- Email: [email protected]
Phenotypic characterization of porcine IFN-γ-producing lymphocytes by flow cytometry
Journal of Immunological Methods 259 Ž2002. 171–179 www.elsevier.comrlocaterjim
Phenotypic characterization of porcine IFN-g-producing lymphocytes by flow cytometry M.P. Rodrıguez-Carreno, C. Revilla, A. Ezquerra, F. Alonso, ´ ˜ L. Lopez-Fuertes, ´ ) J. Domınguez ´ Departamento de Biotecnologıa, ´ INIA, Ctra. de La Coruna ˜ Km 7, 28040 Madrid, Spain Received 27 February 2001; received in revised form 9 July 2001; accepted 28 August 2001
Abstract We have developed a three-colour flow cytometric assay for phenotypic characterization of porcine IFN-g-producing lymphocytes. Analyses of activated swine peripheral blood mononuclear cells ŽPBMC. showed a significant difference in the proportion of IFN-g producing cells between young and adult animals Ž13.2 " 5.8% versus 34.2 " 5.7%.. The majority of IFN-g producing cells were ab T lymphocytes, although there was also an important proportion of gd T cells particularly in young animals. Within the ab T lymphocytes, the double positive CD4qCD8 lo subset, that contains memory T cells, produced high levels of IFN-g, whereas the CD8 hi T cells ranged from low to high levels of IFN-g. Also, consistent with a higher production by memory T cells, the CD45RAy subset of both CD4q and CD8q cells contained higher numbers of IFN-g producing cells than the CD45RAq subset. Finally, no production of IFN-g by either B cells ŽCD21q. or monocytes ŽSWC3q. was detected. This assay may be useful for the assessment of cell-mediated immunity in vaccine trials and may contribute to our understanding of the role of IFN-g in protective immunity against important viral diseases of the pig. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Flow cytometry; Intracellular staining; Lymphocyte subsets; Monoclonal antibodies; Porcine IFN-g
1. Introduction IFN-g is a pleiotropic cytokine that plays important roles in the regulation of inflammatory and immune responses, being particularly relevant in the host defense against intracellular pathogens. Among its multiple biological effects, IFN-g is a major activator of macrophages, enhancing their antimicrobial activity and their capacity for processing and
)
Corresponding author. Fax: q34-91-3572-293. .. E-mail address: [email protected] ŽJ. Domınguez ´
presenting antigens to T lymphocytes. In addition, it regulates Ig isotype switching on B cells and inhibits proliferation of Th2 cells, shifting the balance towards a Th1-type response ŽFarrar and Schreiber, 1993; Billiau, 1996.. In humans and rodents, IFN-g is produced by T lymphocytes and natural killer cells. However, the nature of porcine IFN-g-producing cells has not yet been elucidated. Compared with other species, the porcine immune system shows striking differences in the peripheral T-lymphocyte compartment. Based on the expression of CD4 and CD8 antigens, four Tlymphocyte subsets can be identified in peripheral
0022-1759r02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 5 1 1 - 7
172
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
blood and in lymphoid tissues. In addition to the classical phenotypes of CD4qCD8y helper T cells and CD8qCD4y cytolytic T lymphocytes, there is a double negative CD4yCD8y T-cell subset, also found in ruminants which contains the majority of gd T cells, and a double positive CD4qCD8q T-cell subset, unique to the pig, containing memory ab T cells ŽPescovitz et al., 1985; Saalmuller et al., 1987, ¨ 1990; Yang and Parkhouse, 1996.. The development of monoclonal antibodies ŽmAbs. against porcine IFN-g and their use in ELISA and ELISPOT assays has been recently described ŽLefevre et al., 1998; Mateu de Antonio et al., 1998; Zuckermann et al., 1998., although no attempts to detect porcine IFN-g production using flow cytometry have been reported. The aim of the present study was to characterize the phenotype of IFN-g-producing cells in the pig using multicolour flow cytometry. This assay makes it possible to determine not only the percentage of cells within a particular subset, but also to compare the relative amount of IFN-g produced by different subsets.
2. Materials and methods 2.1. Animals and cells Five Large-White outbred pigs from 3 to 6 months old, and three inbred minipigs of dd haplotype and four Large-White outbred pigs older than 3 years were used as blood donors. Pigs were managed according to the animal care regulations. Peripheral blood mononuclear cells ŽPBMC. were isolated on Percoll discontinuous gradients after blood sedimentation, through dextran as previously described ŽGonzalez ´ et al., 1990.. Cells were resuspended in RPMI 1640 medium containing 10% fetal calf serum ŽFCS., 2 mM L-glutamine, 5 = 10y5 M 2-mercaptoethanol and 30 mgrml gentamicin Žcomplete medium.. 2.2. Monoclonal antibodies Anti-IFN-g mAb G47 ŽLefevre et al., 1998., a generous gift from C. La Bonnardiere ŽINRA, Jouyen-Josas, France., was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose
CL4B ŽPharmacia, Sweden. and labeled with Alexa Fluor 488 dye following the manufacturer’s protocol ŽMolecular Probes, Eugene, OR, USA.. MAbs to porcine CD3 Ž8E6, IgG2b. and CD4 Ž74-12-4, IgG2b. were kindly provided by M. Pescovitz ŽIndiana University, USA., and CD8 Ž762-11, IgG2a. by J. Lunney ŽARS USDA, Beltsville, USA.. MAb to porcine gd TCR ŽPGBL22A, IgG1. was purchased from VMRD ŽPullman, WA, USA. and mAb anti-porcine CD21 Žclone B-ly4, IgG1. was obtained from Pharmingen ŽSan Diego, USA.. MAbs to porcine CD5 Ž1H6, IgG2a., CD45RA Ž3C3r9, IgG1. and SWC3 ŽBL1H7, IgG1. were developed in our laboratory. For the three-colour immunofluorescence assays, CD4, and CD8 mAbs were purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL4B, and labeled with biotin as described previously ŽDomınguez et al., 1990.. ´ 2.3. Immunofluorescent staining of intracellular IFN-g for flow cytometric analysis PBMC were stimulated for 16 h with PMA Ž50 ngrml. and calcium ionophore A23187 Ž500 ngrml. in the presence of the protein transport inhibitor monensin Ž2 mgrml.. Cells were harvested and incubated on ice with mAbs to different cell surface antigens Ž50 ml of hybridoma supernatantr10 6 cells., for 30 min. After two washes in PBS containing 0.1% bovine serum albumin ŽBSA. and 0.01% sodium azide Žfluorescence buffer, FB., cells were incubated for 30 min at 4 8C with phycoerythrin ŽPE.-conjugated rabbit FŽabX . 2 anti-mouse Ig ŽDako, Denmark.. Free binding sites were blocked with 5% normal mouse serum for 15 min prior to fixationr permeabilization with Permeafix ŽOrtho Diagnostic System, USA. for 45 min at room temperature. Cells were then washed in FB and incubated for 30 min with anti-IFN-g mAb G47 labeled with Alexa 488. After three washes, cells were analyzed on a Facscan cytometer ŽBecton-Dickinson, USA.. For three-colour immunofluorescence, cells were first incubated with unlabeled mAbs to cell surface antigens, followed after washing, by phycoerythrin ŽPE.-conjugated rabbit FŽabX . 2 anti-mouse Ig. Free binding sites were blocked with 5% normal mouse serum for 15 min before adding biotin-labeled antiCD4 or anti-CD8 mAb. After two washes in FB, the
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
biotinylated antibodies were detected with streptavidin-Cy-Chrome ŽPharmingen, USA.. Cells were then fixedrpermeabilized with Permeafix and stained with anti-IFN-g mAb G47 conjugated with Alexa 488, as described above. Irrelevant, isotype-matched mAbs, either unlabeled or labeled with biotin or Alexa 488, were used as negative controls. 2.4. Statistical analysis Comparisons of mean differences of IFN-g-producing cells was done using Student’s t-test. Differences were considered significant if p - 0.05.
3. Results 3.1. Flow cytometric detection of IFN-g production by PBMC following PMA r A23187 stimulation Swine PBMC were activated with a combination of PMA plus calcium ionophore A23187, as an optimal stimulus for IFN-g, in the presence or absence of monensin. Sixteen hours after stimulation, cells were stained for intracellular IFN-g with mAb G47 labeled with Alexa Fluor 488. Treatment with monensin, which blocks protein secretion and leads
173
to the accumulation of IFN-g in the Golgi complex, resulted in an increase in the number of fluorescent cells as well as in the intensity of the fluorescence of the stained cells. No fluorescence signal was detected in unstimulated cells Ždata not shown.. The frequency of IFN-g-producing cells differed between animals, with adult pigs having significantly higher values than young pigs Ž34.2 " 5.7%, n s 8, in adult pigs versus 13.2 " 5.8%, n s 8, in young pigs, p - 0.001.. In fact, a gradual increase in the proportion of IFN-g-producing cells was noted in young animals from the time of first sampling Žat 3 months of age. to the end of the experiment, 3 months later Žaged 6–7 months.. No differences were observed between inbred minipigs and outbred Large-White pigs of the same age-group. Cytometric profiles of one representative animal of each agegroup are shown in Fig. 1. 3.2. Phenotypic characterization of peripheral blood mononuclear cell subsets inÕolÕed in IFN-g production IFN-g production by different PBMC subsets was assessed by multi-colour flow cytometry. In adult pigs, the majority of IFN-g producing cells were CD3-positive Ž88.2 " 6.4%. ŽFig. 2a.. Most of these cells, particularly those with strong staining for IFN-
Fig. 1. Differential production of IFN-g by PBMC from adult and young pigs. PBMC were activated with PMA and Ca ionophore in the presence of monensin, fixedrpermeabilized and stained with Alexa 488-anti-porcine IFN-g mAb G47. As a control, cells stained with an irrelevant isotype matched mAb labeled with Alexa 488 were used Žopen histogram.. At least 20,000 cells were analyzed and the percentages of positive IFN-g producing cells are shown. Results are representative of six independent experiments.
174
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
175
Fig. 2. Phenotype of IFN-g producing cells as defined by two-colour flow cytometry analysis. Panel Ža. PBMC from a 3-year-old pig were stimulated as indicated in Fig. 1 in the presence of monensin, and stained with mAbs to surface antigens followed by PE goat anti-mouse Ig ŽFL2.. Cells were then fixedrpermeabilized and incubated with Alexa 488 conjugated anti-IFN-g mAb G47 ŽFL1.. Panel Žb. represents a comparison of the staining of gd TCR versus IFN-g in 6-month-old Žleft. or 3-year-old Žright. pigs. Results are from one representative experiment out of at least four performed. Quadrants were defined by background staining with irrelevant isotype-matched mAbs. The numbers indicate the percentage of cells within the respective regions. For this analysis, 50,000 PBMC were acquired.
g, expressed lower levels of CD3 than IFN-g negative T cells, and high levels of CD5, a phenotype which has been shown in previous reports to correspond to ab T cells ŽSaalmuller et al., 1994; Yang ¨ and Parkhouse, 1996.. About one-third of CD4q T cells produced IFN-g at high levels ŽTable 1, Fig. 2a.. Within the CD8q T cell population, almost 50% synthesized IFN-g. Whereas IFN-g production by CD8 hi T cells ranged from low to high, a subset of high IFN-g producers was identified within the CD8 lo cells. Low amounts of IFN-g production by almost one third of gd T cells ŽFig. 2a. were noted. These IFN-g producing gd T cells only accounted for 16.4 " 2.4% in adult pigs, while in young pigs,
because of their relatively lower proportion of ab T cells were almost a half Ž43.6 " 13.3%, n s 4, p 0.05. of all IFN-g producing cells ŽFig. 2b.. As expected, no production of IFN-g associated with either B cells ŽCD21q cells. or monocytes ŽSWC3q cells. ŽFig. 2a.. The CD8 lo cell subset of high IFN-g producers, identified by two-colour FCM, probably corresponded with the CD4qCD8q double positive cell subset, which have been shown to contain memory cells ŽZuckermann and Husmann, 1996; Summerfield et al., 1996.. To analyze in more detail the IFN-g production by the different T-cell subsets, we performed three-colour immunofluorescent staining
Table 1 Percentages of IFN-g producing cells within different lymphocyte subsets from adult pigs CD3
CD4q CD8y CD4y CD8q CD4q CD8q CD4q CD45RAy CD4q CD45RAq CD8q CD45RAy CD8q CD45RAq
x " S.D. 35.4 " 2.0 19.6 " 3.8 ns3 ns4 p
32.4 " 9.8 ns4
50.3 " 5.8 ns7
36.4 " 8.9 18 " 4.5 ns5 ns5 p - 0.01
41.5 " 8.0 13.5 " 4.2 ns4 ns4 p - 0.01
Values represent mean " standard deviation Ž x " S.D... Number of animals analyzed in each group is given as n. P values were calculated using Student’s t-test.
176
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
ŽFig. 3.. Porcine PBMC were resolved by simultaneous staining with anti-CD4 and anti-CD8 mAbs, into four subpopulations. The CD4qCD8q subset contained the highest percentage of IFN-g producing cells, followed by the CD4yCD8q subset and to a lesser extent by CD4q CD8y T cells ŽFig. 3a.. When CD4q or CD8q T cells were subdivided by CD45RA staining, CD45RAy T cells proved to be the main contributors to IFN-g production ŽTable 1, Fig. 3b..
4. Discussion In this report, we describe a simple and reliable method for the identification of porcine IFN-g producing cells by flow cytometry using mAb G47. The simultaneous examination of surface phenotype and intracytoplasmic cytokine expression permits an objective comparison of the relative amounts of IFN-g produced by the different lymphocyte subsets. Labeling of the anti-IFN-g mAb with the dye Alexa 488 avoids the use of second layer antibodies, reducing the number of controls required for multi-layer protocols as well as the possibilities of non-specific binding. Attempts to establish the specificity of staining by blocking, the binding of anti-IFN-g mAb to intracellular IFN-g by previous co-incubation with a molar excess of recombinant porcine IFN-g ŽrpIFNg . were unsuccessful. Instead of reducing the binding, this treatment increased both the number of positive cells and the fluorescence intensity. A possible explanation is that the rpIFN-g bound to the antibody was still able to bind to the broadly expressed IFN-g receptor, as has been reported to occur with IL-2 ŽMcIntyre et al., 1994.. However, blocking of staining could be achieved using unlabeled antibody. Solid evidence against non-specific binding was provided by the fact that neither monocytes nor B cells were stained in the same experiments.
177
The percentage of porcine peripheral blood lymphocytes capable of producing IFN-g upon stimulation with PMA plus Ca ionophore A23187, was within the range of values reported in humans using polyclonal activators ŽJung et al., 1993; Prussin and Metcalfe, 1995; Mascher et al., 1999; Vitale et al., 2000.. In adult pigs, this percentage is significantly higher than in young pigs Ž34.2 " 5.7% versus 13.2 " 5.8%, p - 0.001.. Because similar values were obtained in adult Large-White pigs and inbred minipigs, we can exclude the possibility of genetic factors contributing to the differences found between these two age groups. The majority of the IFN-g-producing cells were found to be ab T cells, although a substantial proportion of gd T cells were also able to produce IFN-g, although in lower amounts, according to the mean fluorescence intensity. These gd T cells account for 43.6 " 13.3% of IFN-g-producing cells in young animals, falling to 16.4 " 2.4% in adult pigs. These findings are consistent with previous studies which showed a predominance of gd T cells over ab T cells in young pigs and suggested a strong dependence on innate mechanisms for the cellular immunity of young animals ŽYang and Parkhouse, 1996.. Changes with age in the number and phenotype of IFN-g-producing cells appears to be a reflection of the qualitative and quantitative changes that take place in the peripheral blood of aging pigs. These processes are characterized by an increase in the percentage and absolute number of CD4qCD8q double positive T cells, exhibiting features of memory cells, and point to a dominant role for the memory cell population in IFN-g production in adult animals ŽYang and Parkhouse, 1996; Zuckermann and Husmann, 1996; Summerfield et al., 1996.. Indeed, phenotypic analyses showed that within ab T cells, both CD4q and CD8 high T cells have the capacity to produce large amounts of IFN-g and that CD4qCD8q and CD4q CD45RAy subsets, which include memory T cells, produced more IFN-g than
Fig. 3. IFN-g production by porcine T lymphocyte subsets, determined by three-colour FCM. PBMC treated as in Fig. 1 were stained with the mAb combinations shown in the figure and described in the Materials and methods. Ža. IFN-g expression by CD4 versus CD8 defined T-cell subsets. Žb. Analysis of IFN-g production by CD45RAq and CD45RAy subpopulations of CD4q or CD8q T cells. Quadrants are defined by background staining with irrelevant isotype-matched antibodies. The IFN-g fluorescence for cells in the respective quadrants is displayed as histograms. At least 50,000 cells were acquired and this experiment is representative of the four experiments performed.
178
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
CD4q CD8y or CD4q CD45RAq T lymphocytes did. The capacity of CD4qCD8q T cells to produce high levels of IFN-g has also been reported by Zuckermann and Husmann Ž1996. and Zuckermann Ž1999. in an antigen-specific model using pigs immune to pseudorabies. The difference in the ability to synthesize cytokines between naive and memory T cells has also been seen in humans, where naive cells ŽCD45RAq CD45R0y . are mostly restricted to IL-2 production, whereas memory cells ŽCD45R0q CD45RAy . can produce the whole spectrum of T-cell cytokines ŽJung et al., 1993; Kanegane et al., 1996; Hamann et al., 1997; Mascher et al., 1999.. IFN-g production has been observed in the course of the immune response to several porcine viruses, such as ASFV, PRRSV or pseudorabies virus, and might contribute to the control of their replication in the host ŽRevilla et al., 1992; Zuckermann et al., 1998; Lopez-Fuertes et al., 1999.. The assay de´ scribed here may permit the estimates of the frequency and phenotype of viral-specific IFN-g-producing cells, and help our understanding of the relevance of this cytokine in the protective immunity against these important porcine viral diseases. It may also be useful in the assessment of cell-mediated immunity in vaccine trials.
Acknowledgements We thank Drs. Bernard Charley and Franc¸ois Lefevre for critically reading the manuscript, and Dr. Claude La Bonnardiere for providing the G47 antibody. MP Rodrıguez Carreno ´ ˜ was a recipient of an INIA fellowship. This work was supported by Grants CT96-1317 from European Union and PETRI 950298-OP from CICYT.
References Billiau, A., 1996. Interferon-g: biology and role in pathogenesis. Adv. Immunol. 62, 61. Domınguez, J., Hedrick, R.P., Sanchez-Vizcaıno, ´ ´ ´ J.M., 1990. Use of monoclonal antibodies for detection of infectious pancreatic necrosis virus by the enzyme-linked immunosorbent assay. Dis. Aquat. Org. 8, 157. Farrar, M.A., Schreiber, R.D., 1993. The molecular cell biology of interferon-g and its receptor. Annu. Rev. Immunol. 11, 571.
Gonzalez, S., Mendoza, C., Sanchez-Vizcaıno, ´ ´ ´ J.M., Alonso, F., 1990. Inhibitory effect of African swine fever virus on lectindependent swine lymphocyte proliferation. Vet. Immunol. Immunopathol. 26, 71. Hamann, D., Baars, P.A., Rep, M.H.G., Hooibrink, B., KerkhofGarde, S.R., Klein, M.R., van Lier, R.A.W., 1997. Phenotypic and functional separation of memory and effector human CD8q T cells. J. Exp. Med. 186, 1407. Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C., 1993. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197. Kanegane, H., Kasahara, Y., Niida, A., Yachie, A., Sugii, S., Takatsu, K., Taniguchi, N., Miyawaki, T., 1996. Expression of L-selectin ŽCD62L. discrimates Th1- and Th2-like cytokineproducing memory CD4q T cells. Immunology 87, 186. Lefevre, F., Martinat-Botte, F., Locatelli, A., De Niu, P., Terqui, M., La Bonnardiere, C., 1998. Intrauterine infusion of high doses of pig trophoblast interferons has no antiluteolytic effect in cyclic gilts. Biol. Reprod. 58 Ž4., 1026. Lopez-Fuertes, L., Domenech, N., Alvarez, B., Ezquerra, A., ´ Domınguez, J., Castro, J.M., Alonso, F., 1999. Analysis of ´ cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection. Virus Res. 64, 33. Mascher, B., Schlenke, P., Seyfarth, M., 1999. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J. Immunol. Methods 223, 115. Mateu de Antonio, E., Husmann, R.J., Hansen, R., Lunney, J.K., Strom, D., Martin, S., Zuckermann, F.A., 1998. Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen. Vet. Immunol. Immunopathol. 27, 265. McIntyre, C.A., Horne, C.J., Lawry, J., Rees, R.C., 1994. The detection of intracytoplasmic interleukin-2 in Jurkat E6.1 and human peripheral blood mononuclear cells using direct conjugate, two-colour, immunofluorescent flow cytometry. J. Immunol. Methods 169, 213. Pescovitz, M.D., Lunney, J.K., Sachs, D.H., 1985. Murine antiswine T4 and T8 monoclonal antibodies: distribution and effects on proliferative and cytotoxic T cells. J. Immunol. 134, 37. Prussin, C., Metcalfe, D., 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188, 117. Revilla, Y., Pena, L., Vinuela, E., 1992. Interferon-gamma pro˜ duction by African swine fever virus-specific lymphocytes. Scand. J. Immunol. 35, 225. Saalmuller, A., Reddehase, M.J., Buhring, H.J., Jonjic, S., Koszi¨ nowski, U.H., 1987. Simultaneous expression of CD4 and CD8 antigens on a substantial proportion of resting porcine T lymphocytes. Eur. J. Immunol. 17, 1297. Saalmuller, A., Hirt, W., Reddehase, M.J., 1990. Porcine gr d T ¨ lymphocyte subsets differing in their propensity to home to the lymphoid tissue. Eur. J. Immunol. 20, 2343. Saalmuller, A., Hirt, W., Maurer, S., Weiland, E., 1994. Discrimi¨ nation between two subsets of porcine CD8q cytolytic T lymphocytes by the expression of CD5 antigen. Immunology 81, 578.
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179 Summerfield, A., Rziha, H.J., Saalmuller, A., 1996. Functional ¨ characterization of porcine CD4qCD8qextrathymic T lymphocytes. Cell. Immunol. 168, 291. Vitale, M., Caruso, A., Licenziati, S., Rodella, L., Fiorentini, S., Zauli, G., Castelli, F., Manzoli, F.A., Turano, A., 2000. Differential production of IFN-g, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIVq subjects. Cytometry 39, 189. Yang, H., Parkhouse, R.M.E., 1996. Phenotypic classification of porcine lymphocyte subpopulations in blood and lymphoid tissues. Immunology 89, 76.
179
Zuckermann, F.A., 1999. Extrathymic CD4rCD8 double positive T cells. Vet. Immunol. Immunopathol. 72, 55. Zuckermann, F.A., Husmann, R.J., 1996. Functional and phenotypic analysis of porcine peripheral blood CD4rCD8 doublepositive T cells. Immunology 87, 500. Zuckermann, F.A., Husmann, R.J., Schwartz, R., Brandt, J., Mateu de Antonio, E., Martin, S., 1998. Interleukin-12 enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine. Vet. Immunol. Immunopathol. 63, 57.
Phenotypic characterization of porcine IFN-g-producing lymphocytes by flow cytometry M.P. Rodrıguez-Carreno, C. Revilla, A. Ezquerra, F. Alonso, ´ ˜ L. Lopez-Fuertes, ´ ) J. Domınguez ´ Departamento de Biotecnologıa, ´ INIA, Ctra. de La Coruna ˜ Km 7, 28040 Madrid, Spain Received 27 February 2001; received in revised form 9 July 2001; accepted 28 August 2001
Abstract We have developed a three-colour flow cytometric assay for phenotypic characterization of porcine IFN-g-producing lymphocytes. Analyses of activated swine peripheral blood mononuclear cells ŽPBMC. showed a significant difference in the proportion of IFN-g producing cells between young and adult animals Ž13.2 " 5.8% versus 34.2 " 5.7%.. The majority of IFN-g producing cells were ab T lymphocytes, although there was also an important proportion of gd T cells particularly in young animals. Within the ab T lymphocytes, the double positive CD4qCD8 lo subset, that contains memory T cells, produced high levels of IFN-g, whereas the CD8 hi T cells ranged from low to high levels of IFN-g. Also, consistent with a higher production by memory T cells, the CD45RAy subset of both CD4q and CD8q cells contained higher numbers of IFN-g producing cells than the CD45RAq subset. Finally, no production of IFN-g by either B cells ŽCD21q. or monocytes ŽSWC3q. was detected. This assay may be useful for the assessment of cell-mediated immunity in vaccine trials and may contribute to our understanding of the role of IFN-g in protective immunity against important viral diseases of the pig. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Flow cytometry; Intracellular staining; Lymphocyte subsets; Monoclonal antibodies; Porcine IFN-g
1. Introduction IFN-g is a pleiotropic cytokine that plays important roles in the regulation of inflammatory and immune responses, being particularly relevant in the host defense against intracellular pathogens. Among its multiple biological effects, IFN-g is a major activator of macrophages, enhancing their antimicrobial activity and their capacity for processing and
)
Corresponding author. Fax: q34-91-3572-293. .. E-mail address: [email protected] ŽJ. Domınguez ´
presenting antigens to T lymphocytes. In addition, it regulates Ig isotype switching on B cells and inhibits proliferation of Th2 cells, shifting the balance towards a Th1-type response ŽFarrar and Schreiber, 1993; Billiau, 1996.. In humans and rodents, IFN-g is produced by T lymphocytes and natural killer cells. However, the nature of porcine IFN-g-producing cells has not yet been elucidated. Compared with other species, the porcine immune system shows striking differences in the peripheral T-lymphocyte compartment. Based on the expression of CD4 and CD8 antigens, four Tlymphocyte subsets can be identified in peripheral
0022-1759r02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 5 1 1 - 7
172
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
blood and in lymphoid tissues. In addition to the classical phenotypes of CD4qCD8y helper T cells and CD8qCD4y cytolytic T lymphocytes, there is a double negative CD4yCD8y T-cell subset, also found in ruminants which contains the majority of gd T cells, and a double positive CD4qCD8q T-cell subset, unique to the pig, containing memory ab T cells ŽPescovitz et al., 1985; Saalmuller et al., 1987, ¨ 1990; Yang and Parkhouse, 1996.. The development of monoclonal antibodies ŽmAbs. against porcine IFN-g and their use in ELISA and ELISPOT assays has been recently described ŽLefevre et al., 1998; Mateu de Antonio et al., 1998; Zuckermann et al., 1998., although no attempts to detect porcine IFN-g production using flow cytometry have been reported. The aim of the present study was to characterize the phenotype of IFN-g-producing cells in the pig using multicolour flow cytometry. This assay makes it possible to determine not only the percentage of cells within a particular subset, but also to compare the relative amount of IFN-g produced by different subsets.
2. Materials and methods 2.1. Animals and cells Five Large-White outbred pigs from 3 to 6 months old, and three inbred minipigs of dd haplotype and four Large-White outbred pigs older than 3 years were used as blood donors. Pigs were managed according to the animal care regulations. Peripheral blood mononuclear cells ŽPBMC. were isolated on Percoll discontinuous gradients after blood sedimentation, through dextran as previously described ŽGonzalez ´ et al., 1990.. Cells were resuspended in RPMI 1640 medium containing 10% fetal calf serum ŽFCS., 2 mM L-glutamine, 5 = 10y5 M 2-mercaptoethanol and 30 mgrml gentamicin Žcomplete medium.. 2.2. Monoclonal antibodies Anti-IFN-g mAb G47 ŽLefevre et al., 1998., a generous gift from C. La Bonnardiere ŽINRA, Jouyen-Josas, France., was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose
CL4B ŽPharmacia, Sweden. and labeled with Alexa Fluor 488 dye following the manufacturer’s protocol ŽMolecular Probes, Eugene, OR, USA.. MAbs to porcine CD3 Ž8E6, IgG2b. and CD4 Ž74-12-4, IgG2b. were kindly provided by M. Pescovitz ŽIndiana University, USA., and CD8 Ž762-11, IgG2a. by J. Lunney ŽARS USDA, Beltsville, USA.. MAb to porcine gd TCR ŽPGBL22A, IgG1. was purchased from VMRD ŽPullman, WA, USA. and mAb anti-porcine CD21 Žclone B-ly4, IgG1. was obtained from Pharmingen ŽSan Diego, USA.. MAbs to porcine CD5 Ž1H6, IgG2a., CD45RA Ž3C3r9, IgG1. and SWC3 ŽBL1H7, IgG1. were developed in our laboratory. For the three-colour immunofluorescence assays, CD4, and CD8 mAbs were purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL4B, and labeled with biotin as described previously ŽDomınguez et al., 1990.. ´ 2.3. Immunofluorescent staining of intracellular IFN-g for flow cytometric analysis PBMC were stimulated for 16 h with PMA Ž50 ngrml. and calcium ionophore A23187 Ž500 ngrml. in the presence of the protein transport inhibitor monensin Ž2 mgrml.. Cells were harvested and incubated on ice with mAbs to different cell surface antigens Ž50 ml of hybridoma supernatantr10 6 cells., for 30 min. After two washes in PBS containing 0.1% bovine serum albumin ŽBSA. and 0.01% sodium azide Žfluorescence buffer, FB., cells were incubated for 30 min at 4 8C with phycoerythrin ŽPE.-conjugated rabbit FŽabX . 2 anti-mouse Ig ŽDako, Denmark.. Free binding sites were blocked with 5% normal mouse serum for 15 min prior to fixationr permeabilization with Permeafix ŽOrtho Diagnostic System, USA. for 45 min at room temperature. Cells were then washed in FB and incubated for 30 min with anti-IFN-g mAb G47 labeled with Alexa 488. After three washes, cells were analyzed on a Facscan cytometer ŽBecton-Dickinson, USA.. For three-colour immunofluorescence, cells were first incubated with unlabeled mAbs to cell surface antigens, followed after washing, by phycoerythrin ŽPE.-conjugated rabbit FŽabX . 2 anti-mouse Ig. Free binding sites were blocked with 5% normal mouse serum for 15 min before adding biotin-labeled antiCD4 or anti-CD8 mAb. After two washes in FB, the
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
biotinylated antibodies were detected with streptavidin-Cy-Chrome ŽPharmingen, USA.. Cells were then fixedrpermeabilized with Permeafix and stained with anti-IFN-g mAb G47 conjugated with Alexa 488, as described above. Irrelevant, isotype-matched mAbs, either unlabeled or labeled with biotin or Alexa 488, were used as negative controls. 2.4. Statistical analysis Comparisons of mean differences of IFN-g-producing cells was done using Student’s t-test. Differences were considered significant if p - 0.05.
3. Results 3.1. Flow cytometric detection of IFN-g production by PBMC following PMA r A23187 stimulation Swine PBMC were activated with a combination of PMA plus calcium ionophore A23187, as an optimal stimulus for IFN-g, in the presence or absence of monensin. Sixteen hours after stimulation, cells were stained for intracellular IFN-g with mAb G47 labeled with Alexa Fluor 488. Treatment with monensin, which blocks protein secretion and leads
173
to the accumulation of IFN-g in the Golgi complex, resulted in an increase in the number of fluorescent cells as well as in the intensity of the fluorescence of the stained cells. No fluorescence signal was detected in unstimulated cells Ždata not shown.. The frequency of IFN-g-producing cells differed between animals, with adult pigs having significantly higher values than young pigs Ž34.2 " 5.7%, n s 8, in adult pigs versus 13.2 " 5.8%, n s 8, in young pigs, p - 0.001.. In fact, a gradual increase in the proportion of IFN-g-producing cells was noted in young animals from the time of first sampling Žat 3 months of age. to the end of the experiment, 3 months later Žaged 6–7 months.. No differences were observed between inbred minipigs and outbred Large-White pigs of the same age-group. Cytometric profiles of one representative animal of each agegroup are shown in Fig. 1. 3.2. Phenotypic characterization of peripheral blood mononuclear cell subsets inÕolÕed in IFN-g production IFN-g production by different PBMC subsets was assessed by multi-colour flow cytometry. In adult pigs, the majority of IFN-g producing cells were CD3-positive Ž88.2 " 6.4%. ŽFig. 2a.. Most of these cells, particularly those with strong staining for IFN-
Fig. 1. Differential production of IFN-g by PBMC from adult and young pigs. PBMC were activated with PMA and Ca ionophore in the presence of monensin, fixedrpermeabilized and stained with Alexa 488-anti-porcine IFN-g mAb G47. As a control, cells stained with an irrelevant isotype matched mAb labeled with Alexa 488 were used Žopen histogram.. At least 20,000 cells were analyzed and the percentages of positive IFN-g producing cells are shown. Results are representative of six independent experiments.
174
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
175
Fig. 2. Phenotype of IFN-g producing cells as defined by two-colour flow cytometry analysis. Panel Ža. PBMC from a 3-year-old pig were stimulated as indicated in Fig. 1 in the presence of monensin, and stained with mAbs to surface antigens followed by PE goat anti-mouse Ig ŽFL2.. Cells were then fixedrpermeabilized and incubated with Alexa 488 conjugated anti-IFN-g mAb G47 ŽFL1.. Panel Žb. represents a comparison of the staining of gd TCR versus IFN-g in 6-month-old Žleft. or 3-year-old Žright. pigs. Results are from one representative experiment out of at least four performed. Quadrants were defined by background staining with irrelevant isotype-matched mAbs. The numbers indicate the percentage of cells within the respective regions. For this analysis, 50,000 PBMC were acquired.
g, expressed lower levels of CD3 than IFN-g negative T cells, and high levels of CD5, a phenotype which has been shown in previous reports to correspond to ab T cells ŽSaalmuller et al., 1994; Yang ¨ and Parkhouse, 1996.. About one-third of CD4q T cells produced IFN-g at high levels ŽTable 1, Fig. 2a.. Within the CD8q T cell population, almost 50% synthesized IFN-g. Whereas IFN-g production by CD8 hi T cells ranged from low to high, a subset of high IFN-g producers was identified within the CD8 lo cells. Low amounts of IFN-g production by almost one third of gd T cells ŽFig. 2a. were noted. These IFN-g producing gd T cells only accounted for 16.4 " 2.4% in adult pigs, while in young pigs,
because of their relatively lower proportion of ab T cells were almost a half Ž43.6 " 13.3%, n s 4, p 0.05. of all IFN-g producing cells ŽFig. 2b.. As expected, no production of IFN-g associated with either B cells ŽCD21q cells. or monocytes ŽSWC3q cells. ŽFig. 2a.. The CD8 lo cell subset of high IFN-g producers, identified by two-colour FCM, probably corresponded with the CD4qCD8q double positive cell subset, which have been shown to contain memory cells ŽZuckermann and Husmann, 1996; Summerfield et al., 1996.. To analyze in more detail the IFN-g production by the different T-cell subsets, we performed three-colour immunofluorescent staining
Table 1 Percentages of IFN-g producing cells within different lymphocyte subsets from adult pigs CD3
CD4q CD8y CD4y CD8q CD4q CD8q CD4q CD45RAy CD4q CD45RAq CD8q CD45RAy CD8q CD45RAq
x " S.D. 35.4 " 2.0 19.6 " 3.8 ns3 ns4 p
32.4 " 9.8 ns4
50.3 " 5.8 ns7
36.4 " 8.9 18 " 4.5 ns5 ns5 p - 0.01
41.5 " 8.0 13.5 " 4.2 ns4 ns4 p - 0.01
Values represent mean " standard deviation Ž x " S.D... Number of animals analyzed in each group is given as n. P values were calculated using Student’s t-test.
176
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
ŽFig. 3.. Porcine PBMC were resolved by simultaneous staining with anti-CD4 and anti-CD8 mAbs, into four subpopulations. The CD4qCD8q subset contained the highest percentage of IFN-g producing cells, followed by the CD4yCD8q subset and to a lesser extent by CD4q CD8y T cells ŽFig. 3a.. When CD4q or CD8q T cells were subdivided by CD45RA staining, CD45RAy T cells proved to be the main contributors to IFN-g production ŽTable 1, Fig. 3b..
4. Discussion In this report, we describe a simple and reliable method for the identification of porcine IFN-g producing cells by flow cytometry using mAb G47. The simultaneous examination of surface phenotype and intracytoplasmic cytokine expression permits an objective comparison of the relative amounts of IFN-g produced by the different lymphocyte subsets. Labeling of the anti-IFN-g mAb with the dye Alexa 488 avoids the use of second layer antibodies, reducing the number of controls required for multi-layer protocols as well as the possibilities of non-specific binding. Attempts to establish the specificity of staining by blocking, the binding of anti-IFN-g mAb to intracellular IFN-g by previous co-incubation with a molar excess of recombinant porcine IFN-g ŽrpIFNg . were unsuccessful. Instead of reducing the binding, this treatment increased both the number of positive cells and the fluorescence intensity. A possible explanation is that the rpIFN-g bound to the antibody was still able to bind to the broadly expressed IFN-g receptor, as has been reported to occur with IL-2 ŽMcIntyre et al., 1994.. However, blocking of staining could be achieved using unlabeled antibody. Solid evidence against non-specific binding was provided by the fact that neither monocytes nor B cells were stained in the same experiments.
177
The percentage of porcine peripheral blood lymphocytes capable of producing IFN-g upon stimulation with PMA plus Ca ionophore A23187, was within the range of values reported in humans using polyclonal activators ŽJung et al., 1993; Prussin and Metcalfe, 1995; Mascher et al., 1999; Vitale et al., 2000.. In adult pigs, this percentage is significantly higher than in young pigs Ž34.2 " 5.7% versus 13.2 " 5.8%, p - 0.001.. Because similar values were obtained in adult Large-White pigs and inbred minipigs, we can exclude the possibility of genetic factors contributing to the differences found between these two age groups. The majority of the IFN-g-producing cells were found to be ab T cells, although a substantial proportion of gd T cells were also able to produce IFN-g, although in lower amounts, according to the mean fluorescence intensity. These gd T cells account for 43.6 " 13.3% of IFN-g-producing cells in young animals, falling to 16.4 " 2.4% in adult pigs. These findings are consistent with previous studies which showed a predominance of gd T cells over ab T cells in young pigs and suggested a strong dependence on innate mechanisms for the cellular immunity of young animals ŽYang and Parkhouse, 1996.. Changes with age in the number and phenotype of IFN-g-producing cells appears to be a reflection of the qualitative and quantitative changes that take place in the peripheral blood of aging pigs. These processes are characterized by an increase in the percentage and absolute number of CD4qCD8q double positive T cells, exhibiting features of memory cells, and point to a dominant role for the memory cell population in IFN-g production in adult animals ŽYang and Parkhouse, 1996; Zuckermann and Husmann, 1996; Summerfield et al., 1996.. Indeed, phenotypic analyses showed that within ab T cells, both CD4q and CD8 high T cells have the capacity to produce large amounts of IFN-g and that CD4qCD8q and CD4q CD45RAy subsets, which include memory T cells, produced more IFN-g than
Fig. 3. IFN-g production by porcine T lymphocyte subsets, determined by three-colour FCM. PBMC treated as in Fig. 1 were stained with the mAb combinations shown in the figure and described in the Materials and methods. Ža. IFN-g expression by CD4 versus CD8 defined T-cell subsets. Žb. Analysis of IFN-g production by CD45RAq and CD45RAy subpopulations of CD4q or CD8q T cells. Quadrants are defined by background staining with irrelevant isotype-matched antibodies. The IFN-g fluorescence for cells in the respective quadrants is displayed as histograms. At least 50,000 cells were acquired and this experiment is representative of the four experiments performed.
178
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179
CD4q CD8y or CD4q CD45RAq T lymphocytes did. The capacity of CD4qCD8q T cells to produce high levels of IFN-g has also been reported by Zuckermann and Husmann Ž1996. and Zuckermann Ž1999. in an antigen-specific model using pigs immune to pseudorabies. The difference in the ability to synthesize cytokines between naive and memory T cells has also been seen in humans, where naive cells ŽCD45RAq CD45R0y . are mostly restricted to IL-2 production, whereas memory cells ŽCD45R0q CD45RAy . can produce the whole spectrum of T-cell cytokines ŽJung et al., 1993; Kanegane et al., 1996; Hamann et al., 1997; Mascher et al., 1999.. IFN-g production has been observed in the course of the immune response to several porcine viruses, such as ASFV, PRRSV or pseudorabies virus, and might contribute to the control of their replication in the host ŽRevilla et al., 1992; Zuckermann et al., 1998; Lopez-Fuertes et al., 1999.. The assay de´ scribed here may permit the estimates of the frequency and phenotype of viral-specific IFN-g-producing cells, and help our understanding of the relevance of this cytokine in the protective immunity against these important porcine viral diseases. It may also be useful in the assessment of cell-mediated immunity in vaccine trials.
Acknowledgements We thank Drs. Bernard Charley and Franc¸ois Lefevre for critically reading the manuscript, and Dr. Claude La Bonnardiere for providing the G47 antibody. MP Rodrıguez Carreno ´ ˜ was a recipient of an INIA fellowship. This work was supported by Grants CT96-1317 from European Union and PETRI 950298-OP from CICYT.
References Billiau, A., 1996. Interferon-g: biology and role in pathogenesis. Adv. Immunol. 62, 61. Domınguez, J., Hedrick, R.P., Sanchez-Vizcaıno, ´ ´ ´ J.M., 1990. Use of monoclonal antibodies for detection of infectious pancreatic necrosis virus by the enzyme-linked immunosorbent assay. Dis. Aquat. Org. 8, 157. Farrar, M.A., Schreiber, R.D., 1993. The molecular cell biology of interferon-g and its receptor. Annu. Rev. Immunol. 11, 571.
Gonzalez, S., Mendoza, C., Sanchez-Vizcaıno, ´ ´ ´ J.M., Alonso, F., 1990. Inhibitory effect of African swine fever virus on lectindependent swine lymphocyte proliferation. Vet. Immunol. Immunopathol. 26, 71. Hamann, D., Baars, P.A., Rep, M.H.G., Hooibrink, B., KerkhofGarde, S.R., Klein, M.R., van Lier, R.A.W., 1997. Phenotypic and functional separation of memory and effector human CD8q T cells. J. Exp. Med. 186, 1407. Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C., 1993. Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197. Kanegane, H., Kasahara, Y., Niida, A., Yachie, A., Sugii, S., Takatsu, K., Taniguchi, N., Miyawaki, T., 1996. Expression of L-selectin ŽCD62L. discrimates Th1- and Th2-like cytokineproducing memory CD4q T cells. Immunology 87, 186. Lefevre, F., Martinat-Botte, F., Locatelli, A., De Niu, P., Terqui, M., La Bonnardiere, C., 1998. Intrauterine infusion of high doses of pig trophoblast interferons has no antiluteolytic effect in cyclic gilts. Biol. Reprod. 58 Ž4., 1026. Lopez-Fuertes, L., Domenech, N., Alvarez, B., Ezquerra, A., ´ Domınguez, J., Castro, J.M., Alonso, F., 1999. Analysis of ´ cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection. Virus Res. 64, 33. Mascher, B., Schlenke, P., Seyfarth, M., 1999. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J. Immunol. Methods 223, 115. Mateu de Antonio, E., Husmann, R.J., Hansen, R., Lunney, J.K., Strom, D., Martin, S., Zuckermann, F.A., 1998. Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen. Vet. Immunol. Immunopathol. 27, 265. McIntyre, C.A., Horne, C.J., Lawry, J., Rees, R.C., 1994. The detection of intracytoplasmic interleukin-2 in Jurkat E6.1 and human peripheral blood mononuclear cells using direct conjugate, two-colour, immunofluorescent flow cytometry. J. Immunol. Methods 169, 213. Pescovitz, M.D., Lunney, J.K., Sachs, D.H., 1985. Murine antiswine T4 and T8 monoclonal antibodies: distribution and effects on proliferative and cytotoxic T cells. J. Immunol. 134, 37. Prussin, C., Metcalfe, D., 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188, 117. Revilla, Y., Pena, L., Vinuela, E., 1992. Interferon-gamma pro˜ duction by African swine fever virus-specific lymphocytes. Scand. J. Immunol. 35, 225. Saalmuller, A., Reddehase, M.J., Buhring, H.J., Jonjic, S., Koszi¨ nowski, U.H., 1987. Simultaneous expression of CD4 and CD8 antigens on a substantial proportion of resting porcine T lymphocytes. Eur. J. Immunol. 17, 1297. Saalmuller, A., Hirt, W., Reddehase, M.J., 1990. Porcine gr d T ¨ lymphocyte subsets differing in their propensity to home to the lymphoid tissue. Eur. J. Immunol. 20, 2343. Saalmuller, A., Hirt, W., Maurer, S., Weiland, E., 1994. Discrimi¨ nation between two subsets of porcine CD8q cytolytic T lymphocytes by the expression of CD5 antigen. Immunology 81, 578.
M.P. Rodrıguez-Carreno ´ ˜ et al.r Journal of Immunological Methods 259 (2002) 171–179 Summerfield, A., Rziha, H.J., Saalmuller, A., 1996. Functional ¨ characterization of porcine CD4qCD8qextrathymic T lymphocytes. Cell. Immunol. 168, 291. Vitale, M., Caruso, A., Licenziati, S., Rodella, L., Fiorentini, S., Zauli, G., Castelli, F., Manzoli, F.A., Turano, A., 2000. Differential production of IFN-g, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIVq subjects. Cytometry 39, 189. Yang, H., Parkhouse, R.M.E., 1996. Phenotypic classification of porcine lymphocyte subpopulations in blood and lymphoid tissues. Immunology 89, 76.
179
Zuckermann, F.A., 1999. Extrathymic CD4rCD8 double positive T cells. Vet. Immunol. Immunopathol. 72, 55. Zuckermann, F.A., Husmann, R.J., 1996. Functional and phenotypic analysis of porcine peripheral blood CD4rCD8 doublepositive T cells. Immunology 87, 500. Zuckermann, F.A., Husmann, R.J., Schwartz, R., Brandt, J., Mateu de Antonio, E., Martin, S., 1998. Interleukin-12 enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine. Vet. Immunol. Immunopathol. 63, 57.