Differential requirements for proliferation of CD4+ and γδ+ T cells to spirochetal antigens

Cellular Immunology 224 (2003) 38–46 www.elsevier.com/locate/ycimm

Differential requirements for proliferation of CD4þ and cdþ T cells to spirochetal antigens Raquel Hontecillasa,*,1 and Josep Bassaganya-Rierab,1 b

a Immunobiology Program, Veterinary Medical Research Institute, Iowa State University, Ames, IA 50010, USA Nutritional Immunology & Molecular Nutrition Laboratory, Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50010, USA

Received 28 May 2003; accepted 28 July 2003

Abstract abþ and cdþ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4þ and cdþ T cells in mixed lymphocyte cultures. CD4þ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas cdþ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-a (IL-2Ra). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking cdþ than CD4þ T cell proliferation. Immunization induced interferon-c (IFN-c) production by CD4þ T cells, whereas only a small fraction of the antigen-stimulated cdþ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4þ T cell functions are more tightly regulated when compared to cdþ T cells. We conclude that these differences are due, in part, to the enhanced cdþ T cell responsiveness to IL-2. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Cellular immunology; IL-2; cdþ T cells; CD4þ T cells and spirochetes

1. Introduction CD4þ and cdþ T cells constitute an important fraction of the circulating lymphocyte pool in pigs [4,11,32,36,51,53]. This makes the pig an ideal model for examining functional properties and interactions between these two cell subsets in the context of an immune response. cdþ T cells differ from abþ T cells in several aspects. Through the T cell receptor (TCR) abþ T cells recognize peptides presented in association with MHCclass I or II molecules on the surface of target cells (i.e., CD8þ cytotoxic T cells) or antigen presenting cells (APC) (i.e., CD4þ T cells). Conversely, cdþ T cells react to antigens with chemical composition other than pro* Corresponding author. Fax: +1-540-231-3916. E-mail address: [email protected] (R. Hontecillas). 1 Present address: Nutritional Immunology and Molecular Nutrition Laboratory, Department of Human Nutrition Foods and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA.

0008-8749/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0008-8749(03)00172-2

teins (e.g., phosphoantigens, alkilamines) [10,40,43], and antigen recognition does not require processing and presentation by professional APC [39]. In addition, the cd TCR is MHC-I and II-unrestricted; instead, nonclassical MHC molecules, such as T10 and T22 [39], or CD1 [41] are ligands for the cd TCR. Functional studies of porcine CD4þ T cells have revealed that antigen-experienced CD4þ T cells from peripheral blood up-regulate CD8aa following a subsequent antigenic exposure [55,56]. CD4þ CD8aa
T cells are the precursors of CD4þ CD8aaþ T cells, which represent memory/effector T helper cells. With regard to cdþ T cells, two major subsets have been identified based on the expression of the molecule SWC6 [7]. Porcine SWC6þ cells, which constitute the majority of the circulating cdþ T cells, are capable of inducing non-specific cytotoxicity in vitro [13,30]. The SWC6
cdþ T cells are phenotypically characterized as CD2þ CD6þ [51], and a fraction of this subpopulation additionally expresses CD8aa [52]. SWC6
cdþ T cells are found in spleen, secondary lymphoid organs and they are also detectable

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as circulating lymphocytes, although in lower numbers than SWC6þ cdþ T cells [51]. Porcine CD4þ and cdþ T cells contribute to cellmediated immune responses to extracellular bacterial antigens. We previously demonstrated that immunization with a pepsin-digested bacterin of Brachyspira hyodysenteriae, a Gram-negative spirochete, increases the pool of circulating CD4þ and cdþ T cells, which also proliferate to B. hyodysenteriae-specific antigen in vitro [2]. In addition, CD4þ T cell depletion or blockade of antigen presentation through MHC-II greatly diminished the proliferative responses of peripheral blood mononuclear cells (PBMC) [47]. Based on these results, immunization with this bacterin induces a MHC-II-dependent CD4þ T cell response. However, because the vaccine also affected cdþ T cells, additional mechanisms of activation might exist besides antigen presentation through MHC-II. We used immunization with B. hyodysenteriae as a model to investigate differential functional aspects of CD4þ and cdþ T cells. In the present study, it was determined that the requirements for the proliferation of CD4þ and cdþ T cells are different. Specifically, while CD4þ T cell responses were strictly B. hyodysenteriae antigen-dependent, cdþ T cells also proliferated to stimulation with exogenous IL-2. Additionally, most of the IFN-c produced in culture originated from CD4þ T cells, whereas cdþ T cells showed reduced capacity of IFN-c production.

2. Materials and methods 2.1. Animals and immunization Four-week-old, cross-bred pigs were immunized with either a squalene-adjuvanted protease-digested preparation of B. hyodysenteriae, or adjuvant alone as previously described [2,3]. B. hyodysenteriae strain B204 was grown in tripticase soy broth (Becton–Dickinson; Cockesville, MD) supplemented with 5% horse serum (Hyclone, Logan, UT), 0.5% yeast extract (Difco, Detroit, MI), and VPI salts under anaerobic conditions. Lyophilized bacterial cells were digested by incubation with pepsin (10
3 g, per g of lyophilized B. hyodysenteriae) at pH 1.9– 2.2 for 25 h at 37 °C. The vaccine was prepared by mixing at 1:1 (v:v) the pepsin-digested B. hyodysenteriae with the adjuvant (10% of squalene/pluronic acid (80:20 v:v) in 2% Tween 80-Saline). Pigs received three intramuscular doses of either vaccine or adjuvant alone at 15 days intervals. All animal experiments were approved by the Institutional Animal Care Committee. 2.2. Isolation of peripheral blood mononuclear cells One week after the last immunization, PBMC were recovered from whole blood by gradient centrifugation.

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Briefly, a 1:4 dilution of blood in PBS was overlaid onto 4 mL of 1.077 lymphocyte separation media (Mediatec, Herdon, VA), and centrifuged at 800 g for 40 min. PBMC from the plasma/media interface were aspirated with a pasteur pipete, washed three times in PBS, and resuspended in complete RPMI 1640 medium (10% fetal bovine serum (Hyclone, Logan, UT), 25 mM Hepes buffer (Sigma, St. Louis, MO), 100 U/mL penicillin (Sigma), 0.1 m/mL streptomycin (Sigma), 5  10
5 2-mercaptoethanol (Sigma), 1 mM sodium pyruvate (Sigma), 1 mM non-essential amino acids (Sigma), and 2 mM essential amino acids (Mediatech)). Cells were enumerated with a Coulter Z1 Single Particle Counter (Beckman Coulter, Miami, FL). 2.3. Proliferation assay A total of 20  106 PBMC were labeled with the green fluorescent dye PKH67 (Sigma) following manufacturerÕs instructions. Briefly, PBMC were aliquoted into 15-mL conical tubes and centrifuged at 400 g for 5 min. After eliminating the supernatant, cells were resuspended in 1 mL of Diluent C (Sigma). The cell suspension was immediately transferred to a 15 mL conical tube containing 1 mL of a 2 mM solution of PKH67 dye in Diluent C (Sigma). Cells were incubated with the dye for 5 min and the reaction was stopped by the addition of 2 mL of fetal bovine serum (Hyclone) and, after 1 min, 4 mL of RPMI 1640. To eliminate excess fluorescent dye, cells were washed three times in RPMI 1640 and resuspended in complete RPMI to a final concentration of 2  106 cells/mL. PBMC at 106 cells/mL were seeded in 96-well, flatbottomed microtiter plates and incubated with complete RPMI only, the pepsin-digested preparation of B. hyodysenteriae B204 antigen at 5 lg/mL, Concanavalin A at 5 lg/mL (Sigma) or 5 ng/mL of recombinant porcine IL2 (Biosource, Camarillo, CA), in a 200 lL final volume. After 5 days in culture, PBMC were harvested and labeled for the analysis of cell surface marker expression by flow cytometry as previously described [47]. For IL-2 neutralization experiments, polyclonal anti-porcine IL-2 (R&D Systems, Minneapolis, MN) was added at the beginning of the culture period at 3 lg/mL. The optimal dose of recombinant IL-2 and the inhibitory concentration of polyclonal anti-porcine IL-2 were determined in a preliminary titration experiment in which PBMC were incubated with increasing amounts of recombinant IL-2, ranging from 0 to 20 ng/mL. Neutralizing IL-2 antibodies were added at 0, 0.75, 1.5 or 3 lg/mL to triplicate cultures of the various amounts of recombinant IL-2. Proliferation was assessed by [3 H] thymidine incorporation. Results showed that addition of more than 5 ng/mL of recombinant IL-2 did not result in stronger stimulation and that 3 lg/mL of polyclonal anti-IL-2 completely inhibited exogenous IL-2-induced proliferation.

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2.4. T cell immunophenotyping by flow cytometry

3. Results

The following antibodies were used for lymphocyte immunophenotyping: mouse IgG2b anti-porcine CD4 (clone 74-12-1), biotinylated mouse IgG2a anti-porcine CD8a (clone 76-2-11), mouse IgG1 anti-porcine TCRd chain (PGBL22A, VMRD, Pullman, WA), mouse IgG1 anti-porcine CD3 (8E6, VMRD), mouse IgG2a antiporcine CD8b (PG145A, VMRD), and mouse IgG1 anti-porcine CD25 (PGBL25A, VMRD). For the determination of CD25 expression in cdþ T cells, the mouse IgG1 anti-porcine TCRd chain monoclonal antibody was conjugated to FITC using the Fluoreporter Protein Labeling Kit (Molecular Probes, Eugene, OR). Cell surface staining was conducted by incubating cells in 100 ll of combined primary antibodies (CD3/ CD8a, CD4/CD8a, TCRd/CD8a or CD8b) in FACS buffer (PBS containing 5% of fetal bovine serum (Hyclone) and 0.01% of sodium azide (Sigma)) for 20 min. Following two washes in FACS buffer, cells were incubated with the secondary antibodies or streptavidinCyChrome (Pharmingen, San Diego, CA) for the biotinylated CD8a antibody. Cells were analyzed by three-color flow cytometry (PKH67, phycoerythrin and CyChrome) in a FACScan (Becton–Dickinson). Five to ten thousand events were acquired, and data were analyzed using the Cell Quest software (Becton–Dickinson).

3.1. Lymphocyte activation and proliferation in vitro

2.5. Intracellular IFN-c detection PBMC were incubated in 96-well, flat-bottomed microtiter plates at 106 cells/mL with either medium alone, or the pepsin-digested preparation of B. hyodysenteriae B204 antigen at 5 lg/mL, in a final volume of 200 lL. On day 5, cultures were stimulated with 50 ng/mL of phorbol myristate acetate (PMA) (Sigma) and 500 ng/mL ionomycin (Sigma). Golgi transport was blocked by the addition of 4 lL of Golgi Stop (Pharmingen) for every 6 mL of cell culture. After CD4/CD8a and TCRcd/ CD8a cell surface staining, cells were fixed and permeabilized with the Cytofix-cytoperm Kit (Pharmingen) following manufacturerÕs instructions. PE-labeled mouse IgG1 anti-porcine IFN-c (P2G10, Pharmingen) was used for intracellular cytokine detection. Equally stained non-PMA and ionomycin-stimulated cultures were used as negative controls, and mouse IgG1-PE (Sigma) was used as isotype control. 2.6. Statistical analysis Data were analyzed as a randomized complete block design. Analysis of variance (ANOVA) was used to investigate the main effects of vaccination followed by StudentÕs t test or ScheffeÕs method. ANOVA was performed using the general linear model procedure of SAS, differences with P < 0:05 were considered significant.

Previous studies demonstrated that immunization with a B. hyodysenteriae bacterin induced antigen-specific T cell proliferation [2,48]. In order to further investigate these responses, PBMC from naive and immunized animals were cultured with medium alone, or with B. hyodysenteriae antigens. As it had been reported before, T cells from immunized animals proliferated in vitro in the presence of B. hyodysenteriae antigens as determined by the decrease in PKH67 mean fluorescence intensity of CD3þ PBMC (Fig. 1A). In addition, a fraction of CD3
cells from immunized pigs also proliferated to antigenic stimulation, although we did not phenotypically characterize the responding CD3
lymphocytes. Anti-CD25 staining of PBMC from immunized pigs cultured with B. hyodysenteriae antigens (Fig. 1B) indicates that antigenic stimulation up-regulated cell-surface IL-2Ra. Based on the expression of CD25 on cells from immunized pigs that had proliferated, IL-2Ra up-regulation correlated with cell division. However, by day 5, a small fraction of non-stimulated PBMC from immunized pigs, or PBMC from control pigs cultured with either medium or B. hyodysenteriae antigens stained positive for IL-2Ra; although these cells failed to proliferate. 3.2. Subset analysis of T cells responding to B. hyodysenteriae In order to dissect the pool of T cells that had proliferated, CD4þ (i.e., helper abþ T cells), CD8abþ (i.e., cytotoxic abþ T cells), and cdþ T cells were individually analyzed. An additional T cell subset (i.e., abþ TCR CD4
CD8aaþ ) has been identified in porcine PBMC; however, due to the lack of monoclonal antibodies specific for either the porcine TCRa or b chains, it was not possible to analyze this subset individually. Proliferation results show that after 5 days in culture, CD4þ and cdþ T cells isolated from immunized pigs had proliferated to B. hyodysenteriae antigens (Fig. 2). Thus, these two subsets account for the vast majority of the T cells responding to antigenic stimulation. No proliferative responses were detected in CD8abþ T cells (Fig. 2). Additionally, when non-specific T cell responses were detected (i.e., PBMC from immunized pigs cultured with medium, or PBMC from controls cultured with B. hyodysenteriae antigens), it was due to proliferation of cdþ T cells. Both CD4þ and cdþ T cells can be further subdivided based on the expression of CD8a. In order to determine differential responses within CD4þ or cdþ T cell subsets, cells were double-stained with anti-CD4 and anti-CD8a,

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Fig. 1. Proliferation (A) and CD25 expression (B) of PBMC from an immunized (left panel) and a non-immunized control pig (right panel) after 5 days in culture with or without 5 lg/mL of B. hyodysenteriae (B. hyo) antigens. PBMC were isolated 1 week after three intramuscular immunizations with a pepsin-inactivated B. hyodysenteriae bacterin. CD3 staining indicates that T cells from immunized pigs specifically responded to stimulation with spirochetal antigens. The forward versus side scatter plot shows the cells included in the analysis (i.e., live mononuclear cells). Plots are representative from two experiments with a total of five pigs per group.

Fig. 2. Proliferation of CD4þ , CD8abþ , and cdþ T cells from an immunized (left panel) and a naive pig (right panel) after 5 days in culture with or without 5 lg/mL of B. hyodysenteriae (B. hyo) antigens. PBMC were isolated 1 week after the third immunization with a pepsin-inactivated B. hyodysenteriae bacterin. CD4þ and cdþ T cells from vaccinated pigs account for the majority of the CD3þ proliferating cells. The forward versus side scatter plot shows the cells included in the analysis (i.e., live mononuclear cells). Plots are representative from two experiments with a total of 5 pigs per group.

or anti-TCRd chain and anti-CD8a for the phenotypic analysis of cells that had proliferated after 5 days in culture. Results show that when PBMC from immu-

nized pigs were stimulated with B. hyodysenteriae antigens, the CD4þ T cells that had proliferated were both CD8aþ and CD8a
(Table 1).With regard to the cdþ T

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Table 1 Cell surface phenotype of proliferating peripheral blood T cell subsets from control or immunized pigsa Phenotypec

þ

Mediumb




lod

CD4 CD8aa PKH67 CD4þ CD8aaþ PKH67lo cd CD8aa
PKH67loe cd CD8aaþ PKH67lo

B. hyodysenteriae antigen

Control

Immunized

Control

Immunized

2.228  0.998 1.263  0.709 3.804  2.061 0.438  0.279

2.244  0.826 0.961  0.261 7.226  4.226 0.326  0.161

3.345  1.422 1.831  0.891 7.366  2.414 0.689  0.401

9.894  3.971 f 12.394  6 17.131  8.063 1.383  0.487

a

Results are expressed as means (n ¼ 5) percentage of CD4þ or TCRdþ cells that had proliferated (i.e., PKH67lo ) after 5 days in culture. Peripheral blood mononuclear cells isolated from control and B. hyodysenteriae-immunized pigs were labeled with PKH67 and cultured for 5 days with or without 5 lg/mL of B. hyodysenteriae (B. hyo) antigens. c Cells were harvested on day 5 and double-stained with monoclonal antibodies specific for CD4 and CD8a or TCRd and CD8a, and analyzed by three-color flow cytometry. Ten thousand events within the viable cell gate were acquired. d CD4þ cells within the live mononuclear gate were analyzed by plotting CD8a versus PKH67. e TCRdþ cells within the live monocyte gate were analyzed by plotting CD8a versus PKH67. f P value <0.05 when compared to the non-immunized control group. b

cell subset, most of the proliferating cells belonged to the CD8a
fraction (Table 1). Also, non-specific background proliferation was observed for TCRcdþ CD8aa
T cells isolated from non-immunized animals in cultures, where bacterial antigens were added.

T cells to IL-2, the presence of the receptor was analyzed in each subset in non-cultured PBMC. A fraction of both CD4þ and cdþ T cells expressed IL-2Ra, although the percentage of IL-2Raþ cells was higher in the latter (Fig. 4).

3.3. Differential requirements for proliferation of CD4þ and cdþ T cells

3.4. CD4þ T cells are the major source of IFN-c

Lymphocytes that had proliferated during 5 days in culture with spirochetal antigens up-regulated and maintained cell surface CD25 expression (Fig. 1). In order to determine the contribution of IL-2 to the activation and proliferation of the responding T lymphocytes, PBMC were cultured with either B. hyodysenteriae pepsin-digested antigen or medium supplemented with recombinant porcine IL-2. This cytokine alone was an insufficient stimulus for abþ T cells (i.e., CD4þ and CD8abþ ) to proliferate (Fig. 3A). In contrast to abþ T cells, a significant proportion of cdþ T cells responded to the addition of IL-2 (Fig. 3A). Furthermore, proliferation of cdþ T cells correlated with up-regulation of IL2Ra in IL-2-stimulated PBMC (Fig. 3A). The majority of the cdþ T cells that proliferated were CD8a
(Fig. 3B). No significant differences were found between PBMC isolated from control or B. hyodysenteriae-immunized pigs in their responses to exogenous IL-2 (data not shown). Treatment of cultures with neutralizing anti-IL-2 antibodies at the beginning of the culture period completely abrogated proliferation of cdþ CD8aa
T cells to exogenous IL-2, and resulted in the reduction of the proliferative response of this cell type when PBMC from immunized pigs were stimulated with antigen (Fig. 3B). With regards to the CD4þ pool, proliferation of CD4þ CD8aaþ T cells to B. hyodysenteriae was diminished by the addition of IL-2-neutralizing antibodies (Fig. 3B). To investigate whether baseline IL-2Ra expression accounted for the differential responses of CD4þ and cdþ

Previously, we reported the presence of IFN-c-producing cells in B. hyodysenteriae-stimulated PBMC from vaccinated pigs. Additionally, we showed that the IFN-c response was CD4þ T cell-dependent because in vitro CD4þ T cell depletion decreased the numbers of IFN-cproducing cells [49]. In the present study, intracellular IFN-c staining results indicate that CD4þ T cells are the major source of IFN-c in PBMC (Fig. 5). In addition, when CD4þ T cells were stimulated with B. hyodysenteriae, the percentage of IFN-c-producing cells decreased, along with the mean fluorescence intensity of the cytokine staining. This effect was observed in all pigs, irrespective of the in vivo treatment, although it was more pronounced in CD4þ cells from immunized pigs. On the other hand, very few cdþ T cells were able to produce IFN-c in vitro. However, the percentage of IFN-cþ cdþ T cells increased in both vaccinated and controls after stimulation with B. hyodysenteriae antigens.

4. Discussion Our results show that both CD4þ and cdþ T cells respond to antigenic stimulation in vitro, although the requirements for proliferation are different for the two subsets. The fact that cells that had divided expressed high levels of IL-2Ra, and that IL-2 neutralization decreased proliferation, suggests that IL-2 is necessary for the proliferation of both CD4þ and cdþ T cells. However, based on the responses to exogenous IL-2, this cytokine alone was insufficient to induce CD4þ T cell division. The antigen used in these experiments is a

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Fig. 3. Effect of IL-2 in T cell proliferation and IL-2Ra expression. PBMC isolated from immunized and control pigs were incubated for 5 days with exogenous IL-2 (A), or with exogenous IL-2 or B. hyodysenteriae antigens, with or without neutralizing anti-IL-2 polyclonal antibodies (B). Cells included in the analysis in (A) are represented in the forward versus side scatter plot. CD25 expression in PBMC on day 0 (thin line) and after 5 days in culture with IL-2 (thick line) is represented in histogram format. CD4þ and TCRdþ T cells were gated as indicated in the CD4/CD8a and TCRd/ CD8a for their individual analysis (B).

Fig. 4. IL-2Ra expression in freshly isolated CD4þ and cdþ T cells. PBMC isolated from non-immunized pigs were stained with CD4/ CD25 or TCRd/CD25 and analyzed by two-color flow cytometry.

protease-digested B. hyodysenteriae. This antigenic preparation was chosen because preliminary results demonstrated that the pepsin-digested B. hyodysenteriae was more efficient than a whole cell sonicated antigen inducing CD4þ and cdþ T cell responses. As it would be expected for an extracellular antigen processed trough an MHC-II-dependent pathway, CD4þ (i.e, T helper) but not CD8abþ T cells (i.e., cytotoxic T cells) responded to antigenic stimulation. Thus, both IL-2Raand TCR-derived signals are necessary for abþ T cell activation. In contrast to the more stringent requirements for CD4þ T cell proliferation, cdþ T cells proliferated in cultures stimulated with either spirochetal antigens or IL-2. Earlier studies have shown that blockade of antigen presentation through MHC-class II, or culture of PBMC depleted from CD4þ T cells diminished cdþ T cell proliferation to B. hyodysenteriae antigens [47]. It could

be interpreted that cdþ T cell proliferation is secondary to CD4þ T cell activation. We propose that B. hyodysenteriae-antigen-specific cells are within the CD4þ pool, and cdþ T cell proliferation is bystander to the paracrine action of IL-2 secreted by activated CD4þ T cells. This hypothesis is consistent with what it has been reported in studies using human lymphocytes. More specifically, it has been demonstrated that cdþ T cells produce low amounts of IL-2 [26,27], and their proliferation depends on the presence of activated CD4þ T cells [15,22,31]. Also, a subset of cdþ T cells could have responded independently from CD4þ T cells. This can be explained by non-MHC-class-II-restricted specific recognition of spirochetal epitopes by cdþ T cells. In this regard, lipoproteins and lipidated hexapeptides from the spirochete Borrelia burgdorferi induced proliferation and IL-2Ra expression on cdþ T cells isolated from Lyme disease patients. These responses were optimal in the presence of dendritic cells (DC) and exogenous IL-2 [46]. Moreover, in mice, adoptive transfer of B. burgdorferipulsed, MHC-class II-deficient DC provided a protective immune response in challenged recipients mediated by cdþ T cells [25]. Also, cd T cell responses to leptospiral antigens have been reported in cattle [28,29]. This mechanism would further support the observation that cdþ T cells from non-immunized pigs proliferated at background levels when stimulated with B. hyodysenteriae antigens. Very recently, Klimpel et al. [23] have shown that na€ıve peripheral blood cdþ T cells isolated

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R. Hontecillas, J. Bassaganya-Riera / Cellular Immunology 224 (2003) 38–46

Fig. 5. IFN-c production by CD4þ and cdþ T cells from an immunized (top row) and a non-immunized control pig (bottom row). PBMC were isolated from controls and B. hyodysenteriae-immunized pigs 1 week after the third immunization. After 5 days in culture with or without 5 lg/mL of B. hyodysenteriae (B. hyo) antigens, cells were stimulated with PMA and ionomycin for 4 h. Initial gate was set on the mononuclear cell population as indicated in the forward versus side scatter plot. Plots are representative from one experiment of three pigs per treatment.

from healthy humans were activated in vitro by Leptospira interrogans. Interestingly, although IL-2Ra expression was detected in unstimulated CD4þ and cdþ T cells, only the latter responded to IL-2. This finding suggests that an additional CD4þ - and B. hyodysenteriae antigen-independent mechanism exists for the activation of cdþ T cells. The expression of IL-2Ra has been previously described in cdþ T cells from na€ıve and Mycobacterium bovis-infected cattle [9,50]. Moreover, stimulation of purified cdþ T cells up-regulated IL-2Ra [50]. In the absence of CD4þ T cells, human cdþ T cells can be activated in vitro by cytokines that signal through the IL-2R [15]. In addition, a CD4þ -independent pathway of cdþ T cell activation in vitro by an unknown ligand present on monocytes and granulocytes has been described in cattle [37]. Several experimental models of infectious diseases have shown an increase in cdþ T cells following activation of abþ T cells [5,6,14]. Such changes in the cdþ T cell population initially depend on the production of IL2 [8,16]. Others have suggested that a negative feedback loop is established between cdþ and abþ T cells by which antigen-specific abþ T cell responses are downregulated by the expanded cdþ T cells. Activated cdþ T cells isolated from synovial fluid of Lyme arthritis patients express high levels of Fas ligand and can induce apoptosis of Fasþ CD4þ T cells [34,45,46]. Porcine cdþ T cells have non-specific cytotoxic activity in vitro [12,13]. Thus, it is possible that the activated cdþ T cells proliferating in B. hyodysenteriae-stimulated cultures down-regulated CD4þ T cell responses. This mechanism would explain the decreased IFN-c production following stimulation with B. hyodysenteriae antigens. Our results also indicate that the capacity of IFN-c production by CD4þ T cells is higher than that of cdþ T

cells. Similar findings have been previously reported in cattle and in pigs [1,33]. In contrast, murine and human cdþ T cells, which expand significantly following bacterial or parasitic infections [18,21,24,35,38], have higher capacity than CD4þ T cells for IFN-c production [44,54]. Hence, it appears that the IFN-c production by peripheral blood cdþ T cells is lower than in CD4þ T cells in species in which this subset constitutes a large fraction the circulating lymphocyte pool. In summary, we have shown that immunization with a spirochetal bacterin generated antigen-dependent CD4þ T cell responses that resulted in proliferation and IFN-c production. In contrast, proliferation of cdþ T cells did not correlate with a strong IFN-c response, and was not solely associated with antigen-specific stimulation. Pigs along with ruminants and chickens are characterized by having large numbers of circulating cdþ T cells when compared to humans and mice [17]. Interestingly, studies on the different species have revealed functional similarities, such as their cytotoxic potential, sensitivity to IL-2, or their capacity to undergo clonal expansion overtime [13,17,19,20,42]. The use of pig models could be of great advantage for investigating the reciprocal regulation between CD4þ and cdþ T cells.

Acknowledgments The authors acknowledge Dr. J. Espadamala and Mr. S.C. Jobgen for their assistance in the animal studies.

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