PETER C. DOHERTY,* AND MARCIA A. BLACKMAN. Department ofImmunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Vol. 68, No. 5
JOURNAL OF VIROLOGY, May 1994, p. 3065-3070 0022-5 38X/94/$04.00 +O Copyright ©D 1994, American Society for Microbiology
Virus-Specific CD8+ T-Cell Responses in Mice Transgenic for a T-Cell Receptor I Chain Selected at Random CHRISTINE EWING, WILLIAM ALLAN, KIERAN DALY, SAM HOU, GEOFFREY A. COLE, PETER C. DOHERTY,* AND MARCIA A. BLACKMAN Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 Received 3 November 1993/Accepted 15 February 1994
The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR a-chain gene (V08.1DP2J,12.3CPI2). Analyzing the prevalence of VP8.1' CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3E showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR ji chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.
influenza virus nucleoprotein peptide plus H-2Dh emphasizes CD8+ T cells expressing V18.3DI2J,B2.2, but there is no obvious pattern in TCR cx-chain usage and mice clear the virus with substantially normal kinetics when depleted of all VI8+ T cells (7). The indications from the influenza virus experiments are thus that there is considerable redundancy in the T-cell repertoire, even when the analysis is limited to a single peptide-plus-class I MHC glycoprotein complex (6, 29). We show here that the CD8+ T-cell response in H-2k mice transgenic (5, 24) for a single TCR f chain (V38.1Dr32JPi2. 3C12) can promote recovery from the pneumonias caused by an influenza A virus or a parainfluenza type 1 virus (Sendai), though this TCR is not used by normal mice to control either
Virus-specific CD8+ T cells, assayed in vitro as cytotoxic T lymphocytes (CTL), are generally considered to be the main effectors mediating recovery in the acute response to viruses (8). The CD4+ T-cell subset can function in the absence of CD8+ T cells to terminate respiratory infections caused by influenza A viruses and by a parainfluenza type 1 virus (Sendai), though clearance may be delayed and the incidence of lethal disease may be substantially increased (4, 10, 18). Removing the CD4+ T-cell population from mice with intact CD8+ T-cell function abrogates the immunoglobulin G and A class antibody responses but has relatively little effect on either the development of the virus-specific CTL activity in bronchoalveolar lavage (BAL) populations or on the kinetics of virus elimination from the lungs of C57BL/6J (B6) mice (3, 10, 11, 18). Considerable attention has been given to the possibility that eliminating responder T cells that express a dominant T-cell receptor (TCR) 1-chain variable region (V,B) may cause selective immunosuppression, an effect that could be useful for the treatment of autoimmune diseases (1, 26). The implication is that the range of clonotypes specific for a particular major histocompatibility complex (MHC) glycoprotein-plus-peptide determinant (1, 25) is extremely constrained and that a state of nonresponsiveness can be induced by treatment with the appropriate monoclonal antibody (MAb) to TCR VP. The question of CD8+ TCR repertoire usage in viral immunity has, to date, been largely addressed for H-2' mice infected either with lymphocytic choriomeningitis virus or with an influenza A virus (6, 7, 20, 28, 29). A single TCR ac4 pair is used prominently (6, 20), though not exclusively (28, 29), to recognize a lymphocytic choriomeningitis virus glycoprotein peptide presented in the context of H-2Dh. The response to an
infection. MATERIALS AND METHODS Mice. The V,8.1 transgenic mouse was generated by using a
Vf8.1D,B2J,B2.3 P chain from a T-cell hybridoma, 3DT-52.5, that has reactivity for H-2 &' and Mls-1 (5, 12). These mice
express the transgenic 3 chain in >95% of peripheral T cells and have been extensively characterized (5). The transgene, which is carried heterozygously, has been repeatedly (>5 years) backcrossed onto the CBA/CaJ (H-2k) strain. Nontransgenic (non-TG) littermates are used as control mice in these experiments. The transgenic breeding colony is maintained at St. Jude Children's Research Hospital. CBA/J mice were purchased from the Jackson Laboratory, Bar Harbor, Maine. Females were used unless otherwise specified. All mice were between 8 and 12 weeks of age at the time of challenge and, apart from exposure to the particular virus, were maintained as specific pathogen free throughout. Virus infection and titration. The A/HKx31 (H3N2) influenza A virus is a recombinant between A/PR8 (HINI) and A/Aichi (H3N2) which has many of the internal components of A/PR8 (19). The HKx31 and A/PR8/31 (PR8) influenza A
* Corresponding author. Mailing address: Department of Immunology, 332 N. Lauderdale, Memphis, TN 38105. Phone: (90)1) 522-0470. Fax: (901) 522-8570. 3065
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viruses and the Enders strain of Sendai virus (21) were grown in the allantoic cavity of 10-day-old embryonated hen's eggs. Virus stocks, stored at - 70°C as allantoic fluid, were shown to be free of mycoplasma and endotoxin. Mice were infected intranasally (i.n.) under (2,2,2-tribromoethanol) anesthesia with 50 pLI of phosphate-buffered saline (PBS) containing 240 hemagglutinating units of HKx31 virus, 60 hemagglutinating units of PR8, or 30 to 50 50% egg infective doses (EID50) of Sendai virus (2, 3, 18). Reisolation and titration of virus from infected mouse lung tissue was done by using chicken embryos
(3, 18). Sampling the lung and regional lymph node. Pooled BAL cells were obtained from the lungs of 2 to 8 anesthetized, virus-infected mice as described previously (3). The regional mediastinal lymph nodes (MLN) were then removed, and whole lungs were frozen for later virus isolation and titration. The MLN were disrupted by using tissue grinders, and the BAL and MLN samples were washed and counted. The BAL population was also adhered on plastic for 90 min at 37°C to remove most of the macrophages. The nonadherent BAL lymphocytes and MLN cells were then processed for flow cytometric analysis or for in vitro culture. The lungs were homogenized in PBS containing antibiotics, an aliquot (100 ,ul) was injected into each of four embryonated hen's eggs, and evidence of virus growth was detected by hemagglutinating activity in allantoic fluid (3). Virus titers are expressed as loglo EID50 per milliliter of lung homogenate. In vitro culture and CTL assays. Established (9, 18) protocols were used to stimulate spleen or lymph node cells in culture for 5 or 6 days, by using irradiated spleen cells infected with either Sendai virus or the HKx31 influenza virus, or an irradiated hybridoma cell line that produces a MAb to CD3s for polyclonal expansion (22). Standard 5 Cr release assays were used with virus-infected L cells and MHC-recombinant
target cells (3, 18). The redirected CTL assay with FcR-bound MAb to CD3s (or the lectin phytohemagglutinin) was also used to screen for the presence of any CD3+ T cells with activated lytic machinery (9, 22). The assays were incubated for 6 to 8 h at 37°C, and results are expressed as specific 5 Cr release. Virus-specific CTL precursor (CTLp) frequencies were determined by limiting dilution analysis as described previously (2, 17). A level of specific 51Cr release equivalent to three standard deviations was taken as positive. Minimal estimates of precursor frequency according to the Poisson distribution were obtained as the slope of the line relative to the number of responder cells per well (plotted on a linear x axis) and the percentage of nonresponder cultures (plotted on a logarithmic y axis) fitting the data by the least-squares method. In vivo T-cell depletion. Mice were treated with sequential doses of the GK1.5 MAb to CD4 or the F23.1 MAb to V18. The effectiveness of the T-cell depletion was monitored by flow cytometry, by using a FACScan (Becton Dickinson, Mountain View, Calif.) and the RM4-5 and 53.6.7 MAbs to CD4 and CD8 (Pharmingen, San Diego, Calif.). These techniques, together with the source of the MAbs, have been described in detail previously (2, 7). Phenotyping TCR+ lymphocytes. The TCR phenotypes of virally stimulated lymphocyte populations were determined by two- or three-color fluorescence-activated cell sorter (FACS) analysis with fluorescein isothiocyanate-conjugated MAbs or avidin in association with biotinylated MAbs, as described
previously (7).
cRu
70
a)
HKx31 L Cells P815 + PHA
El
p-1
() 1-_ 60 a) Lcc
50 0
o -
40
0.-0I:j
30
LO
o)-
20 10
0
ID
HKx31
aCD3
Non-TG
HKx31
aCD3
TG 8.1
FIG. 1. Virus-specific and lectin (phytohemagglutinin)-dependent CTL activity of cultured MLN populations from mice primed i.n. with the HKx31 influenza virus 42 days previously. The two bars in each group represent 5:1 and 10:1 effector/target (E:T) ratios. The lymphocytes were stimulated in vitro for 6 days either with HKx31-infected spleen cells or with antibody (aCD3) to the CD3s component of the TCR which stimulates all T cells. Specific procedures are described in the Materials and Methods.
RESULTS
Survival and influenza virus-specific CD8+ CTLp in the TG8.1 mice. Groups of 10 non-TG and TG8.1 mice were infected i.n. with a dose of the HKx31 virus which, in B6 mice (3), causes substantial (but nonfatal) pneumonia. All survived, and MLN cells were taken for in vitro culture at 42 days after infection. The lymphocytes were stimulated either specifically with influenza virus or nonspecifically with a MAb to CD3s (9). Cultures from both the non-TG and TG8.1 mice generated potent CTL populations following exposure to virus (Fig. 1). The level of effector function was comparable for influenza virus-infected class I MHC+ class II MHC- HKx31-infected L cells and for lectin-dependent (phytohemagglutinin) lysis of normal P815 cells (9), indicating that substantial cytotoxic populations were not being missed in the virus-specific assay. Stimulation with the MAb to CD3s gave a much less potent CTL response (Fig. 1). Virus clearance and in vivo CD4+ T-cell depletion in mice with influenza. Groups of non-TG and TG8.1 mice were selectively immunosuppressed with the GK1.5 MAb to CD4 (or left untreated) and infected i.n. with the HKx31 virus (3). The inflammatory exudate in the respiratory tract is, as with H-2' mice infected with this virus (2), dominated by the CD8+ aot TCR+ set (Table 1). All mice had eliminated the virus from the lungs within 15 days (Table 1) though, whether CD4 depleted or not. The virus titers for the TG8.1 mice were much more variable than those for the non-TG controls on day 10 and gave the impression that clearance was slightly delayed. Analyzing the phenotypes of freshly isolated BAL T cells showed that >90% of the otB TCR+ cells were CD8+ in the mice treated with the GK1.5 MAb to CD4. The CD8+ BAL T cells from the TG8.1 mice were, as would be expected, uniformly V,8+. The expression of Vox2 was monitored as a measure of internal shifts in TCRo usage: the data indicate that other TCR (x chains are being used by the influenza virus-specific T cells in the TG8.1 mice. Also, as described previously for TCR VP8.2 transgenic mice in a different experimental system (13), there were essentially no y8 TCR+ lymphocytes in BAL populations from the TG8.1 groups,
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VIRAL IMMUNITY IN V,B8.1 TRANSGENIC MICE
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TABLE 1. Influenza virus titers in lungs and phenotypes of freshly isolated BAL T lymphocytesa
c3/-ybcells TCR+
% of CD8+ T cells that are:
% of afx TCR+ cells that are CD8+
Miceb
CD4 depletion
Day of sampling
(logl0 EID50)
Non-TG Non-TG
+
10 10
