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Cell mediated immune responses in ponies following infection with equine influenza virus (H3N8): the influence of induction culture conditions on the properties of cytotoxic effector cells
Veterinary Immunology and Immunopathology, 21 (1989) 327-337 Elsevier Science Publishers B.V., AmsterdAm-- Printed in The Netherlands
327
Cell Mediated Immune Responses in Ponies following Infection with Equine Influenza Virus (H3N8): the Influence of Induction Culture Conditions on the Properties of Cytotoxic Effector Cells D. HANNANT* and JENNIFER A. MUMFORD Department of Infectious Diseases, Animal Health Trust, Lanwades Park, Nr. Newmarket, Suffolk CB8 7PN (GreatBritain) (Accepted14 November 1988)
ABSTRACT Hannant, D. and Mumford, J.A., 1989. Cell mediated immune responses in ponies following infection with equine influenza virus (H3N8): the influence of induction culture conditions on the properties of cytotoxic effectorcells.Vet.Immunol. Immunopathol., 21: 327-337. Cytotoxic cellprecursors and/or cytotoxic memory cellswere demonstrated in the peripheral blood of ponies afteraerosol infection with influenza A/equine/Newmarket/79 (H3N8). In order to reveal their cytotoxic potential,peripheral blood mononuclear cellsrequired a secondary antigenic stimulation. In vitroinduced cytotoxic cellsshowed activityagainst influenza infected target cellsin a 3-4 h SICr-releaseassay. The reactivityof cytotoxic cellswas markedly influenced by the conditions of the secondary induction culture. If high concentrations of exogenous crude equine IL-2 were used, virus infected target cellswere susceptible to lysis by autologous or allogeneic effector cells.However, if IL-2 concentration was reduced, cytotoxic cellswere generated which showed featuresconsistentwith cytotoxicT cellsin that target-cellkillingwas geneticallyrestricted.
INTRODUCTION
Exposure of Welsh mountain ponies to a standardised dose of influenza A / e q u i n e / N e w m a r k e t / 7 9 (H3NS) by nebulised aerosol has been shown to induce severe but self-limiting pulmonary infection (Mumford et al., 1989). Clinical immunity to rechallenge with homotypic virus lasted up to I year and resistance did not correlate absolutely with levels of circulating antibody ( H a n n a n t et al., 1988a). This feature contrasted markedly with immunity induced by vaccination with inactivated whole virus vaccines, where immunity *To w h o m correspondence should be addressed.
0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
328 to rechallenge correlated very strongly with levels of circulating antibody to the haemagglutinin (Mumford et al., 1983). In man and mouse, local (respiratory tract) and serum antibody has been associated with protection (Liew et al., 1984; Ada and Jones, 1986). In horses, local antibody was stimulated by influenza infection and was rather shortlived, although local antibody memory was demonstrated in the nasopharynx and trachea by rapid secondary responses to rechallenge (Hannant et al., 1988b). The contribution of cell-mediated immune responses to protection and recovery from equine influenza has not been studied in depth. The importance of these mechanisms in equine influenza may be inferred from studies on human influenza which suggested that cell-mediated immune responses were important in cross protection (heterotypic immunity) between influenza A subtypes and in limiting infection (Webster and Askonas, 1980; Askonas et al., 1982; Pala et al., 1986). The purpose of the present study was to examine cell-mediated immune reactions in horses after infection with influenza virus and to identify some features of the cytotoxic effector cells. MATERIALSAND METHODS
Challenge virus The equine influenza virus used for aerosol infection of Welsh mountain ponies was the second egg passage of A/equine/Newmarket/D64/79 (H3N8) recovered from the nasopharynx of an unvaccinated pony.
Infection protocol Ponies seronegative for H3N8 viruses, were infected by exposure to an aerosol generated from 20 ml of allantoic fluid containing a total of 107gEIDso of the challenge virus using a De-Vilbiss nebuliser (model 65) as described (Mumford et al., 1989).
Peripheral blood mononuclear cells (PBMC) Whole blood was collected into heparinised tubes (¥acutainer, Becton Dickinson) and diluted 1:1 with 0.85% NaC1 before PBMC were isolated by Ficoll-Hypaque (Pharmacia) centrifugation (Hudson and Hay, 1980). Washed PBMC were used as virus infected target cells and as effector cells for cytotoxic assays.
Cytotoxic ef[ector cells Preliminary studies (unpublished) established that cytotoxic effector cells could not be demonstrated directly in the peripheral blood of ponies after infection with influenza virus. Therefore, it was necessary to stimulate cytotoxic cells by exposure to virus antigen in vitro. Induction culture of cytotoxic effector cells was carried out for 5 days at 37 ° C, 5% CO2 in complete medium which
329
comprised RPMI 1640 (Gibco) containing 10% fetal calf serum, 10-SM 2-mercaptoethanol (Sigma), penicillin (100 IU ml- 1) streptomycin ( 100/tg ml-1) and influenza virus (5 haemagglutination units ml-1).
Target cells PBMC were infected with influenza A/equine/Newmarket/79 or A/equine/ Miami/63 (H3N8) in serum-free RPMI 1640 before labelling with 51Cr as described (McMichael and Askonas, 1978). Cytotoxic assay Virus infected, 51Cr labelled target cells were plated at 2 × 104 cells 100 pl- 1 well- ~ in round-bottomed 96-well microplates (Nunc) in triplicate. Effector cells (100/~l) were added at killer/target ratios ranging from 100:1 to 25:1. Percentage cytotoxicity after 3-4h incubation was calculated using standard formulae (McMichael and Askonas, 1978). Virus antigen expression on infected cells Equine PBMC were infected with influenza in serum-free medium for 1.5 h, washed and resuspended in complete medium at 2 × 106 cells m l - 1. The development of cell surface virus antigens was detected by the binding of a xenogeneic monospecific polyclonal antiserum to 100/tl aliquots of cells in roundbottomed disposable microwells (Removawells, Dynatech). After washing in complete medium, antiserum binding was revealed by uptake of [125I]labelled Staphylococcus aureus protein A prepared as described (Hannant et al., 1985). Antigen specific lymphocyte proliferation assay Specific lymphocyte reactivity to influenza virus antigens was assessed by proliferation assays using PBMC prepared from 5 ponies 14 days after infection. PBMC were cultured for 5 days in the presence of varying amounts of H3N8 virus antigens and lymphocyte proliferation was assessed by uptake of radiolabelled nucleic acid precursor [3H]thymidine (Amersham International) over the final 18 h. Crude equine interleukin-2 (IL-2) Washed PBMC from ponies seronegative to H3N8 virus, were cultured in the presenceof 15/tg m1-1 concanavalin A (ConA; Sigma) for 48 h at 37°C in the presence of 5% CO2. The culture supernatant was mixed with 25 % suspension of Sepharose-4B (Pharmacia) to remove the lectin and filtered through 0.22 ttm membranes (Millipore). This sterile reagent was referred to as crude equine IL-2. It was not mitogenic for fresh equine PBMC but enhanced the proliferation responses to sub-optimal concentrations of Con A and phytohaemagglutinin (PHA; Sigma) (data not presented).
330 RESULTS
Virus antigen expression on infected equine PBMC in vitro The kinetics of virus antigen expression on equine P B M C is shown in Table 1. Antigen was first detected at about 1.5 h after infection and the levels of antigen expression increased over the following 24 h. Cell viability (as assessed by trypan blue exclusion) was approximately 75% at 24 h. This experiment confirmed the suitability of in vitro infected P B M C as targets for cytotoxicity studies.
Generation of virus specific T lymphocytes in ponies after influenza infection Sensitised lymphocytes were demonstrated in ponies 14 days after infection with equine influenza by proliferation assays to virus antigens in vitro (Fig. 1). Although the identity of the responding cells was not confirmed unequivocally, there was no antibody to H3N8 virus detected in the culture supernatants using a sensitive radioassay ( H a n n a n t et al., 1988a). Therefore, it was assumed that the proliferating cells were at least non-B and probably of T lymphocyte origin by analogy with similar studies in other species (Dolin et al., 1978; Askonas et al., 1982; Ada and Jones, 1986). The in vitro induced, antigen sensitised lymphocytes described above comprised the cytotoxic effector cells.
Cytotoxic activity of equine lymphocytes for virus infected target cells Autologous and allogeneic combinations of effector cells with uninfected and virus infected target cells were studied using a 3-4 h 51Cr-release assay for cytotoxicity. It was found t h a t if the culture conditions were optimised for the production of large numbers of in vitro induced cytotoxic cells, then their cytotoxic activity was not genetically restricted. Thus, cytotoxicity activity was demonstrated in autologous as well as allogeneic combinations of effector and target cells (Table 2 ) but only if the target cells were expressing virus antigens. Similar results were obtained in repeated experiments which were all optimTABLE1 Detection of H3N8 virus antigens on the surface of in vitro infected equine PBMC Time after infection (h) 1.5 + 1.0 1.5+ 24.0 1.5 + 48.0
Antibody binding to cells (mean counts rain- l ± s.d.)"
H3N8 infected
H7N7 infected
Uninfected
1726_ 448 4148 ± 1119" 6044 ± 788*
2403 ± 213 2404 ± 415 2610 ± 287
913 ± 129 1157± 113 1042± 93
"Antibodybinding detected by [~25I]labelledprotein A. *Significantincrease in antibody binding comparedwith 1.5+ 1.0 h infection period; P < 0.05.
331 25. ~, 20. o
Tz 15-
== 10" 8 5"
O"
I
0.00
0.05
!
0.50
5.00
!
!
50.00
500.00
ANTIGEN CONCENTRATION HAU/m] Fig. 1. Dose-response curves for lymphocyte proliferation to H3N8 virus antigens. Results are the mean counts rain- i _+s.d. [3H ] thymidine uptake of 2 X 105cells after 5 days culture in the presence
of inactivated whole virus preparations. Results of five ponies are illustrated and the dotted line represents the mean + 2 s.d. of control lymphocyte proliferations. HAU = haemagglutination units. TABLE 2
Cytotoxic activity of in vitro induced lymphocytes from influenza infected ponies for uninfected and virus infected target cells Combination of effector/targetcell
% Cytotoxicity for target cells~
(n=3)
Uninfected
Infected
Autologous
- 1.0 _+1.8 -4.8_+ 1.0
34.5 ± 5.2 15.5_+2.0
1.0 ± 0.5 5.6±0.5
22.0 _+1.6 21.5_+7.1
Allogeneic
~Meanpercentagecytotoxicity_+standarddeviation. ised to produce high levels of cytotoxicity (data not presented) by the inclusion of 25% crude equine IL-2 in the effector-cell-induction cultures. The obvious interpretation of these results was that the cytotoxic effector cell populations contained large numbers of natural killer (NK) cells. Attempted purification of T lymphocytes from other effector cells by nylon wool adherence also failed to reveal genetic restriction of cytotoxic effector cells (data not shown). This was emphasised by the fact that two cycles of passage over nylon wool was not sufficient to purify equine T lymphocytes. The cells purified by this method responded remarkably well to the polyclonal T cell activator PHA in the absence of added accessory cells (Fig. 2). The well known requirement for accessory cells in polyclonal T cell activation by mitogens suggested that nylon wool purified cells contained populations other than T lymphocytes.
332 30
25
20 -T z 15--
S z
IO--
5--
O-O
5 PHA
lO 15 20 CONCENTRATION pg/ml
25
Fig. 2. Mitogenic responses of equine PBMC to phytohaemagglutinin. Results are the mean +_s.d. of three ponies. Closed circles--unfractionated cells; closed triangles = nylon wool non-adherent cells; closed squares = plastic adherent cells.
Genetic restriction of cytotoxic lymphocytes In order to control for possible activators of NK cells in vitro, some experiments were carried out to modify the induction process for cytotoxic effector cells. The effect of reducing the concentration of crude equine IL-2 on the
TABLE 3 Effect of crude equine IL-2 concentration on the induction of cytotoxic lymphocytes for equine influenza infected target cells Crude IL-2 concentration (%) during in vitro induction cultures
Killer: target ratio
25
100:1
25
50:1
15
100:1
5
100:1
5
50:1
Mean % cytotoxicity~ Autologous
Allogeneic
55.4 (6b) (68-48) ~ 35.1 (4) {60-31) 32.0 (3) (52-25) 29.8 (4) (41-25) 15.5 (2) (19-12)
34.1 (3) (40-31) 24.3 (3) (27-19) 27.4 (3) (33-25) 9.5 (2) (11- 8) 7.9 (1) (-)
aCytotoxic effector cellstaken from ponies 5-7 days after infection. bNumber of observations. CRange.
333 subsequent activity of in vitro induced cytotoxic cells is shown in Table 3. Genetically restricted cytotoxic activity was revealed with killer: target ratios as low as 50:1 and showed features consistent with cytotoxic T lymphocyte activity. Therefore, a population of the effector cells taken from ponies after infection with equine influenza was shown to be genetically restricted. DISCUSSION The results of this study have established that cytotoxic cell precursors and/or cytotoxic memory cells may be demonstrated in ponies after infection with equine influenza. The kinetics of cytotoxic cell generation have not been studied in detail, but unpublished experiments indicated that specifically sensitised cells (as revealed by in vitro proliferation assays) were present in the circulation 4 days after primary infection with equine influenza. In order to reveal their cytotoxic potential, PBMC from infected ponies required a secondary antigenic stimulation. In vitro induced cytotoxic effector cells showed activity against influenza-infected target cells in a 3-4 h 51Cr-release assay. A significant finding was that the reactivity of cytotoxic cells was markedly influenced by the conditions of the secondary induction culture. Thus, virus infected target cells were shown to be susceptible to lysis by autologous or allogeneic effector cells (Table 2). However, a reduction in equine IL-2 concentration of the induction culture medium resulted in the generation of cytotoxic effector cells which showed features consistent with cytotoxic T lymphocytes, i.e., target cell killing was genetically restricted (Table 3 ). At the time of writing, monoclonal antibody markers for equine lymphocyte subsets were not widely available and analysis of cell-mediated immune responses were carried out on the basis of functional activity. The genetically non-restricted cytotoxic activity described above was thought to be of NK origin. Two cycles of purification by non-adherence to nylon wool failed to isolate a pure population of T cells because the resulting cells responded well to PHA in the absence of exogenous accessory cells (Fig. 2). The activity of NK cells has been shown to be greatly augmented by interferon and other lymphokines, resulting in an expanded target cell range including the ability to kill a second or third time (Welsh, 1984). In contrast to normal horses, PBMC from horses with the autosomal recessive disorder severe-combined immunodeficiency (SCID) have been shown not to respond to polyclonal activation with phytolectins (Magnuson et al., 1984) which suggested a possible defect in lymphocyte differentiation. However, unfractionated PBMC from SCID horses were very sensitive to IL-2 induction which resulted in potent cytotoxic activity for target cells across MHC barriers (Magnuson et al., 1987). This NK-like cytotoxicity was not inducible
334 in normal PBMC with IL-2. These data reflect the high frequency (greater than 50% ) of cells with NK phenotypes in the unfractionated PBMC of SCID horses compared with that found in normal horses (approximately 5% ). In the present studies, monocyte/macrophage mediated cytotoxicity was considered unlikely because of the short time of the assay ( 3-4 h). Moreover, the fact that there was very little killing of non-infected target cells suggested that nutrient depletion by macrophages was not a feature of this cytotoxic system. Clearly, the genetically non-restricted cytotoxic lymphocytes demonstrated in this study require further characterisation. The genetic restriction phenomenon, typical of cytotoxic T cell activity (Zinkernagel and Doherty, 1974) was demonstrated with equine cellular reagents in vitro when the cytotoxic induction phase was modified. It has been a widely held view that cytotoxic lymphocytes recognised and killed virus-infected target cells that shared MHC class I antigens, although some influenzaspecific cytotoxic T cells have also been described which show restriction for class II molecules of the MHC (Morrison et al., 1986). In the present experiments (Table 3), a component of the cytotoxic activity of PBMC was shown to be genetically restricted to autologous combinations of target and effector cells. On the basis of this feature, the identity of the effector cell in this situation was considered to be a cytotoxic T cell. Therefore, cells with the properties of genetically restricted T lymphocytes and cytotoxic cells which function across a histocompatibility barrier have been identified in the peripheral blood of horses after infection with equine influenza. However, the identity of effector cells in the respiratory tract remains to be established. Although PBMC were prepared from ponies seronegative for H3N8 viruses prior to infection, no steps were taken to monitor their status with regard to equid herpesvirus 1 (EHV-1, equine rhinopneumonitis virus) or equid herpesvirus 2 (EHV-2, slow growing equid herpesvirus } other than routine measurements for antibody to EHV-1. No significant changes in antibody levels were detected through the course of these experiments (data not shown). EHV-2 and EHV-1 are ubiquitous viruses in the equine population (Bryans, 1969). EHV-1 spreads throughout the host via the bloodstream as a cell-associated viraemia (Burrows and Goodridge, 1984). A state of latency may exist which can be reactivated by stress (Edington et al., 1985). Therefore, it is possible that latent EHV-1 and/or EHV-2 virus may have recrudesced during the influenza infection and culture phase of target cell preparation. Some target cells may have expressed these virus antigens and some effector cells may have shown EHV-1/EHV-2 specificity. This probability was considered remote however, because target cells not expressing H3N8 antigens were not lysed, whether by cytotoxic cells which were genetically restricted or by NK-like effectors. Cross protection between serologically distinct subtypes of influenza A virus has been shown to be a property of cytotoxic T lymphocytes in other species
335 (Zweerink et al., 1977; McMichael and Askonas, 1978; Webster and Askonas, 1980). The viral nucleoprotein has been suggested as the major target for this reaction (Townsend et al., 1984; Yewdell et al., 1985) although there is a marked variation in responsiveness to this antigen between individuals (Pala and Askonas, 1986; Pala et al., 1986). In the present experiments, cytotoxic lymphocyte studies were confined to the homologous (H3N8) virus. The demonstration of cytotoxic effector cells of at least two types (functionally) using different culture methods is not without precedent. Recent studies have suggested that genetically restricted cytotoxic T cells represent a differentiation step in the development of non-restricted cytotoxic cells of "promiscuous" target specificity (Brooks et al., 1985; Havele et al., 1986). Cloned cytotoxic T cells have been shown to expand their target cell range from autologous to xenogeneic cells and show features consistent with NK cells (Brooks and Holscher, 1987). The changes in target cell specificity were induced by exposure of cloned T cells to lymphokines in vitro such as IL-2 and interferon. These studies have some parallels with the data presented herein where nonrestricted cytotoxic cells were generated in vitro in the presence of high concentrations of crude equine IL-2. However, a differentiation pathway of nonrestricted cytotoxic cells via genetically restricted cytotoxic T cells is not supported by the observation of lymphoid cell differentiation in SCID horses (Magnuson et al., 1984) which are known to have large numbers of NK-like cells in the PBMC. In combination, these data suggest that NK-like cells may be products of a pathway separate from that of lymphocytes. The significance of the present results can be assessed more fully as reagents become available to differentiate cell surface phenotypes for the equine species. However, one important aspect which needs to be addressed is the target antigen which stimulates strong cytotoxic activity for virus infected cells in vivo. ACKNOWLEDGEMENTS This study was supported by the Horserace Betting Levy Board and the Animal Health Trust. We thank Dr. Neil Edington for helpful discussions on equine cytotoxic lymphocytes, Mr. H. Sawyer and his staff for excellent management of the pony herd and Mrs. Denise Burkett for preparation of the manuscript. Some of the results in this paper were presented at the 5th International Conference on Equine Infectious Diseases, Kentucky, 1987. REFERENCES Ads, G. and Jones, P.D., 1986.The immuneresponseto influenzainfection.Curr. Top. Microbiol. Immunol.,128: 1-54.
336 Askonas, B.A., McMichael, A.J. and Webster, R.G., 1982. The immune response to influenza viruses and the problem of protection against infection. In: A.S. Beare (Editor), Basic and Applied Influenza Research. CRC Press. Boca Raton, FL, pp. 157-188. Brooks, C.G. and Holscher, M., 1987. Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes. J. Immunol., 138: 1331-1337. Brooks, C.G., Holscher, M. and Urdall, D., 1985. Natural killer activity in cloned cytotoxic T lymphocytes:regulation by interleukin 2, interferon and specific antigen. J. Immunol., 135: 1145-1152. Bryans, J.T., 1969. On immunity to disease caused by equine herpesvirus-1. J. Am. Vet. Med. Assoc., 155: 294-300. Burrows, R. and Goodridge, R., 1984. Studies of persistent and latent equid herpesvirus-1 and herpesvirus-3 infections in the Pirbright pony herd. In: Latent Herpesvirus Infection in Veterinary Medicine. Martinus Nijhoff, Boston, pp. 307-319. Dolin, R., Murphy, B.R. and Caplan, E,A., 1978. Lymphocyte blastogenic responses to influenza virus antigens after influenza infection and vaccination in humans. Infect. Immun., 19: 867874. Edington, N., Bridges, C.J. and Huckle, A., 1985. Experimental reactivation of equid herpesvirus 1 following the administration of corticosteroids. Equine Vet., J., 17: 369-372. Hannant, D., Donaldson, K. and Bolton, R.E., 1985. Immunomodulatory effects of mineral dusts. I - Effect of intraperitoneal dust inoculation on splenic lymphocyte function and humoral immune responses in vivo. J. Clin. Lab. Immunol., 16: 81-85. Hannant, D., Mumford, J.A. and Jessett, D.M., 1988a. Duration of circulating antibody and immunity following infection with equine influenza virus. Vet. Rec., 122: 125-128. Hannant, D., Jessett, D.M., O'Neill, T., Sundqvist, B. and Mumford, J.A., 1988b. Nasopharyngeal, tracheobronchial and systemic immune responses to vaccination and aerosol infection with equine influenza virus (H3N8). In: D.G. Powell (Editor), Proceedings 5th International Conference on Equine Infectious Diseases. University press, Kentucky (in press). Havele, C., Bleakley, R.C. and Paetkau, V., 1986. Conversion of specific to non-specific cytotoxic T lymphocytes. J. Immunol., 137: 1448-1454. Hudson, L. and Hay, F.C., 1980. Practical Immunology. Blackwell Scientific Publications, Oxford, pp. 256-258. Liew, F.Y., Russell, S.M., Appleyard, G., Brand, C.M. and Beale, J., 1984. Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur. J. Immunol., 14: 350-356. Magnuson, N.S., Perryman, L.E., Wyatt, C.R., Ishizaka, T., Mason, P.H., Namen, A.E., Banks, K.L. and Magnuson, J.A., 1984. Continuous cultivation of equine lymphocytes : evidence for occasional T cell-like maturation events in horses with hereditary severe combined immunodeficiency. J. Immunol., 133: 2518-2524. Magnuson, N.S., Perryman, L.E., Wyatt, C.R., Mason, P.H. and Talmadge, J.E., 1987. Large granular lymphocytes from SCID horses develop potent cytotoxic activity after treatment with human recombinant interleukin-2. J. Immunol., 139: 61-67. McMichael, A.J. and Askonas, B.A., 1978. Influenza virus-specific cytotoxic T cells in man; induction and properties of the cytotoxic cell. Eur. J. Immunol., 8: 705-711. Morrison, L.A., Lukacher, A.E., Braciale, V.L., Fan, D. and Braciale, T.J., 1986. Differences in antigen presentation to MHC Class I- and Class II-restricted influenza virus-specific cytolytic T lymphocyte clones. J. Exp. Med., 163: 903-909. Mumford, J.A., Wood, J.M., Scott, A.M., Folkers, C. and Schild, G.C., 1983. Studies with inactivated equine influenza vaccines. 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). J. Hyg., 90: 385-395. Mumford, J.A., Hannant, D. and Jessett, D.M., 1989. Experimental infection of ponies with equine influenza (H3N8) virus by intranasal inoculation or exposure to aerosols. Equine Vet. J., (in press).
337 Pala, P. and Askonas, B.A., 1986. Low responder MHC alleles for Tc recognition of influenza. Immunogenetics, 23; 379-384. Pala, P., Townsend, A.R.M. and Askonas, B.A., 1986. Viral recognition by influenza A virus crossreactive cytotoxic T (Tc) cells:the proportion of Tc cells that recognise nucleoprotein varies between individual mice. Eur. J. Immunol., 16: 193-198. Townsend, A.R.M., McMichael, A.J., Carter, N.P., Huddleston, J.A. and Brownlea, G.G., 1984. Cytotoxic T cell recognition of the influenza nucleoprotein and haemagglutinin expressed in transfected mouse L cells. Cell, 39: 13-25. Yewdell, J.W., Bennink, R.J., Smith, G.L. and Moss, B., 1985. Influenza A virus nucleoprotein is a major target antigen for cross-reactive anti-influenza A virus cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. U.S.A., 82: 1785-1789. Webster, R.G. and Askonas, B.A., 1980. Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice. Eur. J. Immunol., 10: 396-401. Welsh, R.M., 1984. Natural killer cells and interferon. CRC Crit. Rev. Immunol., 5: 55-93. Zinkernagel, R.M. and Doherty, P.C., 1974. Restriction of in vitro T cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature, 248: 701702. Zweerink, H.J., Courteneidge, S.A., Shehel, J.J., Crumpton, M.J. and Askonas, B.A., 1977. Cytotoxic T cells kill influenza infected cells but do not distinguish between serologically distinct type A viruses. Nature, 267: 354-356.
327
Cell Mediated Immune Responses in Ponies following Infection with Equine Influenza Virus (H3N8): the Influence of Induction Culture Conditions on the Properties of Cytotoxic Effector Cells D. HANNANT* and JENNIFER A. MUMFORD Department of Infectious Diseases, Animal Health Trust, Lanwades Park, Nr. Newmarket, Suffolk CB8 7PN (GreatBritain) (Accepted14 November 1988)
ABSTRACT Hannant, D. and Mumford, J.A., 1989. Cell mediated immune responses in ponies following infection with equine influenza virus (H3N8): the influence of induction culture conditions on the properties of cytotoxic effectorcells.Vet.Immunol. Immunopathol., 21: 327-337. Cytotoxic cellprecursors and/or cytotoxic memory cellswere demonstrated in the peripheral blood of ponies afteraerosol infection with influenza A/equine/Newmarket/79 (H3N8). In order to reveal their cytotoxic potential,peripheral blood mononuclear cellsrequired a secondary antigenic stimulation. In vitroinduced cytotoxic cellsshowed activityagainst influenza infected target cellsin a 3-4 h SICr-releaseassay. The reactivityof cytotoxic cellswas markedly influenced by the conditions of the secondary induction culture. If high concentrations of exogenous crude equine IL-2 were used, virus infected target cellswere susceptible to lysis by autologous or allogeneic effector cells.However, if IL-2 concentration was reduced, cytotoxic cellswere generated which showed featuresconsistentwith cytotoxicT cellsin that target-cellkillingwas geneticallyrestricted.
INTRODUCTION
Exposure of Welsh mountain ponies to a standardised dose of influenza A / e q u i n e / N e w m a r k e t / 7 9 (H3NS) by nebulised aerosol has been shown to induce severe but self-limiting pulmonary infection (Mumford et al., 1989). Clinical immunity to rechallenge with homotypic virus lasted up to I year and resistance did not correlate absolutely with levels of circulating antibody ( H a n n a n t et al., 1988a). This feature contrasted markedly with immunity induced by vaccination with inactivated whole virus vaccines, where immunity *To w h o m correspondence should be addressed.
0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
328 to rechallenge correlated very strongly with levels of circulating antibody to the haemagglutinin (Mumford et al., 1983). In man and mouse, local (respiratory tract) and serum antibody has been associated with protection (Liew et al., 1984; Ada and Jones, 1986). In horses, local antibody was stimulated by influenza infection and was rather shortlived, although local antibody memory was demonstrated in the nasopharynx and trachea by rapid secondary responses to rechallenge (Hannant et al., 1988b). The contribution of cell-mediated immune responses to protection and recovery from equine influenza has not been studied in depth. The importance of these mechanisms in equine influenza may be inferred from studies on human influenza which suggested that cell-mediated immune responses were important in cross protection (heterotypic immunity) between influenza A subtypes and in limiting infection (Webster and Askonas, 1980; Askonas et al., 1982; Pala et al., 1986). The purpose of the present study was to examine cell-mediated immune reactions in horses after infection with influenza virus and to identify some features of the cytotoxic effector cells. MATERIALSAND METHODS
Challenge virus The equine influenza virus used for aerosol infection of Welsh mountain ponies was the second egg passage of A/equine/Newmarket/D64/79 (H3N8) recovered from the nasopharynx of an unvaccinated pony.
Infection protocol Ponies seronegative for H3N8 viruses, were infected by exposure to an aerosol generated from 20 ml of allantoic fluid containing a total of 107gEIDso of the challenge virus using a De-Vilbiss nebuliser (model 65) as described (Mumford et al., 1989).
Peripheral blood mononuclear cells (PBMC) Whole blood was collected into heparinised tubes (¥acutainer, Becton Dickinson) and diluted 1:1 with 0.85% NaC1 before PBMC were isolated by Ficoll-Hypaque (Pharmacia) centrifugation (Hudson and Hay, 1980). Washed PBMC were used as virus infected target cells and as effector cells for cytotoxic assays.
Cytotoxic ef[ector cells Preliminary studies (unpublished) established that cytotoxic effector cells could not be demonstrated directly in the peripheral blood of ponies after infection with influenza virus. Therefore, it was necessary to stimulate cytotoxic cells by exposure to virus antigen in vitro. Induction culture of cytotoxic effector cells was carried out for 5 days at 37 ° C, 5% CO2 in complete medium which
329
comprised RPMI 1640 (Gibco) containing 10% fetal calf serum, 10-SM 2-mercaptoethanol (Sigma), penicillin (100 IU ml- 1) streptomycin ( 100/tg ml-1) and influenza virus (5 haemagglutination units ml-1).
Target cells PBMC were infected with influenza A/equine/Newmarket/79 or A/equine/ Miami/63 (H3N8) in serum-free RPMI 1640 before labelling with 51Cr as described (McMichael and Askonas, 1978). Cytotoxic assay Virus infected, 51Cr labelled target cells were plated at 2 × 104 cells 100 pl- 1 well- ~ in round-bottomed 96-well microplates (Nunc) in triplicate. Effector cells (100/~l) were added at killer/target ratios ranging from 100:1 to 25:1. Percentage cytotoxicity after 3-4h incubation was calculated using standard formulae (McMichael and Askonas, 1978). Virus antigen expression on infected cells Equine PBMC were infected with influenza in serum-free medium for 1.5 h, washed and resuspended in complete medium at 2 × 106 cells m l - 1. The development of cell surface virus antigens was detected by the binding of a xenogeneic monospecific polyclonal antiserum to 100/tl aliquots of cells in roundbottomed disposable microwells (Removawells, Dynatech). After washing in complete medium, antiserum binding was revealed by uptake of [125I]labelled Staphylococcus aureus protein A prepared as described (Hannant et al., 1985). Antigen specific lymphocyte proliferation assay Specific lymphocyte reactivity to influenza virus antigens was assessed by proliferation assays using PBMC prepared from 5 ponies 14 days after infection. PBMC were cultured for 5 days in the presence of varying amounts of H3N8 virus antigens and lymphocyte proliferation was assessed by uptake of radiolabelled nucleic acid precursor [3H]thymidine (Amersham International) over the final 18 h. Crude equine interleukin-2 (IL-2) Washed PBMC from ponies seronegative to H3N8 virus, were cultured in the presenceof 15/tg m1-1 concanavalin A (ConA; Sigma) for 48 h at 37°C in the presence of 5% CO2. The culture supernatant was mixed with 25 % suspension of Sepharose-4B (Pharmacia) to remove the lectin and filtered through 0.22 ttm membranes (Millipore). This sterile reagent was referred to as crude equine IL-2. It was not mitogenic for fresh equine PBMC but enhanced the proliferation responses to sub-optimal concentrations of Con A and phytohaemagglutinin (PHA; Sigma) (data not presented).
330 RESULTS
Virus antigen expression on infected equine PBMC in vitro The kinetics of virus antigen expression on equine P B M C is shown in Table 1. Antigen was first detected at about 1.5 h after infection and the levels of antigen expression increased over the following 24 h. Cell viability (as assessed by trypan blue exclusion) was approximately 75% at 24 h. This experiment confirmed the suitability of in vitro infected P B M C as targets for cytotoxicity studies.
Generation of virus specific T lymphocytes in ponies after influenza infection Sensitised lymphocytes were demonstrated in ponies 14 days after infection with equine influenza by proliferation assays to virus antigens in vitro (Fig. 1). Although the identity of the responding cells was not confirmed unequivocally, there was no antibody to H3N8 virus detected in the culture supernatants using a sensitive radioassay ( H a n n a n t et al., 1988a). Therefore, it was assumed that the proliferating cells were at least non-B and probably of T lymphocyte origin by analogy with similar studies in other species (Dolin et al., 1978; Askonas et al., 1982; Ada and Jones, 1986). The in vitro induced, antigen sensitised lymphocytes described above comprised the cytotoxic effector cells.
Cytotoxic activity of equine lymphocytes for virus infected target cells Autologous and allogeneic combinations of effector cells with uninfected and virus infected target cells were studied using a 3-4 h 51Cr-release assay for cytotoxicity. It was found t h a t if the culture conditions were optimised for the production of large numbers of in vitro induced cytotoxic cells, then their cytotoxic activity was not genetically restricted. Thus, cytotoxicity activity was demonstrated in autologous as well as allogeneic combinations of effector and target cells (Table 2 ) but only if the target cells were expressing virus antigens. Similar results were obtained in repeated experiments which were all optimTABLE1 Detection of H3N8 virus antigens on the surface of in vitro infected equine PBMC Time after infection (h) 1.5 + 1.0 1.5+ 24.0 1.5 + 48.0
Antibody binding to cells (mean counts rain- l ± s.d.)"
H3N8 infected
H7N7 infected
Uninfected
1726_ 448 4148 ± 1119" 6044 ± 788*
2403 ± 213 2404 ± 415 2610 ± 287
913 ± 129 1157± 113 1042± 93
"Antibodybinding detected by [~25I]labelledprotein A. *Significantincrease in antibody binding comparedwith 1.5+ 1.0 h infection period; P < 0.05.
331 25. ~, 20. o
Tz 15-
== 10" 8 5"
O"
I
0.00
0.05
!
0.50
5.00
!
!
50.00
500.00
ANTIGEN CONCENTRATION HAU/m] Fig. 1. Dose-response curves for lymphocyte proliferation to H3N8 virus antigens. Results are the mean counts rain- i _+s.d. [3H ] thymidine uptake of 2 X 105cells after 5 days culture in the presence
of inactivated whole virus preparations. Results of five ponies are illustrated and the dotted line represents the mean + 2 s.d. of control lymphocyte proliferations. HAU = haemagglutination units. TABLE 2
Cytotoxic activity of in vitro induced lymphocytes from influenza infected ponies for uninfected and virus infected target cells Combination of effector/targetcell
% Cytotoxicity for target cells~
(n=3)
Uninfected
Infected
Autologous
- 1.0 _+1.8 -4.8_+ 1.0
34.5 ± 5.2 15.5_+2.0
1.0 ± 0.5 5.6±0.5
22.0 _+1.6 21.5_+7.1
Allogeneic
~Meanpercentagecytotoxicity_+standarddeviation. ised to produce high levels of cytotoxicity (data not presented) by the inclusion of 25% crude equine IL-2 in the effector-cell-induction cultures. The obvious interpretation of these results was that the cytotoxic effector cell populations contained large numbers of natural killer (NK) cells. Attempted purification of T lymphocytes from other effector cells by nylon wool adherence also failed to reveal genetic restriction of cytotoxic effector cells (data not shown). This was emphasised by the fact that two cycles of passage over nylon wool was not sufficient to purify equine T lymphocytes. The cells purified by this method responded remarkably well to the polyclonal T cell activator PHA in the absence of added accessory cells (Fig. 2). The well known requirement for accessory cells in polyclonal T cell activation by mitogens suggested that nylon wool purified cells contained populations other than T lymphocytes.
332 30
25
20 -T z 15--
S z
IO--
5--
O-O
5 PHA
lO 15 20 CONCENTRATION pg/ml
25
Fig. 2. Mitogenic responses of equine PBMC to phytohaemagglutinin. Results are the mean +_s.d. of three ponies. Closed circles--unfractionated cells; closed triangles = nylon wool non-adherent cells; closed squares = plastic adherent cells.
Genetic restriction of cytotoxic lymphocytes In order to control for possible activators of NK cells in vitro, some experiments were carried out to modify the induction process for cytotoxic effector cells. The effect of reducing the concentration of crude equine IL-2 on the
TABLE 3 Effect of crude equine IL-2 concentration on the induction of cytotoxic lymphocytes for equine influenza infected target cells Crude IL-2 concentration (%) during in vitro induction cultures
Killer: target ratio
25
100:1
25
50:1
15
100:1
5
100:1
5
50:1
Mean % cytotoxicity~ Autologous
Allogeneic
55.4 (6b) (68-48) ~ 35.1 (4) {60-31) 32.0 (3) (52-25) 29.8 (4) (41-25) 15.5 (2) (19-12)
34.1 (3) (40-31) 24.3 (3) (27-19) 27.4 (3) (33-25) 9.5 (2) (11- 8) 7.9 (1) (-)
aCytotoxic effector cellstaken from ponies 5-7 days after infection. bNumber of observations. CRange.
333 subsequent activity of in vitro induced cytotoxic cells is shown in Table 3. Genetically restricted cytotoxic activity was revealed with killer: target ratios as low as 50:1 and showed features consistent with cytotoxic T lymphocyte activity. Therefore, a population of the effector cells taken from ponies after infection with equine influenza was shown to be genetically restricted. DISCUSSION The results of this study have established that cytotoxic cell precursors and/or cytotoxic memory cells may be demonstrated in ponies after infection with equine influenza. The kinetics of cytotoxic cell generation have not been studied in detail, but unpublished experiments indicated that specifically sensitised cells (as revealed by in vitro proliferation assays) were present in the circulation 4 days after primary infection with equine influenza. In order to reveal their cytotoxic potential, PBMC from infected ponies required a secondary antigenic stimulation. In vitro induced cytotoxic effector cells showed activity against influenza-infected target cells in a 3-4 h 51Cr-release assay. A significant finding was that the reactivity of cytotoxic cells was markedly influenced by the conditions of the secondary induction culture. Thus, virus infected target cells were shown to be susceptible to lysis by autologous or allogeneic effector cells (Table 2). However, a reduction in equine IL-2 concentration of the induction culture medium resulted in the generation of cytotoxic effector cells which showed features consistent with cytotoxic T lymphocytes, i.e., target cell killing was genetically restricted (Table 3 ). At the time of writing, monoclonal antibody markers for equine lymphocyte subsets were not widely available and analysis of cell-mediated immune responses were carried out on the basis of functional activity. The genetically non-restricted cytotoxic activity described above was thought to be of NK origin. Two cycles of purification by non-adherence to nylon wool failed to isolate a pure population of T cells because the resulting cells responded well to PHA in the absence of exogenous accessory cells (Fig. 2). The activity of NK cells has been shown to be greatly augmented by interferon and other lymphokines, resulting in an expanded target cell range including the ability to kill a second or third time (Welsh, 1984). In contrast to normal horses, PBMC from horses with the autosomal recessive disorder severe-combined immunodeficiency (SCID) have been shown not to respond to polyclonal activation with phytolectins (Magnuson et al., 1984) which suggested a possible defect in lymphocyte differentiation. However, unfractionated PBMC from SCID horses were very sensitive to IL-2 induction which resulted in potent cytotoxic activity for target cells across MHC barriers (Magnuson et al., 1987). This NK-like cytotoxicity was not inducible
334 in normal PBMC with IL-2. These data reflect the high frequency (greater than 50% ) of cells with NK phenotypes in the unfractionated PBMC of SCID horses compared with that found in normal horses (approximately 5% ). In the present studies, monocyte/macrophage mediated cytotoxicity was considered unlikely because of the short time of the assay ( 3-4 h). Moreover, the fact that there was very little killing of non-infected target cells suggested that nutrient depletion by macrophages was not a feature of this cytotoxic system. Clearly, the genetically non-restricted cytotoxic lymphocytes demonstrated in this study require further characterisation. The genetic restriction phenomenon, typical of cytotoxic T cell activity (Zinkernagel and Doherty, 1974) was demonstrated with equine cellular reagents in vitro when the cytotoxic induction phase was modified. It has been a widely held view that cytotoxic lymphocytes recognised and killed virus-infected target cells that shared MHC class I antigens, although some influenzaspecific cytotoxic T cells have also been described which show restriction for class II molecules of the MHC (Morrison et al., 1986). In the present experiments (Table 3), a component of the cytotoxic activity of PBMC was shown to be genetically restricted to autologous combinations of target and effector cells. On the basis of this feature, the identity of the effector cell in this situation was considered to be a cytotoxic T cell. Therefore, cells with the properties of genetically restricted T lymphocytes and cytotoxic cells which function across a histocompatibility barrier have been identified in the peripheral blood of horses after infection with equine influenza. However, the identity of effector cells in the respiratory tract remains to be established. Although PBMC were prepared from ponies seronegative for H3N8 viruses prior to infection, no steps were taken to monitor their status with regard to equid herpesvirus 1 (EHV-1, equine rhinopneumonitis virus) or equid herpesvirus 2 (EHV-2, slow growing equid herpesvirus } other than routine measurements for antibody to EHV-1. No significant changes in antibody levels were detected through the course of these experiments (data not shown). EHV-2 and EHV-1 are ubiquitous viruses in the equine population (Bryans, 1969). EHV-1 spreads throughout the host via the bloodstream as a cell-associated viraemia (Burrows and Goodridge, 1984). A state of latency may exist which can be reactivated by stress (Edington et al., 1985). Therefore, it is possible that latent EHV-1 and/or EHV-2 virus may have recrudesced during the influenza infection and culture phase of target cell preparation. Some target cells may have expressed these virus antigens and some effector cells may have shown EHV-1/EHV-2 specificity. This probability was considered remote however, because target cells not expressing H3N8 antigens were not lysed, whether by cytotoxic cells which were genetically restricted or by NK-like effectors. Cross protection between serologically distinct subtypes of influenza A virus has been shown to be a property of cytotoxic T lymphocytes in other species
335 (Zweerink et al., 1977; McMichael and Askonas, 1978; Webster and Askonas, 1980). The viral nucleoprotein has been suggested as the major target for this reaction (Townsend et al., 1984; Yewdell et al., 1985) although there is a marked variation in responsiveness to this antigen between individuals (Pala and Askonas, 1986; Pala et al., 1986). In the present experiments, cytotoxic lymphocyte studies were confined to the homologous (H3N8) virus. The demonstration of cytotoxic effector cells of at least two types (functionally) using different culture methods is not without precedent. Recent studies have suggested that genetically restricted cytotoxic T cells represent a differentiation step in the development of non-restricted cytotoxic cells of "promiscuous" target specificity (Brooks et al., 1985; Havele et al., 1986). Cloned cytotoxic T cells have been shown to expand their target cell range from autologous to xenogeneic cells and show features consistent with NK cells (Brooks and Holscher, 1987). The changes in target cell specificity were induced by exposure of cloned T cells to lymphokines in vitro such as IL-2 and interferon. These studies have some parallels with the data presented herein where nonrestricted cytotoxic cells were generated in vitro in the presence of high concentrations of crude equine IL-2. However, a differentiation pathway of nonrestricted cytotoxic cells via genetically restricted cytotoxic T cells is not supported by the observation of lymphoid cell differentiation in SCID horses (Magnuson et al., 1984) which are known to have large numbers of NK-like cells in the PBMC. In combination, these data suggest that NK-like cells may be products of a pathway separate from that of lymphocytes. The significance of the present results can be assessed more fully as reagents become available to differentiate cell surface phenotypes for the equine species. However, one important aspect which needs to be addressed is the target antigen which stimulates strong cytotoxic activity for virus infected cells in vivo. ACKNOWLEDGEMENTS This study was supported by the Horserace Betting Levy Board and the Animal Health Trust. We thank Dr. Neil Edington for helpful discussions on equine cytotoxic lymphocytes, Mr. H. Sawyer and his staff for excellent management of the pony herd and Mrs. Denise Burkett for preparation of the manuscript. Some of the results in this paper were presented at the 5th International Conference on Equine Infectious Diseases, Kentucky, 1987. REFERENCES Ads, G. and Jones, P.D., 1986.The immuneresponseto influenzainfection.Curr. Top. Microbiol. Immunol.,128: 1-54.
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