Journal of Reproductive
Immunology,
17 (1990) 69-78
69
Elsevier Scientific Publishers Ireland Ltd. JR1 00635
Role of T cell subsets in the maternal-to-neonatal transmission of immunity against Trichinella spiralis during lactation in rats Shantha N. Kumara, George L. Stewartb, William M. Stevena and Leonard L. Seelig, Jr” aDepartment of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, Louisiana 71l30, and “Center for Parasitology, University of Texas at Arlington, Arlington, TX 76019 (U.S.A.)
(Accepted for publication 30 October)
Summary We have previously demonstrated the maternal-to-neonatal transfer of immunity to T. spirulis during lactation and have shown that antigen-specific T lymphocytes, when injected into the mother or orally fed to neonates, can mediate this transfer. To further analyze the T cell subsets involved in conferring this protection, T lymphocytes were isolated from the mesenteric lymph nodes of syngeneic donor rats infected 4-6 days earlier with ZYspiralis. The T cells were incubated in vitro with either mouse-anti-rat OX8 or W3/25 monoclonal antibody, “panned” on plates coated with goat-anti-mouse Ig, and the non-adherent T helper or T cytotoxic/suppressor cells harvested. 100 x lo6 T helper cells were injected i.v. into mothers once in early lactation and again two days prior to challenging their pups (200 T. spirulis larvae) at 2 weeks of age. This resulted in significant passage of immunity from the mothers to their suckling neonates, worm counts being 59% and 73% of control values 3 and 8 days post-challenge (P -C0.01). Injection of T-cytotoxic/suppressor cells using the same regimen resulted in significant suppression of immunity in challenged pups, who retained worm counts that were 105% and 145% of control values at 3 and 8 days post-challenge. Synergy between recombined panned T-helper and T cytotoxic/suppressor cells without Lyl’2+3+amplifier cells was tested by recombining non-adherent panned OX8 and W3/25 cells. This resulted in no significant expressions of immunity in the pups when compared to controls. Correspondence to: Dr. Leonard L. Seelig, Jr. Department of Cellular Biology and Anatomy LSU Medical Center P.O. Box 33932 Shreveport, LA 71130-3932
0165-0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
70
The presence of transferred maternal T cells within the neonate was evidenced by the fact that neonates (nursing on immune mothers) had significant (P< 0.01) delayed footpad reactions to a crude T. spiralis antigen preparation, as compared with neonates nursing on non-immune controls. Key words: T cell subsets; lactation; immune transfer; Trichinella spiralis.
Introduction
The presence of viable cellular elements like lymphocytes and macrophages has been reported in colostrum and milk of many animal species (Smith and Schultz, 1977; Head et al., 1977), and there is evidence to support the theory that these cells can transmit immunity to the neonate during lactation. Studies by Ogra et al. (1977) showed that infants breastfed by tuberculin-positive mothers developed positive peripheral blood proliferative responses to tuberculin, and Archambault et al. (1988) demonstrated that orally fed colostral lymphocytes from immune animals could transfer protection against rotavirus to newborn calves. Immunity to T. spiralis has been shown to be passaged from immune mothers to their suckling offspring, with both serum IgG (Appleton and McGregor, 1987) and sensitized T lymphocytes (Kumar et al., 1989) being implicated as mediating this process. In adult mice, immunity to T. spiralis can be adoptively transferred by mesenteric lymph node cells (L3T4 +, Ly2-helper phenotype) present during the period of infection (Grencis et al., 1985). It has also been shown that the in vitro cell proliferation response against T. spiralis antigen is mediated by helper T cells and dependent on Lyl +2+3 + amplifier cells and the immune response is MHC-restricted with respect to genes mapping to the I region (Krco et al., 1982). While the exact mechanisms of immunity to T. spiralis are not well understood, it has been postulated that it is T cell-dependent, with the helper phenotype playing a major role in the response (Manson-Smith et al., 1979; Grencis et al., 1985). These experiments were designed to further analyze the role of specific T cell subsets in the transfer of immunity to T. spiralis from mother to offspring during lactation and to functionally assess the ability of these cells to enter the neonatal circulation and respond to a peripheral challenge with T. spiralis antigens. Materials and methods
Animals Rats of the Fischer (FI) strain, housed in temperature- and light-controlled rooms and provided laboratory chow and water ad libitum, were used in these experiments.
71
Antibodies
Affinity-purified goat-anti-rat and goat-anti-mouse immunoglobulins used for panning were obtained from Hyclone Labs, UT. For immunofluorescent staining and FACS analysis of cell suspensions, mouse-anti-rat monoclonal antibody OX19 was used for T cells, W3/25 for T helper cells, OX8 for T cytotoxic/suppressor cells, and a mixture of EDl, ED2, and ED3 for macrophages (Bioproducts for Science, IN). Mouse IgG against human FSH was used as an irrelevant antibody control. FITC-conjugated goat-anti-mouse IgG (Jackson Labs, PA) was used as the secondary antibody. B cells were stained using FITC-conjugated sheep-anti-rat Ig (Bioproducts for Science, IN), while FITC-conjugated sheep-anti-horse ferritin (Jackson Labs, PA) served as its non-specific control,
T spiralis infection
The strain of parasites employed and methods of isolation have been previously described (Stewart et al., 1985). Donor rats for lymph node cells were infected with 2000 Ll stage larvae of i? spiralis.
Antigens
Crude saline extracts of 7: spiralis were obtained from infective muscle larvae suspended in saline, by homogenizing them in a Ten-Broeck glass tissue grinder at 4°C for lo-15 min. The protein content of the parasite extract was determined by the spectrophotometric Coomassie blue G250 technique of Sedmak and Grossberg (1977). Crude i’Yspiralis antigens were stored at - 20°C until use.
Separation of T cells and subsets
The methods employed for lymph node cell extraction and T cell separation by “panning” are described in detail elsewhere (Kumar et al., 1985). The separated T cell suspension obtained after “panning” was resuspended at a concentration of 50 x lo6 cells/ml in minimal essential media (MEM) containing OX8 or W3/25 antibody at a dilution of 1:200 and the cells incubated at 4°C for 45 min, shaking every 15 min. Cells were washed twice in Hank’s balanced salt solution (HBSS) and resuspended at a concentration of 25 x lo6 cells/ml in HBSS containing 5% fetal bovine serum (FBS). The T cells were sequentially panned twice at 4°C on culture flasks precoated with 10% goat-anti-mouse immunoglobulins (Hyclone Labs, UT) in normal goat IgG (250 &ml). The corresponding non-adherent T cell subsets, enriched for T helper or T cytotoxic/suppressor cells were harvested. Aliquots of the cell populations were stained by immunofluorescence and analyzed by FACS for the percentage of various cell types.
12
Assessment of immunity to i? spiralis
Pups were challenged with 200 Ll larvae of T. spiralis when they were 2-3 weeks of age. At 3 days post-challenge, half of the littermates were autopsied and the adult worms present within the small intestine were recovered and counted. The remaining pups continued to nurse and were sacrificed 8 days after challenge. To recover the enteric stages of T. spiralis, animals were killed and processed individually. The entire small intestine (from the pyloric sphincter to the cecum) was removed and the luminal contents rinsed out with saline. The gut was split longitudinally and immersed in a beaker containing HBSS (pH 7.4) for 4 h at 37°C. After removal of the intestine, the HBSS solution was poured into a grid-marked petri dish and the adult worms counted under a dissecting microscope. Any residual worms in the intestine were released after digestion of the mucosa in 1% pepsin-HCL and counted. Experimental
design
For the adoptive transfer, T cells were obtained by “panning” mesenteric lymph node cells obtained from 4-6 day i? spiralis-infected syngeneic donor rats. The harvested T cells 00 x 106/inj./ani.) were transferred by i.v. tail vein injection into lactating recipients once in early lactation (1-3 days post-partum) and again 2 days prior to challenging the pups. Pups were between 2 and 3 weeks of age at the time of worm challenge (oral intubation with 200 i? spiralis Ll larvae) and at least 4 litters containing 8 pups each were used in each group. For transferring T cell subsets, 100 x 106/inj./ani. of T helper or T cytotoxic/suppressor cells, separated from i? spiralisprimed donor lymph node cells, were injected into lactating recipients using a similar protocol. In addition, 200 x 106/inj./ani. of a combination of nonadherent cells of panned cells for W3/25 and OX8 were also injected into lactating recipients, thus eliminating the Ly1+2+3+ amplifier cell population. Immunity was assessed in pups as described above. Also, to preclude the possibility that immunization via contaminating antigen was occurring, neonates were orally intubated with 60 x lo6 T cells (obtained from T. spiralis-infected donors) that were killed by freeze thawing and exposure to ultraviolet radiation for 15 min. Delayed footpad reaction
DTH was assessed in 3-week-old neonates nursing on T. spiralis-immune or naive mothers. Mothers were rendered immune by injecting 200 x lo6 T cells obtained from T. spiral&infected donors in early lactation and resensitized at 18 days postpartum by administering 75 kg of is spiralis crude saline extract in (MPL + TDM) emulsion (Ribi Immunochemical Research) intraperitoneally and in each of four footpads. Two days later, neonates were challenged in the left hind footpad with 75 kg T. spiralis
73
antigen by subcutaneous injection and saline in the right footpad as a nonspecific control. Age-matched control neonates nursing on naive mothers were similarly challenged. The change in footpad thickness was measured with a micrometer 24 and 48 h after neonatal challenge and used as a measure of delayed-type hypersensitivity. Statistical analysis
All worm counts and thickness measurements were made without prior knowledge of the experimental manipulations, and data was entered into a computer. Differences between the various groups were analyzed using the Student’s t-test (Statistix II, NG Analytical Software, Roseville, MN). Results T lymphocytes separated after “panning” contained less than 1.5% B cells and 1% macrophages as determined by FACS analysis. The purified subsets (T helper and T cytotoxic/suppressor phenotypes) contained less than 5% of the reciprocal cell population. Control neonates nursing on naive mothers harbored 78 and 57 worms 3 and 8 days after challenge with 200 T. spiralis larvae (Fig. l), representing baseline values of worm establishment in control neonates. These numbers are similar to worm establishment in neonatal rats reported by Appleton and McGregor (1985). When mothers were rendered immune by adoptive
m
Day 3 post-challenge
Day 8 post-challenge
Fig. 1 T. spiralis worms recovered from neonatal intestine 3 and 8 days after challenge with 200 LI stage larvae. (A) Neonates nursing on naive control mothers; (B) Neonates nursing on mothers injected with T. spiralis-primed T cells; (C) Neonates nursing on mothers injected with 7 spiralis-primed T helper cells and (D) Neonates nursing on mothers injected with T. spiralis-primed T cytotoxickuppressor cells. Control group A is the mean value obtained from approximately 12 litters while groups B-D were from 4 litters. Numbers of pups are indicated within bars. Statistical differences were computed between test and control groups using Student’s r-test, *P < 0.05 **P -C O.Ol***P < 0.001 NS, not significant. Standard error of the mean is indicated for each experimental group.
transfer of T spiralis-primed T cells, the pups nursing on them showed significant immunity on challenge (Fig. l), evidenced by a significant reduction in worm numbers compared to control neonates (immune pups retained 26% and 12% of the worm numbers present in control pups 3 and 8 days after infection, P -C0.001). However, when neonates were fed killed T cells, they harbored 109% of cells in controls at 3 days post-challenge. Further analysis of the T cell subsets involved in conferring protection against T. spirulis in immune pups revealed that adoptive transfer of T helper cells (Fig. 1) into the mother resulted in significant immune passage to her young compared to control neonates nursing on naive mothers (immune pups retained 59% and 73% of the counts in control neonates 3 and 8 days after challenge, P < 0.01). However, transfer of T cytotoxic/suppressor cells into the mother resulted in a slight increase in the number of worms retained in the challenged neonates at day 3 and a significant immune suppression compared to control pups by day 8 post-challenge (immune-suppressed pups retained 105% and 145% of the worm numbers present in controls at 3 and 8” days post-challenge, *P< 0.05). When panned T helper and T cytotoxic/suppressor cells depleted of any amplifer cells were recombined and injected into lactating rats, no immunity was observed in their pups at 3 or 8 days following r spiralis challenge (92% and 105% of control, respectively). Neonates nursing on mothers who had received i.v. injections of i? spiralis-specific T cells and were further boosted with T. spirulis crude antigen at day 18 of lactation showed significant footpad swelling (Table 1) as compared to control neonates nursing on naive mothers (P-C 0.01 at 24 h and P < 0.05 at 48 h after footpad challenge). Pups nursing on immune mothers also had significant swelling on the left antigen-challenged footpad compared to the right footpad which received saline as a control. Control neonates showed no significant difference between right and left footpads. Discussion Cellular factors such as lymphocytes and macrophages are a constant component of the milk of many animal species (Lee et al., 1980; Head and Seelig, 1983). Although it has been shown that the ratio of T helper to T cytotoxic/suppressor cells in human milk parallels that of peripheral blood (Keller et al., 1986), the functional roles of these subsets has not been investigated. In this study, we have used infection with T. spiralis as a model to demonstrate the maternal-to-neonatal transmission of cell-mediated immunity during lactation and the role played by individual T cell subsets in effecting the immune transfer. Studies by other investigators and in our laboratory have demonstrated the passage of immunity to T. spiralis from mothers immunized to the
75
TABLE 1 Change in neonatal footpad thickness as a measure of delayed type hypersensitivity. Treatment
Threeweek old neonates challenged with antigen
Time after challenge
Change in footpad thickness (mm) Mean + S.E.M.*
(h)
Right: saline
Left : antieen
Significance Rieht vs. left
Immune mothers
0.054 eo.033
0.387 e-o.065
P < 0.001**
Control mothers
0.054 +0.046
0.125
N.S.
Immune mothers
0.084 +0.044
0.223 kO.059
P < 0.001
Control mohers
0.031 *0.040
0.068 20.041
N.S.
Neonates nursed by
Significance Immune vs. control
P < 0.01
kO.032
P < 0.05
*Mean values obtained from 4 litters in each experimental group. S.E.M., standard error of the mean. **Differences computed between right and left foot and between immune and control groups using Student’s t-test. NS, not significant.
parasite i? spiralis to their suckling offspring (Perry, 1974; Appleton and McGregor, 1987; Kumar et al., Immunology, 1989). We further showed by foster-nursing studies that the transfer of immunity occurred during lactation and not via placental transfer. Also, mothers injected with T lymphocytes primed to i? spiralis were able to transfer immunity to their neonates; the specificity of the immune transfer was shown by the inability of mothers injected with KLH-primed T cells to transfer immunity against T. spiralis to neonates. Neonates who were administered 7: spiralis primed T cells orally or intravenously were also rendered immune. -Neonates who were administered killed T cells obtained from T. spiralis-infected donors did not show any differences in worm numbers as compared to naive controls, indicating that neonatal immune transfer to i? spiralis did not occur via contaminating antigen carried over with the T cells. These findings present evidence that antigen-specific T lymphocytes present in the mother, and subsequently in the neonate, can elicit immunity against 7: spiralis in the neonate. In the present study, analysis of the T cell subsets involved in immune transfer revealed that the helper cells played a major role in protection, while the T cytotoxic/suppressor class of cells suppressed the immune response in the neonates. Further, our results have shown some evidence that the transferred T cells are present within the neonatal circulation, since the immune neonates demonstrated delayed-type hypersensitivity when challenged peripherally (in the footpad) with antigen. It has been reported that in adult animals, lymphocytes of the helper
76
subclass confer protection against i? spiralis (Riedlinger et al., 1986; Bell et al., 1987). CD4+ helper T cells represent a heterogeneous array of cells which can mediate a broad spectrum of activities including antigen-specific or polyclonal B cell activation, delayed-type hypersensitivity, killing of appropriate target cells and induction of CD8+ killer or suppressor T cells (Bottomly, 1988). It remains to be established which of these mechanisms may be involved in protective immunity in this host-parasite model. Liew et al. (1989), working with Leishmaniasis infection in mice, proposed that induction of specific Ta%elper subclasses and their balance of lymphokine secretion determine the outcome of this infection. In their system, T helper cells secreting gamma interferon resulted in protection, while those elaborating IL-3 and IL-4 were not effective. Studies using T cell lines specific against i? spirdis show that they secrete gamma interferon, IL-2 and IL3 (Riedlinger et al., 1986). It is thus possible that antigen challenge could initiate events leading to activation of specific lymphocyte subclasses present locally in the neonatal intestine, which can mediate worm expulsion in the neonate. For instance, the ability of T helper cells and their cytokines to stimulate mucus secretion has received much attention (Miller et al., 1979). The immunity transferred using W3/25+ T helper cells was not as strong as when the whole T cell population was used. It has been reported by Krco et al., (1982) that besides Lyl+ T helper cells, an additional Lyl’2+3’ amplifier cell type, that separates with the panned 0X8’ or W3/25+ subset was needed for maximal protection and could explain the lower level of immunity seen when the separated T helper cells were used for adoptive transfer. By recombining the T helper and T cytotoxic/supressor cells depleted of Ly1+2+3+ amplifier cells, and injecting these sensitized cells into lactating females, their pups showed no immunity to 7: spiralis when compared to controls. It can be concluded that the loss of synergy between these two populations of cells was not the cause of the reduction in transferred immunity to pups that were suckled by dams that had received only T helper cells. However, the implication that the amplifier cells depleted in both experiments, but not when total T cells were given, may be necessary for complete transfer of immunity to T. spirulis in suckling pups. These experiments will be the focus of future studies. We speculate the following sequence of events in the maternal-to-neonatal transfer of cell-mediated immunity to 1 spiralis during lactation. Antigen-specific T lymphocytes are primed after immunization in the mother, and some of these migrate to the mammary gland and milk during lactation. These cells may remain in the neonatal gut for a short period of time and it is possible that a few of these even enter the neonatal tissues and remain there transiently. Data supporting the ability of ingested lymphocytes to traverse the gastric epithelium into neonatal tissues has emanated from our
77
laboratory (Seelig and Billingham, 1981; Seelig and Head, 1987). Upon neonatal infection, specific T cell subsets encounter antigen and mediate the cascade of events (antibody production and/or local cell-mediated inflammatory responses) which lead to worm expulsion. It has been shown by Appleton and McGregor (1984) that mucus secretion is part of the neonatal response in the maternal-to-neonatal transfer of immunity against T. spiralis and T cell lymphokines may help mediate this reaction. Continuing studies in our laboratory are focusing on the ultimate fate of the maternal T cells transmitted in milk and the mechanisms by which they mediate immunity in the neonate. Acknowledgements The authors wish to thank Mr. Chris Duggan and Larry Smart for their excellent technicial assistance. This work was supported in part by grants USPHS HD14358 and AA07381. References Appleton, J.A. and McGregor, D.D. (1987) Characterization of the immune mediator of rapid expulsion to Trichinella spiralis in suckling rats. Immunology 62, 477-484. Archambault, D., Morin, G., Elazhary, Y., Roy, R.S. and Joncas, J.H. (1988) Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes. Am. J. Vet. Res. 49(7), 1084-1091. Bell, R.G., Korenaga, M. and Wang, C.H. (1987) Characterization of a cell population in thoracic duct lymph that adoptively transfers rejection of adult Trichinella spiralis to normal rats. Immunology 61, 221-227. Bottomly, K. (1988) A functional dichotomy in CD4+ T lymphocytes. Immunol. Today. 9, 268-274. Grencis, R.K., Riedlinger, J. and Wakelin, D. (1985) L3T4-positive T lymphoblasts are responsible for transfer of immunity against Trichinella spiralis in mice. Immunology 56, 213-218. Head, J.R., Beer, A.E. and Billingham, R.E. (1977) Significance of the cellular component of the maternal immunologic endowment in milk. Transplant. Proc. 9, 1465-1471. Head, J.R. and Seelig, L.L. Jr. (1983) Autoradiographic analysis of lymphocyte migration into the mammary epithelium and milk of lactating female rats. J. Reprod. Immunol. 5, 61-72. Keller, M.A., Faust, J., Relevic, L.J. and Stewart, D.D. (1986) T-cell subsets in human colostrum. J. Gastroenterol. Nutr. 5, 439-443. Krco, C.J., David, C.S. and Wassom, D.L. (1982) Solubilized Trichinella spiralis antigens, Role of Ia antigens and Ly - l+ cells. Cell. Immunol. 68, 359-367. 10 Kumar, S.N., Seelis, L.L. Jr. and Head. J.R. (1985) Mieration of radiolabeled. adontivelv transferred T-lymphocytes into the mammary gland and milk if lactating rats. J. Reprod.‘Immunol. 8, 235-248. Kumar, S.N., Stewart, G.L., Steven, W.M. and Seelig, L.L. Jr. (1989) Maternal-to-neonatal transmission of T cell-mediated immunity to Trichinella spiralis during lactation. Immunology 68, 87-92. Lee, C.S., Wooding, F.B.D. and Kemp, P. (1980) Identification, properties and differential counts of cell populations using electron microscopy, of dry cows secretions, colostrum and milk from normal cows. J. Dairy Res. 47, 39-50. Liew, F..Y...(l989), Functional heterogeneity of CD4 + T cells in leishmaniasis. Immunol. Today, 40-45.
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14 Manson-Smith, D.F., Bruce, R.G. and Parrott, D.M.V. (1979) Villous atrophy and expulsion of intestinal Trichinellu spiralis are mediated by T cells. Cell. Immunol. 47, 285-292. 15 Miller, H.R.P., Nawa, Y. and Parish, C.R. (1979) Intestinal goblet cell differentiation in Nipposrrongylus-infected rats after transfer of fractionated thoracic duct lymphocytes. Int. Arch. All Appl Immunol. 59, 281-285. 16 Ogra, S.S., Weintraub, D. and Ogra, P.L. (1977) Immunologic aspects of human colostrum and milk III. Fate and absorption of cellular and soluble components in the gastrointestinal tract of the newborn. J. Immunol. 119(l), 245-248. 17 Perry, R.H. (1974) Transfer of immunity to Trichindu spiralis from mother to offspring J. Parasitol. 60(3), 460-465. 18 Riedlinger, J., Grencis, R.K. and Wakelin, D. (1986) Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo. Immunology 58, 57-61. 19 Sedmak, J.J. and Grossberg, S.G. (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue 9-250. Anal. Biochem. 79, 544-552 20 Seelig, L.L., Jr. and Billingham, R.E. (1981) The capacity of “transplanted” lymphocytes to traverse the intestinal epithelium of adult rats. Transplantation 32, 308-314. 21 Seelig, L.L. Jr. and Head, J.R. (1987) Uptake of lymphocytes fed to suckling rats. An autoradiographic study of the transit of labeled cells through the neonatal gastric mucosa. J. Reprod. Immunol 10, 285-297. 22 Smith, J.W. and Schultz, R.D. (1977) Mitogen- and antigen-responsive milk lymphocytes. Cell. Immunol. 29, 165-173. 23 Stewart, G.L., Wood, B.G. and Boley, R.B. (1985) Modulation of host response by Trichinella pseudospirdis. Parasite Immunol. 7, 223-233.
Immunology,
17 (1990) 69-78
69
Elsevier Scientific Publishers Ireland Ltd. JR1 00635
Role of T cell subsets in the maternal-to-neonatal transmission of immunity against Trichinella spiralis during lactation in rats Shantha N. Kumara, George L. Stewartb, William M. Stevena and Leonard L. Seelig, Jr” aDepartment of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, Louisiana 71l30, and “Center for Parasitology, University of Texas at Arlington, Arlington, TX 76019 (U.S.A.)
(Accepted for publication 30 October)
Summary We have previously demonstrated the maternal-to-neonatal transfer of immunity to T. spirulis during lactation and have shown that antigen-specific T lymphocytes, when injected into the mother or orally fed to neonates, can mediate this transfer. To further analyze the T cell subsets involved in conferring this protection, T lymphocytes were isolated from the mesenteric lymph nodes of syngeneic donor rats infected 4-6 days earlier with ZYspiralis. The T cells were incubated in vitro with either mouse-anti-rat OX8 or W3/25 monoclonal antibody, “panned” on plates coated with goat-anti-mouse Ig, and the non-adherent T helper or T cytotoxic/suppressor cells harvested. 100 x lo6 T helper cells were injected i.v. into mothers once in early lactation and again two days prior to challenging their pups (200 T. spirulis larvae) at 2 weeks of age. This resulted in significant passage of immunity from the mothers to their suckling neonates, worm counts being 59% and 73% of control values 3 and 8 days post-challenge (P -C0.01). Injection of T-cytotoxic/suppressor cells using the same regimen resulted in significant suppression of immunity in challenged pups, who retained worm counts that were 105% and 145% of control values at 3 and 8 days post-challenge. Synergy between recombined panned T-helper and T cytotoxic/suppressor cells without Lyl’2+3+amplifier cells was tested by recombining non-adherent panned OX8 and W3/25 cells. This resulted in no significant expressions of immunity in the pups when compared to controls. Correspondence to: Dr. Leonard L. Seelig, Jr. Department of Cellular Biology and Anatomy LSU Medical Center P.O. Box 33932 Shreveport, LA 71130-3932
0165-0378/90/$03.50 0 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
70
The presence of transferred maternal T cells within the neonate was evidenced by the fact that neonates (nursing on immune mothers) had significant (P< 0.01) delayed footpad reactions to a crude T. spiralis antigen preparation, as compared with neonates nursing on non-immune controls. Key words: T cell subsets; lactation; immune transfer; Trichinella spiralis.
Introduction
The presence of viable cellular elements like lymphocytes and macrophages has been reported in colostrum and milk of many animal species (Smith and Schultz, 1977; Head et al., 1977), and there is evidence to support the theory that these cells can transmit immunity to the neonate during lactation. Studies by Ogra et al. (1977) showed that infants breastfed by tuberculin-positive mothers developed positive peripheral blood proliferative responses to tuberculin, and Archambault et al. (1988) demonstrated that orally fed colostral lymphocytes from immune animals could transfer protection against rotavirus to newborn calves. Immunity to T. spiralis has been shown to be passaged from immune mothers to their suckling offspring, with both serum IgG (Appleton and McGregor, 1987) and sensitized T lymphocytes (Kumar et al., 1989) being implicated as mediating this process. In adult mice, immunity to T. spiralis can be adoptively transferred by mesenteric lymph node cells (L3T4 +, Ly2-helper phenotype) present during the period of infection (Grencis et al., 1985). It has also been shown that the in vitro cell proliferation response against T. spiralis antigen is mediated by helper T cells and dependent on Lyl +2+3 + amplifier cells and the immune response is MHC-restricted with respect to genes mapping to the I region (Krco et al., 1982). While the exact mechanisms of immunity to T. spiralis are not well understood, it has been postulated that it is T cell-dependent, with the helper phenotype playing a major role in the response (Manson-Smith et al., 1979; Grencis et al., 1985). These experiments were designed to further analyze the role of specific T cell subsets in the transfer of immunity to T. spiralis from mother to offspring during lactation and to functionally assess the ability of these cells to enter the neonatal circulation and respond to a peripheral challenge with T. spiralis antigens. Materials and methods
Animals Rats of the Fischer (FI) strain, housed in temperature- and light-controlled rooms and provided laboratory chow and water ad libitum, were used in these experiments.
71
Antibodies
Affinity-purified goat-anti-rat and goat-anti-mouse immunoglobulins used for panning were obtained from Hyclone Labs, UT. For immunofluorescent staining and FACS analysis of cell suspensions, mouse-anti-rat monoclonal antibody OX19 was used for T cells, W3/25 for T helper cells, OX8 for T cytotoxic/suppressor cells, and a mixture of EDl, ED2, and ED3 for macrophages (Bioproducts for Science, IN). Mouse IgG against human FSH was used as an irrelevant antibody control. FITC-conjugated goat-anti-mouse IgG (Jackson Labs, PA) was used as the secondary antibody. B cells were stained using FITC-conjugated sheep-anti-rat Ig (Bioproducts for Science, IN), while FITC-conjugated sheep-anti-horse ferritin (Jackson Labs, PA) served as its non-specific control,
T spiralis infection
The strain of parasites employed and methods of isolation have been previously described (Stewart et al., 1985). Donor rats for lymph node cells were infected with 2000 Ll stage larvae of i? spiralis.
Antigens
Crude saline extracts of 7: spiralis were obtained from infective muscle larvae suspended in saline, by homogenizing them in a Ten-Broeck glass tissue grinder at 4°C for lo-15 min. The protein content of the parasite extract was determined by the spectrophotometric Coomassie blue G250 technique of Sedmak and Grossberg (1977). Crude i’Yspiralis antigens were stored at - 20°C until use.
Separation of T cells and subsets
The methods employed for lymph node cell extraction and T cell separation by “panning” are described in detail elsewhere (Kumar et al., 1985). The separated T cell suspension obtained after “panning” was resuspended at a concentration of 50 x lo6 cells/ml in minimal essential media (MEM) containing OX8 or W3/25 antibody at a dilution of 1:200 and the cells incubated at 4°C for 45 min, shaking every 15 min. Cells were washed twice in Hank’s balanced salt solution (HBSS) and resuspended at a concentration of 25 x lo6 cells/ml in HBSS containing 5% fetal bovine serum (FBS). The T cells were sequentially panned twice at 4°C on culture flasks precoated with 10% goat-anti-mouse immunoglobulins (Hyclone Labs, UT) in normal goat IgG (250 &ml). The corresponding non-adherent T cell subsets, enriched for T helper or T cytotoxic/suppressor cells were harvested. Aliquots of the cell populations were stained by immunofluorescence and analyzed by FACS for the percentage of various cell types.
12
Assessment of immunity to i? spiralis
Pups were challenged with 200 Ll larvae of T. spiralis when they were 2-3 weeks of age. At 3 days post-challenge, half of the littermates were autopsied and the adult worms present within the small intestine were recovered and counted. The remaining pups continued to nurse and were sacrificed 8 days after challenge. To recover the enteric stages of T. spiralis, animals were killed and processed individually. The entire small intestine (from the pyloric sphincter to the cecum) was removed and the luminal contents rinsed out with saline. The gut was split longitudinally and immersed in a beaker containing HBSS (pH 7.4) for 4 h at 37°C. After removal of the intestine, the HBSS solution was poured into a grid-marked petri dish and the adult worms counted under a dissecting microscope. Any residual worms in the intestine were released after digestion of the mucosa in 1% pepsin-HCL and counted. Experimental
design
For the adoptive transfer, T cells were obtained by “panning” mesenteric lymph node cells obtained from 4-6 day i? spiralis-infected syngeneic donor rats. The harvested T cells 00 x 106/inj./ani.) were transferred by i.v. tail vein injection into lactating recipients once in early lactation (1-3 days post-partum) and again 2 days prior to challenging the pups. Pups were between 2 and 3 weeks of age at the time of worm challenge (oral intubation with 200 i? spiralis Ll larvae) and at least 4 litters containing 8 pups each were used in each group. For transferring T cell subsets, 100 x 106/inj./ani. of T helper or T cytotoxic/suppressor cells, separated from i? spiralisprimed donor lymph node cells, were injected into lactating recipients using a similar protocol. In addition, 200 x 106/inj./ani. of a combination of nonadherent cells of panned cells for W3/25 and OX8 were also injected into lactating recipients, thus eliminating the Ly1+2+3+ amplifier cell population. Immunity was assessed in pups as described above. Also, to preclude the possibility that immunization via contaminating antigen was occurring, neonates were orally intubated with 60 x lo6 T cells (obtained from T. spiralis-infected donors) that were killed by freeze thawing and exposure to ultraviolet radiation for 15 min. Delayed footpad reaction
DTH was assessed in 3-week-old neonates nursing on T. spiralis-immune or naive mothers. Mothers were rendered immune by injecting 200 x lo6 T cells obtained from T. spiral&infected donors in early lactation and resensitized at 18 days postpartum by administering 75 kg of is spiralis crude saline extract in (MPL + TDM) emulsion (Ribi Immunochemical Research) intraperitoneally and in each of four footpads. Two days later, neonates were challenged in the left hind footpad with 75 kg T. spiralis
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antigen by subcutaneous injection and saline in the right footpad as a nonspecific control. Age-matched control neonates nursing on naive mothers were similarly challenged. The change in footpad thickness was measured with a micrometer 24 and 48 h after neonatal challenge and used as a measure of delayed-type hypersensitivity. Statistical analysis
All worm counts and thickness measurements were made without prior knowledge of the experimental manipulations, and data was entered into a computer. Differences between the various groups were analyzed using the Student’s t-test (Statistix II, NG Analytical Software, Roseville, MN). Results T lymphocytes separated after “panning” contained less than 1.5% B cells and 1% macrophages as determined by FACS analysis. The purified subsets (T helper and T cytotoxic/suppressor phenotypes) contained less than 5% of the reciprocal cell population. Control neonates nursing on naive mothers harbored 78 and 57 worms 3 and 8 days after challenge with 200 T. spiralis larvae (Fig. l), representing baseline values of worm establishment in control neonates. These numbers are similar to worm establishment in neonatal rats reported by Appleton and McGregor (1985). When mothers were rendered immune by adoptive
m
Day 3 post-challenge
Day 8 post-challenge
Fig. 1 T. spiralis worms recovered from neonatal intestine 3 and 8 days after challenge with 200 LI stage larvae. (A) Neonates nursing on naive control mothers; (B) Neonates nursing on mothers injected with T. spiralis-primed T cells; (C) Neonates nursing on mothers injected with 7 spiralis-primed T helper cells and (D) Neonates nursing on mothers injected with T. spiralis-primed T cytotoxickuppressor cells. Control group A is the mean value obtained from approximately 12 litters while groups B-D were from 4 litters. Numbers of pups are indicated within bars. Statistical differences were computed between test and control groups using Student’s r-test, *P < 0.05 **P -C O.Ol***P < 0.001 NS, not significant. Standard error of the mean is indicated for each experimental group.
transfer of T spiralis-primed T cells, the pups nursing on them showed significant immunity on challenge (Fig. l), evidenced by a significant reduction in worm numbers compared to control neonates (immune pups retained 26% and 12% of the worm numbers present in control pups 3 and 8 days after infection, P -C0.001). However, when neonates were fed killed T cells, they harbored 109% of cells in controls at 3 days post-challenge. Further analysis of the T cell subsets involved in conferring protection against T. spirulis in immune pups revealed that adoptive transfer of T helper cells (Fig. 1) into the mother resulted in significant immune passage to her young compared to control neonates nursing on naive mothers (immune pups retained 59% and 73% of the counts in control neonates 3 and 8 days after challenge, P < 0.01). However, transfer of T cytotoxic/suppressor cells into the mother resulted in a slight increase in the number of worms retained in the challenged neonates at day 3 and a significant immune suppression compared to control pups by day 8 post-challenge (immune-suppressed pups retained 105% and 145% of the worm numbers present in controls at 3 and 8” days post-challenge, *P< 0.05). When panned T helper and T cytotoxic/suppressor cells depleted of any amplifer cells were recombined and injected into lactating rats, no immunity was observed in their pups at 3 or 8 days following r spiralis challenge (92% and 105% of control, respectively). Neonates nursing on mothers who had received i.v. injections of i? spiralis-specific T cells and were further boosted with T. spirulis crude antigen at day 18 of lactation showed significant footpad swelling (Table 1) as compared to control neonates nursing on naive mothers (P-C 0.01 at 24 h and P < 0.05 at 48 h after footpad challenge). Pups nursing on immune mothers also had significant swelling on the left antigen-challenged footpad compared to the right footpad which received saline as a control. Control neonates showed no significant difference between right and left footpads. Discussion Cellular factors such as lymphocytes and macrophages are a constant component of the milk of many animal species (Lee et al., 1980; Head and Seelig, 1983). Although it has been shown that the ratio of T helper to T cytotoxic/suppressor cells in human milk parallels that of peripheral blood (Keller et al., 1986), the functional roles of these subsets has not been investigated. In this study, we have used infection with T. spiralis as a model to demonstrate the maternal-to-neonatal transmission of cell-mediated immunity during lactation and the role played by individual T cell subsets in effecting the immune transfer. Studies by other investigators and in our laboratory have demonstrated the passage of immunity to T. spiralis from mothers immunized to the
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TABLE 1 Change in neonatal footpad thickness as a measure of delayed type hypersensitivity. Treatment
Threeweek old neonates challenged with antigen
Time after challenge
Change in footpad thickness (mm) Mean + S.E.M.*
(h)
Right: saline
Left : antieen
Significance Rieht vs. left
Immune mothers
0.054 eo.033
0.387 e-o.065
P < 0.001**
Control mothers
0.054 +0.046
0.125
N.S.
Immune mothers
0.084 +0.044
0.223 kO.059
P < 0.001
Control mohers
0.031 *0.040
0.068 20.041
N.S.
Neonates nursed by
Significance Immune vs. control
P < 0.01
kO.032
P < 0.05
*Mean values obtained from 4 litters in each experimental group. S.E.M., standard error of the mean. **Differences computed between right and left foot and between immune and control groups using Student’s t-test. NS, not significant.
parasite i? spiralis to their suckling offspring (Perry, 1974; Appleton and McGregor, 1987; Kumar et al., Immunology, 1989). We further showed by foster-nursing studies that the transfer of immunity occurred during lactation and not via placental transfer. Also, mothers injected with T lymphocytes primed to i? spiralis were able to transfer immunity to their neonates; the specificity of the immune transfer was shown by the inability of mothers injected with KLH-primed T cells to transfer immunity against T. spiralis to neonates. Neonates who were administered 7: spiralis primed T cells orally or intravenously were also rendered immune. -Neonates who were administered killed T cells obtained from T. spiralis-infected donors did not show any differences in worm numbers as compared to naive controls, indicating that neonatal immune transfer to i? spiralis did not occur via contaminating antigen carried over with the T cells. These findings present evidence that antigen-specific T lymphocytes present in the mother, and subsequently in the neonate, can elicit immunity against 7: spiralis in the neonate. In the present study, analysis of the T cell subsets involved in immune transfer revealed that the helper cells played a major role in protection, while the T cytotoxic/suppressor class of cells suppressed the immune response in the neonates. Further, our results have shown some evidence that the transferred T cells are present within the neonatal circulation, since the immune neonates demonstrated delayed-type hypersensitivity when challenged peripherally (in the footpad) with antigen. It has been reported that in adult animals, lymphocytes of the helper
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subclass confer protection against i? spiralis (Riedlinger et al., 1986; Bell et al., 1987). CD4+ helper T cells represent a heterogeneous array of cells which can mediate a broad spectrum of activities including antigen-specific or polyclonal B cell activation, delayed-type hypersensitivity, killing of appropriate target cells and induction of CD8+ killer or suppressor T cells (Bottomly, 1988). It remains to be established which of these mechanisms may be involved in protective immunity in this host-parasite model. Liew et al. (1989), working with Leishmaniasis infection in mice, proposed that induction of specific Ta%elper subclasses and their balance of lymphokine secretion determine the outcome of this infection. In their system, T helper cells secreting gamma interferon resulted in protection, while those elaborating IL-3 and IL-4 were not effective. Studies using T cell lines specific against i? spirdis show that they secrete gamma interferon, IL-2 and IL3 (Riedlinger et al., 1986). It is thus possible that antigen challenge could initiate events leading to activation of specific lymphocyte subclasses present locally in the neonatal intestine, which can mediate worm expulsion in the neonate. For instance, the ability of T helper cells and their cytokines to stimulate mucus secretion has received much attention (Miller et al., 1979). The immunity transferred using W3/25+ T helper cells was not as strong as when the whole T cell population was used. It has been reported by Krco et al., (1982) that besides Lyl+ T helper cells, an additional Lyl’2+3’ amplifier cell type, that separates with the panned 0X8’ or W3/25+ subset was needed for maximal protection and could explain the lower level of immunity seen when the separated T helper cells were used for adoptive transfer. By recombining the T helper and T cytotoxic/supressor cells depleted of Ly1+2+3+ amplifier cells, and injecting these sensitized cells into lactating females, their pups showed no immunity to 7: spiralis when compared to controls. It can be concluded that the loss of synergy between these two populations of cells was not the cause of the reduction in transferred immunity to pups that were suckled by dams that had received only T helper cells. However, the implication that the amplifier cells depleted in both experiments, but not when total T cells were given, may be necessary for complete transfer of immunity to T. spirulis in suckling pups. These experiments will be the focus of future studies. We speculate the following sequence of events in the maternal-to-neonatal transfer of cell-mediated immunity to 1 spiralis during lactation. Antigen-specific T lymphocytes are primed after immunization in the mother, and some of these migrate to the mammary gland and milk during lactation. These cells may remain in the neonatal gut for a short period of time and it is possible that a few of these even enter the neonatal tissues and remain there transiently. Data supporting the ability of ingested lymphocytes to traverse the gastric epithelium into neonatal tissues has emanated from our
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laboratory (Seelig and Billingham, 1981; Seelig and Head, 1987). Upon neonatal infection, specific T cell subsets encounter antigen and mediate the cascade of events (antibody production and/or local cell-mediated inflammatory responses) which lead to worm expulsion. It has been shown by Appleton and McGregor (1984) that mucus secretion is part of the neonatal response in the maternal-to-neonatal transfer of immunity against T. spiralis and T cell lymphokines may help mediate this reaction. Continuing studies in our laboratory are focusing on the ultimate fate of the maternal T cells transmitted in milk and the mechanisms by which they mediate immunity in the neonate. Acknowledgements The authors wish to thank Mr. Chris Duggan and Larry Smart for their excellent technicial assistance. This work was supported in part by grants USPHS HD14358 and AA07381. References Appleton, J.A. and McGregor, D.D. (1987) Characterization of the immune mediator of rapid expulsion to Trichinella spiralis in suckling rats. Immunology 62, 477-484. Archambault, D., Morin, G., Elazhary, Y., Roy, R.S. and Joncas, J.H. (1988) Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes. Am. J. Vet. Res. 49(7), 1084-1091. Bell, R.G., Korenaga, M. and Wang, C.H. (1987) Characterization of a cell population in thoracic duct lymph that adoptively transfers rejection of adult Trichinella spiralis to normal rats. Immunology 61, 221-227. Bottomly, K. (1988) A functional dichotomy in CD4+ T lymphocytes. Immunol. Today. 9, 268-274. Grencis, R.K., Riedlinger, J. and Wakelin, D. (1985) L3T4-positive T lymphoblasts are responsible for transfer of immunity against Trichinella spiralis in mice. Immunology 56, 213-218. Head, J.R., Beer, A.E. and Billingham, R.E. (1977) Significance of the cellular component of the maternal immunologic endowment in milk. Transplant. Proc. 9, 1465-1471. Head, J.R. and Seelig, L.L. Jr. (1983) Autoradiographic analysis of lymphocyte migration into the mammary epithelium and milk of lactating female rats. J. Reprod. Immunol. 5, 61-72. Keller, M.A., Faust, J., Relevic, L.J. and Stewart, D.D. (1986) T-cell subsets in human colostrum. J. Gastroenterol. Nutr. 5, 439-443. Krco, C.J., David, C.S. and Wassom, D.L. (1982) Solubilized Trichinella spiralis antigens, Role of Ia antigens and Ly - l+ cells. Cell. Immunol. 68, 359-367. 10 Kumar, S.N., Seelis, L.L. Jr. and Head. J.R. (1985) Mieration of radiolabeled. adontivelv transferred T-lymphocytes into the mammary gland and milk if lactating rats. J. Reprod.‘Immunol. 8, 235-248. Kumar, S.N., Stewart, G.L., Steven, W.M. and Seelig, L.L. Jr. (1989) Maternal-to-neonatal transmission of T cell-mediated immunity to Trichinella spiralis during lactation. Immunology 68, 87-92. Lee, C.S., Wooding, F.B.D. and Kemp, P. (1980) Identification, properties and differential counts of cell populations using electron microscopy, of dry cows secretions, colostrum and milk from normal cows. J. Dairy Res. 47, 39-50. Liew, F..Y...(l989), Functional heterogeneity of CD4 + T cells in leishmaniasis. Immunol. Today, 40-45.
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14 Manson-Smith, D.F., Bruce, R.G. and Parrott, D.M.V. (1979) Villous atrophy and expulsion of intestinal Trichinellu spiralis are mediated by T cells. Cell. Immunol. 47, 285-292. 15 Miller, H.R.P., Nawa, Y. and Parish, C.R. (1979) Intestinal goblet cell differentiation in Nipposrrongylus-infected rats after transfer of fractionated thoracic duct lymphocytes. Int. Arch. All Appl Immunol. 59, 281-285. 16 Ogra, S.S., Weintraub, D. and Ogra, P.L. (1977) Immunologic aspects of human colostrum and milk III. Fate and absorption of cellular and soluble components in the gastrointestinal tract of the newborn. J. Immunol. 119(l), 245-248. 17 Perry, R.H. (1974) Transfer of immunity to Trichindu spiralis from mother to offspring J. Parasitol. 60(3), 460-465. 18 Riedlinger, J., Grencis, R.K. and Wakelin, D. (1986) Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo. Immunology 58, 57-61. 19 Sedmak, J.J. and Grossberg, S.G. (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue 9-250. Anal. Biochem. 79, 544-552 20 Seelig, L.L., Jr. and Billingham, R.E. (1981) The capacity of “transplanted” lymphocytes to traverse the intestinal epithelium of adult rats. Transplantation 32, 308-314. 21 Seelig, L.L. Jr. and Head, J.R. (1987) Uptake of lymphocytes fed to suckling rats. An autoradiographic study of the transit of labeled cells through the neonatal gastric mucosa. J. Reprod. Immunol 10, 285-297. 22 Smith, J.W. and Schultz, R.D. (1977) Mitogen- and antigen-responsive milk lymphocytes. Cell. Immunol. 29, 165-173. 23 Stewart, G.L., Wood, B.G. and Boley, R.B. (1985) Modulation of host response by Trichinella pseudospirdis. Parasite Immunol. 7, 223-233.
