Immunology 1992 77 284-288
Protection against Chlamydia psittaci in mice conferred by Lyt-2 + T cells D. BUZONI-GATEL, L. GUILLOTEAU, F. BERNARD, S. BERNARD, T. CHARDES & A. ROCCA* INRA Unite Genetique et Immunite, Laboratoire de Pathologie Infectieuse et Immunologie, Nouzilly and *INSERM, Unite 93, H6pital St Louis, Paris, France Acceptedfor publication 20 April 1992
SUMMARY A murine model was used to study the respective roles of L3T4+and Lyt-2+ T cells in protection against Chlamydiapsittaci. Donor mice were intravenously (i.v.) infected with I x 105 plaque-forming units (PFU) per mice of live C. psittaci. One month after inoculation, splenic cells from donors were transferred into syngenic recipients (5 x 107 cells/mouse). As measured by splenic colonization on Day 6 after i.v. challenge (1 x 105 PFU/mouse), transfer with primed (untreated) cells conferred a 3 log protection in this model. In vitro treatment, before transfer, of splenic cells with anti-Lyt-2 monoclonal antibody (mAb) and complement, markedly impaired the protection in comparison with control mice transferred with primed untreated cells, whereas treatment with anti-L3T4 mAb did not reduce the transferred protection. Resistance to a reinfection with C. psittaci was also studied after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. One month after primary infection, mice were treated with anti-L3T4 or anti-Lyt-2 mAb and challenged thereafter (i.v., 1 x 105 PFU). The splenic colonization on Day 6 after challenge demonstrated that treatment with anti-Lyt-2 mAb impaired resistance against a subsequent infection with C. psittaci. Treatment with anti-L3T4 mAb in vivo had no effect on protection, as previously described in vitro. The mechanisms by which Lyt-2+ T cells could participate in the elimination of bacteria were discussed.
INTRODUCTION Chlamydiae, which are obligate intracellular bacteria with an extracellular stage for their infectious bodies, infect mostly macrophages and epithelial cells in a wide variety of mammals and birds. Several types of clinical infection may result, depending on bacterial species and serotypes. Chlamydia trachomatis causes mainly mucous membrane localized infections, in particular of the eye (trachoma); C. psittaci may cause either localized infections, such as endometritis and salpingitis, or systemic infections of great clinical importance in veterinary medicine, such as the chlamydial placentitis which causes enzootic abortion in ewes.' Immune mechanisms involved in systemic or mucouslocalized infections may differ greatly, for example trachoma induces only a limited immunity against itself, whereas C. psittaci enzootic abortion confers strong immunity to reinfection. Thus animal models were chosen to study these mechanisms. Correspondence: Dr D. Buzoni-Gatel, INRA, Unite Genktique et Immunit6, Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France.
In C. psittaci infection, T cells can adoptively transfer specific immunity into naive mice,2 but little information is available about the protective role of the different T-cell subsets. Previous studies of cell-mediated cytotoxicity against chlamydial infections have shown conflicting results. The protective role of cytotoxic T lymphocytes was suggested in an assay for Tcell cytotoxicity in vitro, using a mouse model for C. psittaci infection.3 However, no evidence of cell-mediated cytotoxicity towards C. trachomatis-infected target cells has been found by other authors.4'5 By passive transfer experiments, Williams et al.6 suggested that both Lyt-2+ and L3T4+ T-cell subsets play a role in protection. Furthermore, the importance of T-helper epitopes against the major outer membrane protein of chlamydiae was recently underlined for the development of antitrachoma vaccine.7 However, the protective potential of Thelper cells remains to be elucidated. In the present report, the respective role of T-helper and Tcytotoxic cells was studied in a mouse model of systemic C. psittaci infection using selective in vivo and in vitro T-cell subset depletion by anti-L3T4 (CD4) and anti-Lyt-2 (CD8) monoclonal antibodies (mAb). Results show evidence of the superior role of Lyt-2 + T cells in immunity. Cell-mediated cytotoxicity or cytokine release might constitute some of the protective mech-
Cell-mediated immunity against Chlamydia psittaci anisms by which Lyt-2+ T cells are involved in acquired immunity against mouse infection by C. psittaci.
MATERIALS AND METHODS Mice
Eight- to 10-week-old F. (DBA/2 x CBS) female mice raised in our own breeding facilities were used. Bacteria C. psittaci AB7 strain, used in this study, was originally isolated from an aborted lamb8 and since then has been propagated in the yolk sac of developing chick embryos and stored at -70°. When intravenously (i.v.) inoculated into mice, this strain multiplies in the spleen until Day 6. Then the infection decreases progressively to apparently disappear within 2 weeks. It was thus used to induce a strong immunity in mice. One month after the primary infection, the protection induced was transferrable to naive recipient mice by primed splenic cells. As shown by splenic counts, the protection conferred was shown to be very efficient on Day 6 after an i.v. challenge,2 which was therefore chosen as the moment for killing the mice. Using a plaque assay, the titration of chlamydial infectivity was carried out on McCoy monolayer cells under a solid overlay medium containing 15% noble agar.9 Counts of plaqueforming units (PFU) were performed on Day 14, after staining
with 1:10,000 neutral red. mAb Hybridomas GK 1.5 (anti-L3T4, rat IgG2b) and H35 17.2 (antiLyt-2, rat IgG2b) were kindly provided by Dr G. Milon (Pasteur Institut, Paris, France). In vivo depletion experiments and immunofluorescence studies were performed by means of cell culture supernatants concentrated 10-fold (about 100 ,ug/ml of IgG2b antibodies) with polyethylene glycol 20,000 (Serva, New York, NY). Antibody concentrations were determined by a competitive inhibition-blocking enzyme-linked immunosorbent assay with a rat IgG2b mAb (no. 1330, Becton-Dickinson, Paramus, CA) as a standard. In vitro depletions were conducted either with a commercial anti-Lyt-2 mAb (ascite, IgG2a isotype; AMD, Artarmon NSW, Australia) or with the anti-L3T4 mAb from GK 1.5 hybridoma.
Adoptive cell transfer ofanti-chlamydial resistance after in vitro mAb treatment Donor mice were injected i.v. with 1 x 105 PFU per mouse from live strain AB7. One month later, spleens were removed and forced through a stainless steel gauze (12 x 0 3 mm wires). Nonadherent cells were recovered after a 2-hr incubation (5% CO2, 37°) into a tissue culture flask (Nunc, Roskilde, Denmark) and then centrifuged (37°, 2000 g, 15 min) using a ficoll gradient (density= 1 09). In previous experiments, the percentage of Mac- I+ cells that remained among the non-adherent cells was determined by fluorescence-activated cell sorter (FACS) analysis on a Coulter EPICS V flow cytometer (Coulter Electronics, Hialeah, FL) and was shown to be less that 2%. The red cells were lysed at 40 by ionic shock (0155 M, NH4CI, 0-01 M, KHCO3, 0 01 mm EDTA). The cells were then incubated (40, 1 hr, 5 x 107 cells/ml) with non-diluted anti-L3T4 mAb (culture supernatant of GK 1.5 hybridoma) or with 1:20 diluted antiLyt-2 mAb (ascite). The antibody-treated cells were washed
three times and incubated (370, 1 hr) in a 1: 12 dilution of rabbit complement (CL 3051 Low Tox-M Cedarlane, Ontario, Canada). The cells were then washed three times before injection. The depletion efficiency was checked by a viability trypan blue exclusion test. Untreated primed cells were used as a positive control. To adoptively transfer immunity, recipient mice (five per group) were injected i.v. with 5 x 107 cells, which represent cells from about one mouse. The control group received spleen cells from unprimed mice. One day after the transfer, mice were challenged i.v. with strain AB7 (1 x 105 PFU/mouse) and killed by cervical dislocation on Day 6 after challenge. The degree of protection conferred was assessed by the enumeration of chlamydiae in the spleen using the plaque assay method. In vivo mAb treatment of primary infected mice Mice were primary infected i.v. with live strain AB7 (I x 105 PFU/mouse). One month later, individual mice were injected i.v. with 20 yug of anti-L3T4 (GK 1.5) or anti-Lyt-2 (H35 17.2) mAb, every day for four consecutive days. On the fifth day, the last injection was performed intraperitoneally. The depletion efficiency was controlled by FACS analysis as described below. Control mice received cell culture medium without antibody, according to the same schedule. One day after the last injection, mice were reinfected i.v. with strain AB7 (1 x 105 PFU/mouse) and chlamydial splenic counts were performed on Day 6 after
challenge. The normal infection level was given by the number of chlamydiae in the spleens from naive mice. Numbers of PFU per spleen were expressed as logl0 value. Means and standard errors (SE) were calculated from the logarithmic values. Flow cytometer analysis of splenic T-cell subsets after in vivo mAb treatment Spleen cells were incubated (4°, 1 hr) with anti-L3T4 mAb (100 ig/ml) or with anti-Lyt-2 mAb (100 pg/ml). After three washes, a 1: 100 dilution of a fluorescein-conjugated rabbit anti-rat immunoglobulin serum (Nordic Immunology, Tilburg, The Netherlands) was added to the cells. After a 45-min incubation (40) and three washes, cell subsets were analysed on FACS. For each sample, data were collected from 20,000 cells. Data analysis
Comparisons between control and treated groups or between treatments were done by the Student's t-test. RESULTS Effect of in vitro selective T-cell subset depletion on protection following adoptive transfer Transfer of 5 x 107 non-adherent splenic cells from primary infected mice to recipient mice conferred a strong immunity to a subsequent challenge (Fig. 1). On Day 6 after challenge, primed cells lowered splenic colonization by about 3 log PFU (P < 0-01) compared to the control group, which had received unprimed cells. Treatment of primed cells with (i) rabbit complement, (ii) anti-L3T4 mAb with or without rabbit complement, or (iii) with anti-Lyt-2 mAb alone did not modify the protection conferred by untreated primed cells (P > 0 05). In contrast, treatment with
D. Buzonji-Gatel et al.
286 Spleen cells transferred Non-primed Primed Treatment cells cells
Log PFU per spleen 4 3 2
Anti-L3T4 Anti-L3T4 + C Anti-Lyt-2 Anti-Lyt-2 + C
Figure 1. Effect of in vitro T-cell subset depletion on adoptive transfer of resistance to C. psittaci infection. Naive mice were injected i.v. with 5 x 107 spleen cells from uninfected or primary infected mice. Spleen cells from primary infected mice were pretreated in vitro with the indicated mAb and complement (C). Mice were challenged the day after the transfer and bacterial spleen counts were performed 6 days later. Results are expressed by the mean + SE of log PFU from at least five mice.
anti-Lyt-2 mAb and complement drastically reduced the transferred protection: the splenic colonization was almost as high as in the control group transferred with cells from naive mice (4-47 log PFU versus 5 25 log PFU). However, the difference in the level of splenic colonization between these groups remained significant (P< 0-01). Effect of in vivo selective T-cell subset depletion on the resistance to reinfection In order to assess the contribution of Lyt-2+ T cells to protection, primary infected mice were selectively depleted in vivo of Lyt-2+ or L3T4+ T cells. As demonstrated by flow cytometry, in vivo treatment with anti-L3T4 or anti-Lyt-2 mAb resulted in a 95% decrease in splenic L3T4+ or Lyt-2+ T cells (data not shown). Six days post-challenge, spleen counts indicated strong immunity induced by the primary infection that was not modified by treatment with anti-L3T4 mAb, since no colonization had occurred in either group (Fig. 2). In contrast, treatment with anti-Lyt-2 strongly impaired the immunity, since 3-76 log PFU chlamydiae per spleen were isolated. However, this splenic colonization remained lower (P < 0-01) than the one observed in the unprimed group (3-76 log PFU versus 5-15 log PFU in the unprimed control group).
Primary infected mice
In vivo treatment before challenge
Log PFU per spleen 4 3 2
Figure 2. Effect of in vivo T-cell subset depletion on the resistance to reinfection with C. psittaci. Primary infected mice were injected in vivo with the indicated mAb. Mice were challenged the day following the last injection of mAb, and bacterial spleen counts were performed 6 days later. Results are the mean + SE of log PFU from at least five mice.
Our earlier study2 suggested a role for T-cell dependent immunity in the acquired resistance to C. psittaci. The aim of the present study was to assess the protective potential of different T-cell subsets in C. psittaci infection. In the present study, Lyt-2+ T cells were shown to play the major role in the protection following transfer of primed spleen cells. Moreover, the in vivo depletion of Lyt-2+ T cells dramatically decreased the protection conferred by primary infection. In vitro, complement was necessary to observe the anti-Lyt-2 mAb effect, since the treatment of primed cells with anti-Lyt-2 mAb alone was not able to abrogate the protection. However, the protection conferred by primary infection was greater than the protection conferred by adoptive transfer of 5 x 107 primed cells. The number of primed cells transferred seems thus important, which is in concordance with our previous results,2 where a dose effect was observed. As shown by in vitro and in vivo Lyt-2+ T-cell depletion experiments, we failed to abrogate the whole protection. The remaining protection was, however, greater after in vivo depletion owing to the complexity of the in vivo model. Although Lyt-2+ T cells were the main cells responsible for immunity, the lack of a complete reduction in protection after Lyt-2 + T-cell depletion might indicate that other cellular phenotypes were involved in protective mechanisms. These hypothetical cells probably belong to the T phenotype, as treatment with polyclonal anti-T serum completely abrogated the protection.2 However, in our experimental conditions, L3T4+ T cells did not seem to be involved in protection, but further experiments using other anti-L3T4 mAb should be undertaken. In a mouse model for C. trachomatis infection (mouse pneumonitis agent), Williams et al.6 demonstrated the role of Lyt-2+ T lymphocytes, but they also demonstrated a role for Lyt-l + T cells. It must be underlined that L3T4 is a more absolute marker than Lyt-1 for the T-helper subset. However, this may reflect differences in the infecting species (we used C. psittaci). Similar protective mechanisms have been reported for intracellular bacterial and parasitic infections. Lyt-2+ and L3T4+ T subsets were both important for protection of mice against Listeria monocytogenes infections 13 but the Lyt-2+ T subset appeared to play the major role in host resistance against this pathogen. 14 16 Lyt-2 + T cells have also been demonstrated to be involved in protection against Mycobacterium tuberculosis'7 and Toxoplasma gondii.'8 The question that arises is how are Lyt-2+ T cells able to protect infected mice? In certain viral diseases,'9-20 the destruction of infected cells is mediated by cytotoxic lymphocytes that become activated during the infection. As chlamydial infection of cells is similar to viral infection in many ways, and as Lyt-2 + T lymphocytes are often involved in cytotoxic mechanisms, it is rational to think that direct cytotoxicity against infected cells is involved in the protective process. Lammert3 has indeed shown evidence that cells capable of lysing infected targets are present in the spleens of C. psittaci-infected mice. But while it is possible to detect cytotoxic activity of C. psittaci-activated cells,3 several authors4'5 have failed to detect specific cytotoxic activity of C. trachomatis-activated cells. The discordance may be explained by the bacterial species used, but also by the need of effector cell
Cell-mediated immunity against Chlamydia psittaci restimulation with viable bacteria. The involvement of cytotoxic mechanisms in protection against Chlamydia is quite possible. Indeed, chlamydiae have a complex life cycle, involving the orderly alternation of an infectious metabolically inactive stage (elementary body) and a non-infectious metabolically active stage. Thus, premature lysis of infected cells during the multiplication phase, with the release of non-infectious chlamydiae, would prevent development of infection and result in a heightened degree of antigen release that could serve to further stimulate the immune response. Once out of the cells, chlamydiae are exposed again to humoral immunity that has been shown to be efficient.2' Indeed, passive transfer of polyclonal antibodies confer a measurable immunity in infected mice and some mAb have been found having the protection capacity against abortion in a mouse model.2' Moreover, mAb against the major outer membrane protein of chlamydiae have neutralized chlamydiae in vitro.22'23 As commonly admitted, the immune recognition of target cells presenting the antigen by Lyt-2 + T cells is more often MHC (major histocompatibility complex, H-2) class I restricted. We have accumulated indirect evidence of MHC class I antigen involvement in immunity against C. psittaci by infecting inbred congenic and congenic recombinant mice. Firstly, we demonstrated that natural resistance to C. psittaci was linked to the MHC haplotype of mice. Secondly, we showed that the genes controlling resistance to C. psittaci were located in the right hand region of the H-2 complex (H-2D or H-2L) coding for MHC class I antigens (unpublished data). However, direct cytotoxicity is probably not the only mechanism by which T cells and, more specifically, Lyt-2+ T cells are able to confer anti-chlamydial immunity. Lyt-2+ T cells are also able to produce macrophage-activating lymphokines, including interferon-gamma (IFN-y), and their immune function may be related to this potential. IFN-y has been shown to inhibit the growth of C. trachomatis both in vitro and in vivo.24-26 IFN-y both limits infectivity of elementary bodies and functions as a cytotoxic cytokine against chlamydiae-infected fibroblasts.27-30 In our experiments, secretion and protective role of IFN-y remain to be elucidated. Mechanisms of protection involved against C. psittaci are certainly complex. We now know that T cells, and specifically Lyt-2+ T cells, are important factors. But many other components of the immune system may be involved and further studies are required for their elucidation. ACKNOWLEDGMENTS We thank Annie Rodolakis, Michel Plommet, Daniel Bout, Michel Npin and Frederic Lantier for critical review of the manuscript and Janet Hall for English corrections.
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