Memory T lymphocytes in human breast milk
Eur. J. Immunol. 1990. 20: 1877-1880
1877
Short paper Albert0 Bertotto+, Roberto Gerlia, Giulio Fabietti+, Silvana Crupi+, Carla Arcangeli+, Francesco Scalise+ and Renato Vaccaro+ Departments of Paediatrics+ and Internal MedicineA,Perugia University Medical School, Perugia
Human breast milk T lymphocytes display the phenotype and functional characteristics of memory T cells Naive (unsensitized) and memory (antigen-primed) Tcells can be phenotypically distinguished on the basis of the high or low intensity with which they express a number of immunologically relevant lymphocyte membrane antigens, including CD45R, CDw29, UCHL1, LFA-1, LFA-3, CD2 and Pgp-1. Here we report that in contrast to the two major Tcell subsets found in the blood, milk T lymphocytes are almost exclusively composed of the one which exhibits the CD45RIoW,CDw29, UCHL1, LFA-lhigh memory T cell phenotype. In addition, while milk and autologous blood cells expressed similar levels of CD3 surface antigens, CD2 and ICAM-1 expression was approximately twofold greater on the milk T lymphocytes. This agrees with the finding that whereas colostrum Tcells respond poorly to PHA, they proliferate and produce interferon-y normally when stimulated with either the anti-CD3 or anti-CD2 monoclonal antibodies. The selective colonization of the mammary gland during lactation by a population of T lymphocytes which displays the phenotype and functional characteristics of memory T cells may be one of the mechanisms whereby the suckling infant benefits from its mother's immunological experience.
1 Introduction Recent data strongly suggest that adult peripheral blood T lymphocytes can be phenotypically subdivided into naive (unsensitized) and memory (antigen-primed) Tcells on the basis of the intensity (high or low) with which they express a number of cell surface molecules involved in both intercellular adhesion (LFA-1, LFA-3, CD2) and Tcell activation (CD2, CD45R, CDw29, UCHLl, Pgp-1) [l]. Naive T lymphocytes (CD45Rhigh,LFA-1, LFA-3, CD2, CDw29, U C H L - and Pgp-1IoW)proliferate vigorously in response to the mitogen PHA, whereas their responsiveness to activation signals induced by recall antigens and mAb that bind t o either the CD2 or CD3 membrane receptor is depressed. In contrast, memory Tcells (CD45RIoW,LFA-1, LFA-3, CD2, CDw29, UCHLl and Pgp-lhi@) respond poorly to PHA, but are capable of mounting a rapid anamnestic response to antigens to which the donor has previously been immunized [1-31. Furthermore, they are more sensitive to CD2- and CD3-mediated activation [3] and respond much better than naive cells to alloantigens under experimental conditions of restimulation [4]. Naive and memory T lymphocytes functionally differ from each other not only in their activation requirements, but also in lymphokine secretion. mAb-activated memory T cells secrete large amounts of I F N y and IL 3 and IL 4, while the naive counterparts do not. Conversely, naive and memory lymphocytes produce comparable quantities of IL 2 in response to the polyclonal Tcell activator PHA [l-3, 51.
Human colostrum and early milk contain both helperinducer (CD3+, CD4+) and suppressor-cytotoxic (CD3+, CD8+) T lymphocytes [6]. However, the great majority of CD3+ colostral Tcells express the CDw29 surface antigen [7], thereby suggesting that they may be antigen-pulsed T cells capable of mounting a secondary immune response. Evidence supporting this hypothesis includes: the scant proliferative response of milk T lymphocytes to PHA [8, 91, their good responsiveness to a variety of bacterial and viral antigens [8,10-121 and the detection of significant amounts of heat-labile IFN in SN harvested from colostral leukocyte cultures stimulated by mitogens or Newcastle disease virus [13].With these findings in mind, we designed experiments to determine further the phenotypic and functional characteristics of human colostrum and milk T lymphocytes.
2 Materials and methods 2.1 Cell preparations
Paired samples of milk and peripheral blood were collected by sterile techniques from eight lactating healthy mothers between days 4 and 6 after delivery of normal-term infants. Milk and blood T lymphocytes were positively selected by incubation with a mixture of magnetic beads coated with either anti-CD4 or anti-CD8 mAb (Pell-Freez, Brown Deer, WI) according to the manufacturer's suggestions. Magnetic immunoselection yielded a > 95% CD3+ cell population (immunofluorescence analysis) in both milk and blood lymphocyte suspensions. To avoid potential perturbation of cells by bead-coupled anti-CD4 and anti-CD8 mAb [14], milk and blood lymphocytes from four postpartum donors were simultaneously separated by positive [I 83761 immunoselection or equilibrium density gradient centrifuCorrespondence: Albert0 Bertotto, Department of Paediatrics, gation [6]. As both procedures yielded similar results in terms of cell proliferation and IFN-y production (see Perugia University Medical School, 1-06100Perugia, Italy 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0808-1877$3.50+ .25/0
1878
Eur. J. Immunol. 1990. 20: 1877-1880
A. Bertotto, R. Gerli, G. Fabietti et al.
below), only the data obtained with mAb-coated beads are given. 2.2 F C M analysis
Phenotypic studies of milk and blood T cells were performed by an indirect immunofluorescence staining technique. mAb used for staining were Leu-4 (anti-CD3; Becton Dickinson, Mountain View, CA), T11 (anti-CD2), 2H4 (anti-CD45RA), 4B4 (anti-CDw29; Coulter Immunology, Hialeah, FL), anti-LFA-1 (CDlla; Immunotech, Marseille, France), anti-UCHL1 (CD45RO; Dakopatts, Glostrup, Denmark) and anti-ICAM-1 (CD54 84H10, a generous gift from Dr. F? Mannoni, INSERM Unit 119, Marseille, France). Briefly, 2 x lo5 cells are incubated for 30 min at 4°C with saturating concentrations of each antibody or with identical concentrations of isotypematched mAb that do not react with human cells.The cells were then washed twice and reincubated for 30 min at 4 "C with FITC-conjugated goat anti-mouse F(ab')z subclassspecific antisera absorbed with human Ig (Tago, Inc., Burlingame, CA). After two more washes, mAb binding to the cell preparations was assessed by FCM (FACScan, Becton Dickinson). Data were collected on 20000 celldsample and analyzed by a Hewlett Packard (Palo Alto, CA) computer.
Gibco, Grand Island, NY), anti-CD3 antibody (50 ng/ml; OKT3, Ortho, Raritan, NJ) or a combination of anti-CD2 mAb (1/400 dilution of ascites fluid for each reagent; Tllz and T113, kindly supplied by Dr. S. F. Schlossman, Harvard Medical School, Boston, MA) were included in the medium. Proliferation was measured by pulsing cells with 0.5 pCi (= 18.5 kBq) of [3H]dThd (sp. act. 25 CVmmol, Amersham Int., Amersham, GB) added 8 h prior to harvesting. Results are expressed as the arithmetic mean cpm k SEM of triplicate cultures corrected by subtraction of background cpm. 2.4 Assay for IFN-y
IFN-y was measured in SN harvested after 48 h of anti-CD3 or anti-CD2 mAb stimulation with a comercially available radioimmunoassay (Centocor, Malvern, PA) according to the test kit instructions.
3 Results 3.1 Phenotypic characterization of milk and blood T cells
Previous experiments carried out in our laboratories have demonstrated that milk lymphocyte suspensions contain a strikingly low number of T cells expressing the naive 2.3 Prolieration assay phenotype (2H4/CD45R+) and an overwhelming preponderance of CDw29+ memory T lymphocytes. This contrasts Cell proliferation was assayed under standard conditions, with the picture in the blood,where the two populations are as previously described [15]. In brief, milk or blood T of approximately equal size [7]. We have now extended our lymphocytes were cultured at 3 x 104/well in 96-well studies and have investigated the intensity with which milk round-bottom microtiter plates (Nunc, Roskilde, Den- and blood lymphocytes express a number of immunologimark) in a 6% COz humidified incubator for 72 h. Acces- cally relevant Tcell surface markers, including CD45R and sory cells (6 x 103/well)were provided in all cultures by CDw29, involved in the irreversible shift from naive to a addition of irradiated (3000 rad) milk or blood plastic- memory Tcell state [l-31. Results from a representative adherent monocytes. To activate T cells, PHA-M (1% ; experiment are shown in Fig. 1. Histograms of simulta-
CDr29
C OMT ROL
C D 45 RO
(4B4)
( UCHLl )
CD11a (LFA-1)
10
100
1ooo
10
loo
1ooo
lo
100
FLUOREICEMCE
lo00
10
100
1000
(YV )
Figure I. Phenotypic analysis of milk and blood T cells prepared by using positive magnetic immunoselection. Histograms represent superimposedprofiles of milk (brokenline) and autologous blood (solid line) T lymphocytes stained with the mAb of indicated specificity. Data are from a single experiment representative of eight separate experiments.
Eur. J. Immunol. 1990.20: 1877-1880
Memory T lymphocytes in human breast milk
Table 1. Proliferative responses to PHA and anti-CD3(OKT3)or anti-CD2 (Tllz + Tl13) mAb in milk and blood T lymphocyte suspensions obtained from eight post-partum donors") Source of
Mitogenic stimulush)
lymphocytes PHA
*
10.0 2.W) 50.1 f 6.5
Milk
Blood
Anti-CD3
Anti-CD2
7.1 f 1.8 8.6 rt 1.5
12.9 k 2.3
14.0
1879
anti-CD2 mAb were used as mitogenic agents (Table 1). This suggests that the differences documented with PHA were not due to an overall dichotomy in the proliferative capacity of milk and blood T cells. Furthermore, after triggering via either the CD3 or CD2 surface antigens, milk and blood lymphocytes produced similar amounts of IFN-y (Table 2), thereby indicating that T lymphocytes may be one of the cellular sources for the secretion of this lymphokine in human colostrum and milk [13].
2.1
a) T cells were prepared by positive magnetic immunoselection and the cultures supplemented with 20% of milk or blood irradiated accessory cells (plastic-adherentmonocytes). b) Mean SEM of net cpm X (see Sect. 2.3). c) p < 0.001 (Student's t-test for unpaired data).
4 Discussion
Although it is unlikely that milk T lymphocytes are merely contaminating blood-borne Tcells, their origin and biological significance are still a matter of conjecture.The present investigation shows that the phenotype and functional repertoire of milk Tcells are similar t o those of the memory Table2. IFN-y production by milk and autologous blood T cell subsets found not only in blood [l-31 but also in T lymphocytes following anti-CD3 (OKT3) or anti-CD2 certain body microenvironments [19-231. It would, there(Tllz + Tl13) mAb stimulationa) fore, seem reasonable to assume that immune system cells are compartmentalized in the mammary gland during lactation and that T lymphocytes do not accumulate Source of lymphocytes IFN-y (U/ml)b) randomly in colostrum, but rather are directed there by a AntLCD3 Anti-CD2 selective homing process. The fact that cells in mammary secretions display in virro parameters of immunocompe39.5 f 9.6 Milk 20.6 6.5 tence and often possess antigenic reactivities different from 33.8 10.7 22.3 6.2 Blood those of the PBL [9, 11, 121 would seem to support this proposal. However, it is still far from clear whether a) Tcells were prepared and the cultures set up as detailed in antigen-responsive milk lymphocytes, other cells, or lymTable 1. phokines and other subcellular factors are able to convey b) Mean & SEM of six consecutive experiments. specific and nonspecific immunoprotection to the suckling infant. The recent demonstration in experimental animal models that enterally administered human milk leukocytes neously stained .milk and autologous blood T cells have adhere to the gut epithelium or lie intramurally and persist been superimposed to facilitate comparison. Unlike blood in the intestine for at least 60 h after a single breast feed T cells, which manifested a bimodal phenotypic picture, suggests that these cells may be involved in the host's local most milk T lymphocytes were 2H4/CD45RP and displayed immune response [24]. It is worth noting that memory T only a single major fluorescence peak which approached lymphocytes predominate in the bowel wall [19, 22, 231, the high expression seen in the CDw29+, UCHLl+ and skin [20] and on the lung epithelial surfaces [21] of normal LFA-l+ circulating counterparts. In addition, while milk subjects. Such cells could have undergone on-site converand blood cells expressed identical levels of CD3 surface sion to the memory phenotype upon exposure to exogenous antigens, CD2 expression was approximately twofold high- antigens and/or have homed there as fully fledged memory er on the milk T lymphocytes. Similar results were obtained Tcells to aid in eliminating infectious agents. Human milk with a mAb directed against ICAM-1, a putative LFA-1 has been shown to protect the neonate against gastrointesligand molecule [161involved in both intercellular adhesion tinal, systemic and respiratory tract infections [25-291. [ 171 and T cell activation [ 181. These findings demonstrate Whatever the underlying cellular and/or molecular that of the two major T lymphocyte subsets found in adult mechanisms implicated in this adoptive protection are, the blood, the one bearing the CD45R10W,CDw29, UCHLl, demonstration of an overexpanded population of memory LFA-lhighmemory Tcell phenotype predominates in human Tcells in human breast milk provides evidence that breast early milk. This is further supported by the detection of feeding may help to maintain immunological homeostasis higher expression levels of CD2 and ICAM-1 surface during a period of life when the development of the structures on colostral T cells. immune system is incomplete and little information has been committed to the immunological memory.
*
*
**
3.2 Proliferation and IFN-y production by milk and blood T cells
Received February 15, 1990.
Milk and blood T lymphocytes were tested for their ability to proliferate in response to PHA and anti-CD3 or anti-CD2 mAb. As in previously published data [S, 91, the proliferative response of milk T cells to PHA was significantly lower than that of blood lymphocyte cultures. In contrast, milk and blood Tcells displayed comparable levels of reactivity when anti-CD3 or an appropriate pair of
5 References 1 Sanders,M. E., Makgoba, M.W., Sharrow, S. O., Stephany, D., Springer,T.A.,Young, H. A. and Shaw, S., J. Immunol. 1988. 140:
1401.
2 Sanders,M. E., Makgoba, M.W. and Shaw, S., Immunol. Today
1988. 9: 195.
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A. Bertotto, R. Gerli, G. Fabietti et al.
3 Sanders, M. E., Makgoba, M. W., June, C. H.,Young, H. A. and Shaw, S., Eur. J. Immunol. 1989. 19: 803. 4 Akbar, A. N., Terry, L., Timms, A., Beverley, P. C. L. and Janossy, G., J. Immunol. 1988. 140: 2171. 5 Bettens, F.,Walker, C., Gauchat, J. F.,Wyss,T. and Pichler,W. J., Eur. J. Immunol. 1989. 19: 1569. 6 Richie, E. R., Bass, R., Meistrich, M. L. and Dennison, D. K., J. Immunol. 1982. 129: 1116. 7 Bertotto, A.,Vaccaro, R.,Tognellini, R. and Toppetti, L., Acta Paed. Scand. 1990, in press. 8 Ogra, S. S. and Ogra, P. L., J. Pediutr. 1978. 92: 550. 9 Oksemberg, J. R., Persitz, E. and Brautbar, C., A m . J. Reprod. Immunol. Microbiol. 1985. 8: 125. 10 Smith, C. W. and Goldman, A. S., Pediatr. Res. 1968. 2: 103. 11 Parmely, M. J., Beer, A. E. and Billingham, R. E., J. Exp. Med. 1976. 144: 358. 12 Ruben, F. L., Holzman, I. R. and Fireman, P., A m . J. Obstet. Gynecol. 1982. 143: 518. 13 Lawton, J. W. M., Shortridge, K. F. ,Wong, R. L. C. and Ng, M. H., Arch. Dis. Child. 1979. 54: 127. 14 Janeway, C. A., Immunol. Today 1989. 10: 234. 15 Gerli, R., Bertotto, A., Crupi, S., Arcangeli, C., Marinelli, I., Spinozzi, F., Cernetti, C., Angelella, P. and Rambotti, P., J. Immunol. 1989. 142: 2583.
Eur. J. Immunol. 1990.20: 1877-1880 16 Makgoba, M.W., Sanders, M. E., Ginther Luce, G. E., Dustin, M. L., Springer,T. A., Clark, E. A., Mannoni, P. and Shaw, S., Nature 1988. 331: 86. 17 Patarroyo, M. and Makgoba, M.W., Scand. J. Imrnunol. 1989. 30: 129. 18 Dougherty, G. J., Murdoch, S. andHogg, N., Eur. J. Immunol. 1988. 18: 35. 19 James, S. l?, Fiocchi, C., Graeff, A. S. and Strober, W., Gastroenterology 1986. 91: 1483. 20 Bos, J. D., Zonneveld, I. and Das, I? K., J. Invest. Dermatol. 1987. 88: 569. 21 Saltini, C.,Winestock, K., Kirby, M. and Crystal, R. G., Clin. Res. 1988. 36: 446A (abstract). 22 Janossy, G., Bofill, M., Rowe, D., Muir, J. and Beverley, l? C. L., Immunology 1989. 66: 517. 23 Brandtzaeg, P.,Bosnes,V., Halstensen,T. S., Scott, H., Sollid, L. M. and Valnes, K. N., Scand. J. Immunol. 1989. 30: 123. 24 Jain, L. P.,Vidyasagar, D., Xanthou, M., Ghai,V., Shimada, S. and Blend, M., Arch. Dis. Child. 1989. 641s: 930. 25 Winberg, J. and Wessner, G., Lancet 1971. i: 1091. 26 Schlesinger, J. J. and Covelli, H. D., Lancet 1977. ii: 529. 27 Chandra, R. W., Acta Paediatr. Scand. 1979. 68: 691. 28 Rogers, H. J. and Synge, C., Immunology 1979. 34: 19. 29 Cunningham, A. S., J. Pediatr. 1979. 95: 685.
Announcement International Meeting 25 Years of ImmunoenzymaticTechniques Hilton, Athens, Greece September 9-12, 1991 Organizing Committee: S. Avrameas (France), F! K. Nakane (Japan), A. Pesce (USA), M. Papamichael (Greece)
Main topics will include: immunoassays, amplification principles, nucleic acid probes, immunocytochemical procedures, new approaches, contribution of immunodiagnosis in various diseases. Organizing Committee Secretariat: Triaena Congress, 24, Harilaou Trikoupi str., Athens 10679, Greece Tel.: 30-1-3622970; Fax: 30-1-3607962
The meeting will include plenary session lectures providing critical reviews of specific areas in the field of immunoenzymatic techniques, workshops and poster presentations.
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Eur. J. Immunol. 1990. 20: 1877-1880
1877
Short paper Albert0 Bertotto+, Roberto Gerlia, Giulio Fabietti+, Silvana Crupi+, Carla Arcangeli+, Francesco Scalise+ and Renato Vaccaro+ Departments of Paediatrics+ and Internal MedicineA,Perugia University Medical School, Perugia
Human breast milk T lymphocytes display the phenotype and functional characteristics of memory T cells Naive (unsensitized) and memory (antigen-primed) Tcells can be phenotypically distinguished on the basis of the high or low intensity with which they express a number of immunologically relevant lymphocyte membrane antigens, including CD45R, CDw29, UCHL1, LFA-1, LFA-3, CD2 and Pgp-1. Here we report that in contrast to the two major Tcell subsets found in the blood, milk T lymphocytes are almost exclusively composed of the one which exhibits the CD45RIoW,CDw29, UCHL1, LFA-lhigh memory T cell phenotype. In addition, while milk and autologous blood cells expressed similar levels of CD3 surface antigens, CD2 and ICAM-1 expression was approximately twofold greater on the milk T lymphocytes. This agrees with the finding that whereas colostrum Tcells respond poorly to PHA, they proliferate and produce interferon-y normally when stimulated with either the anti-CD3 or anti-CD2 monoclonal antibodies. The selective colonization of the mammary gland during lactation by a population of T lymphocytes which displays the phenotype and functional characteristics of memory T cells may be one of the mechanisms whereby the suckling infant benefits from its mother's immunological experience.
1 Introduction Recent data strongly suggest that adult peripheral blood T lymphocytes can be phenotypically subdivided into naive (unsensitized) and memory (antigen-primed) Tcells on the basis of the intensity (high or low) with which they express a number of cell surface molecules involved in both intercellular adhesion (LFA-1, LFA-3, CD2) and Tcell activation (CD2, CD45R, CDw29, UCHLl, Pgp-1) [l]. Naive T lymphocytes (CD45Rhigh,LFA-1, LFA-3, CD2, CDw29, U C H L - and Pgp-1IoW)proliferate vigorously in response to the mitogen PHA, whereas their responsiveness to activation signals induced by recall antigens and mAb that bind t o either the CD2 or CD3 membrane receptor is depressed. In contrast, memory Tcells (CD45RIoW,LFA-1, LFA-3, CD2, CDw29, UCHLl and Pgp-lhi@) respond poorly to PHA, but are capable of mounting a rapid anamnestic response to antigens to which the donor has previously been immunized [1-31. Furthermore, they are more sensitive to CD2- and CD3-mediated activation [3] and respond much better than naive cells to alloantigens under experimental conditions of restimulation [4]. Naive and memory T lymphocytes functionally differ from each other not only in their activation requirements, but also in lymphokine secretion. mAb-activated memory T cells secrete large amounts of I F N y and IL 3 and IL 4, while the naive counterparts do not. Conversely, naive and memory lymphocytes produce comparable quantities of IL 2 in response to the polyclonal Tcell activator PHA [l-3, 51.
Human colostrum and early milk contain both helperinducer (CD3+, CD4+) and suppressor-cytotoxic (CD3+, CD8+) T lymphocytes [6]. However, the great majority of CD3+ colostral Tcells express the CDw29 surface antigen [7], thereby suggesting that they may be antigen-pulsed T cells capable of mounting a secondary immune response. Evidence supporting this hypothesis includes: the scant proliferative response of milk T lymphocytes to PHA [8, 91, their good responsiveness to a variety of bacterial and viral antigens [8,10-121 and the detection of significant amounts of heat-labile IFN in SN harvested from colostral leukocyte cultures stimulated by mitogens or Newcastle disease virus [13].With these findings in mind, we designed experiments to determine further the phenotypic and functional characteristics of human colostrum and milk T lymphocytes.
2 Materials and methods 2.1 Cell preparations
Paired samples of milk and peripheral blood were collected by sterile techniques from eight lactating healthy mothers between days 4 and 6 after delivery of normal-term infants. Milk and blood T lymphocytes were positively selected by incubation with a mixture of magnetic beads coated with either anti-CD4 or anti-CD8 mAb (Pell-Freez, Brown Deer, WI) according to the manufacturer's suggestions. Magnetic immunoselection yielded a > 95% CD3+ cell population (immunofluorescence analysis) in both milk and blood lymphocyte suspensions. To avoid potential perturbation of cells by bead-coupled anti-CD4 and anti-CD8 mAb [14], milk and blood lymphocytes from four postpartum donors were simultaneously separated by positive [I 83761 immunoselection or equilibrium density gradient centrifuCorrespondence: Albert0 Bertotto, Department of Paediatrics, gation [6]. As both procedures yielded similar results in terms of cell proliferation and IFN-y production (see Perugia University Medical School, 1-06100Perugia, Italy 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
0014-2980/90/0808-1877$3.50+ .25/0
1878
Eur. J. Immunol. 1990. 20: 1877-1880
A. Bertotto, R. Gerli, G. Fabietti et al.
below), only the data obtained with mAb-coated beads are given. 2.2 F C M analysis
Phenotypic studies of milk and blood T cells were performed by an indirect immunofluorescence staining technique. mAb used for staining were Leu-4 (anti-CD3; Becton Dickinson, Mountain View, CA), T11 (anti-CD2), 2H4 (anti-CD45RA), 4B4 (anti-CDw29; Coulter Immunology, Hialeah, FL), anti-LFA-1 (CDlla; Immunotech, Marseille, France), anti-UCHL1 (CD45RO; Dakopatts, Glostrup, Denmark) and anti-ICAM-1 (CD54 84H10, a generous gift from Dr. F? Mannoni, INSERM Unit 119, Marseille, France). Briefly, 2 x lo5 cells are incubated for 30 min at 4°C with saturating concentrations of each antibody or with identical concentrations of isotypematched mAb that do not react with human cells.The cells were then washed twice and reincubated for 30 min at 4 "C with FITC-conjugated goat anti-mouse F(ab')z subclassspecific antisera absorbed with human Ig (Tago, Inc., Burlingame, CA). After two more washes, mAb binding to the cell preparations was assessed by FCM (FACScan, Becton Dickinson). Data were collected on 20000 celldsample and analyzed by a Hewlett Packard (Palo Alto, CA) computer.
Gibco, Grand Island, NY), anti-CD3 antibody (50 ng/ml; OKT3, Ortho, Raritan, NJ) or a combination of anti-CD2 mAb (1/400 dilution of ascites fluid for each reagent; Tllz and T113, kindly supplied by Dr. S. F. Schlossman, Harvard Medical School, Boston, MA) were included in the medium. Proliferation was measured by pulsing cells with 0.5 pCi (= 18.5 kBq) of [3H]dThd (sp. act. 25 CVmmol, Amersham Int., Amersham, GB) added 8 h prior to harvesting. Results are expressed as the arithmetic mean cpm k SEM of triplicate cultures corrected by subtraction of background cpm. 2.4 Assay for IFN-y
IFN-y was measured in SN harvested after 48 h of anti-CD3 or anti-CD2 mAb stimulation with a comercially available radioimmunoassay (Centocor, Malvern, PA) according to the test kit instructions.
3 Results 3.1 Phenotypic characterization of milk and blood T cells
Previous experiments carried out in our laboratories have demonstrated that milk lymphocyte suspensions contain a strikingly low number of T cells expressing the naive 2.3 Prolieration assay phenotype (2H4/CD45R+) and an overwhelming preponderance of CDw29+ memory T lymphocytes. This contrasts Cell proliferation was assayed under standard conditions, with the picture in the blood,where the two populations are as previously described [15]. In brief, milk or blood T of approximately equal size [7]. We have now extended our lymphocytes were cultured at 3 x 104/well in 96-well studies and have investigated the intensity with which milk round-bottom microtiter plates (Nunc, Roskilde, Den- and blood lymphocytes express a number of immunologimark) in a 6% COz humidified incubator for 72 h. Acces- cally relevant Tcell surface markers, including CD45R and sory cells (6 x 103/well)were provided in all cultures by CDw29, involved in the irreversible shift from naive to a addition of irradiated (3000 rad) milk or blood plastic- memory Tcell state [l-31. Results from a representative adherent monocytes. To activate T cells, PHA-M (1% ; experiment are shown in Fig. 1. Histograms of simulta-
CDr29
C OMT ROL
C D 45 RO
(4B4)
( UCHLl )
CD11a (LFA-1)
10
100
1ooo
10
loo
1ooo
lo
100
FLUOREICEMCE
lo00
10
100
1000
(YV )
Figure I. Phenotypic analysis of milk and blood T cells prepared by using positive magnetic immunoselection. Histograms represent superimposedprofiles of milk (brokenline) and autologous blood (solid line) T lymphocytes stained with the mAb of indicated specificity. Data are from a single experiment representative of eight separate experiments.
Eur. J. Immunol. 1990.20: 1877-1880
Memory T lymphocytes in human breast milk
Table 1. Proliferative responses to PHA and anti-CD3(OKT3)or anti-CD2 (Tllz + Tl13) mAb in milk and blood T lymphocyte suspensions obtained from eight post-partum donors") Source of
Mitogenic stimulush)
lymphocytes PHA
*
10.0 2.W) 50.1 f 6.5
Milk
Blood
Anti-CD3
Anti-CD2
7.1 f 1.8 8.6 rt 1.5
12.9 k 2.3
14.0
1879
anti-CD2 mAb were used as mitogenic agents (Table 1). This suggests that the differences documented with PHA were not due to an overall dichotomy in the proliferative capacity of milk and blood T cells. Furthermore, after triggering via either the CD3 or CD2 surface antigens, milk and blood lymphocytes produced similar amounts of IFN-y (Table 2), thereby indicating that T lymphocytes may be one of the cellular sources for the secretion of this lymphokine in human colostrum and milk [13].
2.1
a) T cells were prepared by positive magnetic immunoselection and the cultures supplemented with 20% of milk or blood irradiated accessory cells (plastic-adherentmonocytes). b) Mean SEM of net cpm X (see Sect. 2.3). c) p < 0.001 (Student's t-test for unpaired data).
4 Discussion
Although it is unlikely that milk T lymphocytes are merely contaminating blood-borne Tcells, their origin and biological significance are still a matter of conjecture.The present investigation shows that the phenotype and functional repertoire of milk Tcells are similar t o those of the memory Table2. IFN-y production by milk and autologous blood T cell subsets found not only in blood [l-31 but also in T lymphocytes following anti-CD3 (OKT3) or anti-CD2 certain body microenvironments [19-231. It would, there(Tllz + Tl13) mAb stimulationa) fore, seem reasonable to assume that immune system cells are compartmentalized in the mammary gland during lactation and that T lymphocytes do not accumulate Source of lymphocytes IFN-y (U/ml)b) randomly in colostrum, but rather are directed there by a AntLCD3 Anti-CD2 selective homing process. The fact that cells in mammary secretions display in virro parameters of immunocompe39.5 f 9.6 Milk 20.6 6.5 tence and often possess antigenic reactivities different from 33.8 10.7 22.3 6.2 Blood those of the PBL [9, 11, 121 would seem to support this proposal. However, it is still far from clear whether a) Tcells were prepared and the cultures set up as detailed in antigen-responsive milk lymphocytes, other cells, or lymTable 1. phokines and other subcellular factors are able to convey b) Mean & SEM of six consecutive experiments. specific and nonspecific immunoprotection to the suckling infant. The recent demonstration in experimental animal models that enterally administered human milk leukocytes neously stained .milk and autologous blood T cells have adhere to the gut epithelium or lie intramurally and persist been superimposed to facilitate comparison. Unlike blood in the intestine for at least 60 h after a single breast feed T cells, which manifested a bimodal phenotypic picture, suggests that these cells may be involved in the host's local most milk T lymphocytes were 2H4/CD45RP and displayed immune response [24]. It is worth noting that memory T only a single major fluorescence peak which approached lymphocytes predominate in the bowel wall [19, 22, 231, the high expression seen in the CDw29+, UCHLl+ and skin [20] and on the lung epithelial surfaces [21] of normal LFA-l+ circulating counterparts. In addition, while milk subjects. Such cells could have undergone on-site converand blood cells expressed identical levels of CD3 surface sion to the memory phenotype upon exposure to exogenous antigens, CD2 expression was approximately twofold high- antigens and/or have homed there as fully fledged memory er on the milk T lymphocytes. Similar results were obtained Tcells to aid in eliminating infectious agents. Human milk with a mAb directed against ICAM-1, a putative LFA-1 has been shown to protect the neonate against gastrointesligand molecule [161involved in both intercellular adhesion tinal, systemic and respiratory tract infections [25-291. [ 171 and T cell activation [ 181. These findings demonstrate Whatever the underlying cellular and/or molecular that of the two major T lymphocyte subsets found in adult mechanisms implicated in this adoptive protection are, the blood, the one bearing the CD45R10W,CDw29, UCHLl, demonstration of an overexpanded population of memory LFA-lhighmemory Tcell phenotype predominates in human Tcells in human breast milk provides evidence that breast early milk. This is further supported by the detection of feeding may help to maintain immunological homeostasis higher expression levels of CD2 and ICAM-1 surface during a period of life when the development of the structures on colostral T cells. immune system is incomplete and little information has been committed to the immunological memory.
*
*
**
3.2 Proliferation and IFN-y production by milk and blood T cells
Received February 15, 1990.
Milk and blood T lymphocytes were tested for their ability to proliferate in response to PHA and anti-CD3 or anti-CD2 mAb. As in previously published data [S, 91, the proliferative response of milk T cells to PHA was significantly lower than that of blood lymphocyte cultures. In contrast, milk and blood Tcells displayed comparable levels of reactivity when anti-CD3 or an appropriate pair of
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Announcement International Meeting 25 Years of ImmunoenzymaticTechniques Hilton, Athens, Greece September 9-12, 1991 Organizing Committee: S. Avrameas (France), F! K. Nakane (Japan), A. Pesce (USA), M. Papamichael (Greece)
Main topics will include: immunoassays, amplification principles, nucleic acid probes, immunocytochemical procedures, new approaches, contribution of immunodiagnosis in various diseases. Organizing Committee Secretariat: Triaena Congress, 24, Harilaou Trikoupi str., Athens 10679, Greece Tel.: 30-1-3622970; Fax: 30-1-3607962
The meeting will include plenary session lectures providing critical reviews of specific areas in the field of immunoenzymatic techniques, workshops and poster presentations.
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