HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tuo, W. Articles by Brown, W. C. Search for Related Content PubMed PubMed Citation Articles by Tuo, W. Articles by Brown, W. C.

Differential Effects of Type I IFNs on the Growth of WC1^- CD8⁺ T Cells and WC1⁺ CD8^- T Cells In Vitro¹

Wenbin Tuo^*, Fuller W. Bazer, William C. Davis^*, Daming Zhu^* and Wendy C. Brown^2,*

^* Department of Veterinary Pathology and Microbiology, Washington State University, Pullman, WA 99164; and Center for Animal Biotechnology, Institute of Biosciences and Technology, Texas A&M University, College Station, TX 77843

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type I IFNs have a broad array of immunoregulatory functions that include up-regulation of type 1 immune responses through enhancing differentiation and activation of CD8⁺ T cells and CD4⁺ Th1 cells. Ovine trophoblast IFN- is a recently described type I IFN with the potential for therapeutic use, based on its potent antiviral activity yet low toxicity. Studies were designed to determine the immunoregulatory effects of IFN- on Ag-stimulated T cells, and a novel effect of type I IFNs on T cells was observed. In cultures of parasite Ag-stimulated bovine T cells that contained a mixture of ß and T cells, both IFN- and IFN- suppressed the expansion of WC1⁺ CD2^- CD6^- CD8^- T cells, yet stimulated the growth of WC1^- CD2⁺ CD6⁺ CD8⁺ T cells and CD8⁺ ß T cells. The CD8⁺ T cell subset expressed high levels of the IL-2R -chain. Furthermore, we showed that type I IFN enhanced IL-2 production by these Ag-stimulated T cell lines. In short term cultures of PBMC, IL-2 stimulated an expansion of WC1^- CD6⁺ CD8⁺ T cells, which was significantly increased by IFN-, even though IFN- alone did not support cell survival. These studies demonstrate for the first time that type I IFNs differentially modulate the proliferation of different subsets of T cells, which appears to act in part via IL-2.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Type I IFNs comprise a family of multifunctional cytokines with a broad spectrum of biological functions (1). In addition to their potent antiviral and antiproliferative capabilities, type I IFNs play a critical role in the immune system by modulating the differentiation and effector functions of B cells, NK cells, CD8⁺ CTL, and Th cells (2, 3, 4, 5, 6). Human type I IFNs (IFN- and -ß) have been used in clinical trials for treating patients suffering from cancer, viral infections, and autoimmune diseases (4, 7, 8, 9).

Ovine trophoblast IFN- shares similar functional and structural characteristics with other type I IFNs (10, 11). IFN- suppressed mitogen-induced proliferation of ruminant PBMC and T cells (12), activated ovine and porcine NK cells (13), suppressed viral replication (14), and induced lymphopenia and neutropenia in lambs (78). IFN- was recently shown to prevent experimental allergic encephalomyelitis in mice by inducing CD4⁺ T suppressor cells that produced IL-10 and TGF-ß (15) and by decreasing TNF- production (16). Importantly, IFN- has very low levels of cytotoxicity in vitro compared with IFN- and IFN-ß (16, 17), suggesting that IFN- may be useful as a potent therapeutic drug with fewer side effects. However, the immunomodulatory effects of type I IFNs on different subsets of mammalian T cells have not been studied extensively.

Mammals and birds have evolved two lineages of T cells that express TCR composed of either ß or heterodimers (18). T cells are a minor population in the circulation of rodents and humans (18, 19), although these cells are more prominent in certain epithelial tissues (20). The localization of T cells to the skin and intestinal and reproductive tract epithelia implies their importance as a first line defense against invading pathogens (19, 21). T cells increase in numbers in response to bacterial, viral, and parasitic infection (18, 22, 23). Furthermore, T cells can recognize Ags derived from these microbes and tumor cells (19, 24). The most compelling evidence for a role for T cells in immunity was obtained with T cell-deficient mice that carry an interrupted TCR gene, which were less efficient in eliminating Listeria organisms than wild-type mice (25). Together, these results show that T cells play an important role in host defense by complementing the function of ß T cells.

In contrast to humans and mice, young ruminants have high numbers of T cells (up to 70%) in their peripheral blood, which diminish with age to 5–25% of the total circulating T cells in adults (26, 27). The two major subsets of bovine T cells are distinguished by the expression of a high m.w. dimer designated Workshop Cluster 1 (WC1).³ One subset is WC1⁺ CD3⁺ CD5⁺ CD2^- CD6^- and CD8^- (28), and the second subset is WC1^- CD3⁺ CD5⁺ CD2⁺ CD6⁺ and CD8⁺. WC1, which is not expressed on human or murine T cells, is a member of the scavenger receptor cysteine-rich family of proteins that includes CD5 and CD6 (29). Additionally, WC1⁺ T cells express an unrelated high m.w. molecule designated GD3.5 (30). The functions of WC1 and GD3.5 are not known. In ruminants, WC1⁺ T cells are the predominant subset in the circulation, whereas WC1^- T cells are more abundant in spleen, mammary gland, intestine, skin, and uterus (27, 31, 32, 33, 34, 35).

Bovine WC1⁺ T cells are functionally similar to human and murine T cells, as evidenced by cytolytic activity and expression of IL-2, IL-4, IL-10, IFN-, and TNF- (36, 37). We have observed that WC1⁺ T cells expand in cultures of PBMC repeatedly stimulated with parasite or rickettsial Ags, although WC1⁺ T cell clones and T cells lines do not proliferate in response to Ag and APC (36). Others have shown that proliferation of ruminant T cells requires the presence of APC and ß T cells (38). Human T cells can also proliferate in response to malarial parasite Ag, but only in the presence of CD4⁺ T cells and cytokines that use the IL-2R (39).

In contrast to WC1⁺ T cells, bovine WC1^- CD8⁺ T cells have not been characterized in detail, and their functions are not well defined. In humans and mice, CD8⁺ T cells are a major component of intraepithelial lymphocytes and produce cytokines, notably IFN- (40, 41, 42). CD8⁺ T cells suppressed the adoptive transfer of diabetes by T cells from diabetic mice to nondiabetic mice (43) and appeared to assist ß T cells in the adoptive cell transfer of contact sensitivity (44). CD8⁺ T cells obtained from either thymocytes or PBMC of both humans and rodents were preferentially stimulated by IL-2 (45, 46, 47).

The effects of IFN- on proliferation and cytokine expression by CD8⁺ T cells, CD4⁺ Th cells, and different subsets of T cells have not been reported for any species. Using cattle as a model system in which large numbers of peripheral T cells are readily obtainable, this study determined the effects of IFN- and IFN- on the expansion of CD8⁺ T cells, Ag-specific CD4⁺ T cells, and T cells in cultures of PBMC stimulated with specific Ag. The results of this study provide compelling evidence that type I IFNs selectively suppress expansion of WC1⁺ T cells and stimulate expansion of CD8⁺ and ß T cells by a mechanism that appears to involve IL-2.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antigens

Babesia bovis and B. bigemina membrane CM Ag was prepared by homogenization of cultured merozoites with a French pressure cell (SLM Instruments, Urbana, IL) and ultracentrifugation (48). URBC were similarly fractionated for use as control Ags. Anaplasma marginale organisms were purified from bovine blood infected with the Florida strain (49) and homogenized with the French pressure cell in PBS in the presence of 300 µg/ml PMSF, 25 µg/ml antipain, and 25 µg/ml leupeptin (50). Fasciola hepatica parasite collection and preparation of SWA were performed as previously described (51). Protein concentrations were determined by the Bradford assay (Bio-Rad, Richmond, CA). All Ags were stored at -80°C until used.

Cattle used in this study

Cow C15 was inoculated i.v. with cultured autologous erythrocytes infected with the Mexico strain of B. bovis and became serologically positive for B. bovis following infection (48). Ag-specific T cell lines and T cell clones were successfully established from this animal using B. bovis CM Ag (52). Cow 2216 was immunized with purified native B. bigemina rhoptry-associated protein-1 (RAP-1) protein and was immune to challenge with homologous parasites (53, 54). The RAP-1-specific T cell lines and T cell clones were established using either B. bigemina CM Ag or recombinant RAP-1 (53). Cow G1 was infected orally with metacercariae of the Oregon strain of F. hepatica (Baldwin Aquatics, Monmouth, OR) and became serologically positive for F. hepatica. Ag-specific T cell lines and T cell clones were successfully established from G1 using F. hepatica SWA (51). Animal 96B09 was immunized with outer membranes of the Florida isolate of A. marginale and was protected against challenge infection with homologous A. marginale (50). Ag-specific T cell lines were obtained from animal 96B09 using A. marginale homogenate Ag (50). In addition, 12 healthy Holstein calves, 3–4 mo old, were used for preparation of PBMC to determine the normal proportions of T cell subsets by three-color flow cytometric analysis.

Establishment of T cell lines

T cell lines were established from cattle immune to B. bovis, B. bigemina, F. hepatica, and A. marginale as previously described (50, 51, 52, 53, 54). Briefly, PBMC (3–4 x 10⁶) were isolated with Histopaque-1077 (Sigma, St. Louis, MO) from peripheral blood of immune cattle and cultured in 24-well plates (Costar, Cambridge, MA) in 1.5 ml/well of complete RPMI 1640 medium at 37°C in a humidified atmosphere of 5% CO₂ in air (50, 51, 52, 53). The T cell lines were stimulated with B. bovis CM (25 µg/ml), B. bigemina CM (25 µg/ml), F. hepatica SWA (25 µg/ml), or A. marginale homogenate (1 µg/ml) in the presence or the absence of IFN- or IFN- (500 U/ml). Cells were restimulated weekly for up to 13 wk with autologous irradiated (3000 rad) PBMC as a source of APC and Ag with or without IFN- or IFN-.

Lymphocyte proliferation

Proliferation assays were conducted in replicate wells of round-bottom 96-well plates (Costar) for 3 days at 37°C in a humidified atmosphere of 5% CO₂ in air. Briefly, T cells (3 x 10⁴) were cultured in duplicate wells in a total volume of 100 µl of complete RPMI 1640 medium containing 2 x 10⁵ autologous irradiated PBMC and Ag with or without type I IFNs. Lymphocytes were radiolabeled with [³H]thymidine for the last 16–24 h of the 3-day assay and were harvested using an automatic cell harvester (TomTec, Orange, CT). Results are presented as the mean counts per minute ± 1 SEM.

mAb to bovine leukocytes and analysis by flow cytometry

The PBMC and T cell lines cultured for various weeks were collected and washed before flow cytometric analysis. Specific mAb for bovine leukocyte surface markers, 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, and IL-A29 specific for WC1 (36). Additional mAb obtained from the Monoclonal Antibody Center at Washington State University (Pullman, WA) included MUC2A specific for CD2, MM1A specific for CD3, CACT138A and GC50A specific for CD4, CACT80C and BAQ111A specific for CD8, BAT82A specific for CD8ß, BAQ82A specific for CD6, CACT116A specific for CD25 (IL-2R -chain), GB21A and CACT61A specific for the -chain of TCR , and B7A1 specific for WC1 (27, 55). Second-step reagents included FITC-labeled goat anti-mouse Ig (a mixture of IgG, IgM, and IgA; affinity-purified F(ab')₂, Cappel/Organon Teknika, Malvern, PA) for single-color flow cytometric analysis and FITC-IgM, FITC-IgG2a, PE-IgG2a, PE-IgG2b, and Tri-color-IgG1 conjugates (affinity-purified goat anti-mouse Igs; Caltag, South San Francisco/Burlingame, CA) for three-color flow cytometric analysis (27). Cell phenotypes were determined by indirect immunofluorescence and were analyzed with a Coulter EPICS 741 flow cytometer (Hialeah, FL) (36) or a Becton Dickinson FACSort (Mountain View, CA). The computer software CellQuest was used to collect data, and both CellQuest and Paint-A-Gate Pro (Becton Dickinson Immunocytometry Systems, San Jose, CA) were used to analyze the data.

Recombinant ovine IFN- and human IFN-

Recombinant ovine IFN- was produced in yeast and was purified using ion exchange and gel filtration chromatography (56). The purity of ovine IFN- was assessed by one-dimensional SDS-PAGE and silver staining. Recombinant human IFN- was a gift to Dr. F. W. Bazer from Hoffmann-La Roche (Nutley, NJ). The IFN activities were determined by an antiviral assay (14) and are expressed as antiviral units per milliliter.

Stimulation of PBMC by IL-2 or IL-7 in the presence or the absence of type I IFNs

Bovine PBMC (3–4 x 10⁶/well) were cultured in 24-well plates in complete RPMI 1640 medium for 10 days. Treatments included 500 U/ml IFN- alone, or recombinant human IL-2 or IL-7 (50–100 U/ml; Boehringer Mannheim, Indianapolis, IN) either alone or in the presence of IFN- or IFN- (500 U/ml). Cells were collected, washed, stained for bovine lymphocyte surface markers, and subsequently analyzed by flow cytometry.

RNA and cell supernatant preparation

Total cellular RNA was extracted using the Trizol reagent (Life Technologies, Gaithersburg, MD) from T cells (2 x 10⁶) cultured with APC and 25 µg/ml B. bovis CM, B. bigemina CM, F. hepatica SWA, or 1 µg/ml A. marginale homogenate in 1.5 ml of complete medium for 6 h at 37°C in a humidified atmosphere with 5% CO₂ in air. Supernatants were collected for IL-2 analysis from similarly established cultures incubated for 24 h. RNA samples were stored at -80°C, and supernatants were stored at -20°C until analyzed.

Competitive quantitative RT-PCR analysis of IL-2 mRNA levels

Competitive quantitative RT-PCR analysis of cytokine mRNA was performed using a competitor molecule (mimic) for bovine IL-2 that was provided by Dr. Dante Zarlenga (Department of Immunobiology and Disease Resistance, U.S. Department of Agriculture, Agriculture Research Service, Beltsville, MD) and IL-2 primers (5'-primer, 5'-GTA CAA GAT ACA ACT CTT GTC TTG C-3'; 3'-primer, 5'-TCA AGT CAT TGT TGA GAT GCT T-3'). A bovine ß-actin mimic was generated in our laboratory, and actin primers were designed (5'-primer, 5'-ACC AAC TGG GAC GAC ATG GAG-3'; 3'-primer, 5'-GCA TTT GCG GTG GAC AAT GGA-3'). The resultant IL-2 and ß-actin competitor PCR fragments were distinguishable from native fragments by their smaller size (IL-2: native fragment, 466 bp; competitor fragment, 323 bp; actin: native fragment, 890 bp; competitor fragment, 660 bp). Column-purified, RNA-free plasmids containing IL-2 and actin competitors were serially diluted 5- to 10-fold with sterile double-distilled water and stored at -20°C. Total RNA (0.5–1 µg) was reverse transcribed to cDNA in a 20-µl volume using oligo(dT)₁₆ following the manufacturer’s instructions (Perkin-Elmer, Branchburg, NJ). For each sample tested, PCR reactions were performed with each reaction containing PCR primers, mimic DNA, cDNA (0.05–5 ng cDNA), a master mixture containing 10 x PCR buffer (final concentration, 1x), magnesium (final concentration, 2.5 mM), dNTPs (final concentration, 1 mM), AmpliTaq-Gold (1 U/reaction; Perkin-Elmer), and water in a 50-µl volume. The PCR reaction mixture was preheated to 94°C for 10 min to activate the AmpliTaq-Gold followed by 35 cycles of amplification under the following conditions: 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. Then reactions were completed by an extension at 72°C for 10 min and were stored at 4°C until analyzed. PCR products (20 µl) were electrophoresed on a 1% agarose gel containing ethidium bromide and quantified under UV light using a Digital Imaging System (IS1000, Alpha Innotech, San Leandro, CA). After correcting for differences in the m.w. between native and competitor DNA, the ratios between the amplified products of the target and competitor sequences at each competitor concentration were calculated. The logs of the ratios were plotted against the input concentrations of competitor DNA, and a regression equation was obtained. At the point of equivalence, where the ratios of amplified target:competitor DNA equals 1, the amount of cytokine cDNA/mRNA in the test sample equals the amount of competitor DNA. To compare the amounts of IL-2 mRNA in different samples, IL-2 mRNA levels were normalized to the amount of ß-actin.

Analysis of IL-2 protein by bioassay

An IL-2-dependent bovine CD8⁺ TCR ß⁺ cloned cell line, designated G4.3D1, was used to determine the IL-2 concentration in supernatants of Ag-stimulated T cell lines. This cell line did not proliferate in response to Con A (Sigma) or bovine IL-4 (provided by D. Mark Estes, University of Missouri, Columbia, MO), but proliferated vigorously in response to human IL-2 and bovine T cell growth factor. Serially diluted human rIL-2 (Boehringer Mannheim) was used as a biological standard, and the amount of IL-2 was expressed as picograms per milliliter of cell supernatant.

Statistics

Data were analyzed by analysis of variance with the least significant difference multiple range test, using Statistix version 4.0 (Analytical Software, St. Paul, MN). The value p < 0.05 was used to indicate statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distribution of T cell subsets in peripheral blood

To determine the different subsets of T cells in peripheral blood, PBMC were isolated from 12 calves, 3–4 mo old, and T cells were stained and analyzed by three-color flow cytometry (Table I). The proportion of CD6⁺ CD8⁺ T cells was low (<4% of the total T cells), whereas the proportion of WC1⁺ CD8^- T cells was relatively high (40% of the total T cells), consistent with previous reports (26). The analysis also showed that CD4⁺, CD6⁺, and CD8⁺ cells expressed very low levels of CD25 (from 0.25–2.25%), indicating a resting state of these cells.

IFN--induced phenotypic changes in T cell subsets ex vivo: analysis by single-color flow cytometry

Previous results indicated that Ag-stimulated T cell lines derived from cattle immune to the protozoal parasites B. bigemina (53) or B. bovis (57), the nematode parasite F. hepatica (51), or the pathogenic rickettsia A. marginale (50) were comprised of a mixture of CD4⁺ ß and WC1⁺ T cells. In many of the Ag-specific T cell lines that we have examined, WC1⁺ T cells often eventually predominated the cultures (36, 57). To determine the effects of type I IFNs on the growth of parasite Ag-driven T cells obtained from immune cattle, T cell lines were stimulated with B. bovis, F. hepatica, A. marginale, or B. bigemina Ag with or without 500 U/ml IFN- or IFN- for 11 wk, and the cells were analyzed by single-color flow cytometry. As observed previously, three T cell lines specific for B. bovis, A. marginale, or F. hepatica, when cultured with Ag alone, changed in composition from predominantly CD4⁺ T cells to predominantly WC1⁺ T cells after 5 wk. A representative example of C15 T cells stimulated with B. bovis is shown in Fig. 1. However, when cells were stimulated with Ag in the presence of IFN- (Fig. 1C), the percentage CD4⁺ T cells remained elevated, and WC1⁺ T cells did not expand. In the cell line stimulated with B. bigemina, WC1⁺ T cells from the B. bigemina RAP-1-immune animal did not expand when stimulated with Ag alone (data not shown). However, in all four cell lines, IFN- induced the expansion of T cells expressing CD8 (Fig. 1C). The A. marginale-specific cell line cultured with IFN- lacked WC1⁺ cells, but a similar percentage of cells staining for CD8 cells and the TCR suggested that the CD8⁺ population could be comprised of T cells. In contrast, IFN- stimulated the expansion of CD8⁺ ß T cells in the B. bigemina-stimulated cell line. Similar results were obtained with IFN-, although IFN- appeared more potent at stimulating CD8⁺ T cells (Fig. 1, B and C). Collectively, the results with four different Ag-specific T cell lines suggested that type I IFN directly or indirectly inhibited the outgrowth of the WC1⁺ T cell subset, but supported the growth of CD8⁺ and CD8⁺ ß T cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Single-color flow cytometric analysis of T cell lines established from PBMC of cow C15 and stimulated with B. bovis CM Ag alone (A), B. bovis CM Ag plus IFN- (B), or B. bovis CM Ag plus IFN- (C). T cells lines were analyzed weekly by flow cytometry after staining with Abs against bovine CD2 (data not shown), CD3 (data not shown), CD4, CD8, and WC1. Results are presented as the percentage of CD3+ T cells.

Type I IFNs down-regulate expansion of WC1⁺ T cells and up-regulate expansion of CD6⁺, CD8⁺, T cells ex vivo

Three-color flow cytometric analysis was used to verify the apparent differential effects of IFN- on subsets of WC1⁺, CD8^- T cells and WC1^-, CD8⁺ T cells in cell lines cultured with specific Ag for 12–13 wk. Fig. 2 presents representative data for an A. marginale-specific T cell line obtained from animal 96B09, and the data for all T cell lines are summarized in Table II. The results confirmed that T cell lines stimulated with parasite Ag alone had an expanded population of TCR ⁺ T cells that coexpressed WC1. The CD8⁺ WC1^- TCR ^- T cells were present at very low or undetectable levels in cell lines cultured with parasite Ag alone (Fig. 2A and Table II). Few WC1⁺ T cells coexpressed CD6, whereas 100% of CD4⁺ ß T cells coexpressed CD6. Few CD4⁺ T cells in these cultures coexpressed CD25 (Table II). The low expression of CD25 by CD4⁺ T cells between 12 and 13 wk in culture is consistent with the overall decline in Ag-specific T cell proliferation in such long term cultured cell lines (36) (data not presented).

View larger version (62K): [in this window] [in a new window]   FIGURE 2. Three-color flow cytometric analysis of A. marginale-driven T cell lines derived from cow 96B09 at 13 wk of culture. T cell lines were stimulated and maintained with A. marginale Ag alone (A) or A. marginale Ag plus IFN- (B). Cells were stained for CD3, CD4, CD6, CD8, CD8ß, CD25, WC1, and the -chain of TCR . Results are expressed as the percentage of total T cells gated.

Ag-specific proliferation and inhibition by IFN- and -

As demonstrated previously, T cell lines specific for B. bovis (57), B. bigemina RAP-1 (53), F. hepatica (51), or A. marginale (50) responded to corresponding Ag with high specificity. T cell lines from these animals were also MHC restricted, suggesting that they arose from circulating memory cells (50, 51, 52, 53, 57).To verify that the T cell lines maintained their Ag specificity during the course of in vitro culture with type I IFN, proliferation assays were performed with T cells lines treated with or without type I IFN. Each cell line proliferated specifically and in a dose-dependent manner to the immunizing Ag throughout the whole experiment, whereas a similar response to control URBC Ag was not observed (Table III and data not shown). At most time points, the levels of proliferation in cell lines cultured with type I IFN were lower than those in cells cultured with Ag alone, which is consistent with the antiproliferative properties of type I IFNs (1).

View larger version (14K): [in this window] [in a new window]   FIGURE 3. IFN- and IFN- suppress Ag-induced T cell proliferation. T cell lines were established and maintained with B. bovis CM Ag alone (A) or CM Ag plus IFN- (B) continuously for 5 wk. T cells were stimulated with APC and 10 µg/ml CM Ag in the presence of increasing concentrations of IFN- or IFN- (0.4–2500 U/ml) for 3 days, radiolabeled, and harvested. Results are presented as the mean counts per minute + 1 SEM of triplicate cultures. The counts per minute of Ag-stimulated T cell proliferation were calculated by subtracting the counts per minute induced by control Ag (membrane preparation from URBC) from the counts per minute induced by parasite Ag.

Because the expansion of WC1^-, CD6⁺, CD8⁺ T cells in response to IFN- was associated with high levels of IL-2R expression on these cells (Table II), we postulated that IL-2 produced by Ag-stimulated T cells was driving this response. Therefore, IL-2 transcript expression and protein production were measured in the Ag-stimulated cell lines over time (Fig. 4 and Table IV). Analysis of IL-2 transcript expression by competitive quantitative RT-PCR revealed that in the presence of IFN-, levels of IL-2 mRNA were increased by 69- to 546-fold in the B. bovis-specific T cell line (Fig. 4A), by 7- to 41-fold in the A. marginale-specific T cell line (Fig. 4B), and by 3- to 16-fold in the B. bigemina-specific T cell line (Fig. 4C) between 3 and 6 wk of culture compared with those in T cell lines cultured with Ag alone. When the effects of IFN- and IFN- on B. bovis-stimulated T cells cultured for 6 wk were compared, IFN- induced 8.5-fold higher levels of IL-2 mRNA (data not shown), which is consistent with its superior effects on stimulating the growth of CD8⁺ T cells (Fig. 1).

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Competitive quantitative RT-PCR analysis of IL-2 mRNA levels in reverse transcribed total RNA isolated from parasite-specific bovine T cells lines cultured with or without IFN- or IFN-. T cell lines were derived from animal C15 immune to B. bovis (A) animal 96B09 immune to A. marginale (B), and animal 2216 immune to B. bigemina RAP-1 (C) and cultured continuously for up to 5 or 7 wk. The amount of transcript for IL-2 in the cell lines was calculated based on the known amount of cytokine competitor gene added, as described in Materials and Methods, and is expressed as femtograms per microgram of total cellular RNA.

View this table: [in this window] [in a new window]   Table IV. IL-2 levels in culture supernatants of parasite Ag-stimulated T cells cultured with or without IFN-

Effects of IL-2 and type I IFN on expansion of CD8⁺ T cells

Type I IFN enhanced both the early production of IL-2 by Ag-driven T cell lines and IL-2R expression by bovine CD8⁺ T cells in these lines, leading us to hypothesize that the combination of IL-2 and IFN- was selectively stimulating this T cell subset. Because Ag-stimulated cell lines produce IL-2, freshly isolated PBMC were used to measure the effects of exogenous IL-2 or type I IFN, added alone or in combination, on selective stimulation of the CD8⁺ T cell subset. PBMC were cultured with cytokine for 10 days, and single- or three-color FACS analysis was performed before and after cell culture to define the responding population of cells. Single-color analysis revealed a selective expansion of CD8⁺ T cells from PBMC cultured with IL-2 that was significantly (p < 0.05) enhanced by IFN- (Fig. 5A) or IFN- (data not shown). IFN- alone had no stimulatory effect and failed to support cell proliferation. In contrast, IL-7, which is also a bovine T cell growth factor and one of the T cell cytokines that uses IL-2R, did not share this stimulatory effect on CD8⁺ T cells (Fig. 5B). Thus, the effects of IL-2 were selective for CD8⁺ T cells and differed from those induced by IL-7.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Single-color flow cytometric analysis of CD8+ T cell expansion from freshly isolated PBMC cultured with IL-2 (100 U/ml; A) or IL-7 (50 ng/ml; B) in the presence or the absence of IFN- (500 antiviral units/ml). PBMC were obtained from eight calves and were cultured with IFN-, IL-2 or IL-7 alone, or IL-2 or IL-7 plus IFN- for 10 days. Cells were stained for surface markers CD3, CD4, CD8, and WC1 and were analyzed by flow cytometry. Results are expressed as the percentage of total (CD3+) T cells.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Three-color flow cytometric analysis of the expansion of CD6+ CD8+ T cells by IL-2 and IFN-. PBMC were obtained from five calves and were cultured with medium alone, IFN- alone (500 antiviral units/ml), IL-2 (100 U/ml), or IL-2 plus IFN- for 10 days. Cells were stained for CD3, CD4, CD6, CD8, WC1, and TCR and were analyzed by flow cytometry. Results are expressed as the percentage of total T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Type I IFN-induced and activated CD8⁺ T cells may contribute to host defense by producing inflammatory cytokines (19, 66) and by killing virus- or bacteria-infected cells via an NK-like mechanism (45). The abundance of CD8⁺ T cells in epithelial tissues suggests that the microenvironment (e.g., uterus, skin, and intestine) provides a unique set of signals (e.g., trophoblast IFN-) for these cells to differentiate and proliferate. Recent studies showed that type I IFNs are important for the development of CD8⁺ T cells in mice deficient in IFN regulatory transcription factor-1, a DNA binding protein that regulates the expression of type I IFNs (67). Loss of IFN regulatory transcription factor-1 caused an abnormal induction of type I IFNs, which resulted in impaired development of TCR ß⁺ CD4^- CD8⁺ T cells in the thymus (63, 64). Furthermore, Ag-nonspecific proliferation of murine CD8⁺ T cells in vivo was attributable to type I IFNs (IFN-/ß), which mimicked the effects of virus (65) or LPS (68). Thus, type I IFNs are critical for the development, growth, and maturation of both ß and CD8⁺ T cells. Our data indicate the need to define the role of type I IFNs in regulating tissue-resident CD8⁺ T cells.

Up-regulation of IL-2 by IFN- is consistent with the finding that IFN- augmented the production of IL-2 by PHA-stimulated human T cell clones (69, 70, 71). IFN- and - did not act on CD8⁺ T cells directly by stimulating proliferation, since addition of these cytokines to short term T cell proliferation assays often suppressed Ag-driven proliferation, and IFN- alone had no effect on CD8⁺ T cell expansion in short term cultures. Rather, the expansion of this subset in Ag-stimulated T cell lines appears to be due to the up-regulation of both IL-2 production by the cell lines and selective expression of IL-2R and an enhanced response to IL-2 by the CD8⁺ T cell subset. The high levels of IL-2 and IL-2R expression occurred temporally with the presence of a high percentage of CD4⁺ T cells and the absence of WC1⁺ T cells and preceded the subsequent increase in CD8⁺ T cells in the cultures. Thus, the enhanced expression of IL-2R on CD8⁺ T cells and the response to increased levels IL-2 may partially explain the preferential in vitro growth of the CD8⁺ T cell subset cultured with type I IFN.

The Ag-driven proliferation and IL-2 production by the T cell lines are probably attributable to CD4⁺ T cells in culture. Oligoclonal and monoclonal WC1⁺ T cells obtained from similar parasite Ag-stimulated cell lines failed to proliferate in response to the Ag used for expansion of the parental T cell lines (36, 51) (W. C. Brown, unpublished observations). Furthermore, a WC1^- CD8⁺ T cell clone (G1.3A5) that expressed high levels of IFN- transcript but low levels of IL-2 transcript did not proliferate in response to the F. hepatica Ag used to establish the parental T cell line without or with exogenous IL-2 (W. C. Brown, unpublished observations). Similarly, in mice sensitized with picryl chloride, proliferation and IL-2 production were mediated by CD4⁺ T cells, whereas IFN- was produced by CD8⁺ T cells during contact sensitivity to the hapten (66).

As observed in other species (72), bovine CD8⁺ T cells coexpress CD6 and exist in peripheral blood as a minor population. CD6 mediates T cell activation upon interacting with its ligand, ALCAM (29). ALCAM is a membrane-bound protein expressed by many cell types, including activated T cells, B cells, monocytes, brain cells, thymic epithelial cells, epidermal keratinocytes, and a breast cancer cell line (73, 74). Our results suggest a role for CD6 in T cell activation, since the majority of CD6⁺ cells expressed CD25. The association between CD6 and CD25 expression on CD8⁺ T cells is also consistent with a role for CD6-ALCAM binding in activation and preferential expansion of these cells by type I IFNs. This possibility is strengthened by a report that anti-CD6 mAb preferentially stimulated the proliferation of human T cell clones, but not ß T cell clones (75).

Expansion of WC1⁺ T cells from Ag-stimulated PBMC in the absence of exogenous type I IFN is independent of type of the parasite immunogen, since this has been observed repeatedly with protozoan and metazoan parasite Ags (36, 37, 51) and with Ags from the prokaryotic rickettsia, A. marginale (50). The outgrowth of WC1⁺ T cells in culture occurred at approximately 3–6 wk, after which point the cells expanded rapidly, suggesting that T cell cytokine patterns and/or cell surface costimulatory molecules changed quantitatively or qualitatively during this time. WC1⁺ T cells can be stimulated to grow when APC are physically or chemically altered by irradiation, fixation, or intracellular infection in an autologous mixed lymphocyte reaction (76, 77). It is hypothesized that these alterations may induce or expose a surface molecule expressed by monocytes, which contributes to the expansion of WC1⁺ T cells (77). Interestingly, like CD6 (29), WC1 apparently mediates T cell activation upon binding to its ligand, as shown by using anti-WC1 mAb IL-A29 to induce proliferation (76). The differential regulation of WC1⁺ and CD8⁺ T cell subsets by type I IFNs suggests that different T cell subsets use alternative signaling pathways, leading to proliferation and expansion. Further studies are planned to identify the molecular basis for regulation of CD6 and WC1 ligand expression and their respective roles in T cell activation.

	Acknowledgments

 
We thank Roger Smith III and Betty Rosenbaum for assistance with single-color flow cytometry, Allison Rice-Ficht for providing F. hepatica Ag, and Guy H. Palmer and Beverly Hunter for providing A. marginale Ag.

	Footnotes

 
¹ This work was supported by Grant R01AI30136 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (to W.C.B.) and U.S. Department of Agriculture National Research Initiative Competitive Grants Program Grants 95-37204-2347 (to W.C.B.) and 95-37203-6548 (to F.W.B.).

² Address correspondence and reprint requests to Dr. Wendy C. Brown, Department of Veterinary Pathology and Microbiology, Washington State University, Pullman, WA 99164. E-mail address:

³ Abbreviations used in this paper: WC1, Workshop Cluster 1; CM, crude parasite membrane antigen of Babesia bovis or Babesia bigemina; URBC, uninfected red blood cells; SWA, soluble worm antigen; RAP-1, rhoptry-associated protein-1; PE, phycoerythrin; ALCAM, activated leukocyte cell adhesion molecule.

Received for publication May 11, 1998. Accepted for publication September 22, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Johnson, H. M., F. W. Bazer, B. E. Szente, M. A. Jarpe. 1994. How interferons fight disease. Sci. Am. 270:68.[Medline]
2. Finkelman, F. D., A. Svetic, I. Gresser, C. Snapper, J. Holmes, P. P. Trotta, I. M. Katona, W. C. Gause. 1991. Regulation by interferon- of immunoglobulin isotype selection and lymphokine production in mice. J. Exp. Med. 174:1179.[Abstract/Free Full Text]
3. Biron, C. A.. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9:24.[Medline]
4. Belardelli, F., I. Gresser. 1996. The neglected role of type I interferon in the T-cell response: implications for its clinical use. Immunol. Today 17:369.[Medline]
5. Romagnani, S.. 1992. Induction of TH1 and TH2 responses: a key role for the ‘natural’ immune response?. Immunol. Today 13:379.[Medline]
6. Romagnani, S.. 1997. The Th1/Th2 paradigm. Immunol. Today 18:263.[Medline]
7. Alberti, A., L. Chemello, F. Noventa, L. Cavalletto, G. De-Salvo. 1997. Therapy of hepatitis C: re-treatment with -interferon. Hepatology 26:(Suppl. 1):137S.[Medline]
8. Arnason, B. G., A. Dayal, Z.-X. Qu, M. A. Jensen, K. Genc, A. T. Reder. 1996. Mechanisms of action of interferon-ß in multiple sclerosis. Springer Semin. Immunopathol. 18:125.[Medline]
9. Gutterman, J. U.. 1994. Cytokine therapeutics: lessons from interferon-. Proc. Natl. Acad. Sci. USA 91:1198.[Abstract/Free Full Text]
10. Roberts, R. M., J. C. Cross, D. W. Leaman. 1992. Interferons as hormones of pregnancy. Endocr. Rev. 13:432.[Abstract/Free Full Text]
11. Bazer, F. W., H. M. Johnson. 1991. Type I conceptus interferons: maternal recognition of pregnancy signals and potential therapeutic agents. Am. J. Reprod. Immunol. 26:19.
12. Assal-Meliani, A., G. Charpigny, P. Reinaud, J. Martal, G. Chaouat. 1993. Recombinant ovine trophoblastin (roTP) inhibits ovine, murine and human lymphocyte proliferation. J. Reprod. Immunol. 25:149.[Medline]
13. Tuo, W., T. L. Ott, F. W. Bazer. 1993. Natural killer cell activity of lymphocytes exposed to ovine, type I, trophoblast interferon. Am. J. Reprod. Immunol. 29:26.
14. Pontzer, C. H., J. K. Yamamoto, F. W. Bazer, T. L. Ott, H. M. Johnson. 1997. Potent anti-feline immunodeficiency virus and anti-human immunodeficiency virus effect of IFN-. J. Immunol. 158:4351.[Abstract]
15. Mujtaba, M. G., J. M. Soos, H. M. Johnson. 1997. CD4⁺ T suppressor cells mediate interferon tau protection against experimental allergic encephalomyelitis. J. Neuroimmunol. 75:35.[Medline]
16. Soos, J. M., P. S. Subramaniam, A. C. Hobeika, J. Schiffenbauer, H. M. Johnson. 1995. The IFN pregnancy recognition hormone IFN- blocks both development and superantigen reactivation of experimental allergic encephalomyelitis without associated toxicity. J. Immunol. 155:2747.[Abstract]
17. Subramaniam, P. S., S. A. Khan, C. H. Pontzer, H. M. Johnson. 1995. Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities. Proc. Natl. Acad. Sci. USA 92:12270.[Abstract/Free Full Text]
18. Allison, J. P.. 1993. T-cell development. Curr. Opin. Immunol. 5:241.[Medline]
19. Kronenberg, M.. 1994. Antigens recognized by T cells. Curr. Opin. Immunol. 6:64.[Medline]
20. Kagnoff, M. F., and H. Kiyono 1996. Essentials of Mucosal Immunology. Academic Press, San Diego.
21. Boismenu, R., W. L. Havran. 1997. An innate view of T cells. Curr. Opin. Immunol. 9:57.[Medline]
22. Haas, W., P. Pereira, S. Tonegawa. 1993. T cells. Annu. Rev. Immunol. 11:637.[Medline]
23. Kabelitz, D.. 1992. Function and specificity of human -positive T cells. Crit. Rev. Immunol. 11:281.[Medline]
24. Chien, Y.-H.. 1996. Ligand recognition by T cells. M. F. Kagnoff, and H. Kiyono, eds. Essentials of Mucosal Immunology 169. Academic Press, San Diego.
25. Mombaerts, P., J. Arnoldi, F. Russ, S. Tonegawa, S. H. Kaufmann. 1993. Different roles of ß and T cells in immunity against an intracellular bacterial pathogen. Nature 365:53.[Medline]
26. Hein, W. R., C. R. Mackay. 1991. Prominence of T cells in the ruminant immune system. Immunol. Today 12:30.[Medline]
27. Davis, W. C., W. C. Brown, M. J. Hamilton, C. R. Wyatt, J. A. Orden, A. M. Khalid, J. Naessens. 1996. Analysis of monoclonal antibodies specific for the TcR. Vet. Immunol. Immunopathol. 52:275.[Medline]
28. Wijngaard, P. L., M. J. Metzelaar, N. D. MacHugh, W. I. Morrison, H. C. Clevers. 1992. Molecular characterization of the WC1 antigen expressed specifically on bovine CD4^- CD8^- T lymphocytes. J. Immunol. 149:3273.[Abstract]
29. Aruffo, A., M. A. Bowen, D. D. Patel, B. F. Haynes, G. C. Starling, J. A. Gebe, J. Bajorath. 1997. CD6-ligand interactions: a paradigm for SRCR domain function?. Immunol. Today 18:498.[Medline]
30. Jones, W. M., B. Walcheck, M. A. Jutila. 1996. Generation of a new T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family. J. Immunol. 156:3772.[Abstract]
31. Wyatt, C. R., C. Madruga, C. Cluff, S. Parish, M. J. Hamilton, W. Goff, W. C. Davis. 1994. Differential distribution of T-cell receptor lymphocyte subpopulations in blood and spleen of young and adult cattle. Vet. Immunol. Immunopathol. 40:187.[Medline]
32. Park, Y. H., L. K. Fox, M. J. Hamilton, W. C. Davis. 1992. Bovine mononuclear leukocyte subpopulations in peripheral blood and mammary gland secretions during lactation. J. Dairy Sci. 75:998.[Abstract]
33. Wyatt, C. R., E. J. Brackett, L. E. Perryman, W. C. Davis. 1996. Identification of T lymphocyte subsets that populate calf ileal mucosa after birth. Vet. Immunol. Immunopathol. 52:91.[Medline]
34. Hein, W. R., L. Dudler. 1997. TCR ⁺ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors. Immunology 91:58.[Medline]
35. Meeusen, E., A. Fox, M. Brandon, C. S. Lee. 1993. Activation of uterine intraepithelial T cell receptor-positive lymphocytes during pregnancy. Eur. J. Immunol. 23:1112.[Medline]
36. Brown, W. C., W. C. Davis, S.-H. Choi, D. A. Dobbelaere, G. C. Splitter. 1994. Functional and phenotypic characterization of WC1⁺ T cells isolated from Babesia bovis-stimulated T cell lines. Cell. Immunol. 153:9.[Medline]
37. Collins, R. A., P. Sopp, K. I. Gelder, W. I. Morrison, C. J. Howard. 1996. Bovine TcR⁺ T lymphocytes are stimulated to proliferate by autologous Theileria annulata-infected cells in the presence of interleukin-2. Scand. J. Immunol. 44:444.[Medline]
38. Hanrahan, C. F., W. G. Kimpton, C. J. Howard, K. R. Parsons, M. R. Brandon, A. E. Andrews, A. D. Nash. 1997. Cellular requirements for the activation and proliferation of ruminant T cells. J. Immunol. 159:4287.[Abstract]
39. Elloso, M. M., H. C. van der Heyde, A. Troutt, D. D. Manning, W. P. Weidanz. 1996. Human T cell subset-proliferative response to malarial antigen in vitro depends on CD4⁺ T cells or cytokines that signal through components of the IL-2R. J. Immunol. 157:2096.[Abstract]
40. Taguchi, T., W. K. Aicher, K. Fujihashi, M. Yamamoto, J. R. McGhee, J. A. Bluestone, H. Kiyono. 1991. Novel function for intestinal intraepithelial lymphocytes: murine CD3⁺, TCR⁺ T cells produce IFN- and IL-5. J. Immunol. 147:3736.[Abstract]
41. Kiyono, H. K., T. Fujihashi, W. K. Aicher Taguchi, J. R. McGhee. 1991. Regulatory functions for murine intraepithelial lymphocytes in mucosal responses. Immunol. Res. 10:324.[Medline]
42. Morita, C. T., S. Verma, P. Aparicio, C. Martinez, H. Spits, M. B. Brenner. 1991. Functionally distinct subsets of human T cells. Eur. J. Immunol. 21:2999.[Medline]
43. Harrison, L. C., M. Dempsey-Collier, D. R. Kramer, K. Takahashi. 1996. Aerosol insulin induces regulatory CD8⁺ T cells that prevent murine insulin-dependent diabetes. J. Exp. Med. 184:2167.[Abstract/Free Full Text]
44. Askenase, P. W., M. Szczepanik, M. Ptak, V. Paliwal, W. Ptak. 1995. T cells in normal spleen assist immunized ß T cells in the adoptive cell transfer of contact sensitivity: effect of Bordetella pertussis, cyclophosphamide, and antibodies to determinants on suppressor cells. J. Immunol. 154:3644.[Abstract]
45. Satoh, M., S. Seki, W. Hashimoto, K. Ogasawara, T. Kobayashi, K. Kumagai, S. Matsuno, K. Takeda. 1996. Cytotoxic or ß T cells with a natural killer cell marker, CD56, induced from human peripheral blood lymphocytes by a combination of IL-12 and IL-2. J. Immunol. 157:3886.[Abstract]
46. Gotlieb, W. H., L. Takacs, L. R. Finch, W. Kopp, A. M. Weissman, S. K. Durum. 1991. CD8 cells: presence in the adult rat thymus and generation in vitro from CD4^-/CD8^- thymocytes in the presence of interleukin 2. Cytokine 3:598.[Medline]
47. Spetz, A. L., P. Kourilsky, E. L. Larsson-Sciard. 1991. Induction of CD8 molecules on thymic / T cells in vitro is dependent upon /ß T cells. Eur. J. Immunol. 21:2755.[Medline]
48. Brown, W. C., K. S. Logan, G. G. Wagner, C. L. Tetzlaff. 1991. Cell-mediated immune responses to Babesia bovis merozoite antigens in cattle following infection with tick-derived or cultured parasites. Infect. Immun. 59:2418.[Abstract/Free Full Text]
49. Palmer, G. H., T. C. McGuire. 1984. Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol. 133:1010.[Abstract]
50. Brown, W. C., V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, G. H. Palmer. 1998. CD4⁺ T lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge. Infect. Immun. 66:5406.[Abstract/Free Full Text]
51. Brown, W. C., W. C. Davis, D. A. Dobbelaere, A. C. Rice-Ficht. 1994. CD4⁺ T cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles. Infect. Immun. 62:818.[Abstract/Free Full Text]
52. Brown, W. C., S. Zhao, A. C. Rice-Ficht, K. S. Logan, V. M. Woods. 1992. Bovine helper T cell clones recognize five distinct epitopes on Babesia bovis merozoite antigens. Infect. Immun. 60:4364.[Abstract/Free Full Text]
53. Rodriguez, S. D., G. H. Palmer, T. F. McElwain, T. C. McGuire, B. J. Ruef., C. G. Chitko-McKown, W. C. Brown. 1996. CD4⁺ T-helper lymphocyte responses against Babesia bigemina rhoptry-associated protein 1. Infect. Immun. 64:2079.[Abstract]
54. Brown, W. C., T. F. McElwain, T. F. Hotzel, C. E. Suares, G. H. Palmer. 1998. Helper T cell epitopes in polymorphic and conserved regions of Babesia bigemina rhoptry-associated protein-1are identical among parasite strains. Infect. Immun. 66:1561.[Abstract/Free Full Text]
55. Naessens, J., J. Hopkin. 1996. Introduction and summary of workshop findings. Vet. Immunol. Immunopathol. 52:213.[Medline]
56. Ott, T. L., G. Van Heeke, H. M. Johnson, F. W. Bazer. 1991. Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1: purification and antiviral activity. J. Interferon Res. 11:357.[Medline]
57. Brown, W. C., K. S. Logan. 1992. Babesia bovis: bovine helper T cell lines reactive with soluble and membrane antigens of merozoites. Exp. Parasitol. 74:188.[Medline]
58. MacDonald, H. R., M. Schreyer, R. Howe, C. Bron. 1990. Selective expression of CD8 (Ly-2) subunit on activated thymic cells. Eur. J. Immunol. 20:927.[Medline]
59. Totte, P., F. Jongejan, A. L. de Gee, J. Werenne. 1994. Production of interferon in Cowdria ruminantium-infected cattle and its effect on infected endothelial cell cultures. Infect. Immun. 62:2600.[Abstract/Free Full Text]
60. Sprent, J., D. F. Tough, S. Sun. 1997. Factors controlling the turnover of T memory cells. Immunol. Rev. 156:79.[Medline]
61. Eloranta, M. L., K. Sandberg, P. Ricciardi-Castagnoli, M. Lindahl, G. V. Alm. 1997. Production of interferon-/ß by murine dendritic cell lines stimulated by virus and bacteria. Scand. J. Immunol. 46:235.[Medline]
62. Havell, E. A.. 1993. Listeria monocytogenes-induced interferon- primes the host for production of tumor necrosis factor and interferon-/ß. J. Infect. Dis. 167:1364.[Medline]
63. Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T. M. Kundig, R. Amakawa, K. Kishihara, A. Wakeham, et al 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75:83.[Medline]
64. Penninger, J. M., C. Sirard, H. W. Mittrucker, A. Chidgey, I. Kozieradzki, M. Nghiem, A. Hakem, T. Kimura, E. Timms, R. Boyd, et al 1997. The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8⁺ thymocytes. Immunity 7:243.[Medline]
65. Tough, D. F., P. Borrow, J. Sprent. 1996. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 272:1947.[Abstract]
66. Dieli, F., G. L. Asherson, G. Sireci, R. Dominici, E. Scire, A. Salerno. 1997. Development of IFN--producing CD8^{+ +} T lymphocytes and IL-2-producing CD4⁺ ß⁺ T lymphocytes during contact sensitivity. J. Immunol. 158:2567.[Abstract]
67. Miyamoto, M., T. Fujita, Y. Kimura, M. Maruyama, H. Harada, Y. Sudo, T. Miyata, T. Taniguchi. 1988. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-ß gene regulatory elements. Cell 54:903.[Medline]
68. Tough, D. F., S. Sun, J. Sprent. 1997. T cell stimulation in vivo by lipopolysaccharide (LPS). J. Exp. Med. 185:2089.[Abstract/Free Full Text]
69. Holan, V., K. Kohno, J. Minowada J. 1991. Natural human interferon- augments interleukin-2 production by a direct action on the activated IL-2-producing T cells. J. Interferon Res. 11:319.[Medline]
70. Dao, T., V. Holan, J. Minowada. 1993. Interleukin-2 production by T cells: a study of the immunoregulatory actions of interferon-, interferon-, and tumor necrosis factor- in phenotypically different T cell clones. Cell. Immunol. 151:451.[Medline]
71. Holan, V., T. Dao, M. Kosarova, M. Lipoldova, J. Minowada. 1994. Interleukin-1 and interferon- augment interleukin-2 (IL-2) production by distinct mechanisms at the IL-2 mRNA level. Cell. Immunol. 157:549.[Medline]
72. Gobel, T. W., C. H. Chen, M. D. Cooper. 1996. Expression of an avian CD6 candidate is restricted to ß T cells, splenic CD8⁺ T cells and embryonic natural killer cells. Eur. J. Immunol. 26:1743.[Medline]
73. Bowen, M. A., D. D. Patel, X. Li, B. Modrell, A. R. Malacko, W. C. Wang, H. Marquardt, M. Neubauer, J. M. Pesando, U. Francke, et al 1995. Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand. J. Exp. Med. 181:2213.[Abstract/Free Full Text]
74. Wee, S., W. C. Wang, A. G. Farr, A. J. Nelson, D. D. Patel, B. F. Haynes, P. S. Linsley, A. Aruffo. 1994. Characterization of a CD6 ligand(s) expressed on human- and murine-derived cell lines and murine lymphoid tissues. Cell. Immunol. 158:353.[Medline]
75. Pawelec, G., H. J. Buhring. 1991. Monoclonal antibodies to CD6 preferentially stimulate T-cell clones with rather than ß antigen receptors. Hum. Immunol. 31:165.[Medline]
76. Hanby-Flarida, M. D., O. J. Trask, T. J. Yang, C. L. Baldwin. 1996. Modulation of WC1, a lineage-specific cell surface molecule of T cells augments cellular proliferation. Immunology 88:116.[Medline]
77. Okragly, A. J., M. Hanby-Flarida, D. Mann, C. L. Baldwin. 1996. Bovine T-cell proliferation is associated with self-derived molecules constitutively expressed in vivo on mononuclear phagocytes. Immunology 87:71.[Medline]
78. Tuo, W., W. C. Brown, E. Roger, D. Zhu, G. Lin, III R. Smith, F. W. Bazer. 1998. Trophoblast IFN- differentially induces lymphopenia and neutropenia in lambs. J. Interferon Cytokine Res. 18:731.[Medline]

This article has been cited by other articles:

				C. Menge and E. A. Dean-Nystrom Dexamethasone Depletes {gamma}{delta}T Cells and Alters the Activation State and Responsiveness of Bovine Peripheral Blood Lymphocyte Subpopulations J Dairy Sci, June 1, 2008; 91(6): 2284 - 2298. [Abstract] [Full Text] [PDF]

				W. Tuo, R. Fetterer, M. Jenkins, and J. P. Dubey Identification and Characterization of Neospora caninum Cyclophilin That Elicits Gamma Interferon Production Infect. Immun., August 1, 2005; 73(8): 5093 - 5100. [Abstract] [Full Text] [PDF]

				K. K. Lahmers, J. Norimine, M. S. Abrahamsen, G. H. Palmer, and W. C. Brown The CD4+ T cell immunodominant Anaplasma marginale major surface protein 2 stimulates {gamma}{delta} T cell clones that express unique T cell receptors J. Leukoc. Biol., February 1, 2005; 77(2): 199 - 208. [Abstract] [Full Text] [PDF]

				J. F. Hedges, D. Cockrell, L. Jackiw, N. Meissner, and M. A. Jutila Differential mRNA expression in circulating {gamma}{delta} T lymphocyte subsets defines unique tissue-specific functions J. Leukoc. Biol., February 1, 2003; 73(2): 306 - 314. [Abstract] [Full Text] [PDF]

				B. M. Naiman, S. Blumerman, D. Alt, C. A. Bolin, R. Brown, R. Zuerner, and C. L. Baldwin Evaluation of Type 1 Immune Response in Naive and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+{gamma}{delta} and CD4 T Cells Infect. Immun., November 1, 2002; 70(11): 6147 - 6157. [Abstract] [Full Text] [PDF]

				W. Tuo, G. H. Palmer, T. C. McGuire, D. Zhu, and W. C. Brown Interleukin-12 as an Adjuvant Promotes Immunoglobulin G and Type 1 Cytokine Recall Responses to Major Surface Protein 2 of the Ehrlichial Pathogen Anaplasma marginale Infect. Immun., January 1, 2000; 68(1): 270 - 280. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tuo, W. Articles by Brown, W. C. Search for Related Content PubMed PubMed Citation Articles by Tuo, W. Articles by Brown, W. C.