., De Wolf, F., AI, B.J.M., Roos, M., Lange, J.M.A., Danner, S.A., Goudsmit, J. and Schellekens, P.T.A. (1988) Immunological abnormalities in human immunodeficiency virus (HIV)-infected asymptomatic homosexual men: HIV affects the immune system before CD4 + T helper cell depletion occurs. J. Clin. Invest. 82, 1908. Miltenyi, S., Muller, W., Weichel, W. and Radbruch, A. (1990) High gradient magnetic cell separation with MACS. Cytometry 11,231. Morecki, S., Topalian, S.L., Myers, W.W., Okrongly, D., Okarma, T.B. and Rosenberg, S.A. (1990) Separation and growth of human CD4 ÷ and CD8 + tumor-infiltrating lymphocytes and peripheral blood mononuclear cells by direct positive panning on covalently attached monoclonal antibody-coated flasks. J. Biol. Resp. Modif. 9, 463. O'Flynn, K., Krensky, A.M., Beverley, P.C.L., Burakoff, S.J. and Linch, D.C. (1985) Phytohaemagglutinin activation of T cells through the sheep red blood cell receptor. Nature 313, 686. Plaeger-Marshall, S. and Hausner, M.A. (1991) A review of cell separation technology for the research laboratory and evaluation of the AIS MicroCELLector T M T-25 cell culture flask. Monograph for Applied Immune Sciences, Inc., Menlo Park, CA. Plaeger-Marshall, S., Hausner, M.A., Hultin, P.A., and Giorgi, J.V. (1990) CD8/CD57 T cell mediated suppression of HIV replication in CD4 cells from infected individuals.
International Conference on Advances in AIDS Vaccine Development, abstract 48. Prince, H.E. and Czaplicki, C.D. (1989) Preferential loss of Leu8-, CD45R-, HLA-DR + CD8 cell subsets during in vitro culture of mononuclear cells from human immunodeficiency virus type I (HIV)-seropositive former blood donors. J. Clin. Immunol. 9, 421. Prince, H.E. and Jensen, E.R. (1991) HIV-related alterations in CD8 cell subsets defined by in vitro survival characteristics. Cell. Immunol. 134, 276. Redfield, R.R., Birx, D.L., Ketter, N., Tramont, E., Polonis, V., Davis, C., Brundage, J.F., Smith, G., Johnson, S., Fowler, A., Wierzba, T., Shafferman, A., Volvovitz, F., Oster, C. and Burke, D.S. (1991) A phase I evaluation of the safety and immunogenicity of vaccination with recombinant gpl60 in patients with early human immunodeficiency virus infection. N, Engl. J. Med. 324, 1678. Schnittman, S.M., Greenhouse, J.J., Psallidopoulos, M.C., Baseler, M., Salzman, N.P., Fauci, A.S. and Lane, H.C. (1990) Increasing viral burden in CD4 ÷ T cells from patients with human immunodeficiency virus (HIV) infection reflects rapidly progressive immunosuppression and clinical disease, Ann. Intern. Med. 113, 438. Tong-Starksen, S.E., Luciw, P.A. and Peterlin, B.M. (1989) Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat. J. Immunol. 142, 702. Valentine, M.A., Tsoukas, C.D., Rhodes, G., Vaughan, J.H. and Carson, D.A. (1985) Phytohemagglutinin binds to the 20 kDa molecule of the T3 complex. Eur. J. Immunol. 15, 851. Walker, B.D., Chakrabarti, S., Moss, B., Paradis, T.J., Flynn, T., Durno, A.G., Blumberg, R.S., Kaplan, J.C., Hirsch, M.S. and Schooley, R.T. (1987) HIV-specific cytotoxic T lymphocytes in seropositive individuals. Nature 328, 345. Walker, C.M., Moody, D.J., Stites, D.P. and Levy, J.A. (1986) CD8 ÷ lymphocytes can control HIV infection in vitro by suppressing virus replication. Science 234, 1563. Walker, C.M., Moody, D.J., Stites, D.P. and Levy, J.A. (1989) CD8 + T lymphocyte control of HIV replication in cultured CD4 + cells varies among infected individuals. Cell. Immunol. 119, 470. Weiss, A. and Imboden, J.B. (1987) Cell surface molecules and early events involved in T lymphocyte activation. Adv. Immunol. 41, 1. Wysocki, L.J. and Sato, V.L. (1978) Panning for lymphocytes: a method for cell selection. Proc. Natl. Acad. Sci. USA 75, 2844. Zinkernagel, R.M. (1988) Immunopathology by antiviral T cell immunity. In: C. Lopez (Ed.), Immunobiology and Pathogenesis of Persistent Virus Infections. American Society for Microbiology, Washington, DC, p. 454.
181
JIM06536
A reproducible method to detect CD8 T cell mediated inhibition of HIV production from naturally infected CD4 cells Mary A nn H a u s n e r a, Janis V. Giorgi a and Susan Plaeger-Marshall b Departments of ~ Medicine and b Pediatrics, University of California, Los Angeles, School of Medicine, Los Angeles, CA, USA (Received 22 April 1992, revised received 3 August 1992, accepted 3 August 1992)
CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 ceils to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease. Key words: CD8 T cell; HIV infection; Immune response to virus
Introduction
CD8 T cells are important components of the immune response to many persistent viral pathogens (reviewed in Zinkernagel, 1988). In HIV infection, dramatic alterations in CD8 cell number and surface molecule expression suggest that these cells have a central role (Giorgi and Detels, 1989; reviewed in Lewis and Giorgi, 1990). However, whether the CD8 T cell response in HIV infection reflects antiviral immunity or viral pathogenesis has been difficult to discern because
Correspondence to: S. Plaeger-Marshall, Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90024-1752, USA. Tel.: (310) 206-8296; Fax: (310) 206-1318.
of the tropism of HIV for cells of the immune system. To elucidate the role of CD8 T cells in HIV infection, our laboratory has been studying a unique CD8 cell-mediated in vitro phenomenon, i.e., the inhibition of HIV replication in autologous, naturally infected CD4 cells (PlaegerMarshall et al., 1990). As first described by Walker and colleagues (1986), when cultured peripheral blood mononuclear cells from HIV-infected individuals were depleted of CD8 cells, the amount of virus produced in the cultures increased dramatically. Furthermore, CD8 cells added back to cultures were shown to reduce the production of HIV in a dose-dependent manner, indicating that CD8 T cells were able to control viral replication.
182 Characterization of specific immunological aspects of HIV infection that contribute to control of disease is important for understanding the basic disease process and designing effective therapeutic strategies. Moreover, as an in vitro correlate of protective immunity to HIV, CD8 cellmediated inhibition of HIV may be useful clinically for assessing disease progression or prognosis and therapeutic efficacy. However, study of CD8 cell-mediated inhibition of HIV has been hampered by difficulties in the assay system. In particular, it has been difficult to obtain consistent production of HIV from the cultured CD4 cells of infected subjects with little CD4 cell depletion or disease progression and, presumably, low viral load (Brinchmann et al., 1990). These difficulties have led to the development of different assay systems that are not comparable to each other and may reflect different immunologic mechanisms for viral control. We have developed a reproducible method to demonstrate CD8-mediated suppression of HIV production from autologous naturally infected CD4 cells. We have used a commercial cell separation system in which CD4 cells are captured on a monoclonal antibody-coated culture flask. These flasks yield large numbers of purified CD4 cells, which can be cultured in situ without further manipulation. Anti-CD3 monoclonal antibody (MAb) is used to induce HIV replication in the isolated CD4 cells. CD8 cells are further enriched by negative selection with magnetic immunobeads. The technique has broad applicability in the immunology laboratory for studying HIV infection.
Materials and methods
Patient population A total of 27 HIV-1 seropositive adult males without a diagnosis of AIDS were recruited for these studies from among the UCLA participants in the NIH-sponsored Multicenter AIDS Cohort Study (MACS), a natural history study of HIV infection begun in 1984 (Kaslow et al., 1987). CD4 and CD8 cell numbers were obtained routinely on the participants at 3-6 month intervals,
as described elsewhere (Giorgi et al., 1990). The CD4 cell numbers of the 27 men studied ranged from 1094 to 274 cells/mm 3 at the time of testing in the assay described herein, with 19 of the subjects having CD4 cell counts of > 500/mm 3. The study was approved by the UCLA Human Subjects Protection Committee, and informed consent was obtained from all participants after the nature and possible consequences of the studies had been fully explained.
CD4 and CD8 cell separation Peripheral blood mononuclear cells were separated from fresh heparinized blood by centrifugation over Lymphoprep (Nyegaard, Oslo, Norway). Cells were washed and suspended at a concentration of up to 2 x 107 cells in RPMI 1640 medium (Mediatech, Washington, DC) supplemented with 10% pooled human serum (HS, Gemini Bioproducts, Calabasas, CA). The cell suspension was pipetted onto the culture surface of an activated AIS MicroCELLector CD4 capture flask (Applied Immune Sciences, Menlo Park, CA) consisting of a 25 cm 2 tissue culture-grade flask with anti-CD4 MAb covalently attached to the surface (Morecki et al., 1990). The flask was allowed to sit at room temperature on a stable surface for 1 h. An alternative method for rapid cell adherence was the centrifugation of the flask for 5 min at 95 × g at room temperature, reversal of the flask orientation, and repeated centrifugation. The non-adherent cells were removed by rinsing the flask with seven changes of Hanks' balanced salt solution (HBSS) without divalent cations. To induce production of HIV, the adherent CD4 cells were cultured in the capture flask in RPMI 1640 containing 10% HS, 10% purified human IL-2 (Pharmacia, Silver Springs, MD), 40 U / m l recombinant human IL-2 (rlL-2, Cetus, Emeryville, CA), and either 200 ng/ml OKT-3 (Ortho Pharmaceuticals, Raritan, NJ) or a 1/40 final dilution of PHA-P (Difco Laboratories, Detroit, MI). The purified human IL-2 was found to enhance proliferation of peripheral blood cells in culture (unpublished observation), possibly due to the presence of other undefined growth factors. The recombinant IL-2 at this concentration has been shown to enhance lymphocyte viability in the cultures (Prince and Jensen, 1991). The cells
183
were incubated in a humidified chamber at 37°C in 5% CO 2 for 3-5 days. The non-adherent (i.e., CD8-enriched) cells washed from the CD4 capture flask were further enriched for CD8 cells by negative selection with magnetic immunobeads using the magnetic cell sorter (MACS, Miltenyi Biotec, Cologne, Germany) as described elsewhere (Miltenyi et al., 1990). In brief, cells were washed and resuspended at 6 x 107 cells/ml in phosphate-buffered saline (PBS) with 0.01% sodium azide, without divalent cations. To remove residual CD4 cells, B cells, and natural killer cells from the CD8-enriched population, a cocktail of biotinylated MAbs consisting of anti-CD4 (Gentrak, Plymouth Meeting, PA), anti-CD16 ( L e u l l c , Becton Dickinson Immunocytometry Systems, San Jose, CA), and anti-CD19 (Leu12, Becton Dickinson) was added to the cell suspension. After a 10 min incubation on ice, the cells were washed once and incubated for an additional 10 min on ice with avidin-FITC (Becton Dickinson) at 1 /xg/106 cells. A 1/100 dilution of MACS biotinylated beads was added, and the cells incubated on ice for 5 min. The cell suspension was passed through the magnetized MACS column, and the unbound (i.e., CD8) cells collected. The CD8 cells were resuspended at 106/mi in RPMI 1640 supplemented with 10% HS and 40 U / m l rIL-2, and cultured at 37°C for 3-5 days.
Flow cytometric analysis of cell populations The phenotype of the CD4 and CD8 cells that had been cultured for 3-5 days was determined by flow cytometry to assess the purity of the selected cell populations prior to placing them in the inhibition assay. At this time the CD4 cells had detached from the capture flask surface. Cells were analyzed using standard methodology (Lewis and Rickman, 1992). To describe briefly, the cultured cells were washed, counted, and assessed for viability by trypan blue exclusion. 5 × 105 CD4 or CD8 cells were incubated with a two-color combination of murine monoelonal antibody (MAb) consisting of fluorescein isothiocyanate (FITC)-labeled anti-CD8 and phycoerythrin (PE)-labeled anti-CD4 (Leu2 and Leu3, Becton Dickinson) for 20 min at 4°C. The cells were then washed and analyzed for green (FITC)
and orange (PE) fluorescence using a FACScan instrument (Becton Dickinson) with a single argon laser.
CD8-mediated inhibition of HIV production For functional assessment, the CD4 and CD8 cells that had been cultured separately for 3-5 days were resuspended in fresh medium containing 10% HS, 10% purified human IL-2 and 40 U / m l rIL-2. Cells were pipetted into triplicate wells of 24-well plates (Costar, Cambridge, MA) to achieve ratios of CD4 to CD8 of 0.5:1, 1:1, 1 : 2, or 1 : 4 for a final concentration of 1 x 105 CD4 cells/ml/well. Separate wells of CD4 or CD8 cells were plated as controls. The co-cultures were incubated in a humidified chamber at 37°C and 5% CO 2. 0.5 ml of culture supernatants was collected at days 7, 10, 14 and 21 for assessment of virus production and replaced with 0.5 ml fresh medium. Production of HIV in the supernatants was measured as viral p24 antigen by ELISA assay (Abbott Diagnostics, Abbott Park, IL).
Results
Purity and viability of separated cell fractions The results of flow cytometric analysis from a representative experiment are shown in the scatter plot quadrants in Fig. 1. The whole lymphocyte preparation prior to separation is compared to cultured, separated CD4 and CD8 cells. This experiment showed an initial CD4 fraction of 35%, which was purified to 93%. The initial fraction of CD8 cells was 38% and was purified to 86%. The average percent purity for CD4 cell cultures from 15 subjects on whom extensive assessments were done was 94%, with a range of 89-99%. Contaminating cells tended to be double positive C D 4 / C D 8 cells (quadrant 2) or nonCD8 cells (quadrant 3) (i.e., NK or B cells), with an average of < 2% CD8 cells captured in the CD4 flasks. The viability of the cultured CD4 cells averaged 81%, with a range of 53-95%. Recovery of CD4 cells on the capture flasks could not be accurately determined because the cells were stimulated to proliferate in culture, so that in some instances there were more CD4 cells
184 A
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Fig. 1. Quadrant scatter plots from the flow cytometric analysis of lymphocytes from one subject assessed for expression of CD4 and CD8 antigens before and after cell subset separation. A shows 35% CD4 (quadrant 1) and 38% CD8 (quadrant 4) cells in peripheral blood lymphocytes prior to separation of CD4 and CD8 subsets; B shows 93% CD4 cells obtained from a CD4 capture flask after 3 days of culture; panel C shows cells from a CD8 flask with 86% CD8 cells. r e c o v e r e d t h a n t h e original input. T h e p u r i t y o f C D 8 cells a v e r a g e d 86% with a r a n g e o f 5 1 - 1 0 0 % . T h e a v e r a g e viability o f C D 8 ceils was 76% with a r a n g e o f 2 5 - 1 0 0 % . R e c o v e r y of C D 8 cells negatively s e l e c t e d using i m m u n o b e a d s a v e r a g e d 51% o f the original i n p u t o f C D 8 cells. T h e r e a s o n for t h e low viability o f t h e c u l t u r e d C D 4 a n d C D 8 cells in s o m e subjects is u n k n o w n , b u t m a y b e a r e f l e c t i o n o f t h e a c c e l e r a t e d cell d e a t h d u r i n g in vitro c u l t u r e t h a t has b e e n r e p o r t e d for l y m p h o cytes from H I V - i n f e c t e d individuals ( P r i n c e a n d Czaplicki, 1989).
uals, H I V was u n d e t e c t a b l e in 7-day s u p e r n a t a n t s f r o m P H A - s t i m u l a t e d cultures, w h e r e a s a n t i - C D 3 i n d u c e d m e a s u r a b l e p24 a n t i g e n at t h a t time. B a s e d o n t h e s e results, a n t i - C D 3 M A b was u s e d to activate C D 4 cells in all s u b s e q u e n t exp e r i m e n t s . O f 27 H I V seropositive subjects, two d i d not p r o d u c e d e t e c t a b l e p24 a n t i g e n w h e n t h e i r C D 4 cells w e r e c u l t u r e d with a n t i - C D 3 M A b . T h e s e individuals h a d C D 4 cell counts of 807 a n d 1 0 0 2 / m m 3, which are within n o r m a l
TABLE I
Production of H1V from CD4 cell cultures E x p e r i m e n t s w e r e p e r f o r m e d to c o m p a r e t h e efficiency o f t h e m i t o g e n P H A , which has b e e n u s e d r o u t i n e l y to elicit H I V p r o d u c t i o n f r o m p e ripheral lymphocytes of infected individuals ( G a l l o et al., 1984), versus t h e T cell activation stimulus a n t i - C D 3 M A b (Weiss a n d I m b o d e n , 1987) in i n d u c i n g t h e p r o d u c t i o n o f H I V from t h e C D 4 ceils o f i n f e c t e d subjects. T a b l e I shows the results at days 7 a n d 14 of e x p e r i m e n t s testing nine subjects. F o r t h e first t h r e e subjects, p a i r e d s a m p l e s w e r e t e s t e d s i m u l t a n e o u s l y using t h e two d i f f e r e n t stimuli. Six m o r e subjects w e r e t e s t e d using e i t h e r P H A o r a n t i - C D 3 as an activation stimulus. In all cases, C D 4 cells s t i m u l a t e d with a n t i - C D 3 M A b p r o d u c e d consistently h i g h e r levels of p24 a n t i g e n in c u l t u r e s u p e r n a t e s t h a n d i d those c u l t u r e d with P H A . In t h r e e of the individ-
HIV p24 ANTIGEN IN CULTURES STIMULATED WITH PHA OR ANTI-CD3 Day 7 (pg/ml) PHA
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a The first three results were from paired samples from the same three subjects tested simultaneously with PHA or antiCD3.
185
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Fig. 2. Individual results from six individuals for H I V production from CD4 cells cultured alone (dark bars), or CD4 and CD8 cells co-cultured at ratios of 1 : 1 (hashed bars) or 1 : 2 (vertically striped bars) for 7 days.
reference ranges, indicating little if any CD4 cell depletion, which is suggestive of low viral burden (Schnittman, et al., 1990).
CD8-mediated Inhibition of HIV production Fig. 2 shows results of CD8-mediated inhibition assays from six of the 25 subjects tested who produced p24 antigen from their CD4 cell cultures. Viral p24 antigen production on day 10 of culture is shown for CD4:CD8 ratios of 1:1 or 1:2. It is of note that of the four time points tested, viral p24 antigen production was low but generally detectable by day 7 and peaked by day 14. By day 21, the majority of the cells was dead, and this time point provided little additional information (data not shown). Although the levels of viral p24 antigen produced by CD4 cells varied among the 25 individuals tested, in all individuals, less p24 antigen was detected in the CD4/CD8 co-cultures than from CD4 cells cultured alone. The average percent inhibition for all 25 individuals was 83% (range of 38-100%) at the CD8 : CD4 ratio of 1 : 1 and 90% (range of 59-100%) at 2 : 1. The degree of inhibition of HIV replication did not correlate with the
peripheral blood CD4 cell percent, absolute number, or CD4:CD8 ratio (data not shown).
Discussion
The functional competence of T cells in vitro has been used as an indicator of disease progression and prognosis in HIV infection (Lewis and Giorgi, 1990). The restoration or augmentation of T cell function is a surrogate marker for therapeutic efficacy (Redfield et al., 1991). Hence, assay of CD8 cell function has broad applicability in the laboratory assessment of HIV-infected individuals. Although cytotoxic function of CD8 cells has been studied in HIV infection (Walker et al., 1987) and may provide useful information, the requirements for antigen and class I major histocompatibility complex (MHC) specificity limit the general use of cytotoxic T lymphocyte (CTL) assays. Inhibition of HIV replication by CD8 cells, like CTL function, may be an in vitro correlate of antiviral immunity in HIV infection. We have developed a simple and reproducible assay for
186 CD8 cell-mediated inhibition using commercially available reagents for standardization of cell separation and activation. Several problems inherent in the assay system have been addressed. One critical element is the consistent induction of HIV from cultured CD4 cells, which serves as the read-out for the assay. The complexity of this biologic system suggests that there may be multiple immunologic and virologic factors involved in the production of HIV from naturally infected CD4 cells. In extending their original studies, Walker et al. (1989) found that even small numbers of CD8 cells from some subjects were shown to inhibit the production of HIV in CD4 cultures. In the experiments, CD4 cells were isolated by panning (Wysocki and Sato, 1978). There is no standardization of reagents or methodology for panning, leading to variation in the efficiency of cell separation among investigators using this technique. Our use of a commercially available cell capture system has diminished this variability, while maintaining the technical simplicity of a panning-type method. In a comparison of methods for cell purification in our laboratory (Plaeger-Marshall and Hausner, 1991), we have found the capture flasks to yield cell purity and recovery comparable to or better than other cell separation technologies. It is likely that factors other than the purity of CD4 cells also contributed to the lack of virus production in the cultures of some individuals. We have shown that, even when CD4 cells were highly purified, anti-CD3 MAb induced HIV replication when PHA did not. Brinchmann et al. (1990) have reported that MAb to the T cell antigen receptor (TCR) consistently induced virus production in cell cultures from asymptomatic HIV-infected subjects. The reason for this is not known but may relate to the intracellular activation pathways stimulated by different ligand-receptor interactions. T cell stimuli like anti-CD3 or anti-TCR MAbs more closely mimic the interaction of T cells with specific antigen than do mitogens like P H A that have numerous cell ligands (Valentine et al., 1985; O'Flynn et al., 1985). It is possible that MAbs are more efficient activators of cellular pathways critical to the productive replication of HIV, such as activation of the HIV long terminal repeat (Tong-Starksen et al., 1989).
Although a qualitative deficiency in CD3-mediated cellular activation in HIV infection has been reported (Miedema et al., 1988), the CD3-1inked activation pathway inhibited by HIV may be different from that inducing the metabolic events that trigger viral replication. These issues in the biology of virus-cell interactions remain to be resolved. In summary, we have developed a reproducible and simple assay to measure CD8 T cell mediated suppression of HIV production from the CD4 cells of asymptomatic infected individuals. The standardization of the assay allows its application on a large scale for monitoring HIVinfected individuals. Experiments are in progress to assess the usefulness of the assay for following disease progression and the efficacy of antiviral or immunomodulatory therapies.
Acknowledgements We gratefully acknowledge the participants and staff of the UCLA Multicenter AIDS Cohort Study (MACS), especially Drs. Roger Detels and Barbara Visscher and Ms. Jan Dudley and Mr. Dennis Miles. We thank Ms. Patricia Hultin and Drs. Valentin Isacescu and Negoita Neagos for performance of the laser flow cytometric analyses, and Ms. Jeanne Bertolli for expert editorial assistance. The viral p24 antigen assays were performed by Mr. Amadu Diagne in the U C L A HIV Virology Laboratory directed by Dr. Irvin Chen. This work was supported in part by U.S. Public Health Service grant AI 26098, contract AI 72656, and Center for AIDS Research Grant AI 28697. AIS MicroCELLector flasks were generously supplied by Applied Immune Sciences, Inc.
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