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J Virol, February 1998, p. 1627-1631, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Herpesvirus Saimiri-Transformed Human CD4⁺ T-Cell Lines: an Efficient Target Cell System for the Analysis of Human Immunodeficiency Virus-Specific Cytotoxic CD8⁺ T-Lymphocyte Activity

Monika Bauer,¹ Maria Lucchiari-Hartz,^2, Helmut Fickenscher,³ Klaus Eichmann,² Jane McKeating,⁴ and Andreas Meyerhans^1,*

Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, 79104 Freiburg,¹ Max-Planck-Institut für Immunbiologie, 79108 Freiburg,² and Institut für Klinische und Molekulare Virologie, Universität Erlangen Nürnberg, 91054 Erlangen,³ Germany, and School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom⁴

Received 28 July 1997/Accepted 3 November 1997

	ABSTRACT

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Herpesvirus saimiri growth-transformed human CD4⁺ T lymphocytes were examined for their suitability as a target cell system for investigating human immunodeficiency virus (HIV)-specific HLA class I-restricted cytotoxic T-cell activity. Besides CD4, they express the chemokine receptors CCR5 and CXCR4, the common coreceptors of HIV. They are infectible by a range of HIV strains, including primary isolates, becoming efficient targets for CD8-positive HIV-specific cytotoxic T lymphocytes.

	TEXT

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HLA class I-restricted CD8⁺ T lymphocytes (CTL) are believed to play an important role in immune defense against human immunodeficiency virus (HIV) infection (3, 25, 35). For example, this antiviral response is considered a major factor in reducing the virus load during primary infection and in controlling virus production thereafter (5, 6, 13, 15, 16, 22, 29, 30, 37). Several assay systems have been used to evaluate the HIV-specific CTL response (19, 28, 36, 38). Most commonly used as target cells are autologous Epstein-Barr virus (EBV)-transformed B-cell lines that have been infected with recombinant vaccinia viruses expressing various HIV antigens. While such a target cell system allows the detection of a CTL activity restricted by the complete set of a patient's HLA, only a single variant of a single HIV antigen can be examined. However, one of the features of persistent HIV infection is the continuous generation of novel virus variants with altered antigenic properties (13, 20, 21, 26, 27, 37). As the test antigen of the recombinant vaccinia virus usually differs from the patient's HIV strain, the CTL response can be markedly underestimated (13). To overcome such limitations and to measure the CTL activity against the complex HIV populations, so-called quasispecies, present in patient isolates, an HIV-infectible target cell system is necessary. Ideally, it should be based on CD4-positive T lymphocytes, the main cell type infected in HIV-infected patients, as target cells. Second, it should allow testing of the CTL activity restricted by all the patient's HLA, e.g., by using autologous CD4⁺ T lymphocytes. Third, it should express the main HIV coreceptors, in addition to CD4, to allow for infection with a variety of different HIV isolates (2, 7-10, 39). And fourth, it should respond to HIV infection in a manner similar to that of primary CD4⁺ T lymphocytes, at least in respect to surface marker expression, which is important for recognition of the infected cells by CTL (14, 32). In this respect, the use of established T-cell lines is limited due to their infectibility by CXCR4-dependent virus isolates only, while the use of primary CD4⁺ T cells would require new preparation for each assay. However, herpesvirus saimiri (HVS) growth-transformed human CD4⁺ T lymphocytes should be a promising target cell system, as they can be established from primary CD4⁺ T lymphocytes even from AIDS patients (31) and support efficient replication of HIV and simian immunodeficiency virus (1, 23, 34).

In the present study, the suitability of HVS growth-transformed human CD4⁺ T-cell lines to serve as target cells for the analysis of HIV-specific cytotoxicity was examined. It is shown that such cell lines express the commonly used HIV coreceptors CCR5 and CXCR4 and that they may be infected by a variety of different HIV strains, including primary isolates. CTL-mediated lysis of HIV-infected HVS growth-transformed CD4⁺ T cells is HIV specific and HLA-I restricted. Thus, these cell lines might have broad applications for the study of the complex interaction of HIV quasispecies and the CTL response in an autologous system, as well as of other infections with CD4-tropic viruses.

Efficient HIV replication in HVS growth-transformed human CD4⁺ T-cell lines. CD4⁺ T-cell lines were established from peripheral-blood mononuclear cells (PBMC) of HLA-typed normal blood donors by growth transformation with HVS strain C488 as previously described (4, 11). The cell lines designated KAD and CB15 were >98% CD3⁺ CD4⁺, as assessed by flow cytometry. They are HLA-A2, -A3, -B7, -B27 and HLA-A1, -A24, -B8, -B35, respectively, and were used for subsequent experiments.

To determine HIV coreceptor surface expression, KAD and CB15 cells were stained with peptide-purified rabbit anti-CCR5 or anti-CXCR4 immunoglobulin G (IgG) as a first antibody. These were obtained after immunization of rabbits with synthetic peptides comprising the 35 N-terminal amino acids of both chemokine receptors (ADP7039 and ADP7040; AIDS Directed Program, Hertfordshire, United Kingdom). As the second antibody, biotin-labelled anti-rabbit IgG (Coulter-Immunotech, Hamburg, Germany) was used. The reaction was developed with phycoerythrin-labelled streptavidin (PharMingen, Hamburg, Germany). Fluorescence was measured on a FACSsort flow cytometer (Becton Dickinson, Heidelberg, Germany). Chemokine receptor expression levels for KAD and CB15 cells (Fig. 1A through D) were in the range of levels for other human HVS growth-transformed CD4⁺ T-cell lines (data not shown).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   HIV coreceptor expression and HIV replication kinetics in the HVS growth-transformed human CD4+ T-cell lines KAD and CB15. (A through D) Cells were analyzed for surface expression of CXCR4 (A and B) and CCR5 (C and D) by flow cytometry (solid lines). Background fluorescence is shown by dotted lines. (E through H) Cells were infected with HIV derived from the molecular clones HIV-1 pLai-2, HIV-1 pLaiga (pLai-2nef), and HIV-2 pRod (E and F) or with the primary HIV-1 isolates Lai, R/H1865, and S/L2195 (G and H). Culture supernatants were analyzed for RT activity at the time points indicated. Incorporated radioactivity was measured with a Berthold-Inotech Trace-96 (Berthold, Wildbad, Germany). The values obtained are approximately 50-fold lower than those obtained with conventional scintillation counters.

To analyze the replication capacities of various HIV strains in the KAD and CB15 cell lines, 3 × 10⁶ cells were infected with viruses derived from infectious molecular clones HIV-1 pLai-2, HIV-1 pLaiga (pLai-2nef), and HIV-2 pRod or with primary isolates (HIV-1_Lai, HIV-1_R/H1865, and HIV-1_S/L2195). All but HIV-1_S/L2195 were of the syncytium-inducing (SI) phenotype. Infection was carried out with a multiplicity of infection of 0.5, or 5,000 cpm of reverse transcriptase (RT) activity. Virus production was monitored by testing culture supernatants for RT activity as previously described (24). Both cell lines were permissive for all virus strains tested (Fig. 1E through H). Replication kinetics were comparable to those in other HVS growth-transformed CD4⁺ T-cell lines (23, 34), as well as to those in primary PBMC or primary CD4⁺ T cells (data not shown). Even the replication characteristics of slow/low and rapid/high viruses HIV-1_S/L2195 and HIV-1_R/H1865, respectively, were preserved. Supernatants of infected cells were always negative for infectious herpesvirus. Thus, HVS growth-transformed human CD4⁺ cell lines express CCR5 and CXCR4, are susceptible to monocytotropic and T-cell-tropic primary virus isolates, and seem not to alter the growth properties of slow/low and rapid/high viruses.

HIV antigens are expressed in HIV-infected HVS growth-transformed CD4⁺ T-cell lines before the appearance of cytopathic effects. To determine the kinetics of HIV antigen expression after infection of HVS growth-transformed CD4⁺ T cells, HIV-1 pLai-2-infected KAD and CB15 cells were fixed with 2% formaldehyde and permeabilized with 0.5% Nonidet P-40. HIV antigens were stained first with a polyclonal serum that recognized gp41, gp120, p24, p55, and Nef on a Western blot (data not shown). As the second antibody, fluorescein isothiocyanate (FITC)-labelled anti-human IgG (Sigma, Deisenhofen, Germany) was used. Fluorescence intensity was measured by flow cytometry 1 to 4 days after infection (Fig. 2). On day 2, a significant proportion of the infected cells were already antigen positive. Without the appearance of cytopathic effects, their frequency increased further, to more than 90%.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Kinetics of HIV-1 Lai antigen expression in the HIV-infected HVS growth-transformed human CD4+ T-cell lines KAD and CB15. At days 1 to 4 after infection, permeabilized cells were analyzed by flow cytometry for the expression of HIV-1 proteins with a polyclonal patient serum (grey histograms). The fluorescence of uninfected control cells is shown by black histograms. The percentage of HIV antigen-positive cells on each day is indicated.

Effect of HIV infection on the expression of surface antigens on HVS growth-transformed CD4⁺ T-cell lines. Previous reports have shown that HIV alters the expression of cell surface molecules involved in CTL-mediated cytotoxicity, such as HLA class I (14, 32). To analyze such effects on HVS growth-transformed CD4⁺ T cells, KAD and CB15 were infected with HIV-1 pLai-2 and stained 3 days postinfection with W6/32 (anti-HLA-I; Dako, Hamburg, Germany), UB2 (anti-CD95; Coulter-Immunotech, Hamburg, Germany), or MT310 (anti-CD4; Dako) as the first antibody, and FITC-labelled anti-mouse IgG (Sigma) as the second antibody. Uninfected cells were stained accordingly and used as references. At day 3 post-HIV infection, when KAD and CB15 cells showed maximal staining for HIV antigens, CD4 and HLA-I expression was markedly reduced (Fig. 3A and B). Similar observations have been reported for HIV-infected primary CD4⁺ cells and cell lines (12, 14, 33). In contrast, no significant alteration of CD95 was observed (Fig. 3C) Thus, HIV-mediated downregulation of cellular-surface antigens in HVS growth-transformed human CD4⁺ cells is selective for HLA-I and CD4 antigens and is independent of cytopathic effects.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Modulation of surface antigen expression in the HVS growth-transformed human CD4+ T-cell lines KAD and CB15 by HIV-1 pLai-2. Three days after HIV-1 infection, expression of CD4 (A), HLA-I (B), and CD95 (C) was measured by flow cytometry (black histograms) and compared to the respective uninfected cells (grey histograms). Background fluorescence is shown by filled histograms.

HIV-infected HVS growth-transformed CD4⁺ T-cell lines are efficient targets for HIV-specific CTL-mediated lysis. To analyze CTL-mediated recognition of HIV-infected HVS growth-transformed CD4⁺ T-cell lines, 3 × 10⁶ cells were infected with HIV-1 pLai-2. Specific lysis was determined 2 to 5 days postinfection by using a previously described HLA-A2 and HLA-B7-restricted HIV-1 Nef-specific CTL line as effector cells (17, 18). Maximal specific lysis of infected cells of around 75% was observed 3 and 4 days postinfection (Fig. 4A). This correlated well with the kinetics of HIV antigen detection in infected KAD cells (Fig. 2). Target cell lysis was HIV specific, as uninfected control cells showed background lysis of only around 15%. At day 5 postinfection, some cytopathic effects were visible in the culture, which might explain the reduction of the CTL-mediated lysis to 50% (Fig. 4A). Antigen specificity, HLA restriction, and Ca²⁺ dependence of target cell lysis were demonstrated as follows. (i) When KAD cells were infected with HIV-1 pLaiga, which lacks a functional nef gene, lysis by the Nef-specific CTL line was not observed (Fig. 4B). (ii) CTL-mediated lysis of infected KAD cells was abrogated by the addition of antibodies against HLA-I (W6/32) or by the addition of 6 mM EGTA (Fig. 4B). (iii) Likewise, HLA-mismatched HIV-infected CB15 cells were not lysed (Fig. 4C), while CTL-mediated lysis was observed with an HLA-matched, phytohemagglutinin-stimulated HIV-1-oligospecific CTL line (Fig. 4D).

View larger version (23K): [in this window] [in a new window]   FIG. 4.   CTL-mediated HIV-1 Nef-specific lysis of HVS growth-transformed human CD4+ T cells. Either HLA-A2- and HLA-B7-restricted HIV-1 Nef-specific CTL (A through C) or an HLA-A1- and HLA-A24-matched, oligospecific CTL line (D) was incubated with HIV-1 pLai-2-infected () or uninfected () 51Cr-labelled target cells. Specific lysis was determined by a standard 51Cr release assay. (A) Lysis of infected KAD target cells at an effector-to-target cell ratio of 100:1 is shown at days 2, 3, 4, and 5 postinfection. (B) Lysis of infected KAD target cells is Nef specific, HLA-I restricted, and Ca2+ dependent. Only HIV with an intact nef gene (pLai-2 []), not a nef HIV (pLaiga [], sensitized target cells for lysis. Target cell lysis could be inhibited by the anti-HLA-I antibody W6/32 (), and by addition of EGTA (). Lysis was measured 3 days postinfection and is shown for different effector-to-target cell ratios. (C) HIV-infected HLA-mismatched CB15 cells were not lysed. (D) HIV-infected CB15 cells can be lysed by an HLA-matched CTL line.

In conclusion, HVS growth-transformed human CD4⁺ T-cell lines represent an efficient target cell system for the analysis of HIV-specific cellular immune responses. They express the commonly used coreceptors CCR5 and CXCR4, and consequently they can be infected by a variety of HIV strains, including primary isolates. CTL-mediated lysis of infected cells was HIV specific and HLA-I restricted. Because HIV infection downregulates HLA class I in a manner similar to that observed in primary CD4⁺ T lymphocytes but does not affect surface expression of CD95, these cells might be suitable both for testing the effect of HLA expression levels on target cell recognition and for investigating Fas-mediated lysis. Furthermore, as HIV-infected HVS growth-transformed human CD4⁺ T cells express the costimulatory molecules CD80 and CD86 in amounts comparable to those in EBV-transformed B cells (data not shown), they should be useful as stimulator cells in the amplification of HIV-specific CTL responses. These findings, and the recent observations that HVS growth-transformed human CD4⁺ T-cell lines can be established even with cells from AIDS patients (31), indicate that these cell lines may have broad utility for studying the complex interaction of HIV quasispecies and the CTL response.

	ACKNOWLEDGMENTS

We thank Otto Haller and Bernhard Fleckenstein for continuous interest in the work and Simon Wain-Hobson for critical comments.

This work was supported by the Deutsche Forschungsgemeinschaft and Zentrum klinische Forschung (M.B. and A.M.), the Bayerische Forschungsstiftung (H.F.), and the Medical Research Council, ADP, Programme EVA, and the Lister Institute for Preventive Medicine (J.M.).

	FOOTNOTES

^* Corresponding author. Mailing address: Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. Phone: 49 761 203 6614. Fax: 49 761 203 6639. E-mail: andreas{at}sun1.ukl.uni-freiburg.de.

Present address: Laboratory of Virus Immunology, Instituto Butantan, Sao Paulo, Brazil.

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