Mar 11, 1993 - Bovine CD4+ T Cell Clones Activated In Vitro. WENDY ... KATHLEEN S. LOGAN' .... Australia (33), and was provided by Stephen Hines (Wash-.
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IMMUNITY, Aug. 1993,
p.
Vol. 61, No. 8
3273-3281
0019-9567/93/083273-09$02.00/0 Copyright X 1993, American Society for Microbiology
Heterogeneity in Cytokine Profiles of Babesia bovis-Specific Bovine CD4+ T Cell Clones Activated In Vitro WENDY C.
BROWN,`*
VIVIENNE M. WOODS,1 DIRK A. E. DOBBELAERE,2 AND KATHLEEN S. LOGAN'
Department of Veterinary Pathobiology, Texas A & M University, College Station, Texas 77843,1 and Institute for Animal Pathology, University of Bern, Bern, Switzerland2 Received 11 March 1993/Accepted 30 April 1993
The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen were characterized for reactivity against various forms of antigen and against different geographical isolates of B. bovis and B. bigemina and analyzed for cytokine production following mitogenic stimulation with concanavalin A. Biological assays to measure interleukin-2 (IL-2), IL-4, gamma interferon (IFN'y), and tumor necrosis factor alpha or tumor necrosis factor beta and Northern (RNA) blot analysis to verify the expression of IL-2, IL4, IFN-y, and tumor necrosis factor alpha revealed differential production of cytokines by the Th cell clones. The majority of clones expressed the ThO pattern of cytokines: IFN-y, IL-4, and IL-2. One clone expressed the Thl profile (IFN-y and IL-2 but not IL-4), whereas none of the clones expressed the Th2 profile. All of the Th cell clones examined expressed the low-molecular-weight isoform of the leukocyte common antigen associated with a memory cell phenotype (CD45RO), and all expressed the lymph node homing receptor (L-selectin). These results extend our previous finding of differential cytokine expression by B. bovis-specific Th cell clones and confirm the identity of the specific cytokines produced, showing that a ThO response is preferentially induced in a panel of 20 CD4+ T cell clones obtained from immune cattle. Babesia bovis causes a virulent form of babesiosis in cattle that is characterized by fever, anemia, anorexia, cachexia, low-level parasitemia, and a generalized circulatory disturbance and that often results in high fatality rates among nonimmune cattle. Characteristic of this disease is the sequestration of parasitized erythrocytes in the capillary beds of the brain and lungs, leading to cerebral babesiosis and respiratory distress syndrome (56). Similarities in the immunopathological characteristics of diseases caused by this parasite and related malarial parasites have led to the hypothesis that the diseases caused by these hemoparasites share common mechanisms. Cytokines, including gamma interferon (IFN-y) and tumor necrosis factor (TNF), released by parasite antigen-activated T cells and macrophages are implicated in anemia, cytoadherence of parasitized erythocytes to brain microcapillary endothelia, and accumulation of infected erythrocytes and neutrophils in the pulmonary vasculature. Protective immunity in experimental murine malarial and babesial infections is mediated by T cells and macrophages, and the same cytokines involved in immunopathology play a role in immunity. Both subsets of T helper (Th) cells appear to be involved in protective immunity against the intraerythrocytic stage of malarial parasites, with Thl cells appearing early and Th2 cells appearing late in the infection (25). Inflammatory cytokines IFN--y and lymphotoxin, or TNF-P, produced by both CD4+ Thl cells and CD8+ T cells, are important for the resolution of hemoparasitic infections (9, 23, 45, 46, 48, 52), whereas interleukin-4 (IL-4) facilitates the growth of Plasmodium falciparum in vitro by preventing *
macrophage-mediated killing of this parasite (22). Th2 cytokines IL-4, IL-5, and IL-6 probably function to maintain an antiparasitic antibody titer once the initial infection has been controlled (25). In other protozoal infections, the role of Th2 cells is not as benign. For example, Thl cells, producing IFN--y, confer protection, whereas Th2 cells, producing IL-4, exacerbate the disease evoked by infection with Leishmania major (44). Since the initial description of Thl and Th2 subsets (34) additional studies have revealed that other Th subsets, perhaps reflecting different differentiation states, occur. Short-term murine Th cell clones specific for soluble protein antigens were shown to produce an unrestricted pattern of cytokines, and these cells were designated to belong to a ThO subset (15). An analysis of human T cell clones from healthy donors stimulated with mitogens or alloantigens also indicated, in many cases, an unrestricted, or ThO, pattern of lymphokine production (12, 31, 36, 42, 54). In contrast, when T cell clones were obtained from individuals suffering from chronic afflictions, including helminth infection, leprosy, and allergy-related illness, both Thl and Th2 types of clones were identified (10, 13, 24, 30, 32, 38, 41, 57). However, these studies also reported many examples of human T cell clones that exhibited a ThO profile of cytokines in response to the same antigens that induced Thl and Th2 cell responses (30, 32, 38). One popular hypothesis to explain these discordant results is that naive T cells, which produce only IL-2, give rise to ThO cells, which produce all cytokines and which in turn give rise to either Thl or Th2 cells in states involving chronic infection and antigenic stimulation (35, 49). Studies in our laboratory have focused on the characterization of B. bovis-specific Th cell responses and the antigens that induce them. The role of cytokines in immunity or
Corresponding author. 3273
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BROWN ET AL.
immunopathology in B. bovis infections is not known. However, because of the documented relevance of cytokines in other experimentally induced protozoal infections, we have begun to analyze cytokines produced by T cell clones responsive to babesial antigens, with the ultimate goal of correlating cytokine production with a protective or a nonprotective immune response against defined antigens. A Northern (RNA) blot analysis identified the expression of a Thl profile of cytokine mRNA by two CD4+ clones reactive with the 77-kDa merozoite protein of B. bovis, Bb-1 (7). Similar studies of Th cell clones specific for the 42-kDa major merozoite surface antigen of B. bovis, MSA-1, revealed the expression of an unrestricted cytokine profile by two clones (5). In the studies presented here, the majority of over 20 CD4+ Th cell clones specific for unfractionated merozoite antigens of B. bovis expressed both IL-4 and IFN--y, with variable IL-2 expression, suggesting that merozoite antigens preferentially induce ThO cell-like responses when T cells from immune cattle are stimulated in vitro.
MATERIALS AND METHODS Babesial parasite strains and cultivation. The Mexican strain of B. bovis was originally isolated in 1978 from cattle infected with natural, tick-derived isolates from an area of Mexico in which B. bovis is endemic (4). The Texas strain of B. bovis originated in 1978 from an infected animal in southern Texas (37) and was provided by Will Goff (Agricultural Research Service, U.S. Department of Agriculture, Pullman, Wash.). The Australian L strain of B. bovis originated in 1965 from an infected animal in New South Wales, Australia (33), and was provided by Stephen Hines (Washington State University, Pullman). The Mexican strain of B. bigemina was obtained in 1982 from an infected animal in northeastern Mexico (4). In vitro microaerophilous cultures of all babesial strains were maintained in bovine erythrocytes (4). Experimental cattle. Animal C15 was infected with the Mexican strain of B. bovis as described in detail elsewhere (4). In brief, the 2-year-old cross-bred cow was inoculated intravenously four times over the course of 4 years with B. bovis merozoites obtained from autologous, infected erythrocyte cultures. Although animal C15 was not challenged with tick- or blood-derived parasites, challenge inoculation of 109 cultured parasites did not evoke a reduction in packed-erythrocyte volume or an elevation in temperature. Yearling calves G3 and G6 were infected by intravenous inoculation of 1 ml of a fresh blood stabilate, designated S11T3B0, of the Texas strain following passage through a splenectomized calf. Both calves experienced clinical signs of babesiosis, with, respectively, 47 and 35% reductions in packed-erythrocyte volume and elevated temperatures, reaching 106 to 107°F (ca. 41.1 to 41.7°C), on day 6 postinfection, at which time the animals were treated with a nonsterilizing dose (2.5 mg/kg of body weight) of diminazene aceturate (Berenil; Sigma Chemical Co., St. Louis, Mo.). Both calves were found to be completely protected 5 months later against a challenge infection with 1 ml of cryopreserved blood stabilate S11T3B0, whereas a similarly challenged, control calf (G5) showed severe clinical signs of babesiosis and was treated with diminazene aceturate on day 9 postinfection. Serologic responses were monitored by a Western blot (immunoblot) assay with a soluble babesial merozoite antigen and pre- and postinfection sera. Infected and treated calves G3 and G6 showed strong serologic responses, as described earlier for animal C15 (4), with reactivity against
INFECT. IMMUN.
the parasite antigen still present on Western blots with sera diluted 1:10,000, whereas none of the preinfection or control sera reacted on immunoblots with the B. bovis antigen. Parasite antigens. Crude parasite antigens were prepared by homogenization of culture-derived merozoites with a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) and ultracentrifugation to yield a soluble, cytosolic, highspeed supernatant (HSS) fraction and a fraction enriched in crude membranes (CM) as described previously (4). Uninfected erythrocytes were similarly fractionated for use as control antigens. Generation of B. bovis-specific T cell clones. T cell lines specific for B. bovis merozoites were established from peripheral blood mononuclear cells (PBMC) from immune animals C15, G3, and G6 essentially as described previously (3). In brief, a C15 T cell line was established approximately 5 months following the fourth parasite challenge inoculation by stimulation of lymphocytes with the CM fraction of B. bovis (Mexican isolate) merozoites for 5 weeks and then stimulation with the soluble HSS fraction of merozoites for 1 to 3 weeks. G3 and G6 T cell lines were established approximately 3 to 4 months following the primary infection and treatment by stimulation of lymphocytes with the CM fraction prepared from the Texas isolate of B. bovis. One cell line was also established from animal G3 4 months following the challenge inoculation. All cell lines were shown to proliferate in a dose-dependent manner against New World isolates of B. bovis merozoite antigens and were selected for limiting dilution cloning. Cloning was performed with 96well round-bottomed plates (Costar, Cambridge, Mass.) essentially as described previously (6) with the following modifications. A statistical average of 1 or 0.3 cell per well was stimulated with 25 ,ug of the HSS fraction prepared from the Mexican isolate of B. bovis (for the C15 T cell line) or 25 ,ug of the CM fraction prepared from the Texas isolate (for the G3 and G6 T cell lines) per ml of complete RPMI 1640 medium (4) containing 10% bovine T cell growth factor (TCGF; 2) and 5 x 104 irradiated (3,000 rads) autologous PBMC. Proliferating cells were tested for antigen-dependent proliferation. The cloning efficiencies of the clones selected for these experiments ranged from 3 to 33%. Cell surface phenotypic analysis. Antigen-specific clones were stained with a panel of monoclonal antibodies (MAb) by indirect immunofluorescence as described previously (3) and analyzed with a Coulter EPICS 741 flow cytometer. MAb specific for bovine leukocyte surface markers and obtained from the International Laboratory for Research on Animal Diseases, Nairobi, Kenya, included IL-A51, specific for CD8; IL-A12, specific for CD4; IL-A26, specific for CD2; IL-A24, specific for bovine macrophages (14); and IL-A150, considered specific for a polymorphic determinant on bovine CD45RO (27, 28). MAb obtained from Chris Howard at the Agriculture and Food Research Council Institute for Animal Health in Compton, United Kingdom, included CC76, specific for bovine CD45R (19), and CC32, specific for bovine L-selectin (18). Lymphocyte proliferation assays. Proliferation assays were carried out with duplicate wells of half-area 96-well plates (Costar) at 37°C in a humidified atmosphere of 5% CO2 in air for 3 days as described previously (6). Each well (100-,ul total volume) contained complete medium, responder cells added at a final concentration of 3 x 105 cells per ml (obtained 6 or 7 days following the last stimulation of the T cell clones with antigen), and 2 x 106 irradiated autologous or allogeneic PBMC per ml as a source of antigen-presenting cells (APC).
VOL. 61, 1993
Preparation of bovine macrophages. PBMC were cultured in 100-mm petri dishes at a density of 5 x 106 cells per ml for 1 h. The nonadherent cells were removed by gentle washing. The medium was replaced, and the adherent cells were cultured for 6 days at 37°C in 5% CO2 in air. The medium and any nonadherent cells were removed, and the adherent cells were removed by treatment with prewarmed (to 37°C) Ca2+and Mg2+-free Hanks balanced salt solution (GIBCO Laboratories, Grand Island, N.Y.) containing 0.1% disodium EDTA. The purity of the macrophage population was confirmed by flow cytometry after staining of the cells with MAb IL-A24 (14) and by nonspecific esterase staining with a kit from Sigma Chemical Co. Stimulation of cells for cytokine production. T cells obtained 6 or 7 days after the last stimulation with antigen and APC were washed in complete medium and cultured for 17 to 18 h at a concentration of 1.3 x 106 cells per ml of complete medium containing 5 Fg of concanavalin A (ConA; Sigma) per ml in the absence of APC. In one experiment, mitogen-induced T cell clones were cultured with ConA for either 8 or 18 h, and supernatants were compared. In a separate experiment, B. bovis-specific T cell clones C15.1H6 and C97.3C3 (6) were stimulated for 18 h with either ConA or 25 p,g of antigen per ml and autologous APC. As a positive control for IFN--y, IL-2, and IL-4, PBMC were stimulated at a density of 4 x 106 cells per ml for 18 h with ConA. As a positive control for TNF-a, macrophages were harvested after 6 days and cultured for 16 h at a density of 7.5 x 105 cells per ml with 2 p,g of lipopolysaccharide (LPS) (Eschenchia coli 055:B5; Sigma) per ml. Supernatants were harvested by centrifugation and stored at -80°C. Biological assays for cytokines. IFN activity was measured by use of a microtiter cytopathic effect assay with vesicular stomatitis virus and Madin-Darby bovine kidney (MDBK) cells as described elsewhere (6, 7). MDBK cell survival was evaluated on the basis of the 4-h uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye in the cells. IFN titers were compared with that of a human recombinant IFN-a2 reference reagent (National Institute of Allergy and Infectious Diseases, Bethesda, Md.; catalog number, GxaOl-901-535), which was reported to be 9,000, or 3.95 log1o, U/ml. In our assay, this standard had a titer of 3.35 + 0.18 log1o U/ml. For the determination of TNF-a or TNF-,B activity in the supernatants of T cell clones, a TNF-sensitive WEHI-164 subline was incubated with culture supernatants in a 48-h assay and cytopathicity was determined by the MTT dye reduction assay (40). TNF titers were compared with that of a human recombinant TNF-a standard (Upstate Biotechnology Inc., Lake Placid, N.Y.), which was reported to be 106 log1o U/ml; in our assay, this standard had a titer of 5.92 ± 0.15 log1o U/ml. Cloned CD8+, IL-2-dependent bovine T cell lines, designated 99.G1.G3 and 99.2H5, were used to measure IL-2 or IL-4 activity (7). Both CD8+ responder cell lines expressed IL-2 and IL-4 mRNAs and proliferated upon ConA activation (1), so these indicator cell lines do not discriminate IL-4 activity from IL-2 activity. Lectin-depleted culture supernatants of B. bovis-specific T cell clones stimulated with ConA were diluted 1:2 to 1:256 in complete medium and added to duplicate wells of 99.G1.G3 or 99.2H5 cells that had been distributed at a density of 3 x 104 cells per well in half-area 96-well microtiter plates. In all assays, recombinant human IL-2 (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) was serially diluted from 100 to 3.0 times the mean counts per minute of cells cultured in medium alone. The response of all clones to uninfected erythrocytes was negative, with stimulation indices of < 1.3. All T cell clones described in this study to have originated from animal C15 responded to both soluble and membrane forms of the merozoite antigen, whereas only two clones obtained from immune animals G3 and G6 showed reactivity to the soluble form of the antigen. This difference may reflect the choice of antigen used to select the cells in cultures prior to and during cloning; the C15-derived cells were cultured with the CM fraction and then with the HSS
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TABLE 1. Summary of the proliferative responses of B. bovis-specific CD4+ T cell clones Response toa: Th cell clone
bovis antigen
B.
CM
Parasite isolates
B. bovis from:
HSS Mexico
Texas
Australia
+ + + + +
C15.1F11 C15.JD10 C15.lE3 C15.2G10 C15.2G3 C15.3A5b
+ + + + +
+ + + + +
+ + + + +
+ + + + +
+
+
+
C15.2D7b
+ + + + +
+
+ + + + +
+ +
G6.1E5 G6.2C3 G6.2H1 G6.2C2 G6.2D5 G3.2F9 G3.1G2 G3.3F4 G3.2F7
+ + + + +
ND + + -
+ + + + +
+
ND + + + + + + +
-
+ ND + + + + -
_ +
B. bigemina
-
_ + + _ ND -
-
-
_ _
a Proliferative responses of individual Th cell clones to optimal concentrations of the CM or the soluble HSS antigen prepared from the Mexican strain of B. bovis merozoites or to the CM fraction of the indicated B. bovis strains or the Mexican strain of B. bigemina. ND, not determined. b Clones C15.3A5 and C15.2D7 were described in a previous publication (6).
fraction, whereas the G3- and G6-derived cells were cultured only with CM. A differential pattern of responses against different babesial parasite isolates was also observed. Two clones responded to an antigen shared by all B. bovis isolates as well as B. bigemina, nine clones responded to an antigen from all B. bovis isolates, and the remaining clones recognized only an antigen associated with the New World B. bovis isolates. All clones expressed the surface phenotype characteristic of Th cells: CD2+ CD4+ CD8- (Fig. 1 and data not shown). None of the Th cell clones could proliferate in response to a B. bovis antigen when allogeneic APC were used in proliferation assays, whereas the response to TCGF was not affected, showing that the Th cell clones are major histocompatibility complex restricted (data not shown). Surface expression of CD45 isoforms and L-selectin. The Th cell clones from animal C15 were compared with PBMC from this animal for the expression of high- and lowmolecular-weight isoforms of the common leukocyte surface antigen (CD45) and the peripheral lymph node homing receptor (L-selectin) (Fig. 1). All CD4+ Th cell clones expressed high levels of the low-molecular-weight isoform, CD45RO, expressed on memory T cells (27, 28), but expressed very low levels of the high-molecular-weight isoform, CD45R, expressed on naive T cells (19). L-Selectin was also expressed on all T cell clones examined. In contrast, approximately 77% of autologous PBMC expressed CD45R, and 52% of them expressed CD45RO, with L-selectin being present on 45% of the circulating mononuclear cells. Similar results were obtained for B. bovis-specific ThO cell clones obtained from animal C97 and for Bb-1-specific Thl cell clone C97.1C8 (data not shown). Leukocytes from animal G3 did not express the polymorphic CD45RO determinant recognized by MAb IL-A150. However, Thl cell clone G3.2F7 expressed low levels of CD45R, consistent
with a memory cell phenotype. L-Selectin expression was also high on this clone (data not shown). The other ThO cell clones described in this study were not characterized for CD45 isoforms or L-selectin. Analysis of cytokines produced by Th cell clones. Preliminary studies with CD4+ T cell clones derived by stimulation of PBMC with ConA for 3 days and limiting dilution cloning of cells with irradiated PBMC and TCGF revealed that higher levels of IFN were secreted into the culture supernatant by T cells stimulated for 17 h (16 U/ml) than by T cells stimulated for 8 h (2 U/ml) with ConA. Similar results were obtained when IL-2 or IL-4 was tested; no activity wvas detected in 8-h supernatants, whereas 17- and 22-h supernatants contained 6.4 U of activity per ml. When supernatant levels of IL-2 or IL-4, IFN, and TNF were compared for B. bovis-specific T cell clones stimulated for 18 h with either ConA or antigen plus APC, it was found that higher levels of all three cytokines were obtained following ConA activation (Table 2). Therefore, for measurement of cytokines in supernatants of the panel of T cell clones described in this study, cells were collected 6 days following the last stimulation with antigen and APC, washed, and stimulated with ConA for 17 to 18 h, at which time culture supernatants were harvested and assayed for IL-2 or IL-4, IFN, and TNF (Table 3). The majority of the clones secreted detectable levels of IL-2 or IL-4, ranging from 3 to 50 U/ml, whereas the supernatants of five clones did not contain detectable levels of IL-2 or IL-4. IFN titers were generally high (2125 U/ml), with the exception of low or undetectable IFN titers in four clones. Again, most Th cell clones produced low levels of TNF, but five clones did not secrete any detectable TNF. As anticipated, activated macrophages did not secrete detectable IL-2 or IL-4 but did produce IFN (probably IFN-a) and 12 U of TNF per ml. The biological assays used here to measure bovine cytokines do not distinguish between IL-2 and IL-4, TNF-a and TNF-P, or IFN--y and IFN-a (7). For this reason, a Northern blot analysis of total RNA extracted from ConA-activated T cell clones, ConA-stimulated PBMC and LPS-stimulated macrophages (positive controls), or unstimulated MDBK, bovine turbinate, or WEHI-164 cells (negative controls) was performed with cDNA probes specific for bovine IL-2, IL-4, IFN--y, and TNF-ot or for actin as a control. A comparison of cytokine mRNA expression in T cell clones stimulated with ConA for either 8 or 18 h showed stronger expression of both the IL-2 and the IL-4 messages in cells stimulated for the shorter time period (data not shown). For this reason, RNA was routinely obtained from Th cell clones stimulated for 8 h with ConA. An analysis of cytokine mRNA in Th cell clones obtained from hyperimmune animal C15 revealed a differential pattern of cytokine expression (Fig. 2). Clones C15.1F11, C15.1D10, and C15.2G3 expressed IL-2, IL-4, IFN-,y, and TNF-a mRNAs; clone C15.1E3 expressed only IFN--y and TNF-at mRNAs; clone C15.2G10 expressed IL-4 and IFN--y mRNAs and low levels of IL-2 and TNF-a mRNAs; and clones C15.3A5 and C15.2D7 expressed IL-2 mRNA and low levels of IL-4 and IFN--y mRNAs. In some cases, the levels of cytokine mRNAs expressed correlated with the levels of cytokines detected in culture supernatants (e.g., clones C15.3A5 and C15.2D7 expressed low levels of cytokine mRNA and no detectable cytokines in culture supernatants, whereas clones C15.1F11 and C15.1D10 expressed high levels of cytokine mRNA and high levels of cytokines in supernatants). All Th cell clones obtained from animals G3 and G6 prior to a challenge infection expressed IFN-,y and IL-4 mRNAs,
B. BOVWS-SPECIFIC ThO AND Thl CELLS IN CATTLE
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Determinant
C1I5.2Fl0
;
__
-
-
L
/-
C15.2D7
_
C15.1H6
L
L-Selectin
Um.v
t
>2
C15.3A5
CD45RO
CD45R
CD4
Th Clone
S
t
_
_
_
0
-
0 C.)
Cl 5.1 D1 0|
-
L
E z
Cl 5.1 E3
|
_
_
______>
